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

Abstract

Non-typeable Haemophilus influenzae contains an N6-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.

The modA10 phasevarion of nontypeable Haemophilus influenzae R2866 regulates multiple virulence-associated traits

“…These phase variable regulons, or phasevarions [7], control expression of surface antigens and virulence factors, leading to altered phenotypes between Mod ON and OFF variants. Phasevarions have been studied in H. influenzae (ModA; [8,9], N. gonorrhoeae and N. meningitidis (ModA, ModB and ModD; (40-42); H. pylori (ModH; [13] and M. catarrhalis (ModM [14]). The structure of each class of Mod proteins (ModA, B, D, H, and M) are highly conserved, apart from the central DNA recognition domain (DRD) that dictates methylation specificity [15].…”

“…However, over 60% of all NTHi clinical isolates contain just five phase variable modA alleles – modA2 , 4 , 5 , 9 and 10 [8], with modA10 is present in approximately 15% of all clinical isolates, making it second only in prevalence in clinical NTHi samples to modA2 [8]. The modA2 ON state was shown to be preferentially selected for in an in vivo chinchilla model, with all five of the most prevalent modA alleles regulating expression of multiple proteins including a number of current and putative vaccine candidates [8]. Strain R2866 is unusual amongst NTHi in that it was isolated from the blood of a normal child with meningitis [16].…”

“…Strain R2866 is unusual amongst NTHi in that it was isolated from the blood of a normal child with meningitis [16]. We have shown that strain R2866 cultures with 90% modA10 ON cells have increased expression of OMP P6 and OMP P6 in comparison to cultures with modA10 predominantly OFF [8]. …”

Simultaneous Identification of Haemophilus Influenzae and Haemophilus Haemolyticus using Real-Time PCR

“…genomes were available for this study: 316 H. influenzae (145 generated in the current study, 19 unpublished and 152 published elsewhere [6,[18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34]), 59 H. haemolyticus (44 generated in the current study, one unpublished and 14 published elsewhere [6,26,28,[35][36] Table 1). The hypD assay differentiates H. haemolyticus (red) from all other species and the siaT assay differentiates H. influenzae (blue), 'Clade I' H. influenzae (purple) and fucose operon-negative H. influenzae strains (green) from all other species.…”

Immunogenicity of Nontypeable Haemophilus influenzae Outer Membrane Vesicles and Protective Ability in the Chinchilla Model of Otitis Media

“…However, HMW1/ HMW2-and Hia-specific antisera mediate relatively broad-based killing of heterologous NTHi (21), suggesting that the strain heterogeneity present among these proteins would not preclude their potential use as vaccine components. Both the HMW1/HMW2 and Hia proteins are subject to phase variation in the face of immune pressure, resulting in selection for bacterial variants downregulated in HMW1/HMW2 and Hia protein expression (14,22). This property could limit the value of the HMW1/HMW2 and Hia proteins as vaccine components.…”