The role of proteinaceous toxins secreted by Staphylococcus aureus in interbacterial competition

Abstract Staphylococcus aureus is highly adapted to colonization of the mammalian host. In humans the primary site of colonization is the epithelium of the nasal cavity. A major barrier to colonization is the resident microbiota, which have mechanisms to exclude S. aureus. As such, S. aureus has evolved mechanisms to compete with other bacteria, one of which is through secretion of proteinaceous toxins. S. aureus strains collectively produce a number of well-characterized Class I, II, and IV bacteriocins as well as several bacteriocin-like substances, about which less is known. These bacteriocins have potent antibacterial activity against several Gram-positive organisms, with some also active against Gram-negative species. S. aureus bacteriocins characterized to date are sporadically produced, and often encoded on plasmids. More recently the type VII secretion system (T7SS) of S. aureus has also been shown to play a role in interbacterial competition. The T7SS is encoded by all S. aureus isolates and so may represent a more widespread mechanism of competition used by this species. T7SS antagonism is mediated by the secretion of large protein toxins, three of which have been characterized to date: a nuclease toxin, EsaD; a membrane depolarizing toxin, TspA; and a phospholipase toxin, TslA. Further study is required to decipher the role that these different types of secreted toxins play in interbacterial competition and colonization of the host.

Staphylococcus aureus is an opportunistic human pathogen capable of causing disease at many sites in the body.This is mediated by an abundance of virulence factors, which allow the bacterium to invade host tissues and effectively evade the immune system (r e vie wed in T hamma v ongsa et al. 2015, Ho wden et al. 2023 ) ). S. aureus infections most commonly occur in immunocompromised individuals, with the bacterium usually entering the bloodstr eam thr ough a br eac h in the skin barrier, and can be lifethreating.While S. aureus infections are prevalent, particularly within healthcare settings, S. aureus disease is an outcome for only a very small proportion of individuals colonized by these bacteria, with around 30% of the population colonized at any one time (Wertheim et al. 2005 ). S. aureus has e volv ed a number of mechanisms to enable successful colonization of the host including imm une e v asion and nutrient acquisition strategies, and biosynthesis of adhesins that recognize human cell surface receptors.Ho w e v er, one of the first barriers S. aureus must overcome during colonization is the resident microbiota.
Whilst S. aureus can invade and persist in almost e v ery nic he within the human host, it is most commonly found to colonize the nasal cavity, v a ginal tr act and skin (Dancer 2008 ).The anterior nar es ar e a highl y competitiv e envir onment, whic h can harbour diverse bacterial species (Liu et al. 2015, Sc henc k et al. 2016, Kumpitsch et al. 2019 ).Commensal bacteria are highly adapted to compete with S. aureus , controlling S. aureus populations through pr oduction of anta gonistic autoinducing peptides (Se v ern et al. 2022, Williams et al. 2023 ), release of antibacterial compounds such as antibiotics and bacteriocins (Newstead et al. 2020, Zhao et al. 2022 ), and by competing for adhesion sites (Maciag et al. 2023 ).
S. aureus has, ther efor e, e volv ed counter measures to outcompete the r esident micr obiota to enable colonization of the host.S. aureus can compete with commensals for binding sites within the host, expr essing a lar ge n umber of surface-exposed adhesin-lik e molecules , which ha ve incredibly high affinity for host factors (Foster et al. 2014, Sakr et al. 2018 ). S. aureus is also highly adapted to the nutrient-poor environment of the nasal cavity through upregulation of several nutrient uptake systems (Krismer et al. 2017 ).Ho w e v er, S. aureus can also shape the microbiota by the secretion of antibacterial substances.
Antibiotics and bacterial toxins that are secreted by commensal organisms for the control of S. aureus infections have been studied extensiv el y (Ne wstead et al. 2020, Heinzinger et al. 2023 ).By contrast, less is understood about antibacterial compounds secreted by S. aureus and the role that these play in colonization of the host, which will be the focus of this review.

S. aureus secreted bacteriocins
Bacteriocins are antimicrobial proteins, or peptides, that are released by bacteria usually to target closely related bacterial species (Riley andWertz 2002 , Heilbronner et al. 2021 ).Bacteriocins can r ange fr om lar ge pr oteins suc h as colicins and pyocins, to small peptides less than 5 kDa in size.Large bacteriocins such as colicins and p y ocins are commonly produced by Gramnegative bacteria and usually require cell lysis for their release (Michel-Briand andBaysse 2002 , Cascales et al. 2007 ).By contrast, bacteriocins produced by Gram-positive bacteria are commonly secr eted fr om the cell by the gener al secr etory pathw ay or b y specialized tr ansport mac hineries (Ennahar et al. 2000, Gajic et al. 2003, Ishibashi et al. 2014 ).In ad dition, Gram-positi ve bacteriocins often have dedicated regulatory pathwa ys , decoupling host fate from bacteriocin production (Ennahar et al. 2000 , Riley andWertz 2002 ).A likel y r esult of this is the lar ge div ersity of bacteriocins produced by Gram-positive bacteria (Jack et al. 1995, Acedo et al. 2018 ).
Bacteriocins produced by staphylococcal species can be categorized into one of six groups .T he majority of staphylococcal bacteriocins are Class I bacteriocins, which are small ( < 5 kDa) heatstable peptides that ar e post-tr anslationall y modified, and include gr oups suc h as lantibiotics (Bierbaum and Sahl 2009 ).Class II bacteriocins are also small ( < 10 kDa) heat-stable peptides, which are not post-tr anslationall y modified (Nissen-Meyer et al. 2009 ).Class III bacteriocins are much larger ( > 30 kDa) proteins, which are heat-labile and can be subdivided as either lytic or non-lytic (Heng et al. 2007 ).Class IV molecules ar e post-tr anslationall y modified cyclic peptides (Van Belkum et al. 2011 ).
Mor e r ecent discov eries hav e identified two further bacteriocin groups, consisting of sanctipeptides and thiopeptides (Varella Coelho et al. 2017, Zheng et al. 2017 ).Whilst these have not yet been formally classified, it has been proposed that they constitute Class V and Class VI, r espectiv el y (De Fr eir e Bastos et al. 2020 ).A r ecent r e vie w suggested that the classification system for bacteriocins be simplified to two classes-either modified or unmodified peptides (Soltani et al. 2021 ).Ho w e v er, for consistency we will continue to use the classification used in (Newstead et al. 2020 ), which also covered staphylococcal bacteriocins.
It is thought that up to 99% of all bacteria produce at least one bacteriocin (Klaenhammer 1988 ), meaning there is a plethora of as yet unidentified antimicrobial compounds.Numerous studies have identified bacteriocin-like inhibitory substances (BLIS), whic h ar e secr eted by e.g.sta phylococcal species, but unlike bacteriocins, the genetic origin, chemical structure, and mode of action remain poorly characterized (James and Tagg 1991 ).Whilst bacteriocins and BLIS can be found as secr eted pr oducts acr oss staphylococcal species, to date, only Class I, II, and IV bacteriocins have been shown to be secreted by S. aureus .Upwards of 15 different bacteriocins and BLIS have now been identified as being produced by S. aureus , with se v en of these curr entl y classified.

