Browsing by Subject "mycobacteria"

Mycobacterial infection leads to the formation of characteristic immune cell aggregates called granulomas. In humans and animal models, tuberculous granuloma formation is accompanied by dramatic remodeling of host vasculature which ultimately benefits the infecting mycobacteria, suggesting the bacteria may actively drive this host process. First, we sought to identify bacterial factors that promote granuloma vascularization. Using Mycobacterium marinum transposon mutants in a zebrafish infection model, we revealed the enzyme Proximal Cyclopropane Synthase of alpha-Mycolates (PcaA) as an important bacterial determinant of host angiogenesis. We found that PcaA-modified trehalose dimycolate, an abundant glycolipid in the mycobacterial cell wall, drives activation of host VEGF signaling and subsequent granuloma vascularization. To facilitate our continuing investigation of granuloma dynamics, we next sought to expand and improve upon the transgenic tools for studying macrophages in the zebrafish model. I describe two such tools: i) the macrophage-specific zebrafish mfap4 promoter, which allows long-term in vivo visualization and manipulation of macrophages during mycobacterial infection, and ii) the first zebrafish transgenic line with constitutive, ubiquitous Cas9 expression, as well as a transgene design capable of generating sgRNAs using macrophage-specific promoters. These tools allow CRISPR/Cas9 gene editing in vivo in the zebrafish in a macrophage-restricted manner.

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.

Lipids represent an important source of nutrition for infecting mycobacteria, accumulating within the necrotic core of granulomas and present in foamy macrophages associated with mycobacterial infection. In order to better understand the timing, process and importance of lipid accumulation, we developed methods for direct in vivo visualization and quantification of lipid accumulation using the zebrafish-M. marinum model of infection. We find that neutral lipids accumulate cell autonomously in mycobacterium-infected macrophages in vivo during early infection, with detectable levels of accumulation by two days post-infection. Reducing available free cholesterol and neutral lipids during early infection via treatment with ezetimibe, an FDA-approved drug, resulted in a reduction of bacterial growth in vivo. The effect of ezetimibe in reducing bacterial growth was dependent on the mce4 operon, a key bacterial determinant of lipid utilization. Thus, in vivo, lipid accumulation can occur cell autonomously at early timepoints of mycobacterial infection, and this early lipid accumulation confers a growth advantage to infecting mycobacteria.

This accumulation represents a perturbation of the normal homeostatic mechanisms by which intracellular lipids are tightly controlled. Under homeostatic conditions macrophages are central to the function of returning excess lipids to the liver where they are processed for excretion via the digestive tract. This function of macrophages, termed reverse cholesterol transport, results from the uptake of excess extracellular lipids, followed by the coordinated efflux of these lipids through the transport proteins ABCA1 and ABCG1 and packaging into HDL particles containing ApoAI and ApoAII. This process is controlled via the action of nuclear receptors including PPARγ, PPAR-α, and LXRα.

We performed RNA-seq analysis of gene expression in macrophages both uninfected and infected with mycobacteria and observed a profound down-regulation of all the major lipoproteins. The HDL associated lipoproteins ApoAI and ApoAII were the most profoundly down regulated. Based on this observation we sought to investigate the role of nuclear receptors involved in intracellular lipid sensing and control of apolipoprotein expression. We determined that agonism and antagonism of LXRα signaling decrease and increase infection burden during in vivo studies respectively. We further found that the ApoAI agonizing fibrate drugs fenofibrate and gemfibrozil reduce mycobacterial infection in vivo. This work demonstrates that lipid lowering agents already approved for use in humans might function as relevant adjuvant therapies toward treatment of mycobacterial infections.