scholarly journals Proteomics-Based Identification of Anchorless Cell Wall Proteins as Vaccine Candidates against Staphylococcus aureus

2009 ◽  
Vol 77 (7) ◽  
pp. 2719-2729 ◽  
Author(s):  
Eva Glowalla ◽  
Bettina Tosetti ◽  
Martin Krönke ◽  
Oleg Krut
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Survival Rates ◽  
Proteome Analysis ◽  
Surface Proteins ◽  
Human Neutrophils ◽  
Cell Wall Proteins ◽  
Lethal Challenge ◽  
Vaccine Candidates

ABSTRACT Staphylococcus aureus is an important human pathogen with increasing clinical impact due to the extensive spread of antibiotic-resistant strains. Therefore, development of a protective polyvalent vaccine is of great clinical interest. We employed an intravenous immunoglobulin (IVIG) preparation as a source of antibodies directed against anchorless S. aureus surface proteins for identification of novel vaccine candidates. In order to identify such proteins, subtractive proteome analysis (SUPRA) of S. aureus anchorless cell wall proteins was performed. Proteins reacting with IVIG but not with IVIG depleted of S. aureus-specific opsonizing antibodies were considered vaccine candidates. Nearly 40 proteins were identified by this preselection method using matrix-assisted laser desorption ionization—time of flight analysis. Three of these candidate proteins, enolase (Eno), oxoacyl reductase (Oxo), and hypothetical protein hp2160, were expressed as glutathione S-transferase fusion proteins, purified, and used for enrichment of corresponding immunoglobulin Gs from IVIG by affinity chromatography. Use of affinity-purified anti-Eno, anti-Oxo, and anti-hp2160 antibodies resulted in opsonization, phagocytosis, and killing of S. aureus by human neutrophils. High specific antibody titers were detected in mice immunized with recombinant antigens. In mice challenged with bioluminescent S. aureus, reduced staphylococcal spread was measured by in vivo imaging. The recovery of S. aureus CFU from organs of immunized mice was diminished 10- to 100-fold. Finally, mice immunized with hp2160 displayed statistically significant higher survival rates after lethal challenge with clinically relevant S. aureus strains. Taken together, our data suggest that anchorless cell wall proteins might be promising vaccine candidates and that SUPRA is a valuable tool for their identification.

scholarly journals Search of Potential Vaccine Candidates against Trueperella pyogenes Infections through Proteomic and Bioinformatic Analysis

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 314 ◽  
Author(s):  
Ángela Galán-Relaño ◽  
Lidia Gómez-Gascón ◽  
Antonio Rodríguez-Franco ◽  
Inmaculada Luque ◽  
Belén Huerta ◽  
...  
Keyword(s):  
Cell Wall ◽  
Membrane Proteins ◽  
Bioinformatic Analysis ◽  
Surface Proteins ◽  
Live Cells ◽  
Economic Losses ◽  
Diagnostic Tools ◽  
Cell Wall Proteins ◽  
Vaccine Candidates ◽  
Trueperella Pyogenes

Trueperella pyogenes is an opportunistic pathogen, responsible for important infections in pigs and significant economic losses in swine production. To date, there are no available commercial vaccines to control diseases caused by this bacterium. In this work, we performed a comparative proteomic analysis of 15 T. pyogenes clinical isolates, by “shaving” live cells, followed by LC-MS/MS, aiming at the identification of the whole set of surface proteins (i.e., the “pan-surfome”) as a source of antigens to be tested in further studies as putative vaccine candidates, or used in diagnostic tools. A total of 140 surface proteins were detected, comprising 25 cell wall proteins, 10 secreted proteins, 23 lipoproteins and 82 membrane proteins. After describing the “pan-surfome”, the identified proteins were ranked in three different groups based on the following criteria: to be (i) surface-exposed, (ii) highly conserved and (iii) widely distributed among different isolates. Two cell wall proteins, three lipoproteins, four secreted and seven membrane proteins were identified in more than 70% of the studied strains, were highly expressed and highly conserved. These proteins are potential candidates, alone or in combination, to obtain effective vaccines against T. pyogenes or to be used in the diagnosis of this pathogen.

scholarly journals Conservation, Surface Exposure, and In Vivo Expression of the Frp Family of Iron-Regulated Cell Wall Proteins in Staphylococcus aureus

2002 ◽  
Vol 70 (5) ◽  
pp. 2399-2407 ◽  
Author(s):  
Julie A. Morrissey ◽  
Alan Cockayne ◽  
Jane Hammacott ◽  
Keith Bishop ◽  
Amy Denman-Johnson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Growth Conditions ◽  
Open Reading Frames ◽  
Cell Wall Proteins ◽  
Phenotypic Analysis

