Lack of pilE expression in NZCM238 is associated with lower TNF-α secretion by 16HBE cells during coculture. Lack of pilE expression in NZCM238 is associated.

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Lack of pilE expression in NZCM238 is associated with lower TNF-α secretion by 16HBE cells during coculture. Lack of pilE expression in NZCM238 is associated with lower TNF-α secretion by 16HBE cells during coculture. (A) De novo assembly, annotation, and alignment of the pilE-pilS region of the isolates within the NZ97/192 household revealed a large amount of variability. Deletions of 2,933, 2,545, and 1,388 bp were detected in NZCM238, NZCM239, and NZCM240, respectively, compared to the index strain, NZ97/192. Block arrows depict predicted open reading frames. Small arrows below the drawing indicate primers used for genomic PCR. (B) Genomic PCR confirmed the length of the pilE-pilS region in each isolate, using primers indicated above. (C) Quantitative reverse transcriptase PCR (qRT-PCR) confirmation of reduced pilE expression in the carriage isolates, especially NZCM238. Threshold cycle (ΔCT) is presented in the graph, with higher ΔCT corresponding to lower expression. There is no detectable expression in NZCM238 (n = 6), 71% ± 17.5% (SD, n = 6) reduction in NZCM239, and 94% ± 3% (SD, n = 6) reduction in NZCM240, relative to NZ97/192. (D) TNF-α secretion was significantly reduced when 16HBE cells were infected with NZCM238 or a pilE-pilS deletion mutant of NZ97/192 (NZ97192ΔpilE/S) compared with infection with wild-type NZ97/192 (n = 6). Error bars indicate standard deviations. *, P < 0.05; **, P < 0.001; ***, P < 0.0001. (E) Electron micrographs of NZ97/192 (left), NZCM238 (center), and the NZ97/192ΔpilE/S deletion strain. T4P are seen only with wild-type NZ97/192 (arrows). Xiaoyun Ren et al. mSystems 2017; doi:10.1128/mSystems.00127-17