S. aureus Class I bacteriocins
In 1992, a BLIS was identified in the supernatant of S. aureus strain 26, which could inhibit the growth of Streptococcus pyogenes and Micrococcus luteus , and so was named staphylococcin Au-26 (Scott et al. 1992 ).This was later identified as the Class I bacteriocin, Bsa.Bsa is a 21-amino acid lantibiotic encoded downstream of lukD on the Type II νSa β genomic island (Daly et al. 2010 ) (Fig. 1 ).Up to two nonidentical copies can be encoded at this locus, bsaA1 and bsaA2 , follo w ed b y the r equir ed biosynthetic gene cluster.The bsa gene cluster and the Bsa peptide sequence are similar to the epidermin family of lantibiotics (Daly et al. 2010 ).Of these gene products, BsaBCD are predicted to be involved in post-translational modification of Bsa, and BsaEFG are likely involved in providing immunity to this bacteriocin, although this has yet to be confirmed (Daly et al. 2010 ).It was further shown that Bsa has activity against M. luteus amongst other bacterial species (Scott et al. 1992, Daly et al. 2010 ) (Fig. 2 ).Ho w e v er, the antibacterial activity observ ed by Dal y et al. ( 2010) was based on a peptide that was 2 Da smaller than that predicted for BsaA2.It has since been shown that the antimi-cr obial activity observ ed in this assay was due to phenol soluble modulins (PSMs) (Joo et al. 2011 ), which are secreted by S. aureus and hav e pr e viousl y been found to hav e cytol ytic effects on eukaryotic cells (Cheung et al. 2014 ).Ne v ertheless, Bsa was found to hav e antimicr obial activity and a ppears to be a Class I lantibiotic (Scott et al. 1992 ).
A further Class I bacteriocin produced by S. aureus has been c har acterized.BacCH91 was isolated from the supernatant of S. aureus CH91, following observed bacteriocin-like activity.Nterminal Edman degradation and chemical derivatization of the bacteriocin allo w ed for the identification of the 21-residue amino acid sequence, identical to that of BsaA2 from strain ET3-1 (Daly et al. 2010, Wladyka et al. 2013 ).Unlike the Bsa study described abo ve , BacCH91 was purified prior to sequencing and subsequent assa ys , and ther efor e the activity observ ed is unlikel y to be due to alternative antimicrobial molecules.Whilst no activity was observ ed a gainst Gr am-negativ e bacteria, BacCH91 had potent antibacterial activity against most Gr am-positiv e bacteria tested, including M. luteus , Streptococcus spp., and se v er al sta phylococcal species (Fig. 2 ).Inter estingl y, BacCH91 was also able to inhibit the growth of S. aureus CH91, the producing strain.This is uncommon for bacteriocins as the producing strain is often either natur all y immune or has a cognate immunity mechanism for protection (Cotter et al. 2005, Pérez-Ramos et al. 2021 ).
Whilst staphylococcin Au-26, Bsa, and BacCH91 are all likely to be closel y r elated antimicr obial peptides, it is now clear that these Class I lantibiotics have potent activity a gainst se v er al Gr ampositive bacteria, in particular those that are found to commonly colonize the human skin and nasal passages.
Staphylococcin C55 is another Class I bacteriocin produced by S. aureus str ains.Sta phylococcin C55 has antibacterial activity a gainst man y str ains of S. aureus and against some coagulasenegativ e sta phylococci (Nav ar atna et al. 1998 ).Whilst C55 α has low le v els of acti vity alone, acti vity incr eases 128-fold in the pr esence of an equimolar ratio of C55 β (Navaratna et al. 1998 ).C55 α and C55 β contain lanthionine, which is post-tr anslationall y modified, likely mediated by C55M1, a protein encoded by sacM1 found downstream of the C55 peptide-encoding genes ( sac αA and sac βA ), and which shares homology to other lantibiotic modification proteins (Nav ar atna et al. 1999 ) (Fig. 1 ).A putativ e lantibiotic tr ansporter (SacT) is also encoded in this operon.The staphylococcin C55 operon is encoded on a 32-kB plasmid, which has been shown to protect the producer strain from killing by this bacteriocin (Nav ar atna et al. 1998 ).Two open r eading fr ames encoded downstream of this operon are important for immunity against sta phylococcin C55, whic h explains why S. aureus str ains that do not carry this plasmid are susceptible to killing (Kawada-Matsuo et al. 2016 ).
A BLIS, BacR1, identified from S. aureus U0007 has pr e viousl y been shown to have activity against a wide range of Gram-positive bacteria, and against the Gram-negative Neisseria gonorrhoeae (Rogolsky andWiley 1977 , Morriss et al. 1978 ) (Fig. 2 ).It has since been found that BacR1 is identical to the C55 α of staphylococcin C55, both pr oduced fr om the same plasmid in differ ent str ains (Nav ar atna et al. 1998(Nav ar atna et al. , 1999 ) ).It is possible that this could also be the case for other BLIS curr entl y identified.

