Limits...
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.

Show MeSH

Related in: MedlinePlus

Carbonylated protein profiles after exposure to hydrogen peroxide.Protein carbonylation was detected by immunoblotting after coupling to dinitrophenol and protein separation on 12% SDS-PAGE gels. A) Whole cell lysate obtained from the ΔscsABCD mutant reveals a higher concentration of carbonylated proteins after exposure to 0.75 mM hydrogen peroxide (B). Trans-complementation with cloned scsABCD (pNA10) reduces the level of protein carbonylation in the whole cell fraction, as well as in the periplasmic fraction (C). DNPH = Dinitrophenyl hydrazine (+) = presence (−) = absence. p+ indicates the trans-complementing scsABCD genes in pNA10. p- indicates the vector control pSU41.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3672137&req=5

pone-0064948-g003: Carbonylated protein profiles after exposure to hydrogen peroxide.Protein carbonylation was detected by immunoblotting after coupling to dinitrophenol and protein separation on 12% SDS-PAGE gels. A) Whole cell lysate obtained from the ΔscsABCD mutant reveals a higher concentration of carbonylated proteins after exposure to 0.75 mM hydrogen peroxide (B). Trans-complementation with cloned scsABCD (pNA10) reduces the level of protein carbonylation in the whole cell fraction, as well as in the periplasmic fraction (C). DNPH = Dinitrophenyl hydrazine (+) = presence (−) = absence. p+ indicates the trans-complementing scsABCD genes in pNA10. p- indicates the vector control pSU41.

Mentions: Apart from mediating disulphide bond formation, ROS also results in protein carbonylation in S. Typhimurium [17]. Thus, we assayed protein carbonylation in cultures of S. Typhimurium exposed to 0.75 mM H2O2 to detect any discernible differences. To exclude any confounding effects by the ΔscsA single mutant, we continued using the ΔscsABCD mutant only. The rationale for this was that this mutant lacked all the scs genes yet did not reveal sensitization to H2O2. In this experiment we detected a more pronounced accumulation of carbonylated proteins in the ΔscsABCD mutant (Fig. 3A), notably in the periplasmic fraction (Fig. 3C). Expressing the cloned scsABCD genes from pNA10 in the quadruple mutant reduced the H2O2-mediated protein carbonylation (Fig. 3B). That rather few proteins became increasingly carbonylated in the ΔscsABCD mutant could explain why this scs mutant did not display increased loss of viability upon exposure to H2O2. Still, these series of experiments strongly implicated that the Scs proteins are involved in balancing periplasmic oxidative stress in S. Typhimurium.


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

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

Carbonylated protein profiles after exposure to hydrogen peroxide.Protein carbonylation was detected by immunoblotting after coupling to dinitrophenol and protein separation on 12% SDS-PAGE gels. A) Whole cell lysate obtained from the ΔscsABCD mutant reveals a higher concentration of carbonylated proteins after exposure to 0.75 mM hydrogen peroxide (B). Trans-complementation with cloned scsABCD (pNA10) reduces the level of protein carbonylation in the whole cell fraction, as well as in the periplasmic fraction (C). DNPH = Dinitrophenyl hydrazine (+) = presence (−) = absence. p+ indicates the trans-complementing scsABCD genes in pNA10. p- indicates the vector control pSU41.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672137&req=5

pone-0064948-g003: Carbonylated protein profiles after exposure to hydrogen peroxide.Protein carbonylation was detected by immunoblotting after coupling to dinitrophenol and protein separation on 12% SDS-PAGE gels. A) Whole cell lysate obtained from the ΔscsABCD mutant reveals a higher concentration of carbonylated proteins after exposure to 0.75 mM hydrogen peroxide (B). Trans-complementation with cloned scsABCD (pNA10) reduces the level of protein carbonylation in the whole cell fraction, as well as in the periplasmic fraction (C). DNPH = Dinitrophenyl hydrazine (+) = presence (−) = absence. p+ indicates the trans-complementing scsABCD genes in pNA10. p- indicates the vector control pSU41.
Mentions: Apart from mediating disulphide bond formation, ROS also results in protein carbonylation in S. Typhimurium [17]. Thus, we assayed protein carbonylation in cultures of S. Typhimurium exposed to 0.75 mM H2O2 to detect any discernible differences. To exclude any confounding effects by the ΔscsA single mutant, we continued using the ΔscsABCD mutant only. The rationale for this was that this mutant lacked all the scs genes yet did not reveal sensitization to H2O2. In this experiment we detected a more pronounced accumulation of carbonylated proteins in the ΔscsABCD mutant (Fig. 3A), notably in the periplasmic fraction (Fig. 3C). Expressing the cloned scsABCD genes from pNA10 in the quadruple mutant reduced the H2O2-mediated protein carbonylation (Fig. 3B). That rather few proteins became increasingly carbonylated in the ΔscsABCD mutant could explain why this scs mutant did not display increased loss of viability upon exposure to H2O2. Still, these series of experiments strongly implicated that the Scs proteins are involved in balancing periplasmic oxidative stress in S. Typhimurium.

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