The development of novel antimicrobial peptides with activity against MRSA

MRSA is a significant pathogen, which can cause a range of minor and major infections both in the hospital and community environments. MRSA is developing resistance to many antibiotics, including vancomycin, which is now the first choice antibiotic to treat MRSA infections in the UK. This together with the dearth of new antibiotics being introduced could see the emergence of untreatable S. aureus strains. This has led to renewed interest in alternative antimicrobial agents. Lysostaphin is an endopeptidase produced by Staphylococcus simulans biovar staphylolyticus, which cleaves the peptidoglycan cross-bridges of other staphylococcal species. Lysostaphin has been investigated as a potential therapeutic agent and has shown promise in in vitro and in vivo studies and in clinical trials. However, resistance to lysostaphin is likely to emerge and there will be a demand for second generation Iysostaphins and/or other similar novel antimicrobials that can counteract this resistance. This study describes the cloning, purification and assaying of an endolysin of the S. aureus P68 bacteriophage. Lys16 lysin has previously been shown to possess staphylolytic activity. This study demonstrates that the purified recombinant protein is poorly soluble and is inactive against live cells. The Atl autolysin of S. aureus was also investigated as a potential antimicrobial. This study confirmed the hydrolytic profiles of the enzymes, and a chimeric peptide incorporating the lysostaphin targeting domain with the Atl glucosaminidase was designed. This did not confer greater activity against S. aureus, although the targeting domains of each enzyme were shown to utilise different cell surface receptors. Finally, this study reports the development of a novel assay to measure the activity of antimicrobial peptides against S. aureus, using a bioluminescence reporter. This was shown to be a sensitive assay, able to distinguish small differences in the activity of antimicrobial peptides

The development of novel antimicrobial peptides with activity against MRSA

MRSA is a significant pathogen, which can cause a range of minor and major infections both in the hospital and community environments. MRSA is developing resistance to many antibiotics, including vancomycin, which is now the first choice antibiotic to treat MRSA infections in the UK. This together with the dearth of new antibiotics being introduced could see the emergence of untreatable S. aureus strains. This has led to renewed interest in alternative antimicrobial agents. Lysostaphin is an endopeptidase produced by Staphylococcus simulans biovar staphylolyticus, which cleaves the peptidoglycan cross-bridges of other staphylococcal species. Lysostaphin has been investigated as a potential therapeutic agent and has shown promise in in vitro and in vivo studies and in clinical trials. However, resistance to lysostaphin is likely to emerge and there will be a demand for second generation Iysostaphins and/or other similar novel antimicrobials that can counteract this resistance. This study describes the cloning, purification and assaying of an endolysin of the S. aureus P68 bacteriophage. Lys16 lysin has previously been shown to possess staphylolytic activity. This study demonstrates that the purified recombinant protein is poorly soluble and is inactive against live cells. The Atl autolysin of S. aureus was also investigated as a potential antimicrobial. This study confirmed the hydrolytic profiles of the enzymes, and a chimeric peptide incorporating the lysostaphin targeting domain with the Atl glucosaminidase was designed. This did not confer greater activity against S. aureus, although the targeting domains of each enzyme were shown to utilise different cell surface receptors. Finally, this study reports the development of a novel assay to measure the activity of antimicrobial peptides against S. aureus, using a bioluminescence reporter. This was shown to be a sensitive assay, able to distinguish small differences in the activity of antimicrobial peptides

Staphylococcus aureus susceptibility to complestatin and corbomycin depends on the VraSR two-component system

The overuse of antibiotics in humans and livestock has driven the emergence and spread of antimicrobial resistance and has therefore prompted research on the discovery of novel antibiotics. Complestatin (Cm) and corbomycin (Cb) are glycopeptide antibiotics with an unprecedented mechanism of action that is active even against methicillin-resistant and daptomycin-resistant Staphylococcus aureus. They bind to peptidoglycan and block the activity of peptidoglycan hydrolases required for remodeling the cell wall during growth. Bacterial signaling through two-component transduction systems (TCSs) has been associated with the development of S. aureus antimicrobial resistance. However, the role of TCSs in S. aureus susceptibility to Cm and Cb has not been previously addressed. In this study, we determined that, among all 16 S. aureus TCSs, VraSR is the only one controlling the susceptibility to Cm and Cb. Deletion of vraSR increased bacterial susceptibility to both antibiotics. Epistasis analysis with members of the vraSR regulon revealed that deletion of spdC, which encodes a membrane protein that scaffolds SagB for cleavage of peptidoglycan strands to achieve physiological length, in the vraSR mutant restored Cm and Cb susceptibility to wild-type levels. Moreover, deletion of either spdC or sagB in the wild-type strain increased resistance to both antibiotics. Further analyses revealed a significant rise in the relative amount of peptidoglycan and its total degree of cross-linkage in ΔspdC and ΔsagB mutants compared to the wild-type strain, suggesting that these changes in the cell wall provide resistance to the damaging effect of Cm and Cb.This work was financially supported by the Spanish Ministry of Science and Innovation grant PID2020-113494RB-I00 to I.L. (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) and a Canadian Institutes of Health Research grant (FRN-148463; to G.D.W.). C.G.A. was supported by a predoctoral contract from the Public University of Navarra. Research in the Cava lab was supported by The Swedish Research Council (VR), The Knut and Alice Wallenberg Foundation (KAW), The Laboratory of Molecular Infection Medicine Sweden (MIMS) and The Kempe Foundation

