Doctoral Degrees (Medical Microbiology)

Permanent URI for this collectionhttps://hdl.handle.net/10413/9619

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Calymmatobacterium granulomatis: culture, electron microscopic studies and molecular analysis.
(1997) Kharsany, Ayesha Bibi Mahomed.; Hoosen, Anwar Ahmed.; Kiepiela, Photini.
Abstract available in PDF.
A pharmacokinetic study of rifabutin and its interaction with antiretrovirals in African patients with TB-HIV co-infection.
(2012) Naiker, Suhashni.; Pym, Alexander S.; McIlleron, Helen.
The management of HIV-associated tuberculosis (TB) is complicated by the pharmacokinetic interactions between rifampicin (RMP) and co-administered protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors. Rifabutin (RBT) is an alternative rifamycin, preferred in patients requiring PIs. Recent studies suggest the current recommended dose of RBT in combination with boosted lopinavir (LPV/r) is suboptimal and there are insufficient pharmacokinetic data evaluating the interaction between RBT coadministered with efavirenz (EFV) and nevirapine (NVP). Pharmacogenomic studies have shown that RMP concentrations are lower in patients from sub-Saharan Africa with polymorphisms of the SLCO1B1gene but there is currently no data on the pharmacogenetic determinants of RBT exposure. The pharmacokinetics of RBT were evaluated at two different doses in HIV co-infected patients before and after the introduction of LPV/r, EFV and NVPbased antiretroviral therapy (ART). After six weeks of standard TB therapy, RBT 300 mg daily was started for four weeks. Thereafter patients were randomized to receive either RBT 150 mg daily or RBT 150 mg three times a week (TPW) with LPV/r, RBT 300mg or 450mg with NVP or RBT- 450mg or 600mg with efavirenz. After four weeks on the first RBT dose, patients switched to the alternate dose and continued until the end of TB treatment. Serial RBT and 25-O-desacetylrifabutin (dRBT) concentrations were measured during a dose interval before patients switched RBT doses. The median AUC0-24 and Cmax, of RBT in patients taking 150mg RBT TPW was significantly reduced when compared to the other treatment arms. 86% of patients whilst on this intermittent RBT arm had an AUC0-24 < 4.5 μg.h/mL, level that has been associated with acquired rifamycin resistance. Rifabutin exposure was maintained within the range of AUCs that have been shown to prevent acquired rifamycin resistance (ARR) with 150mg daily dosing in combination with LPV/r. In addition, the combination of RBT with NVP 300mg resulted in significantly increased exposure of RBT, with significantly higher exposure observed with 600mg RBT. However, the combination of RBT 450mg with EFV resulted in RBT exposure lower than 300mg RBT given alone in the same patients, whereas RBT 600mg plus NVP results in bioavailability of RBT equivalent to 300mg given alone. Rifabutin was well tolerated at all doses. Only three grade 4 laboratory toxicities, elevated transaminases, neutropenia, and uveitis, possibly related to RBT were reported in patients taking NVP. SLCO1B1 rs4149032 C>T polymorphism occurs frequently in African patients in Durban and may be associated with low RBT bioavailability. These findings support recommendations for the higher dose of RBT in combination with LPV and EFV but not with NVP.
Host induced microevolution of ESX secretion systems of M. Tuberculosis.
(2013) Sukkhu, Melisha.; Pym, Alexander S.
The ESX family of genes (esxA-W) in Mycobacterium tuberculosis (Mtb) encodes 23 effector molecules influencing immunogenicity and pathogenicity. This study was aimed at identifying and evaluating variations in ESX sequence and protein expression profiles in clinical isolates and examining how diversity might influence immune responses. 23 ESX genes from 55 clinical isolates (20 Beijing, 25 KZN and 10 Other) and 3 Laboratory strains (H37Rv, H37Ra and BCG) were sequenced. 482 single nucleotide polymorphisms (SNPs) were identified in 12 ESX genes relative to H37Rv. Majority of the identified 363 nsSNPs occured in Beijing isolates. No mutations were observed in esxA, B, C, E, G, H, J, R, S and T. Six unique nsSNPs were identified in the Beijing isolates: esxI (Q20L), esxO (E52G), 2 in esxP (T3S; N83D), esxU (P63S) and esxW (T2A). Three unique nsSNPs were identified in the KZN isolates: esxK (A58T), esxL (R33S). The esxL polymorphism resulted from a dinucleotide change. ESX gene transcription levels were evaluated using RT-qPCR. Varying expression levels were observed for esxA, B, C, F, M and Q across all clinical isolates with lowest levels seen amongst the Beijing isolates. This correlated with immunoblots with confirmed decreased esxAB protein expression relative to the other strains. The Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) spectral protein profiles were quantitatively compared within and between Mtb clinical and laboratory isolates. Protein spectral profiles within the mass range of the CFP-10 protein with variations in peak intensities were observed across all isolates. QILSS and Mtb9.9 peptides were tested individually for immune responses in TB infected patients. Healthy patients displayed no responses to QILSS and Mtb9.9, strong but variable immune responses were detected for specific regions of QILSS and Mtb9.9 in TB infected patients. These findings demonstrate that differences in sequence, transcriptional profiles and protein expression patterns in ESX secreted proteins exist between clinical isolates, and may translate into differences in human immune responses. Further research is needed to correlate human host immune responses to the phenotype and genotype of the infecting strain of Mtb to determine the consequences of specific variations of the other ESX members. These studies are important for the development of improved immune diagnostics and vaccines.
Development of novel reagents for tuberculosis detection.
(2013) Ngubane, Nqobile Angel Cebile.; Pym, Alexander S.; Khati, Makobetsa.; Rubin, Eric.
