Browsing by Author "Pillay, Manormoney."

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Clinical strains of mycobacterium tuberculosis induce strain-specific patterns of cytokine production, gene expression and pathway changes in pulmonary alveolar epithelial cells.
(2016) Mvubu, Nontobeko Eunice.; Pillay, Manormoney.; Pillay, Balakrishna.
The epidemiological success of M. tuberculosis strains, dominant in different geographic regions globally, may be ascribed to a subversion of the host‟s protective immune response. The increasing prevalence of F15/LAM4/KZN, Beijing, F11 and F28 Mycobacterium tuberculosis strain families, coupled with rapidly evolving drug resistance within the KwaZulu-Natal province of South Africa population has resulted in a need to characterize host response associated with infection by these strains. Therefore, in this study, cytokine/chemokine production and host transcriptomics were investigated in A549 pulmonary epithelial cells infected with the F15/LAM4/KZN, Beijing, F28, F11, Unique and H37Rv strains. Cytokines/chemokines were quantified using the Bio-Plex Pro Human Cytokine 27-Plex assay at 0, 24, 48 and 72 hr post-infection. Changes in host gene expression were determined by whole genome RNA Sequencing (RNA-Seq) using the Illumina HiSeq 2000 platform. The 50 bp reads were mapped to the human genome (hg19) using Tophat (2.0.10). Differential expression was quantified using Cufflinks (2.1.0) with false discovery rate (FDR) of 0.05 and a log fold change cutoff of ≥2. R commands (Bioconductor), MeV and Ingenuity Pathway Analysis (IPA) were used to generate heat maps, network and pathways analysis. Twenty-three out of 27 analytes were detected. All strains, except the F28 strain induced an increased production of 18, and a decrease in 5 cytokines/chemokines at 24, 48 and 72 hr post-infection, compared to the uninfected control. Increased production of all 23 analytes by the F28 strain occurred at 48 and 72 hr. Among the 23 cytokines/chemokines that were detected, anti-inflammatory and pro-inflammatory cytokines, as well as chemokines were produced at the different time intervals. Compared to the other strains, high cytokine levels were induced by the F28 strain at 48 hr and F15/LAM4/KZN strain at 72 hr for most analytes. A lower cytokine production was induced by the Beijing and Unique strains at all time intervals. In the case of the laboratory strain H37Rv either a higher or lower cytokine/chemokine production was observed, compared to the clinical strains. RNA-Seq revealed differential gene expression that varied among the strains with respect to both up- and down-regulated genes: F15/LAM4/KZN (1187), Beijing (1252), F11 (1639), F28 (870), Unique (886) and H37Rv (1179). A total of 292 genes were commonly induced by all strains, of which 52 were down-regulated and 240 were up-regulated. Different strain combinations induced different genes that were involved in a variety of pathways, including immune response and apoptosis pathways. Furthermore, strain specific genes were activated by each strain as follows: F15/LAM4/KZN (138), Beijing (52), F11 (255), F28 (55), Unique (185) and H37Rv (125). The F15/LAM4/KZN, Unique and H37Rv were the only strains that had molecular signatures with overlapping functional Kegg and Reactome pathways for their specific genes. IPA analysis revealed canonical pathways that differed among the strains, with the interferon signalling and hepatic fibrosis/hepatic stellate cell activation pathways being among the top 5 pathways in all the strains. Cholesterol biosynthesis and immune related pathway enrichment was similar in the Beijing and Unique strains whilst the F15/LAM4/KZN strain showed closer relatedness to the F11 strain, and the F28 strain closely clustered to the H37Rv strain. The Beijing and Unique strains highly enriched cholesterol biosynthesis pathways compared to other clinical and laboratory H37Rv strain. The top scoring networks induced by these clinical strains varied among the strains with the associated functions. These gene networks were involved in antimicrobial response, developmental disorder, organismal injury, infectious disease and cellular development. Among the transcriptional factors, only EHL, IRF7, PML, STAT1, STAT2 and VDR were induced by all clinical strains, while other factors were strain specific. In conclusion, low cytokine/chemokine production and activation of immune associated pathways by the Beijing and Unique strains suggest a higher virulence for these strains compared to the F15/LAM4/KZN, F11 and F28 strains. These characteristics may explain the high transmissibility and prevalence of the Beijing strains. A similar pattern exhibited by the less prevalent, non-clustering Unique strain, may suggest some virulence attributes in common with the Beijing strain. Findings in this study have the potential to reveal useful biomarkers that can be used as targets for alternative TB therapeutics including immunomodulators that take into consideration network regulations and strain-specific pathways and molecular signatures.
