Evidence for a non-replicative intracellular stage of nontypable Haemophilus influenzae in epithelial cells

Abstract

Nontypable Haemophilus influenzae (NTHi) is a Gram-negative, non-capsulated human bacterial pathogen, a major cause of a repertoire of respiratory infections, and intimately associated with persistent lung bacterial colonization in patients suffering from chronic obstructive pulmonary disease (COPD). Despite its medical relevance, relatively little is known about its mechanisms of pathogenicity. In this study, we found that NTHi invades the airway epithelium by a distinct mechanism, requiring microtubule assembly, lipid rafts integrity, and activation of phosphatidylinositol 3-kinase (PI3K) signalling. We found that the majority of intracellular bacteria are located inside an acidic subcellular compartment, in a metabolically active and non-proliferative state. This NTHi-containing vacuole (NTHi-CV) is endowed with late endosome features, co-localizing with LysoTracker, lamp-1, lamp-2, CD63 and Rab7. The NTHi-CV does not acquire Golgi-or autophagy-related markers. These observations were extended to immortalized and primary human airway epithelial cells. By using NTHi clinical isolates expressing different amounts of phosphocholine (PCho), a major modification of NTHi lipooligosaccharide, on their surfaces, and an isogenic lic1BC mutant strain lacking PCho, we showed that PCho is not responsible for NTHi intracellular location. In sum, this study indicates that NTHi can survive inside airway epithelial cells. INTRODUCTIONHaemophilus influenzae is a Gram-negative human pathogen that colonizes asymptomatically the nasopharynx of healthy individuals. Disease occurs when the bacterium reaches privileged anatomical sites under various predisposing conditions, including, among others, age, viral infections or a constant exposure to pollutants, leading to otitis media, sinusitis, meningitis, septicaemia or respiratory infections (Foxwell et al., 1998;Rao et al., 1999). Nontypable H. influenzae (NTHi) is the most frequently isolated bacterial pathogen in the lungs of patients suffering from chronic obstructive pulmonary disease (COPD), and furthermore, NTHi accounts for the majority of bacterial COPD exacerbation episodes (Sethi et al., 2002;Sethi & Murphy, 2001.One of the NTHi virulence factors that has been most extensively analysed is its lipooligosaccharide (LOS). It consists of lipid A, an inner core consisting of a Abbreviations: COPD, chronic obstructive pulmonary disease; EEA1, early endosome antigen 1; FISH, fluorescent in situ hybridization; LOS, lipooligosaccharide; MbCD, methyl-b-cyclodextrin; NHBE cells, normal human bronchial epithelial cells; NTHi, nontypable Haemophilus influenzae; NTHi-CV, NTHi-containing vacuole; PCho, phosphocholine; PFA, paraformaldehyde; PI3K, phosphatidylinositol 3-kinase; SCV, Salmonella-containing vacuole; TEM, transmission electron microscopy. phosphorylated 2-keto-3-deoxyoctulosonic acid (KDO) linked to three heptoses and an outer core consisting of a heteropolymer of glucose and galactose (Schweda et al., 2007). Similar to other bacterial pathogens adapted to mucosal surfaces,...

FOXO Transcription Factors Regulate Innate Immune Mechanisms in Respiratory Epithelial Cells

“…In our experiments, we found that activation of the PI3K pathway, which causes FOXO inhibition (26), is capable of enhancing invasion of NTHi in airway epithelial cells as previously described for alveolar epithelial cells (35). Beyond that, we found that silencing of FOXO results in higher intracellular bacterial counts.…”

“…Another approach that we used to evaluate FOXO innate immune functions was an NTHi uptake assay, a common method to detect intracellular bacteria (35,40). In our experiments, we found that activation of the PI3K pathway, which causes FOXO inhibition (26), is capable of enhancing invasion of NTHi in airway epithelial cells as previously described for alveolar epithelial cells (35).…”

“…Degradation and discharge of microbial PAMPs can also be associated with endosomal processing of internalized bacteria. In fact, it has been shown that bacteria internalized by respiratory epithelial cells end up in the endosomal compartment of the cell (35). Considering these points, we assumed that FOXO is involved in an endosomal pathway that requires uptake and degradation of the pathogen.…”

“…One possibility is that NTHi can invade FOXO-deficient cells more easily given their reduced ability to produce antimicrobial factors like defensins, which are likely to affect intracellular killing of bacteria. Thus, internalization may be promoted by the pathogen to evade the immune response, persist intracellularly, and establish colonization (35). The second possibility is that epithelial cells internalize bacteria to endosomally process them.…”

“…NTHi has been shown to invade the alveolar epithelial cell line A549 in a PI3K-dependent manner (35). To examine if PI3K and FOXO play a role in the internalization of NTHi into bronchial epithelial cells, we infected Calu-3 monolayers with viable NTHi in the presence of the PI3K inhibitor LY294002 (Fig.…”