Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b

Lysostaphin is a bacteriolytic enzyme targeting peptidoglycan, the essential component of the bacterial cell envelope. It displays a very potent and specific activity toward staphylococci, including methicillin-resistant Staphylococcus aureus. Lysostaphin causes rapid cell lysis and disrupts biofilms, and is therefore a therapeutic agent of choice to eradicate staphylococcal infections. The C-terminal SH3b domain of lysostaphin recognizes peptidoglycans containing a pentaglycine crossbridge and has been proposed to drive the preferential digestion of staphylococcal cell walls. Here we elucidate the molecular mechanism underpinning recognition of staphylococcal peptidoglycan by the lysostaphin SH3b domain. We show that the pentaglycine crossbridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3b domain, thereby inducing a clustering of SH3b domains. We propose that this unusual binding mechanism allows synergistic and structurally dynamic recognition of S. aureus peptidoglycan and underpins the potent bacteriolytic activity of this enzyme

Bacterial size matters:Multiple mechanisms controlling septum cleavage and diplococcus formation are critical for the virulence of the opportunistic pathogen Enterococcus faecalis

Enterococcus faecalis is an opportunistic pathogen frequently isolated in clinical settings. This organism is intrinsically resistant to several clinically relevant antibiotics and can transfer resistance to other pathogens. Although E. faecalis has emerged as a major nosocomial pathogen, the mechanisms underlying the virulence of this organism remain elusive. We studied the regulation of daughter cell separation during growth and explored the impact of this process on pathogenesis. We demonstrate that the activity of the AtlA peptidoglycan hydrolase, an enzyme dedicated to septum cleavage, is controlled by several mechanisms, including glycosylation and recognition of the peptidoglycan substrate. We show that the long cell chains of E. faecalis mutants are more susceptible to phagocytosis and are no longer able to cause lethality in the zebrafish model of infection. Altogether, this work indicates that control of cell separation during division underpins the pathogenesis of E. faecalis infections and represents a novel enterococcal virulence factor. We propose that inhibition of septum cleavage during division represents an attractive therapeutic strategy to control infections

Production of recombinant Staphylococcus aureus serine protease for proteomic analysis

