Ibrahim AM, Azam MS, Schneewind O, Missiakas D. Processing of LtaS restricts LTA assembly and YSIRK preprotein trafficking into Staphylococcus aureus cross-walls. mBio. 2024 Jan 4; Epub ahead of print. PMID: 38174934.

Bernardino, P. N., Bhattacharya, M., Chen, X., Jenkins, J., Missiakas, D., & Thammavongsa, V. (2023). A humanized monoclonal antibody targeting protein a promotes opsonophagocytosis of Staphylococcus aureus in human umbilical cord blood. Vaccine41(35), 5079-5084.

Bobrovskyy, M., Chen, X., & Missiakas, D. (2023). The Type 7b Secretion System of S. aureus and Its Role in Colonization and Systemic Infection. Infection and Immunity, e00015-23.

Chen, X., Gula, H., Pius, T., Ou, C., Gomozkova, M., Wang, L. X., … & Missiakas, D. (2023). Immunoglobulin G subclasses confer protection against Staphylococcus aureus bloodstream dissemination through distinct mechanisms in mouse models. Proceedings of the National Academy of Sciences120(14), e2220765120.

Bobrovskyy, M., Oh, S. Y., & Missiakas, D. (2022). Contribution of the EssC ATPase to the assembly of the type 7b secretion system in Staphylococcus aureus. Journal of Biological Chemistry298(9).

Chen, X., Schneewind, O., & Missiakas, D. (2022). Engineered human antibodies for the opsonization and killing of Staphylococcus aureus. Proceedings of the National Academy of Sciences119(4).

Shi, M., Chen, X., Sun, Y., Kim, H. K., Schneewind, O., & Missiakas, D. (2021). A protein A based Staphylococcus aureus vaccine with improved safety. Vaccine39(29), 3907–3915. https://doi.org/10.1016/j.vaccine.2021.05.072

Shi, M., Willing, S. E., Kim, H. K., Schneewind, O., & Missiakas, D. (2021). Peptidoglycan Contribution to the B Cell Superantigen Activity of Staphylococcal Protein A. mBio12(2), e00039-21. https://doi.org/10.1128/mBio.00039-21

FIG 6 Model for glycan chain elongation and cleavage in S. aureus. Glycan chain elongation is catalyzed by glycosyltransferase (GT) on the trans side of the plasma membrane. GT initiates the deprotonation of the GlcNAc 4-OH of lipid II via its active site residue. The activated nucleophile attacks the lipid-linked MurNAc carbon C-1 of the growing glycan chain, leading to the formation of a new β-1,4 glycosidic bond and the elongation of the glycan chain (34–36). Cleavage of the extended chain by SagB releases a glycan product that may be incorporated in cell wall peptidoglycan by transpeptidases (6). The second SagB product remains lipid-bound and may serve once more as a GT substrate. In this model, LyrA may help align the substrate into the active site of SagB by binding the lipid moiety of the glycan chain. R: stem peptide with pentaglycine cross bridge. GlcNAc and MurNAc are depicted as blue and red squares or blue and red chemical structures, respectively.

Stephanie Willing , Olaf Schneewind, Dominique Missiakas. Regulated cleavage of glycan strands by the murein hydrolase SagB in S. aureus involves a direct interaction with LyrA (SpdC).J Bacteriol.2021 Feb 16;JB.00014-21. doi: 10.1128/JB.00014-21

Figure 2 Staphylococcal interference with host cell death machineries. All major cell death modes including apoptosis, pyroptosis, and necroptosis may occur in response to extra- or intracellular staphylococci and their exoproducts (see Table 1). While apoptotic cell death is immunologically silent, pyroptosis and necroptosis cause strong inflammatory responses due to the release of pro-inflammatory molecules from injured host cells. Characteristic features and canonical signaling pathways of cell death modalities are indicated.

Dominique Missiakas , Volker Winstel. Selective Host Cell Death by Staphylococcus aureus: A Strategy for Bacterial Persistence. Front Immunol.2021 Jan21;11:621733. doi:10.3389/fimmu.2020.621733. eCollection 2020.

FIG 2 vWbp rather than Coa determines bacterial joint invasiveness. NMRI mice inoculated with S. aureus Newman, Δcoa, Δvwb, and Δcoa Δvwb strains (4.0 × 106 CFU/mouse) were sacrificed on day 10. The data from 3 independent experiments were pooled. (A and B) Severity (A) and frequency (B) of clinical arthritis were observed for 10 days postinfection. (C and D) Cumulative bone erosion scores (C) and frequency of bone destruction (D) of the joints from all 4 limbs of NMRI mice as assessed by microcomputed tomography scan. (E) Histological evaluation of the joints, including synovitis and bone erosion scores from all 4 limbs 10 days after infection. Statistical evaluations were performed using the Mann–Whitney U test (A, C, and E) and Fisher’s exact test (B and D). Data are expressed as mean values ± SEM. *, P < 0.05; **, P < 0.01, ***, P < 0.001.

Manli Na , Zhicheng Hu , Majd Mohammad , Mariana do Nascimento Stroparo , Abukar Ali , Ying Fei , Anders Jarneborn , Peter Verhamme , Olaf Schneewind , Dominique Missiakas , Tao Jin. The Expression of von Willebrand Factor-Binding Protein Determines Joint-Invading Capacity of Staphylococcus aureus, a Core Mechanism of Septic Arthritis. mBio.2020 Nov 17;11(6):e02472-20. doi: 10.1128/mBio.02472-20.

Xinhai Chen, Miaomiao Shi, Xin Tong, Hwan Keun Kim, Lai-Xi Wang, Olaf Schneewind, and Dominique Missiakas. Glycosylation-dependent opsonophagocytic activity of staphylococcal protein A antibodies. PNAS September 15, 2020 117 (37) 22992-23000; first published August 27, 2020

Figure 4FmhC acts as an immunity factor for LytN. A, schematic of dividing daughter cells illustrating the location of the cell wall envelope, membrane (black), and peptidoglycan (gray). The cross-wall structure has been expanded to show LytN cleaving peptidoglycan. The model postulates that FmhC synthesizes peptidoglycan (shaded in green) that is resistant to LytN hydrolysis. The proposed structure of this cross-linked peptidoglycan is shown with sites cleaved by LytN (black arrow) or refractory to LytN cleavage (green inhibition sign). Green and blue hexagons: GlcNAc and N-acetylmuramic acid, respectively. B, the growth rate of S. aureus Newman pCL55 and Newman pCL55-lytN carrying pJK4, pJK4-fmhA, or pJK4-fmhC was interrogated by monitoring changes in A600 over 16 h at 37 °C. Cultures were grown in the presence of anhydrotetracycline and IPTG inducers. C, purified dimeric peptidoglycan fragments obtained from Newman carrying control vector pitet or pitet-fmhC, as shown in Fig. 2B, were treated with buffer (insets a and b) or rLytN (insets c and d). Reaction products were separated by HPLC. Peaks denoted with Roman numerals (i–iv) were subjected to MALDI-TOF analysis and the corresponding m/z values are listed in Table 2. Predicted structures of peptidoglycan fragments i (panels a and b) and ii (panels c–d) are shown on the figure.

