Understanding the role of caveolae in the uptake process of Streptococcus pyogenes into non-phagocytic host cells (2017)

Streptococcus pyogenes, more commonly known as Group A Streptococcus (GAS), is a Gram positive, obligate human bacterial pathogen responsible for two million new cases of infection and more than half a million deaths annually. GAS infections manifest in a variety of diseases ranging from mild superficial conditions such as pharyngitis and impetigo to more severe invasive ones such as bacteraemia, necrotizing fasciitis, and streptococcal toxic shock syndrome. Repeated infections may also result in immune-related sequelae including acute rheumatic fever, rheumatic heart disease and glomerulonephritis. Despite advances in the use of antibiotics to treat bacterial infections, GAS has evolved mechanisms to subvert their hosts’ cellular machinery to escape host immunity, persist, and cause infections. Successful cellular internalization of GAS is inferred to be important for evading host immune responses and antibiotic therapy. GAS has been shown to invade various non-phagocytic host cell types. Although not yet completely understood, a widely accepted model for GAS invasion into non-phagocytic cells occurs via the binding of GAS fibronectin binding proteins (such as SfbI or M protein) to host fibronectins. This engages and activates α5β1 integrins, forming a fibronectin cross-bridge linking GAS to the host extracellular matrix (ECM) to facilitate attachment. In addition to this pathway, SfbI-expressing GAS strains have been described to invade cells via an alternative route that involves host caveolae. Caveolae are specialized lipid rafts that appear as small invaginations in the eukaryotic plasma membrane, and are implicated in a number of cellular functions such as the uptake of various macromolecules and regulation of invasion of some bacterial pathogens into host cells. The scaffolding protein component of caveolae is caveolin-1 (CAV1), which oligomerizes to form the primary caveolae structure. A secondary protein termed Polymerase I and Transcript Release Factor (PTRF) or cavin-1 is also required for formation and stabilization of the caveolae structure. Caveolae have been implicated as an entry port that GAS utilizes during invasion into human epithelial HEp-2 cells. To better understand the role of caveolae in GAS invasion, this thesis examines how loss of the caveolae component proteins CAV1 and PTRF affect GAS invasion using genetic knockout and knockdown approaches. Chapter 3 explores the role of CAV1 in the uptake of GAS. Specific CAV1 knockout and stable CAV1 knockdown cell lines were generated and GAS invasion into these cells was measured by enumeration of colony forming units (CFUs). The invasion process was imaged through immunofluorescence and transmission electron microscopy to provide ultrastructural visualization of invading GAS and interactions with their host cells. Contrary to previous findings, a new protective role of CAV1 against GAS invasion into mouse embryonic fibroblasts (MEFs) and human HEp-2 epithelial cells was identified. Neither CAV1 nor caveolae were physically associated with invading GAS, suggesting a caveolae-independent mechanism in eliciting this protective effect. The constituent proteins of caveolae are CAV1 and PTRF; these proteins together form and stabilize caveolae at the plasma membrane. Expression of CAV1 and PTRF are usually co-dependent and both are required for the formation of caveolae in mammalian cells. Chapter 4 explores the role of PTRF in the uptake of GAS, in which it was hypothesized that the lack of PTRF would similarly enhance GAS uptake. Invasion assays of GAS into PTRF MEFs indicated that the lack of PTRF expression resulted in reduced GAS invasion, which was opposite to the loss of CAV1. This result was replicated in HEp-2 cells, however, issues related to the re-emergence of PTRF expression may have confounded the results from HEp-2 cells. This chapter reports these findings and the caveats associated with the HEp-2 cell experiments in light of the unstable knockdown. To understand the protective role of CAV1 during GAS invasion (Chapter 3), Chapter 5 explores whether three known GAS virulence factors and various host cell components are involved in the mechanism behind caveolae protection against GAS invasion. GAS Streptolysin O (SLO), M1 protein and SpeB protease negative mutants were used in invasion assays into HEp-2 cells. The potential involvement of host cell plasma membrane mobility, lipid droplets and clathrin uptake pathways were also investigated. Of the three GAS virulence factors tested, only SLO appeared to be associated with the regulation of CAV1 over GAS invasion. Host cell plasma membrane mobility experiments indicated that GAS invasion was not altered by increasing membrane fluidity, and OA experiments indicated that GAS survival was enhanced under OA addition which was in contrast to the inferred bactericidal roles of lipid droplets. Clathrin inhibition experiments suggest that GAS invasion was linked to clathrin-mediated uptake, consistent with the accepted model of GAS uptake through a clathrin-dependent process. This was in agreement with electron microscopy images showing the GAS invasion process (Chapter 3) supporting GAS invasion through a “zipper” mechanism. These results are described in this chapter, and tied in to putative roles of CAV1 in protecting against GAS invasion. Understanding GAS pathogenesis and mechanisms underlying host cell invasion is vital for formulating effective countermeasures against GAS infections. The findings presented in this thesis provide targets for promoting vaccine development and where successful, better control of GAS disease worldwide.