Najlepiej poznaną i najliczniejszą grupą proteaz są proteazy serynowe. Odgrywają one znaczącą rolę w funkcjonowaniu organizmów, między innymi u ssaków biorą udział w procesach fizjologicznych takich jak: trawienie białek w układzie trawiennym, kaskadzie krzepnięcia krwi, czy w zapłodnieniu. Glutamyloendopeptydaza (Glu-C) należy do proteaz serynowych produkowanych przez gatunek Staphylococcus aureus. Proteaza ta wykazuje wysoką specyficzność, hydrolizując wiązania peptydowe po karboksylowej stronie reszt kwasu glutaminowego i asparaginowego. Determinantą specyficzności enzymu Glu-C jest N-końcowa reszta waliny 69, która pozwala na lokowanie się w kieszeni reszt aminokwasowych posiadających w łańcuchu bocznym grupę γ-karboksylową. W części doświadczalnej niniejszej pracy przedstawiono sposób konstrukcji szczepu S. aureus BARS4, który wykazywał znaczną nadprodukcję rekombinowanej glutamyloendopeptydazy posiadającej motyw polihistydynowy na C-końcu. Gen proteazy Glu-C znajdował się po kontrolą konstytutywnego promotora P2. Sekwencja promotorowa pochodzi ze szczepu S. aureus CH91, u którego reguluje ekspresję stafopainy A2. Zastosowanie układu homogenicznego zapewniło uzyskanie aktywnej formy rekombinowanej proteazy w płynach pohodowlanych. Zastosowany szczep gronkowcowy posiada wyłączony natywny gen protezy Glu-C (sspA), co umożliwiło uniknięcie zanieczyszczenia uzyskanego preparatu rekombinowanej Glu-C natywną formą tego enzymu. Wprowadzony motyw polihistydynowy pozwolił na jednoetapowe oczyszczenie rekombinowanej glutamyloendopeptydazy. Ze 100 ml hodowli S. aureus BARS4 uzyskano 15 mg białka o wysokiej czystości. Aktywność uzyskanego preparatu rekombinowanego enzymu jest porównywalna z aktywnością komercyjnie dostępnej glutamyloendopeptydazy. Co więcej, rekombinowana proteaza Glu-C wykazuje specyficzność głównie względem reszt kwasu glutaminowego i asparaginowego.Uzyskany preparat rekombinowanej glutamyloendopeptydazy produkowanej przez szczep S. aureus BARS4 nadaje się do zastosowania w badaniach proteomicznych.Serine proteases are the best characterised and the largest group of proteases. Serine proteases play a significant role in organism functioning. In mammals they are involved in physiological processes such as protein digestion in the gastrointestinal tract, coagulation cascade and fertilisation. Glutamyl endopeptidase (Glu-C) is a serine protease produced by Staphylococcus aureus species. Glu-C protease specifically cleaves peptide bonds after glutamic and aspartic acid residues. N-terminal Valine 69 determines substrate-specificity of the enzyme, allowing for bonding of amino acid residues with γ-carboxyl group to active site of the enzyme.In the experimental part of this thesis it was shown how the strain S. aureus BARS4 was constructed. The strain S. aureus BARS4 shows high overproduction of recombinant glutamyl endopeptidase, which has a polyhistidine-tag at its C-terminal end. Gene of recombinant protease Glu-C was under control of the constitutive promoter P2. Promoter sequence was derived from S. aureus strain CH91 where it regulates expression of stafopain A2. Application of the homogeneous system enabled production of the active form of the recombinant protease into liquid medium. The applied S. aureus strain has a knock-out on the native sspA, which allows to avoid contamination of recombinant enzyme preparation with native glutamyl endopeptidase. Polyhistidine-tag attached to C-terminal end of protease enabled for one-step purification of recombinant protein. 15 mg of recombinant protein of high-purity was purified from 100 ml of the cell culture. The activity of obtained recombinant enzyme preparation is comparable to the activity of commercially available glutamyl endopeptidase. Moreover, recombinant protease Glu-C preferentially recognises glutamic and aspartic acid residues. The recombinant glutamyl endopeptidase preparation form S. aureus BARS4 is suitable for applications in proteomic analysis

<i>E</i>. <i>faecalis</i> expressing <i>atlB</i> under the P<i>atlA</i> promoter forms cell chains.

<p><b>A</b>. Western blot detection of His-tagged AtlA and AtlB proteins expressed under the control of the <i>atlA</i> (P<i>atlA</i>) or <i>atlB</i> (P<i>atlB</i>) promoters. Various amounts of exponentially growing cultures were harvested and total extracts corresponding to mixtures of broken cells and supernatants were analyzed by Western blot using anti-His antibodies. The faint band corresponding to AtlB-His is indicated by an arrowhead. <b>B</b>. Comparison of median forward scattered (FSC) light values corresponding to the cell chain lengths of WT, Δ<i>atlA</i> and P<i>atlB</i>::<i>atlB-his</i> strains; ****<i>P</i><0.0001; n = 3; NS, <i>P</i>>0.05; n = 3.</p

AtlA <i>N</i>-acetylglucosaminidase activity is not essential for septum cleavage.