Tuberculosis (TB) is one of the most prevalent infectious diseases worldwide and causes high morbidity and mortality, despite the widespread availability of effective antibiotics against most strains of Mycobacterium tuberculosis, which is the causative agent of TB. One of the primary reasons that hinder TB control is that many cases of active disease go undetected or are discovered late. This is, in large part, due to the relative insensitivity and limited specificity, amongst other limitations, of the current TB diagnostics tests. Moreover, M. tuberculosis infection can be asymptomatic and latent, or cause active disease. Therefore, an ideal or effective TB diagnostic needs to distinguish between these two states. The aim of this study was to develop novel diagnostic reagents for M. tuberculosis using phage displayed peptides and nucleic acid aptamers with a view to discerning latent from active TB. Using a linear (X12) and constrained (CX7C) phage display libraries, five rounds of selection (biopanning) were performed. Ten phage displayed peptides that bind to the mycobacteria surface were selected. These phage clones were identified using both random clone picking and high throughput (HTP) sequencing. A phage clone displaying the CPLHARLPC peptide was identified by HTP sequencing as the most enriched, representing 82.49% of the selected CX7C phage population. Further characterization showed that it bound better to different mycobacteria species, including M. tuberculosis, than the unselected phage library. Moreover, using surface plasmon resonance (SPR) technology, the chemically synthesised CPLHARLPC peptide was shown to bind M. tuberculosis H37Rv whole cell lysate and not non-mycobacteria lysates. In addition, using the systematic evolution of ligands by exponential enrichment (SELEX) protocol and SPR technology, 2'-Fluoro-pyrimidine-RNA aptamers were selected against the mycobacteria ESX-3 secreted protein, ESX-G. At least five aptamers were identified after five rounds of selection. Two of these aptamers, GH43 and GH78, not only bound EsxG with high affinities, KD 8.04 ± 1.90 nM and KD 78.85 ±9.40 nM respectively, but also preferentially bound EsxG better than the EsxA homologue. Taken together, these findings suggest that a combination of phage display, SELEX and HTP sequencing can be a useful tool for the identification of specific detection reagents that can bind to mycobacteria and its associated targets. These reagents could be exploited to develop alternative molecular probes for TB diagnostics.
The role of APOBEC3G in acute and early HIV-1 subtype C infection.
(2014) Reddy, Kavidha.; Winkler, Cheryl Ann.
Introduction APOBEC3G and other related cellular cytosine deaminase family members have potent antiviral activity. In the absence of HIV-1 Vif, APOBEC3G mutates the viral DNA during viral reverse transcription. Our knowledge of the Vif-APOBEC3G interaction in human populations infected with subtype C HIV-1 is limited. Investigation of interactions between HIV and its host is crucial as it can ultimately be exploited in vaccine and therapy design. We hypothesised that certain APOBEC3G haplotypes and/or their expression in peripheral blood mononuclear cells of seroconverters affect viral setpoint and CD4+ T cell counts. We also hypothesised that certain APOBEC3G genetic variants are associated with increased frequency of G to A hypermutations during primary HIV-1 infection and that Vif variability influences disease progression and its ability to neutralise APOBEC3G haplotypes. Methods Our South African study cohort consisted of females at high risk for HIV-1 infection and women with known recent HIV-1 infection. We used quantitative real-time PCR to measure APOBEC3G expression in HIV- and HIV+ samples during primary infection. APOBEC3G variants were identified by DNA sequencing and TaqMan Genotyping. The HIV-1env gene was sequenced to assess Env diversity and the extent of APOBEC3G induced hypermutations. Vif variability was assessed by plasma derived clonal Vif sequences (n= 10-20 per patient) and Vif function was assessed by APOBEC3G degradation assays and HIV-1 infectivity assays. Results We found no correlation between APOBEC3G expression levels and plasma viral loads (r=0.053, p=0.596) or CD4+ T cell counts (r=0.030, p=0.762) in 32 seroconverters. However, APOBEC3G expression levels were significantly higher in HIV- individuals compared to HIV+ individuals (p<0.0001) including matched pre- and post-infection samples from the same individuals (n=13, p<0.0001). Twenty five single nucleotide polymorphisms (SNPs) were identified within the APOBEC3G region. SNP 186R/R was associated with significantly higher viral loads (p=0.0097) and decreased CD4+ T cell levels (p=0.0081), indicating that 186R/R has a negative effect on HIV restriction. Overall HIV-1 env sequences contained a higher number of APOBEC3F compared to APOBEC3G-induced hypermutations and the number of APOBEC3F-induced hypermutations correlated negatively with viral load (r= -0.6, p=0.006) and positively with CD4 T cell counts (r=0.6, p=0.004). We cloned and sequenced a total of 392 subtype C Vifs, which showed an interpatient diversity of 6.2% to 19.2% at the amino acid level. Interestingly, Vif sequence comparison showed a strong preference for a Lysine or a Serine at position 36 for APOBEC3G 186R/R and APOBEC3G 186H/H individuals, respectively. Selected natural subtype C Vif alleles had greater ability to counteract wild type APOBEC3G 186H as compared to the APOBEC3G 186R variant as shown by both functional and HIV infectivity assays. Conclusions In conclusion, APOBEC3G expression in peripheral blood mononuclear cells does not correlate with viral loads or CD4+ T cell counts during primary HIV-1 subtype C infection. However, genetic variants of APOBEC3G may affect HIV-1 pathogenesis. Amino acid changes in Vif may influence its anti-APOBEC3 activity. HIV-1 subtype C Vifs may have adapted to counteract the more active wild type APOBEC3G as compared to the less active APOBEC3G 186R variant. These studies have improved our understanding of viral-host interactions in African populations and HIV-1 subtype C infections.
The impact of sexually transmitted infections (STI) and genital tract inflammation on HIV-1 acquisition and rate of disease progression in subtype C infected women.
(2014) Mlisana, Koleka Patience.; Kharsany, Ayesha Bibi Mahomed.; Passmore, Jo-Ann Shelley.