S. aureus Class II bacteriocins
Class II bacteriocins r epr esent unmodified bacteriocins, of which thr ee hav e been identified to date in S. aureus : aureocin A53, aureocin A70, and aureocin 4181.A summary of all S. aureus -secreted bacteriocins and the genus of bacteria that they have been shown to inhibit.Shading r epr esents sensitivity of species within a given genera to the S. aureus produced bacteriocin and a diagonal dash represents resistance, within a genera, to the bacteriocin.A blank space indicates that a given bacteriocin has not been tested against any species of the corr esponding gener a.As the pr oducer of these bacteriocins, S. aureus (highlighted in red), has been included separ atel y fr om other sta phylococcal species, to highlight the activity of S. aureus -produced bacteriocins against other S. aureus strains.
A study of S. aureus strains isolated from commercial milk identified se v er al BLIS, whic h wer e also found to be encoded on plasmids.Based on the data available, at least two different plasmids encoding phenotypically diverse BLIS have been identified (Giambia gi-Marv al et al. 1990 ).It was found that BLIS produced from plasmids in S. aureus A53 and A70 str ains wer e both able to inhibit Listeria spp., Corynebacterium fimi , Micrococcus sp ., and other S. aureus str ains, whic h did not harbour the plasmids (De Oliv eir a et al. 1998a ) (Fig. 2 ).In addition S. aureus A53 was also able to inhibit the growth of Lactobacillus spp., Lactococcus lactis , and Streptococcus spp.(De Oliv eir a et al. 1998a ).The bacteriocins responsible for the activity observed have since been characterized as members of the Class II family.
Aureocin A70 is produced by S. aureus A70, carrying the pRJ6 plasmid, which harbours the aureocin A70 peptide-encoding genes and associated genes r equir ed for its production and secr etion (Giambia gi-Marv al et al. 1990, Netz et al. 2001 ).Aureocin A70 is composed of four small, related peptides, AurABCD, which ar e str ongl y cationic and highl y hydr ophobic (Netz et al. 2001 ) (Fig. 1 ).It has been shown that unlike staphylococcin C55, the aureocin A70 peptides are not modified prior to secretion (Netz et al. 2001 ).Inter estingl y, at least thr ee of these peptides (AurABC) hav e antimicr obial activity alone, in the absence of other aureocin A70 peptides.AurD is likely to have similar activity, ho w ever, could not be purified (Netz et al. 2001 ).Aureocin A70 is secreted by an ABC transporter, AurT.Exporters such as this can be used in the efflux of toxic compounds from the cell, ho w ever, AurT w as found to be dispensable for imm unity a gainst this bacteriocin.Instead, AurI, a putativ e imm unity pr otein, pr ovides imm unity a gainst aur eocin A70 activity (Coelho et al. 2014 ).AurI is encoded in a two gene oper on, downstr eam of a transcriptional regulator gene aurR .AurR downregulates aureocin A70 production specifically when cells are grown on solid media (Coelho et al. 2016 ).Aureocin A70 has activity against a wide range of Gram-positive bacteria tested (Giambia gi-Marv al et al. 1990, Nascimento et al. 2005, 2006, Varella Coelho et al. 2007, Brito et al. 2011 ), including against species of Staphylococcus and Corynebacterium , which commonly colonize the nasal cavity (Fig. 2 ; Table S1 , Supporting Information ).
A novel bacteriocin with high similarity to aureocin A70 has since been c har acterized.Aur eocin 4181 is composed of three peptides, whic h ar e identical to aur eocin A70 A urABC (Fig. 1 ).A urD, ho w e v er, carries a Leu to Phe substitution at residue 29 and in addition, all four aureocin 4181 peptides are N -formylated (Salustiano Marques-Bastos et al. 2020 ).Aureocin 4181 was found to have 2-4-fold higher activity than aureocin A70 and could inhibit str eptococci, whic h was not pr e viousl y observ ed for aur eocin A70 (Marques-Bastos et al. 2020 , Salustiano Marques-Bastos et al. 2020 , De Oliv eir a et al. 1998a ) (Fig. 2 ).Ne v ertheless, neither aureocin could kill strains, which were producing the other bacteriocin, suggesting cr oss-imm unity (Salustiano Marques-Bastos et al. 2020 ).
Aureocin A53 is produced by S. aureus A53 carrying the pRJ9 plasmid (Giambia gi-Marv al et al. 1990 ).A single structural gene, aucA , encodes this 6-kDa bacteriocin (Netz et al. 2002 ) (Fig. 1 ).Much like aureocin 4181, the bacteriocin has a Nformylmethionine at its N-terminus (Netz et al. 2002, Fagundes et al. 2016, Marques-Bastos et al. 2023 ).Inter estingl y, genes r elating to bacteriocin biosynthesis and modification do not appear to be present on this plasmid.This may be explained by the fact that aureocin A53 is not modified post-translationally, and is highly structured in solution, which has not been observed for amphiphilic bacteriocins pr e viousl y.As suc h, aur eocin A53 is suggested to shar e structur al similarity with the eukaryotic antimicr obial de-fensin peptides (Netz et al. 2002 ).Aureocin A53 is exported by an ABC transporter formed of A ucE, A ucF, and A ucG (Fig. 1 ).Whilst this can provide some immunity against this bacteriocin, two imm unity pr oteins A ucIA and A ucIB ar e r equir ed to pr e v ent killing of S. aureus by aureocin A53 (Nascimento et al. 2012 ).Aureocin A53 causes membrane damage, which leads to eventual lysis of the target cell (Netz et al. 2002 ).The structure of AucA was elucidated by nuclear ma gnetic r esonance, showing that AucA is composed of four short helices, with the outw ar d facing residues being highly cationic, shielding the hydrophobic core (Acedo et al. 2016 ).It is likely the hydrophobic core is responsible for the membrane damaging activity of aureocin A53.Aureocin A53 has activity against a wide range of Gram-positive organisms, including Listeria , Enterococcus , and Streptococcus species (Giambia gi-Marv al et al. 1990, Netz et al. 2002, Nascimento et al. 2006, Varella Coelho et al. 2007, Acedo et al. 2016 ) (Fig. 2 ; Table S1 , Supporting Information ).
Intriguingl y, it was discov er ed that when aur eocin A53 and aur eocin A70 wer e used in combination, activity was m uc h gr eater then when eac h wer e used singly (Varella Coelho et al. 2007 ).In addition, combination use resulted in toxicity against an S. aureus str ain, whic h was r esistant to eac h bacteriocin when used individually.Whilst this may have implications in the development of bacteriocins for ther a peutic pur poses, further work is r equir ed to understand these observations at a molecular le v el (Var ella Coelho et al. 2007 ).Two r ecent studies hav e inv estigated the development of resistance of L. lactis and Enterococcus faecium to aureocin A53.It was found that missense mutations in genes coding for the KinG-LlrG two-component system and the LiaFSR stress response components, r espectiv el y, conferr ed r esistance to aur eocin A53 in these or ganisms (Tymosze wska et al. 2021(Tymosze wska et al. , 2023 ) ).Further studies will be r equir ed to understand the ther a peutic potential of bacteriocins and to understand how we can minimize the risk of resistance developing.

