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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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Related in: MedlinePlus

N. gonorrhoeae O1G1370 association with, and intracellular survival within, primary human cervical epithelial (pex) cells.Pex cells were challenged with N. gonorrhoeae strain O1G1370 as outlined in the text. Data shown represent the invasion index (left panel) or the survival index (right panel) following challenge of pex cells as outlined in the text. The invasion index represents the percentage of pex cell–associated gonococci that survive gentamicin treatment; whereas the survival index is the percentage of invasive gonococci that survive, intracellularly, within pex cells at 3 h post-invasion. There was no significant difference between the naturally occurring O1G1370 modA13 OFF isolate and the O1G1370 modA13::kan “knockout” strain in either the invasion (P = 0.091) or survival (P = 0.23) indices observed. A statistically significant difference (*) was obtained in the invasion (P = 0.046) and survival (P = 0.021) indices when comparing O1G1370 modA13 OFF to O1G1370 modA13 ON, and in the invasion (P = 0.019) and survival (P = 0.004) indices when comparing O1G1370 modA13::kan to O1G1370 modA13 ON. P-values were determined using a Student's t-test. (B) Shows the ratio of O1G1370 modA13 ON to O1G1370 modA13 OFF of the inoculum, and at the invasion and survival time points for O1G1370 modA13 ON and O1G1370 modA13 OFF. †, a statistically significant difference was seen in the ON/OFF ratio between the O1G1370 modA13 OFF inoculum and the O1G1370 modA13 OFF survival sample (P = 0.0234), indicating a selection for OFF organisms over the course of the 3-h assay (for full data, see Table S8).
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ppat-1000400-g008: N. gonorrhoeae O1G1370 association with, and intracellular survival within, primary human cervical epithelial (pex) cells.Pex cells were challenged with N. gonorrhoeae strain O1G1370 as outlined in the text. Data shown represent the invasion index (left panel) or the survival index (right panel) following challenge of pex cells as outlined in the text. The invasion index represents the percentage of pex cell–associated gonococci that survive gentamicin treatment; whereas the survival index is the percentage of invasive gonococci that survive, intracellularly, within pex cells at 3 h post-invasion. There was no significant difference between the naturally occurring O1G1370 modA13 OFF isolate and the O1G1370 modA13::kan “knockout” strain in either the invasion (P = 0.091) or survival (P = 0.23) indices observed. A statistically significant difference (*) was obtained in the invasion (P = 0.046) and survival (P = 0.021) indices when comparing O1G1370 modA13 OFF to O1G1370 modA13 ON, and in the invasion (P = 0.019) and survival (P = 0.004) indices when comparing O1G1370 modA13::kan to O1G1370 modA13 ON. P-values were determined using a Student's t-test. (B) Shows the ratio of O1G1370 modA13 ON to O1G1370 modA13 OFF of the inoculum, and at the invasion and survival time points for O1G1370 modA13 ON and O1G1370 modA13 OFF. †, a statistically significant difference was seen in the ON/OFF ratio between the O1G1370 modA13 OFF inoculum and the O1G1370 modA13 OFF survival sample (P = 0.0234), indicating a selection for OFF organisms over the course of the 3-h assay (for full data, see Table S8).

Mentions: The use of primary human cervical epithelial (pex) cells as a model system of gonococcal cervicitis is well established and has been used in a number of studies, such as the examination of the role of oxidative stress regulators in host-pathogen interactions [29],[32]. To determine the biological significance of O1G1370 modA13 expression using this pex cell culture model, we performed quantitative association, invasion, and survival assays using O1G1370 modA13 ON, O1G1370 modA13 OFF, and O1G1370 modA13::kan mutant gonococci (Figure 8). These data revealed that there was no significant (P≥0.2338) difference in the ability of the O1G1370 modA13 OFF and O1G1370 modA13::kan strains to adhere to, invade, or survive within pex cells. This confirmed that the modA13::kan knockout allele behaves in the same way as a natural phase variant modA13 OFF strain. In contrast, behavior of the O1G1370 modA13 ON strain was significantly (P≤0.001) different from that obtained with the use of either the O1G1370 modA13 OFF or O1G1370 modA13::kan strains in parallel assays. In this regard, a modA13 ON phenotype resulted in the increased ability of gonococci to associate with pex cells, whereas a modA13 OFF configuration augmented the ability of gonococci to invade (Figure 8A, invasion index) and survive within pex cells following invasion (Figure 8A, survival index). These data suggest a possible role for Mod-dependent phase variation in promoting the adaptive changes required for gonococci to switch from an extracellular to an intracellular existence. This idea is supported by our observation of selection for a switch from ON to OFF in the O1G1370 modA13 ON strain. Fragment analysis confirmed that the O1G1370 modA13 ON inoculum, which contains only 5.86% OFF, changes to ∼49.84% OFF by the time the 3 hour intracellular survival sample was taken (Figure 8B, Table S9). An independent N. gonorrhoeae modA13 strain, 1291, displayed the same intracellular survival and modA13 switching phenotype (Figure S6).


