Mycobacterium tuberculosis pili (MTP) modulates pathogen and host metabolomic changes in an A549 epithelial cell model of infection.

Abstract

Background/Aim: Mycobacterium tuberculosis, the causative organism of tuberculosis, continues to drive research efforts in the quest to develop novel diagnostics and therapeutics. The complexities associated with drug-resistant strains (multidrug-resistant (MDR), extensively drug-resistant (XDR) and totally drug-resistant tuberculosis (TDR-TB)) and co-infection with human immunodeficiency virus/acquired immune deficiency virus (HIV/AIDS) further cripple the fight against tuberculosis (TB). Hence, a comprehensive understanding of the M. tuberculosis genome, transcriptome, proteome and metabolome is required to gain different perspectives on potential target points, such as novel biomarkers, for intervention. The M. tuberculosis curli pili (MTP), a surface-located adhesin is involved in the first point of contact with the host cell, and has shown diagnostic and therapeutic potential based on previous genomic, transcriptomic and proteomic findings. Understanding the metabolome of Mycobacterium tuberculosis and its target host cell during infection will provide further insights into the role of MTP and its metabolic influence. This study aimed to determine the role of MTP in modulating bacterial and host metabolic pathways of M. tuberculosis and A549 epithelial cells, respectively, using a two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOFMS) approach coupled with bioinformatic analyses. Methods: The wild-type (WT), mtp deletion mutant (Δmtp) and mtp-complemented strains were confirmed by genomic DNA extraction and PCR. For the pathogen model investigation, ten biological replicates of each of the three strains were individually cultured in supplemented Middlebrook 7H9 broth till an OD600 of 1 was reached. Cultures were centrifuged, subjected to a washing procedure and the resulting pellets were stored at - 80 °C. For the infection model investigation, A549 epithelial cells were grown till confluent, and seeded at a concentration of 5 x 105 cells/mL. A549 cells were infected with each M. tuberculosis strain at a multiplicity of infection of approximately 5. After the 2 hr infection period, cells underwent a washing procedure and the resulting pellets were stored at - 80°C prior to extraction for GCxGC-TOFMS metabolomic analysis. A whole metabolome extraction method was applied to extract metabolites from various metabolite classes using chloroform:methanol:water (1:3:1). The samples were analysed by GCxGC-TOFMS which underwent first and second dimensional separation. ChromaTOF software, MATLAB software along with the Eigenvector PLS_Toolbox 8.7 were used to identify differentiating metabolites. Parametric univariate analysis included independent samples t-test with its associated Cohen’s d-value. Correcting for multiple testing was done by the Benjamini & Hochberg (BH) adjustment to control the rate of false discovery. Multivariate analyses included quality assurance based on Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA). Variable Importance in the Projection (VIP) values, BH-adjusted p-values, Cohen’s d-values and fold changes were considered as criteria for shortlisting metabolites. In order to better understand the metabolomic changes at a transcriptomics level, RT-qPCR was performed on the bacterial strains. The resulting gene expression data was normalised using 16S rRNA and analysed using the relative standard curve method. GraphPad Prism version 8 software was used to determine significance values. Results/Discussion: The results from the bacterial model investigation were significant as 27 metabolites were found to be altered in concentration between the mtp-deficient cells and the WT, while 7 metabolites were deemed significantly different between the WT and mtp-complemented strains. Three of the 4 categories were produced in higher relative concentrations by Δmtp; carbohydrates in cell wall biogenesis, fatty acid metabolism and peptidoglycan synthesis, indicating an overall reduced ability in the utilisation of these metabolites for natural cellular processes in the Δmtp compared to the WT. Metabolites involved in amino acid and protein synthesis were produced in relatively lower concentrations in Δmtp, again suggesting defective pathways in Δmtp. The infection model analysis investigated five different M. tuberculosis infection models, of which only one validated. The first three models, which revealed minimal differences, were the infected and uninfected models of comparison to determine whether any significant differences existed in the M. tuberculosis-infected A549 cell model compared to the respective uninfected model. There were no major differences between the WT-infected and mtp-complement-infected strain showing functional restoration of mtp. Significant differences were observed between the WT-infected and Δmtp-infected A549 cells. These included a total of 46 metabolites produced in significantly lower relative concentrations in the Δmtp-infected cells. The deletion of the MTP adhesin led to a perturbation in nucleic acid metabolism, which was found to be less efficient in the Δmtp-infected cells. A similar observation was seen for lysine metabolism and degradation. Nitrogen assimilation was also found to be less prominent in Δmtp-infected cells arising from aspartate, alanine and glutamate metabolism. Metabolites involved in glutathione metabolism, oxidative stress and lipid metabolism were produced in lower relative concentration in Δmtp-infected cells, potentially resulting in a compromised mycobacterial cell envelope in the deletion mutant. Lanthionine was an unusual metabolite detected in the present study. These metabolic alterations were indicative of lowered pathogenicity of the M. tuberculosis mutant strain, as a result of the absence of MTP. Conclusion: The significant findings of this study confirm previous reports that MTP has potential as a biomarker that can be targeted for intervention. The first investigation revealed a total of 27 metabolites to be biologically significant between the Δmtp and WT strains. These were associated with reduced cell wall biogenesis, fatty acid metabolism, amino acid and protein synthesis, and peptidoglycan synthesis. Between the WT and mtp-complemented strains, seven metabolites were biologically significant and corresponded with various cell envelope functions. In the second investigation, all 46 metabolites were produced in a relatively lower concentration by the Δmtp-infected cells compared to the WT-infected cells and were associated with a decrease in nucleic acid synthesis, amino acid metabolism, glutathione metabolism, oxidative stress, lipid metabolism and a peptidoglycan anomaly. The MTP adhesin is associated with various changes to the pathogen and host metabolome, highlighting its importance as a virulence factor that further substantiates its potential as a suitable biomarker for corrective intervention in the fight against TB.