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Genes Required for the Fitness of Salmonella enterica Serovar Typhimurium during Infection of Immunodeficient gp91-/- phox Mice.

ABSTRACT: Salmonella enterica causes systemic diseases (typhoid and paratyphoid fever), nontyphoidal septicemia (NTS), and gastroenteritis in humans and other animals worldwide. An important but underrecognized emerging infectious disease problem in sub-Saharan Africa is NTS in children and immunocompromised adults. A current goal is to identify Salmonella mutants that are not pathogenic in the absence of key components of the immune system such as might be found in immunocompromised hosts. Such attenuated strains have the potential to be used as live vaccines. We have used transposon-directed insertion site sequencing (TraDIS) to screen mutants of Salmonella enterica serovar Typhimurium for their ability to infect and grow in the tissues of wild-type and immunodeficient mice. This was to identify bacterial genes that might be deleted for the development of live attenuated vaccines that would be safer to use in situations and/or geographical areas where immunodeficiencies are prevalent. The relative fitness of each of 9,356 transposon mutants, representing mutations in 3,139 different genes, was determined in gp91(-/-) phox mice. Mutations in certain genes led to reduced fitness in both wild-type and mutant mice. To validate these results, these genes were mutated by allelic replacement, and resultant mutants were retested for fitness in the mice. A defined deletion mutant of cysE was attenuated in C57BL/6 wild-type mice and immunodeficient gp91(-/-) phox mice and was effective as a live vaccine in wild-type mice.

SUBMITTER: Grant AJ

PROVIDER: S-EPMC4807482 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Genes Required for the Fitness of Salmonella enterica Serovar Typhimurium during Infection of Immunodeficient gp91-/- phox Mice.

Grant Andrew J AJ Oshota Olusegun O Chaudhuri Roy R RR Mayho Matthew M Peters Sarah E SE Clare Simon S Maskell Duncan J DJ Mastroeni Pietro P

Infection and immunity 20160324 4

Salmonella enterica causes systemic diseases (typhoid and paratyphoid fever), nontyphoidal septicemia (NTS), and gastroenteritis in humans and other animals worldwide. An important but underrecognized emerging infectious disease problem in sub-Saharan Africa is NTS in children and immunocompromised adults. A current goal is to identify Salmonella mutants that are not pathogenic in the absence of key components of the immune system such as might be found in immunocompromised hosts. Such attenuate ...[more]

PMID: 26787719

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Project description:The intracellular pathogen Salmonella enterica serovar Typhimurium has emerged as a major cause of foodborne illness, representing a severe clinical and economic concern worldwide. The capacity of this pathogen to efficiently infect and survive inside the host depends on its ability to synchronize a complex network of virulence mechanisms. Therefore, the identification of new virulence determinants has become of paramount importance in the search of new targets for drug development. BolA-like proteins are widely conserved in all kingdoms of life. In Escherichia coli, this transcription factor has a critical regulatory role in several mechanisms that are tightly related to bacterial virulence. Therefore, in the present work we used the well-established infection model Galleria mellonella to evaluate the role of BolA protein in S Typhimurium virulence. We have shown that BolA is an important player in S Typhimurium pathogenesis. Specifically, the absence of BolA leads to a defective virulence capacity that is most likely related to the remarkable effect of this protein on S Typhimurium evasion of the cellular response. Furthermore, it was demonstrated that BolA has a critical role in bacterial survival under harsh conditions since BolA conferred protection against acidic and oxidative stress. Hence, we provide evidence that BolA is a determining factor in the ability of Salmonella to survive and overcome host defense mechanisms, and this is an important step in progress to an understanding of the pathways underlying bacterial virulence.IMPORTANCE BolA has been described as an important protein for survival in the late stages of bacterial growth and under harsh environmental conditions. High levels of BolA in stationary phase and under stresses have been connected with a plethora of phenotypes, strongly suggesting its important role as a master regulator. Here, we show that BolA is a determining factor in the ability of Salmonella to survive and overcome host defense mechanisms, and this is an important step in progress to an understanding of the pathways underlying bacterial virulence. This work constitutes a relevant step toward an understanding of the role of BolA protein and may have an important impact on future studies in other organisms. Therefore, this study is of utmost importance for understanding the genetic and molecular bases involved in the regulation of Salmonella virulence and may contribute to future industrial and public health care applications.