A new bacterial peptidoglycan peptidase LytU and insights into substrate recognition by lysostaphin family

Staphylococcus aureus is a pervasive pathogen, whose infections frequently result in serious medical complications and death. Its encounters are yet more perilous in clinical settings where professional care and financial resources alone do not suffice to ensure successful treatment results. The virulence of the bacteria is enforced by numerous cellular mechanisms that have allowed it to develop resistance to every drug used to this date. The bacterial cell wall (CW) is the primary line of defense, the most common target in treatment strategies, and is likely to remain the prioritized candidate for future therapeutic solutions. The main structural component of bacterial CW is peptidoglycan (PG) that forms protective layers. PG is administered by a large number of enzymes that are involved in its synthesis, maintenance, and cleavage. One family of enzymes, M23 peptidases, cleaves pentaglycine bridges that link chains of PG and are specific to S. aureus. These enzymes can be used by the bacteria to manage its own PG in a controlled manner or, alternatively, by hostile microorganisms and cause cell death. Therefore, M23 peptidases of S. aureus are important as potential targets for drugs as well as pharmacological tools themselves that are already employed by nature. Substrate recognizing SH3b domains enhance the effectiveness of M23 endopeptidases. Previous research had identified a putative M23 peptidase gene, transcription of which is upregulated under S. aureus exposure to compounds harmful to cell wall. We examined and characterized the product of the gene. The protein, which we named LytU, is an M23 family zinc-dependent enzyme that cleaves pentaglycine. It is anchored in plasma membrane and is extracytoplasmic, residing in a periplasm-like space. The physiological role of LytU is not confirmed, but evidence suggest it can recycle PG fragments and participate in daughter cell separation. A distinct feature of the enzyme is its ability to strongly bind a second zinc ion, which incapacitates catalytic residues. We propose that together with pH, the binding of second ion serves a regulatory function in situ. Solution structure of the LytU catalytic domain has been determined. Binding of substrate pentaglycine to catalytic M23 domain is very transient at least in vitro. The binding, nevertheless, is accomplished by SH3b domain of enzymes bearing it. Contrarily to previous beliefs, we found that SH3b domain binding to substrate is primarily driven by interactions with PG branching peptides, rather than by weaker interaction with pentaglycine. The binding of SH3b to substrate is independent of catalytic domain and it targets and binds the PG peptide moieties that are proximal to but different from the pentaglycine cleaved by catalytic domain. In summary, we have introduced and characterized a new M23 family endopeptidase, proposed a regulation mechanism, and changed the paradigm of substrate binding by M23 peptidases. Our results are expected to contribute to a better understanding of S. aureus physiology and provide means for the development of cures.Staphylococcus aureus on yleinen bakteeri, joka aiheuttaa usein vakavia, jopa hengenvaarallisia infektioita. Sairaalaympäristön ammattitaidosta tai hoitoon käytettävistä resursseista huolimatta infektioihin liittyy huomattava kuolleisuus. Bakteeri tuottaa useita mekanistisesti erilaisia virulenssitekijöitä, minkä seurauksena bakteeri on kehittänyt vastustuskyvyn kaikille nykyisille antibiooteille. Bakteerin soluseinä on sen ensisijainen suojautumiskeino. Soluseinä on myös tavallisin lääkehoidon kohde ja todennäköisesti jatkossa edelleen etusijalla lääkekehityksen kohteista. Bakteerin soluseinän päärakennekomponentti on peptidoglykaani, joka muodostaa bakteeria suojaavia kerroksia. Tätä peptidoglykaania tuottaa, ylläpitää ja hajottaa lukuisa joukko entsyymejä, kuten M23-entsyymiperheen peptidaasit, jotka pilkkovat S. aureus-bakteerille ominaisia, peptidoglykaaniketjuja yhdistäviä pentaglysiinisiltoja. Bakteerit voivat käyttää näitä entsyymejä oman soluseinänsä hallittuun ylläpitämiseen tai vaihtoehtoisesti aiheuttamaan kilpailevan mikro-organismin solukuoleman. Tämän takia S. aureus-bakteerin M23-peptidaasit ovat sekä merkittäviä lääkekehityksen kohteita että luonnossakin käytössä oleva farmakologinen työkalu. Substraatin tunnistava SH3b-domeeni tehostaa M23-endopeptidaasien vaikutusta. Aikaisempi tutkimus on tunnistanut mahdollisen M23-peptidaasigeenin, jonka transkriptio aktivoituu kun S. aureus altistuu soluseinää vahingoittaville yhdisteille. Me tutkimme ja karakterisoimme tämän geenituotteen. Proteiini, jonka nimesimme LytU:ksi, on M23-perheen sinkistä riippuvainen pentaglysiiniä pilkkova entsyymi. Se on ankkuroitunut solukalvoon ja on ekstrasytoplasminen, sijoittuen periplasman kaltaiseen tilaan. LytU:n fysiologista roolia ei ole varmistettu, mutta tutkimustulokset viittaavat tehtävään peptidoglykaanifragmenttien kierrätyksessä sekä tytärsolujen erottamisessa toisistaan. Entsyymille omaleimaista on sen kyky sitoa voimakkaasti toinen sinkki-ioni, minkä seurauksena entsyymin katalyyttiset histidiinit muuttuvat toimintakyvyttömiksi. Esitämme, että toisen sinkin sitominen ja pH yhdessä säätelevät entsyymiä in situ. Entsyymin katalyyttisen M23-domeenin sitoutuminen pentaglysiinisubstraattiin on hyvin lyhytaikaista ainakin in vitro. SH3b-domeenin sisältävien entsyymien ja substraatin välinen vuorovaikutus on kuitenkin todennettu. Toisin kuin aiemmin oletettiin, me osoitimme, että SH3b-domeenin sitoutumista substraattiin ohjaa ensisijaisesti sen voimakkaampi vuorovaikutus peptidoglykaanin haarapeptidien kanssa eikä niinkään heikompi vuorovaikutus pentaglysiinin kanssa. SH3b:n sitoutuminen substraattiin ei riipu katalyyttisesta domeenista ja se tunnistaa ja sitoutuu peptidoglykaanin peptidiosaan, joka on proksimaalinen, mutta eri kuin se pentaglysiini, jonka katalyyttinen domeeni pilkkoo. Yhteenvetona väitöskirjassa karakterisoitiin uusi M23-perheen entsyymi, ehdotettiin säätelymekanismi sekä muutettiin näkemystä M23-peptidaasien substraatin sitomisesta. Tuloksemme edistävät S. aureus-bakteerin fysiologian tuntemusta sekä tarjoavat keinoja hoidon kehittämiselle

