Single cell ribonucleic acid sequencing in Tuberculosis research.

Doctoral Degree. University of KwaZulu-Natal, Durban.Tuberculosis (TB) remains a global challenge, with approximately 1,5 million deaths annually. Addressing deficits in our understanding of disease pathology and treatment is needed for the development of new treatment modalities. Despite much effort, prevalence of this disease remains high in resource limited regions, where research capacity is not sufficient to successfully combat the endemic. Research in developed countries has generally been constrained to animal models due lack of access to clinical samples from the site of TB disease, the human lung. Although these animal models have their utility, it is essential that findings from these systems be tested and validated in human tissue. In this thesis, I leveraged a relatively new technology called Seq-well, which is highly portable and low-tech single cell ribonucleic acid sequencing (scRNAseq) platform and access to TB infected lung tissue obtained from lung resections, to generate a single cell atlas of TB affected lung tissue. This involved processing the human tissue immediately post-surgery and loading unprocessed/neat cells or FACS sorted cells (tissue resident t cells) onto a microarray that allowed capture and subsequent sequencing of the cell transcriptomes. In the first part of the thesis, I identified and profiled cellular subsets from TB infected tissue, focussing on a subset of FAP+PDPN + fibroblasts associated with the organisation of tertiary lymphoid organs. I also demonstrated that this dataset can be useful in evaluating current and future TB biomarkers, by superimposing signatures from the literature onto the cellular subsets and localizing them to different parenchymal, stromal and immune cell types. I also profiled tissue resident CD4 T cells from the same lung tissue, identifying canonical marker genes (ITGA1, PRF1) in one specific cluster, together with naive (CCR7, SELL), regulatory (RORA) and activated/myeloid-like T cells (LYZ, S100A9) in separate clusters. Finally, I demonstrated the applicability of this dataset in research involving other pulmonary diseases, by identifying ACE2+ TMPRSS2+ type 2 pneumocytes, a target of the SARS-CoV-2. Taken together, these findings provide new insights into the immunopathology of TB in the human lung together with the impact of HIV on specific immune subsets. It serves as a resource for cross validation of lung immune signatures generated in experimental infections of both mice and non-human primates, which is beneficial for scientists lacking access to the technology and/or tissue. Iqoqa Isifo sofuba (i-TB) silokhu siyinselelo emhlabeni jikelele, ngokufa okuhlobene naso okucishe kufike esigidini esi-1.5 njalo ngonyaka. Ukubhekana nokushoda ekuqondeni kwethu umumosakhiwo wesifo bese kuncishiswa ukufa. Ngaphandle kwemizamo emikhulu, ukudlanga kwalesi sifo kusalokhu kuphezulu ezifundeni ezintula imithombokusiza, lapho umthamokwenza wocwaningo unqindekile. Ucwaningo emazweni asethuthukile, ngakolunye uhlangothi, belwenzeka kuphela kumamodeli asebenzisa izilwane ngenxa yokuntuleka kokufinyelela amasampuleni okwelapha engxenyeni okuqubuke kuyo isifo sofuba, okuyiphaphu lomuntu. Nakuba kunamamodeli ezilwane anomsebenzi, kubalulekile ukuba okutholakele kulezo zinhlelo kuyohlolwa bese kuqinisekiswa ngesigqa somuntu ukuqinisekisa ubunjalo. Kule thesisi, ngiveze ubuchwepheshe obusha obungenayo obubizwa nge-Seq-well, iseli eyodwa e-low-tech ephathekayo ene-ribonucleic acid sequencing (scRNASeq) okuyindawo kanye nokufinyelela esicutshini sephaphu esitheleleke ngesifo sofuba esitholakale ekuhlukanisweni kabusha kwamaphaphu okukhonjwe ngokokwelapha, ukwakha iseli eyodwa yesicutshana sephaphu elitheleleke ngesifo sofuba. Lokhu kwafaka ukusebenzakuhlola isicubu somuntu ngokushesha emva kokuhlinza nokufaka amaseli ahlanzekile angasetshenziwe noma amaseli ahleliwe angama-FACS (ama-T cells asesicutshini) ohlelweni lolibofuzo olwavumela ukufaka ohlwini nokulandelanisa okulandelayo womumofuzo oqondene nezicubu. Engxenyeni yokuqala yethesisi, amaqoqwana ahlonziwe nafakwe kwiphrofayli esicubini esitheleleke ngesifo sofuba kugxilwe eqoqweni le FAP+PDPN + amafayibhroplasti ahlobene nokuhlelwa kwezingxenye zomzimba ezinkulu zamalimfoyidi kanye nemichilwana yamafayibhrodi kanye noma igranyuloma yesifo sofuba. Ngivezile ukuthi lamadathasethi angaba nomsebenzi omkhulu ekuhlaziyeni amabhayomakha amanje nawasesikhathini esizayo esifo sofuba, ngokufaka izinkombabunjalo emaqoqweni amancane nokuwabeka ezinhlotsheni ezehlukene zamaseli angamapharenikhayma nangamastroma. Ngiphinde ngachaza esizindeni sezicutshana ze-CD4 T esicutshini sephaphu elifanayo okuchaza ulibofuzo olukala amakhenoni (i-ITGA1, PRF1) eqoqweni elilodwa eliqondile, kanye namaseli angachazi lutho (CCR7, SELL), alawulayo (RORA) nama-T cell aqaliswe ukusebenza/efana ne-myeloid (LYZ, S100A9) emaqoqweni aseceleni. Okokugcina, ngiveze ukungena kwedathasethi ocwaningweni olufaka izifo zamaphaphu nokuphefumula ngokuhlonza i- ACE2+ TMPRSS2+ type 2 wama-pneumocytes, okuhlosiwe kwe-SARS-CoV- 2. Uma kuhlanganisiwe, lokhu okutholakele kuletha imibono emisha yomumobugciwane bokutheleleka ngesifo sofuba ephashini lomuntu, umthelela we-HIV kokutholakele emumwenikuphila kwephaphu ekuthelelekeni okuyilinga kwakho kokubili amagundane kanye nalokho okungebona abantu

