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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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Biofilm formation by N. gonorrhoeae strain O1G1370 modA13::kan, modA13 OFF, and O1G1370 modA13 ON.(A) Confocal microscopy of the biofilm mass over 2 days of growth for (1) N. gonorrhoeae O1G1370 modA13 ON, (2) O1G1370 modA13::kan, and (3) O1G1370 modA13 OFF. These images are three-dimensional reconstructions of stacked z-series taken at 200× magnification, which were rendered by Volocity. These experiments were performed in quadruplicate on three different occasions, and representative images are shown. (B) Scanning electron microscopy of the surface of the biofilm mass over 2 days of growth on glass taken at 5,000× magnification. It can be noted that there are fewer cells in the O1G1370 modA13 ON biofilm than either the O1G1370 modA13::kan or O1G1370 modA13 OFF biofilms. (C) Scanning electron microscopy of the surface of the biofilm mass over 2 days growth on glass taken at 15,000× magnification. (D) Transmission electron microscopy of 70 nm thin-sections of the biofilm mass over 2 days of growth on glass taken at 10,000× magnification. (E) COMSTAT analysis of biomass, average, and maximum thickness of confocal z-series images of the O1G1370 modA13::kan, O1G1370 modA13 OFF, and O1G1370 modA13 ON biofilms grown for 2 days over glass, which are depicted in (A). COMSTAT was performed for all replicates, and results are as shown. Statistical significance was determined using a Student's t-test. There was no statistically significant difference between the biomass, average, or maximum thickness of the O1G1370 modA13::kan and O1G1370 modA13 OFF strains.
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ppat-1000400-g007: Biofilm formation by N. gonorrhoeae strain O1G1370 modA13::kan, modA13 OFF, and O1G1370 modA13 ON.(A) Confocal microscopy of the biofilm mass over 2 days of growth for (1) N. gonorrhoeae O1G1370 modA13 ON, (2) O1G1370 modA13::kan, and (3) O1G1370 modA13 OFF. These images are three-dimensional reconstructions of stacked z-series taken at 200× magnification, which were rendered by Volocity. These experiments were performed in quadruplicate on three different occasions, and representative images are shown. (B) Scanning electron microscopy of the surface of the biofilm mass over 2 days of growth on glass taken at 5,000× magnification. It can be noted that there are fewer cells in the O1G1370 modA13 ON biofilm than either the O1G1370 modA13::kan or O1G1370 modA13 OFF biofilms. (C) Scanning electron microscopy of the surface of the biofilm mass over 2 days growth on glass taken at 15,000× magnification. (D) Transmission electron microscopy of 70 nm thin-sections of the biofilm mass over 2 days of growth on glass taken at 10,000× magnification. (E) COMSTAT analysis of biomass, average, and maximum thickness of confocal z-series images of the O1G1370 modA13::kan, O1G1370 modA13 OFF, and O1G1370 modA13 ON biofilms grown for 2 days over glass, which are depicted in (A). COMSTAT was performed for all replicates, and results are as shown. Statistical significance was determined using a Student's t-test. There was no statistically significant difference between the biomass, average, or maximum thickness of the O1G1370 modA13::kan and O1G1370 modA13 OFF strains.