Secretion processes as a limiting factor of protein production in Bacillus

Summary Protein secretion involves several important sequential steps. First, proteins to be secreted must be recognized and their translocation-competent conformation must be ensured. This is followed by the overcoming of two barriers, the cell membrane and the cell wall. The active transport across the membrane can occur by several well-studied mechanisms, the most notably of them are known as "general secretory" (Sec) and "twin-arginine translocation" (Tat). For the passage through the cell wall, on the other hand, understanding is still almost completely lacking. In this work, I investigated this process, using super-resolution fluorescence microscopy to visualize AmyE-mCherry during secretion in Bacillus subtilis and Bacillus licheniformis. The overexpressed fusion protein localized as distinct foci in the cell envelope, which were mostly lost upon degradation of the bacterial cell wall through treatment with lysozyme. I could also show that AmyE is released from the cells at discrete zones, similar to the localization of fluorescently labeled AmyE as foci inside the envelope. High-level protein secretion peaked at the transition from exponential growth to the stationary phase and appears to be restricted to a subpopulation of cells, which presumably is also the case for general protein secretion. Time lapse experiments revealed the AmyE-mCherry foci to be statically positioned throughout several minutes, in contrast to the lateral mobility of Secmachinery associated membrane proteins SecA and SecDF, labeled with mNeonGreen. Interestingly, the AmyE-mCherry foci displayed considerable fluctuations of fluorescence intensities within a minutes-time-scale, suggesting visualized diffusion of proteins along the passage through the cell walls meshwork. This idea of diffusion is supported by recent AFM Imaging results of B. subtilis, revealing a heterologous cell wall structure with deep pores its peptidoglycan surface. For large parts of industrial biotechnology, the secretion of microbially produced enzymes and proteins into the culture supernatant is of enormous relevance, due to the lower costs for subsequent processing associated with this method as compared to the disruption of the producing cells. Studies investigating secretion efficiency in Bacillus species, have revealed numerous influencing factors. Since the bacterial cell wall is often overlooked in the search for secretion bottlenecks, I targeted autolysins that can affect cell wall thickness and the density of the meshwork. While absence of LytD had little effect on secretion, deletion of lytC and lytF significantly impaired AmyE transport to the outside of the cell. By introducing additional genes encoding the autolysins LytC and LytF or the cell wall hydrolase PBP5 (dacA), I was able to improve secretion by up to 200%. These findings suggest that cell wall permeability for secreted proteins is modulated by autolysin activity. Flotillins, which are thought to form functional membrane microdomains (FMM) in B. subtilis, are often linked with secretion, although the nature of this connection is not exactly clear. To approach this subject, I used a ΔyuaG (FloT) deletion strain with reduced AmyE secretion and showed that the addition of the membrane fluidizer benzyl alcohol could recover the AmyE secretion level of the wild type. This result indicates, that flotillins affect protein secretion in B. subtilis through the ability to improve membrane fluidity. Furthermore, I was able to double the efficiency of AmyE secretion of B. subtilis by introducing an additional gene encoding FloT

Expanding the Horizons of Enzybiotic Identification

Recently, phage lytic enzymes (also known as endolysins or, simply, lysins) have received considerable attention as potential antibacterial agents. During the infective cycle of double-stranded DNA phage, these peptidoglycan hydrolases are responsible for digesting the cell wall of the host bacterium and freeing newly-assembled viral particles. At the same time, an increasing body of evidence has demonstrated that recombinantly-purified phage lysins—when added exogenously—can potently kill Gram-positive bacteria, whose peptidoglycan is accessible from the extracellular space. Consequently, lysins have been proposed as novel enzybiotic (i.e. enzyme-antibiotic) molecules that could serve as novel weapons in the fight against drug-resistant bacteria. Most lysins characterized to date were initially identified through either recombinant