Stephanie Willing, Emma Dyer, Olaf Schneewind and Dominique Missiakas. FmhA and FmhC of Staphylococcus aureus incorporate serine residues into peptidoglycan cross-bridges. doi: 10.1074/jbc.RA120.014371

FIG 5 Model of macrophage exclusion from staphylococcal abscesses. Diagram illustrating the proposed role of caspase-3 during replication of S. aureus in deep-seated abscesses. Replicating S. aureus cells exploit AdsA to generate dAdo from NETs, thereby triggering caspase-3 activation and macrophage apoptosis. Caspase-3 deficiency promotes macrophage infiltration into infectious foci which affects abscess persistence and prevents the dissemination of bacteria to new foci.

Winstel V, Schneewind O, Missiakas D. Staphylococcus aureus Exploits the Host Apoptotic Pathway To Persist during Infection. MBio. 2019 Nov 12;10(6). pii: e02270-19. doi: 10.1128/mBio.02270-19

Differential mechanisms of S. aureus adhesion to damaged or inflamed heart valves.

Liesenborghs L, Meyers S, Lox M, Criel M, Claes J, Peetermans M, Trenson S, Vande Velde G, Vanden Berghe P, Baatsen P, Missiakas D, Schneewind O, Peetermans WE, Hoylaerts MF, Vanassche T, Verhamme P. Staphylococcus aureus endocarditis: distinct mechanisms of bacterial adhesion to damaged and inflamed heart valves. Eur Heart J. 2019 Oct 14;40(39):3248-3259. doi: 10.1093/eurheartj/ehz175

Schematic illustrating S. aureus cell division, PBP2-mediated synthesis of cross-wall peptidoglycan, LtaS-mediated synthesis of lipoteichoic acid (LTA) in septal membranes, and the trafficking of surface protein precursors with YSIRK-GXXS motif signal peptides (blue circles) to septal membranes. Following translocation, sortase A-mediated cell wall sorting, and incorporation into the cross-wall, the peptidoglycan layer is split and divided cells are separated, exposing cross-wall-incorporated proteins on the bacterial surface. Surface proteins with canonical signal peptides (red circles) are deposited into polar segments of the peptidoglycan layer.

Schneewind O, Missiakas DM. Staphylococcal Protein Secretion and Envelope Assembly. Microbiol Spectr. 2019 Jul;7(4). doi: 10.1128/microbiolspec.GPP3-0070-2019

Putra I, Rabiee B, Anwar KN, Gidfar S, Shen X, Babalooee M, Ghassemi M, Afsharkhamseh N, Bakhsh S, Missiakas D, Nezamabadi A, Milani B, Eslani M, Djalilian AR. Staphylococcus aureus alpha-hemolysin impairs corneal epithelial wound healing and promotes intracellular bacterial invasion.
Exp Eye Res. 2019 Apr;181:263-270. doi: 10.1016/j.exer.2019.02.019. Epub 2019 Feb 27

Putra I, Rabiee B, Anwar KN, Gidfar S, Shen X, Babalooee M, Ghassemi M, Afsharkhamseh N, Bakhsh S, Missiakas D, Nezamabadi A, Milani B, Eslani M, Djalilian AR. Staphylococcus aureus alpha-hemolysin impairs corneal epithelial wound healing and promotes intracellular bacterial invasion. Exp Eye Res. 2019 Apr;181:263-270. doi: 10.1016/j.exer.2019.02.019. Epub 2019 Feb 27

Missiakas D. Staphylococcus aureus TarP: A Brick in the Wall or Rosetta Stone? Cell Host Microbe. 2019 Feb 13;25(2):182-183. doi: 10.1016/j.chom.2019.01.013

Administration of mouse recombinant monoclonal antibody (rMAb) 3F6 promotes Staphylococcus aureus decolonization in mice. A, Enzyme-linked immunosorbent assay examining rMAb 3F6 and mouse hybridoma monoclonal antibody (hMAb) 3F6 affinity for the mouse neonatal Fc receptor. Dissociation constants (Kd) were calculated using GraphPad Prism software. B, Plasma concentration-time profile of rMAb 3F6 following intraperitoneal administration into C57BL/6 mice (n = 5). C, Intraperitoneal administration of rMAb 3F6 prior to nasal inoculation with S. aureus WU1 promotes progressive decolonization of staphylococci from the nasopharynx of C57BL/6 mice (n = 10). The median and standard deviation for each group of animals on a given day are indicated by the horizontal lines and error bars. D, Administration of rMAb 3F6 prior to colonization promotes enhanced antistaphylococcal serum immunoglobulin G responses. Sera of animals shown in (C) were tested for antibodies against the indicated S. aureus antigens. Error bars ± standard error of the mean were calculated from multiple independent repetitions of the experiments. *P < .05. Abbreviations: A450, absorbance at 450 nm; CFU, colony-forming units; hMAb, mouse hybridoma monoclonal antibody; IgG, immunoglobulin G; IgG2a, immunoglobulin G2a; mFcRn, mouse neonatal Fc receptor; PBS, phosphate-buffered saline; rMAb, mouse recombinant monoclonal antibody; SpA, staphylococcal protein A.

Chen X, Sun Y, Missiakas D, Schneewind O. Staphylococcus aureus Decolonization of Mice With Monoclonal Antibody Neutralizing Protein A.
J Infect Dis. 2019 Feb 23;219(6):884-888. doi: 10.1093/infdis/jiy597

Sortase-mediated anchoring to the cell wall envelope of Staphylococcus aureus using SpA as a model substrate. (A) Drawing to illustrate the primary structure of the SpA precursor with its N-terminal signal peptide and signal peptidase cleavage site, the five immunoglobulin binding domains (IgBDs), region X (Xr) LysM domain and C-terminal LPXTG motif sorting signal with cleavage site for sortase A. Cell wall-SpA is linked to peptidoglycan via an amide bond between the carboxyl group of the C-terminal threonine and the amino group of the pentaglycine crossbridge. Released-SpA is liberated from the cell wall envelope via the action of several murein hydrolases. (B) Drawing to illustrate S. aureus secretion of SpA precursor, sortase-mediated cleavage of SpA precursor and acyl-enzyme formation, resolution of the acyl-enzyme by lipid II to generate SpA-linked to lipid II, incorporation of SpA into the cell wall via the transpeptidation and transglycosylation reaction, and release of SpA from the cell wall envelope by murein hydrolases. Released SpA bears the overall structure: L-Ala-D-iGln-L-Lys(SpA-LPET-Gly5)-D-Ala-Gly4.