S. aureus Class IV bacteriocins
Class IV bacteriocins ar e post-tr anslationall y modified peptides, whic h hav e been cir cularized b y cov alent linka ge of the N-to Cterminus .T he supernatant of S. aureus 4185 was found to have bacteriocin-lik e acti vity, with pe ptide fr a gments r esponsible for such activity identified by mass spectrometry (Ceotto et al. 2010 ).The BLIS produced by this strain has activity against M. luteus and Listeria monocytogenes , making these products of potential interest as food preservatives (Ceotto et al. 2010 ) (Fig. 2 ; Table S1 , Supporting Information ).One of the bacteriocins belie v ed to be responsible for this activity is encoded on a plasmid harboured by S. aureus 4185 (Potter et al. 2014 ).Unfortunately this bacteriocin could not be purified, ho w e v er, in silico anal ysis found the encoding biosynthetic cluster to share high homology with that of carnocyclin A from Carnobacterium maltaromaticum (Potter et al. 2014 ).Carnoc yclin A is a c yclic bacteriocin with activity against a diverse range of Gram-positive bacteria (Zipperer et al. 2016 ).As such, the 60 residue, homologous bacteriocin from S. aureus 4185 was named aureocyclin 4185, and is the first putative cyclic bacteriocin identified in S. aureus (Potter et al. 2014 ) (Fig. 1 ).
Aureocyclin 4185 is predicted to have a short leader peptide, which is cleaved, allowing for the covalent linkage of the N-and Ctermini (Potter et al. 2014 ).Homology modelling suggests that the bacteriocin is composed of four short helices, enclosing a highly hydr ophobic cor e. Se v er al Lys r esidues likel y pr ovide the molecule with the positiv e c har ge r equir ed for attr action to the bacterial membrane (Potter et al. 2014 ).Further work is, ho w e v er, r equir ed to confirm that aureocyclin 4185 is responsible for the antibacterial activity imparted by this strain.

