Maisem Laabei

Predicting the virulence of MRSA from its genome sequence

Microbial virulence is a complex and often multifactorial phenotype, intricately linked to a pathogens evolutionary trajectory. Toxicity, the ability to destroy host cell membranes, and adhesion, the ability to adhere to human tissues, are the major virulence factors of many bacterial pathogens, including Staphylococcus aureus. Here, we assayed the toxicity and adhesiveness of 90 MRSA (methicillin resistant S. aureus) isolates and found that while there was remarkably little variation in adhesion, toxicity varied by over an order of magnitude between isolates, suggesting different evolutionary selection pressures acting on these two traits. We performed a genome-wide association study (GWAS) and identified a large number of loci, as well as a putative network of epistatically interacting loci, that significantly associated with toxicity. Despite this apparent complexity in toxicity regulation, a predictive model based on a set of significant single nucleotide polymorphisms (SNPs) and insertion and deletions events (indels) showed a high degree of accuracy in predicting an isolates toxicity solely from the genetic signature at these sites. Our results thus highlight the potential of using sequence data to determine clinically relevant parameters and have further implications for understanding the microbial virulence of this opportunistic pathogen.

Evolutionary Trade-Offs Underlie the Multi-faceted Virulence of Staphylococcus aureus

Bacterial virulence is a multifaceted trait where the interactions between pathogen and host factors affect the severity and outcome of the infection. Toxin secretion is central to the biology of many bacterial pathogens and is widely accepted as playing a crucial role in disease pathology. To understand the relationship between toxicity and bacterial virulence in greater depth, we studied two sequenced collections of the major human pathogen Staphylococcus aureus and found an unexpected inverse correlation between bacterial toxicity and disease severity. By applying a functional genomics approach, we identified several novel toxicity-affecting loci responsible for the wide range in toxic phenotypes observed within these collections. To understand the apparent higher propensity of low toxicity isolates to cause bacteraemia, we performed several functional assays, and our findings suggest that within-host fitness differences between high- and low-toxicity isolates in human serum is a contributing factor. As invasive infections, such as bacteraemia, limit the opportunities for onward transmission, highly toxic strains could gain an additional between-host fitness advantage, potentially contributing to the maintenance of toxicity at the population level. Our results clearly demonstrate how evolutionary trade-offs between toxicity, relative fitness, and transmissibility are critical for understanding the multifaceted nature of bacterial virulence.

From genotype to phenotype: can systems biology be used to predict Staphylococcus aureus virulence?

With the advent of high-throughput whole-genome sequencing, it is now possible to sequence a bacterial genome in a matter of hours. However, although the presence or absence of a particular gene can be determined, we do not yet have the tools to extract information about the true virulence potential of an organism from sequence data alone. Here, we focus on the important human pathogen Staphylococcus aureus and present a framework for the construction of a broad systems biology-based tool that could be used to predict virulence phenotypes from S. aureus genomic sequences using existing technology.

Development of a prototype wound dressing technology which can detect and report colonization by pathogenic bacteria

A new methodology for detecting the microbiological state of a wound dressing in terms of its colonization with pathogenic bacteria such as Staphylococcus aureus or Pseudomonas aeruginosa has been developed. Here we report how stabilized lipid vesicles containing self-quenched carboxyfluorescein dye are sensitive to lysis only by toxins/virulence factors from P. aeruginosa and S. aureus but not by a non-toxic Escherichia coli species. The development of the stabilized vesicles is discussed and their response to detergent (triton), bacterial toxin (α-hemolysin) and lipases (phospholipase A(2)). Finally, fabrics with stabilized vesicles attached via plasma deposited maleic anhydride coupling are shown visibly responding to S. aureus (MSSA 476) and P. aeruginosa (PAO1) but not E. coli DH5α in a prototype dressing.