Evaluation of a synthetic peptide for the detection of anti-Mycobacterium tuberculosis curli pili IgG antibodies in patients with pulmonary tuberculosis.
(Elsevier., 2018) Naidoo, Natasha.; Pillay, Balakrishna.; Bubb, Martin Owen.; Pym, Alexander S.; Chiliza, Thamsanqa Emmanuel.; Naidoo, Kogieleum.; Ndung'u, Peter Thumbi.; Kasprowicz, Victoria.; Pillay, Manormoney.
Abstract available in pdf.
Evaluation of the MTT and MABA assays for rapid screening of the in vitro activity of synthetic chalcones against Mycobacterium tuberculosis.
(2014) Moodley, Suventha.; Pillay, Manormoney.
Background: The chalcone scaffold (1,3-diaryl-2-propen-i-ones) has the advantage of easy chemical modification and has been shown to possess biological activity against a variety of organisms, including a wide range of anti-TB activity. The focus of this study was 2-fold: firstly, to compare the performance of the colorimetric MTT and MABA assays for screening synthetic chalcones, and secondly, to evaluate the activity of fluorinated and non-fluorinated chalcones against drug susceptible and resistant clinical strains of M. tuberculosis. Materials and methods: Twenty seven chalcones and chromenochalcones were screened against the laboratory strain M. tuberculosis H37Rv, using a microtitre plate MTT assay at 7 days. The MIC for 20 active compounds was subsequently determined using the MABA, MTT and the macroscopic broth assays at 7, 14 and 21 days, extracellular activity against clinical isolates of varying drug susceptibility patterns and genotypes using the MTT assay, intracellular activity in a macrophage model and eukaryotic cytotoxicity using Vero cells. Results and discussion: No significant difference in the MICs, or increase in the MICs was observed over time between the MABA (p = 0.209) and the MTT (p = 0.207) assays, in contrast to the gold standard, the macroscopic broth assay (p = 0.000). Fluorinated and non-fluorinated chalcones displayed moderate activity (32- 128 μg/mL) against MDR- and XDR-TB isolates, no significant activity against intracellular H37Rv and low selectivity for M. tuberculosis. The elevated MICs and lack of intracellular activity may be explained by the precipitation of the compounds indicating low solubility, with the exception of IV and XVI. Conclusions: The MTT assay is a more cost effective drug susceptibility testing method than the MABA assay for the rapid in vitro screening of the activity of chalcones against M. tuberculosis. Compound XIX and XI have the most potential for reformulation to improve their biological activity to yield a more potent drug candidate.
Fitness of multi-and extensively drug-resistant mycobacterium tuberculosis clinical strains.
(2015) Naidoo, Charissa Camille.; Pillay, Manormoney.
