Studies on the roles of rffG and rfbB encoding dTDP-glucose 4,6- dehydratase in Salmonella Typhimurium

Abstract

Salmonellosis is a major health concern which causes significant morbidity and mortality worldwide. Infection with Salmonella Typhimurium leads to self-limiting diarrhea in healthy individuals and invasive disease in immune-compromised hosts. Emerging drug resistance in Salmonella is making treatment options increasingly challenging. A key barrier for the development of new classes of antibiotics for Gram negative pathogens is presence of the outer membrane. The presence of the outer membrane prevents the entry of many antibiotics rendering them ineffective in their clinical utility. Consequently, research on finding new potential drug targets in Salmonella will have a great impact on food industry and public health worldwide. The bacterial surface is the first point of contact with the host and hence it’s a target of a variety of antimicrobial defense mechanisms. Lipopolysaccharide (LPS) and enterobacterial common antigen (ECA) are two important virulence determinants which are present on the outer membrane of S. Typhimurium. LPS consists of three components; Lipid A, core oligosaccharide and O-antigen which comprises structural repeating units of four sugar residues. ECA is made up of repeating trisaccharide units composed of 4-acetamide-4,6-dideoxy-D-galactose, N-acetyl-D-mannosaminuronic acid and N-acetyl-D-glucosamine. The gene cluster rfb encodes genes involved in O-antigen biosynthesis whereas rff codes for genes responsible for ECA biosynthesis. The genes rfbB and rffG encode the protein dTDP-glucose 4,6 -dehydratase, an intermediate in the synthesis of both O-antigen and ECA. Since, the enzyme dTDP-glucose- 4,6-dehydratase is involved in the intermittent steps of the synthesis of both O-antigen and ECA, the functional loss of the enzyme renders the cell incapable of synthesizing both O-antigen and the ECA. The function of dTDP-glucose 4,6-dehydratase in organisms such as Candida and Mycobacterium have been well characterized. However, to the best of our knowledge, a detailed study into the major physiological changes associated with the functional loss of both rffG and rfbB in S. Typhimurium is lacking. With this objective in mind, we generated single deletion strains of rffG, rfbB and a double deletion strain of both the genes. First, we studied the growth characteristic of the strains. We did not observe any difference with respect to growth in nutrient rich as well as deficient media. However, we observed that the rffGrfbB strain showed a distinct colony morphology on agar. The colonies formed by the rffGrfbB strain were significantly smaller in size when compared with the wildtype (WT) and the single deletion strains. Also, the colonies formed by the rffGrfbB strain appeared distinct in texture when observed under the microscope. Further, we investigated the individual cell morphology with the help of atomic force microscopy (AFM). Our investigations with AFM revealed that the rffGrfbB strain showed round cell morphology whereas the WT and the single deletion strains displayed rod shaped morphology. Next, we subjected our strains to stresses commonly encountered by S. Typhimurium in the environment or during host colonization. We did not observe any difference in growth in osmolarity, pH and high temperature induced stresses. However, we found that the rffGrfbB strain was highly susceptible to bile as well as antibiotics such as polymyxin B, meropenem and amoxicillin-clavulanic acid but not to ciprofloxacin or cefotaxime. To address the question whether the increased susceptibility to different substances seen in the rffGrfbB strain was due to the loss in the outer membrane integrity, we performed 1-N-phenylnaphthylamine (NPN) dye accumulation assay. We found the outer membrane permeability was markedly increased in the rffGrfbB strain when compared with the WT and the single deletion strains. However, analysis of the outer membrane protein profile of the WT and the rffGrfbB strains did not reveal any noticeable differences. Next, we analyzed the LPS profile of our strains. Our finding revealed that the WT and the single deletion strains displayed a complete LPS profile whereas the rffGrfbB strain displayed a truncated version of the LPS molecule with the absence of the O-antigen repeating units. To obtain mechanistic insights into the observable phenotypic characteristics of the rffGrfbB strain, we performed RNA-seq analysis of the WT and the rffGrfbB strain. Among the key pathways which were downregulated in the rffGrfbB strain, as compared to the WT strain, were genes related to flagellar assembly, chemotaxis, quorum sensing and Salmonella pathogenicity island 1 (SPI-1) pathways. We focused our study on the motility and SPI-1 pathway by performing qRT-PCRs to validate the differentially expressed genes (DEGs) as well as functional responses. Our qRT-PCRs results indicated that the flagellar assembly pathway genes flhD, fliC and fljB were downregulated in the single deletion strains as well as the double deletion strain when compared to the WT, although the extent of downregulation of the genes was more pronounced in the rffGrfbB strain. We also performed swimming and swarming motility assays to assess motility defect in our strains. No difference was observed between the WT and the single deletion strains during swimming motility. On the other hand, in swarming motility, the single deletion strains were less motile when compared to the WT, whereas the rffGrfbB strain was completely non-motile. Next, we performed qRT-PCRs of representative genes of the SPI-1 pathway. We studied the expression of hilD, hilA and sipC and found them to be downregulated in the single deletion strains as well as the double deletion strain compared to the WT strain. However, the extent of down regulation of the genes were more pronounced in the rffGrfbB strain than the single deletion mutants. We also performed in-vitro infection in RAW264.7 and HeLa cells. In HeLa, the single deletion mutants displayed reduced adhesion and invasion ability as compared to the WT, whereas the rffGrfbB strain was highly compromised in both adhesion and invasion. In RAW264.7 cells, we did not detect any difference between the WT and the single deletion strains in terms of adhesion and invasion. However, the rffGrfbB strain displayed reduced adhesion as well as invasion in the RAW264.7 cells as compared to the WT and the single deletion strains. Furthermore, we performed in vivo infection in C57BL/6 with S. Typhimurium WT and rffGrfbB strain through both oral as well as intraperitoneal routes. In both the models, we found that the rffGrfbB strain was less proficient in colonizing different organs than the WT strain. We also observed that the rffGrfbB strain did not generate pro-inflammatory cytokine responses in mice and was highly attenuated during both oral and intraperitoneal infection model systems. Overall, this detailed study highlights the importance of rffG and rfbB in maintaining cell wall integrity, colony and cell morphology, motility and virulence in S. Typhimurium