Investigating resistance to antibody-dependent complement-mediated lysis in invasive Salmonella Typhimurium

Salmonella Typhimurium is a common cause of self–limited foodborne gastroenteritis globally. In contrast, epidemics of fatal, invasive nontyphoidal Salmonella (iNTS) disease have long been reported in sub–Saharan Africa (SSA), accounting for most of the iNTS disease burden worldwide. Despite this, the significance of this bacterium as a cause of disseminated infections, and the specific mechanisms of pathogenesis have only received considerable attention within the last 15 years or so.
Nontyphoidal Salmonellae, particularly serovars Typhimurium and Enteritidis are a leading cause of bacterial bloodstream infections in SSA, associated with high mortality, particularly among young children and HIV–infected adults. High rates of malnutrition, malaria, sickle–cell disease, poor sanitation, and unavailability of clean water are other known contributing factors. Importantly, acquisition and spread of multiple drug resistance genes have led to the emergence of a unique pathovar, S. Typhimurium ST313. Drug resistance is also a challenge for disease management efforts, which are further confounded by a lack of distinguishing clinical features, that makes diagnosis and adequate treatment difficult. In the imminent dearth of treatment and diagnostic options for iNTS disease, it is imperative that an effective vaccine is developed.
Central to the establishment of systemic infection and invasive disease is the ability of the bacteria to overcome host defenses, first by surviving within phagocytic cells, where they mostly reside during extraintestinal infection. Salmonellae also disseminate between phagocytic cells during a transient extracellular phase in the bloodstream, where they encounter potent antibody responses that effect opsonophagocytosis and complement lysis. Antibody and complement are important for immunity to NTS by preventing this extracellular bacterial growth. Promising vaccine candidates based on surface antigens, particularly LPS O–polysaccharide glycoconjugates, confer protection primarily by eliciting antibody responses. Understanding the modalities of these responses, and the characteristics and important determinants of pathogen virulence that may affect antibody immunity among epidemic strains is, therefore, an important consideration for vaccines.
Characterisation of epidemic African S. Typhimurium ST313 strains thus far reveals some traits of adaptation to human infection, and a predilection for extraintestinal disease such as decreased enteric inflammation, increased rates of systemic spread, and have a higher tolerance to complement bactericidal activity than gastrointestinal isolates.
The aim of this study, therefore, was to identify genetic determinants of resistance to antibody–dependent complement–mediated lysis among Salmonella Typhimurium strains and investigate the impact of this phenotype on virulence and potential to contribute to invasive disease. To achieve the study objectives, whole genome sequencing, phylogenetic analyses, saturated transposon mutant library screens in immune human serum coupled with insertion–site sequencing (TraDIS), targeted mutagenesis, transcriptome profiling and phenotypic characterization by serum bactericidal and broth macrodilution assays, in–vivo and in–vitro invasion assays were performed in both laboratory–adapted strains and invasive S. Typhimurium ST313 clinical isolates with stable complement resistance or susceptibility profiles.
No direct correlation between increased resistance to complement lysis and virulence was found in this study. While S. Typhimurium SL1344 sapA and yfgA null mutants were found to be more resistant to complement lysis than the wild–type parent strain, overall virulence measured by growth in vivo, tolerance to detergent (SDS) and osmotic (high NaCl) stress, and invasion of epithelioid cells, was not enhanced, and was observed to be at lower levels in the yfgA– strain. When measured in invasive strains, tolerance to detergent and osmotic stress was not higher among complement–resistant strains. These observations suggest that complement resistance can occur independently of other virulence traits.
Similarly, no genotypic features (at DNA sequence level) were identified as being associated with natural variation in complement resistance among the invasive strains in this study. Phylogenetic analyses of 32 S. Typhimurium ST313 isolates established that fully resistant isolates (with net growth following 3–hour exposure to serum) were found in both ST313 lineages, indicating independent instances or mechanisms of complement resistance. Whole genome analyses of a subset of these strains did not identify a common feature (SNP, indel, rearrangement, or other mobile genetic element) that discriminated resistant and susceptible strains. Characterisation of polymorphisms in individual strains predicted in–silico to have an impact on protein function did not affect viability in immune human serum when transferred to D23580. Since the vast majority of ST313 isolates studied were serum–susceptible, these observations suggest that unlike other traits of ST313 that have co–evolved with multiple drug resistance, complement resistance does not seem to be sustained at a high level among these populations, but instead seems to be selected against in favour of susceptible isolates, and indicates absence of selection due to natural immunity against S. Typhimurium ST313.
Requirements for survival and growth of invasive S. Typhimurium strains in immune human serum were also identified by identifying differentially expressed genes and counter–selected mutants from a D23580 saturating mutant library screen. Genes involved in iron scavenging and metabolism were the most significantly represented common genes in all the six strains profiled, demonstrating the critical role of iron during growth in serum. Additionally, genes involved in extracellular polysaccharide synthesis, particularly of colanic acid were also found to be significantly up–regulated, although colanic acid mutants were not significantly affected when exposed to serum.
Cluster analysis identified transcriptional profiles that distinguished resistant and susceptible strains, with the highest variance seen in genes associated with LPS and colanic acid synthesis. While fold upregulation of colanic acid genes was higher in susceptible strains, the fepE gene, associated with LPS O–antigen modular chain length of over 100 (very long) repeating units, was on average 2–fold more highly upregulated in resistant strains. Other genes known to be associated with complement resistance in S. Typhimurium, such as pgtE, rck, and traT were all present and intact in both susceptible and resistant strains, did not differ in expression between the two phenotypes, and did not affect viability in serum when disrupted. Since no change in DNA sequence was directly associated with complement sensitivity phenotypes, this thesis postulates an epigenetic effect causing differences in expression of membrane components, particularly of extracellular polysaccharides as a mechanism for complement resistance among invasive strains.