Transcriptional profiling of Neisseria meningitidis interacting with human epithelial cells in a long-term in vitro colonization model

Abstract

Neisseria meningitidis is a commensal of humans that can colonize the nasopharyngeal epithelium for weeks to months and occasionally invades to cause life-threatening septicemia and meningitis. Comparatively little is known about meningococcal gene expression during colonization beyond those first few hours. In this study, the transcriptome of adherent serogroup B N. meningitidis strain MC58 was determined at intervals during prolonged cocultivation with confluent monolayers of the human respiratory epithelial cell line 16HBE14. At different time points up to 21 days, 7 to 14% of the meningococcal genome was found to be differentially regulated. The transcriptome of adherent meningococci obtained after 4 h of coculture was markedly different from that obtained after prolonged cocultivation (24 h, 96 h, and 21 days). Genes persistently upregulated during prolonged cocultivation included three genes (hfq, misR/phoP, and lrp) encoding global regulatory proteins. Many genes encoding known adhesins involved in epithelial adherence were upregulated, including those of a novel locus (spanning NMB0342 to NMB0348 [NMB0342-NMB0348]) encoding epithelial cell-adhesive function. Sixteen genes (including porA, porB, rmpM, and fbpA) encoding proteins previously identified by their immunoreactivity to sera from individuals colonized long term with serogroup B meningococci were also upregulated during prolonged cocultivation, indicating that our system models growth conditions in vivo during the commensal state. Surface-expressed proteins downregulated in the nasopharynx (and thus less subject to selection pressure) but upregulated in the bloodstream (and thus vulnerable to antibody-mediated bactericidal activity) should be interesting candidate vaccine antigens, and in this study, three new proteins fulfilling these criteria have been identified: NMB0497, NMB0866, and NMB1882.

Fig 1

Viability of epithelial cells cocultivated with N. meningitidis MC58. Epithelial cell monolayers cocultivated with bacteria for 48 h were washed and stained with Giemsa. (A) COR-L23 cells at a ×400 magnification. The arrow indicates an example of a cell boundary. (B) 16HBE14 cells at a ×1,000 magnification. Arrows indicate examples of cytoplasm, nucleolus, nucleus, and a group of meningococci.

Fig 2

Scanning electron microscopy images of 16HBE14 cells cocultivated with N. meningitidis MC58. Images were taken at 10 h (A), 48 h (B), and 21 days (C) of cocultivation, with white bars indicating 5, 5, and 1 μm, respectively.

Fig 3

Comparison of meningococcal transcriptomes obtained at different cocultivation time points. (A) PCA. Solid dots in the plot represent the relative positions of samples at different cocultivation time points. Dashed arrows represent the passage of time. (B) Gene clustering with similarity between time points measured by Euclidean distance, as indicated.

Fig 4

Venn diagrams showing numbers of differentially regulated genes coidentified at different cocultivation time points. The shaded circles in three-way Venn diagrams represent 230 up- and 164 downregulated genes coidentified at 24 h and 96 h (in two-way Venn diagrams). The total numbers of differentially regulated genes for each time point are indicated in parentheses.

Fig 5

Transcriptional profiles of selected genes. Levels of meningococcal gene expression at different cocultivation time points (as indicated on the x axis) and for DMEM at 3 h were compared and are expressed as fold changes.

Fig 6

Epithelial cell adherence assay of meningococcal mutants. Monolayers of 16HBE14 cells were incubated with wild-type (WT) N. meningitidis MC58 and its isogenic mutants (indicated as NMB0342, NMB0344, NMB0345, NMB0347, and NMB0348, for the genes being disrupted) for 4 h, 24 h, and 96 h. Numbers of adherent meningococcal CFU were obtained from three biological replicates. Error bars indicate standard errors of the means. Asterisks indicate CFU with a significant difference (P < 0.02) between the corresponding mutant and the wild type.

Fig 7

Comparison of gene expression using real-time RT-PCR. Independent biological RNA samples were prepared from MC58 cells harvested after 24 h and 96 h of cocultivation with 16HBE14 epithelial cells and compared to RNA samples from MC58 cells incubated for 3 h in DMEM alone (DMEM3h). The results of comparisons are expressed as fold changes. Error bars indicate standard errors of the means calculated from three biological replicates.