scholarly journals Proteomic and Transcriptomic Profiling of Staphylococcus aureus Surface LPXTG-proteins: Correlation with agr Genotypes and Adherence Phenotypes

2012 ◽  
Vol 11 (11) ◽  
pp. 1123-1139 ◽  
Author(s):  
Mathilde Ythier ◽  
Grégory Resch ◽  
Patrice Waridel ◽  
Alexandre Panchaud ◽  
Aurélie Gfeller ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Regulatory Networks ◽  
Time Course ◽  
Methicillin Resistance ◽  
Binding Protein ◽  
Protein A ◽  
Surface Proteins ◽  
Protein Domain ◽  
Accessory Gene

Staphylococcus aureus infections involve numerous adhesins and toxins, which expression depends on complex regulatory networks. Adhesins include a family of surface proteins covalently attached to the peptidoglycan via a conserved LPXTG motif. Here we determined the protein and mRNA expression of LPXTG-proteins of S. aureus Newman in time-course experiments, and their relation to fibrinogen adherence in vitro. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa), and fibrinogen-binding protein A (ClfA), as well as during growth in iron-rich or iron-poor media. Surface proteins were recovered by trypsin-shaving of live bacteria. Released peptides were analyzed by liquid chromatography coupled to tandem mass-spectrometry. To unambiguously identify peptides unique to LPXTG-proteins, the analytical conditions were refined using a reference library of S. aureus LPXTG-proteins heterogeneously expressed in surrogate Lactococcus lactis. Transcriptomes were determined by microarrays. Sixteen of the 18 LPXTG-proteins present in S. aureus Newman were detected by proteomics. Nine LPXTG-proteins showed a bell-shape agr-like expression that was abrogated in agr-negative mutants including Spa, fibronectin-binding protein A (FnBPA), ClfA, iron-binding IsdA, and IsdB, immunomodulator SasH, functionally uncharacterized SasD, biofilm-related SasG and methicillin resistance-related FmtB. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr- mutant, whereas all other LPXTG-proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in fibrinogen-adherence tests during late growth (24 h), whereas it remained poorly detected by proteomics. On the other hand, iron-regulated IsdA-B-C increased their protein expression by >10-times in iron-poor conditions. Thus, proteomic, transcriptomic, and adherence-phenotype demonstrated differential profiles in S. aureus. Moreover, trypsin peptide signatures suggested differential protein domain exposures in various environments, which might be relevant for anti-adhesin vaccines. A comprehensive understanding of the S. aureus physiology should integrate all three approaches.

scholarly journals Introduction of the mec Element (Methicillin Resistance) into Staphylococcus aureus Alters In Vitro Functional Activities of Fibrinogen and Fibronectin Adhesins

1998 ◽  
Vol 42 (3) ◽  
pp. 564-570 ◽  
Author(s):  
Pierre E. Vaudaux ◽  
Vincenza Monzillo ◽  
Patrice Francois ◽  
Daniel P. Lew ◽  
Tim J. Foster ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistance ◽  
Bacterial Colonization ◽  
Protein A ◽  
Methicillin Resistant ◽  
Functional Activities ◽  
Extracellular Matrix Components ◽  
Resistant Strains ◽  
The Impact

ABSTRACT Some methicillin-resistant strains of Staphylococcus aureus are defective in the production of major surface components such as protein A, clumping factor, or other important adhesins to extracellular matrix components which may play a role in bacterial colonization and infection. To evaluate the impact of methicillin resistance (mec) determinants on bacterial adhesion mediated by fibrinogen or fibronectin adhesins, we compared the in vitro attachment of two genetically distinct susceptible strains (NCTC8325 and Newman) to protein-coated surfaces with that of isogenic methicillin-resistant derivatives. All strains containing an intactmec element in their chromosomes were found to be defective in adhesion to fibrinogen and fibronectin immobilized on polymethylmethacrylate coverslips, regardless of the presence or absence of additional mutations in the femA,femB, or femC gene, known to decrease expression of methicillin resistance in S. aureus. Western ligand affinity blotting or immunoblotting of cell wall-associated adhesins revealed similar contents of fibrinogen- or fibronectin-binding proteins in methicillin-resistant strains compared to those of their methicillin-susceptible counterparts. In contrast to methicillin-resistant strains carrying a mec element in their genomes, methicillin-resistant strains constructed in vitro, by introducing the mecA gene on a plasmid, retained their adhesion phenotypes. In conclusion, the chromosomal insertion of themec element into genetically defined strains of S. aureus impairs the in vitro functional activities of fibrinogen or fibronectin adhesins without altering their production. This effect is unrelated to the activity of the mecA gene.

