High-depth RNA-Seq data sets to investigate the differences in gene expression mediated by phasevarions in non-typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae (NTHi) is a major bacterial pathogen of the human airway. We report high-depth coverage RNA-Seq data from prototype NTHi strains 723 and R2866, encoding two of the most common phase-variable ModA alleles found in NTHi strains, ModA2 and ModA10, respectively.

Phase variable acetylation of lipooligosaccharide modifies antibody production and opsonophagocytic killing of non-typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae (NTHi) causes millions of infections each year. Though it is primarily known to cause otitis media, recent studies have shown NTHi is emerging as a primary pathogen for invasive infection, prompting the need for new vaccines and treatments. Lipooligosaccharide (LOS) has been identified as a potential vaccine candidate due to its immunogenic nature and outer membrane localization. Yet, phase variable expression of genes involved in LOS synthesis has complicated vaccine development. In this study, we used a chinchilla model of otitis media to investigate how phase variation of oafA, a gene involved in LOS biosynthesis, affects antibody production in response to infection. We found that acetylation of LOS by OafA inhibited production of LOS-specific antibodies during infection and that NTHi expressing acetylated LOS were subsequently better protected against opsonophagocytic killing. These findings highlight the importance of understanding how phase variable modifications might affect vaccine efficacy and success.

Identification of Virulence Factors Involved in a Murine Model of Severe Achromobacter xylosoxidans Infection.

Achromobacter xylosoxidans (Ax) is an opportunistic pathogen and causative agent of numerous infections particularly in immunocompromised individuals with increasing prevalence in cystic fibrosis (CF). To date, investigations have focused on the clinical epidemiology and genomic comparisons of Ax isolates, yet little is known about disease pathology or the role that specific virulence factors play in tissue invasion or damage. Here, we model an acute Ax lung infection in immunocompetent C57BL/6 mice and immunocompromised CF mice, revealing a link between in vitro cytotoxicity and disease in an intact host. Mice were intratracheally challenged with sublethal doses of a cytotoxic (GN050) or invasive (GN008) strain of Ax. Bacterial burden, immune cell populations, and inflammatory markers in bronchoalveolar lavage fluid and lung homogenates were measured at different time points to assess disease severity. CF mice had a similar but delayed immune response toward both Ax strains compared to C57BL/6J mice. GN050 caused more severe disease and higher mortality which correlated with greater bacterial burden and increased proinflammatory responses in both mouse models. In agreement with the cytotoxicity of GN050 toward macrophages in vitro, mice challenged with GN050 had fewer macrophages. Mutants with transposon insertions in predicted virulence factors of GN050 showed that disease severity depended on the type III secretion system, Vi capsule, antisigma-E factor, and partially on the ArtA adhesin. The development of an acute infection model provides an essential tool to better understand the infectivity of diverse Ax isolates and enable improved identification of virulence factors important to bacterial persistence and disease.

Adherence of Nontypeable Haemophilus influenzae to Cells and Substrates of the Airway Is Differentially Regulated by Individual ModA Phasevarions.

