Functional Blocking of Staphylococcus Aureus Adhesins Following Growth in Ex Vivo Media

Infection and Immunity. Oct, 2002  |  Pubmed ID: 12228257

Defining the role of Staphylococcus aureus adhesins in disease pathogenesis may depend on the use of bacteria grown in culture media that more closely reflect the human milieu than conventional broth. This study examined the functional effect on S. aureus adhesins following growth in an ex vivo medium containing a complex mixture of human proteins (used peritoneal dialysate) relative to growth in Todd-Hewitt broth. The adherence of S. aureus, cultured in dialysate, to fibronectin and fibrinogen was markedly reduced despite the expresion of full-length ClfA, ClfB, and fibronectin-binding proteins. Growth in dialysate resulted in the acquisition of a surface coat, as visualized by transmission electron microscopy, which was shown to contain fibronectin, fibrinogen, and immunoglobulins. Adherence of S. aureus to fibrinogen following growth in dialysate was significantly reduced by expression of protein A but was restored following growth in immunoglobulin-depleted dialysate. We conclude that bacterial adherence to solid-phase protein is critically dependent on the culture medium, that S. aureus adhesins may become saturated with target protein prior to contact with solid surfaces, and that there is an interaction between fibrinogen-binding proteins and immunoglobulin bound to protein A following contact with host proteins. These findings have important implications for future studies of S. aureus adhesins.

Antibiotic-resistant Sub-populations of the Pathogenic Bacterium Staphylococcus Aureus Confer Population-wide Resistance

Current Biology : CB. Oct, 2002  |  Pubmed ID: 12401183

Staphylococcus Aureus Clumping Factor B (ClfB) Promotes Adherence to Human Type I Cytokeratin 10: Implications for Nasal Colonization

Cellular Microbiology. Nov, 2002  |  Pubmed ID: 12427098

Staphylococcus aureus is an important cause of sepsis in both community and hospital settings, a major risk factor for which is nasal carriage of the bacterium. Eradication of carriage by topical antibiotics reduces sepsis rates in high-risk individuals, an important strategy for the reduction of nosocomial infection in targeted patient populations. Understanding the mechanisms by which S. aureus adheres to nasal epithelial cells in vivo may lead to alternative methods of decolonization that do not rely on sustained antimicrobial susceptibility. Here, we demonstrate for the first time that the S. aureus surface-expressed protein, clumping factor B (ClfB), promotes adherence to immobilized epidermal cytokeratins in vitro. By expressing a range of S. aureus adhesins on the surface of the heterologous host Lactococcus lactis, we demonstrated that adherence to epidermal cytokeratins was conferred by ClfB. Adherence of wild-type S. aureus was inhibited by recombinant ClfB protein or anti-ClfB antibodies, and S. aureus mutants defective in ClfB adhered poorly to epidermal cytokeratins. Expression of ClfB promoted adherence of L. lactis to human desquamated nasal epithelial cells, and a mutant of S. aureus defective in ClfB had reduced adherence compared with wild type. ClfB also promoted adherence of L. lactis cells to a human keratinocyte cell line. Cytokeratin 10 molecules were shown by flow cytometry to be exposed on the surface of both desquamated nasal epithelial cells and keratinocytes. Cytokeratin 10 was also detected on the surface of desquamated human nasal cells using immunofluorescence, and recombinant ClfB protein was shown to bind to cytokeratin K10 extracted from these cells. We also showed that ClfB is transcribed by S. aureus in the human nares. We propose that ClfB is a major determinant in S. aureus nasal colonization.

Environmental Regulation of Mutation Rates at Specific Sites

Trends in Microbiology. Dec, 2002  |  Pubmed ID: 12564995

Recent studies on bacterial adaptation to stress suggest that bacteria can regulate the generation of mutations at specific sites in response to environmental conditions. Here, we review these findings and discuss the circumstances under which these mechanisms might prove advantageous.