<p><b>A</b>. Schematic representation of AtlA variants expressed by recombinant <i>E</i>. <i>faecalis</i> JH2-2 derivatives. All strains were constructed by allele exchange to express AtlA variants under the control of the <i>atlA</i> promoter (arrow). Two restriction sites (NcoI, N and BglII, B) flanking the region encoding the catalytic domain were introduced by site-directed mutagenesis. The resulting allele in strain <i>atlA</i>* encodes an AtlA protein with eleven modified amino-acids (see supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006526#ppat.1006526.s003" target="_blank">S3 Fig</a>). The NcoI-BglII fragment encoding AtlA <i>N</i>-acetylglucosaminidase activity was replaced with a fragment encoding the <i>N</i>-acetylmuramidase activity of <i>E</i>. <i>faecalis</i> AtlB to generate strain <i>atlA</i>_AtlB, the amidase activity of <i>S</i>. <i>aureus</i> Atl to generate strain <i>atlA</i>_Ami or the endopeptidase activity of <i>Streptococcus thermophilus</i> Cse to generate strain <i>atlA</i>_Cse. <b>B</b>. <i>E</i>. <i>faecalis</i> peptidoglycan bonds cleaved by the catalytic domains of <i>E</i>. <i>faecalis</i> AtlA and AtlB, <i>S</i>. <i>aureus</i> AtlA and <i>S</i>. <i>thermophilus</i> Cse. <b>C</b>. Comparison of median forward scattered (FSC) light values corresponding to the cell chain lengths of WT, <i>atlA</i>*, <i>atlA</i>_AtlB, <i>atlA</i>_Ami, <i>atlA</i>_Cse. and Δ<i>atlA</i>. All median FSC values were significantly different from the median FSC value from the Δ<i>atlA</i> strain (**<i>P</i><0.01; n = 3).</p

<i>E</i>. <i>faecalis</i> long cell chains are less virulent in the zebrafish model of infection and more prone to phagocytosis than diplococci.

<p><b>A</b>. Survival of zebrafish larvae (n>20) following infection with <i>E</i>. <i>faecalis</i> OG1RF (WT) and <i>atlA</i> isogenic deletion mutant before (Δ<i>atlA</i>) and after (Δ<i>atlA</i>^S) sonication to disperse long chains. Statistical significance was determined by Log-rank test; **<i>P</i> = 0.0011; *** <i>P</i> = 0.0002; NS, <i>P</i>>0.05. <b>B</b>. Quantification of <i>E</i>. <i>faecalis</i> uptake by zebrafish phagocytes. Embryos were infected with 1,200 <i>E</i>. <i>faecalis</i> cells expressing GFP and fixed in 4% paraformaldehyde 1.5h post infection. Phagocytes were immunolabelled using rabbit anti L-plastin antibodies and detected with goat anti-rabbit antibodies conjugated to Alexafluor 647. Fluorescent bacteria and phagocytes were imaged by scanning confocal microscopy. The area of GFP fluorescence signal outside and inside phagocytes was measured using a dedicated Fiji plugin. The ratio of GFP fluorescence area outside to inside phagocytes was used to quantify bacterial uptake. Phagocytosis was significantly higher for long chains (Δ<i>atlA</i>) when compared to their sonicated counterparts (Δ<i>atlA</i>^S) (**<i>P</i> = 0.0098) or the wild-type cells (*<i>P</i> = 0.0438). No difference in uptake was found between short chains corresponding to the wild-type or sonicated Δ<i>atlA</i> mutant (NS, <i>P</i>>0.05). Representative images of phagocytes (magenta) following infection with Δ<i>atlA</i>, sonicated Δ<i>atlA</i>^S and wild-type OG1RF cells shown. Phagocytes labeled with L-plastin appear in magenta, GFP-producing bacteria in green. Scale bar is 20μm. <b>C</b>. Survival of phagocyte-depleted zebrafish larvae (n>20) following injection with <i>E</i>. <i>faecalis</i> OG1RF (WT) or Δ<i>atlA</i>. <b>D</b>. Pairwise comparisons of phagocytosis indexes corresponding to <i>E</i>. <i>faecalis</i> OG1RF and Δ<i>atlA</i> uptake by human monocyte-derived macrophages (MDM). Statistical significance was determined by paired t-test; Δ<i>atlA</i> cells were more efficiently phagocytosed by MDM than WT cells (<i>**P</i> = 0.0024; n = 7).</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

<i>E</i>. <i>faecalis</i> strains lacking AtlA glycosylation form shorter cell chains.