Adherence of nontypeable Haemophilus influenzae (NTHi) to the host airway is an essential initial step for asymptomatic colonization of the nasopharynx, as well as development of disease. NTHi relies on strict regulation of multiple adhesins for adherence to host substrates encountered in the airway. NTHi encode a phase-variable cytoplasmic DNA methyltransferase, ModA, that regulates expression of multiple genes; a phasevarion (phase-variable regulon). Multiple modA alleles are present in NTHi, in which different alleles methylate a different DNA target, and each controls a different set of genes. However, the role of ModA phasevarions in regulating adherence of NTHi to the host airway is not well understood. This study therefore sought to investigate the role of four of the most prevalent ModA phasevarions in the regulation of adherence of NTHi to multiple substrates of the airway. Four clinical isolates of NTHi with unique modA alleles were tested in this study. The adherence of NTHi to mucus, middle ear epithelial cells, and vitronectin was regulated in a substrate-specific manner that was dependent on the ModA allele encoded. The adhesins Protein E and P4 were found to contribute to the ModA-regulated adherence of NTHi to distinct substrates. A better understanding of substrate-specific regulation of NTHi adherence by ModA phasevarions will allow identification of NTHi populations present at the site of disease within the airway and facilitate more directed development of vaccines and therapeutics. IMPORTANCE Nontypeable Haemophilus influenzae (NTHi) is a predominant pathogen of the human airway that causes respiratory infections such as otitis media (OM) and exacerbations in the lungs of patients suffering from chronic obstructive pulmonary disease (COPD). Due to the lack of a licensed vaccine against NTHi and the emergence of antibiotic-resistant strains, it is extremely challenging to target NTHi for treatment. NTHi adhesins are considered potential candidates for vaccines or other therapeutic approaches. The ModA phasevarions of NTHi play a role in the rapid adaptation of the pathogen to different environmental stress conditions. This study addressed the role of ModA phasevarions in the regulation of adherence of NTHi to specific host substrates found within the respiratory tract. The findings of this study improve our understanding of regulation of adherence of NTHi to the airway, which may further be used to enhance the potential of adhesins as vaccine antigens and therapeutic targets against NTHi.

Characterization of the Phase-Variable Autotransporter Lav Reveals a Role in Host Cell Adherence and Biofilm Formation in Nontypeable Haemophilus influenzae.

Lav is an autotransporter protein found in pathogenic Haemophilus and Neisseria species. Lav in nontypeable Haemophilus influenzae (NTHi) is phase-variable: the gene reversibly switches ON-OFF via changes in length of a locus-located GCAA(n) simple DNA sequence repeat tract. The expression status of lav was examined in carriage and invasive collections of NTHi, where it was predominantly not expressed (OFF). Phenotypic study showed lav expression (ON) results in increased adherence to human lung cells and denser biofilm formation. A survey of Haemophilus species genome sequences showed lav is present in ∼60% of NTHi strains, but lav is not present in most typeable H. influenzae strains. Sequence analysis revealed a total of five distinct variants of the Lav passenger domain present in Haemophilus spp., with these five variants showing a distinct lineage distribution. Determining the role of Lav in NTHi will help understand the role of this protein during distinct pathologies.

A Bacterial Epigenetic Switch in Non-typeable Haemophilus influenzae Modifies Host Immune Response During Otitis Media.

Non-typeable Haemophilus influenzae (NTHi) causes multiple diseases of the human airway and is a predominant bacterial pathogen of acute otitis media and otitis media in which treatment fails. NTHi utilizes a system of phase variable epigenetic regulation, termed the phasevarion, to facilitate adaptation and survival within multiple sites of the human host. The NTHi phasevarion influences numerous disease-relevant phenotypes such as biofilm formation, antibiotic resistance, and opsonization. We have previously identified an advantageous selection for a specific phasevarion status, which significantly affects severity and chronicity of experimental otitis media. In this study, we utilized pure cultures of NTHi variants in which modA was either locked ON or locked OFF, and thus modA was unable to phase vary. These locked variants were used to assess the progression of experimental otitis media and define the specific immune response induced by each subpopulation. Although the initial disease caused by each subpopulation was similar, the immune response elicited by each subpopulation was unique. The modA2 OFF variant induced significantly greater activation of macrophages both in vitro and within the middle ear during disease. In contrast, the modA2 ON variant induced a greater neutrophil extracellular trap response, which led to greater killing of the modA2 ON variant. These data suggest that not only does the NTHi phasevarion facilitate adaptation, but also allows the bacteria to alter immune responses during disease. Understanding these complex bacterial-host interactions and the regulation of bacterial factors responsible is critical to the development of better diagnostic, treatment, and preventative strategies for these bacterial pathogens.

The Nontypeable Haemophilus influenzae Major Adhesin Hia Is a Dual-Function Lectin That Binds to Human-Specific Respiratory Tract Sialic Acid Glycan Receptors.