Genes Encoding a Cellulosic Polymer Contribute Toward the Ecological Success of Pseudomonas Fluorescens SBW25 on Plant Surfaces

Molecular Ecology. Nov, 2003  |  Pubmed ID: 14629390

Pseudomonas fluorescens SBW25 is a Gram-negative bacterium that grows in close association with plants. In common with a broad range of functionally similar bacteria it plays an important role in the turnover of organic matter and certain isolates can promote plant growth. Despite its environmental significance, the causes of its ecological success are poorly understood. Here we describe the development and application of a simple promoter trapping strategy (IVET) to identify P. fluorescens SBW25 genes showing elevated levels of expression in the sugar beet rhizosphere. A total of 25 rhizosphere-induced (rhi) fusions are reported with predicted roles in nutrient acquisition, stress responses, biosynthesis of phytohormones and antibiotics. One rhi fusion is to wss, an operon encoding an acetylated cellulose polymer. A mutant carrying a defective wss locus was competitively compromised (relative to the wild type) in the rhizosphere and in the phyllosphere, but not in bulk soil. The rhizosphere-induced wss locus therefore contributes to the ecological performance of SBW25 in the plant environment and supports our conjecture that genes inactive in the laboratory environment, but active in the wild, are likely to be determinants of fitness in natural environments.

Interference Competition and Parasite Virulence

Proceedings. Biological Sciences / The Royal Society. Apr, 2004  |  Pubmed ID: 15255095

Within-host competition between parasites, a consequence of infection by multiple strains, is predicted to favour rapid host exploitation and greater damage to hosts (virulence). However, the inclusion of biological variables can drastically change this relationship. For example, if competing parasite strains produce toxins that kill each other (interference competition), their growth rates and virulence may be reduced relative to single-strain infections. Bacteriocins are antimicrobial toxins produced by bacteria that target closely related strains and species, and to which the producing strain is immune. We investigated competition between bacteriocin-producing, insect-killing bacteria (Photorhabdus and Xenorhabdus) and how this competition affected virulence in caterpillars. Where one strain could kill the other, and not vice versa, the non-killing strain was competitively excluded, and insect mortality was the same as that of the killing strain alone. However, when caterpillars were multiply infected by strains that could kill each other, we did not observe competitive exclusion and their virulence was less than single-strain infections. The ubiquity and diversity of bacteriocins among pathogenic bacteria suggest mixed infections will be, on average, less virulent than single infections.

Clonal Distribution and Phase-variable Expression of a Major Histocompatibility Complex Analogue Protein in Staphylococcus Aureus

Journal of Bacteriology. Apr, 2005  |  Pubmed ID: 15805539

The mapW gene of Staphylococcus aureus strain N315 contains a poly(A) tract which truncates translation of the protein. This study demonstrates that mapW is an allelic variant of the map/eap genes found in other strains and that the variation in the length of this poly(A) tract suggests that it is a contingency locus.

Antagonistic Coevolution with Parasites Increases the Cost of Host Deleterious Mutations

Proceedings. Biological Sciences / The Royal Society. Jan, 2006  |  Pubmed ID: 16519233

The fitness consequences of deleterious mutations are sometimes greater when individuals are parasitized, hence parasites may result in the more rapid purging of deleterious mutations from host populations. The significance of host deleterious mutations when hosts and parasites antagonistically coevolve (reciprocal evolution of host resistance and parasite infectivity) has not previously been experimentally investigated. We addressed this by coevolving the bacterium Pseudomonas fluorescens and a parasitic bacteriophage in laboratory microcosms, using bacteria with high and low mutation loads. Directional coevolution between bacterial resistance and phage infectivity occurred in all populations. Bacterial population fitness, as measured by competition experiments with ancestral genotypes in the absence of phage, declined with time spent coevolving. However, this decline was significantly more rapid in bacteria with high mutation loads, suggesting the cost of bacterial resistance to phage was greater in the presence of deleterious mutations (synergistic epistasis). As such, resistance to phage was more costly to evolve in the presence of a high mutation load. Consistent with these data, bacteria with high mutation loads underwent less rapid directional coevolution with their phage populations, and showed lower levels of resistance to their coevolving phage populations. These data suggest that coevolution with parasites increases the rate at which deleterious mutations are purged from host populations.