<p><b>A</b>. Schematic representation of the <i>atlA</i>_TEV allele produced by <i>E</i>. <i>faecalis</i>. <b>B</b>. Metal affinity purification of cell surface associated His-tagged AtlA extracted with 8M urea. Two bands indicated by arrowheads were detected on a Coomassie-stained SDS-PAGE (lane 1); the upper band (72 kDa) corresponds to full-length AtlA proteins and the lower band to AtlA without the N-terminal domain (62 kDa). A clear signal corresponding to glycosylated full length AtlA (lane 2) was detected using the ECL glycoprotein detection kit (GE Healthcare). <b>C</b>. Exponentially growing cells from a culture expressing AtlA_TEV were resuspended in buffer in the absence (-) or presence (+) of TEV protease to cleave the N-terminal domain of AtlA. Solubilized proteins were recovered by centrifugation, loaded on an SDS-PAGE and transferred on nitrocellulose to detect glycosylated proteins. Two independent cultures treated with the TEV protease were analysed in parallel. In both cases, a glycosylated polypeptide with the expected molecular weight for the N-terminal domain (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006526#ppat.1006526.g002" target="_blank">Fig 2A</a>) was detected while no signal was observed in the negative control. When a similar experiment was repeated with protein extracts from a Δ<i>gtfAB</i> mutant, no glycosylated protein was detected, indicating that this operon is involved in the post translational modification of AtlA. <b>D</b>. Comparison of median forward scattered (FSC) light values corresponding to the cell chain lengths of WT, Δ<i>gtfAB</i>, <i>atlA</i>_1-4, <i>atlA</i>_1-4Δ<i>gtfAB</i>, Δ<i>atlA</i> and Δ<i>atlA</i>Δ<i>gtfAB</i> strains.</p

Contribution of the LysM domain to septum cleavage.

<p><b>A</b>. Schematic representation of AtlA and AtlB derivatives expressed and purified to test their septum cleavage activity. Full-length AtlA and AtlB (without signal peptides), as well as their counterparts with LysM_B (AtlAB) or LysM_A (AtlBA) domains, were expressed in <i>E</i>. <i>coli</i>. <b>B</b>. SDS-PAGE of AtlA (lane 1), AtlAB (lane 2), AtlB (lane 3) and AtlBA (lane 4) samples showing that all proteins were purified to homogeneity. <b>C</b>. Flow cytometry analysis of septum cleavage activity of recombinant proteins <i>in vitro</i> using OG1RF Δ<i>atlA</i> cell chains as a substrate (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006526#sec011" target="_blank">materials and methods</a>). The Di₅₀ (Dechaining index) value corresponds to the amount of enzyme in pmoles that is able to decrease the median FSC value of Δ<i>atlA</i> cell chains by 50% in 15 minutes at 37°C. <b>D</b>. Schematic representation of <i>atla</i> locus in <i>E</i>. <i>faecalis</i> JH2-2 and isogenic derivatives producing AtlA with a C-terminal LysM domain containing a variable number of LysM repeats (6 in WT; 5 in <i>atlA</i>_1-5; 4 in <i>atlA</i>_1-4; 3 in <i>atlA</i>_1-3; 2 in <i>atlA</i>_1-2; 1 in <i>atlA</i>₁). <b>E</b>. Comparison of median forward scattered (FSC) light values corresponding to the cell chain lengths of WT, <i>atlA</i>_1-5, <i>atlA</i>_1-4, <i>atlA</i>_1-3, <i>atlA</i>_1-2, <i>atlA</i>₁ and Δ<i>atlA</i> strains. <i>P</i> values and significance corresponding to comparisons with the Δ<i>atlA</i> strain are indicated.</p