Schneewind O, Missiakas D. Sortases, Surface Proteins, and Their Roles in Staphylococcus aureus Disease and Vaccine Development. Microbiol Spectr. 2019 Jan;7(1). doi: 10.1128/microbiolspec.PSIB-0004-2018

Bobrovskyy M, Willing SE, Schneewind O, Missiakas D. EssH Peptidoglycan Hydrolase Enables Staphylococcus aureus Type VII Secretion across the Bacterial Cell Wall Envelope.
J Bacteriol. 2018 Sep 24;200(20). pii: e00268-18. doi: 10.1128/JB.00268-18. Print 2018 Oct 15.

Ahmed MM, Aboshanab KM, Ragab YM, Missiakas DM, Aly KA. The transmembrane domain of the Staphylococcus aureus ESAT-6 component EssB mediates interaction with the integral membrane protein EsaA, facilitating partially regulated secretion in a heterologous host. Arch Microbiol. 2018 Sep;200(7):1075-1086. doi: 10.1007/s00203-018-1519-x. Epub 2018 May 8

Diagram illustrating S. aureus killing of macrophages via hENT1-mediated uptake of AdsA/Nuc-derived dAdo, ADK/DCK-catalyzed conversion of dAdo to dAMP, as well as subsequent conversions to dADP and dATP, triggering caspase-3 activation and macrophage apoptosis.

Winstel V, Missiakas D, Schneewind O. Staphylococcus aureus targets the purine salvage pathway to kill phagocytes.
Proc Natl Acad Sci U S A. 2018 Jun 26;115(26):6846-6851. doi: 10.1073/pnas.1805622115. Epub 2018 Jun 11

Yu W, Missiakas D, Schneewind O. Septal secretion of protein A in Staphylococcus aureus requires SecA and lipoteichoic acid synthesis.
Elife. 2018 May 14;7. pii: e34092. doi: 10.7554/eLife.34092

S. aureus WU1 expression of SpA is required for persistent colonization of C57BL/6 mice. (A) Immunoblots of S. aureus lysates derived from strains USA300 LAC, Newman, WU1, and the Δspa variant of WU1 with and without a plasmid for spa expression (pspa) were probed with SpA-specific (αSpA) and sortase A-specific (αSrtA) antibodies. Sizes are shown on the left in kilodaltons. (B) Cohorts of C57BL/6 mice (n = 10) were inoculated intranasally with 1 × 108 CFU of S. aureus WU1 or its Δspa variant, and the oropharynxes of the animals were swabbed at weekly intervals to enumerate the bacterial load. Each dot indicates the number of CFU per mouse. The median and standard deviation for each group of animals on a given day are indicated by the horizontal lines and error bars. The bacterial colonization data sets were analyzed with two-way ANOVA and Sidak multiple-comparison tests; statistically significant differences (*, P < 0.05) between the two groups of animals are indicated.

Sun Y, Emolo C, Holtfreter S, Wiles S, Kreiswirth B, Missiakas D, Schneewind O. Staphylococcal Protein A Contributes to Persistent Colonization of Mice with Staphylococcus aureus. J Bacteriol. 2018 Apr 9;200(9). pii: e00735-17. doi: 10.1128/JB.00735-17. Print 2018 May 1.

Solubilization of EssB from Staphylococcus aureus membranes. (A) Schematic representation of the ESS gene cluster of S. aureus strain USA300 LAC with its four modules (1 to 4). Transmembrane segments were deduced using the topology prediction software TMHMM Server v. 2.0 (32). (B) Proposed topology and domain architecture of four proteins (EsaA, EssA, EssB, and EssC) with predicted transmembrane segments. Structural data are not available for EsaA and EssA; the EssB homodimer is drawn as reported previously (27). The two forkhead-associated and three ATPase-type domains of EssC are shown in light and dark orange, respectively (42). Numbers indicate positions of amino acids in the sequences of the proteins. (C) S. aureus USA300 LAC was grown on tryptic soy agar supplemented with 0.2% horse serum. Staphylococci were suspended in buffer and washed, and cell wall peptidoglycan was digested with lysostaphin. Cell lysate was obtained by removing unbroken cells by centrifugation at 5,000 × g for 15 min. Cell lysate in the supernatant then was subjected to ultracentrifugation at 100,000 × g for 2 h, separating membrane proteins in the sediment (UP1, ultracentrifugation pellet 1) from soluble cytoplasmic proteins (US1, ultracentrifugation supernatant 1). The UP1 sample was suspended in buffer with 2% n-dodecyl-β-d-maltoside (DDM) and again centrifuged at 100,000 × g for 2 h, generating samples UP2 and US2. Proteins in all fractions were separated by 15% SDS-PAGE and visualized with Coomassie blue staining or transferred to PVDF membranes for immunoblotting with rabbit antibodies specific for sortase A (anti-SrtA), EssB (anti-EssB), EsxA (anti-EsxA), and the cytoplasmic ribosomal protein L6 (anti-L6). Numbers indicate the migration of molecular mass markers in kilodaltons.

Aly KA, Anderson M, Ohr RJ, Missiakas D. Isolation of a Membrane Protein Complex for Type VII Secretion in Staphylococcus aureus. J Bacteriol. 2017 Oct 31;199(23). pii: e00482-17. doi: 10.1128/JB.00482-17. Print 2017 Dec 1.

 In silico, molecular docking analysis-based the highest scored docked pose of ANP and Inh2 in the binding pocket of the kinase domain of S. aureus STK1 (PDB ID 4EQM). The protein is shown in the white cartoon. Interaction of (G) ANP (inactivated ATP shown in cyan), (H) small molecule compound Inh2 (shown in yellow) and (I) Inh2-B1 (shown in dark orange) and key residues (only the side-chain non-hydrogenous atoms) around 3.5 Å from the inhibitor are shown in the sticks. The coloring code of the atom type: C (Yellow or green in inhibitors and cyan in ANP), N (blue), and O (red). (J) A summary table is showing the association of ANP, Inh2, and Inh2-B1 with amino acid residues reflecting their affinity for the ATP-binding pocket and a number of conformers clustered in this region during molecular docking analysis. ANP constitutes key residues (blue fonts). The residues around Inh-2 are shown in red fonts and the residues around P-loop that directly interact with Inh2-B1 are shown in purple fonts (See also Fig. S5 for affinity and number of conformers for all Inh2 derivatives). (K) A comparison of the N-terminal sequence of STK1 with those of other Gram-positive pathogens showing the unique nature of the S. aureus STK1 P-loop (Red fonts). The underlined bold letters denote non-conserved amino acid residues.

