Peptide inhibitor FIELD The present disclosure relates to a novel method of inhibition of bacterial virulence factors. There is also provided a novel cohort of inhibitor peptides and uses thereof. BACKGROUND Pseudomonas aeruginosa is an environmental bacterium that colonises a wide range of aquatic and terrestrial habitats. It is a well-known opportunistic human pathogen causing both acute and chronic diseases. Moreover, it is one of the leading causes of chronic infection in cystic fibrosis where its ability to adapt and thrive in different environments makes it incredibly difficult to treat [1, 2]. The survival strategies of P. aeruginosa include its ability to form complex biofilms and secrete numerous virulence factors [3-6]. Secreted virulence factors specifically enhance pathogen survival in a host context and for P. aeruginosa encompass: EPS (extracellular polymeric substances), which include the exopolysaccharides alginate, Pel and Psl; OMV (outer membrane vesicles) [7, 8]; siderophores, such as pyoverdine and pyochelin; toxins such as exotoxin A and pyocyanin; and lytic enzymes, such as proteases and nucleases [9]. Many of these secreted factors cause cell and tissue damage, interfere with host defences and promote bacterial growth and proliferation. Secreted proteases are considered major virulence factors of P. aeruginosa. These include an alkaline protease (AprA); elastases (LasA and LasB); a lysin-specific endopeptidase (Protease IV, also referred to as LysC) [9, 10]. LasB degrades collagens, causing severe tissue damage [11]. It also interferes with host defences by degrading several innate immune components [12, 13]. AprA degrades many host proteins including fibronectin and laminin, playing an important role in invasion and tissue necrosis. It is an important protein to escape bacterial defences by degrading complement proteins and cytokines [14]. Protease IV is mainly associated with virulence in corneal infections [15, 16]. It is involved in tissue damage and invasion by degrading fibrinogen. Recently, attention has been directed to exploring other secreted proteases that may contribute to P. aeruginosa success and complement the activity of the endopeptidases described above. The secreted exopeptidase, Pseudomonas aeruginosa aminopeptidase, PaAP (pepB, PA14_26020), is one of the most abundant proteins in the biofilm matrix [17]. Interestingly, PaAP is also the major protein associated with P. aeruginosa OMVs. These spherical bilayered phospholipids encapsulate proteins, lipopolysaccharides and DNA, and 1 54821242-1 have been implicated in important biological functions such as virulence and nutrient acquisition [7]. PaAP is annotated as an aminopeptidase, which removes amino acids from the N-termini of peptides, with a preference for leucine [18]. It is linked to biofilm development, where it is suggested to play a role in nutrient recycling, leading to its characterisation as a ‘public good’ enzyme (an enzyme which benefits both the producing and non-producing micro-organisms) [19]. Its expression is dependent on the stress sigma factor RpoS and is affected by quorum- sensing (QS) systems [20-22]. Moreover, PaAP requires post-translational processing to become an active enzyme. Firstly, it is secreted through the type-II secretion system (also regulated by the QS system) into the extracellular matrix [4]. Once secreted, PaAP undergoes proteolytic processing by other extracellular matrix proteases, such as truncation of the short C-terminal pro-peptide region likely by LysC [23]. Prior to our work, a precise molecular role for C-terminal truncation was absent. SUMMARY The present disclosure is based on the finding that an aminopeptidase (having a protease associated (PA) domain and a (catalytic) protease domain (PD)) is self-regulated whereby the C-terminal portion of the aminopeptidase sequence binds in a cleft between the PA domain and the PD thereby inhibiting enzyme activity. Additionally, it has now been shown that disrupting the interaction between the C-terminal portion of the aminopeptidase sequence and its binding location between the PA and PD, represents a mechanism by which the aminopeptidase may be switched from being inhibited (and inactive) to uninhibited and active. An active aminopeptidase is capable of, for example, cleaving amino acids from peptides or proteins. In view of the above, an aminopeptidase of the present disclosure may comprise: a protease associated (PA) domain; and a (catalytic) protease domain (PD)). The protease associated domain may be selected from the (bacterial) protease associated domain superfamily; the protease domain may comprise at least one bacterial member of the protease domain superfamily. An aminopeptidase of this disclosure may additionally or further comprise: 2 54821242-1 a C-terminal sequence; and a C-terminal sequence binding region disposed or located between the PA and PD. The aminopeptidase may be a bacterial aminopeptidase. The aminopeptidase may be a Pseudomonas aeruginosa aminopeptidase. The aminopeptidase may be a Vibrio cholera aminopeptidase. The aminopeptidase may be a Bacillus cereus aminopeptidase. The aminopeptidase may be a Bacillus subtilis aminopeptidase. The aminopeptidase may be a Bacillus anthracis aminopeptidase. The aminopeptidase may be a Mycobacteroides abscessus aminopeptidase. The aminopeptidase of the disclosure may not be a Dipeptidyl-peptidase IV The aminopeptidase may be Pseudomonas aeruginosa aminopeptidase, PaAP (pepB, PA2939). PaAP is secreted by Pseudomonas aeruginosa, is one of the most abundant proteins in a P. aeruginosa biofilm matrix and removes amino acids from the N-terminus of peptides with a preference for leucine. Pseudomonas aeruginosa aminopeptidase, PaAP (pepB, PA2939) may have the sequence provided below as SEQ ID NO: 1. SEQ ID NO: 1 MSNKNNLRYALGALALSVSAASLAAPSEAQQFTEFWTPGKPNPSICKSPLLVSTPLGLPR CLQASNVV KRLQKLEDIASLNDGNRAAATPGYQASVDYVKQTLQKAGYKVSVQPFPFTAYYPKGPGSL SATVPQPV TYEWEKDFTYLSQTEAGDVTAKVVPVDLSLGAGNTSTSGCEAEDFANFPAGSIALIQRGT CNFEQKAE NAAAAGAAGVIIFNQGNTDDRKGLENVTVGESYEGGIPVIFATYDNGVAWSQTPDLQLHL VVDVVRKK TETYNVVAETRRGNPNNVVMVGAHLDSVFEGPGINDNGSGSAAQLEMAVLLAKALPVNKV RFAWWGAE EAGLVGSTHYVQNLAPEEKKKIKAYLNFDMIGSPNFGNFIYDGDGSDFGLQGPPGSAAIE RLFEAYFR LRGQQSEGTEIDFRSDYAEFFNSGIAFGGLFTGAEGLKTEEQAQKYGGTAGKAYDECYHS KCDGIANI NQDALEIHSDAMAFVTSWLSLSTKVVDDEIAAAGQKAQSRSLQMQKSASQIERWGHDFIK The C-terminal sequence may comprise residues 513-536. This sequence is extracted below as SEQ ID NO: 2: AQSRSLQMQKSASQIERWGHDFIK 3 54821242-1 The C-terminal sequence may comprise residues 513-531. This sequence is extracted below as SEQ ID NO: 3: AQSRSLQMQKSASQIERWG The C-terminal sequence further comprises a short sequence which binds to a region (e.g. a cleft) located between the PA domain and peptidase domain (PD). Within the context of this disclosure, that region shall be referred to as the C-terminal sequence binding region. The C- terminal sequence which binds the region located between the PA domain and peptidase domain (PD) may comprise residues 527-536. The amino acid sequence of that region may comprise SEQ ID NO: 4 below: SEQ ID NO: 4 IERWGHDFIK The Vibrio cholera aminopeptidase, may have the sequence provided below as SEQ ID NO: 5. SEQ ID NO: 5 YWDDPQWVANKVSQSISRSDVVDHLQTLEGIASPTPDGTSLTRAAGSQGYQESVDYIIAT MKELGYEV TTQEFDFRSWTELGGTKLNVAGVDLISAKQAPEGTEGDFSVLSYAGSSNGELTGELVFIT PDFDFSSP NYDGSDGCEASDFTGIDLQGKIAVIQRGTCGFSDKVVNAQKAGAKAVIVFNQGNSAGRTG LFSGTLSN TSTATIPAFGVTFQLGKNWFDAAQSAAIPVTLTLNVDDKMIVTQNVLAETRKGNPDQIVM LGAHLDSV PEGPGINDNGSGTAGLLEYAEKLAKLKVPVKNKVRFAWWAAEEAGLVGSNHYTKTLFEPI YQQAQQQI MDELGISDPAQLTEAQKDLVEARYTQLNKIKLYLNFDMIGSPNYIFGVMDGDLSDTKDSP DNAYTGDF KPPFGTSDIELRFNQFFTDKGEGTIPQALSKRSDYAGFADWGVAFGGLFTGAEKTKTAEE VVKFGGEI DVAYDHCYHQACDDLNNISQKALYVNTQALAYVTTYYAMSKTLFPAEVSAQPKTMAKQSF RIQPVEKH QIGTTLKAAHSESDHGHFHGDFDQEKF* The C-terminal sequence may comprise residues 551-571. This sequence is extracted below as SEQ ID NO: 6: KAAHSESDHGHFHGDFDQEKF The C-terminal sequence further comprises a short sequence which binds to a region (e.g. a cleft) located between the PA domain and peptidase domain (PD). Within the context of this 4 54821242-1 disclosure, that region shall be referred to as the C-terminal sequence binding region. The C- terminal sequence which binds the region located between the PA domain and peptidase domain (PD) may comprise residues 556-570. The amino acid sequence of that region may comprise SEQ ID NO: 7 below: SEQ ID NO: 7 ESDHGHFHGDFDQEK The Bacillus cereus aminopeptidase, have the sequence provided below as SEQ ID NO: 8. SEQ ID NO: 8 MKKSLKQKIVSSLLAVSLAVSLAPIGQAKADSTSEITQTSSITKQVDASRAIEHIRFLSE TIGPRPGG TKSEEWASRYVGMQLKSMGYEVEYQPFQVPDQYVGFIESPLSTKRNWQAGAAPNALISTE SVTAPLIF VQGGTKLEDIPNEVNGKIVLFERGTTVADYNKQVENAVSKGAKGVLLYSLIGGRGNYGQT FNPRLTKK QSIPVFGLAYAQGNAFKEEIAKKGTTILSLKARHESNLTSLNVIAKKKPKNSTGNEKAVV VSSHYDSV VGAPGANDNASGTGLVLELARAFQNVETDKEIRFIAFGSEETGLLGSDYYVNSLSQKERD RILGVFNA DMVATNYDKAKNLYAMTPNGSPNLVTDAALQAGKQLNNDLVLQGKFGSSDHVPFAEVGIP AALFIWMG VDSWNPLIYHIEKVYHTPQDNVFENISPERMKMALEVIGTGVYNTLQKPVAQTEQKAA The C-terminal sequence may comprise residues 440-466. This sequence is extracted below as SEQ ID NO: 9: KMALEVIGTGVYNTLQKPVAQTEQKAA The C-terminal sequence further comprises a short sequence which binds to a region (e.g. a cleft) located between the PA domain and peptidase domain (PD). Within the context of this disclosure, that region shall be referred to as the C-terminal sequence binding region. The C- terminal sequence which binds the region located between the PA domain and peptidase domain (PD) may comprise residues 453-466. The amino acid sequence of that region may comprise SEQ ID NO: 10 below: SEQ ID NO: 10 TLQKPVAQTEQKAA The Bacillus subtilis aminopeptidase, have the sequence provided below as SEQ ID NO: 11. SEQ ID NO: 11 5 54821242-1 MKKLLTVMTMAVLTAGTLLLPAQSVTPAAHAVQISNSERELPFKAKHAYSTISQLSEAIG PRIAGTAA EKKSALLIASSMRKLKLDVKVQRFNIPDRLEGTLSSAGRDILLQAASGSAPTEEQGLTAP LYNAGLGY QKDFTADAKGKIALISRGDLTYYEKAKNAEAAGAKAVIIYNNKESLVPMTPNLSGNKVGI PVVGIKKE DGEALTQQKEATLKLKAFTNQTSQNIIGIKKPKNIKHPDIVYVTAHYDSVPFSPGANDNG SGTSVMLE MARVLKSVPSDKEIRFIAFGAEELGLLGSSHYVDHLSEKELKRSEVNFNLDMVGTSWEKA SELYVNTL DGQSNYVWESSRTAAEKIGFDSLSLTQGGSSDHVPFHEAGIDSANFIWGDPETEEVEPWY HTPEDSIE HISKERLQQAGDLVTAAVYEAVKKEKKPKTIKKQMKAKASDIFEDIK The C-terminal sequence may comprise residues 437-455. This sequence is extracted below as SEQ ID NO: 12: KTIKKQMKAKASDIFEDIK The C-terminal sequence further comprises a short sequence which binds to a region (e.g. a cleft) located between the PA domain and peptidase domain (PD). Within the context of this disclosure, that region shall be referred to as the C-terminal sequence binding region. The C- terminal sequence which binds the region located between the PA domain and peptidase domain (PD) may comprise residues 444-466 or 446-466. The amino acid sequence of those regions may comprise SEQ ID NO: 13 or 14 below: SEQ ID NO: 13 and 14 KAKASDIFEDIK/KASDIFEDIK The Bacillus anthracis aminopeptidase, have the sequence provided below as SEQ ID NO: 15. SEQ ID NO: 15 MSVRENRRIGEEKMKKSLKQKIVSSLLAVSLAVSLAPIGQANADSTSEIKQTSSITKQVD ASRAIEHI RFLSETIGPRPGGTKSEEWASRYVGMQLKSMGYEVEYQPFQVPDQYVGFIESPLSTKRNW QTGAAPNA LISTESVTAPLIFVQGGTKLEDIPNEVNGKIVLFERGTTVADYNKQVENAVSKGAKGVLL YSLIGGRG NYGQTFNPRLTKKQSIPVFGLAYAQGNAFKEEIAKKGTTILSLKARHESNLTSLNVIAKK KPKNSTGN EKAVVVSSHYDSVVGAPGANDNASGTGLVLELARAFQNVETDKEIRFIAFGSEETGLLGS DYYVNSLS 6 54821242-1 PKERDRILGVFNADMVATNYDKAKNLYAMMPNGSPNLVTDAALQAGKQLNNDLVLQGKFG SSDHVPFA EVGIPAALFIWMGVDSWNPLIYHIEKVYHTPQDNVFENISPERMKMALEVIGTGVYNTLQ QSVTQTEQ KAA The C-terminal sequence may comprise residues 466-479. This sequence is extracted below as SEQ ID NO: 16: TLQQSVTQTEQKAA SEQ ID NO: 16 is also comprises the C-terminal sequence which binds to a region (e.g. a cleft) located between the PA domain and peptidase domain (PD). Within the context of this disclosure, that region shall be referred to as the C-terminal sequence binding region. Mycobacteroides abscessus aminopeptidase, have the sequence provided below as SEQ ID NO: x SEQ ID NO: x MATNRVLYAVAVSAACALATVTACDRDAAEAPPRSAPQAGSEEAVGFAHALHEKVTVDNV VKHLSALQEI ADKNNNTRAAGTAGFDQSVDYVVKALKDKGFDVQTPEFSFKYFQAKSLDLTVGPKKVDAG VLSYSPGGRV EGRLVTARAEESPGCTVEDYDGLDVKGAVVLVDRGSCPFADKERVAAERGAAAVIIADNV DENKTSGTLG EDSSPKIPVVSVTKSVGADLRAHPDKVVLNVDAETKDVKARNVIAQTKTGATTDVVMAGA HLDSVPEGPG INDNGTGTAAVLETALQLGPSPDVKNAVRFAFWGAEEEGLIGSTDYVKSLDVDALKNIAL YLNYDMLGSP NAAYLTYDGDQSDEPDPNEVPVRIPEGSAGIERTEVAYLAEQGKKAHDTGYDGRSDYDAF SRAGIPTGGI FSGAEDKMSDEEAKQWGGKAGQPFDPNYHQAGDTLANVNKDALKINAGGVAYTVGLYAQS IDGRNGVPVH EDRTRHQLKG The C-terminal sequence may comprise residues 491-500. This sequence is extracted below as SEQ ID NO: y: EDRTRHQLKG Any of the aminopeptidases disclosed herein may comprise an isolated aminopeptidase. Any of the disclosed aminopeptidases may comprise a recombinant aminopeptidase. As stated, binding between the C-terminal sequence and the C-terminal sequence binding region inhibits the aminopeptidase and/or renders it inactive. Without wishing to be bound by theory, it is suggested that this binding event (between the C-terminal sequence and the C- terminal sequence binding region) inhibits the aminopeptidase by inducing a conformational change, stabilising a closed conformation and blocking access to the active site. This effectively locks the aminopeptidase into an inhibited (or inactive) conformation. 