NTHi is a human-adapted pathogen that colonizes the human respiratory tract. Strains of NTHi express multiple adhesins; however, there is a unique, mutually exclusive relationship between the major adhesins Hia and HMW1 and HMW2 (HMW1/2). Approximately 25% of NTHi strains express Hia, a phase-variable autotransporter protein that has a critical role in colonization of the host nasopharynx. The remaining 75% of strains express HMW1/2. Previous work has shown that the HMW1 and HMW2 proteins mediate binding to 2-3- and 2-6-linked sialic acid glycans found in the human respiratory tract. Here, we show that the high-affinity binding domain of Hia, binding domain 1 (BD1), is responsible for binding to α2-6-sialyllactosamine (2-6 SLN) glycans. BD1 is highly specific for glycans that incorporate the form of sialic acid expressed by humans, N-acetylneuraminic acid (Neu5Ac). We further show that Hia has lower-affinity binding activity for 2-3-linked sialic acid and that this binding activity is mediated via a distinct domain. Thus, Hia with its dual binding activities functionally mimics the combined activities of the HMW1 and HMW2 adhesins. In addition, we show that Hia has a role in biofilm formation by strains of NTHi that express the adhesin. Knowledge of the binding affinity of this major NTHi adhesin and putative vaccine candidate will direct and inform development of future vaccines and therapeutic strategies for this important pathogen.IMPORTANCE Host-adapted bacterial pathogens like NTHi have evolved specific mechanisms to colonize their restricted host niche. Relatively few of the adhesins expressed by NTHi have been characterized as regards their binding affinity at the molecular level. In this work, we show that the major NTHi adhesin Hia preferentially binds to Neu5Ac-α2-6-sialyllactosamine, the form of sialic acid expressed in humans. The receptors targeted by Hia in the human airway mirror those targeted by influenza A virus and indicates the broad importance of sialic acid glycans as receptors for microbes that colonize the human airway.

The TRPM2 Ion Channel Regulates Inflammatory Functions of Neutrophils During Listeria monocytogenes Infection.

During infection, phagocytic cells pursue homeostasis in the host via multiple mechanisms that control microbial invasion. Neutrophils respond to infection by exerting a variety of cellular processes, including chemotaxis, activation, phagocytosis, degranulation and the generation of reactive oxygen species (ROS). Calcium (Ca2+) signaling and the activation of specific Ca2+ channels are required for most antimicrobial effector functions of neutrophils. The transient receptor potential melastatin-2 (TRPM2) cation channel has been proposed to play important roles in modulating Ca2+ mobilization and oxidative stress in neutrophils. In the present study, we use a mouse model of Listeria monocytogenes infection to define the role of TRPM2 in the regulation of neutrophils' functions during infection. We show that the susceptibility of Trpm2-/- mice to L. monocytogenes infection is characterized by increased migration rates of neutrophils and monocytes to the liver and spleen in the first 24 h. During the acute phase of L. monocytogenes infection, Trpm2-/- mice developed septic shock, characterized by increased serum levels of TNF-α, IL-6, and IL-10. Furthermore, in vivo depletion of neutrophils demonstrated a critical role of these immune cells in regulating acute inflammation in Trpm2-/- infected mice. Gene expression and inflammatory cytokine analyses of infected tissues further confirmed the hyperinflammatory profile of Trpm2-/- neutrophils. Finally, the increased inflammatory properties of Trpm2-/- neutrophils correlated with the dysregulated cytoplasmic concentration of Ca2+ and potentiated membrane depolarization, in response to L. monocytogenes. In conclusion, our findings suggest that the TRPM2 channel plays critical functional roles in regulating the inflammatory properties of neutrophils and preventing tissue damage during Listeria infection.

Regulation and Maturation of the Shewanella oneidensis Sulfite Reductase SirA.

