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A biphasic epigenetic switch controls immunoevasion, virulence and niche adaptation in non-typeable Haemophilus influenzae.

Atack JM, Srikhanta YN, Fox KL, Jurcisek JA, Brockman KL, Clark TA, Boitano M, Power PM, Jen FE, McEwan AG, Grimmond SM, Smith AL, Barenkamp SJ, Korlach J, Bakaletz LO, Jennings MP - Nat Commun (2015)

Bottom Line: Here, we use single molecule, real-time (SMRT) methylome analysis to identify the DNA-recognition motifs for all five of these modA alleles.Phase variation of these alleles regulates multiple proteins including vaccine candidates, and key virulence phenotypes such as antibiotic resistance (modA2, modA5, modA10), biofilm formation (modA2) and immunoevasion (modA4).Our results indicate that a biphasic epigenetic switch can control bacterial virulence, immunoevasion and niche adaptation in an animal model system.

View Article: PubMed Central - PubMed

Affiliation: Institute for Glycomics, Griffith University, Gold Coast, Queensland 4222, Australia.

ABSTRACT
Non-typeable Haemophilus influenzae contains an N(6)-adenine DNA-methyltransferase (ModA) that is subject to phase-variable expression (random ON/OFF switching). Five modA alleles, modA2, modA4, modA5, modA9 and modA10, account for over two-thirds of clinical otitis media isolates surveyed. Here, we use single molecule, real-time (SMRT) methylome analysis to identify the DNA-recognition motifs for all five of these modA alleles. Phase variation of these alleles regulates multiple proteins including vaccine candidates, and key virulence phenotypes such as antibiotic resistance (modA2, modA5, modA10), biofilm formation (modA2) and immunoevasion (modA4). Analyses of a modA2 strain in the chinchilla model of otitis media show a clear selection for ON switching of modA2 in the middle ear. Our results indicate that a biphasic epigenetic switch can control bacterial virulence, immunoevasion and niche adaptation in an animal model system.

No MeSH data available.


Related in: MedlinePlus

Western blots showing ModA presence in modA ON OFF and kanamycin knockout strains and an example of the fragment analysis methodology.(a) Western blot analysis using ON/OFF/kan cells from all the five modA alleles; anti-modA primary antibody at 1:5,000; AP conjugated anti-rabbit secondary at 1:20,000. A modA band is only present in those cells possessing the ON variant of each strain. The number of repeats (reps) represents the major number of repeats present in that particular isolate, and the percentage ON of that particular strain is noted underneath each relevant blot. Full western blots are presented in Supplementary Fig. 4; (b) an illustration of the methodology used during fragment analysis to ascertain modA ON/OFF state and percentages of cell populations. A 6-carboxyflourescein (FAM)-labelled forward primer (green hexagon) and unlabelled reverse primer was used to generate a PCR product over the repeat tract (AGCCn; grey box) that was analysed using GeneScan technology; and (c) an example trace produced by GeneScan analysis on a representative genomic sample from an NTHi strain containing a phase variable modA gene.
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f2: Western blots showing ModA presence in modA ON OFF and kanamycin knockout strains and an example of the fragment analysis methodology.(a) Western blot analysis using ON/OFF/kan cells from all the five modA alleles; anti-modA primary antibody at 1:5,000; AP conjugated anti-rabbit secondary at 1:20,000. A modA band is only present in those cells possessing the ON variant of each strain. The number of repeats (reps) represents the major number of repeats present in that particular isolate, and the percentage ON of that particular strain is noted underneath each relevant blot. Full western blots are presented in Supplementary Fig. 4; (b) an illustration of the methodology used during fragment analysis to ascertain modA ON/OFF state and percentages of cell populations. A 6-carboxyflourescein (FAM)-labelled forward primer (green hexagon) and unlabelled reverse primer was used to generate a PCR product over the repeat tract (AGCCn; grey box) that was analysed using GeneScan technology; and (c) an example trace produced by GeneScan analysis on a representative genomic sample from an NTHi strain containing a phase variable modA gene.

Mentions: We generated modA natural ON- and OFF-enriched populations (>90% ON or OFF) from the five NTHi strains containing the modA2, 4, 5, 9 and 10 alleles (Fig. 1e). All ON strains contain a number of AGCC repeats that places the modA open reading frame (ORF) in frame, and therefore express a full-length functional protein, as confirmed by western blot using an anti-ModA antibody (Fig. 2a). All the natural ON and OFF strains were continually verified using our well-established FAM-labelled primer PCR screen coupled to fragment length analysis (an example of the methodology and results is given in Fig. 2b,c)610. Natural modAOFF strains contain a number of AGCC repeats that place the modA ORF out of frame, leading to a frame shift mutation and premature termination at stop codons in the alternate reading frames (Fig. 2a). Kanamycin knockout modA::kan mutants were also generated in all the five NTHi strains bearing these alleles (Fig. 1e) by disruption of the modA gene via insertion of a kanamycin resistance cassette, as described previously610.