ABSTRACT Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis identified two conserved, immunogenic Staphylococcus aureus cell wall proteins, of 40 and 87 kDa, expressed under iron-restricted growth conditions in vitro and in vivo. N-terminal sequencing and subsequent genome analysis showed that these proteins are encoded by adjacent monocistronic open reading frames designated frpA and frpB, respectively. Studies with an S. aureus fur mutant confirmed that expression of FrpA and FrpB is regulated by Fur but that there also appears to be differential expression of these proteins in different iron-restricted media in vitro. FrpA and FrpB share some amino acid sequence homology with each other and with a putative S. aureus membrane protein, FrpC. frpC is the first gene of a Fur-regulated operon encoding four proteins of unknown function (FrpC, -D, -G, and -H) and the binding protein (FrpE) and permease (FrpF) of a putative iron transporter. Antisense mutagenesis and bioassays showed that FrpA and FrpB are not required for growth of S. aureus under iron-restricted conditions in vitro and do not appear to be involved in the transport of iron from siderophores or in binding of hemin. Further phenotypic analysis suggested that FrpA may be involved in adhesion of S. aureus to plastic in vitro. Binding of S. aureus to microtiter wells was found to be iron regulated, and iron-restricted S. aureus containing antisense frpA or frpAB but not frpB constructs showed reduced binding compared to vector construct controls.

scholarly journals Effects of Tedizolid Phosphate on Survival Outcomes and Suppression of Production of Staphylococcal Toxins in a Rabbit Model of Methicillin-Resistant Staphylococcus aureus Necrotizing Pneumonia

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Vien T. M. Le ◽  
Hoan N. Le ◽  
Marcos Gabriel Pinheiro ◽  
Kenneth J. Hahn ◽  
Mary L. Dinh ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Bacterial Production ◽  
Protective Efficacy ◽  
Survival Rates ◽  
Rabbit Model ◽  
Rank Test ◽  
Necrotizing Pneumonia ◽  
Time Curve ◽  
Content Type

ABSTRACT The protective efficacy of tedizolid phosphate, a novel oxazolidinone that potently inhibits bacterial protein synthesis, was compared to those of linezolid, vancomycin, and saline in a rabbit model of Staphylococcus aureus necrotizing pneumonia. Tedizolid phosphate was administered to rabbits at 6 mg/kg of body weight intravenously twice daily, which yielded values of the 24-h area under the concentration-time curve approximating those found in humans. The overall survival rate was 83% for rabbits treated with 6 mg/kg tedizolid phosphate twice daily and 83% for those treated with 50 mg/kg linezolid thrice daily (P = 0.66 by the log-rank test versus the results obtained with tedizolid phosphate). These survival rates were significantly greater than the survival rates of 17% for rabbits treated with 30 mg/kg vancomycin twice daily (P = 0.003) and 17% for rabbits treated with saline (P = 0.002). The bacterial count in the lungs of rabbits treated with tedizolid phosphate was significantly decreased compared to that in the lungs of rabbits treated with saline, although it was not significantly different from that in the lungs of rabbits treated with vancomycin or linezolid. The in vivo bacterial production of alpha-toxin and Panton-Valentine leukocidin, two key S. aureus-secreted toxins that play critical roles in the pathogenesis of necrotizing pneumonia, in the lungs of rabbits treated with tedizolid phosphate and linezolid was significantly inhibited compared to that in the lungs of rabbits treated with vancomycin or saline. Taken together, these results indicate that tedizolid phosphate is superior to vancomycin for the treatment of S. aureus necrotizing pneumonia because it inhibits the bacterial production of lung-damaging toxins at the site of infection.

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.

Covalent Sortase A Inhibitor ML346 Prevents Staphylococcus aureus Infection of Galleria mellonella

2021 ◽  
Author(s):  
Xiang-Na Guan ◽  
Tao Zhang ◽  
Teng Yang ◽  
Ze Dong ◽  
Song Yang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Galleria Mellonella ◽  
Surface Proteins ◽  
Sortase A ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Aureus Infection ◽  
Cell Wall Peptidoglycan

The housekeeping sortase A (SrtA), a membrane-associated cysteine transpeptidase, is responsible for anchoring surface proteins to the cell wall peptidoglycan in Gram-positive bacteria. This process is essential for the regulation...

scholarly journals A Comparative Genome Analysis Identifies Distinct Sorting Pathways in Gram-Positive Bacteria

2004 ◽  
Vol 72 (5) ◽  
pp. 2710-2722 ◽  
Author(s):  
David Comfort ◽  
Robert T. Clubb
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Surface Proteins ◽  
Sequence Motifs ◽  
Comparative Genome Analysis ◽  
Gram Positive ◽  
Microbial Genomes ◽  
Searchable Database ◽  
Gram Positive Bacteria ◽  
Related Proteins

