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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

sigB promotes persistence and SCV-formation.(A) Cultured osteoblasts were infected with S. aureus strain LS1 or the corresponding or complemented mutants and infected cells were analysed for up to 9 days. The numbers of viable intracellular persisting bacteria were determined every 2 days by lysing host cells, plating the lysates on agar plates and counting the colonies that have grown on the following day. (B) The results after 9 days are demonstrated separately. The results shown here are from osteoblast infection experiments, but similar results were obtained after infection of endothelial cells. (C) The percentage of small and very small (SCV) phenotypes (<5 and <10-fold smaller than those of the wild-type phenotypes, respectively) recovered (between 200 and 500 colonies examined in each sample) were determined after 7 days p.i. The values of all experiments represent the means ± SD of at least three independent experiments performed in duplicates. * P≤0.05 ANOVA test comparing the effects induced by the wild-type strains and the corresponding mutants. (D) Photographs of recovered colonies were performed after 7 days of infection of endothelial cells with strains LS1, LS1∆sigB or LS1∆sigB compl. Similar results were obtained when osteoblasts were infected with SH1000 and selected mutants (S7 Fig).
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ppat.1004870.g003: sigB promotes persistence and SCV-formation.(A) Cultured osteoblasts were infected with S. aureus strain LS1 or the corresponding or complemented mutants and infected cells were analysed for up to 9 days. The numbers of viable intracellular persisting bacteria were determined every 2 days by lysing host cells, plating the lysates on agar plates and counting the colonies that have grown on the following day. (B) The results after 9 days are demonstrated separately. The results shown here are from osteoblast infection experiments, but similar results were obtained after infection of endothelial cells. (C) The percentage of small and very small (SCV) phenotypes (<5 and <10-fold smaller than those of the wild-type phenotypes, respectively) recovered (between 200 and 500 colonies examined in each sample) were determined after 7 days p.i. The values of all experiments represent the means ± SD of at least three independent experiments performed in duplicates. * P≤0.05 ANOVA test comparing the effects induced by the wild-type strains and the corresponding mutants. (D) Photographs of recovered colonies were performed after 7 days of infection of endothelial cells with strains LS1, LS1∆sigB or LS1∆sigB compl. Similar results were obtained when osteoblasts were infected with SH1000 and selected mutants (S7 Fig).

Mentions: To test the function of the global regulatory systems in the course from acute to chronic infection, we infected osteoblast and endothelial cell cultures with wild-type and mutant strains and analyzed their ability to persist intracellularly for 9 days. All strains were invasive in osteoblasts to a similar extent (S5 Fig) and induced cell death ranging around 50% immediately after infection (S6A and S6B Fig). Yet, in the following 2–3 days the integrity of the infected cell monolayers were fully recovered and the rate of cell death was reduced to control levels (S6C Fig). In general the numbers of intracellular bacteria were decreased during the whole infection course (Figs 3A and S7A), but considerable differences between the strains appeared after several days (9 days, Figs 3B and S7B). The agr/sarA- and the sigB/sarA-double mutants as well as the triple mutant were able to persist within the intracellular location at significantly higher numbers (up to 100-fold) than the corresponding wild-type strain (Fig 3A and 3B). By contrast, the sigB-mutants were completely cleared from the host cells within 7–9 days, whereas this effect could be fully reversed by the complementation of sigB (Figs 3A and S7A). To test whether this effect is specific for sigB-mutants, we further tested mutants for other virulence or regulatory factors such as for sae and hla which did not reveal any differences in the numbers of intracellular persisting bacteria compared with the wild-type strain (S8 Fig). From our previous work we know that bacterial persistence is associated with dynamic SCV-formation. As recently described [9] we discovered an increased rate of SCV-formation after several days of intracellular bacterial persistence, whereby the recovered SCV were not stable, but the majority reverted back to the wild-type phenotype upon 2 to 5 subcultivating steps on agar plates. Interestingly, in the present study we found that all sigB-mutants completely failed to develop SCV phenotypes after 7 days of intracellular persistence (Fig 3C and 3D and S7C and S7D Fig). By analyzing the recovered colonies from sigB-mutants, we observed much less phenotypic diversity than in the wild-types and other mutants, as the plates revealed only uniform large white colonies. Again these effects could be reversed by complementation of the sigB-mutations with an intact sigB-operon, thus proving a clear and specific connection between the bacterial ability to form dynamic SCVs and the SigB-system.