7 54821242-1 In view of the above, the disclosure provides a method for generating an active aminopeptidase, wherein the aminopeptidase comprises a protease associated (PA) domain, a catalytic protease domain (PD) and a C-terminal sequence, said method comprising modifying the C-terminal sequence to disrupt, inhibit or prevent binding between the C- terminal sequence and a C-terminal sequence binding region between the PA domain and the PD. In one teaching, the C-terminal sequence is modified relative to a reference sequence, for example any one of the sequences disclosed herein (including any of SEQ ID NOS 1-16 disclosed above). The modification made to the C-terminal sequence of the aminopeptidase disrupts, inhibits or prevents the C-terminal sequence from binding to the C-terminal binding region located or disposed between the PA domain and the PD of the amino peptidase. The abovementioned method may yield a constitutively active aminopeptidase. The C-terminal sequence may be modified by inclusion of one or more mutations to the all or part of the C-terminal sequence. These mutations may be made relative to a reference or wild type sequence. The reference sequence may comprise the wild type sequence of an aminopeptidase. For example, where the amino peptidase is the Pseudomonas aeruginosa aminopeptidase, PaAP (pepB, PA2939), the reference sequence may comprise the sequence provided herein as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. Where the aminopeptidase is a Vibrio cholera aminopeptidase the reference sequence may comprise the sequence provided herein as SEQ ID NOS: 5-7. Where the aminopeptidase is a Bacillus cereus aminopeptidase the reference sequence may comprise the sequence provided herein as SEQ ID NOS: 8-10 Where the aminopeptidase is a Bacillus subtilis aminopeptidase the reference sequence may comprise the sequence provided herein as SEQ ID NOS: 11-14 Where the aminopeptidase is a Bacillus anthracis aminopeptidase the reference sequence may comprise the sequence provided herein as SEQ ID NOS: 15-16. By way of example, and relative to a reference sequence, a modified or mutated C-terminal sequence of an aminopeptidase may comprise: (i) one or more amino acid substitution(s); (ii) one or more amino acid deletion(s); (iii) one or more amino acid addition(s)/insertion(s); (iv) one or more amino acid/sequence inversions; and/or 8 54821242-1 (v) one or more amino acid/sequence duplications. As stated, whatever the modification, the functional effect of the modification is to (i) prevent the C-terminal sequence of the aminopeptidase from occupying the C-terminal sequence binding site located between the PA domain and the PD and (ii) to yield an active aminopeptidase (because the modified aminopeptidase is unable to self-regulate). The modified the C-terminal sequence may be truncated. One of skill will appreciate that there are many ways in which an amino acid sequence can be truncated. For example, the aminopeptidase sequence may be modified by the inclusion of a protease cleavage site within the relevant part of the sequence. Use of the appropriate protease will then result in cleavage of the protein sequence resulting in a truncated or shortened fragment. For example, an aminopeptidase sequence may be modified by inclusion of a thrombin cleavage site. The protease (e.g. thrombin) cleavage site may be located at the appropriate point of the C- terminal sequence and can be used to remove the relevant part of the C-terminus to produce a truncated aminopeptidase sequence. A modified Pseudomonas aeruginosa aminopeptidase with a thrombin cleavage site may have the following sequence (SEQ ID NO: 17): SEQ ID NO: 17 MSNKNNLRYALGALALSVSAASLAAPSEAQQFTEFWTPGKPNPSICKSPLLVSTPLGLPR CLQASNVV KRLQKLEDIASLNDGNRAAATPGYQASVDYVKQTLQKAGYKVSVQPFPFTAYYPKGPGSL SATVPQPV TYEWEKDFTYLSQTEAGDVTAKVVPVDLSLGAGNTSTSGCEAEDFANFPAGSIALIQRGT CNFEQKAE NAAAAGAAGVIIFNQGNTDDRKGLENVTVGESYEGGIPVIFATYDNGVAWSQTPDLQLHL VVDVVRKK TETYNVVAETRRGNPNNVVMVGAHLDSVFEGPGINDNGSGSAAQLEMAVLLAKALPVNKV RFAWWGAE EAGLVGSTHYVQNLAPEEKKKIKAYLNFDMIGSPNFGNFIYDGDGSDFGLQGPPGSAAIE RLFEAYFR LRGQQSEGTEIDFRSDYAEFFNSGIAFGGLFTGAEGLKTEEQAQKYGGTAGKAYDECYHS KCDGIANI NQDALEIHSDAMAFVTSWLSLSTKVVDDEIAAAGQKLVPRGSQMQKSASQIERWGHDFIK SEQ ID NO: 17 contains a thrombin cleavage site (LVPRGS (SEQ ID NO: 6)) at residues 513- 518. As such, the present disclosure provides a method for generating an active, for example constitutively active, aminopeptidase, wherein the aminopeptidase is the Pseudomonas aeruginosa aminopeptidase, PaAP (pepB, PA2939), said method comprising modifying the C- terminal sequence of the aminopeptidase to disrupt, inhibit or prevent binding between the C- terminal sequence and the C-terminal sequence binding region located or disposed between 9 54821242-1 the aminopeptidase PA domain and the PD.In one teaching, the C-terminal sequence is modified by deletion of one or more amino acid residues in order to yield a truncated C-terminal sequence. As stated, the C-terminal sequence of the Pseudomonas aeruginosa aminopeptidase, PaAP (pepB, PA2939) occupies residues 513-536 of SEQ ID NO: 1. The C-terminal sequence further comprises a short sequence which binds to the C-terminal sequence binding region (e.g. a cleft) located between the PA domain and peptidase domain (PD). The sequence which binds the C-terminal sequence binding region located between the PA domain and peptidase domain (PD) may comprise residues 527-536 (SEQ ID NO: 4) In view of the above, the disclosure provides a modified Pseudomonas aeruginosa aminopeptidase, wherein the amino peptidase comprises a protease associated (PA) domain and a (catalytic) peptidase domain (PD) and a truncated C-terminal sequence. The modified aminopeptidase may be a modified Pseudomonas aeruginosa aminopeptidase, PaAP (pepB, PA2939); a modified Pseudomonas aeruginosa PaAP (pepB, PA2939) aminopeptidase may comprise a truncated C-terminal sequence. The present disclosure also provides a method for generating an active, for example constitutively active, aminopeptidase, wherein the aminopeptidase is a Vibrio cholera aminopeptidase, said method comprising modifying the C-terminal sequence of the Vibrio cholera aminopeptidase to disrupt, inhibit or prevent binding between the C-terminal sequence and the C-terminal sequence binding region located or disposed between the aminopeptidase PA domain and the PD. In one teaching, the C-terminal sequence is modified by deletion of one or more amino acid residues in order to yield a truncated C-terminal sequence. As stated, the C-terminal sequence of the Vibrio cholera aminopeptidase occupies residues 551-571 of SEQ ID NO: 5. In view of the above, the disclosure provides a modified Vibrio cholera aminopeptidase, wherein the amino peptidase comprises a protease associated (PA) domain and a (catalytic) peptidase domain (PD) and a truncated C-terminal sequence. The modified Vibrio cholera aminopeptidase may be constitutively active. The present disclosure provides a method for generating an active, for example constitutively active, aminopeptidase, wherein the aminopeptidase is a Bacillus aminopeptidase, said method comprising modifying the C-terminal sequence of the Bacillus aminopeptidase to disrupt, inhibit or prevent binding between the C-terminal sequence and the C-terminal sequence binding region located or disposed between the aminopeptidase PA domain and the PD. 10 54821242-1 The present disclosure provides a method for generating an active, for example constitutively active, aminopeptidase, wherein the aminopeptidase is a Bacillus cereus aminopeptidase, said method comprising modifying the C-terminal sequence of the Bacillus cereus aminopeptidase to disrupt, inhibit or prevent binding between the C-terminal sequence and the C-terminal sequence binding region located or disposed between the aminopeptidase PA domain and the PD. In one teaching, the C-terminal sequence is modified by deletion of one or more amino acid residues in order to yield a truncated C-terminal sequence. As stated, the C-terminal sequence of the Bacillus cereus aminopeptidase occupies residues 440-466 of SEQ ID NO: 8. In view of the above, the disclosure provides a modified Bacillus cereus aminopeptidase, wherein the amino peptidase comprises a protease associated (PA) domain and a (catalytic) peptidase domain (PD) and a truncated C-terminal sequence. The modified Bacillus cereus aminopeptidase may be constitutively active. The present disclosure provides a method for generating an active, for example constitutively active, aminopeptidase, wherein the aminopeptidase is a Bacillus subtilis aminopeptidase, said method comprising modifying the C-terminal sequence of the Bacillus cereus aminopeptidase to disrupt, inhibit or prevent binding between the C-terminal sequence and the C-terminal sequence binding region located or disposed between the aminopeptidase PA domain and the PD. In one teaching, the C-terminal sequence is modified by deletion of one or more amino acid residues in order to yield a truncated C-terminal sequence. As stated, the C-terminal sequence of the Bacillus subtilis aminopeptidase occupies residues 437-455 of SEQ ID NO: 11. In view of the above, the disclosure provides a modified Bacillus subtilis aminopeptidase, wherein the amino peptidase comprises a protease associated (PA) domain and a (catalytic) peptidase domain (PD) and a truncated C-terminal sequence. The modified Bacillus subtilis aminopeptidase may be constitutively active. The present disclosure provides a method for generating an active, for example constitutively active, aminopeptidase, wherein the aminopeptidase is a Bacillus anthracis aminopeptidase, said method comprising modifying the C-terminal sequence of the Bacillus anthracis aminopeptidase to disrupt, inhibit or prevent binding between the C-terminal sequence and the C-terminal sequence binding region located or disposed between the aminopeptidase PA domain and the PD. In one teaching, the C-terminal sequence is modified by deletion of one or more amino acid residues in order to yield a truncated C-terminal sequence. As stated, the C-terminal sequence of the Bacillus anthracis aminopeptidase occupies residues 466-479 of SEQ ID NO: 15. In view of the above, the disclosure provides a modified Bacillus anthracis aminopeptidase, wherein the amino peptidase comprises a protease associated (PA) domain 11 54821242-1 and a (catalytic) peptidase domain (PD) and a truncated C-terminal sequence. The modified Bacillus anthracis aminopeptidase may be constitutively active. Any of the modified aminopeptidases of this disclosure may be constitutively active. Any of the modified aminopeptidases may be produced by recombinant means. As such, the disclosure provides an isolated modified recombinant aminopeptidase. A modified Pseudomonas aeruginosa PaAP (pepB, PA2939) aminopeptidase may lack all or part of its C-terminal sequence – in other words, all or part