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Oxidoreductases that act as conditional virulence suppressors in Salmonella enterica serovar Typhimurium.

Anwar N, Sem XH, Rhen M - PLoS ONE (2013)

Bottom Line: However, we noted a significant copper chloride sensitivity of SPI1 T3SS mediated invasiveness that strongly depended on the presence of the scs genes.Our findings thus demonstrate that the scs gene cluster conditionally affects virulence and underscore the complex interactions between oxidoreductases of the thioredoxin superfamily in maintaining host adaptation of S.Typhimurium.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
In Salmonella enterica serovar Typhimurium, oxidoreductases of the thioredoxin superfamily contribute to bacterial invasiveness, intracellular replication and to the virulence in BALB/c mice as well as in the soil nematode Caenorhabditis elegans. The scsABCD gene cluster, present in many but not all enteric bacteria, codes for four putative oxidoreductases of the thioredoxin superfamily. Here we have analyzed the potential role of the scs genes in oxidative stress tolerance and virulence in S. Typhimurium. An scsABCD deletion mutant showed moderate sensitization to the redox-active transition metal ion copper and increased protein carbonylation upon exposure to hydrogen peroxide. Still, the scsABCD mutant was not significantly affected for invasiveness or intracellular replication in respectively cultured epithelial or macrophage-like cells. However, we noted a significant copper chloride sensitivity of SPI1 T3SS mediated invasiveness that strongly depended on the presence of the scs genes. The scsABCD deletion mutant was not attenuated in animal infection models. In contrast, the mutant showed a moderate increase in its competitive index upon intraperitoneal challenge and enhanced invasiveness in small intestinal ileal loops of BALB/c mice. Moreover, deletion of the scsABCD genes restored the invasiveness of a trxA mutant in epithelial cells and its virulence in C. elegans. Our findings thus demonstrate that the scs gene cluster conditionally affects virulence and underscore the complex interactions between oxidoreductases of the thioredoxin superfamily in maintaining host adaptation of S. Typhimurium.

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β-galactosidase and β-lactamase activities and expression measurements.A) and B) β-galactosidase activities for hilA and prgH promoter fusions from overnight LB culture are slightly reduced for scsABCD mutant in the presence of CuCl2. C) and D) β-galactosidase activities for hilA and prgH promoter fusions from 4 hrs DMEM-based invasive culture are unaffected irrespective of scsABCD mutation. β-lactamase activities from supernatants and cell lysates of the invasive cultures of wild type and in ΔscsABCD mutant bacteria in the absence (E) and presence (F) of CuCl2. G) β-lactamase as assayed from the same cultures by immunoblotting for β-lactamase. ns = non-significant; ** = p≤0.01; *** = p≤0.001. Error bars indicate standard error of the mean.
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pone-0064948-g005: β-galactosidase and β-lactamase activities and expression measurements.A) and B) β-galactosidase activities for hilA and prgH promoter fusions from overnight LB culture are slightly reduced for scsABCD mutant in the presence of CuCl2. C) and D) β-galactosidase activities for hilA and prgH promoter fusions from 4 hrs DMEM-based invasive culture are unaffected irrespective of scsABCD mutation. β-lactamase activities from supernatants and cell lysates of the invasive cultures of wild type and in ΔscsABCD mutant bacteria in the absence (E) and presence (F) of CuCl2. G) β-lactamase as assayed from the same cultures by immunoblotting for β-lactamase. ns = non-significant; ** = p≤0.01; *** = p≤0.001. Error bars indicate standard error of the mean.

Mentions: That the scsABCD mutant retained invasiveness upon CuCl2 stress in a SPI1-mediated manner led us to test whether the scs gene cluster affected SPI1 gene expression. For this we measured SPI1 gene expression using the two forth mentioned SPI1 Tn5::lacZY constructs. When grown over night in LB we noted clear reading of the hilA::lacZY and prgH::lacZY transcriptional fusions by assaying beta-galactosidase activities (Fig. 5A and B). Addition of CuCl2 to the culture prior to the inoculation resulted in an overall decreased reporter activity with a slight relative decrease in both fusions for the scsABCD mutant (Fig. 5A and B). However, this difference disappeared when bacteria were grown for invasiveness in cull culture medium supplemented with CuCl2 (Fig 5C and D).