Light forge : a microfluidic high throughput platform for rapid and affordable detection of drug resistant strains of tuberculosis.

Master of Medical Sciences in Medical Microbiology. University of KwaZulu-Natal, Medical School 2015.Tuberculosis is one of the most deadly infectious diseases currently plaguing the global community. Unfortunately, lack of accessible, reliable and affordable diagnostic tools in the high disease burden, and resource poor regions such as Sub-Saharan Africa has hampered efforts to eradicate the epidemic. This study documents the development of a microfluidic platform called Light Forge, which is capable of detecting genetic drug resistance signatures in M.tuberculosis DNA. The first phase of this study involved a molecular drug susceptibility assay on 7 strains of M.tuberculosis using the high resolution melt analysis at the rpoB, katG, mab-inhA and gyrA loci with the Light Cycler96 . These findings compared with phenotypic drug susceptibility testing and Sanger sequencing. The results from the preliminary tests showed that the commercial system could detect positive strains at sensitivity estimates of 86%, 17% , 0% and 100% for rpoB, katG, mab-inhA and gyrA respectively. Detection of non-synonymous mutation in gyrA region for all test strains halted further testing. The rpoB gene was selected for on chip profiling with the Light Forge system due to the higher sensitivity. The results from the Light Forge showed that the system was capable of detecting test strains with 100% sensitivity, with modest reproducibility and correspondence with the phenotypic drug susceptibility profiles and the sequencing results. A microfluidic TB assay based on the Light Forge system is on the horizon based on the findings of the study. However, more work is required to incorporate other genes and ultimately design the best-equipped device for the clinical setting