of the C-terminal sequence of SEQ ID NO: 1 or all or part of SEQ ID NO: 2. A modified Pseudomonas aeruginosa PaAP (pepB, PA2939) aminopeptidase with a truncated C-terminal sequence may comprise a C terminal sequence having any of the following sequences (that is a sequence comprising residues 1-512 of SEQ ID NO: 1 and any of the following sequences (given as SEQ ID NOS: 18-37 (and 37a/b and c) as the C-terminal sequence. SEQ ID NO: 18 AQSRSLQMQKSASQIERWGHDFI SEQ ID NO: 19 AQSRSLQMQKSASQIERWGHDF SEQ ID NO: 20 AQSRSLQMQKSASQIERWGHD SEQ ID NO: 21 AQSRSLQMQKSASQIERWGH SEQ ID NO: 22 AQSRSLQMQKSASQIERWG SEQ ID NO: 23 AQSRSLQMQKSASQIERW SEQ ID NO: 24 AQSRSLQMQKSASQIER SEQ ID NO: 25 AQSRSLQMQKSASQIE SEQ ID NO: 26 AQSRSLQMQKSASQI SEQ ID NO: 27 AQSRSLQMQKSASQ SEQ ID NO: 28 AQSRSLQMQKSAS SEQ ID NO: 29 AQSRSLQMQKSA SEQ ID NO: 30 AQSRSLQMQKS SEQ ID NO: 31 AQSRSLQMQK SEQ ID NO: 32 AQSRSLQMQ SEQ ID NO: 33 AQSRSLQM SEQ ID NO: 34 AQSRSLQ SEQ ID NO: 35 AQSRSL SEQ ID NO: 36 AQSRS 12 54821242-1 SEQ ID NO: 37 AQSR SEQ ID NO: 37a AQS SEQ ID NO: 37b AQ SEQ ID NO: 37c A A modified Pseudomonas aeruginosa PaAP (pepB, PA2939) aminopeptidase of this disclosure may lack the full length of the C-terminal sequence. Such a molecule may have (comprise, consist of or consist essentially of) the following sequence: SEQ ID NO: 38 MSNKNNLRYALGALALSVSAASLAAPSEAQQFTEFWTPGKPNPSICKSPLLVSTPLGLPR CLQASNVV KRLQKLEDIASLNDGNRAAATPGYQASVDYVKQTLQKAGYKVSVQPFPFTAYYPKGPGSL SATVPQPV TYEWEKDFTYLSQTEAGDVTAKVVPVDLSLGAGNTSTSGCEAEDFANFPAGSIALIQRGT CNFEQKAE NAAAAGAAGVIIFNQGNTDDRKGLENVTVGESYEGGIPVIFATYDNGVAWSQTPDLQLHL VVDVVRKK TETYNVVAETRRGNPNNVVMVGAHLDSVFEGPGINDNGSGSAAQLEMAVLLAKALPVNKV RFAWWGAE EAGLVGSTHYVQNLAPEEKKKIKAYLNFDMIGSPNFGNFIYDGDGSDFGLQGPPGSAAIE RLFEAYFR LRGQQSEGTEIDFRSDYAEFFNSGIAFGGLFTGAEGLKTEEQAQKYGGTAGKAYDECYHS KCDGIANI NQDALEIHSDAMAFVTSWLSLSTKVVDDEIAAAGQK A modified Vibrio cholera aminopeptidase may lack all or part of its C-terminal sequence – in other words, all or part of the C-terminal sequence of SEQ ID NO: 5 or all or part of SEQ ID NO: 6. A modified Vibrio cholera aminopeptidase with a truncated C-terminal sequence may comprise a C terminal sequence having any of the following sequences (that is a sequence comprising residues 1-550 of SEQ ID NO: 1 and any of the following sequences (given as SEQ ID NOS: 39-55 (and 55a/b and c)) as the C-terminal sequence. SEQ ID NO: 39 KAAHSESDHGHFHGDFDQEK SEQ ID NO: 40 KAAHSESDHGHFHGDFDQE SEQ ID NO: 41 KAAHSESDHGHFHGDFDQ SEQ ID NO: 42 KAAHSESDHGHFHGDFD SEQ ID NO: 43 KAAHSESDHGHFHGDF SEQ ID NO: 44 KAAHSESDHGHFHGD SEQ ID NO: 45 KAAHSESDHGHFHG SEQ ID NO: 46 KAAHSESDHGHFH 13 54821242-1 SEQ ID NO: 47 KAAHSESDHGHF SEQ ID NO: 48 KAAHSESDHGH SEQ ID NO: 49 KAAHSESDHG SEQ ID NO: 50 KAAHSESDH SEQ ID NO: 51 KAAHSESD SEQ ID NO: 52 KAAHSES SEQ ID NO: 53 KAAHSE SEQ ID NO: 54 KAAHS SEQ ID NO: 55 KAAH SEQ ID NO: 55a KAA SEQ ID NO: 55b KA SEQ ID NO: 55c K A modified Bacillus cereus aminopeptidase may lack all or part of its C-terminal sequence – in other words, all or part of the C-terminal sequence of SEQ ID NO: 8 or all or part of SEQ ID NO: 9. A modified Bacillus cereus aminopeptidase with a truncated C-terminal sequence may comprise a C terminal sequence having any of the following sequences (that is a sequence comprising residues 1-439 of SEQ ID NO: 1 and any of the following sequences (given as SEQ ID NOS: 56-78 (including 78a/b and c)) as the C-terminal sequence. SEQ ID NO: 56 KMALEVIGTGVYNTLQKPVAQTEQKA SEQ ID NO: 57 KMALEVIGTGVYNTLQKPVAQTEQK SEQ ID NO: 58 KMALEVIGTGVYNTLQKPVAQTEQ SEQ ID NO: 59 KMALEVIGTGVYNTLQKPVAQTE SEQ ID NO: 60 KMALEVIGTGVYNTLQKPVAQT SEQ ID NO: 61 KMALEVIGTGVYNTLQKPVAQ SEQ ID NO: 62 KMALEVIGTGVYNTLQKPVA SEQ ID NO: 63 KMALEVIGTGVYNTLQKPV SEQ ID NO: 64 KMALEVIGTGVYNTLQKP SEQ ID NO: 65 KMALEVIGTGVYNTLQK SEQ ID NO: 66 KMALEVIGTGVYNTLQ SEQ ID NO: 67 KMALEVIGTGVYNTL SEQ ID NO: 68 KMALEVIGTGVYNT 14 54821242-1 SEQ ID NO: 69 KMALEVIGTGVYN SEQ ID NO: 70 KMALEVIGTGVY SEQ ID NO: 71 KMALEVIGTGV SEQ ID NO: 72 KMALEVIGTG SEQ ID NO: 73 KMALEVIGT SEQ ID NO: 74 KMALEVIG SEQ ID NO: 75 KMALEVI SEQ ID NO: 76 KMALEV SEQ ID NO: 77 KMALE SEQ ID NO: 78 KMAL SEQ ID NO: 78a KMA SEQ ID NO: 78b KM SEQ ID NO: 78c K A modified Bacillus subtilis aminopeptidase may lack all or part of its C-terminal sequence – in other words, all or part of the C-terminal sequence of SEQ ID NO: 11 or all or part of SEQ ID NO: 12. A modified Bacillus subtilis aminopeptidase with a truncated C-terminal sequence may comprise a C terminal sequence having any of the following sequences (that is a sequence comprising residues 1-436 of SEQ ID NO: 11 and any of the following sequences (given as SEQ ID NOS: 79-93 (including 93a/b and c) as the C-terminal sequence. SEQ ID NO: 79 KTIKKQMKAKASDIFEDI SEQ ID NO: 80 KTIKKQMKAKASDIFED SEQ ID NO: 81 KTIKKQMKAKASDIFE SEQ ID NO: 82 KTIKKQMKAKASDIF SEQ ID NO: 83 KTIKKQMKAKASDI SEQ ID NO: 84 KTIKKQMKAKASD SEQ ID NO: 85 KTIKKQMKAKAS SEQ ID NO: 86 KTIKKQMKAKA SEQ ID NO: 87 KTIKKQMKAK SEQ ID NO: 88 KTIKKQMKA SEQ ID NO: 89 KTIKKQMK SEQ ID NO: 90 KTIKKQM 15 54821242-1 SEQ ID NO: 91 KTIKKQ SEQ ID NO: 92 KTIKK SEQ ID NO: 93 KTIK SEQ ID NO: 93a KTI SEQ ID NO: 93b KT SEQ ID NO: 93c K A modified Bacillus anthracis aminopeptidase may lack all or part of its C-terminal sequence – in other words, all or part of the C-terminal sequence of SEQ ID NO: 15 or all or part of SEQ ID NO: 16. A modified Bacillus anthracis aminopeptidase with a truncated C-terminal sequence may comprise a C terminal sequence having any of the following sequences (that is a sequence comprising residues 1-465 of SEQ ID NO: 1 and any of the following sequences (given as SEQ ID NOS: 94-104 (including 104a/b and c) as the C-terminal sequence. SEQ ID NO: 94 TLQQSVTQTEQKA SEQ ID NO: 95 TLQQSVTQTEQK SEQ ID NO: 96 TLQQSVTQTEQ SEQ ID NO: 97 TLQQSVTQTE SEQ ID NO: 98 TLQQSVTQT SEQ ID NO: 99 TLQQSVTQ SEQ ID NO: 100 TLQQSVT SEQ ID NO: 101 TLQQSV SEQ ID NO: 102 TLQQS SEQ ID NO: 103 TLQQ SEQ ID NO: 104a TLQ SEQ ID NO: 104b TL SEQ ID NO: 104c T Additionally, the disclosure provides a cohort of peptides, which inhibit aminopeptidase activity. For convenience, these peptides shall be collectively referred to as ‘inhibitor peptides’. An inhibitor peptide of this disclosure may comprise a sequence derived from or having sequence identity, homology or similarity to all or part of a C-terminal sequence of the 16 54821242-1 aminopeptidase, including any of the aminopeptidase, or amino peptidase C-terminal sequences, described herein. Inhibitor peptides of this disclosure may exhibit 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with all or part, for example the C-terminal part, of an aminopeptidase sequence of this disclosure. The amount of sequence identity between an inhibitor peptide of this disclosure and an aminopeptidase sequence (e.g. a C-terminal aminopeptidase sequence) of this disclosure may be calculated by aligning the sequences and determining the number of identical residues as a proportion or percentage of the total number of residues. No matter what the level of sequence identity, the inhibitor peptide should be functional, that is it should display or exhibit aminopeptidase inhibitor activity. An inhibitor peptide of this disclosure may comprise the following sequence: X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 (SEQ ID NO:105) Or X1 X2 X3 X4 X5 X6 X7 X8 X9 X10, X11, X12, X13, X14, X15 (SEQ ID NO:106) Wherein the variable residues X2-(optional) X10 may be selected according to the following tabulated options: The disclosure provides the following inhibitor peptides: 17 54821242-1 In another teaching, an inhibitor peptide of this disclosure may comprise any of the following sequences: In view of the above, the term “inhibitor peptide” embraces all of the peptides disclosed as SEQ ID NOS: 105-124. The term further includes fragments thereof having aminopeptidase inhibiting activity. Moreover, the term includes variants of any of SEQ ID NOS: 105-124 which variants also exhibit aminopeptidase inhibitor activity. Useful variants may exhibit 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of the inhibitor peptides described herein. The amount of sequence identity between an inhibitor peptide of this disclosure and a variant therefore may be calculated by aligning the sequences and determining the number of identical residues as a proportion or percentage of the total number of residues. No matter what the level of sequence identity, the variant inhibitor peptide should be functional, that is it should display or exhibit aminopeptidase inhibitor activity. An inhibitor peptide of this disclosure may comprise one or more modifications or structural alterations preventing the inhibitor from becoming a substrate for the aminopeptidase it 18 54821242-1 inhibits. One such modification or alteration may comprise using a cyclic form of the peptide and methods of cyclising peptides are well known in the art. In one embodiment, the cyclic peptide may comprise a lactam bridge. The peptides of the present embodiments may be cyclised via the formation of a lactam bridge between, for example, a Lys and a Glu amino acid side chain. In some embodiments, the peptide may be cyclised via an isopeptide bond between a Lys and a Glu amino acid side chain. In an alternative embodiment, the cyclic peptide may be a stapled peptide. Thus, any of the inhibitor peptides of the present disclosure may be cyclised via the formation of an all-hydrocarbon “staple.” Any variant inhibitor peptides and/or any inhibitor peptides designed to have some level of sequence identity or homology may be tested for inhibitor activity in any suitable assay. For example, an aminopeptidase may be contacted with a test inhibitor peptide, under conditions permiting an interaction (for example a binding event) between the test inhibitor peptide and the aminopeptidase. The user may then assess the level of aminopeptidase activity and if, in the presence of the test inhibitor peptide the aminopeptidase activity is lower than expected or lower as compared to some control value of aminopeptidase activity, the test agent may be identified as one which may have aminopeptidase inhibitor activity. It should be noted that a control value of aminopeptidase activity, may provide a value which is representative of a standard or normal amount of aminopeptidase activity (for example a level of activity reported in the absence of an inhibitor peptide of this disclosure). As such, the invention provides not only linear forms of any of the inhibitor peptides described herein but also cyclised forms thereof. In one teaching, the disclosure provides linear or cyclic peptide having the sequence ERWGHDFIK (SEQ ID NO: 125). In one teaching, the disclosure provides linear- ERWGHDFIK (SEQ ID NO: 125). In another teaching, the disclosure provides cyclic- ERWGHDFIK (SEQ ID NO: 125). In the case of cyclic-ERWGHDFIK, this has been shown to be a potent inhibitor of PaAP with a Ki of 22.8 nM. When compared to linear-ERWGHDFIK (SEQ ID NO: 125) which is also a PaAP inhibitor (Ki of 9.98 µM), cyclic-ERWGHDFIK has a Ki which is ~440 fold lower. The disclosure also provides linear or cyclic peptide having the sequence EDRTRHQLK (SEQ ID NO: 126). In one teaching, the disclosure provides linear- EDRTRHQLK (SEQ ID NO: 126). In another teaching, the disclosure provides cyclic- EDRTRHQLK (SEQ ID NO: 126). In the case of linear/cyclic- EDRTRHQLK, this has been shown to be a potent inhibitor of Vibrio cholera aminopeptidase. 