Nontypeable Haemophilus influenzae challenge during gammaherpesvirus infection enhances viral reactivation and latency.

Gammaherpesviruses are ubiquitous, lifelong pathogens associated with multiple cancers that infect over 95% of the adult population. Increases in viral reactivation, due to stress and other unknown factors impacting the immune response, frequently precedes lymphomagenesis. One potential stressor that could promote viral reactivation and increase viral latency would be the myriad of infections from bacterial and viral pathogens that we experience throughout our lives. Using murine gammaherpesvirus 68 (MHV68), a mouse model of gammaherpesvirus infection, we examined the impact of bacterial challenge on gammaherpesvirus infection. We challenged MHV68 infected mice during the establishment of latency with nontypeable Haemophilus influenzae (NTHi) to determine the impact of bacterial infection on viral reactivation and latency. Mice infected with MHV68 and then challenged with NTHi, saw increases in viral reactivation and viral latency. These data support the hypothesis that bacterial challenge can promote gammaherpesvirus reactivation and latency establishment, with possible consequences for viral lymphomagenesis.

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.