The biological fitness of a pathogen is defined as its ability to reproduce, survive, cause disease and be transmitted. Drug-resistant M. tuberculosis isolates often exhibit reduced competitive ability against susceptible isolates in the absence of drug selection. However, compensatory mutations may, to some extent, offset fitness costs associated with resistance-conferring mutations. Most studies to date have focused on the fitness of isogenic laboratory strains or globally-relevant strains. Fewer studies have addressed the fitness of endemic strains which propagate in multidrug and extensively drug-resistant forms (MDR and XDR, respectively). In this study, the fitness of four genotypes which drive the transmission of tuberculosis (TB) in KwaZulu-Natal (KZN), South Africa was explored. This included F15/LAM4/KZN genotype strains, some of which were involved in the notorious XDR-TB outbreak in Tugela Ferry, KZN as well as Beijing, F11 and F28 genotype strains. Drug-resistant F15/LAM4/KZN and F28 genotype strains demonstrated increased in vitro fitness, whilst Beijing and F11 MDR strains had markedly reduced fitness. These findings correlated with whole-genome and Sanger sequencing data which revealed the presence of low/no-cost resistance-conferring mutations as well as intra- and intergenic compensatory mutations in drug-resistant F15/LAM4/KZN and F28 strains, respectively. In contrast, high cost katG mutations and no accompanying compensatory mutations were identified in Beijing and F11 MDR strains. During co-culture experiments, a novel observation was made whereby susceptible and resistant strains exhibited synergistic growth compared with axenic cultures i.e. in vitro trans-complementation. Drug-resistant F15/LAM4/KZN strains did not undergo fitness costs in THP-1 macrophages and produced increasing levels of TNF-α which may enhance tissue destruction and dissemination to other hosts. Although demonstrating similar intracellular fitness, susceptible and MDR Beijing, F11 and F28 strains induced heterogeneous cytokine responses. Thus, the lack of a direct relationship between bacillary burden and cytokine responses indicates that this diversity results from strain heterogeneity. Relapse isolates, including those from F15/LAM4/KZN and Beijing genotypes, may reactivate without any changes in biological fitness, thereby retaining the potential to re-transmit. Taken together, the enhanced fitness of drug-resistant F15/LAM4/KZN and F28 genotype strains is due to the presence of beneficial mutations, while the reduced fitness of MDR Beijing and F11 strains is associated with high cost mutations.
Global transcriptome analysis of THP-1 cells infected with an rpfB gene knockout strain of Mycobacterium tuberculosis.
(2021) Moti, Deepika.; Pillay, Manormoney.; Mvubu, Nontobeko Eunice.
Background: Since the initial isolation of Mycobacterium tuberculosis (Mtb), tuberculosis (TB) remains among the top 10 causes of death. In 2019, 10 million people globally were burdened with TB, of which 25 %were from Africa. A fundamental process in determining the outcome of Mtb infection is host-pathogen interactions. These interactions may lead to eradication of bacteria via the innate immune response or latent TB infection (LTBI) in which the bacilli reside in a non-replicating, low metabolic state (dormancy) within alveolar macrophages (AM). Ten percent of the global population develops LTBI leaving them at greater risk of developing active TB. Initiation of an immune response requires effective antigenic stimulation that induces multiple signalling cascades and the production of pro-inflammatory and anti-inflammatory cytokines, effective against or beneficial to Mtb. The pathogen has a family of 5 resuscitation promoting factors (rfps A-E) that have been previously characterized in Mtb growth, persistence and reactivation, in vitro and in vivo, using single and multiple rpf gene knockouts. Among the other Rpf proteins, RpfB was shown to be highly antigenic and immunogenic, with the ability to induce a Th1 phenotype immune response in dendritic cells (DC) through binding of toll-like receptor 4 (TLR4). Since macrophages are the target cell of Mtb and the reservoir of dormant bacilli, this study investigated the effect of rpfB in THP-1 macrophages at a late phase of replication (72 hours post-infection) using an Mtb rpfB gene knockout strain, through global transcriptomic analysis. Material and Methods: THP-1 macrophages seeded at 7.5 x 105 cells/ mL, were infected at a MOI of 1 with wildtype (WT), ΔrpfB mutant and rpfB-complemented strains. To elucidate host transcriptomic changes attributed to rpfB, mammalian RNA was isolated 72 hours post-infection (P.I) and sequenced using the Illumina High Seq platform. Bioinformatics analysis was performed using the tuxedo suite and Ingenuity Pathway Analysis (IPA). THP-1 cytokine production was analysed at 24-, 48-, and 72 hours P.I using the human GM-CSF and IL-1β multiplex panel (Bio-Rad Laboratories) in a Bio-Plex 200 System (Bio-Rad Laboratories). RNA sequencing data was validated using quantitative real-time PCR (qRT-PCR). Results: Global transcriptomic analysis revealed that rpfB induced differential transcriptional regulation in THP-1 macrophages. The total number of significantly enriched genes (SDEGs) induced by the WT strain was 5 times more than the mutant. Furthermore, the WT also induced a greater number of networks and upstream regulators. In addition this study showed rpfB stimulates, but is not essential to, IFN signalling, Role of JAK family kinases in IL-6 type cytokine signalling and Activation of IRF by Cytosolic PRRs, enhances IL-10 signalling and DC maturation, and enriches Acute phase response signalling, Phagosome formation, LXR/RXR activation, CAMP-mediated signalling, Gαi signalling and GADD45 signalling. In summary, the findings suggest rpfB contributes to the host protective immune response against Mtb infection. Upstream regulators induced by both infection models were all cytokines, however, they regulated genes that were exclusive to each pathway and mostly interferon stimulated genes (ISGs) in WT infected macrophages. Discussion and conclusion: Transcriptomic analysis showed rpfB induced recognition of host immunity and enhanced the THP-1 macrophage host immune response during late stages of Mtb infection. These findings provide basic evidence, which requires further interrogation, that rpfB regulates the immune response through interferon signalling and possibly plays a role in IL-10 signalling and DC maturation. Collectively, these findings indicate rpfB contributes to protective immunity of THP-1 macrophages against Mtb and may be used as a recombinant in vaccines against active TB, and in combination with LTBI antigens, against LTBI. However, since these observations are reported for the first time, further investigation to validate these findings would be valuable.
