Bacterial sphingolipid synthesis
Caulobacter crescentus is a Gram-negative oligotrophic organism that is well adapted to life in low phosphate environment. In response to phosphate limitation, the cells and its stalk appendage elongate dramatically leading to an overall increase in cellular surface area of 6-7 fold. Since most membranes, including C. crescentus, are composed of phosphoplipids, how do you make all of this new membrane material in the absence of phosphate? Working with Ziqiang Guan (Duke University), Dominic Campopiano (University of Edinburgh), and Howard Goldfine (University of Pennsylvania), we found that C. crescentus can make glycosphingolipids in response to phosphate starvation. These ceramide-based lipids were thought to be fairly unusual in bacteria, though they are ubiquitous among eukaryotes. In C. crescentus, these lipids play a role in resistance to phage by reducing their adsorption to the cell surface. Interestingly, we don’t understand how these lipids are made in bacteria. While the eukaryotic pathway is well characterized, most of the eukaryotic biosynthesis enzymes do not have bacterial homologues?? This suggests that either 1) bacteria use the same the synthetic pathway, however the enzymes diverged long ago making it difficult to find the orthologues, or 2) bacteria evolved an completely different pathway for ceramide synthesis. We are actively working on uncovering this bacterial pathway.
Regulation of bacterial cell shape
Bacteria grow in a variety of cell shapes and often, these shapes can be dynamically regulated. In the case of Caulobacter crescentus, a long polar stalk is elongated in response to phosphate starvation. Despite decades of research, the composition of the stalk and the enzymes required for its synthesis remain unknown. Our lab is using genetic, microscopic, and mass spectrometry techniques to unravel the mechanisms of stalk elongation.
Our lab is currently investigating how adaptation to phosphate starvation leads to changes in peptidoglycan organization, lipid synthesis, and metabolism. In addition to our targeted approaches, we have isolated a set of stalk-elongation mutants that we are characterizing in an effort to dissect the molecular pathways required for stalk synthesis.
Mechanical regulation of bacterial pathogenesis
Uropathogenic E. coli (UPEC) are intracellular pathogens with a complex life cycle. After entering the bladder cell host via an endocytic process, the bacteria escape the endosome a proliferate rapidly in the cytoplasm forming an Intracellular Bacterial Community (IBC). While these IBCs have been observed in animal and human UTIs, they are not easily recapitulated in tissue culture models.
The inability to form IBCs in vitro is often attributed to the fact that the standard lab bladder cell lines are tumor-derived and do not faithfully mimic the fully differentiated umbrella epithelial cells which line the bladder in vivo. While it is certainly true that gene expression patterns between cell lines and in vivo tissue are different, it is also true that the physical/mechanical environment of tissue culture is distinct from the physiological environment. Using hydrogels as a culture substrate, our lab is investigating how tissue mechanical properties influence the intracellular behavior of UPEC.
We recently published our work showing that UPEC infection of epithelial cells grown on physiologically relevant substrates (elastic modulus ~300 Pa) leads to endosomal escape and cytoplasmic proliferation. Furthermore, we observed UPEC filamentation which is a mechanism to evade the immune system following host-cell efflux. This endosomal escape is mediated by mechanical regulation of RhoB expression. Inhibition or RhoB, or its effector PRK1, leads to escape even on stiff substrates and we also found that soft substrates led to post-transcriptional downregulation of RhoB expression.