Agr Interference Between Clinical Staphylococcus Aureus Strains in an Insect Model of Virulence

Journal of Bacteriology. Nov, 2006  |  Pubmed ID: 16936013

Repression of virulence by Staphylococcus aureus strains from different Agr groups has been demonstrated in vitro and is proposed as a means of competitive interference. Here, using the insect Manduca sexta, we show for the first time that this interference also occurs in vivo within a mixed population.

The Evolution and Maintenance of Virulence in Staphylococcus Aureus: a Role for Host-to-host Transmission?

Nature Reviews. Microbiology. Dec, 2006  |  Pubmed ID: 17109032

Despite progress in our understanding of infectious disease biology and prevention, the conditions that select for the establishment and maintenance of microbial virulence remain enigmatic. To address this aspect of pathogen biology, we focus on two members of the Staphylococcus genus - Staphylococcus aureus and Staphylococcus epidermidis - and consider why S. aureus has evolved to become more virulent than S. epidermidis. Several hypotheses to explain this phenomenon are discussed and a mathematical model is used to argue that a complex transmission pathway is the key factor in explaining the evolution and maintenance of virulence in S. aureus. In the case of S. epidermidis, where skin contact affords easier transmission between hosts, high levels of virulence do not offer an advantage to this pathogen.

Use of Peptide-major Histocompatibility Complex Tetramer Technology to Study Interactions Between Staphylococcus Aureus Proteins and Human Cells

Infection and Immunity. Dec, 2007  |  Pubmed ID: 17938227

In this study, we report the use of peptide-major histocompatibility complex tetramer technology to study the interactions that occur between Staphylococcus aureus proteins and human leukocytes. We demonstrated that this technology can be used to study the activity of superantigens such as toxic shock syndrome toxin 1 and also found that despite similarities to known proteins (i.e., major histocompatibility complex [MHC] class II molecules and superantigens), the S. aureus Eap protein does not block MHC-T-cell receptor interactions and is not a superantigen. Instead, it has nonspecific cross-linking activity that is dependent upon having at least two of its six 110-amino-acid repeats.

Interspecific Competition and Siderophore-mediated Cooperation in Pseudomonas Aeruginosa

The ISME Journal. Jan, 2008  |  Pubmed ID: 18180746

Both intra- and interspecific interactions between microbes are likely to play an important role in determining the severity of microbial infections. Here, we study the impact of interactions between coinfecting opportunistic pathogens Staphylococcus aureus and Pseudomonas aeruginosa on both phenotypic and genetic changes in a P. aeruginosa social trait, the production of iron-scavenging siderophores. Siderophores are facultatively upregulated in response to iron limitation and play a key role in determining the virulence of microbial infections. Siderophore production is metabolically expensive to individual producers but benefits the group as a whole because siderophores can be used by all cells in the vicinity with siderophore receptors. Hence, populations of siderophore producers can be invaded by nonproducing cheats. Previous work has shown that P. aeruginosa can lyse S. aureus, supplying a source of free iron. We therefore hypothesized that the presence of S. aureus might result in facultative downregulation of siderophore production, and in turn, reduced selection for siderophore cheats. We tested this hypothesis by evolving P. aeruginosa in the presence and absence of free iron and S. aureus, in a fully factorial design. Iron had the expected effect: siderophore production was downregulated and cheats evolved less readily, but the presence of S. aureus instead increased facultative siderophore production and selection for cheats. This is probably because the S. aureus had the net effect of competing for iron, rather than acting as an iron source. This study demonstrates that interspecific competition can have a marked effect on intraspecific social interactions.