Investigating the in vitro roles played by the major adhesins HBHA and MTP in the pathogenesis of M. tuberculosis, in a novel double gene knock-out mutant strain.
(2018) Govender, Viveshree Shalom.; Pillay, Manormoney.
Abstract available in pdf.
Mycobacterium tuberculosis pili (MTP) modulates pathogen and host metabolomic changes in an A549 epithelial cell model of infection.
(2020) Reedoy, Kajal Soulakshana.; Pillay, Manormoney.; Pillay, Balakrishna.
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.
Primary capreomycin resistance is common and associated with early mortality in patients with extensively drug-resistant tuberculosis in KwaZulu-Natal, South Africa.
(Wolters Kluwer., 2015) O’Donnell, Max Roe.; Pillay, Melendhran.; Pillay, Manormoney.; Werner, Lise.; Master, Iqbal.; Wolf, Allison.; Mathema, Barun.; Coovadia, Yacoob Mahomed.; Mlisana, Koleka Patience.; Horsburgh, Charles Robert.; Padayatchi, Nesri.
Abstract available in pdf.
Rapid prediction of multi-drug resistance in clinical specimens of Mycobacterium tuberculosis.
(2011) Ndimande, Bongiwe Olga.; Pillay, Manormoney.
Conventional drug susceptibility testing techniques, the ‘gold standard’ for M. tuberculosis are slow, requiring about 3-6 weeks from a positive culture. This diagnostic delay, before initiation of appropriate treatment, contributes to increased transmission rates. Molecular techniques provide rapid results and therefore present an alternative to conventional tests. The aim of this project was to develop an inhouse reverse line blot hybridization assay (RIFO assay) that could detect mutations associated with Rifampicin resistance directly in clinical specimens of patients in KwaZulu Natal. A 437 bp region of the rpoB gene was sequenced to ascertain the most frequently occurring mutations conferring resistance to rifampicin in isolates in KwaZulu-Natal. Wildtype and mutant probes designed to target these mutations, were immobilized on a Biodyne C membrane. Hybridization conditions were optimized using biotin labeled PCR products from culture. Detection was performed with peroxidase labeled streptavidin using enhanced chemiluminescence. Four DNA extraction methods were evaluated on sputum specimens to determine the one with the least inhibitory effect on amplification. A total of 11 mutations were found in 236 clinical isolates: 531TTG (109, 58.3%), 516GTC (26, 13%), 533CCG/516GGC (20, 10%), 533CCG (18, 9.6%), other mutations < 5% each. The chelex extraction method was found to be optimal for removing inhibitors in sputum specimens. Sputum specimens of 404 patients hospitalized at King George V Hospital between 2005 and 2006 were rifoligotyped. The RIFO assay was optimised on clinical isolates and then applied to sputum specimens. The RIFO assay on culture and sputum correlated well with the DST (sensitivity 92% and 94% respectively). However, the specificity was very low in both culture and sputum specimens compared to DST (38% and 35% respectively). This could be attributed to the presence of silent mutations, mixed infections, mixed populations of bacteria or the small number of susceptible strains used in this study. The in-house RIFO assay can be used directly on sputum specimens to predict Rifampicin resistance and therefore MDR-TB in less than a week compared to the gold standards. A total of 43 samples can be tested simultaneously at low cost and the membrane is reusable compared to commercial kits such as the Hains test that is expensive and strips are not reusable. A similar assay can be designed to target mutations for the detection of XDR-TB. Future studies should be conducted in a clinical setting on patients with sensitive strains to increase the specificity.