Kant S, Asthana S, Missiakas D, Pancholi V. A novel STK1-targeted small-molecule as an “antibiotic resistance breaker” against multidrug-resistant Staphylococcus aureus. Sci Rep. 2017 Jul 11;7(1):5067. doi: 10.1038/s41598-017-05314-z.

Adhesion of S. aureus to the vessel wall in vivo is mediated by the ternary complex VWF-vWbp-ClfA. (A) In vivo venous mesenteric perfusion model with WT mice. A total of 5 μL of the Ca2+ -ionophore A23187 (10 mm) was applied to the region of the visualized vascular bed to trigger endothelial cell activation and VWF release. A suspension of fluorescent-labeled WT, clfA and vwb strains was injected through the jugular catheter. Where indicated, 20 μg mL−1 rvWbp was added to the bacterial perfusate (n ≥ 14). All results are expressed as mean ± SEM. **P < 0.01, ***P < 0.001. (B) Fluorescence image (× 630) of S. aureus (green) adhering to activated murine vessel wall with immuno-staining for VWF (red) and 4',6-diamidino-2-phenylindole-staining of the cell nucleus (blue). White bar is 50 μm. VWF, von Willebrand factor; vWbp, von Willebrand factor-binding protein; ClfA, Clumping factor A; WT, wild type.

Claes J, Liesenborghs L, Peetermans M, Veloso TR, Missiakas D, Schneewind O, Mancini S, Entenza JM, Hoylaerts MF, Heying R, Verhamme P, Vanassche T. Clumping factor A, von Willebrand factor-binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall. J Thromb Haemost. 2017 May;15(5):1009-1019. doi: 10.1111/jth.13653. Epub 2017 Mar 23

Immune evasive attributes of SpA. SpA coats the surface of S. aureus and binds the Fcγ domain of antibodies to block their effector functions and inhibit opsonophagocytosis. Cell wall–anchored SpA is released from the bacterial surface by murein hydrolases and cross-links B cell receptors of VH3 clonal B lymphocytes. SpA induces B cell proliferation and maturation into antibody-secreting cells (ASC) via a pathway requiring CD4 T cells, MHC class II activation, activation-induced cytidine deamination (AID), and somatic hypermutation (SHM) for the secretion of mature antibodies. SpA induces the secretion VH3 clonal antibodies that are nonreactive to staphylococcal antigens. The B cell superantigen activity is dependent on the peptidoglycan modification of SpA and on RIPK2 signaling by immune cells.

Missiakas D, Schneewind O. Staphylococcus aureus vaccines: Deviating from the carol. J Exp Med. 2016 Aug 22;213(9):1645-53. doi: 10.1084/jem.20160569. Epub 2016 Aug 15. Review

(A) Average abscess volume for rabbits infected subcutaneously with S. aureus Newman wild-type (WT) or isogenic mutant strains as indicated. The volume of 10 abscesses per bacterial strain was measured daily following inoculation. (B) Individual abscesses plotted for selected days are depicted in panel A.

Malachowa N, Kobayashi SD, Porter AR, Braughton KR, Scott DP, Gardner DJ, Missiakas DM, Schneewind O, DeLeo FR. Contribution of Staphylococcus aureus Coagulases and Clumping Factor A to Abscess Formation in a Rabbit Model of Skin and Soft Tissue Infection. PLoS One. 2016 Jun 23;11(6):e0158293. doi: 10.1371/journal.pone.0158293. eCollection 2016.

Pathogenic conversion of S. simulans with S. aureus coa, vwb and clfA. Mice were injected in the peri-orbital venous plexus with 5×107 of S. simulans carrying either vector, pcoa-vwb, pclfA or pcoa-vwb-clfA, and euthanized 5 days following challenge. (A) Following necropsy, renal tissues were assessed for bacterial load. (B) Lesions observed in panels C through F were enumerated using 20–25 slides per infected group. The data in panels A and B represent the cumulative result of three independent experiments and were analyzed using the one-way ANOVA on ranks (Kruskal-Wallis test) with Dunn's post-hoc test (***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant). (C-F) Microscopy images of hematoxylin-eosin-stained, thin-sectioned renal tissue. Whole kidney sections are shown and areas boxed in black are shown as a 20× enlarged images. Images and insets in panels C and D did not reveal pathological features in renal tissues. In panels E and F, arrows point to replicating bacteria and immune cell infiltrates.

Yu W, Kim HK, Rauch S, Schneewind O, Missiakas D. Pathogenic conversion of coagulase-negative staphylococci. Microbes Infect. 2017 Feb;19(2):101-109. doi: 10.1016/j.micinf.2016.12.002. Epub 2016 Dec 21.

essE mutants exhibit altered abscesses. Staphylococcal replication in kidneys was measured 5 days postinfection after injection of 5 × 106 CFU of USA300 or its isogenic essE mutant in mice (groups of 8 to 10). (A and B) Kidneys were removed during necropsy to enumerate surface abscesses and subsequently homogenized in 1% Triton X-100 for plating and enumeration of CFU. Statistical analyses were performed using an unpaired two-tailed Student's t test with Welch's correction (A) and a two-tailed Mann-Whitney test (B). (C to E) Kidney sections were taken at 12-μm intervals, and abscesses were first stained with H&E to identify abscesses with staphylococcal communities (dark purple and denser stain in the center of slides) surrounded by immune cells. Next, the sections were incubated with anti-RANTES antibody or F4/80 antibody, a macrophage-specific marker. Following incubation with a secondary HRP-conjugated antibody and development, brown staining reflected the presence of macrophages outside the fibrin cuffs and surrounding USA300 lesions while the staining penetrated ΔessE lesions. Representative images are shown. Anti–RANTES signals were quantified using Fiji software, and values are reported as average darkness. Statistical analysis was performed using a two-tailed Mann-Whitney test.

Anderson M, Ohr RJ, Aly KA, Nocadello S, Kim HK, Schneewind CE, Schneewind O, Missiakas D. EssE Promotes Staphylococcus aureus ESS-Dependent Protein Secretion To Modify Host Immune Responses during Infection. J Bacteriol. 2016 Dec 13;199(1). pii: e00527-16. Print 2017 Jan 1.

