The discovery that mechanical forces regulate microbial virulence, stress responses and gene expression was made using log phase cultures of Salmonella Typhimurium (S. Typhimurium) grown under low fluid shear (LFS) conditions relevant to those encountered in the intestine. However, there has been limited characterization of LFS on other growth phases. To advance the growth-phase dependent understanding of the effect of LFS on S. Typhimurium pathogenicity, this dissertation characterized the effect of LFS on the transcriptomic and phenotypic responses in both stationary and lag phase cultures. In response to LFS, stationary phase cultures exhibited alterations in gene expression associated with metabolism, transport, secretion and stress responses (acid, bile salts, oxidative, and thermal stressors), motility, and colonization of intestinal epithelium (adherence, invasion and intracellular survival). Many of these characteristics are known to be regulated by the stationary phase general stress response regulator, RNA polymerase sigma factor S (RpoS), when S. Typhimurium is grown under conventional conditions. Surprisingly, the stationary phase phenotypic LFS stress response to acid and bile salts, colonization of human intestinal epithelial cells, and swimming motility was not dependent on RpoS. Lag phase cultures exhibited intriguing differences in their LFS regulated transcriptomic and phenotypic profiles as compared to stationary phase cultures, including LFS-dependent regulation of gene expression, adherence to intestinal epithelial cells, and high thermal stress. Furthermore, the addition of cell-free conditioned supernatants derived from either stationary phase LFS or Control cultures modulated the gene expression of lag phase cultures in a manner that differed from either growth phase, however, these supernatants did not modulate the phenotypic responses of lag phase cultures. Collectively, these results demonstrated that S. Typhimurium can sense and respond to LFS as early as lag phase, albeit in a limited fashion, and that the lag phase transcriptomic and phenotypic responses differ from those in stationary phase, which hold important implications for the lifecycle of this pathogen during the infection process.

Invasive salmonellosis caused by Salmonella enterica serovar Typhimurium ST313 is a major health crisis in sub-Saharan Africa, with multidrug resistance and atypical clinical presentation challenging current treatment regimens and resulting in high mortality. Moreover, the increased risk of spreading ST313 pathovars worldwide is of major concern, given global public transportation networks and increased populations of immunocompromised individuals (as a result of HIV infection, drug use, cancer therapy, aging, etc). While it is unclear as to how Salmonella ST313 strains cause invasive disease in humans, it is intriguing that the genomic profile of some of these pathovars indicates key differences between classic Typhimurium (broad host range), but similarities to human-specific typhoidal Salmonella Typhi and Paratyphi. In an effort to advance fundamental understanding of the pathogenesis mechanisms of ST313 in humans, I report characterization of the molecular genetic, phenotypic and virulence profiles of D23580 (a representative ST313 strain). Preliminary studies to characterize D23580 virulence, baseline stress responses, and biochemical profiles, and in vitro infection profiles in human surrogate 3-D tissue culture models were done using conventional bacterial culture conditions; while subsequent studies integrated a range of incrementally increasing fluid shear levels relevant to those naturally encountered by D23580 in the infected host to understand the impact of biomechanical forces in altering these characteristics. In response to culture of D23580 under these conditions, distinct differences in transcriptional biosignatures, pathogenesis-related stress responses, in vitro infection profiles and in vivo virulence in mice were observed as compared to those of classic Salmonella pathovars tested.

Collectively, this work represents the first characterization of in vivo virulence and in vitro pathogenesis properties of D23580, the latter using advanced human surrogate models that mimic key aspects of the parental tissue. Results from these studies highlight the importance of studying infectious diseases using an integrated approach that combines actions of biological and physical networks that mimic the host-pathogen microenvironment and regulate pathogen responses.