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Phase variation of poly-N-acetylglucosamine expression in Staphylococcus aureus.

Brooks JL, Jefferson KK - PLoS Pathog. (2014)

Bottom Line: Inactivation of IcaC results in a PIA/PNAG-negative phenotype.There was also a survival advantage for an icaC-negative strain harboring intact icaADB genes relative to an isogenic icaADBC deletion mutant.Together, these results suggest that inactivation of icaC is a mode of phase variation for PIA/PNAG expression, that high-level production of PIA/PNAG carries a fitness cost, and that icaADB may contribute to bacterial fitness, by an unknown mechanism, in the absence of an intact icaC gene and PIA/PNAG production.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, Virginia, United States of America.

ABSTRACT
Polysaccharide intercellular adhesin (PIA), also known as poly-N-acetyl-β-(1-6)-glucosamine (PIA/PNAG) is an important component of Staphylococcus aureus biofilms and also contributes to resistance to phagocytosis. The proteins IcaA, IcaD, IcaB, and IcaC are encoded within the intercellular adhesin (ica) operon and synthesize PIA/PNAG. We discovered a mechanism of phase variation in PIA/PNAG expression that appears to involve slipped-strand mispairing. The process is reversible and RecA-independent, and involves the expansion and contraction of a simple tetranucleotide tandem repeat within icaC. Inactivation of IcaC results in a PIA/PNAG-negative phenotype. A PIA/PNAG-hyperproducing strain gained a fitness advantage in vitro following the icaC mutation and loss of PIA/PNAG production. The mutation was also detected in two clinical isolates, suggesting that under certain conditions, loss of PIA/PNAG production may be advantageous during infection. There was also a survival advantage for an icaC-negative strain harboring intact icaADB genes relative to an isogenic icaADBC deletion mutant. Together, these results suggest that inactivation of icaC is a mode of phase variation for PIA/PNAG expression, that high-level production of PIA/PNAG carries a fitness cost, and that icaADB may contribute to bacterial fitness, by an unknown mechanism, in the absence of an intact icaC gene and PIA/PNAG production.

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Survival in minimal media.MN8m, JB12, and MN8Δica were grown in TSBG. The cells were collected and resuspended in minimal media lacking a carbon source to mimic starvation conditions. Points represent the mean percent value of cells enumerated at each time point relative to the starting populations with n = 3, and error bars indicate the standard deviations. Statistical comparison (unpaired t test) of MN8m versus JB12 and MN8Δica versus JB12 at 8 hours and 10 hours gave a P value of <0.0001. Comparison of MN8m versus JB12 and MN8Δica versus JB12 at 18 hours and 24 hours gave a P value of <0.001.
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ppat-1004292-g007: Survival in minimal media.MN8m, JB12, and MN8Δica were grown in TSBG. The cells were collected and resuspended in minimal media lacking a carbon source to mimic starvation conditions. Points represent the mean percent value of cells enumerated at each time point relative to the starting populations with n = 3, and error bars indicate the standard deviations. Statistical comparison (unpaired t test) of MN8m versus JB12 and MN8Δica versus JB12 at 8 hours and 10 hours gave a P value of <0.0001. Comparison of MN8m versus JB12 and MN8Δica versus JB12 at 18 hours and 24 hours gave a P value of <0.001.

Mentions: We did not directly measure function to confirm that the IcaA, IcaD, and/or IcaB proteins were functional in JB12; however, complementation of the mucoid phenotype in trans by a copy of the icaC gene alone suggests that only the function of IcaC is altered in JB12 and that IcaA, IcaD, and IcaB are still present and functional. We hypothesized that one or more of these proteins may have alternative roles within the cell that contribute to bacterial fitness and that this was the reason why icaC was the target for phase variation. Under starvation conditions, when the bacteria were switched to minimal media containing no carbon source, JB12 survived significantly longer than MN8Δica::tet (data not shown). To confirm that this difference was not due to a secondary mutation introduced during strain passage, three isogenic strains that differed only at the ica locus were made. We performed allelic exchange in strain MN8Δica::tet to replace the tetracycline resistance cassette and the interrupted ica locus with the entire MN8m (MN8icaC_On) or the entire JB12 (MN8icaC_Off) ica locus on the chromosome. The allelic exchange mutants exhibited survival profiles similar to MN8m and JB12 and both strains survived longer in the minimal media than MN8Δica::tet (Fig. 7). Production of intact, and presumably functional IcaA, IcaD, and IcaB appeared to significantly increase survival of JB12 under these growth-limiting conditions.


