The ModA2 Phasevarion of nontypeable Haemophilus influenzae Regulates Resistance to Oxidative Stress and Killing by Human Neutrophils

Abstract

Nontypeable Haemophilus influenzae (NTHI) is the causative agent of multiple respiratory tract infections. Several human pathogens, including NTHI, possess a novel genetic system, termed the phasevarion, which mediates a rapid and reversible change in the expression of many genes throughout the chromosome. This occurs by phase variation of a single gene (modA) that encodes a DNA methyltransferase and results in two phenotypically distinct subpopulations, ON and OFF. NTHI encounters many pressures within the various microenvironments of its human host as the disease course evolves from one of asymptomatic nasopharyngeal carriage to overt disease. These include oxidative stresses, which are present throughout the respiratory tract. To persist in the human nasopharynx and as a pathogen throughout the airways, NTHI must be able to mitigate toxic levels of oxidative stress. Here we show that expression of ModA2, modA2 ON status, resulted in increased sensitivity to oxidative stress. Furthermore, the modA2 ON status resulted in decreased resistance to neutrophil-mediated killing, which resulted in selection for the modA2 OFF subpopulation in an ex vivo survival assay. These findings highlight the importance of the ModA2 phasevarion in adaptation to innate host defences and reveal an additional microenvironmental pressure that selected for a specific ModA2 subpopulation.

Epigenetic Regulation Alters Biofilm Architecture and Composition in Multiple Clinical Isolates of Nontypeable Haemophilus influenzae

“…To identify key phasevarion-regulated factors that contributed to the observed differences in biofilm structure, we first investigated biofilm formation by NTHI strain 723. NTHI strain 723 carries the modA2 allele and has been studied in vitro as well as in the chinchilla model of OM ( 12 , 13 , 29 ). Locked versions of NTHI strain 723, in which modA2 is unable to phase vary, were utilized, and biofilm formation was assessed after both 16 and 24 h ( 29 ).…”

“…NTHI strain 723 carries the modA2 allele and has been studied in vitro as well as in the chinchilla model of OM ( 12 , 13 , 29 ). Locked versions of NTHI strain 723, in which modA2 is unable to phase vary, were utilized, and biofilm formation was assessed after both 16 and 24 h ( 29 ). As was observed with the enriched populations that were free to phase vary ( Fig.…”

“…Phasevarions have been identified in multiple human mucosa-associated pathogens, all of which are known to form biofilms during pathogenesis and disease. The phasevarions of several species have been shown to regulate disease-relevant phenotypes such as the ability to acquire nutrients and resistance to oxidative stress ( 29 ). Here we show for the first time that the phasevarions of multiple NTHI clinical isolates regulated biofilm formation under host-specific microenvironmental conditions, yet others, such as the ModA9 phasevarion of strain 1209, had no effect on biofilm formation.…”

“…NTHI strain 723 modA2 locked ON and modA2 locked OFF populations that were engineered to constitutively expressed GFP ( 29 ) were inoculated into the chambers of an eight-well-chambered cover glass, as described above. After 16 h, biofilms were rinsed twice with DPBS to remove nonadherent bacteria and then incubated with anti-NTHI HU polyclonal rabbit sera (1:200) for 30 min or commercially available murine antibody against dsDNA (AbCam) (1:200) for 1 h. Biofilms were then rinsed and incubated for 15 min with goat anti-rabbit or goat anti-mouse IgG secondary antibody conjugated to Alexa Fluor 647 (Thermo Fisher, Waltham, MA).…”

Subversion of host immune responses by otopathogens during otitis media

“…Further, biofilms formed in alkaline environments have an associated increase in HMW adhesins in the modA2 ON phase version group . ModA2 ON is also associated with greater susceptibility to oxidative stress and less resistance to neutrophil‐directed killing . Protection against oxidative stress is also seen in other OM pathogens, such as S. pneumoniae by manipulation of the thiol‐specific antioxidant (TlpA/TSA), which is an additional example of host immune evasion…”