Shewanella oneidensis, a metal reducer and facultative anaerobe, expresses a large number of c-type cytochromes, many of which function as anaerobic reductases. All of these proteins contain the typical heme-binding motif CXXCH and require the Ccm proteins for maturation. Two c-type cytochrome reductases also possess atypical heme-binding sites, the NrfA nitrite reductase (CXXCK) and the SirA sulfite reductase (CX12NKGCH). S. oneidensis MR-1 encodes two cytochrome c synthetases (CcmF and SirE) and two apocytochrome c chaperones (CcmI and SirG). SirE located in the sir gene cluster is required for the maturation of SirA, but not NrfA. Here we show that maturation of SirA requires the combined function of the two apocytochrome c chaperones CcmI and SirG. Loss of either protein resulted in decreased sulfite reductase. Furthermore, SirA was not detected in a mutant that lacked both chaperones, perhaps due to misfolding or instability. These results suggest that CcmI interacts with SirEFG during SirA maturation, and with CcmF during maturation of NrfA. Additionally, we show that CRP regulates expression of sirA via the newly identified transcriptional regulatory protein, SirR.

Biofilm biology and vaccine strategies for otitis media due to nontypeable Haemophilus influenzae.

Otitis media (OM) is one of the most common diseases of childhood, and nontypeable Haemophilus influenzae (NTHI) is the predominant causative agent of chronic and recurrent OM, as well as OM for which treatment has failed. Moreover, NTHI is now as important a causative agent of acute OM as the pneumococcus. NTHI colonizes the human nasopharynx asymptomatically. However, upon perturbation of the innate and physical defenses of the airway by upper respiratory tract viral infection, NTHI can replicate, ascend the Eustachian tube, gain access to the normally sterile middle ear space, and cause disease. Bacterial biofilms within the middle ear, including those formed by NTHI, contribute to the chronic and recurrent nature of this disease. These multicomponent structures are highly resistant to clearance by host defenses and elimination by traditional antimicrobial therapies. Herein, we review several strategies utilized by NTHI in order to persist within the human host and interventions currently under investigation to prevent and/or resolve NTHI-induced diseases of the middle ear and uppermost airway.

Antibodies against the Majority Subunit (PilA) of the Type IV Pilus of Nontypeable Haemophilus influenzae Disperse Moraxella catarrhalis from a Dual-Species Biofilm.

Otitis media (OM) is often polymicrobial, with nontypeable Haemophilus influenzae (NTHI) and Moraxella catarrhalis (Mcat) frequently cocultured from clinical specimens. Bacterial biofilms in the middle ear contribute to the chronicity and recurrence of OM; therefore, strategies to disrupt biofilms are needed. We have focused our vaccine development efforts on the majority subunit of NTHI type IV pili, PilA. Antibodies against a recombinant, soluble form of PilA (rsPilA) both disrupt and prevent the formation of NTHI biofilms in vitro. Moreover, immunization with rsPilA prevents and resolves NTHI-induced experimental OM. Here, we show that antibodies against rsPilA also prevent and disrupt polymicrobial biofilms. Dual-species biofilms formed by NTHI and Mcat at temperatures that mimic the human nasopharynx (34°C) or middle ear (37°C) were exposed to antiserum against either rsPilA or the OMP P5 adhesin of NTHI. NTHI+Mcat biofilm formation was significantly inhibited by antiserum directed against both adhesin proteins at either temperature. However, only anti-rsPilA disrupted NTHI+Mcat preestablished biofilms at either temperature and actively dispersed both NTHI and Mcat via interspecies quorum signaling. Newly released NTHI and Mcat were significantly more susceptible to killing by antibiotics. Taken together, these results revealed new opportunities for treatment of biofilm-associated diseases via a strategy that combines vaccine-induced antibody-mediated biofilm dispersal with traditional antibiotics, at a significantly reduced dosage to exploit the newly released, antibiotic-sensitive phenotype. Combined, our data strongly support the utility of rsPilA both as a preventative and as a therapeutic vaccine antigen for polymicrobial OM due to NTHI and Mcat.IMPORTANCE Middle ear infections (or otitis media [OM]) are highly prevalent among children worldwide and present a tremendous socioeconomic challenge for health care systems. More importantly, this disease diminishes the quality of life of young children. OM is often chronic and recurrent, due to the presence of highly antibiotic-resistant communities of bacteria (called biofilms) that persist within the middle ear space. To combat these recalcitrant infections, new and powerful biofilm-directed approaches are needed. Here, we describe the ability to disrupt a biofilm formed by the two most common bacteria that cause chronic and recurrent OM in children, via an approach that combines the power of vaccines with that of traditional antibiotics. An outcome of this strategy is that antibiotics can more easily kill the bacteria that our vaccine-induced antibodies have released from the biofilm. We believe that this approach holds great promise for both the prevention and treatment of OM.