screening or DNA-sequencing of phage genomes. Recent technological and methodological advances, however, have drastically increased the potential avenues for lysin identification. The goal of the work presented here to exploit and expand upon these advances so that the identification of new lysins is increasingly rapid and straightforward. This thesis is subdivided into four interrelated sections, each of which represents a distinct study into a novel approach/method for cloning phage lysins. The first study (Chapter 2) addresses the issue of bacterial genomic sequencing and how the rapidly expanding database of bacterial genomes represents a vast source of proviral lysins. Focusing on the anaerobic pathogen Clostridium perfringens, the genomes of 9 recently-sequenced strains were computationally mined for prophage lysins and lysin-like ORFs (open reading frames), revealing several dozen proteins of various enzymatic classes. Of these lysins, a muramidase (termed PlyCM) from strain ATCC 13124 was chosen for recombinant analysis based on its dissimilarity to the only other previouslycharacterized C. perfringens lysin. Following expression and purification, various biochemical properties of PlyCM were determined in vitro, including pH/saltdependence and temperature stability. The enzyme exhibited activity at low ï­g/ml concentrations, and it was active against 23/24 strains of C. perfringens tested. Chapters 3 and 4 focus on the emerging field of viral metegenomics, a term which refers to the bulk extraction and analysis of DNA from environmental phage without prior laboratory culture of any particular virus. Phage metagenomes have been shown to be incredibly complex and diverse, and the goal of these chapters was to tap into this diversity through functional metagenomic screens for lytic enzymes. Chapter 3 first addresses a preliminary methodological issue, namely the fact that uncultured phage samples generally do not provide sufficient quantities of DNA for ready screening. A novel ELASL protocol (for expressed linker amplified shotgun library) was developed that combines linker amplification of enzyme-digested DNA with subsequent topoisomerase cloning into linearized expression plasmids. As proof-ofprinciple, genomic and metagenomic E-LASLs were constructed and screened for antibacterial and hemolytic activity in an Escherichia coli host. Six Bacillus anthracis phage lysins were cloned in the process, along with a virulence factor of the aerolysin gene family. Chapter 4 proceeds to address an additional methodological issue surrounding metagenomic lysin identification: the question of how to identify lysin-encoding clones in a functional screen when the targeted bacteria are not pre-defined. A novel two-step screening technique was devised for this purpose. It involves a primary screen in which transformed E. coli clones were identified that demonstrated colony lysis following exposure to nebulized inducing agent. This effect, which can be due to the expression of membrane-permeabilizing phage holins, was discerned by the development a hemolytic-effect in surrounding blood agar. The selected clones were then overlaid with autoclaved Gram-negative bacteria (specifically Pseudomonas aeruginosa) to assay directly for recombinant expression of lytic enzymes, which are often encoded proximally to holins in phage genomes. This method was combined with the aforementioned E-LASL technique and applied to a viral metagenomic library constructed from mixed animal feces. Twenty-six lytic enzymes were cloned in this screen, including both Gram-positive-like and Gram-negative-like enzymes, as well as several atypical lysins whose predicted structures are less common among known phage. Finally, Chapter 5 takes the above techniques and reapplies them outside the context of metegenomics, returning to individual genomes as sources of lytic enzymes. Specifically, 2 lysins were cloned from prophage of Streptococcus suis, an important veterinary and emerging zoonotic pathogen. One of these S. suis enzymes (PlySs1) was identified by applying the two-step screen to the genome of an unsequenced clinical strain. The other (PlySs2) was identified in a manner similar to the clostridial lysin PlyCM, by analyzing the published genomes of various sequenced strains. Finally, PlySs1 was subject to chromatographic purification and in vitro analysis against numerous suis and non-suis strains of streptococci. Currently, both PlySs1 and PlySs2 are involved in a collaborator’s ongoing in vivo trial employing experimentally-infected pigs