The Staphyloccous Aureus Eap Protein Activates Expression of Proinflammatory Cytokines

Infection and Immunity. May, 2008  |  Pubmed ID: 18332207

The extracellular adhesion protein (Eap) secreted by the major human pathogen Staphylococcus aureus is known to have several effects on human immunity. We have recently added to knowledge of these roles by demonstrating that Eap enhances interactions between major histocompatibility complex molecules and human leukocytes. Several studies have indicated that Eap can induce cytokine production by human peripheral blood mononuclear cells (PBMCs). To date, there has been no rigorous attempt to identify the breadth of cytokines produced by Eap stimulation or to identify the cell subsets that respond. Here, we demonstrate that Eap induces the secretion of the proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) by CD14(+) leukocytes (monocytes and macrophages) within direct ex vivo PBMC populations (note that granulocytes are also CD14(+) but are largely depleted from PBMC preparations). Anti-intercellular adhesion molecule 1 (CD54) antibodies inhibited this induction and implicated a role for this known Eap binding protein in cellular activation. IL-6 and TNF-alpha secretion by murine cells exposed to Eap was also observed. The activation of CD14(+) cells by Eap suggests that it could play a significant role in both septic shock and fever, two of the major pathological features of S. aureus infections.

Identification of Factors Contributing to T-cell Toxicity of Staphylococcus Aureus Clinical Isolates

Journal of Clinical Microbiology. Jun, 2008  |  Pubmed ID: 18417658

We examined the ability of 206 clinical isolates of Staphylococcus aureus to lyse T cells and found differences between Agr groups. We found that the beta and delta hemolysins are involved and that methicillin-resistant S. aureus strains are less toxic than methicillin-susceptible S. aureus strains.

Offsetting Virulence and Antibiotic Resistance Costs by MRSA

The ISME Journal. Apr, 2010  |  Pubmed ID: 20072161

The prevalence of diverse MRSA (methicillin-resistant Staphylococcus aureus) types in both hospital and community settings is a major health problem worldwide. Here we compare hospital-acquired MRSAs with large type II SCCmec elements with those prevalent in both hospital and community settings with smaller type IV SCCmec elements. We find that the type II but not the type IV SCCmec element causes the bacteria to reduce their levels of costly toxin expression. We compare the relative growth rates of these MRSA types and show that the type II SCCmec carrying MRSAs are more affected than those carrying type IV elements and from this we hypothesize that offsetting the costs associated with antibiotic resistance and toxin expression is why the type II are confined to hospital environments where antibiotic use, the prevalence of immunocompromised individuals and vector-mediated transmission is high. In contrast, those MRSAs that are also successful in the community can maintain their high levels of toxin expression due to a lower fitness burden associated with the smaller SCCmec element.

Staphylococcus Aureus Host Cell Invasion and Virulence in Sepsis is Facilitated by the Multiple Repeats Within FnBPA

PLoS Pathogens. 2010  |  Pubmed ID: 20585570

Entry of Staphylococcus aureus into the bloodstream can lead to metastatic abscess formation and infective endocarditis. Crucial to the development of both these conditions is the interaction of S. aureus with endothelial cells. In vivo and in vitro studies have shown that the staphylococcal invasin FnBPA triggers bacterial invasion of endothelial cells via a process that involves fibronectin (Fn) bridging to alpha(5)beta(1) integrins. The Fn-binding region of FnBPA usually contains 11 non-identical repeats (FnBRs) with differing affinities for Fn, which facilitate the binding of multiple Fn molecules and may promote integrin clustering. We thus hypothesized that multiple repeats are necessary to trigger the invasion of endothelial cells by S. aureus. To test this we constructed variants of fnbA containing various combinations of FnBRs. In vitro assays revealed that endothelial cell invasion can be facilitated by a single high-affinity, but not low-affinity FnBR. Studies using a nisin-inducible system that controlled surface expression of FnBPA revealed that variants encoding fewer FnBRs required higher levels of surface expression to mediate invasion. High expression levels of FnBPA bearing a single low affinity FnBR bound Fn but did not invade, suggesting that FnBPA affinity for Fn is crucial for triggering internalization. In addition, multiple FnBRs increased the speed of internalization, as did higher expression levels of FnBPA, without altering the uptake mechanism. The relevance of these findings to pathogenesis was demonstrated using a murine sepsis model, which showed that multiple FnBRs were required for virulence. In conclusion, multiple FnBRs within FnBPA facilitate efficient Fn adhesion, trigger rapid bacterial uptake and are required for pathogenesis.

