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Sigma Factor SigB Is Crucial to Mediate Staphylococcus aureus Adaptation during Chronic Infections.

Tuchscherr L, Bischoff M, Lattar SM, Noto Llana M, Pförtner H, Niemann S, Geraci J, Van de Vyver H, Fraunholz MJ, Cheung AL, Herrmann M, Völker U, Sordelli DO, Peters G, Löffler B - PLoS Pathog. (2015)

Bottom Line: Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly.SigB plays a crucial function to promote bacterial intracellular persistence.In fact, ΔsigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Jena University Hospital, Jena, Germany.

ABSTRACT
Staphylococcus aureus is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting S. aureus infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global S. aureus regulators agr, sarA and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different in vitro and in vivo infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the agr and sarA loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence agr and sarA. Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, ΔsigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections.

No MeSH data available.


Related in: MedlinePlus

The combined action of agr and sarA is required for inflammation and cytotoxicity in the acute stage of infection.Cytotoxicity experiments and analysis of inflammatory host response were performed in polymorphonuclear cells (PMNs), human osteoblasts and endothelial cells using wild-type strain LS1 and their derivate mutants. (A, B) PMNs were freshly isolated form human blood (A) and bone marrow of Balb/C mice (B) and 1×106/0.5 ml cells were incubated with 5% v/v of bacterial supernatants for 1 h. Then cells were washed, stained with annexin V and propidium iodide and cell death was measured by flow cytometry. (C, D) Cultured osteoblasts were infected with S. aureus LS1 or their derivate mutants (MOI 50). After bacterial invasion (3 h) extracellular staphylococci were removed by washing and lysostaphin treatment and infected cells were incubated with culture medium for 48 h. To analyze host cell response the changes in the expression of the chemokine CXCL-11 and CCL-5 were measured by real-time PCR. Results demonstrate the relative increase in gene expression, compared to unstimulated cells (control: expression 1). The fold change is the normalized expression of each gene to housekeeping genes (β-actin and GAPDH). The values of all experiments represent the means ± SD of at least three independent experiments performed in duplicates. * P≤0.05 ANOVA comparing the effects induced by the wild-type strains and the corresponding mutants. Similar results were obtained with strain SH1000 and selected mutants (S2 Fig and S3 Fig).
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ppat.1004870.g002: The combined action of agr and sarA is required for inflammation and cytotoxicity in the acute stage of infection.Cytotoxicity experiments and analysis of inflammatory host response were performed in polymorphonuclear cells (PMNs), human osteoblasts and endothelial cells using wild-type strain LS1 and their derivate mutants. (A, B) PMNs were freshly isolated form human blood (A) and bone marrow of Balb/C mice (B) and 1×106/0.5 ml cells were incubated with 5% v/v of bacterial supernatants for 1 h. Then cells were washed, stained with annexin V and propidium iodide and cell death was measured by flow cytometry. (C, D) Cultured osteoblasts were infected with S. aureus LS1 or their derivate mutants (MOI 50). After bacterial invasion (3 h) extracellular staphylococci were removed by washing and lysostaphin treatment and infected cells were incubated with culture medium for 48 h. To analyze host cell response the changes in the expression of the chemokine CXCL-11 and CCL-5 were measured by real-time PCR. Results demonstrate the relative increase in gene expression, compared to unstimulated cells (control: expression 1). The fold change is the normalized expression of each gene to housekeeping genes (β-actin and GAPDH). The values of all experiments represent the means ± SD of at least three independent experiments performed in duplicates. * P≤0.05 ANOVA comparing the effects induced by the wild-type strains and the corresponding mutants. Similar results were obtained with strain SH1000 and selected mutants (S2 Fig and S3 Fig).

Mentions: As secreted virulence factors are particularly directed against professional phagocytes, we tested the effect of bacterial supernatants on neutrophils (PMNs) isolated from humans and mice. In line with the proteomic data, only the strains with high levels of virulence factors (LS1, ΔsigB, ΔsigB compl.) caused cell death, whereas all other mutants with reduced levels of toxins induced significantly less cytotoxicity (Figs 2A and 2B and S2A–S2C). This effect was concentration dependent and revealed highest levels of cell death in response to supernatants of the sigB-mutant (S2C and S2D Fig). Next we analyzed levels of chemokine expression in cultured tissue cells, such as osteoblasts and endothelial cells, 48 h after infection by real time PCR (Fig 2C and 2D and S4C and S4D Fig) and 24 h after infection by ELISA-measurements (S3C and S3D Fig). In contrast to the wild-type strain, all double-and triple-mutants (including mutations in SigB) exhibited reduced cell activating activity, whereas the sigB-single-mutant often caused even more cell activation than the wild-type strain. Furthermore, these effects were independent of the bacterial background and of the infected host cell types, as they were reproduced with selected mutants generated in strain SH1000 (S3D Fig). All effects were equally present in bone and endothelial cells (S4C and S4D Fig) regardless of the observation that endothelial cells took up higher amounts of bacteria than osteoblasts (S4A Fig). Nevertheless, endothelial cells expressed in general lower levels of chemokines than osteoblasts (S4B Fig).


