Thesis (MTech (Consumer Science: Food and Nutrition))--Cape Peninsula University of Technology, 2004 / Concems have been voiced regarding the food practices and dietary intake of students since residences changed from a catered to a self-catering food provision system. In this study, the purchasing, storage, food preparation practices and dietary intake of female students living in self-catering residences at the Cape Technikon in Cape Town, South Africa, were investigated. A sample of 60 students, representative of the female students living in self-catering residences, participated. A structured interview, together with direct observation, using an observational checklist, was used to determine whether food practices complied with food safety guidelines. As self-reported and actual behaviour may differ, the reported food safety behaviour was compared with the observed behaviour. The mean food and beverage intakes were determined using two 24-hour dietary recalls covering a week and weekend day. Intakes were compared with the recommendations of the Daily Food Guide and the South African Food-Based Dietary Guidelines. Nutrient intakes were analysed using dietary analysis software. Nutrient intakes were compared with the Dietary Reference Intakes for their gender and age grouping. Intakes of S 67% of the Recommended Dietary Allowance/Adequate Intakes or below were deemed as inadequate. The weight status of the students was also determined and compared to the health maintenance Body Mass Index range of 20 to 25 kg/m2 . Results indicated that some, but not all food safety guidelines were followed. Both positive and negative practices were reported and observed.

Chapter: 1 Introduction Genus Salmonella is a Gram-negative rod shaped facultative anaerobic bacteria that can survive inside the host macrophages and cause persistent infection. Salmonella Typhimurium, Salmonella Typhi and Salmonella Enteritidis are the serovars belonging to Salmonella enterica. S. Typhi causes typhoid fever in humans. S. Typhimurium is one of the important causes for food poisoning in humans. It causes typhoid like fever in mice and serves as a good model system to study Salmonella pathogenesis. Upon entry Salmonella resides in an intracellular phagosomal compartment called Salmonella containing vacuole (SCV). It eventually uncouples from the endocytic pathway to avoid lysosomal fusion and ultimately reaches the golgi apparatus achieving a perinuclear position. Professional phagocytes like macrophages generate nitric oxide (NO) that acts as a potent agent to limit the growth of many intracellular pathogens including Salmonella. Upon activation of the inducible nitric oxide synthase (iNOS), NO is produced continuously at a high rate in the presence of adequate Larginine supply. Nitric oxide synthases catalyze the oxidation of one of the guanidino nitrogens of larginine to nitric oxide (NO). Of the multiple NOS isoforms that can catalyze NO synthesis, iNOS is mostly associated with antimicrobial activity. Host expression of iNOS is primarily regulated at the transcriptional level and can be stimulated following interaction with microbial products or in response to cytokines such as interleukin 1 (IL1), tumor necrosis factor α (TNFα) and interferon γ (IFNγ). To date, mutations that inactivate iNOS in humans have not been described. The importance of iNOS in human infection can therefore only be understood from indirect evidence and experimental models. Despite initial difficulty in demonstrating iNOS expression and NO production by human mononuclear phagocytes, an increasing body of evidence has identified a number of chronic inflammatory conditions, infectious diseases and in vitro treatments that stimulate iNOS mRNA expression and protein synthesis associated with NO bioactivity in human macrophages. Numerous studies have documented the production of RNIs in rodent models of Salmonella infection. Plasma nitrite and nitrate levels, a measure of RNI generation, have been shown to rise significantly after systemic infection of mice with S. Typhimurium. Chapter:2 Role of nirC in Salmonella infection-Nitrosative stress response. Activation of macrophages by interferon gamma (IFNγ) and the subsequent production of nitric oxide (NO) are critical for the host defense against Salmonella enterica serovar Typhimurium infection. We report here the inhibition of IFNγ induced nitric oxide production in RAW264.7 macrophages infected with the wild type Salmonella. This phenomenon was shown to be dependent on the nirC gene, which encodes a potential nitrite transporter. We observed a higher NO output from the IFNγ treated macrophages infected with the nirC mutant Salmonella. The nirC mutant also showed significantly decreased intracellular proliferation in a NO dependent manner in the activated RAW264.7 macrophages and in liver, spleen and secondary lymph nodes of mice, which was restored by complementing the gene in trans. Under acidified nitrite stress, a 2fold more pronounced NO mediated repression of SPI2 was observed in the nirC knockout strain when compared to the wild type. This enhanced SPI2 repression in the nirC knockout led to a higher level of STAT1 phosphorylation and iNOS expression than the wild type strain. In the iNOS knockout mice, the organ load of the nirC knockout strain was similar to the wild type strain indicating the fact that the mutant is exclusively sensitive towards the host nitrosative stress. Taken together, these results reveal that intracellular Salmonella evade their killing in the activated macrophages by down regulating IFNγ induced NO production and highlights the critical role of nirC as a virulence gene. Chapter:3 Salmonella mediated utilization of the host Arginine pool for intracellular growth -a novel strategy to survive. Cationic amino acid transporters (CAT) are crucial regulators of both the nitric oxide synthase and arginase activity in the host cells as they regulate the Larginine availability. In this study, we show that Salmonella induces arginase activity in both the bone marrow derived macrophages and in dendritic cells in a LPS dependent manner. Further evidence is provided suggesting that the Salmonella mediated arginase induction is accompanied by an enhanced arginine uptake in the infected cells by up regulation of the expression of both mouse cationic amino acid transporters mCAT1 and mCAT2B. The bacterial growth was reduced in the presence of inhibitors of both arginase and arginine transport. We also observed that the argT knockout strain in Salmonella coding for an arginine permease was defective in the Larginine uptake and was also attenuated for growth in the mice model of infection. By utilizing both host and bacterial arginine transporters, Salmonella can access the host Larginine pool in the cytosol. The host CAT transporters co localize with the Salmonella containing vacuole in both the bone marrow derived macrophages and in dendritic cells. Thus the host arginine is channelized to the intracellular Salmonella for its growth and this novel strategy plays a pivotal role to counteract the stringent nutrient condition for the intracellular bacteria. On the other hand this channelization should ultimately decrease the substrate for NO production and serve as a survival strategy of the pathogenic Salmonella under host nitrosative stress.