Oxacillin Alters the Toxin Expression Profile of Community-Associated Methicillin-Resistant Staphylococcus aureus

ABSTRACT The emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a growing cause for concern. These strains are more virulent than health care-associated MRSA (HA-MRSA) due to higher levels of toxin expression. In a previous study, we showed that the high-level expression of PBP2a, the alternative penicillin binding protein encoded by the mecA gene on type II staphylococcal cassette chromosome mec (SCCmec) elements, reduced toxicity by interfering with the Agr quorum sensing system. This was not seen in strains carrying the CA-MRSA-associated type IV SCCmec element. These strains express significantly lower levels of PBP2a than the other MRSA type, which may explain their relatively high toxicity. We hypothesized that as oxacillin is known to increase mecA expression levels, it may be possible to attenuate the toxicity of CA-MRSA by using this antibiotic. Subinhibitory oxacillin concentrations induced PBP2a expression, repressed Agr activity, and, as a consequence, decreased phenol-soluble modulin (PSM) secretion by CA-MRSA strains. However, consistent with other studies, oxacillin also increased the expression levels of alpha-toxin and Panton-Valentine leucocidin (PVL). The net effect of these changes on the ability to lyse diverse cell types was tested, and we found that where the PSMs and alpha-toxin are important, oxacillin reduced overall lytic activity, but where PVL is important, it increased lytic activity, demonstrating the pleiotropic effect of oxacillin on toxin expression by CA-MRSA.

Investigating the lytic activity and structural properties of Staphylococcus aureus phenol soluble modulin (PSM) peptide toxins

The ubiquitous bacterial pathogen, Staphylococcus aureus, expresses a large arsenal of virulence factors essential for pathogenesis. The phenol-soluble modulins (PSMs) are a family of cytolytic peptide toxins which have multiple roles in staphylococcal virulence. To gain an insight into which specific factors are important in PSM-mediated cell membrane disruption, the lytic activity of individual PSM peptides against phospholipid vesicles and T cells was investigated. Vesicles were most susceptible to lysis by the PSMα subclass of peptides (α1-3 in particular), when containing between 10 and 30mol% cholesterol, which for these vesicles is the mixed solid ordered (so)-liquid ordered (lo) phase. Our results show that the PSMβ class of peptides has little effect on vesicles at concentrations comparable to that of the PSMα class and exhibited no cytotoxicity. Furthermore, within the PSMα class, differences emerged with PSMα4 showing decreased vesicle and cytotoxic activity in comparison to its counterparts, in contrast to previous studies. In order to understand this, peptides were studied using helical wheel projections and circular dichroism measurements. The degree of amphipathicity, alpha-helicity and properties such as charge and hydrophobicity were calculated, allowing a structure-function relationship to be inferred. The degree of alpha-helicity of the peptides was the single most important property of the seven peptides studied in predicting their lytic activity. These results help to redefine this class of peptide toxins and also highlight certain membrane parameters required for efficient lysis.

Staphylococcus aureus Extracellular Adherence Protein Triggers TNFα Release, Promoting Attachment to Endothelial Cells via Protein A

Staphylococcus aureus is a leading cause of bacteraemia, which frequently results in complications such as infective endocarditis, osteomyelitis and exit from the bloodstream to cause metastatic abscesses. Interaction with endothelial cells is critical to these complications and several bacterial proteins have been shown to be involved. The S. aureus extracellular adhesion protein (Eap) has many functions, it binds several host glyco-proteins and has both pro- and anti-inflammatory activity. Unfortunately its role in vivo has not been robustly tested to date, due to difficulties in complementing its activity in mutant strains. We previously found Eap to have pro-inflammatory activity, and here show that purified native Eap triggered TNFα release in whole human blood in a dose-dependent manner. This level of TNFα increased adhesion of S. aureus to endothelial cells 4-fold via a mechanism involving protein A on the bacterial surface and gC1qR/p33 on the endothelial cell surface. The contribution this and other Eap activities play in disease severity during bacteraemia was tested by constructing an isogenic set of strains in which the eap gene was inactivated and complemented by inserting an intact copy elsewhere on the bacterial chromosome. Using a murine bacteraemia model we found that Eap expressing strains cause a more severe infection, demonstrating its role in invasive disease.