19 54821242-1 The disclosure also provides linear or cyclic peptide having the sequence ESDHGHFHGDFDQEK (SEQ ID NO: 127). In one teaching, the disclosure provides linear- ESDHGHFHGDFDQEK (SEQ ID NO: 127). In another teaching, the disclosure provides cyclic- ESDHGHFHGDFDQEK (SEQ ID NO: 127) - cyclised via side chain of E to K (isopeptide). In the case of linear/cyclic- ESDHGHFHGDFDQEK, this has been shown to be a potent inhibitor of Vibrio cholera aminopeptidase. The disclosure also provides linear or cyclic peptide having the sequence EGFHGDFDQEK (SEQ ID NO: 128). In one teaching, the disclosure provides linear- EGFHGDFDQEK (SEQ ID NO: 128). In another teaching, the disclosure provides cyclic- EGFHGDFDQEK (SEQ ID NO: 128) - cyclised via side chain of E to K (isopeptide). In the case of linear/cyclic- EGFHGDFDQEK, this has been shown to be a potent inhibitor of Vibrio cholera aminopeptidase. The disclosure also provides linear or cyclic peptide having the sequence KAKASDIFEDIK (SEQ ID NO: 129). In one teaching, the disclosure provides linear- KAKASDIFEDIK (SEQ ID NO: 129). In another teaching, the disclosure provides cyclic- KAKASDIFEDIK (SEQ ID NO: 129) - cyclised via side chain of E to K (isopeptide). In the case of linear/cyclic- KAKASDIFEDIK this has been shown to be a potent inhibitor of Bacillus sp (e.g. Bacillus cereus, Bacillus substilis and/or Bacillus anthracis) aminopeptidase. The disclosure also provides linear or cyclic peptide having the sequence KASDIFEDIK (SEQ ID NO: 130). In one teaching, the disclosure provides linear- KASDIFEDIK (SEQ ID NO: 130). In another teaching, the disclosure provides cyclic- KASDIFEDIK (SEQ ID NO: 130) - cyclised via side chain of E to K (isopeptide). In the case of linear/cyclic- KASDIFEDIK this has been shown to be a potent inhibitor of Bacillus sp (e.g. Bacillus cereus, Bacillus substilis and/or Bacillus anthracis) aminopeptidase. Additionally, inhibitor peptides of this disclosure may include variants of any of the specific inhibitor peptide sequences disclosed herein. For example, a useful variant sequence may include one or more amino acid modifications that do not materially alter their function, such as modifications that improve the pharmacokinetic properties of the peptide without affecting its primary mechanism of action. These modifications may be made to the peptide regardless of whether in a linear or cyclic configuration. In one teaching, an inhibitor peptide sequence of this disclosure may include (relative to a reference sequence, for example any one of SEQ ID NOS: 105-130), one or more conservative amino acid substitutions. One of skill will appreciate that a conservative substitution is an amino acid replacement in a protein that changes a given amino acid to a different amino acid with similar biochemical properties (e.g. charge, hydrophobicity and size). By way of example, any of glycine, alanine, valine, leucine and/or isoleucine may be used in place of one another. Also, any one of serine, cysteine, threonine or methionine may be used in place of one another. Also, phenylalanine, tyrosine and tryptophan may be substituted with one another. Histidine, arginine and lysine may also be substituted with one another as could aspartate, glutamate, asparagine and glutamate residues. One of skill will appreciate that the precise choice of amino acid residue may depend on the exact properties that are to be conserved in the final peptide. 20 54821242-1 Inhibitor peptides of this disclosure may include variants (of any of the specific inhibitor peptide sequences disclosed herein) which variants include the substitution of one or more of the residues with an un-natural amino acid, including for example non-proteinogenic amino acids. For example, the un-natural amino acid ornithine may be used in place of a lysine residue. One of skill will be aware of the various un-natural amino acids and how they may be substituted for at least some of the (natural) amino acids residues of any of the inhibitor peptides described herein. A disclosure of available un-natural amino acids for use as substitutions can be found in Narancic et al (World Journal of microbiology and biotechnology, 35, Article number: 67 (2019)). In one teaching, the peptide of the present disclosure may comprise L- or D- amino acids. In one teaching, the peptide may comprise a combination of L- or D- amino acids. In some embodiments, the peptide of the present disclosure may comprise β-amino acids. In some embodiments, a peptide of the invention may comprise N-methylated amino acid or N-acetyl amino acids. In some embodiments, the peptide of the present disclosure may be, partially or fully, a peptidomimetic or peptoid. In some embodiments, a peptide of the invention may be lipidated and/or PEG-ylated. One of skill will appreciate that it is straightforward to test a fragment or variant for aminopeptidase inhibiting activity by contacting the fragment or variant to an aminopeptidase in the presence of a substrate for the aminopeptidase, and observing or determining whether the aminopeptidase acts on the substrate. If in the presence of a test inhibitor peptide, the aminopeptidase fails to act on its substrate, the test inhibitor peptide may be an aminopeptidase inhibitor. Any of the inhibitor peptides disclosed herein may be used to modulate the activity of an active aminopeptidase – including any of the aminopeptidases described herein. As such, the disclosure provides the use of any of the inhibitor peptides described herein for inhibiting an aminopeptidase. The disclosure further provides any of the inhibitor peptides disclosed herein for use in inhibiting an aminopeptidase. The disclosure further provides a method of inhibiting an aminopeptidase, said method comprising contacting an aminopeptidase with an inhibitor peptide described herein. The inhibitor peptides disclosed herein may be used to modulate the activity of the Pseudomonas aeruginosa aminopeptidase, PaAP (pepB, PA2939). In one teaching the Pseudomonas aeruginosa aminopeptidase, PaAP (pepB, PA2939) is an active Pseudomonas aeruginosa aminopeptidase, PaAP (pepB, PA2939). 21 54821242-1 Also disclosed is a method of modulating the activity of the: Pseudomonas aeruginosa aminopeptidase, PaAP (pepB, PA2939); a Vibrio cholera aminopeptidase; a Bacillus cereus aminopeptidase; a Bacillus subtilis aminopeptidase; a Bacillus anthracis aminopeptidase. said method comprising contacting the aminopeptidase, with an inhibitor peptide of this disclosure. In one teaching the aminopeptidaseis an active aminopeptidase. Also disclosed is an inhibitor peptide of this disclosure for use in medicine or for use as a medicament. In this regard, any of the inhibitor peptides of SEQ ID NOS: 105-130 or aminopeptidase inhibiting variants thereof may also be for use in medicine or for use as a medicament. Accordingly, the disclosure provides linear-ERWGHDFIK (SEQ ID NO: 125); and/or cyclic-ERWGHDFIK (SEQ ID NO: 125) linear-EDRTRHQLK (SEQ ID NO: 126) cyclic-EDRTRHQLK(SEQ ID NO: 126) linear-ESDHGHFHGDFDQEK (SEQ ID NO: 127) cyclic-ESDHGHFHGDFDQEK (SEQ ID NO: 127) linear-EGFHGDFDQEK (SEQ ID NO: 128) cyclic-EGFHGDFDQEK (SEQ ID NO: 128) linear-KAKASDIFEDIK (SEQ ID NO: 129) cyclic-KAKASDIFEDIK (SEQ ID NO: 129) linear-KASDIFEDIK (SEQ ID NO: 130) cyclic-KASDIFEDIK (SEQ ID NO: 130) for use in medicine or for use as a medicament. The disclosure further provides for the use of any of the inhibitor peptides described herein in the manufacture of a medicament for treating a disease or condition associated aminopeptidase expressing bacteria or a biofilm comprising an aminopeptidase expressing bacteria. The disease or condition may be a disease or condition caused or contributed to by strains of Pseudomonas aeruginosa which express the aminopeptidase, PaAP (pepB, PA2939). The disease or condition may be a disease or condition caused or contributed to by strains of Vibrio cholera, Bacillus sp, Bacillus cereus, Bacillus subtilis and/or Bacillus anthracis. 22 54821242-1 Also disclosed is a method of inhibiting a biofilm or for preventing biofilm formation on a surface, said method comprising contacting the biofilm or the surface with any of the inhibitor peptides described herein. Where the method is a method for preventing a biofilm forming on a surface, the method may comprise coating the surface with an inhibitor peptide of this disclosure. In one embodiment, the biofilm to be inhibited or prevented may comprise Pseudomonas aeruginosa, Vibrio cholera, Bacillus sp, Bacillus cereus, Bacillus subtilis and/or Bacillus anthracis. Additionally, the disclosure provides a method of removing a biofilm from a surface, said method comprising contacting the biofilm with an inhibitor peptide of this disclosure. In one embodiment, biofilm to be removed may comprise Pseudomonas aeruginosa, Vibrio cholera, Bacillus sp, Bacillus cereus, Bacillus subtilis and/or Bacillus anthracis. The disclosure further provides compositions comprising any one or more of the inhibitor peptides described herein. Said compositions may further comprise excipients, diluents and/or buffers. The compositions may be pharmaceutical compositions which are, for example, sterile and which may comprise pharmaceutically acceptable excipients, diluents and/or buffers. DETAILED DESCRIPTION The present disclosure is described by way of example with reference to figures, which show: Figure 1. Mechanism of auto inhibition a, The crystal structure of WT PaAP. The secondary structure is coloured in rainbow, blue N- terminus through to red C-terminus. Pink spheres are zinc ions which indicates the location of the active site. A short region of disorder between the C-terminus and the peptidase domain is shown by a dashed red line. b, Structure of PaAP with surface displayed demonstrates the closed inactive conformation. PA domain shown in turquoise and peptidase domain in blue. The active site (yellow residues) is occluded by the C-terminus (orange residues) which binds in a groove between the PA and peptidase domains. c, The structure of WT PaAP (blue) in comparison to the PaAP_T (red) which lacks the C-terminus, illustrates the conformational change of the PA domain which allows access to the active site. d, The activity of WT PaAP vs PaAP_T (solid circles) and PaAP_T mutants (white circles). PaAP shows higher activity upon removal of the inhibitory C-terminus. The inset panel on the left shows zoomed in view of v/Et values to show the reduced activity of other PaAP forms. 23 54821242-1 Figure 2. Kinetics of peptide degradation catalysed by PaAP. Time courses are shown for different peptide substrates. a, Schematic of a fully distributive reaction where PaAP releases a product every reaction cycle (left) or a mixed reaction where the first amino acids from the N-terminus are removed in a processive manner, so that for the first hydrolysis step(s) products remain bond to the enzyme and undergo next amino acid hydrolysis event. b, time course for ERWGHDFIK degradation; c, time course for ERWGHDFIK-NH3 degradation; d, time course for ERLGHDFIK degradation. Lines are fits to single (P0 peptide) or double exponential equations (to reflect formation and decay of intermediates). Figure 3. Structure guided inhibitor design. a, Structure of PaAP_T bound to cyclic ERWGHDFIK inhibitor. Cyclic ERWGHDFIK (yellow) binds between the PA domain (turquoise) and the peptidase domain (blue) directly above the active site (orange), similarly to the C-terminus in WT PaAP structure. b, H-bond network involved in binding cyclic ERWGHDFIK. The side chain of R194 makes an important interaction with the carboxyl group. c, Michaelis-Menten plot of PaAP_T in presence of increasing cyclic ERWGHDFIK concentration. The Ki of cyclic ERWGHDFIK is ~440 fold lower than the Ki of linear ERWGHDFIK. d, Michaelis-Menten plot of PaAP_T in presence of increasing linear ERWGHDFIK concentration. e, IC50 plot for linear ERWGHDFIK in comparison to N-terminally (acetylated) and C-terminally (amidated) modified peptides. Figure 4. PaAP in vivo assays. a, Growth curve of Wt Pseudomonas aeruginosa UCBPP-PA14 (blue) in comparison to ∆PaAP (PaAP deletion mutant, red) and ∆PaAP_PaAPoex (PaAP under the control of an arabinose-inducible PbAD promoter, green). Absorbance at OD 500 was used to monitor growth in casein medium supplemented with arabinose. Growth was carried out in the presence (no fill circle) and absence (solid fill circle) of cyclic ERWGHDFIK inhibitor. ∆PaAP mutant has a growth defect compared to wild type (Wt), whilst ∆PaAP_PaAPoex has a growth advantage compared to WT in the absence of inhibitor. The inhibitor suppresses growth of Wt and ∆PaAP_PaAPoex to the level of ∆PaAP. b, PaAP deletion shows a biofilm development phenotype when grown on a solid medium. Representative images shown of N=4 biological