The Haemophilus influenzae Sap transporter mediates bacterium-epithelial cell homeostasis

Abstract

Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharynx and a causative agent of otitis media and other diseases of the upper and lower human airway. During colonization within the host, NTHI must acquire essential nutrients and evade immune attack. We previously demonstrated that the NTHI Sap transporter, an inner membrane protein complex, mediates resistance to antimicrobial peptides and is required for heme homeostasis. We hypothesized that Sap transporter functions are critical for NTHI interaction with the host epithelium and establishment of colonization. Thus, we cocultured the parent or the sapA mutant on polarized epithelial cells grown at an air-liquid interface, as a physiological model of NTHI colonization, to determine the contribution of the Sap transporter to bacterium-host cell interactions. Although SapA-deficient NTHI was less adherent to epithelial cells, we observed a significant increase in invasive bacteria compared to the parent strain. Upon internalization, the sapA mutant appeared free in the cytoplasm, whereas the parent strain was primarily found in endosomes, indicating differential subcellular trafficking. Additionally, we observed reduced inflammatory cytokine production by the epithelium in response to the sapA mutant strain compared to the parental strain. Furthermore, chinchilla middle ears challenged with the sapA mutant demonstrated a decrease in disease severity compared to ears challenged with the parental strain. Collectively, our data suggest that NTHI senses host environmental cues via Sap transporter function to mediate interaction with host epithelial cells. Epithelial cell invasion and modulation of host inflammatory cytokine responses may promote NTHI colonization and access to essential nutrients.

Fig 1

Adherence and epithelial cell surface remodeling in the absence of SapA. (A and B) CMEE (A) and NHBE (B) cells were inoculated with either the parent strain (white bars) or the sapA mutant strain (black bars). Numbers of bacteria adherent to the epithelial cell monolayers were determined at each time point and are depicted as the mean adherent bacteria ± standard deviation for triplicate wells from three (B) or two (A) independent experiments. The asterisk depicts a significant change in adherence between the sapA mutant and the parent strain (P < 0.05). (C to F) The parent (C and E) or the sapA mutant (D and F) GFP reporter strains (green) were inoculated onto CMEE cell monolayers (red), incubated for 72 h, and monitored for colonization and epithelial cell surface changes by confocal microscopy. Panels E and F are three-dimensional rendered optical sections of the bacterium-epithelial cell interface.

Fig 2

Epithelial cell invasion in the absence of SapA. NHBE cell monolayers (A) were inoculated with the parent strain (B) or the sapA mutant strain (C), incubated for 24 h, and monitored for colonization by fluorescence microscopy. NTHI cells were labeled with a anti-OMP antibody and detected by anti-rabbit FITC antibody (green). Epithelial cell membranes were stained with wheat germ agglutinin conjugated to Alexafluor 594 (red), and DNA was counterstained with Hoechst 34580 (white). Arrows indicate cytoplasmic NTHI.

Fig 3

SapA mediates epithelial cell surface colonization by NTHI. Polarized epithelial cells grown at an air-liquid interface (A and B) were inoculated with the parent strain (C, E, and G) or the sapA mutant strain (D, F, and H) and incubated for 24 h. NTHI-epithelial cell interactions were monitored by scanning electron microscopy.

Fig 4

Loss of SapA promotes a hyperinvasive phenotype. (A to D) Polarized epithelial cells grown at an air-liquid interface were inoculated with the parent strain (A and C) or the sapA mutant strain (B and D) and incubated for 24 h. The subcellular ultrastructural localization of the parent strain or the sapA mutant was determined by transmission electron microscopy. Black arrows indicate bacteria that colonized the epithelial cell surface, and white arrows indicate bacteria present in the cytoplasm of the epithelial cells. (E and F) The parent strain produced outer membrane vesicles at the epithelial cell surface (E), and the sapA mutant strain produced outer membrane vesicles both within and between NHBE cells (F). (G) A gentamicin protection assay indicates an ∼2-fold increase in invasion by the sapA mutant compared to the parent strain when comparing the mean number of bacteria protected from gentamicin lethality ± standard deviation for triplicate wells performed in three independent experiments. The asterisk indicates a significant increase in survival of the sapA mutant compared to that of the parent strain (P < 0.05).

Fig 5

Loss of SapA attenuates NTHI-induced disease severity. Naïve chinchilla middle ears (A) were inoculated transbullarly with the parent strain (B and D) or the sapA mutant strain (C and E). Three days after inoculation, the middle ear inferior bullae were excised, embedded, and sectioned. Sections were H&E stained to monitor middle ear mucosal inflammation and NTHI biofilm formation in the middle ear cavity. L, middle ear lumen; I, infiltrating leukocyte; M, mucosa; B, bone; Bf, biofilm.