Auranofin-loaded nanoparticles as a new therapeutic tool to fight streptococcal infections

Drug-loaded nanoparticles (NPs) can improve infection treatment by ensuring drug concentration at the right place within the therapeutic window. Poly(lactic-co-glycolic acid) (PLGA) NPs are able to enhance drug localization in target site and to sustainably release the entrapped molecule, reducing the secondary effects caused by systemic antibiotic administration. We have loaded auranofin, a gold compound traditionally used for treatment of rheumatoid arthritis,into PLGA NPs and their efficiency as antibacterial agent against two Gram-positive pathogens, Streptococcus pneumoniae and Streptococcus pyogenes was evaluated. Auranofin-PLGA NPs showed a strong bactericidal effect as cultures of multiresistant pneumococcal strains were practically sterilized after 6 h of treatment with such auranofin-NPs at 0.25 μM. Moreover, this potent bactericidal effect was also observed in S. pneumoniae and S. pyogenes biofilms, where the same concentration of auranofin-NPs was capable of decreasing the bacterial population about 4 logs more than free auranofin. These results were validated using zebrafish embryo model demonstrating that treatment with auranofin loaded into NPs achieved a noticeable survival against pneumococcal infections. All these approaches displayed a clear superiority of loaded auranofin PLGA nanocarriers compared to free administration of the drug, which supports their potential application for the treatment of streptococcal infections

Microalgae as a novel production platform for antibacterial proteins

Widespread antibiotic resistance among pathogenic bacteria and the low specificity of these drugs cause a pressing need for the development of novel antibiotics. Endolysins are proteins that are produced by bacteriophage infected cells and digest the bacterial cell wall for phage progeny release. These efficient enzymes are highly specific for the target bacteria without affecting other species. Development of resistance against endolysins is rare, because they evolved to target molecules that are essential for bacterial viability. Taken together, this makes them promising novel antibiotics. The development of recombinant endolysins as antibacterial agents requires an inexpensive and safe production platform. Microalgae emerged as an alternative expression platform in the last years. This study investigated therefore the production of endolysins in two distinct microalgal systems, the eukaryotic green microalga Chlamydomonas reinhardtii and the cyanobacterium Synechocystis sp. PCC 6803. C. reinhardtii is an attractive production platform for therapeutic proteins, due to the lack of endotoxins and infectious agents and its GRAS status (Generally Recognised as Safe). Synechocystis is a prokaryotic system and is natural transformable. Both offer established techniques for the expression of foreign genes and can be cultivated in full containment in simple photobioreactors. Transgenic lines of C. reinhardtii and Synechocystis expressing endolysins specific to the human pathogens Streptococcus pneumoniae and Staphylococcus aureus were created. The S. pneumoniae-specific endolysin Pal has been purified in an active form and its specificity and efficiency in killing the target bacterium demonstrated in vitro. Another endolysin specific to Propionibacterium acnes, a bacterium involved in the skin condition acne vulgaris, was synthesised in Synechocystis. The synthesis of the endolysins in the microalgae was analysed under the influence of different expression elements and at different growth stages, and the yields of recombinant protein were quantified to evaluate microalgae as a production platform for antibacterial and other protein therapeutics. !