Introduction: Women carry more than half the burden of HIV disease globally and this burden is even higher in sub-Saharan Africa (SSA). Young women, in particular, are at disproportionate risk of HIV infection in South Africa. Understanding risk behaviours and factors associated with ability to negotiate safe sex and condom use is one of the key elements in curbing the spread of HIV. Sexually transmitted infections (STI) and bacterial vaginosis (BV), which cause female genital tract inflammation, have been identified as key drivers of the HIV epidemic. This inflammation, which is also present in the absence of symptoms, is associated with increased susceptibility to HIV infection. Although syndromic management of symptomatic STIs or BV at the first encounter with a health care provider is an important public health measure, its effectiveness is minimised because a substantial proportion of individuals have either asymptomatic infections or fail to recognise signs and symptoms of STI and are therefore excluded. Most new HIV infections in SSA occur among young people and particularly among young girls. Prompt diagnosis of acute HIV infection (AHI) is critical and benefits the individual as well as providing opportunities for public health intervention. In South Africa, the majority of HIV infections are due to infection with HIV-1 subtype C for which there is limited data compared to subtype-B HIV-1 infections. The overall aim of this study was to assess the impact of BV, STIs, and associated genital tract inflammation on acquisition of HIV-1 subtype C infections; and evaluate the rate of subsequent disease progression in women. The objectives were: i. to investigate STIs and genital tract inflammation as risk factors for HIV infection in high risk women and adequacy of syndromic management; ii. to evaluate the challenges associated with diagnosing recently acquired (acute) HIV infection in a subtype C prevalent population; iii. and to evaluate the relationship between clinical disease progression and genital and or systemic inflammation in high-risk women who became infected with HIV. We assessed the adequacy of syndromic diagnosis of STIs, compared with laboratory diagnosis of STIs, and evaluated the association between STI diagnosis and the risk of HIV acquisition in a cohort of high-risk women. Genital cytokine profiles and the degree of inflammation associated with common STIs and bacterial vaginosis were assessed. The most common signs and symptoms of acute HIV infection (AHI) were described and a clinical algorithm to identify acute HIV cases was developed. We investigated rates of HIV disease progression of subtype C–infected South African women. Methods: The CAPRISA 002 study was a prospective cohort study established to examine the pathogenesis and natural history of HIV-1 subtype C infection and to describe the immunologic, virologic and clinical characteristics of acute and early infection in KwaZulu Natal, South Africa. A total of 775 high-risk women were screened for HIV infection, and 245 HIV-uninfected women were enrolled into the study. At each monthly visit for a total of 24 months behavioural and clinical data were collected. Cervico-vaginal lavage (CVL) samples were collected at enrolment and at each six month follow-up visits and were tested for STI pathogens (including Chlamydia trachomatis, Neisseria gonorrhoeae, herpes simplex virus type 2 (HSV-2) and Trichomonas vaginalis) and bacterial vaginosis. Forty-two cytokines were measured from the CVL and 13 from the plasma samples at enrolment. All women received monthly HIV-1 antibody and RNA testing and were assessed for AHI. Signs and symptoms at the visit with HIV-1 antibody or HIV-1 RNA positive test were compared to HIV negative visits. Logistic regression identified clinical predictors of AHI and a model-based score was assigned to each predictor to create a risk score for every woman. All women who seroconverted were followed up for more than five years and monitored for HIV disease progression. Rapid disease progression was defined as CD4+ T cell count decline to <350 cells/μl within two years post-infection. Serial clinical and laboratory assessments were compared using survival analysis and logistic regression models. CAPRISA had established several cohorts of HIV negative women to determine the feasibility of establishing cohorts and sites for HIV biomedical prevention trials. Women seroconverting in these studies were referred to the CAPRISA 002 study for longitudinal follow-up for HIV post seroconversion. Results: In this study, the HIV-1 prevalence at screening was 59.6% (95% CI: 55.9% to 62.8%). During a total of 390 person-years of follow-up, 28 new infections occurred, yielding a seroincidence rate of 7.2 (95% CI: 4.5 to 9.8) per 100 person-years. A total of 62 participants, including seroconvertors from other CAPRISA cohorts, were enrolled into the acute HIV infection Women from the HIV-1 negative cohort generally demonstrated a high level of knowledge on HIV/AIDS, and 60.3% reported use of condoms. Reported condom use at last sexual encounter varied slightly by partner type (57.0% with steady versus 64.4% with casual partners; p = 0.36), whilst self-perceived ability to choose to use a condom was significantly lower with steady partners compared to casual partners (20.8% versus 53.9%; p=0.01). An important finding was that vaginal discharge was a poor predictor of laboratory-diagnosed STIs, as only 12.3% of women (25/204) who had a laboratory-confirmed discharge causing pathogen had clinical evidence of a discharge (yielding a sensitivity of 12.3% and a specificity of 93.8%). CVL cytokine concentrations did not differ between women with asymptomatic or symptomatic STIs; and were elevated in women with either asymptomatic or symptomatic STIs compared to women with no STIs or BV. Women with chlamydia or gonorrhoea had the highest genital cytokine concentrations, with 17/42 and 14/42 of the cytokines measured in CVL being up-regulated compared with women with no infections, respectively. While BV was associated with elevated pro-inflammatory cytokine concentrations in CVL, women with BV had lower levels of chemokines and haematopoietic cytokines than women with no infections or BV. HSV-2 reactivation was inflammatory, but yielded a comparatively lower level of inflammation than Chlamydia or gonnorhoea. Trichomoniasis, despite being relatively common in this cohort, did not cause significant changes in genital tract cytokine concentrations compared to women with no infections or BV. Genital infections did not influence plasma cytokine concentrations, suggesting that the compartments were not linked with respect to cytokine responses. Although laboratorydiagnosed STIs were associated with increased risk of HIV infection [hazard ratio, 3.3 (95% confidence interval, 1.5 – 7.2)], clinical symptoms were not. Of the women who became infected, factors