S. aureus BLIS
Due to the complex biosynthesis pathways of many bacteriocins, the identification of novel inhibitory substances has often been carried out phenotypically from culture or culture supernatant, rather than through genetic analysis of putative bacteriocin genes.As a r esult, inhibitory pr oducts commonl y go unc har acterized due to difficulties in the identification process, and this is the case for many BLIS.Up to eight further S. aureus produced bacteriocins have been identified, in addition to those discussed abo ve , ho w ever, they remain poorly characterized ( Table S1 , Supporting Information ).
Some BLIS, such as Bac1829, Bac201, and staphylococcin IYS2, hav e been partiall y c har acterized, with the amino acid composition known, but lack the amino acid sequence and subsequent classification.Bac1829 is a 6.4-kDa peptide with a high proportion of hydr ophobic r esidues that has a bactericidal effect on target cells (Crupper andIandolo 1996 , 1997 ).Staphylococcin IYS2 is a peptide of about 5 kDa, i.e. also bactericidal, and for which the amino acid composition is also known (Nakam ur a et al. 1983 ).Both of these BLIS have activity against a wide range of Actinomycetota and other Gr am-positiv e or ganisms, but Bac1829 also has activity against several Gram-negative bacterial species ( Table S1 , Supporting Information ; Nakam ur a et al. 1983 , Crupper and Iandolo 1996 ) (Fig. 2 ).Bac201 is a m uc h lar ger bacteriocin, comprising a 41-kDa peptide (Iqbal et al. 1999(Iqbal et al. , 2001 ) ).The amino acid composition of Bac201 was found to be similar to that of Bac1829, containing of a high proportion of glycine , proline , and alanine residues (Iqbal et al. 2001 ), ho w ever, the amino acid sequence remains unknown.Again, m uc h lik e Bac1829, Bac201 had acti vity against a range of both Gram-positive and Gram-negative bacteria tested (Fig. 2 ; Table S1 , Supporting Information ), and given the temper atur e and pH stability of this bacteriocin, may warrant further study for therapeutic development (Iqbal et al. 1999(Iqbal et al. , 2001 ) ).
Se v er al BLIS hav e been purified fr om the pr oducing S. aureus strains, but the amino acid composition remains unknown.Staphylococcin 462 was purified from S. aureus 462 and shown to be a peptide of ∼9 kDa (Gagliano andHinsdill 1970 , Hale andHinsdill 1973 ).Staphylococcin 414 was found to be m uc h lar ger, migrating in the v oid v olume after size exclusion c hr omatogr a phy, implying a size of over 200 kDa (Gagliano and Hinsdill 1970 ).This BLIS a ppear ed to be a lipopr otein-carbohydr ate complex that was purified from cell lysate rather than culture supernatant.Staphylococcin 414 appears to target Gram-positive organisms exclusiv el y (Ga gliano and Hinsdill 1970 ) (Fig. 2 ; Table S1 , Supporting Information ).Staphylococcin D91 is another BLIS that could be purified from its producing strain, which has bacteriostatic activity against a range of Gram-positive and Gram-negative organisms (Kader et al. 1984 ) (Fig. 2 ; Table S1 , Supporting Information ).Staphylococcin D91 is also likely to be plasmid encoded as S. aureus D91 loses the ability to produce staphylococcin D91 when grown at 44 • C, suggesting the loss of a plasmid encoding this BLIS (Kader et al. 1984, Iqbal et al. 1999 ).As a sequence and tertiary structure has not been elucidated for any of these BLIS it is not possible for these to be classified.Ho w e v er, giv en the differ ence in size and target range of these bacteriocins it suggests that at least some of these are unique, uncharacterized bacteriocins.
The identification of some BLIS, ho w e v er, has been based solely on activity of S. aureus culture supernatant.Antibacterial activ-ity was observed for staphylococcin 188 from the supernatant of S. aureus 188, ho w e v er, this was solel y observ ed a gainst Gr ampositi ve and Actinom ycetota species (Saeed et al. 2004 ) (Fig. 2 ; Table S1 , Supporting Information ).Likewise, aureocin 215FN from culture supernatant, had activity against Gram-positive species only (Nascimento et al. 2006, Varella Coelho et al. 2007, De Oliveira et al. 1998b ).
A recent bioinformatic study has identified biosynthetic clusters in S. aureus strains for both lactococcin 972 and micrococcin P1, suggesting ther e ar e additional S. aureus -pr oduced bacteriocins yet to be identified.Whilst m uc h work has been carried out to identify the bacteriocins that ar e secr eted by S. aureus , the mechanism by which they inhibit growth remains poorly understood for many of these .Nonetheless , it is clear that S. aureus -produced bacteriocins can have a wide range of targets and are likely used in colonization, to outcompete r esident micr obes, and also to exclude unwanted organisms once established.Howe v er, the sporadic distribution of these antimicrobial compounds suggest they may not be a widespread mechanism used by S. aureus to target competitors.

A role for PSMs in bacterial killing
PSMs are small peptides secreted by staphylococcal species, which hav e cytol ytic activity a gainst eukary otic host cells (P eschel and Otto 2013 ).Ho w e v er, ther e is some e vidence that PSMs can also be used to kill competing bacteria.PSM δ produced by S. epidermidis can inhibit the growth of S. pyogenes on murine skin (Cogen et al. 2010 ), although concentrations required to mediate inhibition are very high, suggesting this may not be the evolved purpose of these PSMs .T he S. aureus strain, USA300 has high levels of antimicrobial activity against S. pyogenes and M. luteus , which is modulated by PSM α and PSM β (Joo et al. 2011 ).Ho w e v er, this activity is only observed when PSM α and PSM β are proteolytically processed at the N-terminus.It has been suggested that this processing is carried out by microbial exoproteases, indicating that targeting of host and bacterial cells may not always be m utuall y exclusiv e. Ne v ertheless, some PSMs appear to be able to kill the Group A Streptococcus , S. pyogenes , possibly playing a role in controlling this pathogen within the host.PSMs do not have activity a gainst sta phylococcal species due to the presence of the phenol-soluble modulin ABC tr ansporter, r esponsible for the export of PSMs from the cell (Chatterjee et al. 2013 ).

The Bacillota type VII secretion system and interbacterial competition
Alongside the study of bacteriocins, a growing field of r esearc h has started to unr av el r oles in interbacterial competition of a protein export pathway encoded by all S. aureus strains, named the Type VII Secretion System (T7SS).The T7SS was first identified in Mycobacterium species, as the system responsible for the secretion of the potent virulence factor , ESA T-6 (Pym et al. 2002, Guinn et al. 2004 ).Structur al anal ysis of a mycobacterial T7SS named ESX-5 shows that it forms a lar ge membr ane complex with a hexameric arrangement of a membrane-bound ATPase, EccC, at the centre, forming a translocation pore (Beckham et al. 2021, Bunduc et al. 2021 ).A distant homologue of the mycobacterial system is found in many Bacillota (previously Firmicutes), including S. aureus and has been designated the T7SSb .
The T7SSb comprises four conserved membrane proteins that likely form a complex.Based on sequence conservation between mycobacterial EccC and the T7SSb EssC component, it is likely that a hexamer of EssC forms the central pore, facilitating substr ate export acr oss the membr ane, driv en by ATP hydr ol ysis (Burts et al. 2005, Rosenberg et al. 2015, Zoltner et al. 2016, Klein et al. 2021 ).The T7SSb of some Bacillota is r equir ed for full virulence, with ess/T7SSb mutant strains of Streptococcus and S. aureus attenuated in murine and zebrafish models of infection (Burts et al. 2005, Anderson et al. 2011, Kneuper et al. 2014, Dai et al. 2017, Ulhuq et al. 2020, Taylor et al. 2021, Spencer and Doran 2022, Schindler et al. 2023 ).Ho w e v er, in other species, such as L. monocytogenes , the T7SSb was found to play no detectable role in virulence (Way andWilson 2005 , Pinheiro et al. 2017 ).In recent years, a growing body of evidence has shown that the T7SSb mediates interbacterial competition, through the secretion of polymorphic protein toxins (Chatterjee et al. 2013, Cao et al. 2016, Whitney et al. 2017, Ulhuq et al. 2020, K oba yashi 2021, Klein et al. 2022, Tassinari et al. 2022, Garrett et al. 2023 ).Polymorphic toxins are commonly used by bacteria in the context of competition, and have a modular arrangement.They comprise a conserved domain responsible for targeting to the appropriate secretion system, and a toxic domain which can carry a range of different toxin functionalities (Zhang et al. 2012 ).In ad dition, to xin domains ar e often highl y v ariable, coevolving with cognate immunity proteins, likely in an attempt to esca pe imm unity mec hanisms of competing bacteria (Koskiniemi et al. 2014, Cao et al. 2016, Garrett et al. 2022 ).Four suc h pol ymorphic toxins have now been identified that are associated with the S. aureus T7SSb, EsaD, TspA, TslA, and EsxX.Whilst a role for EsxX in interbacterial competition has not been tested, the remaining toxin substrates participate in interbacterial competition and are described below.

