Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae

Characterization of the Modular Design of the Autolysin/Adhesin Aaa from Staphylococcus Aureus

BACKGROUND: Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM) that can confer cell wall attachment and a C-terminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain having bacteriolytic activity in many proteins. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA. CONCLUSIONS/SIGNIFICANCE: We identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might promote S. aureus colonization of host extracellular matrix and tissue, suggesting a role for Aaa in the pathogenesis of S. aureus infections

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

Bacteriophage lytic enzymes and their engineering towards improved antibacterial efficacy

Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2015Increasing antibiotic resistance among bacterial pathogens has been promoting the studyof bacteriophage (phage) lytic enzymes (bacterial cell wall hydrolases) asalternatives/complements to antibiotics. Phages can employ two types of these enzymesduring their life cycle: i) virion-associated lysins (VALs), which promote a local cleavageof cell wall bonds to facilitate phage genome entry into the host cell; and ii) endolysinsthat destroy the wall at the end of infection, leading to cell burst and release of virionprogeny. We studied the lytic activity of two enterococcal endolysins, Lys168 andLys170, towards clinical isolates of different Gram-positive bacterial pathogens. In theconditions tested, both enzymes showed broad antimicrobial activity against E. faecalis,including vancomycin-resistant strains, and to less extent against E. faecium.We show that lys170 expression results in the production of the expected full lengthpolypeptide (Lys170FL, 32.6 kDa) and of a C-terminal fragment of the enzyme(CWB170, 12 kDa), with both proteins co-eluting in the purification steps. Furtheranalysis revealed that CWB170 corresponded to the Lys170 cell wall binding domain,which is independently produced from an in-frame, secondary translational start site.Biochemical and biophysical analysis indicated that the fully active Lys170 is a complexmost likely corresponding to one subunit of Lys170FL associated to three of CWB170.Study of Lys170 has thus uncovered a new strategy of increasing the number of CWBdomains in this type of enzymes.A frequently reported problem when working with phage lytic enzymes is theirpropensity to become insoluble. Further, the activity of endolysins is rarely studied inconditions that promote robust growth of target bacteria. With the goal of supplantingthese limitations we engineered a chimerical lysin, EC300, aimed at lysing E. faecalisgrowing in rich culture media. EC300 resulted from the fusion of a M23 endopeptidasedomain of a VAL to the CWB170 domain of Lys170. The bacteriolysin-like proteinexhibited a clear enhanced lytic activity when compared to the parental endolysin,particularly when assayed in a rich culture medium, thus having the potential to be usedas an anti-E. faecalis therapy.Fundação para a Ciência e a Tecnologia (FCT

Cell wall architecture and the role of wall teichoic acid in Staphylococcus aureus

The bacterium Staphylococcus aureus only synthesises peptidoglycan during cell division at the septum using a complex protein biosynthetic apparatus called the divisome. It divides sequentially in three orthogonal planes, using heritable features within the peptidoglycan architecture to maintain this process over generations. The ‘rib’ features that form this ‘memory’ are remnants of a large belt of peptidoglycan called the ‘piecrust’ that is formed at the initiation of septation and before the septal plate. After division, the ribs remain as orthogonal features, which are bisected by further ‘piecrust’ features from ensuing division cycles. This results in a characteristic pattern of different age peptidoglycan sectors, delineated by ribs of a different architecture. As well as maintaining cellular viability and shape the peptidoglycan layer also acts as a scaffold for many other polymers, including wall teichoic acid (WTA). WTA is known to direct and modulate cell wall hydrolase activity. There has been recent debate as to its subcellular localisation. In this study, using the bacterial two-hybrid assay, four putative WTA biosynthesis enzymes were found to interact with numerous members of the divisome. Microscopy techniques localised WTA across the entire cell surface except on the piecrust and rib features. It was hypothesised that WTA blocks the rest of the peptidoglycan thereby directing the localisation of hydrolases and other proteins. The localisation of peptidoglycan hydrolases was studied. Those found to localise to the rib and piecrust features (Atl(glucosaminidase), Atl(amidase), SagB and ScaH) showed a distinct pattern which was completely disrupted in a strain missing WTA. Conversely those (SceD) not associated with rib/piecrust showed no difference to localisation with loss of WTA. The processed forms of Atl (glucosaminidase and amidase) demonstrated different binding properties during the cell cycle and a model to illustrate the cell cycle dependent binding is proposed

Bacteria present mechanisms to evade cellular and humoral responses mediated through peptidoglycan recognition by PGRP-SA and PGRP-LC

The Host presents different innate immune components to fight bacterial infections, most of which are evolutionary conserved strategies. Conversely, Bacteria present numerous mechanisms of virulence and evasion, transversal to different bacterial species, that confer resistance or subvert the activity of the Host components. This thesis presents a study on how the Host perceives the Bacteria and reacts to them and how the Bacteria protect themselves from those responses.(...