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Phasevarions mediate random switching of gene expression in pathogenic Neisseria.

Srikhanta YN, Dowideit SJ, Edwards JL, Falsetta ML, Wu HJ, Harrison OB, Fox KL, Seib KL, Maguire TL, Wang AH, Maiden MC, Grimmond SM, Apicella MA, Jennings MP - PLoS Pathog. (2009)

Bottom Line: Phylogenetic studies on phase-variable mod genes associated with type III restriction modification (R-M) systems revealed that these organisms have two distinct mod genes--modA and modB.ModA11 was only found in N. meningitidis and modA13 only in N. gonorrhoeae.Microarray analysis revealed that in all three modA alleles multiple genes were either upregulated or downregulated, some of which were virulence-associated.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Microbial Sciences, The University of Queensland, St Lucia, Brisbane, Queensland, Australia.

ABSTRACT
Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae, the random switching of the modA gene controls expression of a phase-variable regulon of genes (a "phasevarion"), via differential methylation of the genome in the modA ON and OFF states. Phase-variable mod genes are also present in Neisseria meningitidis and Neisseria gonorrhoeae, suggesting that phasevarions may occur in these important human pathogens. Phylogenetic studies on phase-variable mod genes associated with type III restriction modification (R-M) systems revealed that these organisms have two distinct mod genes--modA and modB. There are also distinct alleles of modA (abundant: modA11, 12, 13; minor: modA4, 15, 18) and modB (modB1, 2). These alleles differ only in their DNA recognition domain. ModA11 was only found in N. meningitidis and modA13 only in N. gonorrhoeae. The recognition site for the modA13 methyltransferase in N. gonorrhoeae strain FA1090 was identified as 5'-AGAAA-3'. Mutant strains lacking the modA11, 12 or 13 genes were made in N. meningitidis and N. gonorrhoeae and their phenotype analyzed in comparison to a corresponding mod ON wild-type strain. Microarray analysis revealed that in all three modA alleles multiple genes were either upregulated or downregulated, some of which were virulence-associated. For example, in N. meningitidis MC58 (modA11), differentially expressed genes included those encoding the candidate vaccine antigens lactoferrin binding proteins A and B. Functional studies using N. gonorrhoeae FA1090 and the clinical isolate O1G1370 confirmed that modA13 ON and OFF strains have distinct phenotypes in antimicrobial resistance, in a primary human cervical epithelial cell model of infection, and in biofilm formation. This study, in conjunction with our previous work in H. influenzae, indicates that phasevarions may be a common strategy used by host-adapted bacterial pathogens to randomly switch between "differentiated" cell types.

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Comparison of wild-type FA1090 modA13 ON and FA1090 modA13::kan mutant in an antimicrobial resistance assay.(A) Wild-type FA1090 modA13 ON and FA1090 modA13::kan mutant cells were serially diluted and spotted onto GC plates containing increasing concentrations of Triton X-100 (x-axis) for determination of viable colony-forming units (y-axis). The white bars correspond to wild-type FA1090 modA13 ON, and the black bars correspond to FA1090 modA13::kan. A Student's t-test showed a significant difference between the two samples (P≤0.021) at each of the following concentrations of Triton X-100; 40 µg/ml, 50 µg/ml, 60 µg/ml, and 80 µg/ml. (B) Shows the ratio of FA1090 modA13 ON to FA1090 modA13 OFF at each of the following concentrations of Triton X-100: 0 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, and 80 µg/ml for FA1090 modA13 ON. †, a statistically significant difference was seen in the ON/OFF ratio between FA1090 modA13 ON 0 µg/ml Triton X-100 and the following FA1090 modA13 ON Triton X-100 concentrations: 40 µg/ml, 50 µg/ml, 60 µg/ml, indicating a selection to OFF organisms at these concentrations. N/D indicates not done. Calculations are shown in Table S8.
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ppat-1000400-g006: Comparison of wild-type FA1090 modA13 ON and FA1090 modA13::kan mutant in an antimicrobial resistance assay.(A) Wild-type FA1090 modA13 ON and FA1090 modA13::kan mutant cells were serially diluted and spotted onto GC plates containing increasing concentrations of Triton X-100 (x-axis) for determination of viable colony-forming units (y-axis). The white bars correspond to wild-type FA1090 modA13 ON, and the black bars correspond to FA1090 modA13::kan. A Student's t-test showed a significant difference between the two samples (P≤0.021) at each of the following concentrations of Triton X-100; 40 µg/ml, 50 µg/ml, 60 µg/ml, and 80 µg/ml. (B) Shows the ratio of FA1090 modA13 ON to FA1090 modA13 OFF at each of the following concentrations of Triton X-100: 0 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, and 80 µg/ml for FA1090 modA13 ON. †, a statistically significant difference was seen in the ON/OFF ratio between FA1090 modA13 ON 0 µg/ml Triton X-100 and the following FA1090 modA13 ON Triton X-100 concentrations: 40 µg/ml, 50 µg/ml, 60 µg/ml, indicating a selection to OFF organisms at these concentrations. N/D indicates not done. Calculations are shown in Table S8.