Nygaard TK, Kobayashi SD, Freedman B, Porter AR, Voyich JM, Otto M, Schneewind O, DeLeo FR. Interaction of Staphylococci with Human B cells. PLoS One. 2016 Oct 6;11(10):e0164410. doi: 10.1371/journal.pone.0164410. eCollection 2016

Expression of essD does not confer any growth advantage and elicits an IL-12 response during infection. (A) Overnight cultures of wild-type USA300 and the isogenic essD::erm variant were normalized to an A600 of 5, diluted 1:100 into fresh medium, and grown with shaking at 37°C. Growth was monitored by recording the A600. (B) Aliquots of cultures shown in panel A were removed at 3, 4, and 6 h (T3, T4, and T6, respectively) and plated to assess viability. (C) S. epidermidis 12228 or S. simulans was cocultured with S. aureus USA300 or the isogenic essD::erm variant. Bacteria were mixed at a 1:1 ratio and plated on TSA. Immediately and 9 h following plating, cocultured bacteria were serially diluted and plated for enumeration of viable target cell counts. The data are presented as the average ± standard error of the mean from three independent competition experiments. (D) Identification of EssI in culture lysates of S. epidermidis (S. epi) 12228, S. simulans, and S. aureus USA300 using Western blotting. Samples were prepared and analyzed as described in the legend to Fig. 6A. (E) Cultures of P. acnes and P. granulosum grown to an A600 of 1.0 were plated on TSA, and the S. epidermidis 14.1.R1, USA300, essD::erm, or essDL546P strain was spotted on top of Propionibacterium lawns. Pictures of plates incubated at 37°C under anaerobic conditions for 72 h are shown. (F) Cohorts of C57BL/6 mice (n = 5) were inoculated intravenously with PBS (control) or with 5 × 107 CFU USA300 or with the essD::erm and essDL546P isogenic variants. Blood was sampled 12 h postinfection, and serum IL-12 (p70) was analyzed by enzyme-linked immunosorbent assay. Data were averaged, repeated for reproducibility, and analyzed by one-way ANOVA using Bonferroni's multiple-comparison test and with an unpaired t test in panel D (*, P < 0.05; ns, not significant).

Ohr RJ, Anderson M, Shi M, Schneewind O, Missiakas D. EssD, a Nuclease Effector of the Staphylococcus aureus ESS Pathway. J Bacteriol. 2016 Dec 13;199(1). pii: e00528-16. Print 2017 Jan 1.

Schneewind O. Classic Spotlight: Molecular Biology of Methicillin Resistance in Staphylococcus aureus.
J Bacteriol. 2016 Jun 27;198(14):1903. doi: 10.1128/JB.00277-16. Print 2016 Jul 15

Survival of Staphylococcus aureus in the bloodstream and dissemination into host tissues. (a) Following intravenous inoculation of mice with ~107 colony-forming units (CFU), S. aureus survives in the bloodstream and disseminates from the vasculature into organ tissues. Bacteria from the initial inoculum cannot be detected in the bloodstream within 24 h. (b–c) Microscopy of Giemsa-stained blood samples from mice infected for 2 h reveals staphylococci that are (b) associated with neutrophils or (c) agglutinated outside of immune cells. Staphylococci phagocytosed by neutrophils deploy several virulence strategies that ensure their survival (see text for details). (d ) Extracellular S. aureus cocci expressing mCherry (red fluorescence) interact with fibrin ( green fluorescence) to generate large bacterial agglutinates covered with a shield of fibrin cables, thereby preventing phagocytosis. The image shows S. aureus mixed for 5 min with anticoagulated human plasma that has been supplemented with 5% Alexa488-conjugated human fibrinogen. Staphylococcal agglutination requires Coa/vWbp (coagulases) and prothrombin as well as ClfA (agglutinin) and fibrinogen. Agglutinins are S. aureus surface proteins that bind coagulase-derived fibrin cables to produce large aggregates of bacteria covered with fibrin. (e) Models for S. aureus exit from the bloodstream. (i ) FnBPA and FnBPB (staphylococcal surface proteins) bind fibronectin and interact with integrin α5β1 on the surface of the vascular endothelium, thereby triggering invasion of cells and transmigration. Wall teichoic acid (WTA) and lipoteichoic acid (LTA) of S. aureus, polymers in the bacterial envelope, have also been proposed to promote staphylococcal invasion of host cells. (ii ) Staphylococci induce formation of fibrin thrombi via Coa/vWbp- and ClfA-mediated agglutination and bind to von Willebrand factor (vWF) on endothelial surfaces, generating Ultra Large vWF (ULVWF) polymers. (iii ) Staphylococci secrete Hla, a toxin that binds to the ADAM10 receptor to disrupt the physiological barrier functions of the vascular endothelium. (iv) The Trojan horse model, whereby neutrophils with intracellular S. aureus extravasate to deliver bacteria into host tissues.

Thomer L, Schneewind O, Missiakas D. Pathogenesis of Staphylococcus aureus Bloodstream Infections. Annu Rev Pathol. 2016 May 23;11:343-64. doi: 10.1146/annurev-pathol-012615-044351. Epub 2016 Feb 25.

VH3 clonal antibody expansion in S. aureus-infected mice requires SpA binding to Ig variant heavy chains. Cohorts of C57BL/6 mice (n = 5) were infected by i.v. inoculation with a sublethal dose (1 × 107 CFUs) of wild-type S. aureus, protein A variants (spaKK, spaAA, and spaKKAA), or left uninfected (naive). On days 5, 12, 19, and 26 post infection, serum samples were collected from mice. IgM (A and B) and IgG (C and D) responses were analyzed against SpAKK (A and C), a protein A variant that specifically recognizes VH3 clonal Fab fragments, and SpAKKAA (B and D), a protein A variant that fails to recognize Ig molecules, to determine the abundance of VH3 clonal antibody and protein A-specific antibody, respectively. Statistical analysis was performed with two-way ANOVA (*P < 0.05; **P < 0.001; ***P < 0.0001). Data points represent the mean ± SEM. Results are representative of three independent analyses.

Kim HK, Falugi F, Missiakas DM, Schneewind O. Peptidoglycan-linked protein A promotes T cell-dependent antibody expansion during Staphylococcus aureus infection. Proc Natl Acad Sci U S A. 2016 May 17;113(20):5718-23. doi: 10.1073/pnas.1524267113. Epub 2016 May 2.

Antibody against the R domain of coagulase protects against S. aureus bloodstream infection. Purified mAbs 5D5, 3B3, or IgG1 isotype control were injected at a concentration of 5 mg kg−1 body weight into the peritoneal cavity of naive BALB/c mice. Animal cohorts (n = 10 per experiment) were challenged by intravenous injection with S. aureus Newman (A), the Δvwb variant of Newman (B), MRSA USA300 (C), MRSA N315 (D), MRSA252 (E), or WIS (F), and survival was monitored over 10 d. Data are representative of two independent analyses; statistical significance was assessed with the log-rank test.