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

Biofilms play a critical role in the colonization, persistence, and pathogenesis of many human pathogens. Multiple mucosa-associated pathogens have evolved a mechanism of rapid adaptation, termed the phasevarion, which facilitates a coordinated regulation of numerous genes throughout the bacterial genome. This epigenetic regulation occurs via phase variation of a DNA methyltransferase, Mod. The phasevarion of nontypeable Haemophilus influenzae (NTHI) significantly affects the severity of experimental otitis media and regulates several disease-related processes. However, the role of the NTHI phasevarion in biofilm formation is unclear. The present study shows that the phasevarions of multiple NTHI clinical isolates regulate in vitro biofilm formation under disease-specific microenvironmental conditions. The impact of phasevarion regulation was greatest under alkaline conditions that mimic those known to occur in the middle ear during disease. Under alkaline conditions, NTHI strains that express the ModA2 methyltransferase formed biofilms with significantly greater biomass and less distinct architecture than those formed by a ModA2-deficient population. The biofilms formed by NTHI strains that express ModA2 also contained less extracellular DNA (eDNA) and significantly less extracellular HU, a DNABII DNA-binding protein critical for biofilm structural stability. Stable biofilm structure is critical for bacterial pathogenesis and persistence in multiple experimental models of disease. These results identify a role for the phasevarion in regulation of biofilm formation, a process integral to the chronic nature of many infections. Understanding the role of the phasevarion in biofilm formation is critical to the development of prevention and treatment strategies for these chronic diseases.IMPORTANCE Upper respiratory tract infections are the number one reason for a child to visit the emergency department, and otitis media (middle ear infection) ranks third overall. Biofilms contribute significantly to the chronic nature of bacterial respiratory tract infections, including otitis media, and make these diseases particularly difficult to treat. Several mucosa-associated human pathogens utilize a mechanism of rapid adaptation termed the phasevarion, or phasevariable regulon, to resist environmental and host immune pressures. In this study, we assessed the role of the phasevarion in regulation of biofilm formation by nontypeable Haemophilus influenzae (NTHI), which causes numerous respiratory tract diseases. We found that the NTHI phasevarion regulates biofilm structure and critical biofilm matrix components under disease-specific conditions. The findings of this work could be significant in the design of improved strategies against NTHI infections, as well as diseases due to other pathogens that utilize a phasevarion.

Dysregulated Calcium Homeostasis in Cystic Fibrosis Neutrophils Leads to Deficient Antimicrobial Responses.

Cystic fibrosis (CF), one of the most common human genetic diseases worldwide, is caused by a defect in the CF transmembrane conductance regulator (CFTR). Patients with CF are highly susceptible to infections caused by opportunistic pathogens (including Burkholderia cenocepacia), which induce excessive lung inflammation and lead to the eventual loss of pulmonary function. Abundant neutrophil recruitment into the lung is a key characteristic of bacterial infections in CF patients. In response to infection, inflammatory neutrophils release reactive oxygen species and toxic proteins, leading to aggravated lung tissue damage in patients with CF. The present study shows a defect in reactive oxygen species production by mouse Cftr-/- , human F508del-CFTR, and CF neutrophils; this results in reduced antimicrobial activity against B. cenocepacia Furthermore, dysregulated Ca2+ homeostasis led to increased intracellular concentrations of Ca2+ that correlated with significantly diminished NADPH oxidase response and impaired secretion of neutrophil extracellular traps in human CF neutrophils. Functionally deficient human CF neutrophils recovered their antimicrobial killing capacity following treatment with pharmacological inhibitors of Ca2+ channels and CFTR channel potentiators. Our findings suggest that regulation of neutrophil Ca2+ homeostasis (via CFTR potentiation or by the regulation of Ca2+ channels) can be used as a new therapeutic approach for reestablishing immune function in patients with CF.

