Transcriptional Response of Mycobacterium tuberculosis on Encounter with Neutrophil In Vitro

Background: Neutrophils are reported to be the dominant phagocytes in sputum from tuberculosis (TB) patients and may explain the distinctive pattern of gene expression of Mycobacterium tuberculosis (Mtb) in these samples. Moreover, neutrophils play an important role during tuberculosis both in collaboration with macrophages as well as antimicrobial functions such as NETosis. Therefore, understanding the transcriptional changes of mycobacteria in response to neutrophils is of general interest. Methods: Following preliminary experiments to develop appropriate methods using BCG and a neutrophil cell line, primary human neutrophils were infected with stationary phase H37Rv preconditioned in RPMI overnight then sampled at 4 and 24 hours. Bacterial RNA samples from these experiments and from TB sputum were subjected to RNA-seq analysis by Illumina NextSeq-500. Reads were normalised by two different methods and differentially expressed genes were detected at q-value < 0.01. Selected results were confirmed by qRT-PCR. Transcription factors associated with detected genes were identified. Results: The colony counts of H37Rv incubated with human neutrophils reduced ~10-fold at 24 hours. Striking up regulation of Mtb tgs1, hspX and icl1 was observed during incubation in RPMI relative to exponentially growing bacilli. RNA-seq revealed 90 genes differentially expressed (28 repressed and 62 induced) in neutrophil encounters at 4 hours relative to RPMI controls. DosR and sigK were the most significantly associated transcription factors with the induced genes. Discussion: Comparison of the neutrophil-stimulated and sputum Mtb transcriptomes did not support the hypothesis that the phagocytes provide a key stimulus underpinning the sputum pattern. SigK regulated expression appears to be a significant aspect of the neutrophil stimulated Mtb transcriptome.