Genome-Wide Screen for Salmonella Genes - Essay Example

Critical review of Genome-wide screen for Salmonella genes required for long-term systemic infection of the mouse. The pathogenic Salmonella species are a group of intracellular gram-negative bacteria that are known to be the cause of a wide range of diseases across the globe. The subspecies enterica in particular is alleged to cause about 99 percent of these pathological conditions. Figures from the World Health Organisation report of 1997 suggested that it is a species responsible for over 600, 000 deaths per annum (WHO 1997). Consequently there is now much directed effort to understand and control the disease transmission process. In this respect animal models of the disease process being caused by Salmonella have proved to be quite useful. The process by which Salmonella invades and then onwards maintains itself in the host species is not only a complicated but indeed and intriguing process. It is also an example of adaptive evolution and selective preservation of a genotype. Given its prevalence across a range of warm blooded hosts it has been possible to study the infection and virulence factors in several species. For instance while, murine infection with Salmonella enterica serovar typhimurium has been used largely to replicate human typhoid (caused by Salmonella enterica serovar Typhi), bovine infection caused by Salmonella enterica serovar Dublin or S. enterica serovar Typhimurium has been the extensively used to model intestinal disease. The ability of Salmonella to exist within its host for months following the initial invasion requires it to resist any innate immunity or more likely evolve/adapt to resist the host resistance. Since these bacteria are well able to establish long term system infection in their hosts, the genetic factors responsible for such ability need to be well established to counteract any pathogeneicity. While, Salmonella enterica serovar Typhi is a strictly human pathogen, Salmonella enterica serovar Typhimurium is a murine pathogen that is increasingly been used to model human typhoid (caused by Salmonella enterica serovar Typhi). The murine pathogen is hence under much investigation to characterise the genes responsible for much of its virulence. If this is accomplished, the model may have some potential to provide useful critical insight into the pathogenesis of disease cause by salmonellae species overall. In the mouse model, S. Typhimurium invades epithelial cells and can survive and proliferate in macrophage before entering the blood stream and causing a systemic infection. The macrophages are ideal places for the bacteria as they are able to elude the host's immune response in this. Indeed it has been evidenced that the longest duration of stay for slamonella within the host occurs in the macrophages. However for the most part understanding of the murine salmonella species has been limited to the Nramp1 susceptible (Nramp1s) mouse strains i.e. the BALB/c and C57Bl/6. Macrophages from Nramp1s mice permit a much higher rate of serovar Typhimurium replication in vitro as well as in vivo as compared to Nramp1r (Nramp1 is functional) macrophages. While this has upuntil date allowed for observation of the diseased condition, the hosts (mice) invariably are most severly affected by the bacteteria considerabily limiting the scope of study. The paper here however focuses on the novel Salmonella persistence model based on the mouse strain 129X1/SvJ (Nramp1r), which are typically resistant to the replication of the bacteria not succumbing easily to the virulence. Consequently the bacteria can be obtained from systemic sites even up to an year post infection which would help to increase the time spectrum of study if need be. Clearly as intricate an adaptive mechanism as seen in the samlonella species would require the contribution of several genes and virulence factors.The invasion-associated genes are required for intestinal secretory and inflammatory disease, that intracellular survival in both the intestinal epithelium and macrophages is essential for systemic pathogenesis as seen in this resistant murine species(rats and mice), if corrected identified and functionally understood could provide a way forward for microbiological control of the species. Purpose of the experiments Here the authors attempt to identify and bring together in a coherent form the combination of genes for the virulence factors that may be helping the bacteria to invade and maintain a long-term systemic infection in the mouse. The contribution of serovar Typhimurium genes has been for the most part unexplored in the mouse model of continual infection. The limited studies so far have concentrated on the experimental infection model based on the Nramp1 susceptible (Nramp1s) mouse strains BALB/c and C57Bl/6. In Nramp1s mice the host macrophages from Nramp1s permit for an increased rate of serovar Typhimurium replication in vitro compared to Nramp1r . The authors attmpt to use a novel (Nramp1r) mice strain here. Since it a new infection model using 129X1/SvJ (Nramp1r) mice that has only recently been introduced by the authors themselves they take a step forward by attempting to characterise the genes. This would help in further validation of their model in effect strengthening the model further. Experimental systems used in the paper For the purpose of the study the authors used a microarray-based negative selection screening process which was performed to identify Salmonella enterica serovar Typhimurium (serovar Typhimurium) genes that contribute to long-term systemic infection in 129X1/SvJ (Nramp1r) mice. The bacterial strain of Serovar Typhimurium used in this study were derived from wild-type strain SL1344. In order to infect the mice a high-complexity transposon-mutagenized library was used; all inoculation was done intraperitoneally with a view to identify serovar Typhimurium genes that contribute to the establishment and maintenance of a long-term infection in mice. Each transposon insertion contained primarily of a T7 transcriptional promoter While the method of inoculation may have been done in order to make results comparable to previous work, it may also be possible that the fecal-oral route of infection in human formed the basis of selection (Darwin and Miller, 1999). It has been suggested previously that i.p administration emulates oral administration closely. Given the constraints of oral dosing as the authors have identified, i.p seems ideal to address those limitations. Nonetheless such a route of inoculation would need some preliminary investigation. With respect to any apprehension concerning the likely impact that the IP route of infection may have on identification of virulence genes, the data were analyzed from two independent passages utilizing the Statistical Analysis of Microarrays software (SAM) tool. This was done so that the data is replicable and substantially consistent in identifing the genes that differ the most. As has been stated by the authors ' In order to identify genes that were significantly different as a function of time, pair-wise groupings of arrays from a dataset that included the input arrays as well as the spleen pool arrays from all time points were analyzed using a number of SAM two-class analyses for every pair-wise combination. Significant genes were then assembled into a single file for further analysis.' The selective disappearance of mutants