Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence

Abstract

Nontypeable Haemophilus influenzae (NTHI) is a leading cause of opportunistic infections of the respiratory tract in children and adults. Although considered an extracellular pathogen, NTHI has been observed repeatedly within and between cells of the human respiratory tract, and these observations have been correlated to symptomatic infection. These findings are intriguing in light of the knowledge that NTHI persists in the respiratory tract despite antibiotic therapy and the development of bactericidal antibodies. We hypothesized that intracellular NTHI avoids, escapes, or neutralizes the endolysosomal pathway and persists within human respiratory epithelial cells and that human IgA1 proteases are required for optimal internalization and persistence of NTHI. Virtually all strains encode a human IgA1 protease gene, igaA, and we previously characterized a novel human IgA1 protease gene, igaB, that is associated with disease-causing strains and is homologous to the IgA1 protease that is unique to pathogenic Neisseria spp. Here, we show that NTHI invades human bronchial epithelial cells in vitro in a lipid raft-independent manner, is subsequently trafficked via the endolysosomal pathway, and is killed in lysosomes after variable durations of persistence. IgaA is required for optimal invasion. IgaB appears to play little or no role in adherence or invasion but is required for optimal intracellular persistence of NTHI. IgaB cleaves lysosome-associated membrane protein 1 (LAMP1) at pHs characteristic of the plasma membrane, early endosome, late endosome, and lysosome. However, neither IgA1 protease inhibits acidification of intracellular vesicles containing NTHI. NTHI IgA1 proteases play important but different roles in NTHI invasion and trafficking in respiratory epithelial cells. Nontypeable Haemophilus influenzae (NTHI) is a Gram-negative, human-exclusive commensal bacterium of the nasopharynx and is a leading cause of opportunistic infections in the upper and lower respiratory tracts, including otitis media, sinusitis, conjunctivitis, community-acquired pneumonia, and exacerbations of chronic obstructive pulmonary disease (COPD) and of cystic fibrosis (1, 2).When a new strain of NTHI is acquired, the outcome depends on a variety of dynamic host and bacterial factors. Colonization and the transition from commensal to opportunistic pathogen require NTHI to resist host innate immune defenses, including nutrient sequestration, mucociliary clearance, antimicrobial peptides, secretory IgA1, and phagocytosis by immune cells or by epithelial cells. Bacterial counterresistance mechanisms under investigation include adhesins, antimicrobial peptide degradation, IgA1 protease, biofilm production, modification of surface-exposed lipooligosaccharide moieties, production of outer membrane vesicles (OMVs), and others (3-12). Understanding these factors will help identify targets for therapeutic intervention, as NTHI infections induce acquired immunity consisting largely of strain-specific bactericidal antibodies that afford littl...

Comprehensive Proteomic and Metabolomic Signatures of Nontypeable Haemophilus influenzae-Induced Acute Otitis Media Reveal Bacterial Aerobic Respiration in an Immunosuppressed Environment

“…5). These data suggest that Arp2/3 participates in NTHI internalization with subsequent intracellular trafficking to the endolysosomal pathway (59).…”

Cellular interaction of nontypeableHaemophilus influenzaetriggers cytotoxicity of infected type II alveolar cells via apoptosis

“…Interestingly, AO and monodansylcadaverine staining have revealed the nature of acidic vacuolar compartments containing large numbers of NTHi to be derived from autophagosomes. Recent studies have suggested the role of lysosomal machinery (lysotracker, lamp-1, lamp-2, CD63, and Rab7) (Morey et al, 2011;Clementi et al, 2014) but no autophagic markers had been linked with NTHi vacuolar uptake. Future studies are required to broadly monitor the role of autophagy during NTHi infection using more rigorous markers.…”

“…We were able to enumerate the viable number of intracellular NTHi until 8 h as monolayer begins to lose its integrity at later time points of infection. Recently, Clementi et al (2014) reported killing of intracellular NTHi in lysosomes 24 h postinfection. Differences observed in terms of time points of infection between our data and previous reports could be due to variation in experimental conditions, procedures, host cell types, NTHi strains, and MOI used.…”

Relationship between Azithromycin Susceptibility and Administration Efficacy for Nontypeable Haemophilus influenzae Respiratory Infection

“…S1 in the supplemental material). We and others previously described that NTHI invades epithelial cells and localizes to an NTHI-containing vacuole (NTHI-CV), a nonproliferative compartment with late endosome features (51,57,58). Given that macrolides present good penetration of lung tissue, are acid resistant, and accumulate in lysosome-like compartments (11), we asked if AZM could modulate NTHI in- .…”