ABSTRACT Surface proteins in gram-positive bacteria are frequently required for virulence, and many are attached to the cell wall by sortase enzymes. Bacteria frequently encode more than one sortase enzyme and an even larger number of potential sortase substrates that possess an LPXTG-type cell wall sorting signal. In order to elucidate the sorting pathways present in gram-positive bacteria, we performed a comparative analysis of 72 sequenced microbial genomes. We show that sortase enzymes can be partitioned into five distinct subfamilies based upon their primary sequences and that most of their substrates can be predicted by making a few conservative assumptions. Most bacteria encode sortases from two or more subfamilies, which are predicted to function nonredundantly in sorting proteins to the cell surface. Only ∼20% of sortase-related proteins are most closely related to the well-characterized Staphylococcus aureus SrtA protein, but nonetheless, these proteins are responsible for anchoring the majority of surface proteins in gram-positive bacteria. In contrast, most sortase-like proteins are predicted to play a more specialized role, with each anchoring far fewer proteins that contain unusual sequence motifs. The functional sortase-substrate linkage predictions are available online (http://www.doe-mbi.ucla.edu/Services/Sortase/ ) in a searchable database.

scholarly journals Three-Dimensional In Vitro Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance

2020 ◽  
Vol 88 (11) ◽  
Author(s):  
Marloes I. Hofstee ◽  
Martijn Riool ◽  
Igors Terjajevs ◽  
Keith Thompson ◽  
Martin J. Stoddart ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Immune Cells ◽  
Early Stage ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Maximum Size ◽  
Human Neutrophils ◽  
Content Type

ABSTRACT Staphylococcus aureus is a prominent human pathogen in bone and soft-tissue infections. Pathophysiology involves abscess formation, which consists of central staphylococcal abscess communities (SACs), surrounded by a fibrin pseudocapsule and infiltrating immune cells. Protection against the ingress of immune cells such as neutrophils, or tolerance to antibiotics, remains largely unknown for SACs and is limited by the lack of availability of in vitro models. We describe a three-dimensional in vitro model of SACs grown in a human plasma-supplemented collagen gel. The in vitro SACs reached their maximum size by 24 h and elaborated a fibrin pseudocapsule, as confirmed by electron and immunofluorescence microscopy. The in vitro SACs tolerated 100× the MIC of gentamicin alone and in combination with rifampin, while planktonic controls and mechanically dispersed SACs were efficiently killed. To simulate a host response, SACs were exposed to differentiated PLB-985 neutrophil-like (dPLB) cells and to primary human neutrophils at an early stage of SAC formation or after maturation at 24 h. Both cell types were unable to clear mature in vitro SACs, but dPLB cells prevented SAC growth upon early exposure before pseudocapsule maturation. Neutrophil exposure after plasmin pretreatment of the SACs resulted in a significant decrease in the number of bacteria within the SACs. The in vitro SAC model mimics key in vivo features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the S. aureus pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.

scholarly journals The purine biosynthesis regulator PurR moonlights as a virulence regulator in Staphylococcus aureus

2019 ◽  
Vol 116 (27) ◽  
pp. 13563-13572 ◽  
Author(s):  
William E. Sause ◽  
Divya Balasubramanian ◽  
Irnov Irnov ◽  
Richard Copin ◽  
Mitchell J. Sullivan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Ex Vivo ◽  
Purine Biosynthesis ◽  
In Vivo Studies ◽  
Human Neutrophils ◽  
Infection Models ◽  
Genes Encoding ◽  
Virulence Regulator

The pathogen Staphylococcus aureus colonizes and infects a variety of different sites within the human body. To adapt to these different environments, S. aureus relies on a complex and finely tuned regulatory network. While some of these networks have been well-elucidated, the functions of more than 50% of the transcriptional regulators in S. aureus remain unexplored. Here, we assess the contribution of the LacI family of metabolic regulators to staphylococcal virulence. We found that inactivating the purine biosynthesis regulator purR resulted in a strain that was acutely virulent in bloodstream infection models in mice and in ex vivo models using primary human neutrophils. Remarkably, these enhanced pathogenic traits are independent of purine biosynthesis, as the purR mutant was still highly virulent in the presence of mutations that disrupt PurR’s canonical role. Through the use of transcriptomics coupled with proteomics, we revealed that a number of virulence factors are differentially regulated in the absence of purR. Indeed, we demonstrate that PurR directly binds to the promoters of genes encoding virulence factors and to master regulators of virulence. These results guided us into further ex vivo and in vivo studies, where we discovered that S. aureus toxins drive the death of human phagocytes and mice, whereas the surface adhesin FnbA contributes to the increased bacterial burden observed in the purR mutant. Thus, S. aureus repurposes a metabolic regulator to directly control the expression of virulence factors, and by doing so, tempers its pathogenesis.

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