scholarly journals Rapid and Broad Immune Efficacy of a Recombinant Five-Antigen Vaccine against Staphylococcus aureus Infection in Animal Models

Vaccines ◽  
2020 ◽  
Vol 8 (1) ◽  
pp. 134 ◽  
Author(s):  
Hao Zeng ◽  
Feng Yang ◽  
Qiang Feng ◽  
Jinyong Zhang ◽  
Jiang Gu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Severe Disease ◽  
Lethal Dose ◽  
Protein A ◽  
Surface Proteins ◽  
Staphylococcal Protein A ◽  
Humoral Immune ◽  
Aureus Infection

Staphylococcus aureus (S. aureus) is a leading cause of both healthcare-and community-associated infections globally, which result in severe disease and readily developing antibiotic resistance. Developing an efficacious vaccine against S. aureus is urgently required. In the present study, we selected five conserved antigens, including the secreted factors α-hemolysin (Hla), staphylococcal enterotoxin B (SEB) and the three surface proteins staphylococcal protein A (SpA), iron surface determinant B N2 domain (IsdB-N2) and manganese transport protein C (MntC). They were all well-characterized virulence factor of S. aureus and developed a recombinant five-antigen S. aureus vaccine (rFSAV), rFSAV provided consistent protection in S. aureus lethal sepsis and pneumonia mouse models, and it showed broad immune protection when challenged with a panel of epidemiologically relevant S. aureus strains. Meanwhile, rFSAV immunized mice were able to induce comprehensive cellular and humoral immune responses to reduce bacterial loads, inflammatory cytokine expression, inflammatory cell infiltration and decrease pathology after challenge with a sub-lethal dose of S. aureus. Moreover, the importance of specific antibodies in protection was demonstrated by antibody function tests in vitro and in vivo. Altogether, our data demonstrate that rFSAV is a potentially promising vaccine candidate for defensing against S. aureus infection.

Contribution of (sub)domains of Staphylococcus aureus fibronectin-binding protein to the proinflammatory and procoagulant response of human vascular endothelial cells

2009 ◽  
Vol 101 (03) ◽  
pp. 495-504 ◽  
Author(s):  
Ruth Heying ◽  
Joke van de Gevel ◽  
Yok-Ai Que ◽  
Lionel Piroth ◽  
Philippe Moreillon ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Binding Protein ◽  
Protein A ◽  
Surface Expression ◽  
Vascular Endothelial ◽  
Non Invasive ◽  
Fibronectin Binding Protein

SummaryThe Staphylococcus aureus fibronectin (Fn) -binding protein A (FnBPA) is involved in bacterium-endothelium interactions which is one of the crucial events leading to infective endocarditis (IE). We previously showed that the sole expression of S. aureus FnBPA was sufficient to confer to non-invasive Lactococcus lactis bacteria the capacity to invade human endothelial cells (ECs) and to launch the typical endothelial proinflammatory and procoagulant responses that characterize IE. In the present study we further questioned whether these bacterium-EC interactions could be reproduced by single or combined FnBPA subdomains (A, B, C or D) using a large library of truncated FnBPA constructs expressed in L. lactis. Significant invasion of cultured ECs was found for L. lactis expressing the FnBPA subdomains CD (aa 604–877) or A4+16 (aa 432–559). Moreover, this correlates with the capacity of these fragments to elicit in vitro a marked increase in EC surface expression of both ICAM-1 and VCAM-1 and secretion of the CXCL8 chemokine and finally to induce a tissue factor-dependent endothelial coagulation response. We thus conclude that (sub)domains of the staphylococcal FnBPA molecule that express Fn-binding modules, alone or in combination, are sufficient to evoke an endothelial proinflammatory as well as a procoagulant response and thus account for IE severity.

scholarly journals Role of σB in the Expression of Staphylococcus aureus Cell Wall Adhesins ClfA and FnbA and Contribution to Infectivity in a Rat Model of Experimental Endocarditis

