The role of Mycobacterium tuberculosis curli pili (MTP) and heparin-binding hemagglutinin adhesin (HBHA) on global in vitro bacterial transcriptomics.

Abstract

Background/Aim: Tuberculosis (TB), is an infectious, airborne disease caused by Mycobacterium tuberculosis (M. tuberculosis). TB remains one of the most devastating bacterial causes of human mortality, especially in low-income countries. Surface located adhesins are crucial for M. tuberculosis survival, as they initiate and perpetuate host-pathogen interactions. The adhesin, M. tuberculosis curli pili (MTP), plays a role in adhesion and invasion of host cells and biofilm formation, whilst heparin-binding hemagglutinin adhesin (HBHA) promotes M. tuberculosis dissemination from the site of infection. The use of transcriptomics promises to enhance current knowledge on MTP and HBHA as virulence factors, thereby substantiating their role as biomarkers for the development of accurate TB diagnostics and therapeutics. Therefore, this study aimed to elucidate the role of MTP and HBHA in the regulation of M. tuberculosis transcriptome, and to identify novel biomarkers. This was achieved by analysing the transcriptomic perturbations in the strains lacking the MTP adhesin, HBHA adhesin or both MTP-HBHA adhesins and the strains containing the aforementioned adhesins. Methods: Polymerase chain reaction (PCR) confirmed strains of M. tuberculosis wild-type (WT), mtp-deletion mutant (Δmtp), hbhA-deletion mutant (ΔhbhA), mtp-hbhA-deletion mutant (Δmtp-hbhA), and the respective complemented strains, were standardized and cultured until log phase. Thereafter, the bacterial cultures were prepared for RNA extraction. RNA was extracted via an optimized TRIzol method and sequenced using the Illumina 2×150 HiSeq ×10 platform. The sequenced reads were analysed by FastQC toolkit (version 0.11.8), pre-processed using Trimmomatic (version 0.36), mapped to the custom-built M. tuberculosis H37Rv genome index using hierarchical indexing for spliced alignment of transcripts (HISAT version 2.1.0), assembled by Stringtie (version 1.2.1), and further annotated and assembled the transcripts into known and novel categories by Gffcompare, located within Stringtie. The output files were annotated in R (version 1.2.1578) using the Ballgown package to generate the respective fold changes (FC) between the deletion mutants and the WT, and q-values and p-values for the differential expression. The generated results were filtered using a FC cut-off value ≥ 1.3 (to indicate a 1.3-fold up-regulation) and ≤ 0.5 (to indicate a 2-fold down-regulation) to identify significant genes and pathways. Thereafter, relevant databases and literature were reviewed to categorize the genes into pathways. Real time quantitative PCR (RT-qPCR) was performed on 10 selected genes, as a genotypic method to functionally confirm

the RNA sequencing data. A bacterial bioluminescence cell viability assay was performed to elucidate the concentration of adenosine triphosphate (ATP) in the deletion mutants and complements, relative to the WT. Results: A total of 43 genes were significantly differentially expressed amongst the deletion mutants. These genes were functionally categorized into: intermediary metabolism and respiration metabolism, cell wall biosynthesis, cell wall transport and processes, lipid metabolism, and virulence; stable RNA’s; conserved hypotheticals; proline-glutamate (PE) or proline-proline-glutamate (PPE); insertion sequences and phages; and information pathways. The bioluminescence assay functionally confirmed the increased utilization of ATP in the absence of MTP and HBHA. Discussion/Conclusion: Adhesin gene deletions caused major perturbations to the central carbon metabolism, cell wall biosynthesis, cell transport process, lipid biosynthesis, and virulence pathways, leading to potentially increased energy requirements; compensatory transport of proteins to the cell wall, altered cell wall biosynthesis and decreased virulence and pathogenicity. Additionally, deletion of these adhesins resulted in the disruption of many processes potentially attenuating growth and replication. Thus, this study further corroborates the adhesins, MTP and HBHA, and associated pathway genes as potential suitable targets for TB diagnostic/therapeutic interventions.