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)

N. gonorrhoeae O1G1370 association with, and intracellular survival within, primary human cervical epithelial (pex) cells.Pex cells were challenged with N. gonorrhoeae strain O1G1370 as outlined in the text. Data shown represent the invasion index (left panel) or the survival index (right panel) following challenge of pex cells as outlined in the text. The invasion index represents the percentage of pex cell–associated gonococci that survive gentamicin treatment; whereas the survival index is the percentage of invasive gonococci that survive, intracellularly, within pex cells at 3 h post-invasion. There was no significant difference between the naturally occurring O1G1370 modA13 OFF isolate and the O1G1370 modA13::kan “knockout” strain in either the invasion (P = 0.091) or survival (P = 0.23) indices observed. A statistically significant difference (*) was obtained in the invasion (P = 0.046) and survival (P = 0.021) indices when comparing O1G1370 modA13 OFF to O1G1370 modA13 ON, and in the invasion (P = 0.019) and survival (P = 0.004) indices when comparing O1G1370 modA13::kan to O1G1370 modA13 ON. P-values were determined using a Student's t-test. (B) Shows the ratio of O1G1370 modA13 ON to O1G1370 modA13 OFF of the inoculum, and at the invasion and survival time points for O1G1370 modA13 ON and O1G1370 modA13 OFF. †, a statistically significant difference was seen in the ON/OFF ratio between the O1G1370 modA13 OFF inoculum and the O1G1370 modA13 OFF survival sample (P = 0.0234), indicating a selection for OFF organisms over the course of the 3-h assay (for full data, see Table S8).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667262&req=5

ppat-1000400-g008: N. gonorrhoeae O1G1370 association with, and intracellular survival within, primary human cervical epithelial (pex) cells.Pex cells were challenged with N. gonorrhoeae strain O1G1370 as outlined in the text. Data shown represent the invasion index (left panel) or the survival index (right panel) following challenge of pex cells as outlined in the text. The invasion index represents the percentage of pex cell–associated gonococci that survive gentamicin treatment; whereas the survival index is the percentage of invasive gonococci that survive, intracellularly, within pex cells at 3 h post-invasion. There was no significant difference between the naturally occurring O1G1370 modA13 OFF isolate and the O1G1370 modA13::kan “knockout” strain in either the invasion (P = 0.091) or survival (P = 0.23) indices observed. A statistically significant difference (*) was obtained in the invasion (P = 0.046) and survival (P = 0.021) indices when comparing O1G1370 modA13 OFF to O1G1370 modA13 ON, and in the invasion (P = 0.019) and survival (P = 0.004) indices when comparing O1G1370 modA13::kan to O1G1370 modA13 ON. P-values were determined using a Student's t-test. (B) Shows the ratio of O1G1370 modA13 ON to O1G1370 modA13 OFF of the inoculum, and at the invasion and survival time points for O1G1370 modA13 ON and O1G1370 modA13 OFF. †, a statistically significant difference was seen in the ON/OFF ratio between the O1G1370 modA13 OFF inoculum and the O1G1370 modA13 OFF survival sample (P = 0.0234), indicating a selection for OFF organisms over the course of the 3-h assay (for full data, see Table S8).
Mentions: The use of primary human cervical epithelial (pex) cells as a model system of gonococcal cervicitis is well established and has been used in a number of studies, such as the examination of the role of oxidative stress regulators in host-pathogen interactions [29],[32]. To determine the biological significance of O1G1370 modA13 expression using this pex cell culture model, we performed quantitative association, invasion, and survival assays using O1G1370 modA13 ON, O1G1370 modA13 OFF, and O1G1370 modA13::kan mutant gonococci (Figure 8). These data revealed that there was no significant (P≥0.2338) difference in the ability of the O1G1370 modA13 OFF and O1G1370 modA13::kan strains to adhere to, invade, or survive within pex cells. This confirmed that the modA13::kan knockout allele behaves in the same way as a natural phase variant modA13 OFF strain. In contrast, behavior of the O1G1370 modA13 ON strain was significantly (P≤0.001) different from that obtained with the use of either the O1G1370 modA13 OFF or O1G1370 modA13::kan strains in parallel assays. In this regard, a modA13 ON phenotype resulted in the increased ability of gonococci to associate with pex cells, whereas a modA13 OFF configuration augmented the ability of gonococci to invade (Figure 8A, invasion index) and survive within pex cells following invasion (Figure 8A, survival index). These data suggest a possible role for Mod-dependent phase variation in promoting the adaptive changes required for gonococci to switch from an extracellular to an intracellular existence. This idea is supported by our observation of selection for a switch from ON to OFF in the O1G1370 modA13 ON strain. Fragment analysis confirmed that the O1G1370 modA13 ON inoculum, which contains only 5.86% OFF, changes to ∼49.84% OFF by the time the 3 hour intracellular survival sample was taken (Figure 8B, Table S9). An independent N. gonorrhoeae modA13 strain, 1291, displayed the same intracellular survival and modA13 switching phenotype (Figure S6).

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