Project description:Enteric pathogens cycle between nutrient-rich host and nutrient-poor external environment. These pathogens compete for nutrients while cycling between host and external environment, and often experience starvation. In this context, we have studied the role of a global regulator (NtrC) of Salmonella Typhimurium. The ntrC knockout mutation caused extended lag phase (8 h) and slow growth in the minimal medium. In lag phase, the wild-type cells showed ~60-fold more expression of ntrC gene. Gene expression studies and biochemical assays showed that the extended lag phase and slow growth is due to slow metabolism, instead of nitrogen transport. Further, we observed that ntrC knockout mutation led extended lag phase and slow growth, made ΔntrC mutant unable to compete with wild-type S. Typhimurium in both static and fluctuating nutrient condition. In addition to this, ΔntrC knockout mutant was unable to survive long-term nitrogen starvation (150 days). The nutrient recycling assays and gene expression studies revealed that ntrC gene is essential for rapid recycling of nutrients from the dead cells. Moreover, in the absence of ntrC gene, magnesium limits the nutrient recycling efficiency of S. Typhimurium. Therefore, the ntrC gene, which is often studied with respect to nitrogen scavenging in a low nitrogen growing condition, is required even in the adequate supply of nitrogen to maintain optimal growth and fast exit from the lag phase. Hence, we conclude that, the ntrC expression is essential for competitive fitness of S. Typhimurium under the low and fluctuating nutrient condition. IMPORTANCE S. Typhimurium, both in host and external environment, faces enormous competition from other microorganisms. The competition may take place either in static or in fluctuating nutrient conditions. Thus, how S. Typhimurium survives under such overlapping stress conditions remained unclear. Therefore, using S. Typhimurium as model organism we report that a global regulator NtrC, found in enteric bacteria like Escherichia coli and Salmonella, activates the set of genes and operons involved in rapid adaptation and efficient nutrient recycling/scavenging. These properties enable cells to compete with other microbes under the characteristic feast-or-famine lifestyle of S. Typhimurium. Therefore, this work helps us to understand the starvation physiology of the enteric bacterial pathogen S. Typhimurium.

Project description:The intracellular pathogen Salmonella enterica serovar Typhimurium causes intestinal inflammation characterized by edema, neutrophil influx and increased pro-inflammatory cytokine expression. A major bacterial factor inducing pro-inflammatory host responses is lipopolysaccharide (LPS). S. Typhimurium ΔmsbB possesses a modified lipid A, has reduced virulence in mice, and is being considered as a potential anti-cancer vaccine strain. The lack of a late myristoyl transferase, encoded by MsbB leads to attenuated TLR4 stimulation. However, whether other host receptor pathways are also altered remains unclear. Nod1 and Nod2 are cytosolic pattern recognition receptors recognizing bacterial peptidoglycan. They play important roles in the host's immune response to enteric pathogens and in immune homeostasis. Here, we investigated how deletion of msbB affects Salmonella's interaction with Nod1 and Nod2. S. Typhimurium Δ msbB-induced inflammation was significantly exacerbated in Nod2-/- mice compared to C57Bl/6 mice. In addition, S. Typhimurium ΔmsbB maintained robust intestinal colonization in Nod2-/- mice from day 2 to day 7 p.i., whereas colonization levels significantly decreased in C57Bl/6 mice during this time. Similarly, infection of Nod1-/- and Nod1/Nod2 double-knockout mice revealed that both Nod1 and Nod2 play a protective role in S. Typhimurium ΔmsbB-induced colitis. To elucidate why S. Typhimurium ΔmsbB, but not wild-type S. Typhimurium, induced an exacerbated inflammatory response in Nod2-/- mice, we used HEK293 cells which were transiently transfected with pathogen recognition receptors. Stimulation of TLR2-transfected cells with S. Typhimurium ΔmsbB resulted in increased IL-8 production compared to wild-type S. Typhimurium. Our results indicate that S. Typhimurium ΔmsbB triggers exacerbated colitis in the absence of Nod1 and/or Nod2, which is likely due to increased TLR2 stimulation. How bacteria with "genetically detoxified" LPS stimulate various innate responses has important implications for the development of safe and effective bacterial vaccines and adjuvants.

Dataset Information

Genes Required for the Fitness of Salmonella enterica Serovar Typhimurium during Infection of Immunodeficient gp91-/- phox Mice.

Publications

Genes Required for the Fitness of Salmonella enterica Serovar Typhimurium during Infection of Immunodeficient gp91-/- phox Mice.

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