Thomer L, Emolo C, Thammavongsa V, Kim HK, McAdow ME, Yu W, Kieffer M, Schneewind O, Missiakas D. Antibodies against a secreted product of Staphylococcus aureus trigger phagocytic killing. J Exp Med. 2016 Mar 7;213(3):293-301. doi: 10.1084/jem.20150074. Epub 2016 Feb 15.

Cell separation defects in S. aureus glucosaminidase mutants. (A) S. aureus Newman strains were fixed, thin sectioned, uranyl acetate stained, and viewed by transmission electron microscopy. Images in the left-most column are representative low-magnification fields of cells for each corresponding frame. The three columns to the right show high-magnification images of representative cell morphologies. Arrowheads identify aberrantly formed septa. Gray scale bars, 2 μm; black scale bars, 0.2 μm.

Chan YG, Frankel MB, Missiakas D, Schneewind O. SagB Glucosaminidase Is a Determinant of Staphylococcus aureus Glycan Chain Length, Antibiotic Susceptibility, and Protein Secretion. J Bacteriol. 2016 Jan 25;198(7):1123-36. doi: 10.1128/JB.00983-15.

a | Staphylococcus aureus infection and its associated inflammatory damage promote the release of ATP, which is converted by adenosine synthase A (AdsA) into the immune suppressive signalling molecule adenosine (A). Adenosine inhibits activation of B cells, T cells, macrophages and dendritic cells via adenosine receptor (AdoR) signalling by acting on four different receptors (AdoR1, AdoR2A, AdoR2B and AdoR3). Under physiological conditions, CD39 and CD73 generate adenosine signals to limit inflammatory responses; CD39 and CD73 are also responsible for the adenosine halo surrounding immune cells and for immune suppressive states involving regulatory T cells (T cells expressing the FOXP3+ marker protein (not shown)). b | S. aureus induced NETosis of infiltrating neutrophils leads to nuclease-mediated degradation of the DNA fibres that are the major components of neutrophil extracellular traps (NETs) and AdsA-mediated conversion of 5′-monophosphate-deoxyadenosine (dAMP) into deoxyadenosine (dAdo), which promotes autocleavage of the apoptosis factor pro-capsase 3 to caspase 3. Caspase 3 induces macrophage death, thereby protecting S. aureus against professional phagocytes. IL-12, interleukin-12; MHC II, major histocompatibility complex class II; Nuc, staphylococcal nuclease; PMN, polymorphonuclear leukocyte.

Thammavongsa V, Kim HK, Missiakas D, Schneewind O. Staphylococcal manipulation of host immune responses. Nat Rev Microbiol. 2015 Sep;13(9):529-43. doi: 10.1038/nrmicro3521. Review.

Guinea pig bloodstream infection with the S. aureus spaKKAA mutant. (A) Guinea pigs (n = 5 or 6) were infected with 5 × 107 CFU of wild-type (WT) S. aureus Newman or its spaKKAA mutant. Body weights of infected animals, as well as naive control animals (mock), were measured for 17 days. (B and C) On days 5 and 17 postinfection, animals were euthanized and necropsied and bacterial loads (log10 CFU per kidney) (B) and numbers of infectious lesions (C) in renal tissues were determined. (D) Representative images of thin-sectioned, hematoxylin-eosin-stained renal tissues on day 17 postinfection. (E) ELISA was performed to determine the abundance of SpAKK-specific (VH3+ clonal) antibodies in serum collected on day 17 postinfection. The data are mean values, and error bars represent the standard errors of the means. Results are representative of two independent analyses.

Kim HK, Falugi F, Thomer L, Missiakas DM, Schneewind O. Protein A Suppresses Immune Responses during Staphylococcus aureus Bloodstream Infection in Guinea Pigs. MBio. 2015 Jan 6;6(1). pii: e02369-14.

Vaccination with SpAKKAA-monoclonal antibody elicits protective immunity against S. aureus infection. (A) Experimental plan for the immunization of neonatal FVB albino mice with mouse SpAKKAA-mAb 3F6 (intraperitoneal injection of 5 mg kg−1), mouse IgG2a mAb (5 mg kg−1 isotype control) or mock treatment, which was followed 24 h later by subcutaneous MRSA challenge with S. aureus USA300 LAC (B); survivors were bled on day 21 and serum analyzed for S. aureus specific antibodies (C) and challenged on day 35 by intravenous injection with S. aureus USA300 LAC (D). (B) Kaplan–Maier survival analysis of neonatal FVB albino mice infected via subcutaneous injection of 1 × 103 CFU S. aureus USA300 LAC to examine the efficacy of antibody treatment in preventing lethal sepsis in neonatal mouse cohorts (n = 16 for each treatment group). Data shown are representative of three independent experiments. Statistical significance was determined with the Mantel-Cox test: mock vs. SpAKKAA-mAb 3F6, P = 0.0374; IgG2a vs. SpAKKAA-mAb 3F6, P = 0.0437. (C) Serum samples of previously infected mice were analyzed for antibodies against ClfA (clumping factor A), ClfB (clumping factor B), IsdB (iron surface determinant B), SpAKKAA, LukF (leukocidin F), Coa (coagulase), and vWbp (von Willebrand binding protein). IRDye 680 conjugated anti-mouse IgG was used to quantify signal intensities with the Odyssey™ infrared imaging system (Li-cor); brackets denote standard error of the means. (D) Kaplan–Meier survival analysis of 35 day old FVB albino mice that had been treated as described in (A&B) and received intravenous injection of 5 × 106 CFU S. aureus USA300 LAC. Data shown are representative of 3 independent experiments. Statistical significance was determined with the Mantel-Cox test: mock vs. naïve, P = 0.1646; mock vs. SpAKKAA-mAb 3F6, P = 0.0054; IgG2a vs. SpAKKAA-mAb 3F6, P = 0.0072.

Thammavongsa V, Rauch S, Kim HK, Missiakas DM, Schneewind O. Protein A-neutralizing monoclonal antibody protects neonatal mice against Staphylococcus aureus. Vaccine. 2015 Jan 15;33(4):523-6.

