Browsing by Subject "Tuberculosis"

Pathological angiogenesis is a widespread biological phenomenon that influences the progression of various diseases, including autoimmune conditions, cancers, and microbial infections. One infection in particular, tuberculosis, is associated with the induction of a potent pro-angiogenic signaling cascade that facilitates bacterial growth and accelerates disease progression. A synthesis of early studies on bacterial factors that drive host angiogenesis with modern genetic findings identified the mycobacterial glycolipid trehalose 6-6'-dimycolate (TDM) as a critical factor driving vascular endothelial growth factor (VEGFA) production and angiogenesis during mycobacterial infection. Despite these recent findings, many of the underlying host response mechanisms remain unknown. The introductory chapter will serve to introduce the reader to the major concepts addressed in this work: Mycobacterium tuberculosis and the disease it causes, the role of macrophages in health and disease, the function of pattern recognition receptors in detecting microbial ligands, the specific downstream intracellular signaling pathway of interest for this work (mediated by the transcription factor, nuclear factor of activated T cells, NFAT), the contributions of angiogenesis to diverse contexts and pathologies, and the promise of host-directed therapies to overcome challenges associated with traditional treatment approaches in infectious disease. Chapter 2 describes the new and existing methodological approaches that were required to complete this work. This work utilizes the zebrafish-Mycobacterium marinum model of tuberculosis infection to facilitate in depth in vivo observation and quantitation of these phenomena. Using this model in tandem with human macrophage cell culture, I was able to model major aspects of the host-pathogen interface, enabling me to identify a critical role for a macrophage-C-type lectin receptor-NFATC2-VEGFA signaling axis required for the angiogenic response to mycobacterial infection and TDM, findings that comprise the core of this work and are detailed at length in Chapter 3. The analysis of the large amounts of data generated in this work required creative approaches to data processing and analysis. To this end, I have developed a set of novel processing modalities in Python and R that are capable of the rapid and reproducible processing of images as well as certain aspects of automated data collection therefrom. These macros, many written for the FIJI/ImageJ programming environment, serve as the infrastructure on which the rest of this work has been built. These will be detailed in Chapter 4. Finally, this body of work leaves many questions as yet unanswered. While it is clear that NFAT signaling is required for VEGFA production, the precise mechanism by which this may work is unclear and could be mediated by either direct DNA binding or indirect activation or cooperative binding with some other transcriptional activator. There also exist a variety of other potential NFAT- and angiogenesis-related phenotypes worthy of exploring using the tools and approaches I have developed. It is my hope that the findings herein stimulate further study on the contributions of NFAT signaling to the host immune response to mycobacterial infection and evaluation of the potential of NFAT inhibition as host-directed therapy to tuberculosis.

Mycobacterium tuberculosis (Mtb) is the causative agent of the disease tuberculosis, which kills more people worldwide than any other infectious disease. In 2017, nearly 2 million people died of tuberculosis. Despite the advent of antibiotics targeting Mtb, the global spread of tuberculosis continues. The development of antibiotic resistance within the bacteria has further complicated the already long and difficult course of treatment for the disease. New therapeutics are necessary to combat tuberculosis. A novel treatment strategy is the use of host-directed therapies, which provide an orthogonal approach to killing intracellular pathogens. Rather than directly targeting bacterial pathways, which may lead to the development of mutations that result in resistance to the drug, host directed therapies (HDTs) target the host immune response to the disease. To uncover these host directed therapies, we have utilized the zebrafish-Mycobacterium marinum model system. Using zebrafish infected with their natural pathogen, Mycobacterium marinum, a close genetic relative to Mtb, we show that we can enhance the ability of the host immune response to kill intracellular bacteria.

In Chapter 1, I introduce tuberculosis as a disease and discuss the past, present and future of treating the disease. I discuss potential host targets for immune modulating therapies, including autophagy, inflammation, and inflammasomes. I highlight the role of calcium signaling in immune cells, specifically neutrophils and macrophages. I briefly describe zebrafish as a model system, emphasizing their use to study immune responses and host-pathogen interactions. In Chapter 2, we show calcium is required for immune cell activity and motility in neutrophils. Calcium is a signal that leads neutrophils not only to wound sites but also to sites of infection and inflammation. We then enhance calcium signaling through potentiation of the membrane channel P2RX7 with the small molecule clemastine, an FDA-approved over-the-counter antihistamine in Chapter 3. We show that clemastine treatment reduces bacterial burden in a P2RX7 –dependent manner in zebrafish larvae. P2RX7 activation leads to assembly of inflammasomes in macrophages, a key immune cell of mycobacterial infection. In human mycobacterial disease, many of the bacteria are contained within structures called granulomas, in which host macrophages and other immune cells have formed a cuff around the bacteria, creating a space that is recalcitrant to treatment with frontline antibiotics. Clemastine is effective in these established infection structures, indicating that it may be a feasible strategy to treat human tuberculosis. We discuss how mycobacteria evade the host immune response and demonstrate how a small molecule can overcome these evasion strategies for improved host outcome.

