The role of specific adhesins in the regulation of other adhesin genes associated with Mycobacterium tuberculosis pathogenicity.

Abstract

Background/Aim: Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), remains one of the most common causes of death throughout the world. The lack of rapid diagnostics, effective vaccines, and drugs contributes to the high number of deaths recorded every year. The presence of multiple surface adhesins is crit ical for M. tuberculosis survival because they initiate and sustain host-pathogen interactions. Amongst other adhesins is the M. tuberculosis curli pili (MTP), which aid in the adhesion/invasion of host cells and the development of biofilms. The heparin-binding haemagglutinin adhesin (HBHA) facilitates the spread of M. tuberculosis away from the site of infection. Since L, D-transpeptidase (Ldt), which is encoded by Rv0309, has been shown to bind to laminin and fibronectin and to be an adhesin, it may serve as a biomarker for the development of novel therapeutic approaches. Studying the impact triggered by the three adhesins, MTP, HBHA, and Rv0309, on the regulation of other adhesins that bind to macrophages; 19-kilodalton (19 kDa), M. tuberculosis Phosphate-binding protein (PstS‐1), Chaperone chaperonin 60.2 (Cpn 60.2), Alanine and proline-rich antigenic glycoprotein (Apa), antigen 85 complexes (Ag85A), chaperone DnaK, and M. tuberculosis type IV pili, will further substantiate their use in biomarker development. Therefore, the purpose of this study was to elucidate the role of MTP, HBHA, and Rv0309 adhesins in regulating adhesins that bind to macrophages during infection. This was achieved using in vitro infection assays with gene knockout and complemented mutant strains of M. tuberculosis, real-time quantitative PCR (RT-qPCR), and a dot blot assay. Methods: M. tuberculosis wild-type, mtp-deletion mutant (Δmtp), hbhA-deletion mutant (ΔhbhA), mtp-hbhA-deletion mutant (Δmtp-hbhA), ΔRv0309 mutant and the respective complemented strains that had been constructed in previous studies, were confirmed using polymerase chain reaction (PCR). The strains were individually cultured in supplemented Middlebrook 7H9 broth till an optical density of 600 (OD)600 of 1 was reached. THP-1 monocytic cells were differentiated into macrophages and infected at a multiplicity of infection (MOI) of five. At the end of 4-h and 24-h post-infection, cells were lysed with TritonX-100. The lysate from the infected cells was collected for RNA extraction and bacterial protein extraction. To quantify the internalized bacteria, serial dilutions of the lysate from the infected cells were plated on 7H11 agar plates for CFUs. To confirm MOI, the bacterial inoculum was serially diluted and plated for CFUs. Intracellular pathogen RNA was extracted using the TriZol method and converted into cDNA using the High Capacity cDNA Reverse Transcription kit. Primers for adhesin genes: Rv0350, Rv0440, Rv0934, Rv1860, Rv3660, Rv3763, and 2 Rv3804 were designed using Primer3plus web. To assess the impact triggered by MTP, HBHA, and Rv0309 adhesin on the expression of Rv0350, Rv0440, Rv0934, Rv1860, Rv3660, Rv3763, and Rv3804, RT-qPCR- was performed using 2X SYBR green supermix in a 7500 RT-qPCR Detection System. The gene expression data was normalized using 16S rRNA and analysed using the absolute quantification method. Proteins were isolated using the TriZol method and resuspended in 0.1 % SDS. Extracted proteins were resolved in SDS-PAGE. Western blot was attempted with Cpn60.2 and PstS-1 primary antibodies used against the Goat anti-mouse HRP secondary antibody. Dot blot was used to determine the optimal antibody dilutions and protein concentration. GraphPad Prism version 8 software was used to determine significance values. Results and Discussion: Infection with mutant mtp resulted in a decrease in the number of bacteria that infected THP-1 cells at both 4-h (p <0.001) and 24-h (p=0.002) time points compared to the wild-type. At 4-h post-infection, four of the seven genes, Rv3763, Rv3804, Rv1860 and Rv0440, were expressed in all three strains. The expression of three of these genes, Rv3763 (p=0.049), Rv3804 (p=0.003) and Rv0440 (p=0.004), was significantly increased in the Δmtp compared to the wild-type strains. Rv3660 was expressed in the Δmtp but not in the wildtype (p=0.012). At 24-h post infection. the expression of five genes was significantly increased in the wild-type compared to the Δmtp strain; Rv3763 (p=0.049), Rv0934 (p=0.006), Rv3804 (p=0.005), Rv0350 (p=0.012) and Rv0440 (p=0.004). The mtp mutant strain induced lower expression of the genes at 24-h (Rv3763, Rv0934, Rv3804, Rv1860 and Rv0440) in contrast to 4-h. Low expression of the genes Rv3763, Rv0934, Rv1860 and Rv3804 in the mutant mtp strain are associated with cell wall activities and are important virulence factors of M. tuberculosis. The decrease in the CFUs and altered gene expression in the mutant suggests that mtp is required for the expression of the genes associated with cell wall processes and that the deletion of the mtp gene may result in a decrease in cell wall activities. The deficiency of mtp gene in the mutant resulted in the reduced capability of the strain in infecting the THP-1 cells implying a decrease in the virulence of the strain. Infection with the hbhA mutant resulted in a decrease in the number of bacteria that infected THP-1 cells at both 4-h (p=0.049) and 24-h (p =0.034) time points compared to the wild-type. At 4-h post-infection, five of the seven genes, Rv3763, Rv0934, Rv3804, Rv1860 and Rv0440, were expressed in all three strains. The hbhA mutant induced the expression of all the genes at both 4-h and 24-h. Rv3660 (p=0.001) and Rv0350 (p<0.001) were expressed in the ΔhbhA but not by the wild-type at 4-h. At 24-h, all seven genes, Rv3763, Rv0934, Rv3804, Rv0350, Rv3660, Rv1860 and Rv0440, were expressed across all strains. The expression of four of these 3 genes was significantly increased in the wild-type compared to the ΔhbhA strain Rv3763 (p=0.023), Rv0934 (p=0.001), Rv3804 (p=0.002), Rv1860 (p=0.017). The deletion of hbhA induced the expression of all the adhesin genes to compensate for the loss of this gene in the mutant. There was a significant reduction in the number of bacteria that infected THP-1 cells in the Δmtp-hbhA compared to the wild-type at the 4-h (p =0.002) and 24-h (p =0.047) time points. The double knockout induced a low expression of Rv3763, Rv0934, Rv0350, Rv3804, and Rv3660 at 4-h. The expression of Rv3763 (p=0.039), Rv0934 (p = 0.002) and Rv3804 (p=0.000) was significantly increased in the wild-type strains compared to the Δmtp-hbhA. There was a higher expression of Rv1860 and Rv0440 in the mutant at 4-h compared to the 24-h time point. At 24-h, all seven genes, Rv3763, Rv0934, Rv3804, Rv0350, Rv3660, Rv1860 and Rv0440, were expressed across all strains. The expression of these genes was significantly increased in the wild-type compared to the Δmtp-hbhA. Rv3763 expression was higher in the mutant at 24- h compared to the 4-h. The observed expression in the mutant suggests the importance of both hbhA and mtp in the virulence of M. tuberculosis. Deletion of mtp-hbhA resulted in a decrease in the capability of M. tuberculosis in initiating infection in THP-1 cells. There was a significant difference in Rv0309 mutant in infecting THP-1 cells in comparison to the wild-type strains at the 4-h and 24-h time points (p = 0.001). This suggests that deletion of the Rv0309 gene in the mutant might have reduced the infecting capability of the strains. There was an upregulation of the Rv3763, Rv0934, Rv1860, Rv3804, and Rv0440 in the mutant at 4- h. Upregulation of these genes suggests that Rv0309 is an important virulence factor of M. tuberculosis. Rv1860 expression at 24-h was doubled compared to the expression of 4-h in the mutant to compensate for the loss of Rv0309 in the mutant. This may suggest that in the absence of Rv0309, the M. tuberculosis expresses Rv1860 which binds to laminin and fibronectin to promote infection. Confirmatory studies by protein detection using anti-Cpn 60.2 and anti-PstS1 antibodies with western blot failed. Numerous attempts for troubleshooting were conducted using the dot blot assay and optimisation of various factors, such as the use of positive control, different antibody dilutions, protein concentration, and optimized buffers. This showed that the purchased antibodies did not work. Conclusion: The findings demonstrated that MTP, HBHA and Rv0309 play a role in the regulation of other adhesin genes, as evidenced by the deletion of the three major genes that

may have disrupted several metabolic and cell wall processes, potentially reducing the virulence of the M. tuberculosis strain. The findings of this work add to the growing evidence that the adhesins, MTP, HBHA, and Rv0309 as well as the related adhesins they interact with during macrophage infection, are promising targets for TB diagnostic or therapeutic interventions.