The Tuberculosis Research Group at Albert Einstein College of Medicine is dedicated to a better understanding of the mechanisms by whichMycobacterium tuberculosis causes tuberculosis (TB), to developing new tools to provide faster diagnosis, and more reliable treatment, and to creating better vaccines. As a team of diverse expertise, we use a multipronged approach to study bacterial physiology, its interaction with host, and host responses to bacteria and to apply this knowledge into developing tools in the fight against tuberculosis. Below is the description of our major research interests.
Mtb genome consists of approximately 4000 genes and the function of many of these genes remains unknown. Studying the role of these genes should provide insights about the unique physiology and virulence of the mycobacteria. One of the major areas of our research is to develop and improve upon methods by which we can precisely manipulate mycobacterial genes. Mtb is particularly resistant to genetic manipulation due to extremely poor efficiency of DNA uptake following electroporation and inherently low rates of homologous recombination. Exploiting the natural infectious ability of mycobacteriophages to deliver packaged DNA and recombination functions, we have developed genetic tools for reliably generating precise null-deletion mutants in pathogenic strains of Mtb. Characterization of mutants generated by specialized transduction by our group has resulted in identification of critical drug targets, generation of safe vaccine strains and elucidation of several biochemical and transport pathways in Mtb.
Over the last few years, we have incrementally improved our vector backbones and protocols for generating engineered transducing phages. These developments have greatly facilitated in streamlining the processes and, in collaboration with Genomics Institute of Novartis Research Foundation, extended to a high-throughput status. We are now close to completion of our goal of attaining an archived collection of mycobacteriophages that can be used for generating deletion mutants corresponding to any open reading frame in the Mtb genome. Using these tools, we are making annotated deletion mutant collections corresponding to individual genes in gene families (transcription factors, amino acid synthesis pathways, transporters etc.) in diverse strains of Mtb, and these are currently being analyzed to identify novel and relevant functions. The latest iteration of our Mtb mutant collections involves the incorporation of unique molecular barcode pairs corresponding to every open reading frame that has been deleted. Barcoded mutants can be analyzed in pooled sets and we have developed a robust in-house next-generation sequencing protocol for quantitative multiplexed phenotyping of mutants in vitro and in vivo. These advancements collectively allow for highly sensitive and cost-effective analysis of our Mtb deletion-mutant sets and ongoing studies will provide unprecedented insight into Mtb physiology during human infection.
Physiology and Metabolism
Mtb is a facultative intra-cellular bacterium contained within an organized collection of immune cells called granuloma. The persistence of bacteria during latent infection and reactivation of bacteria during active disease is governed by a complex interplay of the bacterium sensing and responding to its environment. We study how mycobacteria adapt its physiology and metabolism to face the challenges and opportunities posed by host immune system. Using bacterial culture systems that mimic the hostile environment within the host (eg. scarcity of nutrients, hypoxia, and acidic pH) and cell culture systems, we study how mycobacterial genes and pathways sense and respond to its adverse environment.
Mycobacterial metabolism and energetics
We are particularly interested in nutritional requirements for microbial growth and bacterial systems involved in acquiring major and minor nutrients. We are also interested in studying regulation of metabolic pathways such as TCA cycle under stresses, sigma factors that regulate the bacterial transcriptional network and secretion systems that are involved in secreting virulence factors. For these studies we combine our expertise in genetic engineering with the state of art methods such as transcriptomics, lipidomics, and metabolomics. These studies have already helped us to identify several critical pathways involved in bacterial processes and some of these pathways are being targeted for the development of novel drugs.
Drugs and Diagnostics
Treatment regimen of drug-susceptible TB involves taking 4 drugs for a minimum 6 months. The combination of multiple drugs and long duration of treatment can lead to poor compliance and contributes to drug resistance. In addition, the disease could be caused by Mtb that is already resistant to a few or many anti-TB drugs. The focus of our research is to develop novel drugs and strategies that could reduce the duration of treatment and/or that can be used against multi- or extremely drug-resistant Mtb (MDR and XDR TB respectively). One hurdle in developing novel drugs is that the mechanism of action or mode of development of resistance of currently available drugs remains incompletely understood. Thus, one area of our research is to characterize the mechanism of action and of development of resistance of available anti-TB drugs including first line TB drugs, isoniazid, and pyrazinamide, and second line TB drug, ethionamide. For the identification of novel drug targets, we take advantage of our bacteriological studies that investigate microbial pathways that are critical for the survival of mycobacteria in host. Other efforts to generate novel drugs or treatment strategies include boosting the effects of the known TB drugs (e.g. Vitamin C, Bactrim), testing natural products against Mtb, or repurposing approved FDA drugs.
