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Abstract
Salmonella enterica serovar Typhi (S. Typhi) is a human-adapted pathogen that causes acute and chronic disease. Chronically-infected individuals are the only known biological reservoir of S. Typhi but chronic infections, primarily in the gallbladder, are remarkably difficult to treat. Chronic disease is modeled in mice with Salmonella enterica serovar Typhimurium (S. Typhimurium). The ability of S. Typhi to establish chronic infection is dependent on the modulation of host immunity during early dissemination and on the development of biofilms in the gallbladder.
S. Typhi biofilms consist of extracellular polymeric substances (EPSs) that enhance recalcitrance to host functions and antimicrobials. However, Salmonella spp. produce and regulate multiple EPSs in an environmentally-dependent manner. Furthermore, the organization of each EPS in vivo and role in resisting host immunity is poorly understood. This work investigated the role of each EPS in host and pathogen outcomes relating to innate immunity, hypothesizing one or more EPS has a crucial role for the chronic pathogenicity of S. Typhi biofilms and that identification of these EPSs would advance mechanistic understanding of biofilm tolerance to innate immunity.
Analysis with wild-type and EPS-mutant bacteria in planktonic and biofilm growth states was conducted to identify specific EPSs mediating biofilm phenotypes. Salmonella susceptibility and tolerance to humoral immunity was evaluated, determining that biofilms enhance tolerance to serum and antimicrobial peptides although the major EPSs tested are not responsible. In assessing biofilm modulation of innate phagocytes, biofilm-associated Vi antigen was found to reduce production of macrophage nitric oxide and neutrophil reactive oxygen species (ROS). Using S. Typhimurium, the ROS response to biofilms was shown to be dependent on the regulation and likely location of the other major Salmonella EPSs in biofilm. Furthermore, when ROS intermediates were exogenously supplied, biofilms were found to tolerate hydrogen peroxide (H2O2) but not hypochlorite. Mechanistic evaluation of H2O2 tolerance identified partial dependence on the biofilm growth state. The EPSs Vi antigen and colanic acid contribute to tolerance by a community benefit while O antigen capsule contributes to tolerance but not by community function. Together, the EPS barrier slows diffusion of H2O2 into the biofilm to a rate that is manageable for detoxification by intra-biofilm catalases.
Investigating EPS-associated functions highlighted the complexity of Salmonella biofilm development. To better understand this process, confocal laser scanning microscopy was used to determine the abundance and organization of EPSs in various growth models. This analysis demonstrated S. Typhi and S. Typhimurium produce similar EPS amounts in laboratory conditions but also highlighted key differences between serovar EPSs that should be considered in designing future studies.
Overall, this work identified and contextualized EPS functions important for mitigating innate immunity during chronic infection. It is clear that Salmonella biofilm production is a dynamic process. By demonstrating specific EPS changes in response to environment, the ability of S. Typhi to limit exposure to oxidative stress is explored and a novel community function for EPSs and biofilm catalases in tolerance to H2O2 is reported. Together, these results address how EPSs contribute to Salmonella pathogenesis and perpetuate chronic infections.