Oxidoreductases that act as conditional virulence suppressors in Salmonella enterica serovar Typhimurium.

Anwar N, Sem XH, Rhen M - PLoS ONE (2013)

β-galactosidase and β-lactamase activities and expression measurements.A) and B) β-galactosidase activities for hilA and prgH promoter fusions from overnight LB culture are slightly reduced for scsABCD mutant in the presence of CuCl2. C) and D) β-galactosidase activities for hilA and prgH promoter fusions from 4 hrs DMEM-based invasive culture are unaffected irrespective of scsABCD mutation. β-lactamase activities from supernatants and cell lysates of the invasive cultures of wild type and in ΔscsABCD mutant bacteria in the absence (E) and presence (F) of CuCl2. G) β-lactamase as assayed from the same cultures by immunoblotting for β-lactamase. ns = non-significant; ** = p≤0.01; *** = p≤0.001. Error bars indicate standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672137&req=5

pone-0064948-g005: β-galactosidase and β-lactamase activities and expression measurements.A) and B) β-galactosidase activities for hilA and prgH promoter fusions from overnight LB culture are slightly reduced for scsABCD mutant in the presence of CuCl2. C) and D) β-galactosidase activities for hilA and prgH promoter fusions from 4 hrs DMEM-based invasive culture are unaffected irrespective of scsABCD mutation. β-lactamase activities from supernatants and cell lysates of the invasive cultures of wild type and in ΔscsABCD mutant bacteria in the absence (E) and presence (F) of CuCl2. G) β-lactamase as assayed from the same cultures by immunoblotting for β-lactamase. ns = non-significant; ** = p≤0.01; *** = p≤0.001. Error bars indicate standard error of the mean.
Mentions: That the scsABCD mutant retained invasiveness upon CuCl2 stress in a SPI1-mediated manner led us to test whether the scs gene cluster affected SPI1 gene expression. For this we measured SPI1 gene expression using the two forth mentioned SPI1 Tn5::lacZY constructs. When grown over night in LB we noted clear reading of the hilA::lacZY and prgH::lacZY transcriptional fusions by assaying beta-galactosidase activities (Fig. 5A and B). Addition of CuCl2 to the culture prior to the inoculation resulted in an overall decreased reporter activity with a slight relative decrease in both fusions for the scsABCD mutant (Fig. 5A and B). However, this difference disappeared when bacteria were grown for invasiveness in cull culture medium supplemented with CuCl2 (Fig 5C and D).

Bottom Line: However, we noted a significant copper chloride sensitivity of SPI1 T3SS mediated invasiveness that strongly depended on the presence of the scs genes.Our findings thus demonstrate that the scs gene cluster conditionally affects virulence and underscore the complex interactions between oxidoreductases of the thioredoxin superfamily in maintaining host adaptation of S.Typhimurium.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
In Salmonella enterica serovar Typhimurium, oxidoreductases of the thioredoxin superfamily contribute to bacterial invasiveness, intracellular replication and to the virulence in BALB/c mice as well as in the soil nematode Caenorhabditis elegans. The scsABCD gene cluster, present in many but not all enteric bacteria, codes for four putative oxidoreductases of the thioredoxin superfamily. Here we have analyzed the potential role of the scs genes in oxidative stress tolerance and virulence in S. Typhimurium. An scsABCD deletion mutant showed moderate sensitization to the redox-active transition metal ion copper and increased protein carbonylation upon exposure to hydrogen peroxide. Still, the scsABCD mutant was not significantly affected for invasiveness or intracellular replication in respectively cultured epithelial or macrophage-like cells. However, we noted a significant copper chloride sensitivity of SPI1 T3SS mediated invasiveness that strongly depended on the presence of the scs genes. The scsABCD deletion mutant was not attenuated in animal infection models. In contrast, the mutant showed a moderate increase in its competitive index upon intraperitoneal challenge and enhanced invasiveness in small intestinal ileal loops of BALB/c mice. Moreover, deletion of the scsABCD genes restored the invasiveness of a trxA mutant in epithelial cells and its virulence in C. elegans. Our findings thus demonstrate that the scs gene cluster conditionally affects virulence and underscore the complex interactions between oxidoreductases of the thioredoxin superfamily in maintaining host adaptation of S. Typhimurium.

Show MeSH
Related in: MedlinePlus