EsaD: a nuclease toxin
EsaD was the first large toxin identified as a substrate of the S. aureus T7SSb, encoded downstream of the core components of the secretion system at the ess/T7SSb locus (Fig. 3 A and B).It should be noted that four variants of the T7SSb have been identified in S. aureus , classified based on sequence diversity at the C-terminus of EssC (Warne et al. 2016 ).EsaD is encoded in essC1 variant strains, which account for around 50% of S. aureus sequenced isolates .T he N-terminus of EsaD is composed of an LXG domain follo w ed b y a pretoxin-TG (PT-TG) domain (Cao et al. 2016, Yang et al. 2023 ) (Fig. 4 A).PT-TG domains are linker regions carrying a TG motif, of unknown function, that are found in many bacterial toxins.LXG domains, named for the conserved L-x-G residues found in the amino acid sequence (where x is any amino acid; Zhang et al. 2012 ), are helical and are required for targeting proteins to the T7SSb (Klein et al. 2022 ).Ho w e v er, LXG domains alone ar e not competent for secretion by the T7SSb-instead they require interaction with further small helical partner proteins, termed Laps ( L XG a ccessory p rotein), to form a presecretion complex (Klein et al. 2022(Klein et al. , 2024 ) ). EsaD interacts with three small helix-turn-helix La p pr oteins, whic h ar e necessary for its efficient secr etion by the T7SSb.These Lap proteins, EsxB, EsxC, and EsxD, bind to the LXG domain of EsaD, forming the pr esecr etion complex, and are subsequentl y secr eted by the T7SSb along with EsaD (Yang et al. 2023 ).EsaD also r equir es a c ha per one pr otein, EsaE for its stability and secr etion.EsaE inter acts with the central ATPase subunit of the T7SSb, EssC, and ma y pla y a role in targeting the EsaD complex to the secretion system (Cao et al. 2016 ).It remains unclear whether EsaE is secreted with the complex, or if it dissociates during secr etion and r emains in the cytoplasm (Cao et al. 2016, Yang et al. 2023 ).
The C-terminus of EsaD encodes a Mg 2 + -dependent nuclease domain with a ββα-metal finger motif, which can degrade double stranded DNA (Cao et al. 2016, Wang et al. 2022 ) (Fig. 4 B).This nuclease domain is exceptionally toxic when expressed in Esc heric hia coli or S. aureus (Cao et al. 2016 ).For self-protection, S. aureus produces a cognate immunity protein, EsaG, which is encoded dir ectl y downstr eam of esaD .The pr oduction of EsaG r ecov ers growth of cells that are producing EsaD by binding directly to the nuclease domain (Cao et al. 2016 ).EsaG binding disrupts the active site of EsaD by inserting between two structur all y important beta-sheets (Wang et al. 2022 ) (Fig. 4 C).This results in distortion of the catal ytic site, pr e v enting n uclease acti vity.S. aureus strains also encode strings of nonidentical EsaG homologues, which can diversify by homologous recombination to produce new nonidentical variants (Cao et al. 2016, Garrett et al. 2023 ).Many of these nonidentical EsaG proteins cannot interact with EsaD encoded in the same strain (Cao et al. 2016 ).EsaD proteins encoded across S. aureus strains have highly polymorphic nuclease domains, suggesting that the role of accessory EsaG proteins is to protect from intoxication by EsaD variants secreted from other bacterial cells.
Whilst the absence of EsaD leads to a reduction in abscess formation in a murine model, EsaD plays no detectable role in virulence using a zebrafish embryo model of infection (Ohr et al. 2017, Ulhuq et al. 2020 ).EsaD does, ho w e v er, mediate interbacterial competition.S. aureus cells ov er pr oducing EsaD were found to outcompete prey cells, which were deleted for the cluster of genes coding for EsaG immunity proteins in vitro (Cao et al. 2016 ).Expression of EsaG in the prey cell recovered their growth, suggesting the deficiency in growth was due to intoxication by EsaD.Similarl y, using the zebr afish embryo as a host, in vivo competition was observed between wild type S. aureus used as the attacker strain and a mutant lacking all EsaG immunity proteins as prey (Ulhuq et al. 2020 ).its secretion via the T7SSb (Klein et al. 2022 ).LapD2 is a DUF3958 pr otein, the same famil y as one of the DUF pr oteins encoded at the tspA2 locus (Bowman andPalmer 2021a , Klein et al. 2022 ).Whilst most strains do not encode a copy of tspA at this locus, the genes for the three small DUF proteins appear to be conserved, suggesting they could play a role in targeting TspA to the T7SS.This hypothesis remains to be tested (Bowman and Palmer 2021a ) (Fig. 5 B).