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)

sigB promotes persistence and SCV-formation.(A) Cultured osteoblasts were infected with S. aureus strain LS1 or the corresponding or complemented mutants and infected cells were analysed for up to 9 days. The numbers of viable intracellular persisting bacteria were determined every 2 days by lysing host cells, plating the lysates on agar plates and counting the colonies that have grown on the following day. (B) The results after 9 days are demonstrated separately. The results shown here are from osteoblast infection experiments, but similar results were obtained after infection of endothelial cells. (C) The percentage of small and very small (SCV) phenotypes (<5 and <10-fold smaller than those of the wild-type phenotypes, respectively) recovered (between 200 and 500 colonies examined in each sample) were determined after 7 days p.i. The values of all experiments represent the means ± SD of at least three independent experiments performed in duplicates. * P≤0.05 ANOVA test comparing the effects induced by the wild-type strains and the corresponding mutants. (D) Photographs of recovered colonies were performed after 7 days of infection of endothelial cells with strains LS1, LS1∆sigB or LS1∆sigB compl. Similar results were obtained when osteoblasts were infected with SH1000 and selected mutants (S7 Fig).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414502&req=5

ppat.1004870.g003: sigB promotes persistence and SCV-formation.(A) Cultured osteoblasts were infected with S. aureus strain LS1 or the corresponding or complemented mutants and infected cells were analysed for up to 9 days. The numbers of viable intracellular persisting bacteria were determined every 2 days by lysing host cells, plating the lysates on agar plates and counting the colonies that have grown on the following day. (B) The results after 9 days are demonstrated separately. The results shown here are from osteoblast infection experiments, but similar results were obtained after infection of endothelial cells. (C) The percentage of small and very small (SCV) phenotypes (<5 and <10-fold smaller than those of the wild-type phenotypes, respectively) recovered (between 200 and 500 colonies examined in each sample) were determined after 7 days p.i. The values of all experiments represent the means ± SD of at least three independent experiments performed in duplicates. * P≤0.05 ANOVA test comparing the effects induced by the wild-type strains and the corresponding mutants. (D) Photographs of recovered colonies were performed after 7 days of infection of endothelial cells with strains LS1, LS1∆sigB or LS1∆sigB compl. Similar results were obtained when osteoblasts were infected with SH1000 and selected mutants (S7 Fig).
Mentions: To test the function of the global regulatory systems in the course from acute to chronic infection, we infected osteoblast and endothelial cell cultures with wild-type and mutant strains and analyzed their ability to persist intracellularly for 9 days. All strains were invasive in osteoblasts to a similar extent (S5 Fig) and induced cell death ranging around 50% immediately after infection (S6A and S6B Fig). Yet, in the following 2–3 days the integrity of the infected cell monolayers were fully recovered and the rate of cell death was reduced to control levels (S6C Fig). In general the numbers of intracellular bacteria were decreased during the whole infection course (Figs 3A and S7A), but considerable differences between the strains appeared after several days (9 days, Figs 3B and S7B). The agr/sarA- and the sigB/sarA-double mutants as well as the triple mutant were able to persist within the intracellular location at significantly higher numbers (up to 100-fold) than the corresponding wild-type strain (Fig 3A and 3B). By contrast, the sigB-mutants were completely cleared from the host cells within 7–9 days, whereas this effect could be fully reversed by the complementation of sigB (Figs 3A and S7A). To test whether this effect is specific for sigB-mutants, we further tested mutants for other virulence or regulatory factors such as for sae and hla which did not reveal any differences in the numbers of intracellular persisting bacteria compared with the wild-type strain (S8 Fig). From our previous work we know that bacterial persistence is associated with dynamic SCV-formation. As recently described [9] we discovered an increased rate of SCV-formation after several days of intracellular bacterial persistence, whereby the recovered SCV were not stable, but the majority reverted back to the wild-type phenotype upon 2 to 5 subcultivating steps on agar plates. Interestingly, in the present study we found that all sigB-mutants completely failed to develop SCV phenotypes after 7 days of intracellular persistence (Fig 3C and 3D and S7C and S7D Fig). By analyzing the recovered colonies from sigB-mutants, we observed much less phenotypic diversity than in the wild-types and other mutants, as the plates revealed only uniform large white colonies. Again these effects could be reversed by complementation of the sigB-mutations with an intact sigB-operon, thus proving a clear and specific connection between the bacterial ability to form dynamic SCVs and the SigB-system.

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