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection

Light Forge: A Microfluidic DNA Melting-based Tuberculosis Test

BACKGROUND: There is a well-documented lack of rapid, low-cost tuberculosis (TB) drug resistance diagnostics in low-income settings across the globe. It is these areas that are plagued with a disproportionately high disease burden and in greatest need of these diagnostics. METHODS: In this study, we compared the performance of Light Forge, a microfluidic high-resolution melting analysis (HRMA) prototype for rapid low-cost detection of TB drug resistance with a commercial HRMA device, a predictive nearest-neighbor thermodynamic model, DNA sequencing, and phenotypic drug susceptibility testing (DST). The initial development and assessment of the Light Forge assay was performed with 7 phenotypically drug resistant strains of Mycobacterium tuberculosis (M.tb) that had their rpoB gene subsequently sequenced to confirm resistance to Rifampin. These isolates of M.tb were then compared against a drug-susceptible standard, H37Rv. Seven strains of M.tb were isolated from clinical specimens and individually analyzed to characterize the unique melting profile of each strain. RESULTS: Light Forge was able to detect drug-resistance linked mutations with 100% concordance to the sequencing, phenotypic DST and the nearest neighbor thermodynamic model. Researchers were then blinded to the resistance profile of the seven M.tb strains. In this experiment, Light Forge correctly classified 7 out of 9 strains as either drug resistant or drug susceptible. CONCLUSIONS: Light Forge represents a promising prototype for a fast, low-cost diagnostic alternative for detection of drug resistant strains of TB in resource constrained settings

B cell heterogeneity in human tuberculosis highlights compartment-specific phenotype and functional roles

B cells are important in tuberculosis (TB) immunity, but their role in the human lung is understudied. Here, we characterize B cells from lung tissue and matched blood of TB patients and found they are decreased in the blood and increased in the lungs, consistent with recruitment to infected tissue, where they are located in granuloma associated lymphoid tissue (GrALT). Flow cytometry and transcriptomics identified multiple B cell populations in the lung, including those associated with tissue resident memory, germinal centers, antibody secretion, proinflammatory atypical B cells, and regulatory B cells, some of which are expanded in TB disease. Additionally, TB lungs contained high levels of Mtb-reactive antibodies, specifically IgM, which promoted Mtb phagocytosis. Overall, these data reveal the presence of functionally diverse B cell subsets in TB diseased lung and suggest several potential localized roles that may represent a target for interventions to promote immunity or mitigate immunopathology

HIV infection drives interferon signaling within intestinal SARS-CoV-2 target cells

SARS-CoV-2 infects epithelial cells of the human gastrointestinal (GI) tract and causes related symptoms. HIV infection impairs gut homeostasis and is associated with an increased risk of COVID-19 fatality. To investigate the potential link between these observations, we analyzed single-cell transcriptional profiles and SARS-CoV-2 entry receptor expression across lymphoid and mucosal human tissue from chronically HIV-infected individuals and uninfected controls. Absorptive gut enterocytes displayed the highest coexpression of SARS-CoV-2 receptors ACE2, TMPRSS2, and TMPRSS4, of which ACE2 expression was associated with canonical interferon response and antiviral genes. Chronic treated HIV infection was associated with a clear antiviral response in gut enterocytes and, unexpectedly, with a substantial reduction of ACE2 and TMPRSS2 target cells. Gut tissue from SARS-CoV-2-infected individuals, however, showed abundant SARS-CoV-2 nucleocapsid protein in both the large and small intestine, including an HIV-coinfected individual. Thus, upregulation of antiviral response genes and downregulation of ACE2 and TMPRSS2 in the GI tract of HIV-infected individuals does not prevent SARS-CoV-2 infection in this compartment. The impact of these HIV-associated intestinal mucosal changes on SARS-CoV-2 infection dynamics, disease severity, and vaccine responses remains unclear and requires further investigation

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection

Single-cell meta-analysis of SARS-CoV-2 entry genes across tissues and demographics.

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention