Investigation of Non-typhoidal Salmonella pathogenesis, Biofilm and Vaccine development

Abstract

Salmonella are a diverse group of pathogenic bacteria that remains a serious public health concern worldwide. Understanding the mechanism of pathogenesis and transmission are important for the development of effective vaccines, and strategies to mitigate Salmonella infections. In this thesis I investigate different aspects of Salmonella biology.

Non-typhoidal Salmonella (NTS) associated with gastroenteritis worldwide are the leading cause of bloodstream infections in sub-Saharan Africa. The invasive NTS (iNTS) differ from gastroenteritis causing isolates by more than 700 single nucleotide polymorphisms (SNP). I identified a conserved SNP in invasive S. Typhimurium D23580 that results in a missense mutation in the sensory domain of a diguanylate cyclase enzyme, STM1987. STM1987 catalyzes the formation of c-di-GMP, which positively regulates cellulose production. Previous studies have shown that Salmonella produces cellulose inside macrophages as an antivirulence factor. The mutation in STM1987 results in a 10-fold drop in cellulose production, and increased survival inside human and murine macrophage cell lines. Using competitive index experiments, I showed that compared to wildtype, S. Typhimurium with SNP in stm1987 have increased virulence in mice. My results showed that STM1987 plays a role in Salmonella virulence during infection. Due to the high mortality rate associated with infections a vaccine is urgently needed to reduce incidence of iNTS.

With the rise in antibiotic resistant isolates, there is a growing need for an effective vaccine to reduce the prevalence of diseases caused by NTS and iNTS. Current vaccine development strategies are focued on extracellular polysaccharides (EPS) present on bacterial surface. My first objective was to boost the biosynthesis of EPS O-Antigen capsule and purify large quantities for immunization trials in mice. Using random mutagenesis, colanic acid production was increased instead of O-Antigen capsule. Immunization with colanic acid alone or colanic acid conjugated to carrier proteins did not induce a protective response in mice against Salmonella. However, generalized modules for membrane antigens (GMMAs) purified from colanic acid overproducing strains induced a partially protective response against a lethal Salmonella challenge in mice. My work shows that GMMAs can be developed as potential vaccine candidates against NTS and iNTS infections. In addition to vaccine development, strategies to reduce Salmonella transmission will also reduce the global prevalence of NTS and iNTS.

Biofilm formation is important for the long-term survival of Salmonella in the environment. Similar to sporulation, biofilm formation is induced as a survival strategy under conditions of starvation, however it has not been determined if biofilm formation is also a committed (point of no return) process like sporulation. Salmonella biofilm formation is subject to tight and complex regulation through transcription factor CsgD. Using luciferase reporter assays, I examined the regulation of csgD expression in response to different environmental signals, introduced before and after csgD expression has been induced. In the presence of high osmolarity csgD expression is repressed, however when introduced after induction, increased osmolarity has no effect on csgD expression. In contrast, the introduction of glucose and elevated temperatures represses csgD expression before and after induction. My results showed the existence of a regulatory hierarchy among signals that regulate biofilm formation in Salmonella. I conclude that in the presence of changing osmolarity biofilm formation is a committed process similar to sporulation, however under certain conditions such as increased nutrient availability and elevated temperature, biofilm formation is a reversible process.

When S. Typhimurium is grown under biofilm inducing conditions, the bistable synthesis of CsgD results in the formation of two distinct cell types: multicellular aggregates and planktonic cells. Transcriptome comparison showed that multicellular aggregates had higher expression of genes associated with environmental persistence, while planktonic cells had higher expression of genes involved in virulence. About 798 function unknown (FUN) genes are differentially expressed between multicellular aggregates and planktonic cells. I hypothesized that a proportion of the identified FUN genes are involved in Salmonella environmental persistence and/or virulence. I mapped FUN genes operons and analyzed 23 operons with potential roles in Salmonella virulence and or persistence. Using these FUN operons, I highlight the difficulties associated with the identification of the roles of FUN genes and propose a framework that can aid in this process. My research provides new insights into NTS pathogenesis, transmission, and vaccines development. Understanding different aspects of Salmonella biology is essential for the better management of diseases caused by this pathogen.