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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

Proteomic analysis of strain LS1 and corresponding mutants.(A) Pie chart display of the proportion of secreted/ cell surface proteins displaying alterations in level in the different mutants compared to the wild type strain. Only proteins with SEED annotation are included and they are categorized according to SEED. Proteins displaying reduced and increased levels in the mutants compared to the wild type are displayed in shades of green and red, respectively. (B) Heat map of proteins which belong to the groups of adhesive and surface proteins or to secreted toxins. Log2-values of the ratios of intensity values of the wild type divided by those of the mutants from -4 to +4 are shown. Higher expression in the wild type versus the mutants is displayed in shades of green and higher expression in the mutants compared to the wild type in shades of red. Proteins displaying the same intensities in wild type and mutant are colored black.
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ppat.1004870.g001: Proteomic analysis of strain LS1 and corresponding mutants.(A) Pie chart display of the proportion of secreted/ cell surface proteins displaying alterations in level in the different mutants compared to the wild type strain. Only proteins with SEED annotation are included and they are categorized according to SEED. Proteins displaying reduced and increased levels in the mutants compared to the wild type are displayed in shades of green and red, respectively. (B) Heat map of proteins which belong to the groups of adhesive and surface proteins or to secreted toxins. Log2-values of the ratios of intensity values of the wild type divided by those of the mutants from -4 to +4 are shown. Higher expression in the wild type versus the mutants is displayed in shades of green and higher expression in the mutants compared to the wild type in shades of red. Proteins displaying the same intensities in wild type and mutant are colored black.

Mentions: For our study we generated single mutants for the functions of SigB (ΔsigB), agr (Δagr), SarA (ΔsarA) and the complemented mutant for sigB (ΔsigB compl.), three double mutants for SigB, agr, SarA (Δagr/ΔsarA, ΔsigB/Δagr, ΔsigB/ΔsarA) and a triple mutant (ΔsigB/Δagr/ΔsarA) in S. aureus LS1, a strain derived from mouse osteomyelitis [37]. Several mutants were also generated in the rsbU complemented derivative of the laboratory strain 8325–4, SH1000 [33] (Supp. S1 Table). All strains and mutants were characterized by growth curves, which did not reveal any substantial differences between mutants and parent strains (S1 Fig). Yet, differences in hemolysis were present in many mutants, particularly in the agr-, sarA-, double and triple mutants, which exhibited low hemolysis (S1C Fig). Furthermore, the strain LS1 and the corresponding mutants were analyzed by LC-MS/MS mass spectrometry, which was focused onto the culture supernatants to provide an overview on the levels of virulence factors in each strain (Fig 1A and S3 Table). These data show that particularly the sarA-mutant and even more the double and the triple-mutants released a much reduced number of virulence factors associated with disease development compared with the wild-type strain LS1 (reduced levels of virulence factors associated with disease; deep green areas, Fig 1A); in particular different adhesive proteins and toxins were present in reduced levels in culture supernatants (Fig 1B). Yet, for the FnBPs that are important for host cell invasion we detected similar levels compared with the wild-type LS1 in most mutants that can account for the invasive capacity of the strains in host cells (S5 Fig). Most importantly, the sigB-mutant showed only modest alterations in protein levels (Fig 1A), but increased levels of α-toxin that is known as a strong proinflammatory and cytotoxic factor after host cell invasion (hla, Fig 1B). The other listed toxins, e.g. lukD and lukE, are reported as non-hemolytic and only poorly leucotoxic toxins [37].


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)

Proteomic analysis of strain LS1 and corresponding mutants.(A) Pie chart display of the proportion of secreted/ cell surface proteins displaying alterations in level in the different mutants compared to the wild type strain. Only proteins with SEED annotation are included and they are categorized according to SEED. Proteins displaying reduced and increased levels in the mutants compared to the wild type are displayed in shades of green and red, respectively. (B) Heat map of proteins which belong to the groups of adhesive and surface proteins or to secreted toxins. Log2-values of the ratios of intensity values of the wild type divided by those of the mutants from -4 to +4 are shown. Higher expression in the wild type versus the mutants is displayed in shades of green and higher expression in the mutants compared to the wild type in shades of red. Proteins displaying the same intensities in wild type and mutant are colored black.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004870.g001: Proteomic analysis of strain LS1 and corresponding mutants.(A) Pie chart display of the proportion of secreted/ cell surface proteins displaying alterations in level in the different mutants compared to the wild type strain. Only proteins with SEED annotation are included and they are categorized according to SEED. Proteins displaying reduced and increased levels in the mutants compared to the wild type are displayed in shades of green and red, respectively. (B) Heat map of proteins which belong to the groups of adhesive and surface proteins or to secreted toxins. Log2-values of the ratios of intensity values of the wild type divided by those of the mutants from -4 to +4 are shown. Higher expression in the wild type versus the mutants is displayed in shades of green and higher expression in the mutants compared to the wild type in shades of red. Proteins displaying the same intensities in wild type and mutant are colored black.
Mentions: For our study we generated single mutants for the functions of SigB (ΔsigB), agr (Δagr), SarA (ΔsarA) and the complemented mutant for sigB (ΔsigB compl.), three double mutants for SigB, agr, SarA (Δagr/ΔsarA, ΔsigB/Δagr, ΔsigB/ΔsarA) and a triple mutant (ΔsigB/Δagr/ΔsarA) in S. aureus LS1, a strain derived from mouse osteomyelitis [37]. Several mutants were also generated in the rsbU complemented derivative of the laboratory strain 8325–4, SH1000 [33] (Supp. S1 Table). All strains and mutants were characterized by growth curves, which did not reveal any substantial differences between mutants and parent strains (S1 Fig). Yet, differences in hemolysis were present in many mutants, particularly in the agr-, sarA-, double and triple mutants, which exhibited low hemolysis (S1C Fig). Furthermore, the strain LS1 and the corresponding mutants were analyzed by LC-MS/MS mass spectrometry, which was focused onto the culture supernatants to provide an overview on the levels of virulence factors in each strain (Fig 1A and S3 Table). These data show that particularly the sarA-mutant and even more the double and the triple-mutants released a much reduced number of virulence factors associated with disease development compared with the wild-type strain LS1 (reduced levels of virulence factors associated with disease; deep green areas, Fig 1A); in particular different adhesive proteins and toxins were present in reduced levels in culture supernatants (Fig 1B). Yet, for the FnBPs that are important for host cell invasion we detected similar levels compared with the wild-type LS1 in most mutants that can account for the invasive capacity of the strains in host cells (S5 Fig). Most importantly, the sigB-mutant showed only modest alterations in protein levels (Fig 1A), but increased levels of α-toxin that is known as a strong proinflammatory and cytotoxic factor after host cell invasion (hla, Fig 1B). The other listed toxins, e.g. lukD and lukE, are reported as non-hemolytic and only poorly leucotoxic toxins [37].

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