The role of specific adhesins in the regulation of other adhesin genes associated with Mycobacterium tuberculosis pathogenicity.
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Date
2022
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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
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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
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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.
Description
Masters Degree. University of KwaZulu-Natal, Durban.