Racemases in Salmonella : Insights into the Dexterity of the Pathogen
Chapter -I Introduction Salmonella is a pathogen well-known for its ability to infect a wide variety of hosts and causes disease ranging from mild gastroenteritis to typhoid fever. During infection, it is exposed to a myriad of conditions; from the aquatic environment, the gut lumen to the phagolysosome. The success of Salmonella as a pathogen lies in its ability to sense each of these environments and adapt itself for survival and proliferation accordingly. This is done mainly via the action of specific two-component systems (TCSs) which sense cues specific to each of these niches and trigger the appropriate transcriptional reprogramming. This reprogramming is best studied for the genes directly known to be involved in virulence. In the case of Salmonella, most of these genes are a part of specific clusters, acquired through horizontal gene transfer, known as Salmonella Pathogenicity Islands (SPIs). Of the various SPIs, the two most important are SPI-1 and SPI-2. SPI-1 is classically involved in orchestrating bacterial invasion of non-phagocytic cells in the gut, allowing the pathogen to invade the host. Furthermore, its role is well characterized in the classic inflammation associated with gastroenteritis. On the other hand, SPI-2 is specialized for survival within the harsh intracellular environment of host cells such as macrophages and epithelial cells. Other important virulence determinants include motility, chemotaxis as well as adhesins. The transcription of these virulence genes is under tight regulation and responsive to environmental conditions. Many small molecules such as short chain fatty acids, pp(p)Gpp, bile and acyl homoserine lactones among others are known to be potent regulators of virulence in Salmonella. Furthermore, the metabolic products of the normal flora in the gut also affect its virulence. Thus the metabolic status, of both the host as well as the pathogen, plays an important role in determining the outcome of the infection. Many metabolic enzymes and their products are now known to directly or indirectly affect virulence gene expression. In this study, we explore one such class of metabolic enzymes viz amino acid racemases. They catalyze the chiral conversion of L-amino acids to D-amino acids and vice versa. We have studied the biochemical properties of two such non-canonical racemases as well as their role in bacterial survival and pathogenesis. Chapter-II Identification and characterization of putative aspartate racemases in Salmonella Amino acid racemases, such as alanine and glutamate racemases, are ubiquitously found in all bacteria and they play an essential role in cell wall biosynthesis. Recently it has been found, that bacteria possess other amino acid racemases which produce non-canonical D-amino acids. These D-amino acids, upon secretion, further orchestrate various phenotypes such as cell wall remodeling and biofilm dispersal. In this study, we have explored the ability of Salmonella to produce such non-canonical D-amino acids. The genome of S. Typhimurium possesses genes encoding two putative aspartate racemases; ygeA and aspR. These genes were maximally expressed in mid-log phase of bacterial growth and their corresponding proteins ar localized in the outer membrane of the bacterium. The biochemical characterization of the proteins YgeA and AspR revealed that only the latter is catalytically active under in vitro conditions. AspR could catalyze the conversion of L-Aspartate to D-Aspartate and vice versa, however was unable to use any other amino acid as its substrate. With atleast one of the racemases showing catalytic activity, the profiling of the secreted D-amino acids in Salmonella conditioned medium was undertaken using LC-MS. It was observed that the bacterium actively secreted specific D-amino acids such as D-Ala and D-Met into the culture medium in a growth-phase dependent manner. Furthermore, analysis of the secreted D-amino acid profile of the strains lacking either one or both the racemases revealed that atleast a subset of the secreted D-amino acids were dependent on the activity of YgeA and AspR. Thus, D-amino acids secreted by S. Typhimurium might represent a novel class of signaling molecules. Chapter – III Role of aspartate racemases in growth and survival of S. Typhimurium In order to understand the role of ygeA and aspR in vivo, we created knockouts of these genes (both single as well as double knockout) in S. Typhimurium using λ Red recombinase strategy. These knockouts were then assessed for their growth and morphology. The aspartate racemase knockouts behave similar to the wild type during growth in LB as well as M9 minimal medium. While their gross morphology remained the same as the wild type, the size distribution of the racemase knockouts was slightly different in the stationary phase. Unlike the wild type bacteria, the mutants did not exhibit the characteristic reduction in cell size upon entry into stationary phase. In addition, the survival of the mutants in the presence of cell wall damaging agents such as bile and Triton-X 100 was compromised as compared to the wild type. This can be ascribed to changes in the cell wall of the bacterium, wherein the mutants accumulated peptidoglycan in the stationary phase of growth. This suggests that aspartate racemases might have an effect on cell wall biosynthesis in Salmonella in the stationary phase. Another important strategy employed by bacteria to survive in stress conditions is biofilm formation. It was seen that the mutants were compromised in their ability to form a biofilm at the liquid-air interface in vitro. This defect is due to a transcriptional downregulation of the genes required for biofilm formation. These results demonstrate that, contrary to the established inhibitory effects of D-amino acids on biofilms of various bacteria, the aspartate racemases appear to act as positive regulators of biofilm formation in Salmonella. Chapter – IV Involvement of aspartate racemases in the regulation of Salmonella pathogenesis Salmonella’s success as a pathogen can be broadly assessed by its ability to invade and replicate within two major cell types: epithelial cells and macrophage-like cells. We have studied the fate of the aspartate racemase knockout strains in both these cell types. While the mutants replicate as well as the wild type in macrophage cell lines, their ability to invade epithelial cell lines is highly compromised. This defect can be ascribed to the downregulation of the Salmonella Pathogenicity Island-1 (SPI-1) in the racemase knockouts at the transcriptional level. One of the major pathways that regulate SPI-1 activation is the flagellar pathway. It was observed that in addition to SPI-1, the motility of the racemase mutants was also highly compromised. The mutants did not possess any flagella and showed a high transcriptional downregulation of all the three classes of flagellar genes. Transcriptome analysis revealed a global reprogramming in the aspartate racemase mutants, resulting in the differential regulation of motility, adhesion, amino acid transport, cell wall biosynthesis and other pathways. Of the genes upregulated in the knockouts, FimZ is known for its negative effect on motility and might be responsible for the observed downregulation of the flagellar regulon. This suggests that ygeA and aspR might be repressors of fimbrial gene expression. In totality, the racemases affected the pathogenesis of Salmonella, where the double knockout was severely compromised in the colitis model of infection. Overall the study is the first to identify secretion of non-canonical D-amino acids by Salmonella and suggests that YgeA and AspR might be the source of the same. This is supported in part by in vitro studies with the purified proteins. Studies in vivo further highlight the possible substrates that might be utilized by these enzymes. Physiologically, the aspartate racemases appear to regulate cell wall remodeling and biofilm formation. In contrast to the established literature, aspartate racemases (and their possible D-amino acid products) seem to be essential for formation of biofilms and regulate this phenotype at the transcriptional level. Furthermore, our studies put forth aspartate racemases as novel positive regulators of Flagella and SPI-1, affecting the success of Salmonella in the colitis model of infection in mice. Transcriptome analysis hints at the pleiotropic effects of aspartate racemases in Salmonella, bringing forth hitherto unexplored roles for this class of enzymes in the biology of this pathogen.
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