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

John M Atack,Sean M Grimmond,Michael P Jennings,Stephen J Barenkamp,Freda E.-C Jen,Jonas Korlach,Arnold L Smith,Alastair G Mcewan,Peter M Power,Matthew Boitano,Joseph A Jurcisek,Kate L Fox,Lauren O Bakaletz,Yogitha N Srikhanta,Kenneth L Brockman,Tyson A Clark

doi:10.1038/ncomms8828

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.

Highlights

Non-typeable Haemophilus influenzae contains an N6-adenine DNA-methyltransferase (ModA) that is subject to phase-variable expression
While phase variation is typically associated with genes that encode surface structures, several host-adapted bacterial pathogens, including Non-typeable Haemophilus influenzae (NTHi), have DNA methyltransferases associated with type III restriction modification systems that are subject to phase-variable expression
We conducted a detailed analysis examining the modA allele frequency in a diverse set of NTHi isolates taken from healthy individuals and otitis media (OM) patients (Fig. 1)

Summary

Introduction

Non-typeable Haemophilus influenzae contains an N6-adenine DNA-methyltransferase (ModA) that is subject to phase-variable expression (random ON/OFF switching). 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). In H. influenzae strain Rd, our previous work has demonstrated that random switching of the modA1 gene controls expression of multiple genes via differential methylation of the genome in the modA1 ON and OFF states[10] This novel genetic system, termed the phasevarion (phase-variable regulon) regulates gene expression in four other important human pathogens: Neisseria gonorrhoeae, Neisseria meningitidis[7], Helicobacter pylori[9] and Moraxella catarrhalis[14]. In an in vivo animal model, we demonstrate, for the first time, direct selection for a particular state of modA expression in the major phase variable modA allele present in NTHi, modA2

Methods

Results

Conclusion