Mentions: Previous studies using N. gonorrhoeae strain FA19 demonstrate that mtrF is required for induction of high-level antimicrobial resistance to Triton X-100 by gonococci [33]. Our data show that MtrF expression is up-regulated in the modA13 mutant relative to the wild-type under iron-limiting conditions. To test whether differences in antimicrobial-resistance could be observed between wild-type FA1090 modA13 ON and the FA1090 modA13::kan mutant, an antimicrobial-resistance assay was performed using increasing Triton X-100 concentrations (Figure 6A). The FA1090 modA13::kan mutant was found to be more resistant than wild-type FA1090 modA13 ON, consistent with the higher level of expression of MtrF in this modA13 OFF strain. As modA13 ON is free to phase vary to OFF, and OFF cells appear to be fitter in this assay, the status of modA13 expression was monitored by PCR with fluorescent primers across the repeat region to determine whether ON to OFF phase variants had been selected in the survivor colonies at various Triton X-100 concentrations. This analysis revealed that the FA1090 modA13 ON culture plated on zero Triton X-100 remained ON, with only 11.21% OFF cells. However, cells plated on increasing Triton X-100 concentrations changed to 46.99% OFF, 80.29% OFF and 80.15% OFF over the course of the assay for 40, 50 and 60 µg/ml Triton X-100, respectively (Figure 6B).


Phasevarions mediate random switching of gene expression in pathogenic Neisseria.

Srikhanta YN, Dowideit SJ, Edwards JL, Falsetta ML, Wu HJ, Harrison OB, Fox KL, Seib KL, Maguire TL, Wang AH, Maiden MC, Grimmond SM, Apicella MA, Jennings MP - PLoS Pathog. (2009)

Comparison of wild-type FA1090 modA13 ON and FA1090 modA13::kan mutant in an antimicrobial resistance assay.(A) Wild-type FA1090 modA13 ON and FA1090 modA13::kan mutant cells were serially diluted and spotted onto GC plates containing increasing concentrations of Triton X-100 (x-axis) for determination of viable colony-forming units (y-axis). The white bars correspond to wild-type FA1090 modA13 ON, and the black bars correspond to FA1090 modA13::kan. A Student's t-test showed a significant difference between the two samples (P≤0.021) at each of the following concentrations of Triton X-100; 40 µg/ml, 50 µg/ml, 60 µg/ml, and 80 µg/ml. (B) Shows the ratio of FA1090 modA13 ON to FA1090 modA13 OFF at each of the following concentrations of Triton X-100: 0 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, and 80 µg/ml for FA1090 modA13 ON. †, a statistically significant difference was seen in the ON/OFF ratio between FA1090 modA13 ON 0 µg/ml Triton X-100 and the following FA1090 modA13 ON Triton X-100 concentrations: 40 µg/ml, 50 µg/ml, 60 µg/ml, indicating a selection to OFF organisms at these concentrations. N/D indicates not done. Calculations are shown in Table S8.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2667262&req=5