Mechanisms of action and resistance to the TB drugs isoniazid (INH) and ethionamide (ETH)
The lack of accurate and timely diagnosis of TB in areas where the disease is prevalent is still a major obstacle to global TB control efforts. The gold standard of diagnosis, mycobacterial culture, is neither readily available nor timely reducing its clinical utility. Routine diagnostics such as sputum microscopy or chest X rays are neither accurate nor provide information on the status of drug resistance. Our efforts to develop novel diagnostics is based on the concept that prompt diagnosis of disease and resistance can help to rapidly initiate patients on appropriate regimens, reduce community transmission of resistant strains, and prevent amplification of drug-resistance on treatment. Using our extensive experience with the construction of genetically modified mycobacteriophages, we generated reporter phages to allow direct visualization of individual metabolically active tubercle bacilli using fluorescence microscopy or flow cytometry. We showed that this system allows rapid detection of Mtb and rifampicin sensitivity in sputum samples, including those with paucibacillary concentrations. We are currently assessing the utility of this technique for clinical applications and investigating strategies to extend drug sensitivity testing to anti-TB drugs other than rifampicin.
Immunology and vaccines
Mtb survives the hostile environment within host through an elaborate program of immune evasion, allowing it to persist in the face of normal host defenses. Conversely, host immunity, though incapable of eliminating Mtb, is often successful in controlling the infection and preventing overt disease. Deciphering the microbial and host mechanisms that maintain the delicate balance between host immunity and bacterial virulence is critical for our efforts to develop novel vaccines against tuberculosis. Using primarily mouse models and mammalian cell culture systems, we study the bacterial and host determinants involved in maintaining the structure of tuberculous granuloma, and the nature of innate responses against Mtb. We also study how Mtb modulates cell death pathways and disrupts antigen processing and presentation for its advantage. A substantial effort is directed at the study of mycobacteria-specific T cell responses, and how these responses are altered by changes in bacterial determinants that modulate host innate pathways. We also are working toward a better understanding of the underappreciated role of B-cells and antibodies in tuberculosis pathogenesis.
Targeting mechanisms of immune evasion by Mycobacteria to develop live attenuated vaccines
Developing novel tuberculosis vaccines is a major focus of our research and combines our expertise in bacterial genetics and host pathogen interaction with that in immunology. While the approaches vary significantly, the tenet is to find the right balance between attenuation and immunogenicity. In one of the approaches, we try to improve the currently used vaccine, Mycobacterium bovis Bacillus Calmette-Guèrin (BCG) which, though significantly attenuated, still harbors many pathogenic mechanisms that prevent generation of effective anti-mycobacterial immunity. In another approach, we modify strains of virulent Mtb to generate safe strains that are considerably attenuated while maintaining the ability to generate a strong anti-mycobacterial immune response. We achieve this by creating auxotrophic mutants of mycobacteria or by knocking down virulence factors that has been revealed to suppress host immunity. Latter approaches include generating mycobacterial strains to result in specific outcome such as enhanced antigen presentation, and induction of autophagy and apoptosis. Since mycobacteria are well known inducers of strong Th1 responses, we are also investigating the possibility of using these strains as vectors to present exogenous antigens in the context of other diseases such as HIV, herpes, and cancer. Another venue of our research is to develop novel adjuvants for vaccines. We have extensive experience in working with alpha galactosyl ceramide, a prototype antigen of Natural Killer T cells and well known adjuvant and we test whether we can enhance immunogenicity of our vaccine strains by incorporating alpha galactosyl ceramide or its derivatives. Combining above approaches, we have in pipeline several promising live-mycobacteria vaccine candidates for further studies in non-human primates and humans.