Characterization of a Staphylococcus aureus multi-enzyme complex involved in the phenotypic switch and host-pathogen interactions

Abstract

RNA homeostasis is maintained by synchronizing RNA synthesis and RNA degradation. These processes are synchronized to growth conditions and environmental stimuli, thus enabling bacterial survival in diverse environmental conditions. Intracellular RNA levels are also dictated by mRNA stability, which depends on the nucleotide sequence. The mRNA sequence, in turn, dictates the secondary and tertiary structures and the accessibility of RNA-binding proteins. Compared to mRNA lifetime in eukaryotes, the half-lives of bacterial mRNAs range from 40 seconds to 60 minutes. mRNA decay in bacteria is often triggered by environmental cues. Bacteria possess multiple post-transcriptional mechanisms that regulate mRNA decay. These differ significantly between Gram-positive and Gram-negative bacteria. In the Gram-positive human pathogen, Staphylococcus aureus, the activity of enzymes associated with the degradosome also significantly influences biofilm production and/or virulence. The studies described in this thesis aim to understand the interactions between different enzymes that form the S. aureus degradosome and the functional impact of these complexes. Another aspect that was examined was the S. aureus - host interactions and the factors that influence the outcome regarding bacterial clearance by the host. More specifically, this study aimed to characterize the host-induced effects on S. aureus upon neutrophil phagocytosis. We identified an S. aureus strain that could escape neutrophil-mediated bacterial clearance and performed a transcriptome analysis to evaluate a potential molecular mechanism(s) for this observation. The first chapter introduces the contents of this thesis and provides a brief background and summary of relevant literature. Chapter 2 of the thesis describes studies geared toward understanding conformational features and the association of different enzymes that form the S. aureus degradosome. Of the multiple RNases in S. aureus, PNPase, RNase J1, RNase J2, and RNase Y were suggested to be associated with the RNA degradosome. The experiments and analysis were aimed to evaluate if these enzymes pre-assembled into building blocks, eventually leading to the degradosome complex. In vitro protein interaction experiments suggested no prerequisites of RNA required for their complexes and a gradation of affinity among these interacting partners. These enzymes are known for the presence of disordered or unstructured stretches in the C-terminal domain, and it was proved in this study that these regions are also important for interacting with their partner enzymes. We characterized the new, unreported interaction of PNPase with CshA and RNase J2 in S. aureus. The complexes of these enzymes were successfully assembled in vitro for characterization suitably using the small-angle X-ray scattering (SAXS) technique. SAXS results identified the in-solution shape profiles, quaternary state, and their rearrangement to assemble as different complexes. It was important to note that these enzymes shared similar interface regions for their interactions, and hence, there could be a possible competition among these enzymes for different sub-assemblies. Normal mode analysis on these complexes characterized the local flexibilities and attributes of their multi-conformers in the solution. The functional assays of these complexes on substrate RNA revealed a modulated catalytic activity compared to their enzymes. The results from these studies suggest that the selective association of these enzymes influences the catalytic activity and can modulate intracellular RNA levels. The second section of the work in this thesis focuses on the effect of RNAIII-associated pathogenicity of S. aureus. This study is based on the differential response of two CA-MRSA strains, ST8 (USA300) and ST88, towards human neutrophil phagocytosis. Ex vivo infection assays suggested that the ST88 strain could effectively evade neutrophils, survive, and lyse them, while neutrophils effectively cleared the ST8 strain. Neutrophil response assays showed that the neutrophil response towards the two strains was identical, and the survival response of the two strains towards the neutrophil microbicidal mechanisms was also similar. However, an interesting observation was that the survival response of ST88 is influenced by its population density, suggesting an alternate mechanism involving bacterial quorum could aid their survival inside neutrophils. Confocal microscopy imaging revealed that the ST88 strain is mostly present as aggregates or clusters of cells compared to a homogenously dispersed ST8 strain. Subsequently, this observation could be rationalized by the Agr quorum sensing mechanism enacted in the case of ST88 by its native auto-inducing peptide (AIP III). Imaging studies confirmed that AIP III triggers the ST88 bacteria to disperse the clusters, wherein the overall percentage of clusters and their average size significantly reduced compared to the ST88 strain in the absence of AIP III induction. The transcriptomics-based results on the internalized ST88 identified suppression of major immune modulators (chp, spa) and virulence regulators (agrA, mgrA) during the first hour of phagocytosis while after seven hours of infection, the bacteria could recoup back to proliferative phase possibly due to the upregulation of key virulence factors (Sbi, Coa, HlgA) countering neutrophils defense responses and lyse the neutrophils. The extended analysis of the transcriptome using Weighted Gene Co-expression Networks (WGCNA) highlighted certain virulence regulators and secretory factors as the hub genes expressed during 1st hour and 7th hour of infections. This analysis also extended the findings to a small noncoding RNA- S35 and an uncharacterized hypothetical protein, and these findings could be useful for future studies on ST88 virulence. In summary, the works described in this thesis signify the relevance of transient and dynamic RNA-protein interactions in a cell for the on-time fine-tuning of RNA levels during post-transcriptional regulation and how RNA-mediated signal transduction mechanisms can influence S. aureus growth and pathogenicity.