The Detailed Insight on a Novel rspA Gene of Salmonella Typhimurium and its Role in Modulating Bacterial Pathogenesis

Abstract

Salmonella infection is a major public health concern worldwide, which causes huge mortality and morbidity in developing and developed countries. Even though much has been known about Salmonella pathogenesis and whole genome sequencing is done, functional characterizations of certain genes are yet to be explored. The rspA (STM14_1818) is one such gene with putative dehydratase function, and its role in pathogenesis is unknown. The existing information showed that in the intracellular environment that is inside the macrophages, rspA gene expression of Salmonella enterica serovar Typhimurium (S. Typhimurium) (WT-STM) is significantly upregulated, which led us to investigate its role in Salmonella pathogenesis. In our study when we validated, we also found that the rspA gene expression was upregulated in the MGM-MES media (which mimics the intracellular environment with low Mg2+ and low pH) in a time- dependent manner. Thus, we generated the rspA knockout strain (ΔrspA-STM) and complement strain (ΔrspAc-STM) in S. Typhimurium 14028S. The mutant strain shows enhanced growth in intracellular mimicking media, which suggests that rspA might be regulating the intracellular growth of S. Typhimurium. The rspA gene has no role in motility and flagella number as these phenotypes are unaltered in mutant. The mutant strain is sensitive to higher ROS concentrations and is resistant to various antibiotics and complement system. Gene expression profile revealed that when bacteria grown in LB medium, Salmonella Pathogenecity Island (SPI)-1 genes responsible for Salmonella invasion, SPI-II genes, biofilm forming related genes and other stress related genes are downregulated in the mutant strain. The mutant strain differentially formed the biofilm at different temperatures by altering the expression of genes involved in the synthesis of cellulose and curli. At 20°C, the mutant strain formed less biofilm than the wild type strain, whereas at 25°C and 30°C, the mutant strain produced more biofilm than wild type strain. In the mutant strain, at 20°C master regulator csgD, cellulose synthesis genes (bcsA, bcZ, bcsB, and bcsC), curli synthesis genes (csgA and csgB), and adrA are downregulated but upregulated at 25°C except for bcsC. The cellulose and curli synthesized genes are downregulated in the mutant strain in in-vitro condition and when they reside inside the macrophage. Besides, the mutant strain is less invasive but hyperproliferative intracellularly. The macrophages generated less ROS and RNS when infected with rspA mutant than the wild type strain, resulting in better survival and intracellular hyperproliferation of the mutant. The mutant strain is less adhesive to Jasmin Pradhan (518LS1008) PhD Thesis, NIT Rourkela epithelial cells and macrophages than the wild type, resulting in less entry into the host cells. In in-vivo model of C. elegans, the mutant strain showed an increased bacterial burden than the wild type and is more infectious. The mutant caused faster death of the worms than the wild type and modulates the worm’s innate immunity, favouring its survival. In in-vivo murine model, the organ burden of the mutant strain is less than the WT-STM on early days of post-infection, whereas in the later days of post-infection, the organ burden is comparable between these two strains, which suggests that the mutant strain is able to persist better inside the murine model. Viability of the mutant infected Balb/C mice was significantly reduced than PBS control, but less severe than WT indicating that the rspA deletion mutant was still pathogenic in mice model. Overall, in this study, we concluded that the rspA gene differentially regulates the biofilm formation in a temperature dependent manner by modulating the genes involved in the synthesis of cellulose and curli and negatively regulates the Salmonella virulence for longer persistence inside the host system.