ppat-1000400-g006: Comparison of wild-type FA1090 modA13 ON and FA1090 modA13::kan mutant in an antimicrobial resistance assay.(A) Wild-type FA1090 modA13 ON and FA1090 modA13::kan mutant cells were serially diluted and spotted onto GC plates containing increasing concentrations of Triton X-100 (x-axis) for determination of viable colony-forming units (y-axis). The white bars correspond to wild-type FA1090 modA13 ON, and the black bars correspond to FA1090 modA13::kan. A Student's t-test showed a significant difference between the two samples (P≤0.021) at each of the following concentrations of Triton X-100; 40 µg/ml, 50 µg/ml, 60 µg/ml, and 80 µg/ml. (B) Shows the ratio of FA1090 modA13 ON to FA1090 modA13 OFF at each of the following concentrations of Triton X-100: 0 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, and 80 µg/ml for FA1090 modA13 ON. †, a statistically significant difference was seen in the ON/OFF ratio between FA1090 modA13 ON 0 µg/ml Triton X-100 and the following FA1090 modA13 ON Triton X-100 concentrations: 40 µg/ml, 50 µg/ml, 60 µg/ml, indicating a selection to OFF organisms at these concentrations. N/D indicates not done. Calculations are shown in Table S8.
Mentions: Previous studies using N. gonorrhoeae strain FA19 demonstrate that mtrF is required for induction of high-level antimicrobial resistance to Triton X-100 by gonococci [33]. Our data show that MtrF expression is up-regulated in the modA13 mutant relative to the wild-type under iron-limiting conditions. To test whether differences in antimicrobial-resistance could be observed between wild-type FA1090 modA13 ON and the FA1090 modA13::kan mutant, an antimicrobial-resistance assay was performed using increasing Triton X-100 concentrations (Figure 6A). The FA1090 modA13::kan mutant was found to be more resistant than wild-type FA1090 modA13 ON, consistent with the higher level of expression of MtrF in this modA13 OFF strain. As modA13 ON is free to phase vary to OFF, and OFF cells appear to be fitter in this assay, the status of modA13 expression was monitored by PCR with fluorescent primers across the repeat region to determine whether ON to OFF phase variants had been selected in the survivor colonies at various Triton X-100 concentrations. This analysis revealed that the FA1090 modA13 ON culture plated on zero Triton X-100 remained ON, with only 11.21% OFF cells. However, cells plated on increasing Triton X-100 concentrations changed to 46.99% OFF, 80.29% OFF and 80.15% OFF over the course of the assay for 40, 50 and 60 µg/ml Triton X-100, respectively (Figure 6B).

Bottom Line: Phylogenetic studies on phase-variable mod genes associated with type III restriction modification (R-M) systems revealed that these organisms have two distinct mod genes--modA and modB.ModA11 was only found in N. meningitidis and modA13 only in N. gonorrhoeae.Microarray analysis revealed that in all three modA alleles multiple genes were either upregulated or downregulated, some of which were virulence-associated.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Microbial Sciences, The University of Queensland, St Lucia, Brisbane, Queensland, Australia.

ABSTRACT
Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae, the random switching of the modA gene controls expression of a phase-variable regulon of genes (a "phasevarion"), via differential methylation of the genome in the modA ON and OFF states. Phase-variable mod genes are also present in Neisseria meningitidis and Neisseria gonorrhoeae, suggesting that phasevarions may occur in these important human pathogens. Phylogenetic studies on phase-variable mod genes associated with type III restriction modification (R-M) systems revealed that these organisms have two distinct mod genes--modA and modB. There are also distinct alleles of modA (abundant: modA11, 12, 13; minor: modA4, 15, 18) and modB (modB1, 2). These alleles differ only in their DNA recognition domain. ModA11 was only found in N. meningitidis and modA13 only in N. gonorrhoeae. The recognition site for the modA13 methyltransferase in N. gonorrhoeae strain FA1090 was identified as 5'-AGAAA-3'. Mutant strains lacking the modA11, 12 or 13 genes were made in N. meningitidis and N. gonorrhoeae and their phenotype analyzed in comparison to a corresponding mod ON wild-type strain. Microarray analysis revealed that in all three modA alleles multiple genes were either upregulated or downregulated, some of which were virulence-associated. For example, in N. meningitidis MC58 (modA11), differentially expressed genes included those encoding the candidate vaccine antigens lactoferrin binding proteins A and B. Functional studies using N. gonorrhoeae FA1090 and the clinical isolate O1G1370 confirmed that modA13 ON and OFF strains have distinct phenotypes in antimicrobial resistance, in a primary human cervical epithelial cell model of infection, and in biofilm formation. This study, in conjunction with our previous work in H. influenzae, indicates that phasevarions may be a common strategy used by host-adapted bacterial pathogens to randomly switch between "differentiated" cell types.

Show MeSH
Related in: MedlinePlus