Fortune’s Lab Research

A major focus of the laboratory has been defining the mechanisms by which mycobacterial populations diversify and the functional consequences of this heterogeneity. We have demonstrated that Mtb is relatively genetically stable yet we, and others, have shown that there is dramatic phenotypic heterogeneity at a single cell level.  We have found that this phenotypic variation arises through many mechanisms, including asymmetric growth and division and DNA methylation-based regulation of gene expression and…

Bacterial Determinants of Treatment Response and Failure

It takes months to treat even drug susceptible TB.  Even in culture, most TB cells die quickly in the face of drug but some TB bacteria are persistently tolerant of drug. Here we seek to understand why genetically identical TB bacilli are very difficult to eliminate with antibiotics while others are quite susceptible. In addition, we seek to identify genetic mutations that put some TB strains at risk of acquiring drug resistance and thus…

Drivers of Infection Outcome – From Single Cells to Non-Human Primates

One of the hallmarks of TB infection is the variability in infection outcome.  We see this recapitulated at a lesional level within an individual host and at a single cell level.  We are using single cell approaches including dynamic imaging and single cell RNAseq to identify correlates and drivers of these differences in infection outcome.

Mechanisms of Bacterial Variation

We seek to identify novel mechanisms by which the bacterium establishes and maintains phenotypically distinct subpopulations.   Specifically, we are interested in DNA methylation and post-translational modification of bacterial nucleoid associated proteins as bases of epigenetic inheritance.  Other work focuses on the regulatory roles of ncRNAs and housekeeping proteases.

Protocols and Design Specifications

Pulse-chase labeling of the cell wall in mycobacteria We use this protocol to pulse chase label the cell wall in mycobacteria.  This experiment allows us to establish the sites of new growth in the chase portion of the experiment. For examples, please see above and Aldridge et al. Science 2012. Make up and aliquot the dye (Alexa Fluor® 488 Carboxylic Acid, Succinimidyl Ester, mixed isomers (1 mg) 1.Add 250 uL…

Resources

Harvard University School of Public Health Environmental & Health Services Waste Pickup  (No longer valid) Tuberculist Old New Smegmalist Tuberculosis Database (TbDb) PFAM – protein domain search site NCBI Mtb Whole Genome Sequences BLAST PubMed MIRU/VNTR plus CMR (Comprehensive Microbial Resources) Homepage CRISPR-Jeremy sRNA sequencing aind identification-Eli Barcode sequencing-Forrest Live cell imaging-Jemila