Mentions: The ability of N. gonorrhoeae O1G1370 modA13 ON, O1G1370 modA13 OFF and O1G1370 modA13::kan (OFF) to form a biofilm was evaluated after two days of growth under continuous flow conditions. Three-dimensional images of these biofilms were created in Volocity (Materials and Methods). These images show that O1G1370 modA13::kan and modA13 OFF form a thick and dense biofilm, while O1G1370 modA13 ON forms an extremely weak biofilm with a few sparse patches of cells scattered across the surface of attachment (Figure 7A). The O1G1370 modA13 ON strain also formed biofilms with lower maximum thicknesses than the O1G1370 modA13::kan and O1G1370 modA13 OFF strains. (Figure 7D). Scanning electron microscopy of the surface of the biofilm taken at 5,000× magnification shows that there are gaps between clusters of biofilm in the O1G1370 modA13 ON strain, unlike the O1G1370 modA13 OFF and O1G1370 modA13::kan strain biofilms, where there are no areas where the glass surface of attachment is visible. There are also large areas where no biofilm is present in the O1G1370 modA13 ON samples (Figure 7B). Scanning electron microscopy taken at 15,000× magnification shows that O1G1370 modA13::kan and O1G1370 modA13 OFF form biofilms that are tightly enmeshed in an extracellular material that obscures the structure of individual cells, while cells in the modA13 ON biofilm are clearly distinguishable (Figure 7C). Transmission electron microscopy shows that O1G1370 modA13::kan forms a biofilm where individual cells are shedding copious amounts of membrane, as seen in the numerous enclosed membrane blebs on the surface of the cells, while there is no evidence of blebbing in the O1G1370 modA13 ON biofilm. Cells in the O1G1370 modA13 OFF biofilm also appear to be blebbing, like those in O1G1370 modA13::kan biofilm, as numerous blebs can be seen forming on the surface of the O1G1370 modA13 OFF strain. These electron micrographs suggest that the extracellular matrices of the O1G1370 modA13::kan and O1G1370 modA13 OFF biofilms may be at least partially composed of fused membrane blebs (Figure 7C). COMSTAT [37] was used to quantitatively assess the biomass, and average and maximum thickness of confocal z-series photomicrographs taken for each flow chamber. COMSTAT analysis showed that the O1G1370 modA13::kan and O1G1370 modA13 OFF strains form significantly thicker biofilms with significantly more biomass than the O1G1370 modA13 ON strain. Specifically, O1G1370 modA13 ON had 3.5% of the biomass and 4.2% of the thickness of the O1G1370 modA13::kan mutant on average and 4.7% of the biomass and 5.2% of the thickness of the O1G1370 modA13 OFF (Figure 7E). Similar results were observed using N. gonorrhoeae strains FA1090 modA13 ON and FA1090 modA13::kan (Figure S5).


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)

Biofilm formation by N. gonorrhoeae strain O1G1370 modA13::kan, modA13 OFF, and O1G1370 modA13 ON.(A) Confocal microscopy of the biofilm mass over 2 days of growth for (1) N. gonorrhoeae O1G1370 modA13 ON, (2) O1G1370 modA13::kan, and (3) O1G1370 modA13 OFF. These images are three-dimensional reconstructions of stacked z-series taken at 200× magnification, which were rendered by Volocity. These experiments were performed in quadruplicate on three different occasions, and representative images are shown. (B) Scanning electron microscopy of the surface of the biofilm mass over 2 days of growth on glass taken at 5,000× magnification. It can be noted that there are fewer cells in the O1G1370 modA13 ON biofilm than either the O1G1370 modA13::kan or O1G1370 modA13 OFF biofilms. (C) Scanning electron microscopy of the surface of the biofilm mass over 2 days growth on glass taken at 15,000× magnification. (D) Transmission electron microscopy of 70 nm thin-sections of the biofilm mass over 2 days of growth on glass taken at 10,000× magnification. (E) COMSTAT analysis of biomass, average, and maximum thickness of confocal z-series images of the O1G1370 modA13::kan, O1G1370 modA13 OFF, and O1G1370 modA13 ON biofilms grown for 2 days over glass, which are depicted in (A). COMSTAT was performed for all replicates, and results are as shown. Statistical significance was determined using a Student's t-test. There was no statistically significant difference between the biomass, average, or maximum thickness of the O1G1370 modA13::kan and O1G1370 modA13 OFF strains.
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Related In: Results  -  Collection