Tuberculosis is a devastating disease that has been plaguing humankind for millennia. Co-evolution of humans with Mycobacterium tuberculosis, the causative agent of the disease, has allowed for the pathogen to possess an abundance of survival mechanisms. The outcome of this is the ability of the bacterium to create an intracellular niche lifestyle inside host cells where it can successfully evade the host immune system. While there is a vaccine available, named the BCG vaccine, it confers little protection to adults in the pulmonary form of the disease. The lack of an effective vaccine and the rise of Multidrug-Resistant (MDR) and Extensively Drug-Resistant (XDR) tuberculosis highlight the need for more research into combating Mycobacterium tuberculosis. The purpose of this work is to enhance the field of knowledge of how mycobacterial virulence factors affect host cellular pathways so that the interactions can be exploited for novel therapeutics and vaccine development.

One of the hypotheses for the poor efficacy of the BCG vaccine is that it fails to elicit a strong CD8+ T cell response during infection. Studies have found that vaccinating mice with apoptotic bodies containing mycobacterial antigens were able to protect mice to a greater degree than BCG and that this is dependent on CD8+ T cell activation. Thus, we hypothesized that a pro-apoptotic mutant of M. tuberculosis could be utilized as a novel vaccine candidate. Through screening a library of M. tuberculosis transposon mutants, we identified an Enhanced Cell Death mutant (ECD19) that functions through caspase 3 mediated apoptosis. Sequencing revealed that the mutant has a transposon insertion in Rv2456c, a probable integral membrane transport protein. Immunogenicity testing via Enzyme-Linked ImmunoSpot (ELISPOT) and Intracellular Cytokine Staining (ICS) assays demonstrated that ECD19 induced an altered immune response when compared to the parental strain M. tuberculosis H37Rv. Additionally, ECD19 has reduced survival in an in vitro THP-1 cell model and in an in vivo mouse model. Taken together, our data suggest that Rv2456c is important to the survival of H37Rv in host cells and that deletion of the gene may enhance the immunogenicity of the bacterium.

Inappropriate dosing and poor adherence to antibiotics in the treatment of tuberculosis has led to MDR and XDR, the highest incidences of which can be found in the KwaZulu-Natal (KZN) province of South Africa. Little is known about the virulence of these strains, but it is hypothesized that the drug resistance mechanisms come at a cost to the bacteria. In an in vitro assay, we have found that clinical isolates from the KZN region induce higher levels of necrosis than virulent laboratory strains of M. tuberculosis. Additionally, our in vivo studies show that the drug-resistant isolates do not disseminate as well as susceptible strains, and in both immunocompetent and immunocompromised mouse models, mice infected with the drug-resistant strains are able to live longer than mice infected with drug-sensitive strains. As all strains are highly related on a genetic level, we can say that the drug-resistant mechanisms acquired by the strains come at a cost of reduced virulence. Thus, it is likely that higher prevalence of the MDR and XDR in the KZN province is due to the high rate of HIV+, immunocompromised individuals living in the region.

Lastly, we are interested in building on the knowledge that avirulent mycobacteria are able to induce autophagy in a murine macrophage cell line. Through the use of Mammalian Target of Rapamycin (mTOR) inhibitors and autophagy-deficient macrophages, we were able to show that Mycobacterium smegmatis is able to induce both mTOR and autophagy during infection. Additionally, we found that mycobacterial killing occurs in the absence of autophagy when mTOR is inhibited. This effect is not due to a bactericidal effect of the mTOR inhibitors. From these data, we show that there is an underappreciated role in the induction of mTOR after mycobacterial infection. By studying the interplay of mTOR and autophagy, therapies targeted to favoring host defenses could be developed.

In summary, the insights from this work enhance the knowledge of how mycobacteria are able to be successful pathogens. This data may be useful in the creation of novel vaccine candidates or the identification of potential drug targets to bolster the therapeutic options in treating those afflicted with tuberculosis.

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Background: Indonesia has one of the highest TB burdens in the world and is one of ten countries that accounted for 77% of the global gap between treatment enrollment and the estimated number of new cases of MDR/RR-TB in 2019. However, there are knowledge gaps about how the delay of MDR-TB treatment initiation might affect this situation. Therefore, this study aimed to examine challenges of implementing MDR-TB treatment initiation in a Programmatic Management of Drug-Resistant Tuberculosis (PMDT) national referral hospital in Indonesia. Method: This study used mixed methods to collect both quantitative data through hospital records of MDR-TB patients and qualitative data through interviews with patients and health workers. Result: The median time between diagnosis and treatment initiation was 26 days, and was associated with co-morbidities, MDR-TB knowledge, and support assessment. This study also revealed the complex situation of people affected with MDR-TB with lack of social support and health system challenges during the MDR-TB treatment initiation process. Conclusion: The results of this study revealed the challenges of the treatment initiation process from the complex perspectives of the patients, the aspects of the health system that need to be improved, and the importance of social support starting from diagnosis.