replicates. c, Representative images of LIVE/DEAD® BacLight™-stained and 24- and 48-hour old biofilms, grown with (+) and without (-) cyclic-ERWGHDFIK. Merge of green (live cells) and red (dead cells) channels shown. d, The percentage of fluorescent pixels above the background threshold for green and red channels plotted. In the presence of cyclic- ERWGHDFIK, there are more dead cells than live cells after 48 hours. Significant differences 24 54821242-1 assessed through an unpaired Mann-Whitney test, ** p<0.001, ****p<0.0001. N = 4 biological replicates with 10 random Z-stack images (40 data-points). Figure 5. Summary of PaAP activation. PaAP is secreted from the cell as an inactive enzyme. The C-terminus is a pro-peptide sequence that binds between the PA and peptidase domains, inducing a closed inactive conformation where the active site is occluded. A disordered, protease accessible region linking the C-terminus to the peptidase domain is removed (here showed as cleaved by LysC), allowing the pro-peptidase to be released and degraded. Removal of the C-terminus pro- peptide induces a conformational change where the PA domain rotates outwards, exposing the active site. Activated PaAP can then degrade unstructured peptides, releasing amino acids from the N-terminus. Figure 6: Toxicity test in human HepG2 cells, no toxicity observed up to 100 ^ ^M compound. The peptide (cyclic-ERWGHDFIK) is not toxic to human cells. Figure 7: Survival curves of WT Pseudomonas aeruginosa strain vs a strain in which our target protein was deleted. This is to validate target in an in vivo model. The peptide decreases survival in this in vivo C. elegans infection model. Figure 8: Panel A: Schematic representation of an in vivo C. elegans infection model Zone A (left side) contains Bacteria A and Zone A’ (right side) contains Bacteria B. Nematodes are added to the spot in the centre (in Zone D) and can choose where to go. Worms will avoid infectious bacteria, spending little time on “zone A” from bacteria that cause disease. Panel B: Discrimination index = (number in zone A – number in zone A’)/ total number. Shows that, nematodes prefer zone A’ of the PaAP deletion strain. We tested how much “avoidance” worms have towards a Wild type Pseudomonas aeruginosa strain (WT) vs a strain in which our target protein was deleted (PAAP). There is a strong avoidance in the WT but not on the deletion strain showing our target is signalling infection. Black bars represent a choice between E. coli and wild type P. aeruginosa. Figure 9: Vibrio cholerae’s target protein can also be inhibited via a similar strategy, with an inhibition constant (KD) of 1 ^ ^M. EXAMPLES Structure of PaAP reveals role of C-terminus in self inhibition 25 54821242-1 We solved the structure of PaAP in multiple forms, which revealed an interesting self-inhibitory mechanism involving the PA domain and the pro-peptide C-terminus (Fig.1a). The structure of full-length PaAP was solved to 1.4 Å, and traceable between residues 44 to 536 with a 16 aa region (QKAQSRSLQMQKSASQ) near the C-terminus which lacked density. However, the final 10 aa (IERWGHDFIK) of the C-terminus could be unambiguously traced (Fig.1b). The structure of PaAP is similar to other unpublished aminopeptidases that have been uploaded onto the PDB (PDB 5IB9, 6HC6, 3IIB). PaAP contains an M28 Zn peptidase domain (residues 44-116 and 274-510) and a PA domain (residues 117-273) (Fig.1). The aminopeptidase domain is composed of an 8 stranded ß-sheet surrounded by helices. The smaller mixed α/ß PA domain is attached to the peptidase domain via an extended ß-strand. There are three disulphide bonds; two in the peptidase domain, towards the N- and C- termini; and one in PA domain. The active site contains two zinc ions; Zn1 is coordinated by D306, H296 and D369; whilst Zn2 is coordinated by E341, H467 and D306. Coordinated between both Zn ions is a water molecule, which is proposed to be activated (OH-) during the catalytic mechanism. Residues, E340 and Y466 complete the active site. Interestingly, in the structure of PaAP, following a short region of disorder, the C-terminus (IERWGHDFIK, residues 527-536) binds in the cleft between the PA and peptidase domain (Fig.1b). This short pro-peptide region is structured, folding with a hairpin turn. A short (1-2aa) ß-sheet of 4 strands, runs through the C-terminus and PA domain. The carboxy terminus interacts with the sidechain of R194. Sidechains of H532 and D533 of the ß-hairpin coordinate a water molecule and are positioned directly above the active site pocket. The location of the C-terminus appears to block access to the active site residues. We solved the structure of truncated enzyme form lacking the final C-terminal residues, PaAP_T, to 2.35 Å, which revealed a large conformational change. Superimposition of the peptidase domains of full-length PaAP and PaAP_T demonstrates the different orientation adopted by the PA domain due to the removal of the C-terminus. The PA domain undergoes a ~30° rotational transition in comparison the full-length PaAP (20 Å translation at its furthest point). The conformational change increases the opening between the PA and peptidase domain, causing the active site to be more accessible (Fig.1c). The rotational swing occurs from the base of the two extended beta strands that project from the peptidase domain (starting at P161 and E272). This finding suggests the inhibitory binding of the C-terminus between the PA and peptidase domain traps the enzyme in an inhibited state, where the active site is occluded. Interestingly, the modified C-terminus (thrombin cleavage site, LVPR) is fully traceable in the structure and binds to the PA domain of an adjacent protomer. The interaction 26 54821242-1 of the C-terminus with an adjacent PA domain aids crystal packing, the protomers are linked together, akin to a chain. The interaction between the PA and C-terminus similarly requires R189 to bind the carboxy terminus. To investigate how PaAP interacts with a peptide substrate we attempted to co-crystalise an inactive variant PaAP_TE340A with different peptides. After solving the structures of a number of PaAP_TE340A:peptide complexes, unambiguous density for inhibitory C-terminus was present (located between the PA and peptidase domain, alike the full-length PaAP structure). This was surprising as intact mass spectrometry results confirmed the E340A mutation in PaAP_TE340A was successful and the C-terminus was not present. Because intact mass spectroscopy is performed under denaturing conditions, only the intact protein mass would be determined and not the mass of the protein associated with other factors, such as ions or bound peptides. Therefore, after protease cleavage, the C-terminal pro-peptide remained associated with PaAP_TE340A, which suggests in active PaAP_T the peptide is degraded. Interestingly, we also solved the structure of PaAP_TE340A(trunc), mutant form where the thrombin cleavage step was not required to remove C-terminus (truncated form). In this crystal form PaAP_T E340A forms a crystallographic dimer, where the C-term and PA domains of adjoining promoters are docked head to tail. The PA domain in this crystal form undergoes a larger conformational change compared to PaAP_T (-40° rotation), which suggests the PA domain is free to sample multiple conformations when not restricted by interactions with the C-terminus. Kinetic analysis of PaAP Purified recombinant PaAP, which encompassed residues 27-536 relating to the full-length secreted protein (i.e. lacking the N-terminal signal peptide), was initially used. To assay the activity of PaAP we employed the well-established aminopeptidase assay with p-nitroanilide coupled amino acids (aa-pNA) as substrates. Full-length (FL) PaAP had low activity (Fig.1d), consistent with previous reports that PaAP is secreted as a 58 kDa pro-peptide (Pro-PaAP), which requires C-terminal processing to yield an active enzyme [24]. As a result, we truncated the C-terminus (residues 513-531) by incorporating a C-terminal thrombin cleavage site. This allowed the selective and efficient removal of the C-terminus during purification to produce PaAP_T (a C-terminal truncated form of PaAP). The thrombin cleavage site avoided complications which can arise when attempting to overexpress and purify a constitutively active aminopeptidase from the cytoplasm of E. coli cells. PaAP_T was highly active in comparison to full-length PaAP (increase of -100 fold), suggesting the C-terminus is involved in suppressing PaAP activity, which agreed with previous studies [23, 24]. 27 54821242-1 We tested the activity of PaAP_T against several commercially available aa-pNA substrates . All substrates had similar KM values, whereas kcat values varied considerably. The kcat was highest for Leu-pNA, followed by Lys-pNA, whilst the other substrates tested had much lower kcat values. The efficiency of the enzyme (kcat/KM) for each substrate suggests Leu-pNA and Lys-pNA as the most preferable substrates. Enzyme activity was optimal at pH values > 8.0, with the activity decreasing drastically below neutral pH, in a profile akin to other zinc-dependant metalloproteases [25]. Data fitting yielded two ionizable groups with an apparent pKa of approximately 7.5 both on kcat and kcat/KM. Because this pKa value is likely too low to be the substrate N-terminal amine, this suggests deprotonated active site residues are required for substrate binding and catalysis, in keeping with the generally accepted catalytic mechanism which requires an active site glutamate to act as a general base and deprotonate a water molecule coordinated by the two active site zinc molecules [26, 27]. To probe the catalytic mechanism in PaAP we tested the activity of two active site mutants, E340A and Y466F. No activity was detected for the E340A mutant. On the other hand, the Y466F mutant had a significantly reduced kcat and increased KM, which is reflected in the kcat/KM being considerably lower than PaAP_T (0.69 min-1mM-1 compared to 301 min-1mM-1). These results support E340 to act as the general base, and Y466 to potentially stabilize the negative charge on the peptide tetrahedral intermediate by hydrogen bonding to the carboxylate oxygen atom [26, 27]. The activity of an additional mutant, R189A (located in the PA domain and interacting directly with the C-terminus of the full-length protein) was tested to investigate its role in catalysis. R189A had comparable kcat and KM values to PaAP_T suggesting that, at least in terms of the minimal aa-pNA substrate, it is not involved substrate binding and/or catalysis. PaAP displays catalytic activity in a standard assay buffer, with no requirement to supplement metal ions, suggesting it copurified with metal ions. Structure determination by X-ray crystallography and X-ray fluorescence spectrum of crystals unambiguously confirmed two zinc ions are bound in the active site. Nevertheless, we assayed PaAP in the presence of different metal ions and EDTA. Excess zinc reduced catalytic activity to a similar degree as EDTA treatment. This may be explained by additional zinc ions binding to allosteric sites and having an inhibitory affect. The crystal structure identified additional cation binding sites; of which, site 1 (residues D382, D384, S386 and E400) strongly coordinates a zinc ion in the different crystal forms. PaAP cleaves peptides both processively and distributively 28 54821242-1 The aa-pNA assay is a convenient and simple direct assay, however, the small peptide mimic is less complex and possesses a p-nitro aniline leaving group, not comparable to an amino acid or peptide. Therefore, to better understand the activity of PaAP on substrates more closely resembling physiological conditions, we developed a discontinuous assay using peptide substrates. Several peptides, varying in length and composition, were incubated with PaAP overnight in a standard reaction buffer, alongside a control (PaAP omitted). The samples were analysed by LC-MS or MALDI; samples were deemed to be substrates if there was a mass shift between the control and those incubated with PaAP. Our results demonstrated that PaAP