Evolutionary Adaptations and Host Immune Modulation by Salmonella Typhimurium

Abstract

Salmonella infection remains a major health concern across the world because of the emergence of multiple drug-resistant and hyper-infectious Salmonella strains, leading to disease severity. The outcome of infection is the result of a complex interaction of the bug with the host immune system as well as in the non-host environments with which Salmonella encounters. Adaptation is an important phenomenon for the survival of any organism in various non-host environments as well as in in-vitro and in-vivo conditions leading to their better survival. We studied the evolutionary adaptation in Salmonella by repeatedly exposing it to three different experimental conditions such as in-vitro in LB and F media and in-vivo in Caenorhabditis elegans. We report that this exposure has led to multiple phenotypic and genotypic changes in Salmonella giving the advantage to survive and disseminate better. Compared to unpassaged strain WT-STM, the passaged strains P12-STM, Ce12-STM and F12-STM, showed increased body size, increased number of flagella and increased motility, which are majorly associated with Salmonella virulence. Resistance towards the oxidative and nitrosative stresses has also been increased in these strains. They also showed increased invasion in intestinal epithelial cells INT-407 and proliferation in RAW-264.7 the mouse and U-937 the human macrophage cell lines. Gene expression study revealed that various SPI-I and SPI-II, flagellar, stress response and two-component systems genes have been upregulated. Further, the host colonization of the passaged strains was seen to be increased in metazoan model system C. elegans, causing reduced antimicrobial peptides (abf-2 and spp-1) expression and ultimately leading to their early death. Moving ahead to the vertebrate mouse model, we found that the adapted strains were also capable of colonizing better and also modulating the host immune system by altering pro and anti-inflammatory cytokine response specifically showing upregulation of IL-10 and IFN-β and downregulation of IL-1β and IL-6, reducing antimicrobial peptides cryptdine expression, as well as T cell response by altering CD4+ T cell population. Thus the altered immune response thereby led to increased organ burden in Peyer’s patches, mesenteric lymph nodes, spleen, and liver after 4 and 7 days post-infection. We looked forward to find the mechanism leading to hyper-infection and altered cytokine response making an anti-inflammatory environment in the human and mouse macrophage cells. They were found to evade the host lysosomal degradation as seen with reduced colocalization with the early endosomal marker (Rab5) hence altering phagosome maturation and also reduced the lysosomal number in the macrophage cells. This has led to reduced phagosome lysosomal fusion as indicated via reduced colocalization with LAMP1, the lysosomal marker and hence reduced lysosomal degradation, giving a safe niche for bacterial survival. We also found that the adapted strains caused NF-κB signaling pathway activation more than the unpassaged WT-STM which have helped to provide an anti-inflammatory environment by increased production of IL-10 and IFN-β and reduced the inflammatory response by downregulating IL-1β and IL-6 against them. Another interesting observation we found was higher SopB effector protein gene, sopB expression in these passaged stains which had led to reduced inflammasome activation by downregulating NLRC4 through Akt phosphorylation pathway, hence reduced IL-1β secretion. This might a strategy employed by the adapted strains for reduced exposure to the host inflammatory molecules which would have been detrimental to them. Furthermore, whole-genome sequencing analysis revealed a number of point and frameshift mutations in the passaged strains which might contribute directly or indirectly to the virulence and immune regulation. Collectively our study demonstrated that Salmonella shows adaptation to the exposed conditions which lead to hypervirulence in Salmonella and is capable of modulating host innate and adaptive immune response via various ways for their survival and persistence. These bacterial phenotype has been able to cause disease severity and the genes responsible for the same can be targeted to combat Salmonella infection and the host immune system can also be targeted to combat the infection.