Neisseria cinerea isolates can adhere to human epithelial cells by type IV pilus-independent mechanisms

Abstract

In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host-pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus-pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms.

Fig. 1.

Schematic representation of the pilE locus in nine commensal Neisseria species. Putative pilE genes are highlighted, and predicted ORFs in the regions flanking pilE are shown. A representative isolate is shown for each species and the isolate name is given. The number of isolates analysed within each species is shown in parentheses. Bar, 1 kb.

Fig. 2.

Analysis of pilE1 and pilE2 transcript and protein expression in N. cinerea CCUG 346T. (a) Schematic representation of the WT pilE locus in N. cinerea 346T and isogenic mutants used to analyse transcript expression. Putative promoters are indicated by arrows (σ⁷⁰) and triangles (σ⁵⁴), and Rho-independent terminators by stem–loops. A kanamycin resistance cassette with promoter and bidirectional rrnB terminators was used for the construction of mutants, except for 346TΔpilE1, in which only the ORF was used. Bar, 1 kb. (b) Northern blotting of total RNA from N. cinerea 346T WT and mutants. pilE1 transcript was detected in both the WT and 346TΔpilE2. pilE2 transcript was undetectable in the WT under the conditions tested but was detected when expressed from the pilE1 promoter, in 346TΔpilE1 : : pilE2. tmRNA was used as loading control. (c) Northern blot analysis of total RNA from 346TΔpilE1 : : pilE1-his and 346ΔpilE1 : : pilE2-his. pilE1 and pilE2 transcripts were detected, respectively. tmRNA was used as loading control. (d) Detection of His-tagged PilE1 and PilE2 in supernatant (SN) and cell extracts (C) from strains 346TΔpilE1 : : pilE1-his and 346TΔpilE1 : : pilE2-his. RecA detection was used as loading control. No signal was obtained for PilE2.

Fig. 3.

WT N. cinerea strain 346T and 346TΔpilE2 express Tfp. (a) Negative-staining transmission electron microscopy of Tfp in WT (i, ii) and 346TΔpilE2 mutant (iii, iv). Filaments consistent with Tfp were evident on the surface of both strains. A large number of membranous bleb-like structures were also visible. Bars, 200 nm (i) and 100 nm (ii–iv). (b) N. cinerea strains were transformed using chromosomal DNA isolated from a spectinomycin-resistant 346T ΔpilD mutant. The number of transformants compared with the number of recipient cells is shown as a percentage. Results are the mean+sd of three independent experiments. No transformants were obtained for 346TΔpilE1 and 346TΔpilE1/2. ns, Not significant (unpaired Student's t-test, P = 0.7822).

Fig. 4.

Adherence of N. cinerea CCUG 346T WT and pilus-deficient mutant to biotic and abiotic surfaces. (a) A549 cells, Detroit 562 cells and glass or plastic coverslips were infected for 1.5 h with N. cinerea 346T WT (black bars) or 346TΔpilE1/2 (grey bars). Results are the mean+sd of three experiments. ns, Not significant. (b) Detroit 562 cells were infected for 0.5, 1.5 or 3 h with 346T (chequered bars) or 346TΔpilE1/2 (white bars). Cell-associated bacteria were quantified by plating. N. meningitidis 8013 (black bars) and the corresponding pilE mutant (grey bars) were used as a control for Tfp-dependent adherence. Results are the mean+sd of three experiments. ns, Not significant; asterisks indicate significance (unpaired Student's t-test; *P = 0.01–0.05, **P = 0.001–0.01, ****P < 0.0001).

Fig. 5.

Microscopy analysis of N. cinerea binding to human epithelial cells. (a) SEM analysis of A549 cells infected with WT 346T (i, ii) or 346TΔpilE1/2 (iii, iv) for 1.5 h. Bacterial microcolonies in close association with host cells could be observed for both the WT strain and pilus-deficient mutant. Both strains appear to form intimate contact with cellular protrusions. Bars, 1 μm. (b) A549 cells were infected with WT 346T or 346TΔpilE1/2 expressing GFP at an m.o.i. of 30. At 1.5 h post-infection, cells were fixed and stained for nuclei (blue) and actin (red). Both WT and mutant could be clearly visualized on cells. Images were taken using a CCD3 inverted Zeiss microscope. (c) Quantitative analysis of microcolony formation. At least 300 cells were analysed per experiment and the number of bacteria per microcolony was expressed as a percentage. Black bars correspond to microcolonies consisting of ≤ 5 bacteria; grey bars correspond to microcolonies of >5 bacteria. WT and pilE1/2 mutant formed microcolonies of similar size. ns, Not significant.

Fig. 6.

N. cinerea CCUG 27178A can also bind to A549 cells independently of pilE. The N. cinerea WT isolate 27178A (black/white striped bar) and a mutant lacking both putative pilE genes (27178ΔpilE1/2, grey bar) were used to investigate the adhesion of a second N. cinerea strain to A549 cells. N. meningitidis 8013 (black bar) and isogenic pilus-deficient mutant (8013ΔpilE) were used as a control for Tfp-dependent adherence and 346T (black/white chequered bar) and 346TΔpilE1/2 (white bar) were included for comparison. Results show the mean+sd of data from five independent experiments. Statistical significance was calculated using an unpaired Student's t-test. 8013ΔpilE was impaired for adhesion (**P = 0.001–0.01); ns, not significant.