predictive of AHI included age, 25 years (OR = 3.2; 1.4 – 7.1), rash (OR = 6.1; 2.4 –15.4), sore throat (OR = 2.7; 1.0 – 7.6), weight loss (OR = 4.4; 1.5 – 13.4), genital ulcers (OR = 8.0; 1.6 – 39.5) and vaginal discharge (OR = 5.4; 1.6 – 18.4). A risk score of 2 correctly predicted AHI in 50.0% of cases. The number of signs and symptoms correlated with higher HIV-1 RNA at diagnosis (r = 0.63; p = 0.001). The 62 acutely infected women were identified at a median of 42 days post-infection (IQR = 34 – 59). Mean CD4 count dropped by 39.6% at 3 months and 46.7% at 6 months post-infection in women with pre-infection measurements. CD4 decline to <350 cells/μL occurred in 31%, 44%, and 55% of women at 1, 2, and 3 years post-infection, respectively, and to <500 cells/μL in 69%, 79%, and 81% at equivalent time-points. Predictors of rapid progression were CD4 count at 3 months post-infection (hazard ratio [HR], 2.07; 95% confidence interval [CI], 1.31–3.28; P = .002), setpoint viral load (HR, 3.82; 95% CI, 1.51–9.67; P = .005), and hepatitis B coinfection (HR, 4.54; 95% CI, 1.31–15.69; P = .017). Conversely, presence of any of HLAB*1302, B*27, B*57, B*5801, or B*8101 alleles predicted non–rapid progression (HR, 0.19; 95% CI, .05–.74; P = .016). Discussion/Conclusion: This study showed that syndromic STI diagnosis, which is dependent on clinical signs of vaginal discharge, was poorly predictive of laboratory-diagnosed STIs or BV and missed a significant proportion of women with asymptomatic infections. However, the level of genital inflammation, as measured by cytokine concentrations in CVL, was similar in women with symptomatic and asymptomatic infections and therefore place women at increased risk for HIV infection. While laboratory-diagnosed STIs and the presence of inflammatory cytokines in the genital tract were associated with increased susceptibility to HIV acquisition, vaginal discharge was not. Chlamydial infection was associated with the highest genital cytokine concentrations, followed by gonorrhoea, HSV-2, trichomoniasis, and BV. In regions where HIV is prevalent and STIs are managed syndromically, targeted screening of populations at risk for STIs is critical and urgently needed. Recognition of signs and symptoms of AHI is important for early diagnosis of HIV infection. The proposed algorithm of risk-stratifying individuals for AHI provides a useful clinical tool especially in resource-limited settings where there are limited or no routinely available tests for AHI. However, validation of the algorithm on another cohort is needed to assess its utility further. Point-of-care HIV antigen or viral load testing is needed, to detect asymptomatic, antibody negative cases enabling early interventions and prevention of transmission. This cohort showed high rates of rapid disease progression, with nearly half of these subtype C–infected women progressing to a CD4+ T cell count of below 350 cells/μL within 2 years of infection. Implementing 2013 World Health Organization treatment guidelines (CD4+ T cell counts less than 500cells/μL) would require most individuals to start antiretroviral therapy within 1 year of HIV infection. The economic and health systems planning implicated by these findings need to be explored and addressed to guide policy makers as countries adopt the current WHO guidelines.
Immunopathogenesis of vulvo-vaginal candidiasis in human immunodeficiency virus infected women.
(2014) Apalata, Teke.; Moodley, Prashini.
Vulvovaginal candidiasis (VVC) is an important cause of lower genital tract infections in women. There are currently numerous clinical observations linking increased cases of symptomatic VVC to the progression of HIV epidemic. While the pathogenesis of other commonly encountered mucosal candidiasis (oral and oesophageal) in the context of HIV infection has been well studied, gaps in our knowledge remain regarding candida vaginitis. With increasing degree of immunosuppression, symptomatic VVC in HIV infected women is frequent, severe, recurrent and less responsive to conventional anti-fungal therapy. The quality of life is greatly diminished for women who experience recurrent episodes of symptomatic VVC. Furthermore, the high prevalence of HIV infected women adds to the burden of healthcare. In this study, we sought to further understand the pathogenesis of symptomatic VVC and the associated host defense mechanisms in HIV infected women. The results of this study are presented as a collection of 5 papers, 4 of which are published in peer reviewed journals, 1 is still under review. The initial chapter of this thesis, ‘Introduction and Background’, is followed by the 5 manuscripts which are grouped into 3 consecutive result chapters as follows: I. Impact of HIV on symptomatic VVC. II. Impact of symptomatic VVC on HIV RNA levels in plasma and genital secretions. III. Plasma and vaginal-associated immune responses in women with symptomatic VVC. The final chapter, ‘Discussion and Conclusions’, rounds up the thesis.
Improving the efficacy of bacillus calmette guerin vaccine by concomitant inhibition of T regulatory and T helper 2 cells.
(2015) Kumar, Santosh.; Moodley, Prashini.; Das, Gobardhan.
Tuberculosis (TB) remains a major threat to human population as currently Mycobacterium tuberculosis (MTB) infects nearly 33% of the global population. Annually, about one and a half million deaths are caused by tuberculosis (TB). Current reports suggest that approximately nine million new TB cases are reported every year. There is an available therapy for TB, however it is quite lengthily and consists of numerous antibiotics leading to treatment dropout. This treatment incompletion has been linked to the major cause for the appearance of drug-resistant species of MTB. Consequently, alternate therapies to treat TB are needed. Bacillus Calmette-Guerin (BCG) remains the only vaccine of choice since its inception in 1921. Although BCG mounts host protective T helper1 (Th1) cell activation, which plays a pivotal role in host protection against TB, its efficacy is inadequate, suggesting that additional methods to enhance protective immune responses are needed. We have also shown that simultaneous inhibition of Th2 cells and Tregs by using pharmacological inhibitors (suplatasttosylate and D4476, respectively) dramatically enhance MTB clearance and induces a superior Th1 response. Here we show that treatment with these two immuno-modulators during BCG vaccination dramatically improves vaccine efficacy. Furthermore, we demonstrate that these drugs induce a shift in T cell memory development, towards central memory T (Tcm) cell responses. Collectively, our findings provide evidence that concurrent inhibition of T helper cells type 2 and Tregs during BCG vaccination promotes vaccine efficacy.
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.
Genetic and microrna polymorphisms in young South African Indians with coronary artery disease.