Sigma Factor SigB Is Crucial to Mediate Staphylococcus aureus Adaptation during Chronic Infections.

Tuchscherr L, Bischoff M, Lattar SM, Noto Llana M, Pförtner H, Niemann S, Geraci J, Van de Vyver H, Fraunholz MJ, Cheung AL, Herrmann M, Völker U, Sordelli DO, Peters G, Löffler B - PLoS Pathog. (2015)

The combined action of agr and sarA is required for inflammation and cytotoxicity in the acute stage of infection.Cytotoxicity experiments and analysis of inflammatory host response were performed in polymorphonuclear cells (PMNs), human osteoblasts and endothelial cells using wild-type strain LS1 and their derivate mutants. (A, B) PMNs were freshly isolated form human blood (A) and bone marrow of Balb/C mice (B) and 1×106/0.5 ml cells were incubated with 5% v/v of bacterial supernatants for 1 h. Then cells were washed, stained with annexin V and propidium iodide and cell death was measured by flow cytometry. (C, D) Cultured osteoblasts were infected with S. aureus LS1 or their derivate mutants (MOI 50). After bacterial invasion (3 h) extracellular staphylococci were removed by washing and lysostaphin treatment and infected cells were incubated with culture medium for 48 h. To analyze host cell response the changes in the expression of the chemokine CXCL-11 and CCL-5 were measured by real-time PCR. Results demonstrate the relative increase in gene expression, compared to unstimulated cells (control: expression 1). The fold change is the normalized expression of each gene to housekeeping genes (β-actin and GAPDH). The values of all experiments represent the means ± SD of at least three independent experiments performed in duplicates. * P≤0.05 ANOVA comparing the effects induced by the wild-type strains and the corresponding mutants. Similar results were obtained with strain SH1000 and selected mutants (S2 Fig and S3 Fig).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4414502&req=5

ppat.1004870.g002: The combined action of agr and sarA is required for inflammation and cytotoxicity in the acute stage of infection.Cytotoxicity experiments and analysis of inflammatory host response were performed in polymorphonuclear cells (PMNs), human osteoblasts and endothelial cells using wild-type strain LS1 and their derivate mutants. (A, B) PMNs were freshly isolated form human blood (A) and bone marrow of Balb/C mice (B) and 1×106/0.5 ml cells were incubated with 5% v/v of bacterial supernatants for 1 h. Then cells were washed, stained with annexin V and propidium iodide and cell death was measured by flow cytometry. (C, D) Cultured osteoblasts were infected with S. aureus LS1 or their derivate mutants (MOI 50). After bacterial invasion (3 h) extracellular staphylococci were removed by washing and lysostaphin treatment and infected cells were incubated with culture medium for 48 h. To analyze host cell response the changes in the expression of the chemokine CXCL-11 and CCL-5 were measured by real-time PCR. Results demonstrate the relative increase in gene expression, compared to unstimulated cells (control: expression 1). The fold change is the normalized expression of each gene to housekeeping genes (β-actin and GAPDH). The values of all experiments represent the means ± SD of at least three independent experiments performed in duplicates. * P≤0.05 ANOVA comparing the effects induced by the wild-type strains and the corresponding mutants. Similar results were obtained with strain SH1000 and selected mutants (S2 Fig and S3 Fig).
Mentions: As secreted virulence factors are particularly directed against professional phagocytes, we tested the effect of bacterial supernatants on neutrophils (PMNs) isolated from humans and mice. In line with the proteomic data, only the strains with high levels of virulence factors (LS1, ΔsigB, ΔsigB compl.) caused cell death, whereas all other mutants with reduced levels of toxins induced significantly less cytotoxicity (Figs 2A and 2B and S2A–S2C). This effect was concentration dependent and revealed highest levels of cell death in response to supernatants of the sigB-mutant (S2C and S2D Fig). Next we analyzed levels of chemokine expression in cultured tissue cells, such as osteoblasts and endothelial cells, 48 h after infection by real time PCR (Fig 2C and 2D and S4C and S4D Fig) and 24 h after infection by ELISA-measurements (S3C and S3D Fig). In contrast to the wild-type strain, all double-and triple-mutants (including mutations in SigB) exhibited reduced cell activating activity, whereas the sigB-single-mutant often caused even more cell activation than the wild-type strain. Furthermore, these effects were independent of the bacterial background and of the infected host cell types, as they were reproduced with selected mutants generated in strain SH1000 (S3D Fig). All effects were equally present in bone and endothelial cells (S4C and S4D Fig) regardless of the observation that endothelial cells took up higher amounts of bacteria than osteoblasts (S4A Fig). Nevertheless, endothelial cells expressed in general lower levels of chemokines than osteoblasts (S4B Fig).

Bottom Line: Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly.SigB plays a crucial function to promote bacterial intracellular persistence.In fact, ΔsigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Jena University Hospital, Jena, Germany.

ABSTRACT
Staphylococcus aureus is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting S. aureus infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global S. aureus regulators agr, sarA and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different in vitro and in vivo infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the agr and sarA loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence agr and sarA. Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, ΔsigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections.

No MeSH data available.


Related in: MedlinePlus