The HMW2 adhesin of non-typeable Haemophilus influenzae is a human-adapted lectin that mediates high-affinity binding to 2-6 linked N-acetylneuraminic acid glycans.

Non-typeable Haemophilus influenzae (NTHi) is a human-adapted bacterial pathogen, responsible for infections of the human respiratory tract. This pathogen expresses a range of adhesins that mediate binding to host cells. Most NTHi strains can express the related adhesins HMW1 and HMW2. Expression of HMW proteins is phase-variable: changes in the length of simple-sequence repeats located in the encoding genes promoter regions results in changes in expression levels of these adhesins. HMW expression is also controlled by epigenetic regulation. HMW1 has been previously demonstrated to bind α 2-3 sialyl-lactosamine, but affinity of this interaction has not been investigated. The host receptor(s) for HMW2 is currently unknown. We hypothesized that host glycans may act as receptors for HMW2-mediated adherence. We examined the glycan-binding activity of HMW2 using glycan arrays and Surface Plasmon Resonance (SPR). These studies demonstrate that HMW2 binds 2-6 linked N-acetylneuraminic acid with high affinity. HMW2 did not bind glycan structures containing the non-human form of sialic acid, N-glycolylneuraminic acid. Thus, the specificity of HMW1 and HMW2 have complementary lectin activities that may allow NTHi distinct niches in the human host.

Nontypeable Haemophilus influenzae releases DNA and DNABII proteins via a T4SS-like complex and ComE of the type IV pilus machinery.

Biofilms formed by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and resistance to treatment of multiple human respiratory tract diseases including otitis media, chronic rhinosinusitis, and exacerbations of both cystic fibrosis and chronic obstructive pulmonary disease. Extracellular DNA (eDNA) and associated DNABII proteins are essential to the overall architecture and structural integrity of biofilms formed by NTHI and all other bacterial pathogens tested to date. Although cell lysis and outer-membrane vesicle extrusion are possible means by which these canonically intracellular components might be released into the extracellular environment for incorporation into the biofilm matrix, we hypothesized that NTHI additionally used a mechanism of active DNA release. Herein, we describe a mechanism whereby DNA and associated DNABII proteins transit from the bacterial cytoplasm to the periplasm via an inner-membrane pore complex (TraC and TraG) with homology to type IV secretion-like systems. These components exit the bacterial cell through the ComE pore through which the NTHI type IV pilus is expressed. The described mechanism is independent of explosive cell lysis or cell death, and the release of DNA is confined to a discrete subpolar location, which suggests a novel form of DNA release from viable NTHI. Identification of the mechanisms and determination of the kinetics by which critical biofilm matrix-stabilizing components are released will aid in the design of novel biofilm-targeted therapeutic and preventative strategies for diseases caused by NTHI and many other human pathogens known to integrate eDNA and DNABII proteins into their biofilm matrix.

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

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.

ModA2 Phasevarion Switching in Nontypeable Haemophilus influenzae Increases the Severity of Experimental Otitis Media.

Several human-adapted bacterial pathogens use a phasevarion (ie, a phase-variable regulon) to rapidly and reversibly regulate the expression of many genes, which include known virulence factors, yet the influence of phasevarion-mediated regulation in pathogenesis remains poorly understood. Here we examine the impact of the nontypeable Haemophilus influenzae (NTHI) ModA2 phasevarion on pathogenesis and disease severity in a chinchilla model of experimental otitis media. Chinchillas were challenged with NTHI variant populations that were either inoculated ON and remained ON, inoculated OFF and shifted ON, or inoculated OFF and remained OFF, within the middle ear. We show that populations that shift from OFF to ON within the middle ear induce significantly greater disease severity than populations that are unable to shift. These observations support the importance of phasevarion switching in NTHI pathogenesis and the necessity to considered phasevarion regulation when developing methods to treat and prevent infection.

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

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.