(2015) Ramkaran, Prithiksha.; Chuturgoon, Anil Amichund.; Phulukdaree, Alisa.; Khan, Sajidah.
The global burden of cardiovascular disease (CVD) is on the increase with coronary artery disease (CAD) estimated to become the leading cause of mortality worldwide by 2020. The age of onset of this chronic disorder is on the decline, particularly in the South African Indian population. Indians in South Africa (SA) have a higher prevalence of premature CAD compared to other ethnic groups in SA. Coronary artery disease is a lifestyle and genetic disease, and the inheritance of genetic variation from one or both parents plays an important role in the risk of an individual developing CAD. Genetic and epigenetic studies are being explored as potential tools for therapeutic interventions against CVDs. The role of single nucleotide polymorphisms (SNP) in microRNAs (miR, miRNA) and molecules regulating epigenetic pathways remains poorly understood. This study investigated SNPs in candidate genes; methylenetetrahydrofolate reductase (MTHFR), sirtuin (SIRT) 1, miR-499, and miR-146a; in young SA Indians with CAD. The study population included 106 SA Indian male CAD patients, 100 sex- and age-matched Indian, and 84 sex- and age-matched Black controls. The MTHFR, miR-146a, and miR-499 SNPs were investigated by PCR-RFLP, whilst a TaqMan SNP Genotyping assay assessed the SIRT1 SNPs. MiR-146a expression was measured by qPCR and western blot was used to assess the expression of NF-κB, IRAK-1, and TRAF-6. Interleukin (IL)-6 levels were analysed using an ELISA. All clinical parameters were obtained from pathology reports. Methylenetetrahydrofolate reductase is involved in folate metabolism and methylation pathways. The MTHFR rs1801133 has been associated with increased levels of homocysteine, a well known risk factor for CAD. Sirtuin 1, histone deacetylase, has been identified as a candidate molecule affecting the epigenetic mechanisms of CAD. Two common SNPs in the SIRT1 gene, rs7895833 and rs1467568, have been associated with several well-established risk factors for CAD. MicroRNAs (miRNAs) are small noncoding RNA molecules that inhibit messenger RNA (mRNA) translation or promoting mRNA degradation. MiR-499 and miR-146a are inflammatory-associated miRNAs. Two miRNA SNPs, miR-146a rs2910164 and miR-499 rs3746444, have been implicated in chronic inflammatory diseases. There was a significant association between the MTHFR variant (T) allele and CAD patients compared to Indian controls (p=0.0353, OR=2.105 95% CI 1.077–4.114). Indian controls presented with a higher frequency of the T allele compared to Black controls (7% vs. 2% respectively, p=0.0515 OR=3.086 95% CI 0.9958–9.564). The variant allele for the two SIRT1 SNPs occurred more frequently in the total Indian group compared to the total Black population (rs1467568: 41% vs. 18.5% respectively, p<0.0001, OR=3.190 95% CI 2.058-40943 and rs7895833: 41% vs. 22% respectively, p<0.0001, OR=2.466 95% CI 1.620–3.755). Indian controls presented with a higher frequency for both SNPs compared to Black controls (rs1467568: 40% vs. 18.5% respectively, p<0.0001, OR=2.996 95% CI 1.850–4.853 and rs7895833: 41% vs. 22% respectively, p<0.0001, OR=2.513 95% CI 1.578–4.004). No difference was seen in the distribution of both SNPs between CAD patients and either control group. The MTHFR and SIRT1 SNPs were not associated with any clinical parameters in CAD patients and controls. The miR-499 variant (G) allele was found at a higher frequency in the total Indian group (34%) compared to the total Black population (22%) (p=0.0070, OR=1.796 95% CI 1.182–2.730). Indian cases presented with higher frequency of the rs3746444 G allele compared to Indian controls (38% vs. 29%, p=0.059, respectively). No differences in genotypic frequency for rs2910164 was found (GG: 45 vs. 47 %, GC: 46 vs. 41 %, CC: 9 vs. 12 %) in controls and patients respectively (odds ratio=1.025; 95 % confidence interval 0.6782–1.550; p=0.9164). The lowest levels of NF-κB and C-reactive protein (hsCRP) were found in patients with the homozygous C allele compared to the heterozygous GC and wildtype variants. Higher levels of miR-499 targets, hsCRP and IL-6, were observed in CAD patients with the variant genotypes compared to those with the wild type genotypes (8.92±1.91 vs. 6.73±0.87 mg/L; p=0.299, 3.02±0.77 vs. 2.18±0.57 pg/mL; p=0.381 respectively). A 6.25-fold increase in miR-146a levels was observed in CAD patients with the CC genotype (relative to controls and patients with the wildtype variant, p<0.0001). These (CC genotype) patients had significantly lower levels of miR-146a targets, IRAK-1 (0.38±0.02; p=0.0072) and TRAF-6 (0.44±0.02; p=0.0146). Taken together, this study provides the frequency distribution of the SNPs in four candidate genes for CAD in young South African Indians compared to Indian and Black controls. The frequency of variant alleles of rs1801133, rs3746444, rs7895833 and rs1467568 was greater in the Indian population compared to Black South Africans. Although no difference in frequency was observed for rs2010164, our results suggest a role for miR-146a in toll-like receptor (TLR) signalling via a negative feedback mechanism involving the attenuation of NF-κB by downregulation of IRAK-1 and TRAF-6. Thus miR-146a can act as a target for therapy towards lowering inflammation in CAD patients.
Susceptibility of Trichomonas vaginalis to metronidazole and other compounds.
(2015) Naidoo, Sarita.; Sturm, Adriaan Willem.
