The molecular mechanisms of mycobacterium tuberculosis curli pili (MTP) in regulation of fatty acid metabolism, carbon metabolism, and bioenergetics of the pathogen and host during infection.

Abstract

Background/Aim: The initial contact between pathogen and host is a vital step in the establishment of infection. Mycobacterium tuberculosis (M. tuberculosis) mediates adhesion to target hosts cells such as macrophages via various proteinaceous molecules known as adhesins, found on the cell surface. A distinguished adhesin of M. tuberculosis is M. tuberculosis curli pili (MTP). Previous functional genomics studies have described the involvement of MTP in bacterial aggregation and biofilm formation in addition to its role as an adhesin and invasin of epithelial cells and macrophages. Transcriptomic studies elucidated the importance of MTP in modulating host immune response in vitro and in vivo. Moreover, MTP was found to be present exclusively in the M. tuberculosis complex, and to bind to antibodies in patient sera. The use of metabolomics, bioenergetics and molecular biology will further improve current understanding of MTP as a virulence factor, thereby corroborating its role as a biomarker for the development of better tuberculosis diagnostics and therapeutics. Therefore, this study aimed to elucidate the effect of MTP on M. tuberculosis metabolism and expression of selected genes involved in fatty acid transport, β-oxidation, lactate oxidation, and gluconeogenic carbon flow. Furthermore, this study aimed to investigate the metabolomic and bioenergetic role that MTP plays in the pathogen-host infection model. Methods: Confirmed wildtype (WT) M. tuberculosis, mtp-deletion mutant (Δmtp) and mtp-complemented strains were used throughout this study. Bacterial cultures were standardised prior to harvesting of wet cell mass for metabolite and ribonucleic acid (RNA) extraction. The bacterial metabolite extraction was performed using a whole metabolome extraction method and the metabolites were detected using untargeted two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS) (Chapter 2). The RNA was extracted via the TriZol method for reverse transcription quantitative polymerase chain reaction (RT-qPCR) on selected genes to support metabolomic data (Chapters 2 and 6). Host THP-1 monocytes were differentiated into macrophages prior to infection. Each of the bacterial strains were used to infect macrophages at a multiplicity of infection (MOI) of five. After four hours of incubation, host cells were used for: a) metabolomics via whole metabolome extraction for either detection with untargeted GC×GC-TOFMS (Chapters 3 and 4) or targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) with stable isotope carbon-13 (13C6)-glucose tracing (Chapter 5); b) bioenergetics through cell mitochondrial stress tests (CMSTs) and glycolytic rate assays (GRAs) performed in an extracellular flux analyser (Chapter 5); and c) lysis to harvest intracellular bacteria for RNA extraction to perform RT-qPCR (Chapter 6). All experiments were repeated to ensure reproducibility of the data, which were analysed by the appropriate statistical tests to highlight significant differences or similarities amongst the models, groups and samples. Results/Discussion: Bacterial RT-qPCR and functional metabolomics through GC×GC-TOFMS revealed that the Δmtp was associated with an altered cell wall composition. Of the 28 metabolites shortlisted, 19 of them were detected in elevated concentrations in the Δmtp compared to the WT, thus indicating that the lack of MTP reduces the overall ability of the pathogen to utilise these metabolites for biological processes. This was deduced from the high concentrations of carbohydrates involved in cell wall biogenesis and functions, and fatty acid metabolism. Additionally, alterations to amino acid concentrations further explained the previously reported growth retardation compared to the WT. Mycobacterium tuberculosis infected THP-1 macrophage models revealed significant alterations to central carbon metabolism (CCM), fatty acid metabolism and amino acid metabolism in the absence of MTP. In particular, the Δmtp infected THP-1 macrophages demonstrated that nine of the GC×GCTOFMS detected metabolites were significantly higher relative to the WT infected THP-1 macrophages. These altered concentrations were observed for metabolites involved in glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, fatty acid and amino acid metabolic pathways that feed into or are fed by CCM. Given that M. tuberculosis establishes contact with both macrophages and epithelial cells during infection, a comparison of two different host cells were performed. Metabolite concentrations compared between the THP-1 macrophage infection model and A549 epithelial infection model demonstrated that the macrophages are more likely to advocate for pathogen clearance through a more efficient pro-inflammatory response, particularly in the absence of MTP, relative to the epithelial cells. Data presented here demonstrated that the MTP adhesin plays a role in securing a protective environment for M. tuberculosis during initial stages of infection. Therefore, the THP-1 macrophage infection model was further investigated by targeted LC-MS/MS and bioenergetics to ascertain a more comprehensive understanding of the carbon flux, energy profiles and macrophage polarity in the absence and presence of MTP. The Δmtp infection model mimicked the oxygen consumption rate (OCR) for basal respiration, adenosine triphosphate (ATP) production, maximal respiration, and spare respiratory capacity of the uninfected THP-1 macrophages and had the highest compensatory glycolytic rate. Furthermore, the tracing experiments revealed that MTP has no effect on the flux of glucose, but rather has an impact on the total concentrations of the macrophage infection model. These findings indicate that MTP plays a role in modulating the macrophage respiratory parameters and dampens the pro-inflammatory response of the host. Hence, the macrophage host will be more proficient at launching an immune response if the adhesin is absent. The RT-qPCR performed on selected genes from pure bacterial culture and intracellular bacteria demonstrated that removal of MTP from M. tuberculosis alters lipid import, β-oxidation, and lactate oxidation of M. tuberculosis. Moreover, in all models in which the mtp-complement was used, partial restoration of the phenotype was observed as a result of the overexpressing episomal plasmid. Cumulatively, MTP was evidenced to play a multifunctional role for M. tuberculosis. Conclusion: The surface-located adhesin, MTP, is a virulence factor of M. tuberculosis as it initiates and facilitates attachment to, and invasion of hosts in addition to providing structural integrity to the pathogen. This adhesin enhances bacterial survival as it reprograms the M. tuberculosis and THP-1 macrophage metabolism to manipulate host effector functions. Collectively, this study provided metabolomic, bioenergetic and molecular evidence that MTP significantly contributes to the success of M.tuberculosis as an intracellular pathogen. Hence, the focus of TB intervention strategies should include intercepting the MTP interaction with the host to offset M. tuberculosis pathogenicity and confer an advantage to the host.