Figure 2.
Figure 2. S. aureus infection–induced plasmablasts are VH3-biased and respond to SpA but lack reactivity to other S. aureus antigens. (A) Illustration of wild-type and mutated SpA protein. (B) PBs were isolated from patients infected with S. aureus (n = 10) or influenza (n = 14), or from uninfected controls (n = 7). The percentage of cells containing VH3 idiotypes was determined in naive B cells or responding PBs by sequencing BCR genes. Each point represents a single patient. Statistical analysis was performed using the Mann-Whitney test. Red (055–11077) and blue data (055–11080) points represent two blood draws from the same subject on different days. Statistics were calculated from the mean value of these two data points. Eleven total blood samples were analyzed from the ten patients. Red lines represent mean value. Pie charts represent the overall percentage of VH3 versus non-VH3 BCRs used in the repertoire. Center number of the pie chart represents the total number of sequences analyzed. (C) The percentages of VH3 and non-VH3–containing B cells among PBs and naive B cells were calculated in three individual S. aureus–infected patients as in B. Number in the center of the pie chart indicates the total number of sequences analyzed. Number above the pie charts is the patient designation. Statistical analysis performed using the χ2 test. (D) mAbs were generated by expression cloning the antibody genes of responding PBs in infected individuals and transiently transfecting the cloned antibody genes into HEK293 cells. 134 infection-induced mAbs from a total of seven patients were screened for reactivity against the indicated S. aureus virulence factors by ELISA. Dots represent individual mAb clones. Antigens tested: Coagulase (Coa), von Willebrand factor Binding Protein (vWbp), Clumping Factor A (ClfA), Iron-regulated surface determinant A (IsdA), IsdB, ESAT-6 secretion system extracellular A (EsxA), EsxB, Serine-Aspartate repeat protein C (SdrC), SdrD, SdrE, α-hemolysin (Hla), Luekotoxin (LukD), LukE, Fibronectin-binding protein A (FnbpA), FnbpB, and Fc-binding deficient S. aureus protein A (SpAKK). The experiment was performed twice with similar results. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
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S. aureus infection–induced plasmablasts are VH3-biased and respond to SpA but lack reactivity to other S. aureus antigens. (A) Illustration of wild-type and mutated SpA protein. (B) PBs were isolated from patients infected with S. aureus (n = 10) or influenza (n = 14), or from uninfected controls (n = 7). The percentage of cells containing VH3 idiotypes was determined in naive B cells or responding PBs by sequencing BCR genes. Each point represents a single patient. Statistical analysis was performed using the Mann-Whitney test. Red (055–11077) and blue data (055–11080) points represent two blood draws from the same subject on different days. Statistics were calculated from the mean value of these two data points. Eleven total blood samples were analyzed from the ten patients. Red lines represent mean value. Pie charts represent the overall percentage of VH3 versus non-VH3 BCRs used in the repertoire. Center number of the pie chart represents the total number of sequences analyzed.

Pauli NT, Kim HK, Falugi F, Huang M, Dulac J, Henry Dunand C, Zheng NY, Kaur K, Andrews SF, Huang Y, DeDent A, Frank KM, Charnot-Katsikas A, Schneewind O, Wilson PC. Staphylococcus aureus infection induces protein A-mediated immune evasion in humans. J Exp Med. 2014 Nov 17;211(12):2331-9.

Staphylococcus aureus bloodstream infection in leukopenic mice. (A and B) Cohorts of 7-week-old female CD-1 mice (n = 10) mock treated (A) or treated with cyclophosphamide in 48-h intervals (B) were infected by injection of S. aureus Newman (1 × 105, 1 × 106, 1 × 107, 5 × 107, or 1 × 108 CFU) into the periorbital venous plexus, and animal survival was recorded over 10 days. (C and D) At day 10 (C) or 4 (D) postinfection, kidney, liver, and lung tissues of mock-treated (C) or cyclophosphamide-treated (D) CD-1 mice infected with S. aureus Newman bloodstream infection (1 × 106 or 1 × 107 CFU) were removed during necropsy; homogenized tissues were analyzed for staphylococcal load by plating serially diluted samples on agar plates and enumerating CFU. (E and F) Hematoxylin and eosin-stained thin sections of kidney tissues from mock-treated (E) and cyclophosphamide-treated (F) mice that had been euthanized on day 10 following bloodstream infection with 1 × 107 and 1 × 106 CFU S. aureus Newman, respectively, were viewed by light microscopy and images were acquired. Bars indicate length measurements. See the text for details.

Rauch S, Gough P, Kim HK, Schneewind O, Missiakas D. Vaccine protection of leukopenic mice against Staphylococcus aureus bloodstream infection. Infect Immun. 2014 Nov;82(11):4889-98.

Mouse models for S. aureus infections. Mice are susceptible to S. aureus infections. Staphylococci can be administered by four different routes: intravenous, intraperitoneal, subcutaneous and intranasal inoculation. Intravenous delivery of staphylococci generates metastatic infectious lesions in multiple internal organs including heart and kidneys. In heart, agglutinated staphylococci (staphylococcal abscess community; SAC) are surrounded by necrotic cardiac myocytes (red). In renal tissue, SAC are surrounded by fibrin deposits (brown – eosinophilic pseudocapsule) which separate bacteria from massive immune cell infiltrates (purple). Injection of staphylococci into the peritoneum creates a lesion comprised of a large number of immune cells with staphylococci. This lesion is attached to the peritoneal lining and surrounded by an inner layer of fibrin deposits (brown) and an outer layer of collagen (blue). Subcutaneous injection of staphylococci generates a subcutaneous abscess and dermonecrotic (red) lesions on the overlying skin. Lower respiratory tract infection caused by intranasal inoculation of staphylococci is characterized by obstruction of airspace (red) by inflammatory cell infiltrates and aggregates of S. aureus.

Kim HK, Missiakas D, Schneewind O. Mouse models for infectious diseases caused by Staphylococcus aureus. J Immunol Methods. 2014 Aug;410:88-99.

Model for S. aureus capsular polysaccharide synthesis. Enzymes unique to CP5 or CP8 synthesis are denoted. Enzyme names in italic refer to those whose roles are predicted based on homology and bioinformatics, and whose functions have not been experimentally validated. CapK is hypothesized to serve as a flippase, transporting the lipid-linked capsule intermediate from the cytoplasm to the extracellular milieu, whereas the function of CapC (not depicted) is unknown.

Chan YG, Kim HK, Schneewind O, Missiakas D. The capsular polysaccharide of Staphylococcus aureus is attached to peptidoglycan by the LytR-CpsA-Psr (LCP) family of enzymes. J Biol Chem. 2014 May 30;289(22):15680-90.

SpA release during staphylococcal growth. S. aureus Newman (sbi) cells were washed and diluted into fresh TSB medium to A600 0.05 and incubated with rotation at 37 °C. At 30-min intervals, the absorbance (600 nm) was measured and cfus were enumerated. SpA release was quantified by immunoblotting of culture supernatant samples and recorded as signal intensity divided by cfu (×10−3). Experiments were performed in triplicate to calculate average values and SEM (error bars).

Becker S, Frankel MB, Schneewind O, Missiakas D. Release of protein A from the cell wall of Staphylococcus aureus. Proc Natl Acad Sci U S A. 2014 Jan 28;111(4):1574-9.