was monitored after 7, 14, 21, and 28 days following inoculation. Results Overall, the data from this study revealed that long-term systemic infection caused by Salmonella in the mouse involves a large and varied range of virulence factors. One hundred and eighteen genes were identified which could potentially contribute to serovar Typhimurium infection of the spleens of mice by 28 day following infection. Although the bulk of genes identified were of here to unidentified function, there were many which have a well recognized role known in the Salmonella physiology and pathogenesis. It was identified that approximately 30% of the negatively selected genes corresponded to horizontally acquired regions such as those within Salmonella pathogenicity islands (SPI 1-5), prophages (Gifsy-1 and 2 and remnant), and the pSLT virulence plasmid. In addition, mutations in genes responsible for outer membrane structure and remodeling, such as LPS- and PhoP-regulated and fimbrial genes, were also selected against. It was also seen that there was a progressive selection against serovar Typhimurium mutants in a time dependent manner largely based on the duration of the infection. This evidently suggested that different classes of genes play a pivotal role in the salmonella infection at separate stages of the infection process. A most interesting finding of the study was the recognition that SPI1-encoded type III secretion system effectors/translocases were selected against. This would indicate that the SPI1 would be implicated in the invasion and quite unpredictably the persistence of the systemic infection. This is a novel finding at odds with previously available literature on the subject of Salmonella pathogenesis. Significance of the result /Advances The results form this study demnstarte that the Typhimurium mutant model in the (Nramp1r) mice is a potentially useful model organism which can be used to dissect complex gene regulatory networks. It provides for validation of the Nramp1r mice model which would have much use to provie a temporal profile of the salmonella infection. This may have far reaching consequences for the understanding and indeed control of the human typhoid virus Salmonella enterica serovar Typhi. Previous murine models so far are somewhat limited in their efficacy since the mice are likely to succumb to an overwhelming infection by the bacteria which makes it difficult to obtain cultures for a time period that may be needed. To mimic the human systemic infection a resistant starin of mice would be ideal. In this study we see preliminary evidence of the same. Furthermore the validity of the microarray techniques was established by data demonstarting mutations in 13 SPI2 genes . This is consistent with the well recognized role of this genomic island in intracellular replication and survival of serovar Typhimurium within the host. Consequently it can be assumed that the model is well suited for such an analysis of pathogenic mechanism and indeed physiology. Typhimurium mutant model in the (Nramp1r) mice is revelaed to be an potentially efficacious and tractable model organism in which to study complex gene regulatory networks. As can be seen in this study it is particularly useful as it permits drawing from known regulatory molecular studies to be applied to genes that are directly involved in pathogenesis. Weakness/Strength of study Undoubtedly, Salmonella pathogenesis is a composite occurrence which requires the coordination regulation of several bacterial virulence factors in order to function adequately at different anatomical sites within the animal host. However, several genes previously associated with systemic disease and persistence were not identified in this screen. Most noteworthy in this list are the mig-14and sifA. Since the role of these factor in salmonella pathogenesis is well established, it is likely that the list of candidate virulence genes obtained in this study although most comprehensive is still numerically limited. It may be useful to investigate why certain common genetic factors were not picked up by the array and if it may be in anyway relating to methodological factors. In addition it may be worthwhile to note that a not so stringent criterion of 46|% overlap has been used for the study. However at the same time it has been justified by high complexity of the library. Further as has been identified by the authors, the choice of the muse as a model also adds to the acceptability of overlap percentage selection Role of SPI1 would also need further work as this is a new model using 129X1/SvJ (Nramp1r) mice. It would be interesting to note if the results reported by the authors are replicatble and persistent. This is a relatively new work and not many follow up studies have yet been performed. It may be prudent to make further investigation prior to drawing conclusive hypothesis. However there are some significant contributions that the study makes. The demonstration that SPI1 is important for systemic infection, challenges the current model that SPI1 only contributes to the gastrointestinal phase of Salmonella pathogenesis and that it may not be required to establish systemic disease in mice and that SPI1 may have alternative functions during infection. The exact function(s) of SPI1 during long-term systemic Salmonella infection and persistence still remains unknown but there may be some indications here of the way to go with it with the suggestion that SPI1 may play a role in mediating the increase in the number of infection foci. Observations from this study suggest that Salmonella persistence relies upon both intracellular and extracellular to sustain the long-term infection Conclusion This is a relatively new study and not many follow up work have been forth coming so far. However taking on from the finding here with respect to the multiplicity of genes which are presumably useful for the bacteria to sequentially evolve to respond to the variety of host environments . Evidently it requires over at least over 150 genes an dpossibly more to express a pathogenic phenotype. Some of the important virulence genes are located within A+T-rich pathogenicity islands on the S. Typhimurium chromosome, of which the best studied are SPI-1 and SPI-2. The SPI-1 pathogenicity island encodes the Inv/Spa type III secretion system and effector proteins for invasion of epithelial cells. Other example sof the novel invasive process identified here are the role of MgtC virulence factor. This has been first described in Salmonella enterica serovar Typhimurium (S. Typhimurium) where it is apparnetly needed for intramacrophage survival and long-term systemic infection in mice. A follow up study by Rang et al in 2007 has further investigated the role of MgtC virulence factor in host and non-host environments, in line with this study. While it is a fact that this paer suggest several novel genes that may be implicated in the pathogenesis, concentrated studies should be carried out as has been done by Rang et al to take these finding further. Furthermore the identities and more importantly functionality of many of these genes are still unknown. Further efforts to understand the mechanisms by which Salmonella invasion and maintenance in the host environment occurs need to bear in mind these considerations. In terms of microbiological pathogenesis this would be a step forward indeed. 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