Trichomonas vaginalis is the most common sexually transmitted infection caused by a single known organism worldwide; and has been associated with an increased risk of HIV acquisition and transmission. Despite its high prevalence in South Africa, limited information is available on the extent of T. vaginalis metronidazole resistance and genotypic variation in this setting. We therefore tested the susceptibility of local T. vaginalis isolates against metronidazole and drugs prescribed in combination in the context of syndromic management of vaginal discharge syndrome. Susceptibility testing of 40 isolates demonstrated that metronidazole as well as some of the other drugs tested showed inhibiting effect on T. vaginalis. We recommend that these drugs be tested for synergistic effect with metronidazole. In a different set of 160 isolates the minimum inhibitory concentrations (MIC) of metronidazole ranged from 1.1 μg/ml to > 34.2 μg/ml (6.25 μM to > 200 μM) in the aerobic assay. Interpretation of these MICs differed based on the different resistance breakpoints applied. There was no correlation between MIC and treatment outcome in the subset of 56 patients that returned for follow-up. The expected association between MIC and clinical outcome was only observed in one of eight patients with unsatisfactory treatment outcome. This patient‘s isolate had the highest MIC. In the remaining seven patients with unsatisfactory treatment outcome, no relation with the susceptibility test result was found. A possible reason for the poor correlation may be inadequate concentration of metronidazole at the site of infection. In view of this, we assessed a self-administered and collected vaginal tampon specimen for the investigation of metronidazole concentration in the vagina of five healthy volunteers, using high performance liquid chromatography (HPLC). Maximum values of metronidazole concentrations detected in both serum and vaginal fluid were obtained at two hours following oral administration of 2 g of the drug. This method can be applied in future clinical studies to correlate treatment outcome and MICs with metronidazole concentration at the site of infection. This may lead to the development of susceptibility assays and interpretation criteria that are better able to predict treatment outcome than the current methods. Another reason for the poor correlation between treatment outcome and in vitro resistance may be early reinfection. We used PCR-RFLP, targeting a 650-bp repeat region in the T. vaginalis genome, to genotype T. vaginalis isolates. Four genotypes were found in 100 T. vaginalis isolates using this method. Both the vaginal secretion of metronidazole and the strain typing methodology needs to be further investigated before a comprehensive study as outlined above can be executed.
TylA has an essential virulence role in mycobacterium tuberculosis pathogenesis.
(2015) Sobia, Parveen.; Moodley, Prashini.
Mycobacterium tuberculosis (M.tb), the causative agent of the disease tuberculosis, is an ancient pathogen and a major cause of death worldwide. Although various virulence factors of M.tb have been identified, its pathogenesis remains incompletely understood. TlyA is a virulence factor that is evolutionarily conserved in many gram-positive bacteria, but its function in the pathogenesis of infection with M.tb has not been elucidated. Here, we report that TlyA cause translocation of M.tb from phagolysosome into the cytosol in murine macrophages, which is the key to mycobacterial pathogenesis. In this study we also showed that TlyA mutant M.tb strain induces increased IL-12 and reduced IL-1β and IL-10 cytokine responses, which is in contrast to the immune responses induced by wild type M.tb. Mice infected with TlyA deficient mutant M.tb organisms exhibited increased host protective immune responses, reduced bacillary load, and increased survival compared with animals infected with wild type M.tb. Therefore it is likely that M.tb employs TlyA as a host evasion factor, thereby contributing to its virulence.
Effectiveness of a monovalent human rotavirus vaccine among children of 5 years and under in KwaZulu-Natal.
(2016) Asowata, Osaretin Emmanuel.; Moodley, Prashini.
Human rotavirus infection is the leading cause of gastroenteritis in infants and young children worldwide. In South Africa, gastroenteritis is a major cause of childhood morbidity and mortality in children less than 5 years, and rotavirus infection has been documented as causing one-third of all gastroenteritis related hospital admissions. Vaccination is the major public health intervention to control rotavirus disease. The Rotarix® is the only rotavirus vaccine included in the national immunization program of South Africa. The effectiveness of this vaccine is questionable due to the continual outbreaks of rotavirus infection in South Africa, including KwaZulu-Natal, regardless of the high vaccination coverage. This study focused on evaluating the factors influencing the effectiveness of the Rotarix® vaccine in children 5 years and under in KwaZulu-Natal, South Africa. After obtaining written informed consent from parents or guardians, stool and blood specimens where collected from children 5 years and under presenting to King Edward VIII hospital (KEH VIII) in Durban, South Africa. The study was conducted between June 2014 and June 2015. Demographic and clinical information was collected using a well-structured questionnaire. Enzyme immunoassay (EIA) was performed to detect rotavirus antigen in the stool and rotavirus immunoglobulin G (IgG) in the serum. Selected EIA positive and negative samples were confirmed using G-types and P-types consensus primers in a Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). The RT-PCR positives were genotyped using genotypes specific primers. The avidity of the rotavirus specific IgG was determined using the urea elution technique. Rotarix® vaccines stored at optimum temperatures were collected from the provincial pharmaceutical store. The effect of sub-optimal temperatures on the potency of the Rotarix® vaccine were determined using the plaque assay. Three hundred and sixty-five (365) stool specimens were collected. Rotavirus antigen was detected in 83 (22.7%) patients from stool specimens. The stratification of rotavirus cases by vaccination status was not significant (p=0.4). The distribution of rotavirus was not significantly associated with HIV status of the children (p=0.7). We observed that seasonality was a significant driving force influencing the prevalence of rotavirus infection in our setting (p<0.001). We recorded the highest rotavirus prevalence in the winter months of the year with 79 (45.9%) positive cases of rotavirus associated diarrhoea. Blood specimens were only collected in 35 patients. From the corresponding stool specimens [21 (60%) EIA positives and 14 (40%) EIA negatives)], 29 (82.9%) were positive for rotavirus using conventional RT-PCR. Genotyping revealed G9P[8] (20.7%) to be the most prevalent genotype followed by G9P[4] (13.8%), G12P[4] (10.3%), G9P[6] (6.9%) and a 3.4% prevalence was recorded for each of G4/G8P[6], G4P[6], G12P[6], G8P[10] and G9P[10]. We were unable to fully genotype some of the rotavirus strains (non-typeable) by the available primers. 2 (6.9%) and 4 (13.8%) were non-typeable for the G and P types respectively. However, all 35 serum samples were positive for rotavirus IgG. We observed that the rotavirus specific IgG had no significant effect on the prevalence of rotavirus detection in stool (p=0.8). There was no significant difference in the mean avidity of IgG in the 3 vaccination strata (p=0.3). Exposure of the Rotarix® vaccine to the seasonal temperatures and to extreme temperatures of 40oC for 3 to 72 hours as well as -20oC and -80oC for 12 hours did not affect the potency of the vaccine beyond its expected standard. Our study highlighted the genetic diversity of rotaviruses and poor immunogenicity of the vaccine as key factors affecting the effectiveness of the rotavirus vaccine. Whether the vaccine is able to induce homotypic and heterotypic protection in immunized children is critical in predicting the long range effectiveness of this vaccine against uncommon regional rotavirus strains. Interventions targeted at improving socio-economic conditions in low income countries might be a starting point towards the control and prevention of rotavirus infection in these settings.
