Biological roles of nontypeable Haemophilus influenzae type IV pilus proteins encoded by the pil and com operons

Abstract

We previously demonstrated that one or more products of the genes in the pil and com gene clusters of the opportunistic human respiratory pathogen nontypeable Haemophilus influenzae (NTHI) are required for type IV pilus (Tfp) biogenesis and function. Here, we have now demonstrated that the pilABCD and comABCDEF gene clusters are operons and that the product of each gene is essential for normal pilus function. Mutants with nonpolar deletions in each of the 10 pil and com genes had an adherence defect when primary human airway cells were used as the target. These mutants were also diminished in their ability to form a biofilm in vitro and, additionally, were deficient in natural transformation. Collectively, our data demonstrate that the product of each gene within these operons is required for the normal biogenesis and/or function of NTHI Tfp. Based on the similarity of PilA to other type IV pilins, we further predicted that the product of the pilA gene would be the major pilin subunit. Toward that end, we also demonstrated by immunogold labeling and mass spectrometry that PilA is indeed the majority type IV pilin protein expressed by NTHI. These new observations set the stage for experiments designed to dissect the function of each of the proteins encoded by genes within the pil and com gene clusters. The ability to characterize individual proteins with vital roles in NTHI colonization or pathogenesis has the potential to reduce the burden of NTHI-induced diseases through development of a Tfp-derived vaccine or a pilus-directed therapeutic.

Fig 1

Data demonstrated that the NTHI pil and com gene clusters were indeed operons. (A) Organization of the NTHI strain 86-028NP pil operon and RT-PCR across the junctions between the pil genes. (B) Organization of the NTHI strain 86-028NP com operon and RT-PCR across the junctions between the com genes.

Fig 2

NTHI pil and com mutant biofilms had reduced thickness and decreased biomass. COMSTAT analysis of Z-stack composite images of NTHI biofilms formed after 24 h in vitro. (A) Average biofilm thickness. (B) Biofilm biomass. Error bars represent standard errors of the means of 3 or more biological replicates. *, statistical significance at P < 0.05.

Fig 3

NTHI pil and com mutants had adherence defects on normal human bronchial epithelial cells. Relative adherence of NTHI parent strains, mutants, and complemented mutant strains to normal human bronchial epithelial cells. Error bars represent standard errors of the means of 3 or more biological replicates. *, statistical significance at P < 0.05.

Fig 4

Mutations in NTHI pil and com genes result in a loss of transformability. Transformation of the pil and com mutants (M) was below our level of detection. Complementation (C) of each mutation restored transformation, although not necessarily to parent levels. Therefore, the expression of each product of the pil and com loci was required for natural transformation. Error bars represent standard errors of the means of 3 or more biological replicates.

Fig 5

PilA is the major subunit of NTHI Tfp. (A) Western blot of whole-cell extracts from strains 86-028NPrpsL(pGZRS-39A), 86-028NPrpsL(pSXY), and 86-028NPrpsLΔpilA(pSXY) grown on chocolate agar, MIV medium, or a chemically defined agar. (B) Silver-stained gel of strain 86-028NPrpsLΔpilA(pSXY) and 86-028NPrpsL(pSXY) sheared-cell supernatants. (C) Western blot of strain 86-028NPrpsLΔpilA(pSXY) and 86-028NPrpsL(pSXY) sheared-cell supernatants using anti-rsPilA antibody. (D) Sequence of PilA with mass spectrometry-identified peptides from strain 86-028NPrpsL(pSXY) highlighted. (E) Transformation efficiency of strains 86-028NPrpsL, 86-028NPrpsL(pSXY), and 86-028NPrpsLΔpilA(pSXY).

Fig 6

Immunolabeling of NTHI Tfp with rabbit anti-rsPilA antibody. (A) 86-028NPrpsL(pSXY), an sxy overexpression strain. (B) 86-028NPrpsL(pGZRS-39A), empty-vector control strain. (C) 86-028NPrpsLΔpilA(pSXY), pilA mutant overexpressing sxy.