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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

Phagosomal escape of wild-type strain LS1 and corresponding mutants after infection of the host cell construct A549YFP-CWT.Phagosomal escape was measured 2 hours (A, B, C, D) and 24 hours (E) after infection with the strain LS1 or corresponding mutants in genetically engineered A549 cells. This reporter recruitment technique has been recently described [13]. To quantify the phagosomal escape for each strain 10 fields of view were evaluated respectively and the numbers of escaping bacteria were counted. The values represent the mean ± SD of 5 independent experiments measured.* P≤0.05 ANOVA comparing levels of gene expression in the acute and chronic stage.
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ppat.1004870.g005: Phagosomal escape of wild-type strain LS1 and corresponding mutants after infection of the host cell construct A549YFP-CWT.Phagosomal escape was measured 2 hours (A, B, C, D) and 24 hours (E) after infection with the strain LS1 or corresponding mutants in genetically engineered A549 cells. This reporter recruitment technique has been recently described [13]. To quantify the phagosomal escape for each strain 10 fields of view were evaluated respectively and the numbers of escaping bacteria were counted. The values represent the mean ± SD of 5 independent experiments measured.* P≤0.05 ANOVA comparing levels of gene expression in the acute and chronic stage.

Mentions: Only recently, phagosomal escape to the cytoplasm was reported for different S. aureus strains early after host cell invasion [13,40]. In a further approach we analyzed whether an early phagosomal escape is a prerequisite for persistence. Therefore we used a reporter recruitment technique based on the host cell line A549 genetically engineered to produce a phagosomal escape marker [13]. Within the first 2 h after host cell infection we detected phagosomal escape for the wild-type strain LS1, the sigB-, the sigB compl.- and the sigB/agr-mutants (Fig 5A–5D). These strains showed only weak changes and down-regulation of virulence factor expression by proteomic analysis compared with the wild-type (Fig 1A) and were not able to persist at high bacterial numbers (Fig 3B). As the single agr and sarA mutants readily lost their ability to translocate to the cytoplasm, apparently both agr- and sarA-regulated factors are required for the escape mechanism. The activity of SarA alone is sufficient only in case of a non-functional SigB-system, indicating a modulating role of SigB in virulence factor expression. Our results show that an early phagosomal escape is not required for persistence. Further on, mutants that persisted at high bacterial numbers did not escape to the cytoplasm. As phagosomal escape could not be detected at later stages of infection (up to 24 h; as shown in for the triple mutant; Fig 5E), it must be assumed that these mutants are not degraded within phagolysosomes and thus persist in phagosomes at high bacterial numbers.


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)

Phagosomal escape of wild-type strain LS1 and corresponding mutants after infection of the host cell construct A549YFP-CWT.Phagosomal escape was measured 2 hours (A, B, C, D) and 24 hours (E) after infection with the strain LS1 or corresponding mutants in genetically engineered A549 cells. This reporter recruitment technique has been recently described [13]. To quantify the phagosomal escape for each strain 10 fields of view were evaluated respectively and the numbers of escaping bacteria were counted. The values represent the mean ± SD of 5 independent experiments measured.* P≤0.05 ANOVA comparing levels of gene expression in the acute and chronic stage.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004870.g005: Phagosomal escape of wild-type strain LS1 and corresponding mutants after infection of the host cell construct A549YFP-CWT.Phagosomal escape was measured 2 hours (A, B, C, D) and 24 hours (E) after infection with the strain LS1 or corresponding mutants in genetically engineered A549 cells. This reporter recruitment technique has been recently described [13]. To quantify the phagosomal escape for each strain 10 fields of view were evaluated respectively and the numbers of escaping bacteria were counted. The values represent the mean ± SD of 5 independent experiments measured.* P≤0.05 ANOVA comparing levels of gene expression in the acute and chronic stage.
Mentions: Only recently, phagosomal escape to the cytoplasm was reported for different S. aureus strains early after host cell invasion [13,40]. In a further approach we analyzed whether an early phagosomal escape is a prerequisite for persistence. Therefore we used a reporter recruitment technique based on the host cell line A549 genetically engineered to produce a phagosomal escape marker [13]. Within the first 2 h after host cell infection we detected phagosomal escape for the wild-type strain LS1, the sigB-, the sigB compl.- and the sigB/agr-mutants (Fig 5A–5D). These strains showed only weak changes and down-regulation of virulence factor expression by proteomic analysis compared with the wild-type (Fig 1A) and were not able to persist at high bacterial numbers (Fig 3B). As the single agr and sarA mutants readily lost their ability to translocate to the cytoplasm, apparently both agr- and sarA-regulated factors are required for the escape mechanism. The activity of SarA alone is sufficient only in case of a non-functional SigB-system, indicating a modulating role of SigB in virulence factor expression. Our results show that an early phagosomal escape is not required for persistence. Further on, mutants that persisted at high bacterial numbers did not escape to the cytoplasm. As phagosomal escape could not be detected at later stages of infection (up to 24 h; as shown in for the triple mutant; Fig 5E), it must be assumed that these mutants are not degraded within phagolysosomes and thus persist in phagosomes at high bacterial numbers.

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