Browsing by Author "Mvubu, Nontobeko Eunice."

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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.
The effect of prenatal Mycobacterium tuberculosis infection on offspring neurodevelopment and autistic-like behaviours in a valproic acid mouse model of autism.
(2021) Manjeese, Wadzanai.; Mpofana, Thabisile.; Mvubu, Nontobeko Eunice.; Steyn, Adrie J.C.
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by restricted repetitive patterns, communication challenges and lack of social skills. ASD has no distinct biomarkers, with symptoms overlapping with related developmental disorders like Schizophrenia. Maternal immune activation (MIA) is when the maternal immune system is invaded by a pathogen causing an immune response that interferes with the normal fetal brain development process. Mycobacterium tuberculosis (Mtb) infections are common during pregnancy and are known to affect fetal health, often causing spontaneous abortions and low birth weights. Valproic acid (VPA) is an anticonvulsant and mood stabilizer associated with ASD when administered during pregnancy. Gestational VPA exposure of mice on Embryonic day 12.5 (E12.5) induces ASD-traits in offspring, as such, this study employed VPA as a positive control. This study investigated the effects of prenatal exposure to Mycobacterium tuberculosis (Mtb) (singularly and in combination with VPA) on developmental delays and offspring behaviour. Pregnant mice were divided into saline, VPA, Mtb, and VPA+Mtb; treatments were administered on E12.5. Developmental milestones were measured between post-natal day 7 (PND 7) and 28. Offspring were subjected to neurobehavioural studies to test for social interaction and repetitive behaviours on PND 35. Ionised calcium binding molecule 1 (IBA-1) and Glial Fibrillary acid protein (GFAP) expression in the prefrontal cortex (PFC) and cerebellum regions were analysed using immunohistochemistry (IHC). The effect on the BBB’s function was determined using Evans blue dye-albumin extravasation method on PND 35. Additionally, cerebellar tissues were homogenized and processed for molecular analyses of NRXN1, NRXN2, NLGN1, NLGN2 and SHANK3 expression. Changes in expression patterns of NRXNs and NLGNs causes an imbalance in the excitation and inhibition of neurons, a feature associated with ASD. The Mtb treated group had significantly low litter count and high fetal resorption compared to saline treated group. Neuroinflammation was evident in the Mtb offspring at PND 35 as shown by a significant increase in GFAP and IBA-1 expressing astrocytes and microglia in the PFC and cerebellum compared to saline group. The BBB’s integrity was compromised as shown by the increased permeability to EB-dye in the PFC and cerebellum of Mtb, VPA and VPA+Mtb offspring. The Mtb offspring also displayed systemic inflammation and altered ASD-linked behaviours. NRXN1 and NLGN1 were overexpressed in the cerebellum of Mtb-induced MIA offspring compared to saline offspring. Dual exposure to VPA and Mtb restored NRXN1 expression levels, reduced astrocyte and microglia injury in the PFC, rescued social behaviours and restored normal eye-opening patterns in offspring. The study demonstrates impaired fetal development which persists into the post-natal period. The impaired development was accompanied by neuroanatomical changes and behavioural patterns consistent with ASD pathophysiology. These findings might be attributed to Mtb-induced maternal system inflammation in pregnancy that induces fetal inflammation via the placenta and BBB of a developing fetus causing insult in the brain. Immune dysregulation and synaptic defects are hallmarks of ASD. We therefore conclude that prenatal Mtb infection predisposes offspring to a higher risk of neurodevelopmental challenges later in life and dual exposure to VPA and Mtb rescues some of these challenges.
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 role of small RNAS in transcriptome regulation of genetically diverse clinical strains of mycobacterium tuberculosis.
(2021) Govender, Divenita.; Mvubu, Nontobeko Eunice.
Tuberculosis (TB), caused by the human adapted members of the Mycobacterium tuberculosis complex (MTBC), is a threat to global health. Understanding the regulatory network of the MTBC members may reveal novel vaccine candidates and drug targets. The small RNAs (sRNAs) have only recently been investigated for their role in Mycobacterium tuberculosis (M. tb) transcriptome regulation with none being explored in clinical strains or within the MTBC lineages. The present study aimed to investigate the regulatory role of sRNAs on the M. tb transcriptome in a lineage-specific manner, with emphasis on the clinical strains most prevalent in South Africa. In silico whole genome sequence alignment of strains belonging to the eight MTBC lineages was performed to identify sRNAs containing lineage-specific mutations and their respective potential targets. To elucidate transcriptome regulation in clinical strains of M. tb belonging to the Beijing and F15/LAM4/KZN lineages, mRNA and sRNA sequencing were performed followed by Hisat-Ballgown Bioinformatics analysis to identify novel sRNAs and their respective targets. The sRNAs discovered from sRNA sequencing were confirmed through real time qPCR. The in silico data revealed several sRNAs that may play a role in transcriptome regulation at a lineage-specific level, such as those involved in macrophage entry, lipid biosynthesis pathway, adaptation mechanisms during antibiotic exposure, and environmental stress. They may also be able to disrupt genes that are detrimental and restore functions to those that are beneficial. The mutated and consensus sRNAs were identified to target the same function, but one pathway may be more efficient than the other. Novel sRNAs were discovered from sRNA sequencing of the Beijing and F15/LAM4/KZN clinical strains, with their predicted targets absent from the mRNA sequencing results, indicating these sRNAs may elicit an inhibitory function. Real time-PCR analysis revealed significant fold change differences between the clinical strains belonging to the Beijing, F15/LAM4/KZN, F11 and Unique families suggesting an underlying regulation of these transcripts at a family level. This data could explain the underlying phenotypic differences observed within the MTBC and understanding of the regulatory function of these sRNAs, may identify novel alternative strategies in the fight against M. tb.