Background: Ending the global tuberculosis (TB) epidemic by 2035 will substantially depend on the effective control of the “lost to follow-up” (LTFU) from TB treatment. Myanmar is one of the 14 countries with high burden of TB, TB/HIV, and Multidrug-Resistant TB (MDR-TB). The aim of the study is to identify the factors associated with LTFU from TB treatment among migrant and mobile populations in Mon and Kayin States in Myanmar.

Methods: This was a prospective cohort study with a convergent mixed methods design. 146 new TB patients were surveyed and 14 “treatment after LTFU” patients were interviewed between June and September, 2016. Upon the treatment outcome data made available in February 2017, the survival analysis was conducted to measure the effect of potential predictors on time to LTFU during the full duration of treatment using Stata 14.0 version for Mac. Thematic networks analysis was applied to the qualitative data analysis by NVivo software 11.3.2 version for Mac.

Results: Of the 146 patients included, 10 (6.85%) new patients were LTFU from treatment. Having a family and/or community member support during the six to eight months’ treatment was a protective factor (Hazard Ratio (HR) 0.146; 95% CI 0.037 - 0.576; p = 0.0075), whereas the intention to stay for less than three months at the current place was a potential risk factor (HR 6.323; 95% CI 1.403 – 28.499; p = 0.0075) for getting LTFU from TB treatment. Having a lack of knowledge, but a positive attitude towards TB predisposed migrant TB patients to look for health education. However, financial constraint and social stigma of TB reinforced them to get LTFU from TB treatment. Poor provider-to-patient communication and barriers to accessibility of services were the enabling factors for the delay seeking care and treatment.

Conclusions: People on the move who are intended to stay in working area for less than 3 months are the high-risk group for TB treatment default. Having no family and/or community member support is a risk factor associated with tuberculosis treatment default among the migrant and mobile populations in Mon and Kayin States of Myanmar. National Tuberculosis Program should strengthen the existing multilateral community-based TB care with an integrated referral system inclusive of people on the move who are intended to stay in working area for less than three months. Fostering self-efficacy of TB patients by patient-centered communication and informed decision-making in the clinical setting as well as in the community will enable the better adherence to TB treatment among the migrant and mobile populations.

In the past 200 years, tuberculosis (TB) has caused more deaths than any other infectious disease and currently infects more people than it has at any other time in human history. Mycobacterium tuberculosis (Mtb), the etiological agent of TB, is an obligate human pathogen that has evolved through the millennia to become the archetypal human-adapted pathogen. This work focuses on the evolutionary framework by which Mtb emerged as a specialized human pathogen, and applying this perspective to outbreak strains and the strategies Mtb deploys to manipulate its host environment.

There are seven major lineages that define the human-adapted species of Mtb. Beijing lineage strains, also known as Lineage 2 strains, have emerged as important drivers of global Mtb burden due to the elevated rates of drug-resistance, rapid disease progression, and increased transmission characteristics they display. Beijing lineage strains are endemic to East Asia, but have recently expanded globally. In Chapter 2, we investigate circulating Beijing strains in Guatemala, a country with limited data regarding the molecular epidemiology of Mtb and few reported cases of disease caused by Beijing strains. We report the first whole genome sequencing of Central American Beijing-lineage strains of Mtb. We find that multiple Beijing-lineage strains, derived from independent founding events, are currently circulating in Guatemala, but overall still represent a relatively small proportion of disease burden. Finally, we identify a specific Beijing-lineage outbreak centered on a poor neighborhood in Guatemala City.

Pairing whole genome sequencing with outbreak strains displaying unusual disease phenotypes provides compelling insight into the genetic changes underlying the corresponding disease presentation. Mtb most commonly causes lung disease, but can also disseminate to other tissue sites. We identified an outbreak strain of Mtb that presented clinically with unusually high rates of extrapulmonary and bone disease in seemingly immunocompetent individuals. We find that the outbreak was caused by an ancient strain of Mtb that, like other ancestral strains and animal-adapted mycobacterial pathogens, carries a full-length version of the Type VII secreted effector EsxM. Here we show that EsxM is required for the full disseminative properties of mycobacterium-infected macrophages and is sufficient to directly enhance macrophage motility. Moreover, we find that EsxM has been inactivated in all modern strains of Mtb, suggesting a potential selective advantage to limiting dissemination as Mtb adopted and adapted to its modern pulmonary niche.

Pathogenic mycobacteria have long been known to utilize an array of effectors to manipulate their host environment. Mycobacterial infection initiates the assembly of granulomas, which are discrete host structures composed of tightly associated immune cell aggregates. Macrophages within the granuloma have been described as "epithelioid" due to the morphological transformation they undergo as they interdigitate with their neighboring cells. The epithelial transformation macrophages undergo is central to the formation of the granuloma, yet this transformation has not been well characterized at the molecular level during mycobacterial infection. Our laboratory has utilized the zebrafish to dissect the epithelial transformation of macrophages during mycobacterial infection. In Chapter 4, I outline the transcriptional reprogramming that occurs in granuloma macrophages that underlies this transformation. We find that granulomas exhibit distinct transcriptional profiles from macrophages, and, surprisingly, that immune cells deploy developmental programs to construct the granuloma.