Expression of the Nontypeable Haemophilus influenzae Type IV Pilus Is Stimulated by Coculture with Host Respiratory Tract Epithelial Cells.

The type IV pilus (Tfp) of nontypeable Haemophilus influenzae (NTHI) mediates adherence, colonization, motility, and biofilm formation, and the major protein subunit, PilA, is a promising vaccine candidate. Thus, it is crucial to understand how Tfp expression is regulated within the microenvironments of the human nasopharynx, which NTHI colonizes asymptomatically, and the more distal regions of the respiratory tract where NTHI-induced diseases occur. Here, we examined the effects of coculture of NTHI with human airway epithelial cells and heme availability on Tfp expression at temperatures typical of the human nasopharynx (34°C) or warmer anatomical sites during infection (37°C). Tfp expression was estimated by pilA promoter activity, pilA gene expression, and relative abundances of PilA and pilin protein. The results revealed that at both temperatures, NTHI cocultured with airway epithelial cells demonstrated significantly greater expression of pilA, PilA/pilin protein, and likely, fully assembled Tfp than NTHI cultured on an abiotic surface. Because NTHI is a heme auxotroph, we hypothesized that availability of heme from host cells might be a signal for Tfp expression. Thereby, we cultured NTHI in iron-limited medium, and we observed that supplementation with heme significantly increased pilA promoter activity. Collectively, our data suggested that NTHI Tfp expression was stimulated by soluble factor(s) released by epithelial cells, which are present in all microenvironments of the respiratory tract. The expression of this target antigen under conditions that mimic the human airway strongly supports the rationale for the use of PilA as a vaccine immunogen to prevent NTHI-induced diseases of the respiratory tract.