was capable of processing peptides of varying lengths (2 aa up to 25 aa) and composition. This includes a casein derived peptide (α-casein (90-95), RYLGYL) and its own C-terminus (ERWGHDFIK). However, more complex peptides (containing secondary structure and disulphide bonds), such as human defensins, were not processed. Peptides with modified termini (N-terminal acetylation and C-terminal amidation) were tested as substrates. Whilst a peptide with an acetylated N-terminus was not a substrate of PaAP, a peptide harbouring a C-terminal amide (ERWGHDFIK-NH3) was a substrate. To investigate the activity of PaAP further, we performed a timecourse assay with a selection of peptide substrates. Curves for ERWGHDFIK, ERWGHDFIK-NH3 and ERLGHDFIK are shown in Fig.2, while curves for KWLGYL, KA-AMC (Lys-Ala-methylcoumaryl-7-amide) and HCATIPAFDG are not shown. The assay was quenched at different time-points and samples analysed by LC-MS. The initial substrate was broken down sequentially into smaller peptide products which could be detected by LC-MS using extracted ion chromatograms for each peptide products/intermediates. The time course showed a complex pattern in which parent peptide decreased over time, while intermediates (akin to truncation products) appeared and were consumed throughout the time course. This suggests that after cleaving the first N- terminal amino acid (P0), PaAP could utilize the product (P-1) as substrate for further rounds of hydrolysis, and this process occurred for subsequent peptide intermediates until single amino acids were generated. Because of the pattern and rates of formation and decay of each peptide intermediate are different for peptides with varying sequence, we conclude PaAP does not function processively, at least not exclusively. A processive enzyme can catalyse consecutive reaction cycles without releasing its substrate. In contrast, a distributive enzyme releases a product every reaction cycle. [28] We hypothesised that the PA domain may be involved in binding and presenting substrates to the active site during catalysis besides its regulatory role. We tested a peptide substrate containing a C-terminal amide (ERWGHDFIK-NH3) in the time course assay. Surprisingly, ERWGHDFIK-NH3 was processed at a faster rate than unmodified ERWGHDFIK, suggesting that the interaction between the PA domain and the peptide substrate (R189 of PA domain 29 54821242-1 interacts directly with carboxy terminus) does not exclude substrates that lack a free C- terminus. However, linear ERWGHDFIK, which is a substrate (Fig. 2b) also inhibits PaAP activity (Fig.3d). Therefore, the peptide can bind in an inverted orientation (EI complex) but can also bind in a standard orientation in which the peptide N-terminus residue binds to the active site zinc ions (ES complex). We hypothesize that carboxy terminus amidation reduces the likelihood of forming an inverted (EI) complex. Indeed, ERWGHDFIK-NH3 showed no inhibition at concentrations up to 50 µM in the Leu-pNA assay, much higher than observed for other peptide inhibitors (Fig. 3e). We also performed time courses with the R189A mutant alongside PaAP_T to compare the kinetics of peptide substrates. R189A mutant degrades both ERWGHDFIK and KWLGYL peptides to slower degree compared to PaAP_T. Moreover, the R189A mutant accumulates mid stage breakdown products which had been completely turned over by PaAP_T (WGHDFIK and GHDFIK) after 24 hours. This reduction in rate and accumulation of mid stage breakdown products suggests R189 may have a role in binding and/or catalysis of peptide substrates. Comparison of time courses with different peptide substrates (Fig.2) reveals a distinct pattern of substrate consumption and accumulation of intermediates. Fitting data to exponential equations revealed that while some substrates behave exclusively in a distributive manner (RWGHDFIK), meaning each reaction cycle releases a peptide product which then competes with other potential substrates for enzyme binding and for the next catalytic cycle, others did not. In a processive reaction, peptide intermediates do not accumulate over time, as there is one binding event for P0 followed by peptide bond cleavage without product release in subsequent cycles. Here, for most peptides investigated, the first few amino acids were removed likely in a processive manner, since intermediates for those were not observed during the time course. After a few amino acids were removed, intermediates accumulated. For example, P-1 did not accumulate for ERWGHDFIK and ERWGHDFIK-NH3, but all later products (P-2 to P¬7) accumulated later on. Alternatively, ERLGHDFIK didn’t accumulate P- 1 or P-2 but did accumulate products from P-3 to P-7. HCATIPAFDG mainly accumulated products from P-5. This is consistent with a mixed system, in which the first few amino acids are removed processively and subsequent amino acids removed in a distributive manner. Processive kinetics was observed in a Xaa-Pro aminopeptidase involved in protein degradation in yeast. [29] Proteolysis of proteins Previous studies using refolded PaAP determined it could perform autoprocessing in cis, meaning one molecule of PaAP could cleave its own N-terminal residues, but not the N- terminal residues from another PaAP molecule [24]. Assertions about substrate preference 30 54821242-1 were based on assays conducted using aa-pNA substrates [23]. We hypothesised that PaAP could prune residues from proteins with accessible N-termini, since peptides of longer lengths were substrates. To test this, we incubated PaAP with several proteins with known ordered and disordered N-termini and analysed by intact protein mass spectrometry. PaAP preferentially removes the N-terminal methionine residue from a few of the proteins tested (WP_010598044 and WP_077070634). In one instance, PaAP was able to liberate a few residues (GMQQ) from the N-terminus of BtCDPS up to the start of known secondary structure (PDB: 6ZTU). By comparing the intact mass of PaAP_T and PaAP_TE340A, we determined the N-terminus of PaAP is autoprocessed in the active protein. Intact mass spec results showed PaAP_T had three mass peaks (m/z = 51881.0, 51504.6 and 51319.3), corresponding to species starting with TEFWTPGKP, WTPGKP and TPGKP respectively (removal of 7, 10 and 11 residues from N-terminus (- GSEAQQF, - GSEAQQFTEF and - GSEAQQFTEFW, respectively) and the same thrombin cleaved C-terminus (ending VPR). On the other hand, inactive PaAP_T_E340A had a single peak (m/z = 52570.2), corresponding to a species with a thrombin cleaved C-terminus (ending VPR) and a TEV cleaved N-terminus (starting GSE), with a E340A mutation. Although PaAP could cleave the unstructured N-termini of some proteins, our data supports that its own N-terminal processing occurred in cis as previously described [24]. Structure guided inhibitor design The activity of PaAP is proposed to complement other secreted proteases P. aeruginosa and recycle nutrients. Additionally, PaAP is abundantly expressed by isolates from cystic fibrosis lungs and promotes vesicle association [30]. Therefore, we used our structural findings to develop an inhibitor that mimicked the self-inhibitory conformation adopted by the full-length protein. We first tested a linear peptide (linear-ERWGHDFIK), based on the C-terminus of PaAP. We hypothesised linear-ERWGHDFIK would inhibit PaAP_T activity (active form). Indeed, linear-ERWGHDFIK inhibited the activity of PaAP in the Leu-pNA assay with a KI of 9.98 µM (Fig. 3d). However, linear-ERWGHDFIK would not be a viable inhibitor as we previously showed it also acts as a substrate for PaAP. Guided by the structure of PaAP, we designed a cyclic peptide (cyclic-ERWGHDFIK) cyclised via an isopeptide bond between the glutamate and lysine sidechains. Cyclisation between the sidechains was preferred to preserve the carboxy terminus of the lysine residue and introduce minimal stress on the backbone angles. Cyclic-ERWGHDFIK was a potent inhibitor of PaAP with a KI of 22.8 nM, which is ~440 fold lower than the linear peptide (Fig.3c). Moreover, cyclic-ERWGHDFIK was not degraded when incubated overnight with PaAP. 31 54821242-1 The structure of PaAP_T in complex with cyclic-ERWGHDFIK (PaAP_T(ERWGHDFIK)) was solved to confirm enzyme:inhibitor interactions. The structure of PaAP_T(ERWGHDFIK) is comparable to the structure of full-length PaAP, adopting the same closed/inactive conformation (RMSD = 0.3610 Å over 934 residues). Cyclic-ERWGHDFIK was predictably bound in the groove between the PA and peptidase domain, almost identically to the C- terminus bound in the full-length structure (Figs.3a and 3b). Complete electron density was visible for the inhibitor, including the sidechain linkage between K and E. The inhibitor complex structure suggests inhibitor binding induces a conformational change, stabilising the closed conformation, and blocking access to the active site. Next, we hypothesised the interaction between R194 and the peptide carboxy terminus to be important for inhibition. To test this, we produced the PaAP mutant R194A, to eliminate a likely salt bridge between the peptide COOH and the guanidinium of the protein arginine sidechain. Indeed, the IC50 for linear-ERWGHDFIK with R194A mutant was determined as 306 μM, a 100-fold increase compared to full-length PaAP. Furthermore, no inhibition was detected for cyclic-ERWGHDFIK within the inhibitor range tested (0.05 μM to 100 μM), and an IC50 value could not be determined. This suggests the interaction between R194 and the carboxy terminus is crucial for inhibition, in contrast to the little effect seen on substrate binding. In addition, the ERWEGHDFIK-NH3 peptide was not an inhibitor of PaAP_T within the concentration of peptide tested, although it was shown to be a good substrate. This finding suggests the carboxy terminus is key for the interaction with the PA domain, which likely has a regulatory function. PaAP expression and activity impact growth on complex nutrient sources and biofilm development PaAP was previously reported to play a role in biofilm development and growth on a complex nutrient source. A PaAP deletion mutant (L1PaAP) showed increased propidium iodide staining and moderately reduced CFU/ml in a pellicle biofilm assay and reduced growth in minimal medium with casein as a sole carbon and nitrogen source [31]. We generated a clean deletion of the PA14_26020 locus in the UCBPP-PA14 strain background of P. aeruginosa (L1PaAP) to determine the baseline behaviour in the complete absence of PaAP activity, and also reintroduced the pa14_26020 gene under the control of the arabinose-inducible PBAD promoter at the attB locus(PBAD-PaAP). We then investigated the effects of modulating PaAP expression levels or inhibiting its activity using the cyclic-ERWGHDFIK peptide on cellular phenotypes in P. aeruginosa. First, we investigated the impact of PaAP expression and activity on growth when casein was the sole source of carbon and nitrogen (Fig.4a). As expected, the 4PaAP strain showed a 32 54821242-1 substantial growth defect in this medium. The PBAD-PaAP strain grew slightly better than the 4PaAP strain even in the absence of arabinose, likely due to leaky expression from the PBAD promoter. The addition of arabinose caused growth of the PBAD-PaAP strain to surpass that of the wild type, suggesting that PaAP expression and activity may be limiting for growth under this condition. In the presence of 10 µM cyclic-ERWGHDFIK and arabinose, growth of both the wild type and the PBAD-PaAP strain were inhibited (although not to the same degree as the 4paAP strain) while the 4PaAP strain was unaffected. In the presence of 100 µM cyclic- ERWGHDFIK, the wild type and PBAD-PaAP strain grew only slightly more than the 4PaAP strain, indicating nearly complete inhibition of PaAP for providing amino acids from casein to support growth. Next, we investigated the roles of PaAP in a more complex biofilm context. We grew the wild type and 4PaAP strains on 1% tryptone agar supplemented with Congo Red and Coomassie Blue to observe colony morphology as described previously [32]. We observed subtle differences, with reduced