The role of heparin binding haemagglutinin adhesin and curli pili on the pathogenicity of Mycobacterium tuberculosis.
(2018) Moodley, Suventha.; Pillay, Manormoney.
Background: Phagocytic host cells drive both the innate and adaptive arms of the host immune response during Mycobacterium tuberculosis (M. tuberculosis) infection. M. tuberculosis modulates the host immune responses and is able to proliferate in macrophages. The structures that mediate M. tuberculosis adherence (Adhesins) to macrophages are of particular interest for therapeutic development due to their cell surface localisation and immunogenic characteristics. M. tuberculosis produces numerous antigens that display adhesin functionality, including heparin-binding haemagglutinin adhesin (HBHA) and M. tuberculosis curli pili (MTP) that are critical for adherence to host cells. Recently, the independent elucidation of the immunogenic potential of each suggested that HBHA and MTP may represent a novel combination as a biomarker for future therapeutic development. This study aimed to elucidate the effect of HBHA and MTP in combination on adhesion, invasion, replication, cytokine production and transcription regulation of macrophages infected with HBHA and MTP proficient and deficient strains in an attempt to assess their immunogenic capacity. Materials and methods: THP-1 monocytic cells were differentiated into macrophages and infected at a multiplicity of infection of 5 with single mutants (ΔhbhA and Δmtp), single complements of double mutant (hbhA comp and mtp comp), MTP and HBHA deficient double mutant ΔhbhA-mtp (DM) and MTP and HBHA proficient wild-type (WT) strain. The relative percentage adhesion/ invasion of the mutant and complemented strains was calculated at 1 h and 2 h post-infection respectively and compared to wild-type. Intracellular replication was quantified by colony forming units at 4 h, day 3 and day 6 post-infection. To assess host transcriptomic changes elicited during early infection of THP-1 differentiated macrophages by WT and DM, RNA was extracted from host cells at 4 h post-infection. For the biological adhesion data set, raw data were filtered for genes in common with the Gene Ontology biological adhesion dataset sourced from EntrezGeneIds using the molecular signatures database with a False Discovery Rate q-value <1 (Chapter 1). Significantly differentially expressed genes with a p value <0.05 were used for further enrichment analysis (Chapter 2 and 3). Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, USA) upstream regulator, canonical pathway and biofunctions enrichment analysis were used to further investigate the differential regulation of molecular signatures by MTP and HBHA proficient and deficient strains. Macrophage cytokine/chemokine production was quantified at 24, 48 and 72 h post-infection using the Bio-Plex Pro Human Cytokine Multi-Plex Panel (Bio-Rad). Real-time quantitative RT-PCR was used to validate RNA sequencing findings and investigate transcriptional regulation of HBHA and MTP of following genes: CD80, DLX3, NLRP3, TGM5 and TLR2 at 1 h, 2 h and 4 h post-infection Results: During adhesion, DM induced a similar decrease in percentage adhesion (33.16%) to Δmtp (39.4%), ΔhbhA (22.78%), mtp comp (24.72%), but statistically lower decrease in percentage adhesion than hbhA comp (53.85%). During invasion, DM displayed a significant decrease in percentage invasion (36.49%) compared to Δmtp (61.49%) and hbhA comp (53.85%); and significantly higher decrease in percentage invasion than ΔhbhA (22.29%) and mtp comp (24.72%). Δmtp demonstrated a 39.4% and 61.49% decrease in percentage adhesion and invasion compared to WT respectively. The HBHA-MTP proficient strain induced greater transcriptional changes resulting in enhanced adhesion to phagocytes and invasion of cells. Furthermore, the HBHA-MTP proficient strain displayed the sole ability to induce activation of phagocytosis. Further investigation of canonical pathway differential regulation by HBHA-MTP proficient strain demonstrated greater induction of canonical pathways. The most differentially regulated pathway was Gαq signalling canonical pathway, which is vital for migration of phagocytes. In addition, the HBHA-MTP proficient strain also enhanced activation of the acute phase response, role of pattern recognition receptors in recognition of bacteria and viruses, and