Nontyphoidal Salmonella (NTS) causes invasive, and frequently fatal, disease in African children. The burden of disease secondary to NTS reflects inadequacy of Salmonella-control strategies in Africa, with expanding antibiotic resistance, and no licensed anti-NTS vaccine. The delivery of improved interventions to prevent, diagnose, and treat invasive NTS (iNTS) infection, will be facilitated by an improved understanding of the biological determinants of susceptibility to iNTS, including host genetic factors. To identify host genetic determinants of iNTS disease, we performed a GWAS and replication analysis of NTS bacteraemia in African children. This analysis identified and validated a common genetic variant in STAT4 associated with increased iNTS risk. To characterise the function of the NTS-associated STAT4 variant, we utilised a genotype-selectable bioresource of healthy European adults and samples from African children with iNTS disease. In these experiments, the risk genotype at STAT4 is associated with reduced STAT4 RNA expression in stimulated leukocytes, and reduced IFNγ production in both ex vivo stimulated natural killer cells and in the serum of African children with acute NTS bacteraemia. To validate genetic variation suggestively associated with NTS bacteraemia in the GWAS, NTS-associated loci with evidence of regulatory function were prioritised for functional characterisation. Using in vitro models of intracellular Salmonella infection and RNA interference, I characterise the role of a candidate NTS-susceptibility determinant, EVI5L, in Salmonella infections. Finally, applying a pathway enrichment analysis to the NTS bacteraemia GWAS demonstrated that NTS-associated genetic variation in African children is enriched for methionine salvage enzymes. I further investigate the potential for host-pathogen interaction in this pathway, generating and characterising Salmonella mutants deficient in methionine metabolism. Taken together, these data represent the first unbiased assessment of genetic susceptibility to iNTS disease in unselected populations. These results have important implications for the design of Salmonella-control strategies for use in Africa.

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.