wrinkling of the 4PaAP strain in the centre of the colony, supporting a role for PaAP activity in organisation and development of biofilm structure (Fig.4b). Finally, we repeated the propidium iodide staining of pellicle biofilms that previously showed increased staining for a 4PaAP strain in the PAO1 background [31]. As expected, we also observed increased propidium iodide staining in our 4PaAP strain in 48-hour old biofilms. Importantly, wild type biofilms grown in the presence of cyclic-ERWGHDFIK also increased propidium iodide staining after 48 hours to a similar extent as the 4PaAP strain, consistent with substantial inhibition of PaAP activity even in this complex environment and over a long time period (Figs.4c and 4d). PaAP activity Until this study, all investigations into the activity of PaAP were conducted with small molecule substrates, aa-pNA. Although these substrates provide a convenient way to assay peptidase activity, they employ minimalistic substrate mimics, and do not necessarily recapitulate trends observed with peptide substrates. Our results using aa-pNA substrates agree somewhat with previous reports, where Leu and Lys-pNA are preferred over other amino acids. However, the protein used in previous studies was not homogeneous and multiple species were present depending on extracellular processing [18, 23, 24, 35]. Instead, we purified recombinant PaAP and mutant forms, ensuring enzyme homogeneity. Leu-pNA was preferred compared to the other minimal substrate tested, which is consistent with previous studies. Moreover, we tested the activity of PaAP against a variety of peptide substrates. We observed the removal of amino acids from the N-terminus of peptides over time by LC-MS. Some peptide intermediates accumulated to a larger extent than others (those with Trp or His at the N-terminus) suggesting 33 54821242-1 these residues are unfavourable substrates. Ultimately theses peptide intermediates with N- terminal Trp or His residues were also degraded. This was also exemplified by the ERLGHDFIK peptide (W replaced by L) which was degraded at a faster rate than ERWGHDFIK. We tested PaAP activity against different protein substrates and concluded PaAP can liberate amino acids from proteins with accessible N-termini, although not all proteins tested could be processed. Moreover, it appears that PaAP preferentially removes the N-terminal methionine from these proteins. However, in two cases additional residues were removed; these include the removal of GMQQ from the N-terminus of BtCDPS and the auto-processing of PaAP’s own N-terminus to remove GSEAQQFTEFW. Auto-processing was identified by comparing intact mass spec results between active PaAP_T and inactive PaAP_TE340A, whereby a 10aa sequence is cleaved in PaAP_T. Interestingly, the thrombin cleaved C-terminus of PaAP_TE340A (confirmed by intact MS) remained as a bound ligand in the crystal structure, suggesting the liberated C-terminus pro-peptide is degraded by active PaAP. Regulation of PaAP occurs at transcriptional and post-translational level PaAP transcription is tightly regulated. It is directly affected by three global regulators: the stress sigma factor RpoS [22]; the quorum sensing regulator LasR [21, 36]; and an RNA polymerase-binding protein that is upregulated under anoxic conditions, SutA [37]. All of these regulators are active in the high-density, nutrient-and oxygen-limited context of a biofilm, where total levels of gene expression activity may be low but efficient utilisation of limited resources is important. As a result, PaAP is one of the most abundant proteins in the biofilm matrix and in OMV, which are a major component of the P. aeruginosa biofilm matrix [17, 30]. PaAP has been proposed to function in biofilm development [31], biofilm remodelling [7] and acquisition of nutrients from complex sources [19]. As a result PaAP has been termed a “public good” enzyme, secreted by some members of the biofilm to benefit the bacterial community more broadly [19]. Since P. aeruginosa forms high-density aggregates in many environments including infections, a better understanding of factors contributing to metabolism and developmental decisions in such contexts is needed. PaAP activity is also post-translationally regulated (Fig. 5). It is first secreted by the type II secretion system, which is also under QS control [4, 38]. A further level of regulation is imposed by the C-terminal pro-peptide sequence of PaAP, which must be proteolytically removed [24]. Removal of the C-terminus is believed to be performed by LysC, although elastase and alkaline phosphatase may also be involved, by indirect activation of LysC [23]. Here, we demonstrate, at the molecular level, how the C-terminal pro-peptide regulates the activity of PaAP. The structure of PaAP reveals the C-terminus binds in a groove between the 34 54821242-1 PA and peptidase domains, which blocks access to the active site and locks these 2 domains into a closed conformation. When the C-terminus was truncated, PaAP adopted an open conformation, where the PA domain is free to rotate, uncovering the active site. We also considered the PA domain may be involved in binding and delivering peptides into the active site, analogous to PDZ domain function in carboxy terminal processing proteases [35], due to its propensity to interact with the carboxy terminus of peptides in our different crystal forms. Indeed, the PaAP_TR189A mutant showed a modest reduction in the rate of peptide degradation but retained a similar kcat upon the minimal Leu-pNA substrate. Additionally, ERWGHDFIK degradation accumulates the intermediates WGHDFIK and GHDFIK, when incubated with PaAP_TR194A, but not the WT enzyme. This further suggests the PA domain is involved in interacting with peptide substrates. In contrast, the ERWGHDFIK C-terminus NH3 modified peptide, was degraded faster than the peptide containing the free carboxyl group. This could be due to ERWGHDFIK-NH3 peptide not interacting with PaAP as an inhibitor, while linear-ERWGHDFIK does. This is supported by the increased IC50 value of ERWGHDFIK-NH3 compared to ERWGHDFIK. Moreover, N-terminally modified Ac- ERWGHDFIK (which is not a substrate) is a stronger inhibitor (lower IC50) than unmodified linear substrate. This regulatory mechanism involving the C-terminus pro-peptide and the PA domain is likely to be a common feature of this class of aminopeptidases. A homologue deposited to the PDB (6HC6), shows a similar interaction between the C-terminus and the PA domain to what is seen in PaAP, suggesting that the insight gained here is generalisable to secreted aminopeptidases from other organisms. Potent inhibitor of PaAP impacts growth on casein and late-stage biofilms We rationally developed a potent inhibitor of PaAP based on the interaction between the C-terminus pro- peptide (ERWGHDFIK) and the PA and peptidase domains. The peptide was cyclised to increase stability and decrease conformational flexibility [36]. Indeed, cyclic-ERWGHDFIK was not cleaved by PaAP after prolonged incubation. In addition, cyclic-ERWGHDFIK was far more potent with a KI of 22.8 nM. Although higher concentrations were required in the more complex environments of bacterial growth and biofilm assays where cell density is high, we were able to show that the inhibitor affected phenotypes in liquid media and biofilms similarly to the deletion mutant lacking PaAP (Fig.4). PaAP has recently attracted attention as a complementary enzyme to other well-characterised major virulence factors. PaAP is one of the most abundant OMV proteins and is implicated in association of vesicles with lung cells [7, 30]. OMVs have diverse roles but are believed to contribute to deployment of virulence factors and nutrient acquisition for P. aeruginosa in 35 54821242-1 infections [8]. Given its demonstrated role in facilitating growth on a protein substrate (Fig.4a), PaAP may be a dominant contributor to the nutrient acquisition function of OMVs. Anti-virulence therapy is a relatively new strategy that has the ability to control the emergence and spread of resistant pathogens [39-41]. Several aminopeptidases have been described as virulence factors in multiple organisms. Examples are the arginine-specific aminopeptidase from Pseudomonas [42]; PepP from Campylobacter jejuni [43]; and aminopeptidase T from Listeria monocytogenes [44]. Moreover, the leucine aminopeptidase (LAP) from Staphylococcus aureus has been investigated as a drug target, where its suppression led to improved mouse survivability [45]. To our knowledge only the commercially available drug, Balsalazide, has been shown impact the activity of PaAP in vivo, where it displayed an antibiofilm phenotype [46]. Here we demonstrated our inhibitor, cyclic-ERWGHDFIK, also impacted pellicle biofilm development and growth on complex media. CONCLUSION Post translational processing is required for activation, as a C-terminal truncation mutant possessed ~100-fold higher activity than the full-length enzyme. PaAP is a promiscuous aminopeptidase acting on unstructured regions of peptides and proteins. High-resolution crystal structures of wild type enzyme and mutants revealed the mechanism of autoinhibition, whereby the C-terminal pro-peptide locks the protease-associated (PA) domain and the catalytic peptidase domain, into a closed, inhibited conformation. Inspired by this self- regulatory mechanism and guided by structural data, a highly potent small cyclic-peptide inhibitor (Ki in the nM range) was designed. This inhibitor recapitulates the deleterious phenotype observed with a PaAP deletion mutant in liquid cultures and biofilm assays and presents a path towards targeting secreted proteins in a biofilm context. METHODS Materials, chemicals and general Chemicals used in this investigation were purchased from Fisher and Sigma. Peptides tested as substrates and inhibitors were commercially obtained (Peptide Synthetics). Longer peptide substrates (HCATIPAFDG, VTATIAFPAYDGE, VGAGIGWPWTAEHVDQTLASGNDIC) were a kind gift from James Naismith (Oxford University). Peptides RYLGYL (α-casein(90-95)), 8- defensin 1 Human and 8-defensin 2 Human, where purchased from Sigma. Kinetic data were fitted using GraphPad Prism. Strains, media, and growth conditions 36 54821242-1 The clean deletion of PA14_26020 from the UCBPP-PA14 genome was carried out as previously described with minor variations [47]. Briefly, 600 bp upstream and downstream of the gene were cloned into the pMQ30 suicide vector using Gibson assembly and this was introduced by conjugation from E. coli into P. aeruginosa, where it integrated by homologous recombination into the genome adjacent to PA14_26020. Subsequently, colonies that lost the plasmid and the wild-type copy of the gene by homologous recombination were selected by plating on sucrose and confirmed by colony PCR using check primers that flanked the gene. To reintroduce the PA14_26020 gene sequence under control of the arabinose-inducible PBAD promoter at the attB site, the coding sequence and ribosome binding site for PA14_26020 were cloned into a previously described pUC18-miniTn7T-GmR variant encoding the PBAD promoter using Gibson assembly [33]. This plasmid was then conjugated from E. coli into P. aeruginosa along with a helper strain to catalyse recombination into the attB site as previously described [48] Media and growth conditions Lysogeny broth (LB) contained 10 g/L NaCl, 10 g/L tryptone, and 5 g/L yeast extract, solidified with 15 g/L agar for solid media. LB was supplemented with 100 µg/ml carbenicillin, 20 µg/ml gentamicin, or 50 µg/ml kanamycin as appropriate for selection of plasmids in E. coli. Phosphate-buffered minimal media with casein as a sole carbon and nitrogen source contained 35.9 mM K2HPO4, 14.2 mM KH2PO4, 42.8 mM NaCl, 1.0 mM MgSO4, 0.1 mM CaCl2, 0.5% (w/v) casein from bovine milk (Sigma Aldrich catalogue number C5890) and trace metals (7.5 μM FeCl2·4H2O, 0.8 μM CoCl2·6H2O, 0.5 μM MnCl2·4H2O, 0.5 μM ZnCl2, 0.2 μM Na2MoO4·2H2O, 0.1 μM NiCl2·6H2O, 0.1 μM H3BO3, and 0.01 μM CuCl2·2H2O). Media was sterile-filtered with a 0.2 mm filter but also autoclaved for 15 minutes to achieve