One of the major areas of our research is investigating the role of mycobacterial genes in growth, virulence and drug resistance. We have available more than 5000 square feet Bio-Safety Level (BSL)-3 laboratories for the safe containment and manipulation of various strains of Mycobacterium tuberculosis including Multi-Drug Resistant (MDR) and Extremely-Drug Resistant (XDR) clinical isolates. Of particular significance are the tools that we developed to generate precise changes in mycobacterial genome. We achieve this by exploiting the natural infectious ability of mycobacteriophages to deliver DNA. This system of genetic manipulation in combination with other molecular biology techniques forms the foundation of the bacteriological studies performed in our laboratories. Genetically modified bacteria are then studied for the changes in their characteristics such as growth, virulence and drug susceptibility. For growing mycobacteria, we use DASGIP DASbox Chemostat which allows fully automated monitoring and manipulation of parameters such as temperature, agitation, pH, dissolved oxygen, redox, optical density and off-gas analysis. This system allows us simulate growth conditions that mycobacteria may encounter in the host environment and is particularly important in metabolic and growth kinetic studies where high reproducibility is a requisite. In addition to this culture system, our BSL-3 facility is also equipped with techniques that allow visualization of mycobacteria or mycobacteriophages such as fluorescent microscopes including a real-time live-cell imaging system (see under Cell Biology) and BD FACSCalibur flow cytometer.
Growing Mycobacterium tuberculosis in BSL-3
Using various cell biology techniques, we study at the cellular level mechanisms by which Mycobacterium tuberculosis survive in the host cell and evade and subvert host immunity for its advantage. The processes we study include fusion of phagosome with lysosome, antigen processing and presentation, autophagy and apoptosis. While we predominantly use host cell lines that represent various relevant host cells, primary cells are also routinely used. We have several cell culture systems available that can control of oxygen levels allowing us the use of physiological levels of oxygen for cell culture. Among the techniques we use, light microscopy remains as the most frequently used technique. We also employ other related techniques such as flow cytometry and various protein chemistry techniques with and without cell fractionation. In our BSL-3 facility, real-time live-cell imaging is made possible by a Nikon’s Eclipse Ti-E Inverted Microscope with Perfect Focus System. In addition, we take advantage of the state of the art imaging methods available at the Analytical Imaging Facility (AIF) at Einstein. We frequently use confocal microscopy which allows reconstruction of three-dimensional structure of the cell. Ultra-structural features are studied using Transmission Electron Microscopy which is also available at the facility. The facility also provides with several image analysis packages that can be used for visualization, quantification and restoration of light or electron microscopy images.
Autophagy in mycobacteria-infected cells
We employ a variety of immunological tools and techniques to study innate and adaptive immune responses to mycobacteria. Soluble mediators of inflammation such as cytokines and antibodies are measured using standard ELISA technique. For high throughput measurement of these mediators, we use QuickPlex SQ 120 (Meso Scale Delivery (MSD)) which allows multiplex detection of up to 10 mediators from a single sample. Immunophenotyping and intracellular cytokine measurements are performed at the state of the art Flow Cytometry Core Facility at Einstein. The facility also hosts high-speed cell sorter flow cytometers for sorting cells of interest, Miltenyi Biotec SuperMACS for large-scale cell sorting and a laser scanning cytometer which is a combination of microscopy and cytometry. Enumeration of cytokine producing T cells and antibody-producing B cells are performed by ELISPOT assays using AID EliSpot Reader. In addition to the above resources that can be used at BSL-1 and 2, we have resources in place to interrogate immune responses at BSL-3 (see under Animal Studies).
Analysis of multi-functional CD4+ T cells
Mouse models remain an integral component of tuberculosis research and allow us to explore mechanisms of host-pathogen interactions in multiple phases of infection in vivo, develop novel vaccines, and discover diagnostic markers and new drugs. At Einstein, animal experiments involving mycobacteria are performed at dedicated BSL-2 or BSL-3 laboratories. Experiments involving “safe” mycobacterial strains such as Mycobacterium bovis Bacille Calmette Guerin or mycobacterial auxotrophs are performed in the BSL-2 laboratories while experiments involving virulent mycobacterial strains are performed in the BSL-3 laboratories. At BSL-3 laboratories, the most frequently used model is the low-dose aerosol infection model which is believed to mimic the natural infection in humans. The low dose infection model is achieved by the delivery of precise numbers of bacteria into mouse lung using a unique enclosed system called Einstein Contained Aerosol Pulmonizer (ECAP) which integrates a Madison Aerosol Exposure Chamber (MAEC) with a Baker Class III ventilated glove box and a control box. We have two of these infection systems in our BSL-3 laboratories. Each system has a capacity of infecting 90 mice at a time and is compatible with clinical strains including Multi-Drug Resistant (MDR) and Extremely-Drug Resistant (XDR) clinical isolates. In addition, our animal laboratories are equipped with systems that allow assessment of bacterial burden and pathology after infection. These systems include a BD FACSCalibur flow cytometer [L4] that enables analysis of immune responses in the infected organs at a single cell level.
Effect of vaccination on lung histology
An array of “omics” resources are available to us to investigate mycobacteria or host at the systems level. For mycobacteria, we have technologies in place to perform transcriptomic, genomic, metabolomic and lipidomic studies. These resources allow us to characterize responses of the mycobacterium to genetic and environmental perturbations in an unbiased, high throughput manner. Oligonucleotide microarrays remain our favored method for the analysis of mycobacterial transcriptome. For genomics studies, we use the Illumina MiSeq Desktop Sequencer which allows sequencing of whole bacterial genomes at the rate of 20 genomes every 3 days. MiSeq is also useful for counting applications such as ChIP-seq or RNA-seq or for quantitation of barcoded bacteria from organs harvested from infected animals. These transcriptomic and genomic analysis are complemented with metabolomic and lipidomic studies which are performed using an Acuity UPLC system coupled to aSynapt G2 qTOF hybrid mass spectromter. For host studies at the systems level, we take advantage of Shared Scientific Facilities and Cores supported by Einstein. These facilities include Genomics Facilitythat allows both genomic and transcriptomic studies, Proteomics Facility and Computational Genomics Facility which helps us with the analysis and interpretation of large data sets.
Transcriptome and metabolome data painted on Mtb metabolic pathways
Shared Scientific Facilities and Cores
In addition to the resources described here, Albert Einstein College of Medicine supports a broad array of Shared Scientific Facilities and Cores designed to cater the need of biomedical researchers at Einstein. Details of these facilities are found here.