TslA: an antibacterial 're v erse' lipase toxin
All the T7SS antibacterial toxins that have been identified in Bacillota to date show a conserved domain arc hitectur e, comprising a C-terminal toxin domain, and an N-terminal LXG or LXG-like domain (Zhang et al. 2011, Cao et al. 2016, Whitney et al. 2017, Ulhuq et al. 2020, Bowman and Palmer 2021a,b , Teh et al. 2023 ), with the N-terminal domain involved in targeting the effector to the T7SSb, in complex with two or three Lap proteins (Klein et al. 2022, Yang et al. 2023 ).Recently, a ne w class of T7SSb substr ate has been identified, which has a reversed domain architecture.
TslA is the first 'r e v erse' substr ate of the T7SSb to be c har acterized.It has a helical LXG-like domain similar to other T7SSb substrates, but in TslA this is found at the C-terminus rather than the N-terminus (Garrett et al. 2023 ) (Fig. 6 A).Nevertheless, it has been shown that this LXG-like domain is r equir ed for secr etion of TslA by the T7SSb.Secretion is facilitated by the binding of two small La p pr oteins to this domain, as has been observed for other T7SSb substrates (Klein et al. 2022, Yang et al. 2023, 2024 ).This suggests that the T7SSb can recognize a secretion signal present at either end of a substrate protein, which is a highly unusual feature for a pr otein secr etion system.
TslA has phospholipase A activity and can cleav e a wide r ange of bacterial membrane phospholipids, mediated by its N-terminal lipase domain (Garrett et al. 2023 ) (Fig. 6 A).This results in accum ulation of l yso-acyl phospholipids, whic h hav e deter gent-like properties and lead to disruption of the cell membrane .T his significantly perturbs the growth of susceptible S. aureus cells.Toxicity can be abrogated by the production of a lipidated immunity protein, TilA, embedded in the outer leaflet of the cytoplasmic membr ane (Garr ett et al. 2023 ).TilA binds with 14.2 nM affinity to the N-terminal lipase domain of TslA to inhibit this activity.It is unclear how the two proteins interact with one another, howe v er, an AlphaFold Colab model of TilA indicates that there are h ydrophobic, h ydrophilic, and charged residues present on the conca ve face , which ma y be in volv ed in this inter action (Fig. 6 B  and C).Whilst the mechanism of secretion of substrates by the T7SSb is still not understood, it has been proposed that substrates are tr anslocated acr oss the entir e cell env elope in a single step.The crystal structure of the large extracellular loop of the EsaA T7SSb component of Streptococcus r e v eals that it is of sufficient length to fully span the peptidoglycan cell wall (Klein et al. 2018(Klein et al. , 2021 ) ).Given that TilA binding to TslA is very tight, it suggests that during secretion, the toxin must bypass the periplasm environment wher e the TilA imm unity pr otein r esides as complex formation would result in the toxin being retained by the producing cell (Fig. 6 D).
Homologues of TslA can be encoded at up to two additional loci on the S. aureus genome, depending on the particular strain (Garrett et al. ).This includes EsxX, which is encoded downstream of essC in essC2 variant strains (Fig. 1 A).The toxin domain of EsxX has been proposed to share structural similarity with colicin IA, indicating that it may be pore-forming (Dai et al. 2017 ).Further work of EsxX is r equir ed to understand the role it may play in interbacterial competition.
Future studies should begin to shed light on how these toxins ar e deliv er ed to the tar get cell, and in the case of intracellularly acting toxins such as EsaD, to provide an understanding of how they access the cytoplasm of target bacteria.A role in virulence has also been cited for EsaD, EsxX, and TspA (Dai et al. 2017, Ohr et al. 2017, Ulhuq et al. 2020 ), at least for some strains and virulence models.Whilst no statistically significant role in murine skin abscess formation was observed for TslA, there was a trend to w ar ds reduced virulence when the encoding gene was deleted, suggesting that there may be a cum ulativ e effect of these secr eted pr oteins on the host (Garrett et al. 2023 ).Further work into understanding toxin delivery to targets will provide important context to these phenotypes, as there is curr entl y a ga p in kno wledge betw een the virulence phenotypes associated with the T7SS of S. aureus and the molecular mechanisms by which they work.

Concluding remarks
In recent years bioinformatic studies have identified additional putativ e pol ymor phic toxin systems acr oss a br oad spectrum of bacterial species, including in S. aureus (Zhang et al. 2012, Li et al. 2022 ), suggesting ther e ar e further antibacterial compounds that may be used by S. aureus in a competition setting.Ne v ertheless, ther e ar e two major mec hanisms curr entl y described that are emplo y ed b y S. aureus .The T7SS is responsible for the secretion of se v er al pol ymor phic toxins, whic h ar e known to kill other S. aureus species in the absence of a cognate immunity protein.Bacteriocins produced by S. aureus have been shown to inhibit the growth of a diverse range of bacterial species, ho w ever, appear to be less conserved across S. aureus strains in comparison to the highly conserved T7SS.These mechanisms of interbacterial competition likely work in tandem to control competitors in the pol ymicr obial envir onments that they colonize.As S. aureus strains of different clonal complexes are commonly found to colonize different niches (Howden et al. 2023 ), understanding the distribution of bacteriocins and the pol ymor phic T7SS toxins in the context of S. aureus lineage may illuminate the predominant role of some of these compounds in interbacterial competition.Some S. aureus -produced bacteriocins show great promise in the production of alternatives to antibiotics, ho w ever, much w ork is still need to determine the molecular mechanism of killing emplo y ed b y many of these bacteriocins and to further assess the possibility of resistance development in bacterial populations.The resistance mechanisms utilized against bacteriocins are often associated with the presence of the bacteriocin gene cluster, with transporters usually providing resistance to toxicity (Ennahar et al. 2000, Gajic et al. 2003, Ishibashi et al. 2014 ).Ho w e v er, it has been found that escape mutations in target cells, including in their two-component systems and ABC transporters, can pr ovide r esistance to bacteriocin killing (Tymoszewska et al. 2021(Tymoszewska et al. , 2023 ) ).For toxins secreted by the T7SS, strings of immunity proteins ar e commonl y found in both pr oducing and closel y r elated, T7SS-deficent bacterial species, implying a continual coevolution between toxin and immunity genes (Bowman andPalmer 2021 , Garrett et al. 2022 ).As such, a firm understanding of acquired resistance to these compounds is essential for the de v elopment of suc h pr oteins for ther a peutic uses.
The S. aureus T7SSb has been shown to play roles in both virulence and interbacterial competition.As further r esearc h uncovers the molecular mechanisms of protein secretion and the role of secr eted substr ates it is anticipated that we will have a clearer understanding of how this system contributes to virulence and colonization.It will be interesting to decipher how antibacterial toxins tr av erse the env elope of susceptible cells to access their molecular targets .T he continued work on S. aureus -produced antibacterial molecules will hopefull y pr ovide a clearer understanding of how this major human pathogen has e volv ed to colonize the host with such success.