production of nitric oxide and reactive oxygen species in macrophages canonical pathways. RNA sequencing analysis showed that the M. tuberculosis adhesins, HBHA and MTP, elicited differential transcriptional regulation in macrophages, and demonstrated that predicted upstream regulators were associated with cytokine production. Further investigation of canonical pathways associated with these upstream regulators and cytokine quantification revealed that HBHA and MTP activate NF-κB, toll-like receptor, p38 MAPK and PI3-K/AKT canonical signalling pathways. HBHA and MTP elicited greater production of IL-4 and IL-10 at 24 h; G-CSF, GM-CSF, IL-2, IL-4, IL-5, IL-10, IL-12, IL-17, IFN-γ and TNF-α at 48 h and G-CSF, GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, IFN-γ and TNF-α at 72 h respectively, compared to DM infection. IL-1β, IL-2, IL-6, IL-12(p70), IL-17, TNF-α, IFN-γ, colony-stimulating factors G-CSF, GM-CSF and chemokines MCP-1 and MIP-1β were produced in higher concentrations by M. tuberculosis infection than anti-inflammatory cytokines IL-4, IL-5, IL-10 and IL-13. The bacillary load of M was significantly less than WT at all time intervals and similar to DM. The decreased replication ability of the HBHA-MTP mutant was attributed to MTP and not HBHA, suggesting that MTP facilitates replication during infection of macrophages. A transcriptional response common to both WT and DM, independent of HBHA-MTP, as well as unique responses induced by HBHA-MTP presence and deficiency were observed. The common transcriptional pattern exhibited the most enrichment for granulocyte adhesion and diapedesis canonical pathway, TNF upstream regulation and migration of cells biological function. The HBHA-MTP uniquely induced transcripts were associated with the most significant enrichment of the Adipogenesis pathway, whilst HBHA-MTP deficiency induced the most significant enrichment of T helper cell differentiation. Unique transcripts elicited by HBHA-MTP deficiency induced less enrichment of NF-κB upstream regulator and were associated with migration of cells. The top 10 canonical pathways enriched by all transcripts were similar between both infections, but differed in molecules involved and their significance. HBHA-MTP enriched the TREM1 signalling pathway to a greater degree than HBHA-MTP deficiency in macrophages. HBHA-MTP deficiency, but not presence, enriched Th1 and Th2 Activation, Th1, Th2, Melatonin degradation, Sumoylation, Methylglyoxal degradation III, Granzyme A signalling, PCP pathways. Discussion and conclusion: MTP played a greater role in adhesion and invasion during independent knockout and complementation in the double knockout strain than HBHA. HBHA and MTP together induced transcriptional changes that favour adhesion and invasion of macrophages. In addition, these 2 adhesins serve as pathogen-associated molecular patterns that enable host immune recognition during early infection of macrophages. HBHA and MTP activate intracellular signalling pathways that result in the longitudinal enhancement of a pro-inflammatory response during M. tuberculosis infection of macrophages. HBHA and MTP predominately induced a pro-inflammatory cytokine profile instead of an anti-inflammatory cytokine profile. This suggests that HBHA and MTP play a role in protective immunity and immunopathology as a consequence of pro-inflammatory cytokines such as TNF-α and minimal anti-inflammatory cytokines during M. tuberculosis infection. HBHA and MTP deficiency led to advanced immune activation and decreased intracellular growth. This suggests in the absence of HBHA and MTP, the presence of multiple, alternate antigens stimulate the intracellular signalling and transcriptional regulation in vitro. This advanced immune activation would potentially be detrimental to M. tuberculosis establishing a successful infection and would suggest that HBHA and MTP play a role in host immune response modulation as a protective measure during initial infection. Further investigation into the identity of these antigens would possibly result in a more successful, novel therapeutic target combination in addition to HBHA and MTP.
The role of Mycobacterium tuberculosis curli pili (MTP) and heparin-binding hemagglutinin adhesin (HBHA) on global in vitro bacterial transcriptomics.
(2021) Naidoo, Tarien Jael.; Pillay, Manormoney.; Pillay, Balakrishna.
