Limits...
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

Differential expression of the regulators agr, sarA and sigB during the course of host cell infection in wild-type LS1 and the corresponding mutants.Endothelial cells were infected with S. aureus strain LS1 or the corresponding mutants as described in Fig 3A and infected cells were analysed for up to 7 days. Host cells infected with wild-type strain LS1 were lysed after 2 (acute phase) and 7 days (chronic phase) and the whole RNA was extracted and was used to determine changes in bacterial gene expression for agrA/hla (α-hemolysin) (A), sarA/aur (aureolysin) (B) and sigB/asp23 (C) during the course of infection by real-time PCR. The values of all experiments represent the mean ± SD of 5 independent experiments measured in triplicate. * P≤0.05 ANOVA comparing levels of gene expression in the wild-type strains and the corresponding mutants at each time point. The fold change is the result of normalized expression to the housekeeping genes gyr, aroE and gmk. Uninfected cells were used as controls (Control = 1).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4414502&req=5

ppat.1004870.g004: Differential expression of the regulators agr, sarA and sigB during the course of host cell infection in wild-type LS1 and the corresponding mutants.Endothelial cells were infected with S. aureus strain LS1 or the corresponding mutants as described in Fig 3A and infected cells were analysed for up to 7 days. Host cells infected with wild-type strain LS1 were lysed after 2 (acute phase) and 7 days (chronic phase) and the whole RNA was extracted and was used to determine changes in bacterial gene expression for agrA/hla (α-hemolysin) (A), sarA/aur (aureolysin) (B) and sigB/asp23 (C) during the course of infection by real-time PCR. The values of all experiments represent the mean ± SD of 5 independent experiments measured in triplicate. * P≤0.05 ANOVA comparing levels of gene expression in the wild-type strains and the corresponding mutants at each time point. The fold change is the result of normalized expression to the housekeeping genes gyr, aroE and gmk. Uninfected cells were used as controls (Control = 1).

Mentions: To further explain the differential ability of the mutants to persist, we evaluated the expression of the global regulatory systems during the long course of the infection. To accomplish this, we extracted RNA from infected host cells (HUVEC; as they can host higher numbers of bacteria, S4A Fig) at day 2 and day 7 p.i. and measured the expression of agr, sarA and sigB and the related factors hla (regulated by agrA), aur (repressed by sarA [38]) and asp23 (regulated by sigB [39]) in the wild-type strain and corresponding mutants by quantitative real-time PCR (Fig 4). As expected high levels of both agrA and sarA were only expressed by the strains LS1, ΔsigB and ΔsigB compl. that resulted in high levels of hla expression in the acute phase of the infection. By contrast, the agr/sarA-double mutant expressed sigB and asp23 at significant higher levels than the wild-type strain during chronic infection (Fig 4) and was able to form higher numbers of SCV phenotypes (Figs 3C and S7C and S7D). Taken together, our results show that a concomitant downregulation of agrA and sarA promotes long-term intracellular persistence of S. aureus. SigB promotes chronic infections and is highly associated with the bacterial ability to form SCVs.


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)

Differential expression of the regulators agr, sarA and sigB during the course of host cell infection in wild-type LS1 and the corresponding mutants.Endothelial cells were infected with S. aureus strain LS1 or the corresponding mutants as described in Fig 3A and infected cells were analysed for up to 7 days. Host cells infected with wild-type strain LS1 were lysed after 2 (acute phase) and 7 days (chronic phase) and the whole RNA was extracted and was used to determine changes in bacterial gene expression for agrA/hla (α-hemolysin) (A), sarA/aur (aureolysin) (B) and sigB/asp23 (C) during the course of infection by real-time PCR. The values of all experiments represent the mean ± SD of 5 independent experiments measured in triplicate. * P≤0.05 ANOVA comparing levels of gene expression in the wild-type strains and the corresponding mutants at each time point. The fold change is the result of normalized expression to the housekeeping genes gyr, aroE and gmk. Uninfected cells were used as controls (Control = 1).
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004870.g004: Differential expression of the regulators agr, sarA and sigB during the course of host cell infection in wild-type LS1 and the corresponding mutants.Endothelial cells were infected with S. aureus strain LS1 or the corresponding mutants as described in Fig 3A and infected cells were analysed for up to 7 days. Host cells infected with wild-type strain LS1 were lysed after 2 (acute phase) and 7 days (chronic phase) and the whole RNA was extracted and was used to determine changes in bacterial gene expression for agrA/hla (α-hemolysin) (A), sarA/aur (aureolysin) (B) and sigB/asp23 (C) during the course of infection by real-time PCR. The values of all experiments represent the mean ± SD of 5 independent experiments measured in triplicate. * P≤0.05 ANOVA comparing levels of gene expression in the wild-type strains and the corresponding mutants at each time point. The fold change is the result of normalized expression to the housekeeping genes gyr, aroE and gmk. Uninfected cells were used as controls (Control = 1).
Mentions: To further explain the differential ability of the mutants to persist, we evaluated the expression of the global regulatory systems during the long course of the infection. To accomplish this, we extracted RNA from infected host cells (HUVEC; as they can host higher numbers of bacteria, S4A Fig) at day 2 and day 7 p.i. and measured the expression of agr, sarA and sigB and the related factors hla (regulated by agrA), aur (repressed by sarA [38]) and asp23 (regulated by sigB [39]) in the wild-type strain and corresponding mutants by quantitative real-time PCR (Fig 4). As expected high levels of both agrA and sarA were only expressed by the strains LS1, ΔsigB and ΔsigB compl. that resulted in high levels of hla expression in the acute phase of the infection. By contrast, the agr/sarA-double mutant expressed sigB and asp23 at significant higher levels than the wild-type strain during chronic infection (Fig 4) and was able to form higher numbers of SCV phenotypes (Figs 3C and S7C and S7D). Taken together, our results show that a concomitant downregulation of agrA and sarA promotes long-term intracellular persistence of S. aureus. SigB promotes chronic infections and is highly associated with the bacterial ability to form SCVs.

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