Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of complementation of JB12 with icaC in trans on colony morphology, PNAG production, and biofilm formation.A. Colony morphology of labeled S. aureus strains on Congo red agar (CRA) plates. B. PNAG slot blot of cell surface fractions probed with polyclonal anti-PNAG antiserum (MN8m and JB12+pCL15-icaC samples were diluted 1∶100 prior to immobilization on membrane whereas JB12 and JB12+pCL15 were not diluted). C. Biofilms formed on microtiter plate wells were stained with safranin and the safranin was quantified by resuspending in acetic acid and measuring OD562 nm. Bars represent the mean OD562 nm of 6 replicates, and error bars indicate the standard deviations. Statistical comparison (unpaired t test) of JB12+pCL15 (empty vector) versus JB12+pCL15-icaC gave a P value of <0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4117637&req=5

ppat-1004292-g004: Effect of complementation of JB12 with icaC in trans on colony morphology, PNAG production, and biofilm formation.A. Colony morphology of labeled S. aureus strains on Congo red agar (CRA) plates. B. PNAG slot blot of cell surface fractions probed with polyclonal anti-PNAG antiserum (MN8m and JB12+pCL15-icaC samples were diluted 1∶100 prior to immobilization on membrane whereas JB12 and JB12+pCL15 were not diluted). C. Biofilms formed on microtiter plate wells were stained with safranin and the safranin was quantified by resuspending in acetic acid and measuring OD562 nm. Bars represent the mean OD562 nm of 6 replicates, and error bars indicate the standard deviations. Statistical comparison (unpaired t test) of JB12+pCL15 (empty vector) versus JB12+pCL15-icaC gave a P value of <0.001.

Mentions: To confirm that the nucleotide insertion was responsible for loss of the mucoid phenotype, we complemented the icaC gene in trans. Expression of the intact icaC gene in strain JB12 from the IPTG-inducible plasmid pCL15, lead to restoration of the mucoid colony morphology on CRA plates (Fig.4A), PIA/PNAG synthesis (Fig. 4B), and biofilm formation (Fig. 4C). Introduction of the empty vector into the variant had no effect.


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

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

Effect of complementation of JB12 with icaC in trans on colony morphology, PNAG production, and biofilm formation.A. Colony morphology of labeled S. aureus strains on Congo red agar (CRA) plates. B. PNAG slot blot of cell surface fractions probed with polyclonal anti-PNAG antiserum (MN8m and JB12+pCL15-icaC samples were diluted 1∶100 prior to immobilization on membrane whereas JB12 and JB12+pCL15 were not diluted). C. Biofilms formed on microtiter plate wells were stained with safranin and the safranin was quantified by resuspending in acetic acid and measuring OD562 nm. Bars represent the mean OD562 nm of 6 replicates, and error bars indicate the standard deviations. Statistical comparison (unpaired t test) of JB12+pCL15 (empty vector) versus JB12+pCL15-icaC 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-g004: Effect of complementation of JB12 with icaC in trans on colony morphology, PNAG production, and biofilm formation.A. Colony morphology of labeled S. aureus strains on Congo red agar (CRA) plates. B. PNAG slot blot of cell surface fractions probed with polyclonal anti-PNAG antiserum (MN8m and JB12+pCL15-icaC samples were diluted 1∶100 prior to immobilization on membrane whereas JB12 and JB12+pCL15 were not diluted). C. Biofilms formed on microtiter plate wells were stained with safranin and the safranin was quantified by resuspending in acetic acid and measuring OD562 nm. Bars represent the mean OD562 nm of 6 replicates, and error bars indicate the standard deviations. Statistical comparison (unpaired t test) of JB12+pCL15 (empty vector) versus JB12+pCL15-icaC gave a P value of <0.001.
Mentions: To confirm that the nucleotide insertion was responsible for loss of the mucoid phenotype, we complemented the icaC gene in trans. Expression of the intact icaC gene in strain JB12 from the IPTG-inducible plasmid pCL15, lead to restoration of the mucoid colony morphology on CRA plates (Fig.4A), PIA/PNAG synthesis (Fig. 4B), and biofilm formation (Fig. 4C). Introduction of the empty vector into the variant had no effect.

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