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ppat-1000400-g007: Biofilm formation by N. gonorrhoeae strain O1G1370 modA13::kan, modA13 OFF, and O1G1370 modA13 ON.(A) Confocal microscopy of the biofilm mass over 2 days of growth for (1) N. gonorrhoeae O1G1370 modA13 ON, (2) O1G1370 modA13::kan, and (3) O1G1370 modA13 OFF. These images are three-dimensional reconstructions of stacked z-series taken at 200× magnification, which were rendered by Volocity. These experiments were performed in quadruplicate on three different occasions, and representative images are shown. (B) Scanning electron microscopy of the surface of the biofilm mass over 2 days of growth on glass taken at 5,000× magnification. It can be noted that there are fewer cells in the O1G1370 modA13 ON biofilm than either the O1G1370 modA13::kan or O1G1370 modA13 OFF biofilms. (C) Scanning electron microscopy of the surface of the biofilm mass over 2 days growth on glass taken at 15,000× magnification. (D) Transmission electron microscopy of 70 nm thin-sections of the biofilm mass over 2 days of growth on glass taken at 10,000× magnification. (E) COMSTAT analysis of biomass, average, and maximum thickness of confocal z-series images of the O1G1370 modA13::kan, O1G1370 modA13 OFF, and O1G1370 modA13 ON biofilms grown for 2 days over glass, which are depicted in (A). COMSTAT was performed for all replicates, and results are as shown. Statistical significance was determined using a Student's t-test. There was no statistically significant difference between the biomass, average, or maximum thickness of the O1G1370 modA13::kan and O1G1370 modA13 OFF strains.
Mentions: The ability of N. gonorrhoeae O1G1370 modA13 ON, O1G1370 modA13 OFF and O1G1370 modA13::kan (OFF) to form a biofilm was evaluated after two days of growth under continuous flow conditions. Three-dimensional images of these biofilms were created in Volocity (Materials and Methods). These images show that O1G1370 modA13::kan and modA13 OFF form a thick and dense biofilm, while O1G1370 modA13 ON forms an extremely weak biofilm with a few sparse patches of cells scattered across the surface of attachment (Figure 7A). The O1G1370 modA13 ON strain also formed biofilms with lower maximum thicknesses than the O1G1370 modA13::kan and O1G1370 modA13 OFF strains. (Figure 7D). Scanning electron microscopy of the surface of the biofilm taken at 5,000× magnification shows that there are gaps between clusters of biofilm in the O1G1370 modA13 ON strain, unlike the O1G1370 modA13 OFF and O1G1370 modA13::kan strain biofilms, where there are no areas where the glass surface of attachment is visible. There are also large areas where no biofilm is present in the O1G1370 modA13 ON samples (Figure 7B). Scanning electron microscopy taken at 15,000× magnification shows that O1G1370 modA13::kan and O1G1370 modA13 OFF form biofilms that are tightly enmeshed in an extracellular material that obscures the structure of individual cells, while cells in the modA13 ON biofilm are clearly distinguishable (Figure 7C). Transmission electron microscopy shows that O1G1370 modA13::kan forms a biofilm where individual cells are shedding copious amounts of membrane, as seen in the numerous enclosed membrane blebs on the surface of the cells, while there is no evidence of blebbing in the O1G1370 modA13 ON biofilm. Cells in the O1G1370 modA13 OFF biofilm also appear to be blebbing, like those in O1G1370 modA13::kan biofilm, as numerous blebs can be seen forming on the surface of the O1G1370 modA13 OFF strain. These electron micrographs suggest that the extracellular matrices of the O1G1370 modA13::kan and O1G1370 modA13 OFF biofilms may be at least partially composed of fused membrane blebs (Figure 7C). COMSTAT [37] was used to quantitatively assess the biomass, and average and maximum thickness of confocal z-series photomicrographs taken for each flow chamber. COMSTAT analysis showed that the O1G1370 modA13::kan and O1G1370 modA13 OFF strains form significantly thicker biofilms with significantly more biomass than the O1G1370 modA13 ON strain. Specifically, O1G1370 modA13 ON had 3.5% of the biomass and 4.2% of the thickness of the O1G1370 modA13::kan mutant on average and 4.7% of the biomass and 5.2% of the thickness of the O1G1370 modA13 OFF (Figure 7E). Similar results were observed using N. gonorrhoeae strains FA1090 modA13 ON and FA1090 modA13::kan (Figure S5).

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