Probing the Internalization Mechanism of a Bacteriophage-encoded Endolysin that can Lyse Extracellular and Intracellular Streptococci

Bacteriophage-encoded peptidoglycan hydrolases, or endolysins, have been investigated as an alternative to antimicrobials due to their ability to lyse the bacterial cell wall upon contact. However, pathogens are often able to invade epithelial cells where they can repopulate the mucosal surface after antibiotic or endolysin prophylaxis. Thus, there is growing interest in endolysins that can be engineered, or inherently possess, a capacity to internalize in eukaryotic cells such that they can target extracellular and intracellular pathogens. Previously, one streptococcal specific endolysin, PlyC, was shown to control group A Streptococcus localized on mucosal surfaces as well as infected tissues. To further evaluate the therapeutic potential of PlyC, a streptococci/human epithelial cell co-culture model was established to differentiate extracellular vs. intracellular bacteriolytic activity. We found that a single dose (50 μg/ml) of PlyC was able to decrease intracellular streptococci by 96% compared to controls, as well as prevented the host epithelial cells death. In addition, the internalization and co-localization of PlyC with intracellular streptococci was captured by confocal laser scanning microscopy. Further studies revealed the PlyC binding domain alone, termed PlyCB, with a highly positive-charged surface, was responsible for entry into epithelial cells. By applying site-directed mutagenesis, several positive residues (Lys-23, Lys-59, Arg-66 and Lys-70&71) of PlyCB were shown to mediate internalization. We then biochemically demonstrated that PlyCB directly and specifically bound to phosphatidic acid, phosphatidylserine and phosphatidylinositol through a phospholipid screening assay. Computational modeling suggests that two cationic residues, Lys-59 and Arg-66, form a pocket to help secure the interaction between PlyC and specific phospholipids. Internalization of PlyC was found to be via caveolae-mediated endocytosis in an energy-dependent process with the subsequent intracellular trafficking of PlyC regulated by the PI3K pathway. To the best of our knowledge, PlyC is the first endolysin reported that can penetrate through the eukaryotic lipid membrane and retain biological binding and lytic activity against streptococci in the intracellular niche

Studies on the biochemistry of the Targeting domain of Lysostaphin

Lysostaphin from S. simulans was cloned in expression vector pET21a and expressed and purified in E .coli. A spot test and a broth dilution assay indicated that the minimum inhibitory concentration of lysostaphin required to kill EMRSA-16 was 40 nM. Lysostaphin consists of an endopeptidase and a trargeting domain the former of which codes for catalytic activity while the latter is responsible for substrate specificity. In order to find out whether the targeting domain of lysostaphin is an individually functional domain, it was cloned in vector pET21d and expressed in E. coli. The purified protein was assayed against EMRSA-16 in the presence of mature lysostaphin and it was found that the targeting domain alone can protect EMRSA-16 cells. This further indicated the potential of the targeting domain of lysostaphin to be used in future domainswapping studies with other proteins. Random PCR mutagenesis was used to identify putative active site residues in the C-terminal targeting domain of lysostaphin. One mutation was isolated, where a phenylalanine was replaced by a serine at position 172 of the mature protein. Sequence alignment with other lysostaphin homologues indicated the presence of three more conserved amino acids, two tyrosines at positions 203 and 226 and a tryptophan at position 214. Site-directed mutagenesis was employed to mutate all conserved residues to alanine and FI 72 to tyrosine to distinguish between important from unimportant sites. All six mutants (F172S, F172A, F172Y, Y203A, Y226A and W214A) were cloned and their proteins expressed and purified in E. coli. Their activity was assayed in an agar diffusion assay, a broth dilution assay, turbidimetrically and in a FRET assay. The results indicated that mutants F172S, F172Y, Y203A and Y226A remained bacteriolytic while mutants F172A and W214A had lost most of their activity, suggesting their significance in the activity of lysostaphin. Finally, a reversion experiment carried out with FI72A confirmed the importance of phenylalanine at position 172 of the mature protein