A biphasic epigenetic switch controls immunoevasion, virulence and niche adaptation in non-typeable Haemophilus influenzae.

Atack JM, Srikhanta YN, Fox KL, Jurcisek JA, Brockman KL, Clark TA, Boitano M, Power PM, Jen FE, McEwan AG, Grimmond SM, Smith AL, Barenkamp SJ, Korlach J, Bakaletz LO, Jennings MP - Nat Commun (2015)

Western blots showing ModA presence in modA ON OFF and kanamycin knockout strains and an example of the fragment analysis methodology.(a) Western blot analysis using ON/OFF/kan cells from all the five modA alleles; anti-modA primary antibody at 1:5,000; AP conjugated anti-rabbit secondary at 1:20,000. A modA band is only present in those cells possessing the ON variant of each strain. The number of repeats (reps) represents the major number of repeats present in that particular isolate, and the percentage ON of that particular strain is noted underneath each relevant blot. Full western blots are presented in Supplementary Fig. 4; (b) an illustration of the methodology used during fragment analysis to ascertain modA ON/OFF state and percentages of cell populations. A 6-carboxyflourescein (FAM)-labelled forward primer (green hexagon) and unlabelled reverse primer was used to generate a PCR product over the repeat tract (AGCCn; grey box) that was analysed using GeneScan technology; and (c) an example trace produced by GeneScan analysis on a representative genomic sample from an NTHi strain containing a phase variable modA gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525171&req=5

f2: Western blots showing ModA presence in modA ON OFF and kanamycin knockout strains and an example of the fragment analysis methodology.(a) Western blot analysis using ON/OFF/kan cells from all the five modA alleles; anti-modA primary antibody at 1:5,000; AP conjugated anti-rabbit secondary at 1:20,000. A modA band is only present in those cells possessing the ON variant of each strain. The number of repeats (reps) represents the major number of repeats present in that particular isolate, and the percentage ON of that particular strain is noted underneath each relevant blot. Full western blots are presented in Supplementary Fig. 4; (b) an illustration of the methodology used during fragment analysis to ascertain modA ON/OFF state and percentages of cell populations. A 6-carboxyflourescein (FAM)-labelled forward primer (green hexagon) and unlabelled reverse primer was used to generate a PCR product over the repeat tract (AGCCn; grey box) that was analysed using GeneScan technology; and (c) an example trace produced by GeneScan analysis on a representative genomic sample from an NTHi strain containing a phase variable modA gene.
Mentions: We generated modA natural ON- and OFF-enriched populations (>90% ON or OFF) from the five NTHi strains containing the modA2, 4, 5, 9 and 10 alleles (Fig. 1e). All ON strains contain a number of AGCC repeats that places the modA open reading frame (ORF) in frame, and therefore express a full-length functional protein, as confirmed by western blot using an anti-ModA antibody (Fig. 2a). All the natural ON and OFF strains were continually verified using our well-established FAM-labelled primer PCR screen coupled to fragment length analysis (an example of the methodology and results is given in Fig. 2b,c)610. Natural modAOFF strains contain a number of AGCC repeats that place the modA ORF out of frame, leading to a frame shift mutation and premature termination at stop codons in the alternate reading frames (Fig. 2a). Kanamycin knockout modA::kan mutants were also generated in all the five NTHi strains bearing these alleles (Fig. 1e) by disruption of the modA gene via insertion of a kanamycin resistance cassette, as described previously610.

Bottom Line: Here, we use single molecule, real-time (SMRT) methylome analysis to identify the DNA-recognition motifs for all five of these modA alleles.Phase variation of these alleles regulates multiple proteins including vaccine candidates, and key virulence phenotypes such as antibiotic resistance (modA2, modA5, modA10), biofilm formation (modA2) and immunoevasion (modA4).Our results indicate that a biphasic epigenetic switch can control bacterial virulence, immunoevasion and niche adaptation in an animal model system.

View Article: PubMed Central - PubMed

Affiliation: Institute for Glycomics, Griffith University, Gold Coast, Queensland 4222, Australia.

ABSTRACT
Non-typeable Haemophilus influenzae contains an N(6)-adenine DNA-methyltransferase (ModA) that is subject to phase-variable expression (random ON/OFF switching). Five modA alleles, modA2, modA4, modA5, modA9 and modA10, account for over two-thirds of clinical otitis media isolates surveyed. Here, we use single molecule, real-time (SMRT) methylome analysis to identify the DNA-recognition motifs for all five of these modA alleles. Phase variation of these alleles regulates multiple proteins including vaccine candidates, and key virulence phenotypes such as antibiotic resistance (modA2, modA5, modA10), biofilm formation (modA2) and immunoevasion (modA4). Analyses of a modA2 strain in the chinchilla model of otitis media show a clear selection for ON switching of modA2 in the middle ear. Our results indicate that a biphasic epigenetic switch can control bacterial virulence, immunoevasion and niche adaptation in an animal model system.

No MeSH data available.


Related in: MedlinePlus