Microchemostat technologies for characterization of efflux pumps associated with multidrug resistance in mycobacterium tuberculosis.
(2016) Mackenzie, Jared Stuart.; Balagaddé, Frederick.
Abstract available in PDF file.
Antibiotic resistance in mycobacterium tuberculosis : the role of genetic mutations in resistance conferring genes and efflux transporters.
(2016) Dookie, Navisha.; Moodley, Prashini.
Two decades after the World Health Organisation (WHO) declaration of tuberculosis (TB) as a global emergency, the disease remains a public health crisis of epic proportions. The emergence of drug resistant strains of Mycobacterium tuberculosis, the etiologic agent of TB, and the convergent human immunodeficiency virus (HIV) epidemic places a devastating burden on an already weakened public health care system in South Africa. Rapid and accurate detection of drug resistance to first and second line drugs to guide effective treatment of TB is central to control of the disease and in preventing further dissemination of drug resistant strains. Knowledge of the underlying resistance mechanisms driving drug resistance in M.tuberculosis is pivotal in the design of rapid molecular based assays and will impact of the development of novel drugs and regimens for the disease. The manuscript in chapter 2 of this thesis, entitled Dynamics of antimicrobial resistance in Multi-Drug and Extensively Drug resistant strains of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa, demonstrated the diversity of the resistance mechanisms amongst the multidrug resistant (MDR) TB strains currently circulating in the KwaZulu-Natal province of South Africa by the analysis of the rpoB, katG, inhA, pncA and embB genes associated with resistance to key drugs used in the treatment of TB. Multiple drug resistance mechanisms in the MDR-TB isolates suggests that the strains emerged separately and acquired resistance mutations independently. The findings of this study also confirms the clonality of the XDR-TB epidemic demonstrated by the predominance of the F15/LAM4/KZN strain family and reveals that MDR-TB strains are evolving and spreading via transmission. The manuscript in chapter 3 of this thesis, entitled Streptomycin resistance in the F15/LAM4/KZN strain of Mycobacterium tuberculosis is mediated by lineage-specific alteration of the gidB gene, demonstrated that streptomycin (STR) resistance in the F15/LAM 4/KZN MDR and XDR-TB strains was mediated by a rare, 130bp deletion within the gidB gene of M.tuberculosis leading to a complete disruption of the gene. Classical mutations in the rpsL gene mediated STR resistance in the remaining strain families. Widespread STR resistance has resulted in the exclusion of the drug from current treatment regimens. The findings of this study support the decision of policymakers and cautions the application of the drug in the absence of drug susceptibility testing. The manuscript in chapter 4 of this thesis, entitled Moxifloxacin resistance in the F15/LAM4/KZN extensively drug-resistant strain of Mycobacterium tuberculosis, demonstrated that the F15/LAM4/KZN XDR strain harboured the A90V gyrA mutation associated with high level ciprofloxacin (CPX) and ofloxacin (OFX) resistance and correlated with increased minimum inhibitory concentrations (MIC) for moxifloxacin (MXF). The results of this study cautions the utilization of MXF as part of empiric treatment protocols in the absence of moxifloxacin MIC data of the circulating XDR strains in an area. It also raises concerns regarding the regarding the use of moxifloxacin in KwaZulu-Natal. Furthermore, the current breakpoint defining resistance to MXF is of concern and requires revision. The manuscript in chapter 5 of this thesis, entitled Evaluation of Capreomycin in the treatment of the F15/LAM4/KZN extensively drug-resistant strain of Mycobacterium tuberculosis demonstrated that the A1401G rrs mutation was the main mechanism mediating resistance to the aminoglycosides, kanamycin (KAN) and amikacin (AMIK); and to capreomycin (CAP). CAP was reintroduced into TB treatment protocols without prior drug susceptibility testing. This results of this study demonstrates high level resistance to CAP and urges careful consideration in the application of CAP the KwaZulu-Natal province. Furthermore, concerns regarding the high breakpoint value that defines CAP resistance as compared to wild-type MICs for the drug results in misdiagnosis of resistance that results inadequate patient treatment and amplifies resistance. The manuscript in chapter 6 of this thesis, entitled KZN Multidrug and Extensively drug resistant strains of Mycobacterium tuberculosis remain susceptible to Linezolid and para-Amino salicylic Acid, demonstrated that the mechanisms most commonly associated with resistance to the linezolid (LIN) and para-amino salicylic acid (PAS) were absent in the MDR and XDR-TB strains in this study. Mutations detected in the drug targets were lineage specific markers rather than resistance mechanisms. This study also highlights the poor understanding of resistance to these drugs and the need for further study to allow for resistance detection to be incorporated into diagnostic assays, thus prolonging the utility of these drugs. The manuscript in chapter 7 of this thesis, entitled Efflux mediated drug resistance in clinical isolates of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa, demonstrated the role of efflux pumps in mediating low level resistance. The results of this study supports the hypothesis that efflux activity leads to decreased intracellular antibiotic concentrations, thereby allowing the survival of a sub-population of bacteria under the sub-inhibitory level of the antibiotic, from which resistant mutants emerge, leading to clinically significant levels of resistance. The results of this study strongly supports the application of efflux pump inhibitors as adjunctive to the current treatment protocols. The results emanating from this thesis has contributed to the body of knowledge of drug resistance in M.tuberculosis, especially in the KwaZulu-Natal province of South Africa. Furthermore, the results can be used to guide treatment protocols and contributes to the future development of molecular based assays aimed at detecting resistance.
