Host-pathogen interactions in invasive Staphylococcus aureus infections

Abstract

Staphylococcus aureus is a versatile human pathogen causing a wide range of diseases from uncomplicated skin and soft tissue infections to life-threatening invasive diseases like endocarditis, bacteremia, necrotizing pneumonia, and fasciitis. The pathogen has become increasingly resistant to -lactam antibiotics, and of special concern is the rise in community- acquired (CA)-MRSA strains, as specific CA-MRSA clones have been associated with highly aggressive infections. The ability of S. aureus to cause such a multitude of infections is linked to the production of a wide array of virulence factors. Several virulence factors have been implicated in disease pathogenesis, including the exotoxins Panton-Valentine Leukocidin (PVL), alpha-toxin (α-toxin), superantigens and phenol soluble modulins. This thesis project aimed to characterize S. aureus strains in the community as well as through use of clinical invasive isolates and human lung/skin organotypic tissue models explore the role of specific staphylococcal toxins and virulence regulation in the pathologic events leading to the destructive infections in lung and skin.

In paper I, molecular characterization of Indian community S. aureus isolates to determine their lineage and to analyze their virulence and immune-evasion factors was conducted. The percentage of methicillin resistance was 26% in carrier isolates while 60% among disease isolates. 69% of the isolates were positive for PVL genes along with combinations of many other toxins. The patterns of presence and absence of virulence and immune evasion factors strictly followed the sequence type (ST). We are reporting several new STs, which have not been reported earlier, along with factors influencing virulence and host-pathogen interactions. Next, we demonstrate in paper II that community S. aureus strains displayed stable phenotypic response profiles, defined by either proliferative or cytotoxic responses. The cytotoxic supernatants contained significantly higher levels of α-toxin as compared to proliferative supernatants. Furthermore, a significant association between agr type and phenotypic response profile was found, with agr I and agr IV strains being predominantly cytotoxic whereas agr II and III strains were proliferative. This differential response profiles associated with certain S. aureus strains with varying toxin production abilities could have an impact on disease outcome and may reflect upon the existence of specific pathotypes.

In paper III we focused on the pathogenesis of CA S. aureus severe pneumonia, in particular, the impact of exotoxins produced by strains isolated from varying severity of lung infections on human host cells and in human 3D organotypic lung tissue. α-toxin had a direct damaging effect on the epithelium whereas PVL contributed indirectly to the tissue pathology by triggering lysis of neutrophils. We demonstrated that severe tissue pathology is associated with a combination and high levels of both α-toxin and PVL, and fatal outcome correlated with higher toxin production in pneumonia. Notably, both α-toxin and PVL mediated cytotoxic effect and epithelial disruption was significantly abrogated by addition of polyclonal intravenous immunoglobulins.

In paper IV we focused on skin and soft tissue infections caused by ST22 strains, one of the most critically expanding MRSA clones world-wide. Here we identified a mechanism for which new variants, cytotoxic vs. persistent phenotype, can emerge. We link this phenotype switch to a specific mutation of receptor histidine kinase AgrC. The phenotypic switch to a persistence phenotype is associated with upregulation of bacterial surface proteins, less severe skin tissue damage, resistance to antimicrobials, and induction of autophagy. In contrast, cytotoxic phenotype strains showed upregulated exotoxin expression and caused infections characterized by inflammasome activation and severe skin tissue pathology. This study shows a strong effect of a single amino acid substitution in AgrC as a critical factor contributing to virulence properties and infection outcome.

Together, the studies in this thesis demonstrate that several different toxins will contribute to tissue pathology, but they target different cells and their impact may be tissue-specific. Also, distinct functional differences between the isolates were identified that are likely to contribute to disease outcome. Such insight should promote the development of novel diagnostics or therapeutic strategies.