How Does Staphylococcus Aureus Escape the Bloodstream?

Trends in Microbiology. Apr, 2011  |  Pubmed ID: 21227700

Staphylococcus aureus is a major cause of bacteraemia, which frequently leads to infective endocarditis, osteomyelitis, septic arthritis and metastatic abscess formation. The development of these secondary infections is due to bacterial dissemination from the blood into surrounding tissues and is associated with significantly increased morbidity and mortality. Despite the importance of S. aureus extravasation in disease progression, there is relatively little understanding of the molecular mechanisms by which this pathogen crosses the endothelial barrier and establishes new sites of infection. Recent work has identified a number of putative routes by which S. aureus can escape the bloodstream. In this article we review these new developments and set them in the context of strategies used by other established pathogens to traverse cellular barriers.

Staphylococcus Aureus Keratinocyte Invasion is Dependent Upon Multiple High-affinity Fibronectin-binding Repeats Within FnBPA

PloS One. 2011  |  Pubmed ID: 21526122

Staphylococcus aureus is a commensal organism and a frequent cause of skin and soft tissue infections, which can progress to serious invasive disease. This bacterium uses its fibronectin binding proteins (FnBPs) to invade host cells and it has been hypothesised that this provides a protected niche from host antimicrobial defences, allows access to deeper tissues and provides a reservoir for persistent or recurring infections. FnBPs contain multiple tandem fibronectin-binding repeats (FnBRs) which bind fibronectin with varying affinity but it is unclear what selects for this configuration. Since both colonisation and skin infection are dependent upon the interaction of S. aureus with keratinocytes we hypothesised that this might select for FnBP function and thus composition of the FnBR region. Initial experiments revealed that S. aureus attachment to keratinocytes is rapid but does not require FnBRs. By contrast, invasion of keratinocytes was dependent upon the FnBR region and occurred via similar cellular processes to those described for endothelial cells. Despite this, keratinocyte invasion was relatively inefficient and appeared to include a lag phase, most likely due to very weak expression of α(5)β(1) integrins. Molecular dissection of the role of the FnBR region revealed that efficient invasion of keratinocytes was dependent on the presence of at least three high-affinity (but not low-affinity) FnBRs. Over-expression of a single high-affinity or three low-affinity repeats promoted invasion but not to the same levels as S. aureus expressing an FnBPA variant containing three high-affinity repeats. In summary, invasion of keratinocytes by S. aureus requires multiple high-affinity FnBRs within FnBPA, and given the importance of the interaction between these cell types and S. aureus for both colonisation and infection, may have provided the selective pressure for the multiple binding repeats within FnBPA.

Methicillin Resistance Reduces the Virulence of Healthcare-Associated Methicillin-Resistant Staphylococcus Aureus by Interfering With the Agr Quorum Sensing System

The Journal of Infectious Diseases. Mar, 2012  |  Pubmed ID: 22301683

The difficulty in successfully treating infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has led to them being referred to as highly virulent or pathogenic. In our study of one of the major healthcare-associated MRSA (HA-MRSA) clones, we show that expression of the gene responsible for conferring methicillin resistance (mecA) is also directly responsible for reducing the ability of HA-MRSA to secrete cytolytic toxins. We show that resistance to methicillin induces changes in the cell wall, which affects the bacteria's agr quorum sensing system. This leads to reduced toxin expression and, as a consequence, reduced virulence in a murine model of sepsis. This diminished capacity to cause infection may explain the inability of HA-MRSA to move into the community and help us understand the recent emergence of community-associated MRSA (CA-MRSA). CA-MRSA typically express less penicillin-binding protein 2a (encoded by mecA), allowing them to maintain full virulence and succeed in the community environment.