solubilisation of the casein. This may have resulted in some partial degradation of casein. Cultures were incubated at 37°C with shaking unless otherwise indicated. Cloning, mutagenesis and constructs The gene encoding PaAP [PA2939 (PAO1 genome) / PA14_26020 (UCBPP-PA14 genome)] from Pseudomonas aeruginosa was synthesised as a gBlock from IDT and cloned into the pJ414 vector in-frame with a TEV cleavable N-terminal His6 tag using the Gibson assembly method. PaAP was cloned without the N-terminal signal peptide sequence (MSNKNNLRYALGALALSVSAASLAAP), so the full-length PaAP construct in this study starts from S27. Mutant and truncated proteins were produced by standard site directed mutagenesis. Constructs and mutations were confirmed by sequencing before transformation into the E. coli expression strain SHuffle® T7 by NEB.5 constructs of PaAP were used in this study: PaAP (WT, residues 27-536); PaAP_T (Thrombin cleavage site (LVPRGS) substituted 37 54821242-1 into position 513-518, Thrombin used to remove C-terminus); PaAP_TE340A (E340A mutation incorporated into PaAP_T construct); PaAP_TE340A(trunc) (stop codon TGA incorporated into PaAP_TE340A construct, so the C-terminus was truncated without requiring thrombin cleavage). Protein production and purification Cells transformed with PaAP constructs were grown at 37 °C (shaken at 180 rpm) in LB media supplemented with 100 μg/ml ampicillin until an OD600 of 0.8 was reached. Gene expression was induced with 1 mM IPTG and incubated over night at 18 °C (shaken at 180 rpm). Cells were harvested by centrifugation at 6000 RPM (Beckman JLA 8.1000 rotor) for 10 min and the pellets stored at −20 °C. Cells were re-suspended in fresh lysis buffer (50 mM HEPES pH 8.0, 20 mM Imidazole pH 8.0, 250 mM NaCl) and incubated with ~1 mg/ml lysozyme for 30 min at 4 °C. Cells were lysed using a high-pressured cell disruptor. Insoluble cell debris was removed by centrifugation for 30 minutes at 33,000 g (Beckman JA 25.50). The supernatant was loaded onto a 5 ml HisTrap HP column (GE Healthcare) pre-equilibrated with lysis buffer. The column was washed with 20 column volumes (CV) of lysis buffer and 10 CV 10% elution buffer solution (50 mM HEPES pH 8.0, 250 mM NaCl, 300 mM Imidazole pH 8.0) to remove non-specific interacting proteins.10 CV of elution buffer was pasted over the column to elute the His6-tagged PaAP. Fractions containing PaAP were pooled and an appropriate amount of TEV protease was added (1:50, PaAP:TEV) to remove the N-terminal tag. In constructs containing an incorporated thrombin cleavage site, thrombin was added (1:100, PaAP:thrombin), to remove the C-terminus. Pooled protease treated samples were dialysed over night against 2 L of dialysis buffer (25 mM Tris pH 8.5, 200 mM NaCl). Cleaved protein was separated from residual tagged protein by a second passage over the HisTrap column. Flow through and wash solution containing unbound protein were concentrated to 5ml and injected onto a 10/16 S200 size exclusion chromatography column for polishing (and to remove thrombin contamination when used). Fractions containing PaAP were pooled and concentrated to 10 mg/ml for further experiments. Structure determination Crystals were grown at 20°C using sitting drop vapour diffusion technique, with a drop size of 0.3 μl in a ratio of 2:1, reservoir:protein solution. Crystals were cryoprotected in mother liquor supplemented with 20% (v/v) ethylene glycol before being flash cooled in liquid nitrogen. Diffraction data was collected at the Diamond Light Source in Oxford, UK, on I03 and I04 beamlines. Data reduction and processing was completed using XDS and the xia2 suite. The structure was solved by molecular replacement with PHASER searching for 2 individual components (peptidase and PA domain). Search components comprised an ensemble of 3 38 54821242-1 PDBs (6HC6, 5IB9 and 1TKJ), which had been modified sculptor and manually truncated in COOT. Mutant and ligand complex structures [PaAP_T, PaAP_T(ERWGHDFIK), PaAP_TE340A and PaaP_TE340A(trunc)] were solved using the structure of WT PaAP as the search model in PHASER. Protein structures were built/ modified using COOT, with cycles of refinement in PHENIX. Enzymatic assay with aa-pNA substrates Aminopeptidase activity was determined spectrophotometrically primarily using amino Leu-p- nitroanilide (Leu-pNA). Other commercially available pNA substrates (isoleucine, methionine, alanine, valine, proline, arginine, lysine and phenylalanine) were also assayed. Leu-pNA was incubated with enzyme (100 nM) in assay buffer (50 mM Tris pH 8.5, 200 mM NaCl) at 25°C. p-nitroanilide release caused an absorbance increase at 405 nm, monitored using a BMG Labtech plate reader. Kinetic constants KM and kcat were determined from assays varying substrate concentration (between 5 mM to 31.25 μM). Data were plotted in Graphpad Prism and the Michalis Menten equation was used for fitting. Inhibitor studies Inhibition assays were carried out with the following peptides: linear ERWGHDFIK (PaAP C- terminal residues), cyclic ERWGHDFIK, N-acetylated and C-amidated ERWGHDFIK. IC50 values were determined by varying peptide concentration at a fixed substrate concentration (fixed at KM, [S] = 2mM, PaAP concentration was 100nM). KI was determined by assaying multiple fixed inhibitor concentrations (1000 nM, 500 nM, 250 nM, 100 nM, 50 nM, 25 nM and 10 nM) at varying substrate concentrations under standard reaction conditions with Leu-pNA as a substrate (outlined above). Ki values were determined by fitting the data to Eq. 1 (Morrison’s equation): Equation 1 where v is the velocity, Et is the total enzyme concentration, I is the concentration of inhibitor, and Ki is the equilibrium dissociation constant for the inhibitor. IC50 values increased when measured at increasing [S], consistent with competitive inhibition. Peptide digestion assay 39 54821242-1 A variety of peptides of varying length and composition were incubated with PaAP at room temperature. Assay were performed by incubating (over-night at room temperature) 500 μM of substrate (peptides) with 1 μM enzyme (PaAP) in reaction buffer 50 mM Tris pH 8.5, 100 mM NaCl. The reactions were quenched by heating to 100°C for 10 minutes followed by centrifugation at 14K rpm in a benchtop centrifuge to remove precipitate. The samples were analysed by LC-MS. Initial trial assays were carried out in the presence and absence (control) of enzyme to determine if the substrate was degraded (single or triplicate experiments). Timecourse assays were performed on selected peptide substrates in triplicate. A 300 μL reaction (including 500 μM of substrate) was initiated by addition of 1 μM enzyme. The reaction was incubated at room temperature. The reaction was quenched at set timepoints by the removal of 30 μL aliquots which were immediately heated to 100 C for 10 minutes. The samples were analysed by LC-MS using a Xevo Q-ToF (Waters) as described below, searching for masses of all peptide breakdown products. Data analysis for enzyme kinetics Peptide degradation time courses were fitted to exponential equations as follows: P0 peptide consumption was fitted to a single exponential equation (Equation 2) with the format Equation 2 Time courses for formation and decay of peptide intermediates were fitted to equations double exponential equations (3), single exponential equations followed by a linear phase (4) Equation 3 4 where y(t) is the product formed at time t, y0-Plateau is the amplitude of the exponential phase, k is the observed rate constant for the exponential phase, y0 is the value of y at time 0, Plateau is the value of y after exponential phase, C is the offset, and v is the slope of the linear phase. For pH dependence of kinetic parameters, data were fitted to two models, n=1 accounting for a single ionizable group that needs to be deprotonated for binding/catalysis and n=2 for two ionizable groups that need to be deprotonated for binding/catalysis. In Equation 5, y is the kinetic parameter, C is the pH-independent value of y, pH is the experimental pH, and pKa is the apparent acid dissociation constants for ionizing groups. 40 54821242-1 Equation 5 Protein digestion assay A variety of proteins (1 mg/ml), purified in house, were incubated in the presence or the absence of PaAP (0.01 mg/ml) O/N at room temperature in standard assay buffer (50 mM Tris pH 8.5, 100 mM NaCl). After incubation, samples were analysed by intact mass. Mass differences between +/- PaAP samples were evaluated by sequentially truncating the N- terminus. Intact mass spectrometry analysis ESI Protein samples were analysed by intact mass spec at the University of St. Andrews mass spectrometry and proteomics facility. The protein sample (10 µL, 1 µM) was desalted on-line through a MassPrep On-Line Desalting Cartridge 2.1 x 10 mm, using a Waters Acquity H class UPLC plus, eluting at 200 µL/min, with an increasing acetonitrile concentration (2 % acetonitrile, 98 % aqueous 1 % formic acid to 98 % acetonitrile, 2 % aqueous 1 % formic acid) and delivered to a Waters Xevo electrospray ionisation mass spectrometer which was calibrated using horse heart myoglobin. An envelope of multiply charged signals was obtained and deconvoluted using MaxEnt1 software to give the molecular mass of the protein. MALDI Peptide samples incubated in the presence and absence of PaAP, were initially analysed by MALDI. The peptide sample (0.5 µL, 10 µM) was spotted along with matrix (0.5 µL 10 mg/ml α-cyano-4-hydroxycinnamic acid in 50% ACN 50% 0.1%TFA) on a stainless steel MALDI plate and left to dry. The sample plate was loaded into a Sciex 4800 MALDI mass spectrometer. The laser voltage was adjusted to give a good signal to noise strength, and 50 shots were acquired in a random pattern across the spot, and spectra combined. The spectra was externally calibrated with Sciex Pepcal mix (500-4000 m/z range). Biofilm assay 41 54821242-1 Biofilms (pellicles) of Wt Pseudomonas aeruginosa PA14 were grown in an 8 well chambered cover glass slide (NuncTM Lab-TekTM II Chambered Coverglass). P. aeruginosa overnight culture was diluted 1/100 into Jensen’s medium [49] +/- 100 μM cyclic-ERWGHDFIK.200 μL was dispensed into each well and the chambered glass slides were placed in a static incubator set to 37 C for 24 h, 48 h to allow biofilms to form. After biofilms were formed the media was gently removed and the biofilms washed with 200 μL PBS. The biofilms were then stained using LIVE/ DEAD® BacLightTM Bacterial Viability Kits (Molecular Probes, Invitrogen) for 30 mins, in the dark. The stained biofilms were then washed twice with PBS to remove excess stain. To assess the number of live/dead cells, stained biofilms were imaged using a Leica SP8 confocal microscope (Leica Microsystems, Mannheim, Germany) with a 63x objective (HC PL APO 1.4 Oil). A 488 nm Argon laser was set to 20% power, PMT detectors set to 510- 540 nm and 620-650 nm with the gain set to 500V were used to detect SYTO 9 (all cells, green channel) and propidium iodide (dead cells, red channel), respectively. Four biological replicates were imaged by collecting 10 z-stacks (from bottom to top of biofilm; z-level interval 0.3 µm) from random areas of each well. The stacks were maximum intensity z-projected, and the resulting images analysed with the red and green channels treated separately. Images were thresholded to above the limit of background signal (i.e. the average fluorescence intensity in unstained control samples (8-bit grey value=30, N=10)). For both the red and the green channel, the percentage of pixels above the background threshold was calculated for each image. Images were analysed in ImageJ [50]. Data was plotted using GraphPad Prism 9.3. Colony morphology assay Cultures were grown overnight in LB medium and spotted in a 10-μL volume on solid media (1% tryptone, 1% agar, 20 μg/mL Coomassie blue, and 40 μg/mL Congo red) [29]. Plates were incubated at room temperature for 6 days and then photographed with a Canon EOS 700D SLR camera fitted to a Leica MZ12 stereoscope. Growth curves Growth curves were performed on a 96-well format. 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