Clonal differences in Staphylococcus aureus bacteraemia-associated mortality

The bacterium Staphylococcus aureus is a major human pathogen for which the emergence of antibiotic resistance is a global public health concern. Infection severity, and in particular bacteraemia-associated mortality, has been attributed to several host-related factors, such as age and the presence of comorbidities. The role of the bacterium in infection severity is less well understood, as it is complicated by the multifaceted nature of bacterial virulence, which has so far prevented a robust mapping between genotype, phenotype and infection outcome. To investigate the role of bacterial factors in contributing to bacteraemia-associated mortality, we phenotyped a collection of sequenced clinical S. aureus isolates from patients with bloodstream infections, representing two globally important clonal types, CC22 and CC30. By adopting a genome-wide association study approach we identified and functionally verified several genetic loci that affect the expression of cytolytic toxicity and biofilm formation. By analysing the pooled data comprising bacterial genotype and phenotype together with clinical metadata within a machine-learning framework, we found significant clonal differences in the determinants most predictive of poor infection outcome. Whereas elevated cytolytic toxicity in combination with low levels of biofilm formation was predictive of an increased risk of mortality in infections by strains of a CC22 background, these virulence-specific factors had little influence on mortality rates associated with CC30 infections. Our results therefore suggest that different clones may have adopted different strategies to overcome host responses and cause severe pathology. Our study further demonstrates the use of a combined genomics and data analytic approach to enhance our understanding of bacterial pathogenesis at the individual level, which will be an important step towards personalized medicine and infectious disease management.A genome-wide association approach identifies differential biofilm and virulence attributes associated with mortality in two Staphylococcus aureus clonal complexes.

Manipulation of Autophagy in Phagocytes Facilitates Staphylococcus aureus Bloodstream Infection

ABSTRACT The capacity for intracellular survival within phagocytes is likely a critical factor facilitating the dissemination of Staphylococcus aureus in the host. To date, the majority of work on S. aureus-phagocyte interactions has focused on neutrophils and, to a lesser extent, macrophages, yet we understand little about the role played by dendritic cells (DCs) in the direct killing of this bacterium. Using bone marrow-derived DCs (BMDCs), we demonstrate for the first time that DCs can effectively kill S. aureus but that certain strains of S. aureus have the capacity to evade DC (and macrophage) killing by manipulation of autophagic pathways. Strains with high levels of Agr activity were capable of causing autophagosome accumulation, were not killed by BMDCs, and subsequently escaped from the phagocyte, exerting significant cytotoxic effects. Conversely, strains that exhibited low levels of Agr activity failed to accumulate autophagosomes and were killed by BMDCs. Inhibition of the autophagic pathway by treatment with 3-methyladenine restored the bactericidal effects of BMDCs. Using an in vivo model of systemic infection, we demonstrated that the ability of S. aureus strains to evade phagocytic cell killing and to survive temporarily within phagocytes correlated with persistence in the periphery and that this effect is critically Agr dependent. Taken together, our data suggest that strains of S. aureus exhibiting high levels of Agr activity are capable of blocking autophagic flux, leading to the accumulation of autophagosomes. Within these autophagosomes, the bacteria are protected from phagocytic killing, thus providing an intracellular survival niche within professional phagocytes, which ultimately facilitates dissemination.

Staphylococcus aureus interaction with phospholipid vesicles--a new method to accurately determine accessory gene regulator (agr) activity.

The staphylococcal accessory gene regulatory (agr) operon is a well-characterised global regulatory element that is important in the control of virulence gene expression for Staphylococcus aureus, a major human pathogen. Hence, accurate and sensitive measurement of Agr activity is central in understanding the virulence potential of Staphylococcus aureus, especially in the context of Agr dysfunction, which has been linked with persistent bacteraemia and reduced susceptibility to glycopeptide antibiotics. Agr function is typically measured using a synergistic haemolysis CAMP assay, which is believe to report on the level of expression of one of the translated products of the agr locus, delta toxin. In this study we develop a vesicle lysis test (VLT) that is specific to small amphipathic peptides, most notably delta and Phenol Soluble Modulin (PSM) toxins. To determine the accuracy of this VLT method in assaying Agr activity, we compared it to the CAMP assay using 89 clinical Staphylococcus aureus isolates. Of the 89 isolates, 16 were designated as having dysfunctional Agr systems by the CAMP assay, whereas only three were designated as such by VLT. Molecular analysis demonstrated that of these 16 isolates, the 13 designated as having a functional Agr system by VLT transcribed rnaIII and secreted delta toxin, demonstrating they have a functional Agr system despite the results of the CAMP assay. The agr locus of all 16 isolates was sequenced, and only the 3 designated as having a dysfunctional Agr system contained mutations, explaining their Agr dysfunction. Given the potentially important link between Agr dysfunction and clinical outcome, we have developed an assay that determines this more accurately than the conventional CAMP assay.