Ebh contributes to S. aureus methicillin resistance. Growth of S. aureus USA300 LAC and its ebh variants ΦNΞ9044 and ΦNΞ10853 was determined for samples diluted from overnight cultures in TSB without (A) or with (B) 2 μg/ml oxacillin. Growth was measured as increased absorbance at 600 nm (A600). S. aureus USA300 LAC (C) and its ebh variant ΦNΞ9044 (D) were grown to mid-log phase in 2 μg/ml oxacillin, fixed, thin sectioned, and viewed by transmission electron microscopy.

Cheng AG, Missiakas D, Schneewind O. The giant protein Ebh is a determinant of Staphylococcus aureus cell size and complement resistance. J Bacteriol. 2014 Mar;196(5):971-81.

Thomer L, Becker S, Emolo C, Quach A, Kim HK, Rauch S, Anderson M, Leblanc JF, Schneewind O, Faull KF, Missiakas D. N-acetylglucosaminylation of serine-aspartate repeat proteins promotes Staphylococcus aureus bloodstream infection. J Biol Chem. 2014 Feb 7;289(6):3478-86.

Kim HK, Emolo C, Missiakas D, Schneewind O. A monoclonal antibody that recognizes the E domain of staphylococcal protein A. Vaccine. 2014 Jan 16;32(4):464-9.

Macrophages are excluded from staphylococcal abscesses.(A to J) BALB/c mice were infected by intravenous injection with S. aureus wild type or variants with mutations in genes for nuclease (nuc) or adenosine synthase A (adsA). Animals were euthanized 5 days postinfection and kidneys removed for histopathology. (A and J) WT staphylococci are distinguishable as central nidus (yellow arrowheads) surrounded by a fibrin capsule, a zone of infiltrating neutrophils (green boxes, immune cell cuffs), and macrophages at the periphery of these lesions [red box in (J), hematoxylin-and-eosin stained tissue]. Cryo-sections of renal tissues were examined by immunohistochemistry with αLy-6G antibodies [neutrophils stained as brown pigment (A to C)] or αF4/80 antibodies [macrophages (D to I)] and counterstained with hematoxylin. (G to I) Magnifications of the areas framed by the black boxes in (D) to (F). Macrophages (red boxes) are excluded from the neutrophil cuff of abscesses formed by WT S. aureus (green boxes), but not from those formed by the nuc or adsA mutants. Images are representative of similar data from kidneys of each cohort (N = 4) of infected mice. Scale bars: (A to F) 200 μm; (G to I) 20 μm; (J) 100 μm.

Thammavongsa V, Missiakas DM, Schneewind O. Staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death. Science. 2013 Nov 15;342(6160):863-6.

Genetic requirements for secretion of Esx proteins.
A. Schematic representation of the ESS gene cluster. Proteins with defined functions are indicated as follows: secreted proteins (red), FtsK SpoIIIE-like ATPase (yellow), proposed secretion machinery (grey), not known (white). The locus is annotated for strain USA300. Acronyms Esx and Ess refer to ESAT-6 like secretion extracellular and ESAT-6 like secretion system. Genes previously referred as ESAT-6 secretion accessory (EsaC, EsaD) have been renamed to better reflect their contribution to the secretion system.
B. Bacterial cultures of S. aureus USA300 (WT) or isogenic mutants, esxA, esxB, esxC, esxD and essA, essB, essB, essC were fractionated into cells and medium. Proteins in all fractions were precipitated with trichloroacetic acid, separated by SDS-PAGE and detected by immunoblotting with antibodies specific for secreted substrates (α-EsxA, α-EsxB, α-EsxC, α-EsxD) and cytosolic and secreted protein controls (α-L6, α-Hla).

Anderson M, Aly KA, Chen YH, Missiakas D. Secretion of atypical protein substrates by the ESAT-6 secretion system of Staphylococcus aureus. Mol Microbiol. 2013 Nov;90(4):734-43.

Falugi F, Kim HK, Missiakas DM, Schneewind O. Role of protein A in the evasion of host adaptive immune responses by Staphylococcus aureus. MBio. 2013 Aug 27;4(5):e00575-13.

Model illustrating S. aureus atl-dependent biofilm formation and the impact of serine proteases, i.e., S. epidermidis Esp or S. aureus V8 (SspA), on controlling Atl activity and biofilm disassembly. The model distinguishes five steps in the biofilm developmental process: attachment, eDNA release, maturation, detachment, and dissemination. Three surface proteins (Eap, FnbA, and FnbB) are thought to promote S. aureus attachment to fibronectin (attachment). The secretion of Atl promotes the release of eDNA as an extracellular matrix for biofilm formation (eDNA release). Activation of secreted SspA (V8 protease) inactivates Atl, thereby promoting staphylococcal replication in the newly formed matrix (biofilm maturation). The continued activation of SspA promotes the detachment of staphylococcal cells from the biofilm (detachment). Detached staphylococci disseminate and adhere elsewhere by binding to fibronectin and establishing another biofilm. S. aureus biofilm formation is perturbed by the S. epidermidis secreted protease Esp. We propose that exuberant expression of S. epidermidis Esp (unlike S. aureus SspA) perturbs biofilm formation of S. aureus.

Chen C, Krishnan V, Macon K, Manne K, Narayana SV, Schneewind O. Secreted proteases control autolysin-mediated biofilm growth of Staphylococcus aureus. J Biol Chem. 2013 Oct 11;288(41):29440-52.

Mapping the functional domains of vWbp. A, diagram illustrating the primary translational products of mature full-length vWbp and truncated variants. Each variant includes an N-terminal six histidyl tag (H6) and a C-terminal strep tag (WSHPQFEK). The binding sites for prothrombin, fibrinogen, FXIII, and fibronectin are delineated. Numbers indicate amino acid residues within the mature protein. B and C, human plasma (500 μl) was flowed over Strep-Tactin resin uncharged (control) or charged with 100 nmol of proteins shown in A. Samples were eluted by boiling the resin in sample buffer and examined as described in Fig. 1. Immunoblotting was performed for prothrombin (PT), FXIII subunit A (FXIII A) and B (FXIIIB), fibronectin, and vWF.

Thomer L, Schneewind O, Missiakas D. Multiple ligands of von Willebrand factor-binding protein (vWbp) promote Staphylococcus aureus clot formation in human plasma. J Biol Chem. 2013 Sep 27;288(39):28283-92.

Chan YG, Frankel MB, Dengler V, Schneewind O, Missiakas D. Staphylococcus aureus mutants lacking the LytR-CpsA-Psr family of enzymes release cell wall teichoic acids into the extracellular medium. J Bacteriol. 2013 Oct;195(20):4650-9.