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.
The role of mycobacterium tuberculosis pili in pathogenesis : growth and survival kinetics, gene regulation and host immune response, and in vitro growth kinetics.
(2016) Nyawo, Georgina Rumbidzai.; Pillay, Manormoney.
Abstract available in PDF file.
The role of specific adhesins in the regulation of other adhesin genes associated with Mycobacterium tuberculosis pathogenicity.
(2022) Mthembu, Johannes Nkanyiso Thandabantu.; Pillay, Manormoney.; Senzani, Sibusiso.
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.
Selection and identification of novel Mycobacterium tuberculosis phage-displayed biomarkers by immunoscreening against patients’ serum samples.
(2018) Chiliza, Thamsanqa Emmanuel.; Pillay, Balakrishna.; Pillay, Manormoney.
Abstract available in pdf.
A standardised approach to the treatment and management of significant acinetobacter species infection at academic complex hospitals in KwaZulu-Natal.
(2017) Swe Swe-Han, Khine.; Mlisana, Koleka Patience.; Baba, Kamaldeen.; Pillay, Manormoney.
Introduction: Carbapenem-resistant Acinetobacter species (Acinetobacter spp.) are increasingly recognised as important pathogens, whose resistance patterns present a high-risk global challenge. However, there is limited scientific data and a lack of a standardised approach to help the clinician select optimal therapy in local setting. This study aimed to provide a standardised approach for the management of significant Acinetobacter spp. infection based on phenotypic and genotypic characterisation of local isolates, as well as clinical characteristics and outcomes of patients at academic complex hospitals in KwaZulu-Natal. Objectives: The significance of Acinetobacter spp. infections and the most effective drug combinations for optimal therapy were determined. Acinetobacter spp. isolates were phenotypically and genotypically characterised. This was followed by the development of a standard management guideline for local use, based on the data obtained in the different objectives. Methods: The research consisted of a retrospective and prospective observational and experimental laboratory component. The laboratory component included synergy testing of colistin, susceptibility to antimicrobial agents in use at local hospitals, polymerase chain reaction and sequencing for analysis of the resistant genes related to carbapenem, colistin and amikacin. Phenotypic, genotypic, and clinical characterisation were utilised to develop a standardised management approach of significant Acinetobacter spp. infection. Results: Acinetobacter spp. was identified as a significant cause of sepsis and mortality among patients in a surgical intensive care unit (ICU). Cases of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Acinetobacter spp. increased over seven years, together with the emergence of pandrug-resistant (PDR) isolates. The results of synergy testing of colistin combinations with amikacin, carbapemens (imipemen, meropenem), ciprofloxacin, tazocin, linezolid, rifampicin and vancomycin against Acinetobacter spp. was highly diverse and speciesdependent. Characterisation of Acinetobacter spp. isolates showed that oxacillinase β-lactamase (OXA-23)-producing MDR isolates correlated with their antibiogram. Pulsed field gel electrophoresis (PFGE) showed horizontal transfer between seven clusters, each containing two patients each, totalling 14 patients. However, the PFGE typing revealed a diverse collection of MDR Acinetobacter spp. clones, and that isolates from not more than two patients were related. This suggests, therefore, that no outbreak had occurred based on the PFGE typing interpretation. Further genetic investigation revealed that the aphA6 gene were associated with amikacin resistance and IpxA gene may be associated with colistin resistance in our local setting. Conclusion: The results highlighted the importance of antibiotic stewardship in the treatment of Acinetobacter spp. infection. Individual-specific antibiograms are recommended as the best 2 approach for treatment in KwaZulu-Natal (KZN) and synergy testing should be performed for individualised direct therapy. The clinical and microbiological indicators of significant infection are crucial when establishing the decision to treat. The study provided a valuable standardised approach, including a flow chart of criteria for sepsis and colonisation; a standardised algorithm for the management; and synergy test at academic complex hospitals, Medical Microbiology laboratory, National Health Laboratory Service (NHLS) in KZN.
Whole transcriptome analysis to elucidate the role of mtp in gene regulation of pulmonary epithelial cells infected with Mycobacterium tuberculosis.
(2016) Dlamini, Mlungisi Thabiso.; Pillay, Manormoney.
Abstract available in PDF file.