Phase variation of poly-N-acetylglucosamine expression in Staphylococcus aureus.

Brooks JL, Jefferson KK - PLoS Pathog. (2014)

Survival in minimal media.MN8m, JB12, and MN8Δica were grown in TSBG. The cells were collected and resuspended in minimal media lacking a carbon source to mimic starvation conditions. Points represent the mean percent value of cells enumerated at each time point relative to the starting populations with n = 3, and error bars indicate the standard deviations. Statistical comparison (unpaired t test) of MN8m versus JB12 and MN8Δica versus JB12 at 8 hours and 10 hours gave a P value of <0.0001. Comparison of MN8m versus JB12 and MN8Δica versus JB12 at 18 hours and 24 hours gave a P value of <0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117637&req=5

ppat-1004292-g007: Survival in minimal media.MN8m, JB12, and MN8Δica were grown in TSBG. The cells were collected and resuspended in minimal media lacking a carbon source to mimic starvation conditions. Points represent the mean percent value of cells enumerated at each time point relative to the starting populations with n = 3, and error bars indicate the standard deviations. Statistical comparison (unpaired t test) of MN8m versus JB12 and MN8Δica versus JB12 at 8 hours and 10 hours gave a P value of <0.0001. Comparison of MN8m versus JB12 and MN8Δica versus JB12 at 18 hours and 24 hours gave a P value of <0.001.
Mentions: We did not directly measure function to confirm that the IcaA, IcaD, and/or IcaB proteins were functional in JB12; however, complementation of the mucoid phenotype in trans by a copy of the icaC gene alone suggests that only the function of IcaC is altered in JB12 and that IcaA, IcaD, and IcaB are still present and functional. We hypothesized that one or more of these proteins may have alternative roles within the cell that contribute to bacterial fitness and that this was the reason why icaC was the target for phase variation. Under starvation conditions, when the bacteria were switched to minimal media containing no carbon source, JB12 survived significantly longer than MN8Δica::tet (data not shown). To confirm that this difference was not due to a secondary mutation introduced during strain passage, three isogenic strains that differed only at the ica locus were made. We performed allelic exchange in strain MN8Δica::tet to replace the tetracycline resistance cassette and the interrupted ica locus with the entire MN8m (MN8icaC_On) or the entire JB12 (MN8icaC_Off) ica locus on the chromosome. The allelic exchange mutants exhibited survival profiles similar to MN8m and JB12 and both strains survived longer in the minimal media than MN8Δica::tet (Fig. 7). Production of intact, and presumably functional IcaA, IcaD, and IcaB appeared to significantly increase survival of JB12 under these growth-limiting conditions.

Bottom Line: Inactivation of IcaC results in a PIA/PNAG-negative phenotype.There was also a survival advantage for an icaC-negative strain harboring intact icaADB genes relative to an isogenic icaADBC deletion mutant.Together, these results suggest that inactivation of icaC is a mode of phase variation for PIA/PNAG expression, that high-level production of PIA/PNAG carries a fitness cost, and that icaADB may contribute to bacterial fitness, by an unknown mechanism, in the absence of an intact icaC gene and PIA/PNAG production.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, Virginia, United States of America.

ABSTRACT
Polysaccharide intercellular adhesin (PIA), also known as poly-N-acetyl-β-(1-6)-glucosamine (PIA/PNAG) is an important component of Staphylococcus aureus biofilms and also contributes to resistance to phagocytosis. The proteins IcaA, IcaD, IcaB, and IcaC are encoded within the intercellular adhesin (ica) operon and synthesize PIA/PNAG. We discovered a mechanism of phase variation in PIA/PNAG expression that appears to involve slipped-strand mispairing. The process is reversible and RecA-independent, and involves the expansion and contraction of a simple tetranucleotide tandem repeat within icaC. Inactivation of IcaC results in a PIA/PNAG-negative phenotype. A PIA/PNAG-hyperproducing strain gained a fitness advantage in vitro following the icaC mutation and loss of PIA/PNAG production. The mutation was also detected in two clinical isolates, suggesting that under certain conditions, loss of PIA/PNAG production may be advantageous during infection. There was also a survival advantage for an icaC-negative strain harboring intact icaADB genes relative to an isogenic icaADBC deletion mutant. Together, these results suggest that inactivation of icaC is a mode of phase variation for PIA/PNAG expression, that high-level production of PIA/PNAG carries a fitness cost, and that icaADB may contribute to bacterial fitness, by an unknown mechanism, in the absence of an intact icaC gene and PIA/PNAG production.

Show MeSH
Related in: MedlinePlus