2005 ◽  
Vol 73 (2) ◽  
pp. 990-998 ◽  
Author(s):  
Jose-Manuel Entenza ◽  
Philippe Moreillon ◽  
Maria Magdalena Senn ◽  
Jan Kormanec ◽  
Paul M. Dunman ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Protein ◽  
Protein A ◽  
Northern Blotting ◽  
Cell Matrix ◽  
Fibrinogen Binding ◽  
Fibronectin Binding Protein ◽  
Coated Surfaces ◽  
Fibrin Clots

ABSTRACT Isogenic Staphylococcus aureus strains with different capacities to produce σB activity were analyzed for their ability to attach to fibrinogen- or fibronectin-coated surfaces or platelet-fibrin clots and to cause endocarditis in rats. In comparison to the σB-deficient strain, BB255, which harbors an rsbU mutation, both rsbU-complemented and σB-overproducing derivatives exhibited at least five times greater attachment to fibrinogen- and fibronectin-coated surfaces and showed increased adherence to platelet-fibrin clots. No differences in adherence were seen between BB255 and a ΔrsbUVWsigB isogen. Northern blotting analyses revealed that transcription of clfA, encoding fibrinogen-binding protein clumping factor A, and fnbA, encoding fibronectin-binding protein A, were positively influenced by σB. σB overproduction resulted in a statistically significant increase in positive spleen cultures and enhanced bacterial densities in both the aortic vegetations and spleens at 16 h postinoculation. In contrast, at 72 h postinoculation, tissues infected with the σB overproducer had lower bacterial densities than did those infected with BB255. These results suggest that although σB appears to increase the adhesion of S. aureus to various host cell-matrix proteins in vitro, it has limited effect on pathogenesis in the rat endocarditis model. σB appears to have a transient enhancing effect on bacterial density in the early stages of infection that is lost during progression.

scholarly journals Functional Analysis of Putative Adhesion Factors in Lactobacillus acidophilus NCFM

2005 ◽  
Vol 71 (12) ◽  
pp. 8344-8351 ◽  
Author(s):  
B. Logan Buck ◽  
Eric Altermann ◽  
Tina Svingerud ◽  
Todd R. Klaenhammer
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Binding Protein ◽  
Protein A ◽  
Surface Proteins ◽  
Intestinal Epithelial ◽  
Cell Surface Proteins ◽  
Fibronectin Binding Protein ◽  
Fibronectin Binding

ABSTRACT Lactobacilli are major inhabitants of the normal microflora of the gastrointestinal tract, and some select species have been used extensively as probiotic cultures. One potentially important property of these organisms is their ability to interact with epithelial cells in the intestinal tract, which may promote retention and host-bacterial communication. However, the mechanisms by which they attach to intestinal epithelial cells are unknown. The objective of this study was to investigate cell surface proteins in Lactobacillus acidophilus that may promote attachment to intestinal tissues. Using genome sequence data, predicted open reading frames were searched against known protein and protein motif databases to identify four proteins potentially involved in adhesion to epithelial cells. Homologous recombination was used to construct isogenic mutations in genes encoding a mucin-binding protein, a fibronectin-binding protein, a surface layer protein, and two streptococcal R28 homologs. The abilities of the mutants to adhere to intestinal epithelial cells were then evaluated in vitro. Each strain was screened on Caco-2 cells, which differentiate and express markers characteristic of normal small-intestine cells. A significant decrease in adhesion was observed in the fibronectin-binding protein mutant (76%) and the mucin-binding protein mutant (65%). A surface layer protein mutant also showed reduction in adhesion ability (84%), but the effect of this mutation is likely due to the loss of multiple surface proteins that may be embedded in the S-layer. This study demonstrated that multiple cell surface proteins in L. acidophilus NCFM can individually contribute to the organism's ability to attach to intestinal cells in vitro.

scholarly journals Penicillin-binding protein 4 overproduction increases beta-lactam resistance in Staphylococcus aureus.