Molecular epidemiology of antibiotic resistant ESKAPE pathogens isolated from public sector hospitals in uMgungundlovu District, KwaZulu-Natal, South Africa.
(2017) Zangue, Raspail Carrel Founou.; Essack, Sabiha Yusuf.
Multi-drug resistant Enterococcus faecium, staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp termed ESKAPE pathogens are commonly implicated in difficult-to-treat infectious diseases in developed and developing countries. The prevalence, risk factors, phenotypic and genotypic profiles including but not limited to clonal relatedness, genetic diversity, resistance and virulence associated with ESKAPE bacteria were investigated in carriage and clinical isolates from patients in a rural, district and an urban tertiary hospital in the public health sector in uMgungundlovu District, Kwazulu- Natal, South Africa. The overall carriage of MDR ESKAPE Gram-negative bacteria in both hospitals was 37.21%, 42.31% and 57.14% at admission, after 48 hours and at discharge, respectively. The prevalence of MDR ESKAPE Gram-negative bacteria in faecal carriage (46%) was higher than clinical samples (28%) and colonization was mainly associated with referral from the district to the tertiary hospital with high statistical significance (OR: 14.40, 95% CI 0.98-210.84). blaCTX-M-group-9, blaCTX-M-group-1 and blaSHV were the main resistance genes identified. Similarly, the overall prevalence of faecal VRE carriage was 53% with patients at the district hospital being more likely to be colonized by VRE at admission (44%), after 48 hours (64%) and discharge (100%) than those of the tertiary level. Fifteen (39%) E. faecium and 23 (61%) E. faecalis, were detected and displayed high level of antibiotic resistance. Extensive genetic diversity of E. faecalis and E. faecium and clonal dissemination of various lineages were observed across wards and within hospitals. The high levels of resistance in S. aureus were attributed to the multi-drug resistant efflux pumps mepA, mexE, AcrB, MATE, qac and qacA. Whole genome analysis revealed that the circulating S. aureus isolates belonged to the extremely virulent ST121 clone that harboured a total of 18 virulence genes. The high prevalence, genetic diversity and virulence of antibiotic-resistant ESKAPE bacteria elucidated in this study necessitates routine screening and surveillance in communities and hospitals, stringent infection prevention and control measures and antibiotic stewardship to monitor epidemiological changes, to contain their spread and inform appropriate antibiotic treatment options respectively.
Investigating schistosomiasis markers of inflammation and immune responses in school children on repeated mass drug administration in Bandanyenje, Zimbabwe.
(2017) Chisango, Tawana Jonathan.; Mduluza, Takafira.; Ndlovu, Blanche Ntombizodwa.
Abstract available in PDF file.
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.
Investigations into the morphometrics, uterine tissue adaptation, maternal fluid biochemistry, heavy metal offloading and early embryonic teeth development impacting the reproductive strategy of the female Ragged-tooth shark (Carcharias taurus)
(2018) Naidoo, Kristina.; Chuturgoon, Anil Amichund.; Gregory, Michael Alfred.
Vulnerable” status of ragged-tooth sharks (Carcharias taurus) in South Africa caused by overexploitation, late maturity and low fecundity suggests an intervention to increase the size of this population is needed. Achieving this will require an understanding of all aspects linked to this species maternal-embryonic relationship. Morphometric relationships, uterine histology and maternal fluid biochemistry were 8 assessed in C. taurus through all the respective reproductive stages (RS) from non-gravid (immature to mature-sexually active; RS1-3) to gravid (i.e. only capsules found; RS4 or capsules and pups found; RS5A-5E) females. Examination of metals in the maternal fluids and embryonic dentition were only examined in early-staged gravid females (i.e. RS5A). Haematoxylin/Eosin and Periodic Acid Schiff-Alcian Blue stains in conjunction with light microscopy was used to assess the uterine epithelium and wall while scanning electron microscopy further evaluated the epithelium. These techniques revealed an increase of the uterine lamellae (folds) protruding into the lumen lined with micro-ridges containing blood vessels. The close proximity of blood vessels to the lumen filled with uterine fluid and the decrease in wall thickness as pregnancy progressed suggests an adaption for the exchange of respiration and osmoregulation in the developing aplacental embryos. Although there is no evidence for uterine secretion through structural adaptations, the female supports the embryos nutritional requirement through embryonic tissue (intrauterine cannibalism) and yolk (oophagy) provisions. The pivotal interplay of the liver and ovary, during vitellogenesis, that impact on yolk formation was evident during the morphometric evaluation of hepatosomatic and gonadosomatic indices. Length, weight, uterine width, capsule production and migration trends of the females as well as length and weight relationship of the embryos were tabulated. Reproductive hormones, assessed in maternal fluids (i.e. plasma (in RS1-5D females), uterine fluid (in RS4-5D females) and intracapsular fluid (in RS5A females), showed that follicle-stimulating-, progesterone- and oestradiol hormones were responsible for promoting vitellogenesis and encapsulation which led to three main stages where the rate of ovulation increases in the female. Clinical biochemistry analysers confirmed the composition and concentration of biochemical analytes in same maternal fluids, which were found to be higher in the plasma. Finally, heavy metals were found to be present in all three fluids, but found highest in the plasma, using inductively coupled mass spectrophotometry. In addition, variable pressure (VP) SEM confirmed the dental composition of the embryonic teeth found in the jaws of some embryos that appeared to escape encapsulation earlier than previously documented. It would appear that the embryos are creating adaptive ways to survive the intracannibalistic stage (RS5C) by escaping encapsulation early. However, the presence of heavy metals in the maternal fluids that surround the embryos could compromise their development over time; creating concern for a species that is Vulnerable. This study, which serves as the first detailed analysis of the maternal-embryonic relationship, may serve as areas to model in forthcoming programmes aimed at increasing the numbers of this species.
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.

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