Figure 2 .
Figure2.Bacteriocins secreted by S. aureus strains.A summary of all S. aureus -secreted bacteriocins and the genus of bacteria that they have been shown to inhibit.Shading r epr esents sensitivity of species within a given genera to the S. aureus produced bacteriocin and a diagonal dash represents resistance, within a genera, to the bacteriocin.A blank space indicates that a given bacteriocin has not been tested against any species of the corr esponding gener a.As the pr oducer of these bacteriocins, S. aureus (highlighted in red), has been included separ atel y fr om other sta phylococcal species, to highlight the activity of S. aureus -produced bacteriocins against other S. aureus strains.

Figure 3 .
Figure 3. Distribution of known T7SSb toxin-encoding genes on the S. aureus c hr omosome .(A) T he ess / T7SSb locus from NCTC8325 and ST398.A cluster of genes encoding immunity proteins against T7SS toxins is found downstream of the ess/T7b locus (grey box).Gene dia gr ams wer e visualized using Clinker (Gilchrist and Chooi 2021 ).(B) Distribution of genes coding for T7SSb LXG substrates on the S. aureus NCTC8325 chromosome .T he tslB gene found in NCTC8325 is annotated as a pseudogene, ho w e v er, full-length homologues are found in other S. aureus strains.Note that tspA2 and tslC are not found in the S. aureus NCTC8325 strain.The encoding genes have been included on this figure to indicate the position in which they are found on the S. aureus c hr omosome in encoding strains.

Figur e 4 .
Figur e 4. T he EsaD substrate of the S. aureus T7SS.(A) Domain organization of EsaD predicted by InterProScan (Yang et al. 2023 ).PT-TG-pretoxin-TG domain; dis-disordered region.(B) An AlphaFold Colab model EsaD (SAOUHSC_00268) from NCTC8325 (Jumper et al. 2021 , Varadi et al. 2022 ).(inset) The active site of the EsaD nuclease domain with the catalytic H528 highlighted.(C) The crystal structure of EsaG (PDB: 8GUO) binding to the EsaD nuclease domain resulting in deformation of the active site.ChimeraX was used to visualize all structural models (Petterson el al. 2020 ).

Figure 5 .
Figure 5.An AlphaFold Colab model of TspA in complex with TsaI.(A) Alphafold Colab was used to model TspA (SAOUHCS_00584) in complex with its imm unity pr otein, TsaI (SAOUHSC_00585) (Jumper et al. 2021 , Varadi et al. 2022 ).(inset) The pr edicted inter action between TspA and TsaI fr om S. aureus NCTC8325 is highlighted by illustrating the surface of TsaI.ChimeraX was used to visualize all structural models (Petterson el al. 2020) .(B) Genetic organization of the tspA1 and tspA2 loci in the indicated strains.Gene diagrams were visualized using Clinker (Gilchrist and Chooi 2021 ).

Figure 6 .
Figure 6.TilA immunity protein binding to TslA.(A) An AlphaFold Colab model of TslA in complex with its immunity protein, TilA (Jumper et al. 2021 , Varadi et al. 2022 ).(Inset) The catalytic triad of TslA is composed of S164, D224, and H251 in TslA (SAOUHSC_00406) from S. aureus NCTC8325.ChimeraX was used to visualize all structural models (Petterson el al. 2020) .(B) and (C) An AlphaFold Colab model of TilA with (B) lipophilicity and (C) electrostatic potential mapped onto the surface.(D) A model of TslA export out of the cell if the T7SS spans the entire cell wall (left) or if the T7SS spans only the cytoplasmic membrane (right).(E) Genetic organization of the tslA , tslB , and tslC loci in the indicated strains.Gene diagrams were visualized using Clinker (Gilchrist and Chooi 2021 ).
2023 ) (Figs 3 B and 6 E).Whilst these homologues have not been studied to date, their conservation suggests that these 'reverse' to xins mak e up a significant part of the S. aureus T7SSb to xin r epertoir e .Unlike bacteriocins , whic h a ppear to be spor adicall y encoded by S. aureus , the T7SSb is encoded by all strains, suggesting this r epr esents a widespr ead competitiv e mec hanism.Mor eover, both TslA and TspA, or homologues of these, are encoded almost ubiquitously by S. aureus .Although EsaD is only carried by str ains, whic h harbour the essC1 variant of the T7SSb (Warne et al. 2016 ), other essC types encode putative LXG or LXG-like toxins at their T7SSb loci, which are predicted to have similar antibacterial activities (Bowman and Palmer 2021b