1996 ◽  
Vol 40 (9) ◽  
pp. 2121-2125 ◽  
Author(s):  
U U Henze ◽  
B Berger-Bächi
Keyword(s):  
Staphylococcus Aureus ◽  
Promoter Region ◽  
Methicillin Resistance ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Beta Lactam ◽  
In The Wild ◽  
Mutant Promoter

The Staphylococcus aureus mutant strain PVI selected in vitro for methicillin resistance overexpressed penicillin-binding protein (PBP) 4. In the wild-type parent strain the pbp4 gene was separated by 419 nucleotides from a divergently transcribed abcA locus coding for an ATP-binding cassette transporter. The mutant PVI was shown to have a deletion in the pbp4-abcA promoter region that affected pbp4 transcription but not expression of abcA. Introduction of the pbp4 gene plus the mutant promoter region into different genetic backgrounds revealed that PBP 4 overproduction was sufficient to increase in vitro-acquired methicillin resistance independently of other chromosomal genes. The role of the AbcA transporter in methicillin resistance remained unknown.

scholarly journals The Fibrinogen- and Fibronectin-Binding Domains of Staphylococcus aureus Fibronectin-Binding Protein A Synergistically Promote Endothelial Invasion and Experimental Endocarditis

2008 ◽  
Vol 76 (8) ◽  
pp. 3824-3831 ◽  
Author(s):  
Lionel Piroth ◽  
Yok-Ai Que ◽  
Eleonora Widmer ◽  
Alexandre Panchaud ◽  
Stéphane Piu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Binding Protein ◽  
Protein A ◽  
Binding Domains ◽  
Fibrinogen Binding ◽  
Fibronectin Binding Protein ◽  
Fibronectin Binding

ABSTRACT Staphylococcus aureus experimental endocarditis relies on sequential fibrinogen binding (for valve colonization) and fibronectin binding (for endothelial invasion) conferred by peptidoglycan-attached adhesins. Fibronectin-binding protein A (FnBPA) reconciles these two properties—as well as elastin binding—and promotes experimental endocarditis by itself. Here we attempted to delineate the minimal subdomain of FnBPA responsible for fibrinogen and fibronectin binding, cell invasion, and in vivo endocarditis. A large library of truncated constructs of FnBPA was expressed in Lactococcus lactis and tested in vitro and in animals. A 127-amino-acid subdomain spanning the hinge of the FnBPA fibrinogen-binding and fibronectin-binding regions appeared necessary and sufficient to confer the sum of these properties. Competition with synthetic peptides could not delineate specific fibrinogen- and fibronectin-binding sites, suggesting that dual binding arose from protein folding, irrespective of clearly defined binding domains. Moreover, coexpressing the 127-amino-acid subdomain with remote domains of FnBPA further increased fibrinogen binding by ≥10 times, confirming the importance of domain interactions for binding efficacy. In animals, fibrinogen binding (but not fibronectin binding) was significantly associated with endocarditis induction, whereas both fibrinogen binding and fibronectin binding were associated with disease severity. Moreover, fibrinogen binding also combined with fibronectin binding to synergize the invasion of cultured cell lines significantly, a feature correlating with endocarditis severity. Thus, while fibrinogen binding and fibronectin binding were believed to act sequentially in colonization and invasion, they appeared unexpectedly intertwined in terms of both functional anatomy and pathogenicity (in endocarditis). This unforeseen FnBPA subtlety might bear importance for the development of antiadhesin strategies.

scholarly journals The Synergistic Effect of Nicotine and Staphylococcus aureus on Peri-Implant Infections

2021 ◽  
Vol 9 ◽  
Author(s):  
Yao Hu ◽  
Wen Zhou ◽  
Chengguang Zhu ◽  
Yujie Zhou ◽  
Qiang Guo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Synergistic Effects ◽  
Protein A ◽  
Treated Group ◽  
Combination Group ◽  
Staphylococcal Protein A ◽  
Receptor Activator ◽  
And Staphylococcus Aureus

Smoking is considered a key risk factor for implant survival; however, how it interacts with the pathogens in peri-implant infections is not clear. Here, we identified that nicotine, the key component of cigarette smoking, can interact with Staphylococcus aureus and synergistically induce peri-implant infections in a rat osteolysis model. The nicotine–S. aureus combination group increased the gross bone pathology, osteolysis, periosteal reactions, and bone resorption compared to the nicotine or S. aureus single treated group (p < 0.05). Nicotine did not promote the proliferation of S. aureus both in vitro and in vivo, but it can significantly upregulate the expression of staphylococcal protein A (SpA), a key virulence factor of S. aureus. The nicotine–S. aureus combination also synergistically activated the expression of RANKL (receptor activator of nuclear factor-kappa B ligand, p < 0.05) to promote the development of peri-implant infections. The synergistic effects between nicotine and S. aureus infection can be a new target to reduce the peri-implant infections.

scholarly journals A genomic and proteomic analysis of surface proteins in bovine-adapted lineages of Staphylococcus aureus

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Shauna D. Drumm ◽  
Rebecca Owens ◽  
Jennifer Mitchell ◽  
Orla M. Keane
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Surface Proteins ◽  
Host Cells ◽  
Mass Spectrometry Analysis ◽  
In Vitro Assays ◽  
Immune Recognition ◽  
Independent Manner ◽  
Genes Encoding

In Ireland, Staphylococcus aureus is the most common cause of intramammary infection (IMI) in cattle with the bovine-adapted lineages CC151 and CC97 most commonly found. Surface proteins play a major role in establishing and maintaining the infection. A previous study revealed that a strain from the CC151 lineage showed significant decay in genes encoding predicted surface proteins. Twenty-three S. aureus strains, twelve belonging to CC151 and eleven belonging to CC97, isolated from clinical IMI, were sequenced and genes encoding cell wall anchored (CWA) proteins predicted. Analysis showed that a minority of genes encoding putative CWA proteins were intact in the CC151 strains compared to CC97. Of the 26 known CWA proteins in S. aureus, the CC151 strains only encoded 10 intact genes while CC97 encoded on average 18 genes. Also within the CC97 lineage, the repertoire of genes varied depending on individual strains, with strains encoding between 17-20 intact genes. Although CC151 is reported to internalize within bovine host cells, it does so in a fibronectin-binding protein (FnBPA and FnBPB) independent manner. In-vitro assays were performed and results showed that strains from CC151, and surprisingly also CC97, weakly bound bovine fibronectin and that the FnBPs were poorly expressed in both these lineages. Mass spectrometry analysis of cell wall extracts revealed that SdrE and AdsA were the most highly expressed CWA proteins in both lineages. These results demonstrate significant differences between CC151 and CC97 in their repertoire of genes encoding CWA proteins, which may impact immune recognition of these strains and their interactions with host cells.

scholarly journals Prevalence of Methicillin Resistant and Virulence Determinants in Clinical Isolates of Staphylococcus aureus

2018 ◽  
Vol 10 (1) ◽  
pp. 108-115
Author(s):  
Manjunath Chavadi ◽  
Rahul Narasanna ◽  
Ashajyothi Chavan ◽  
Ajay Kumar Oli ◽  
Chandrakanth Kelmani. R
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Morphology ◽  
Methicillin Resistance ◽  
Protein A ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Virulence Determinants ◽  
Methicillin Resistant ◽  
Alternative Medicines ◽  
Mrsa Strains

Introduction:Methicillin-resistantStaphylococcus aureus(MRSA) is the major threat that is a result of the uncontrolled use of antibiotics causing a huge loss in health, so understanding their prevalence is necessary as a public health measure.Objective:The aim of this study was to determine the prevalence of methicillin-resistant MRSA and virulence determinant among associatedS. aureusfrom the clinical samples obtained from various hospital and health care centers of the Gulbarga region in India.Materials and Methods:All the collected samples were subjected for the screening ofS. aureusand were further characterized by conventional and molecular methods including their antibiotic profiling. Further, the response of methicillin antibiotic on cell morphology was studied using scanning electron microscopy.Results:A total 126S. aureuswas isolated from the clinical samples which showed, 100% resistant to penicillin, 55.5% to oxacillin, 75.3% to ampicillin, 70.6% to streptomycin, 66.6% to gentamicin, 8.7% to vancomycin and 6.3% to teicoplanin. The selected MRSA strains were found to possessmecA(gene coding for penicillin-binding protein 2A) andfemA(factor essential for methicillin resistance)genetic determinants in their genome with virulence determinants such as Coagulase (coa) and the X region of the protein A (spa)gene. Further, the methicillin response in resistantS. aureusshowed to be enlarged and malformed on cell morphology.Conclusion:The molecular typing of clinical isolates ofS. aureusin this study was highly virulent and also resistant to methicillin; this will assist health professionals to control, exploration of alternative medicines and new approaches to combat Staphylococcal infections more efficiently by using targeted therapy.

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