The bacterial envelope is composed of a typical phospholipid bilayer common to all cells that is encased within a unique peptidoglycan polymer mesh consisting of glycan strands crosslinked by oligopeptides. In Gram negative bacteria, there is an additional asymmetric outermembrane lipid bilayer containing the saccharolipid lipopolysaccharide. Beyond being a requisite structural element that imparts structural rigidity, the cell envelope regulates the uptake of nutrients, the exclusion of toxins, and is at the forefront of host-pathogen interactions. We use a combination of biochemistry, bacterial genetics, and functional genomics to understand how the cell envelope is assembled, maintained, and altered by cellular programs in response to complex environmental stimuli. By uncovering and characterizing cell envelope related genetic determinants responsible for cell envelope biogenesis, we aim to establish strategies for new antibiotic development, characterize antibiotic resistance mechanisms, and uncover factors that enhance virulence.
Functional genomics in Staphylococcus aureus
Methicillin resistant Staphylococcus aureus (MRSA) is a leading cause of hospital acquired infections, and ever increasingly outside of the clinic with the rise of hyper virulent community acquired S. aureus lineages. With nearly 30% of the ~3 Mb genome subject to genetic exchange (via plasmids, transposons, bacteriophage, etc.), S. aureus demonstrates remarkable genomic plasticity that confers a high level of adaptability. Certain members within clonal complexes account for a disproportionate number of infections. We are interested in understanding the molecular determinants that impart increased fitness, focusing on contemporary clinical isolates.
We have developed a comprehensive phage based delivery transposon system in S. aureus to generate high coverage libraries in diverse strains belonging to widely circulating clonal complexes. Each cassette contains a distinct promoter or transcriptional terminator element of varying intrinsic strength, so a gradient of gene expression levels can be achieved proximal to the insertion site.
By installing a unique 4-bp DNA barcode on each cassette, the different cassettes can be pulled in a single transposon library, probed for phenotype, and then de-multiplexed using the DNA bar code tag in a massively parallel fashion using next generation sequencing. In collaboration with Suzanne Walker’s laboratory (Department of Microbiology and Immunobiology, Harvard Medical School), we have identified a number of uncharacterized cell envelop related genes involved in resistance to clinically relevant cell envelope targeting antibiotics, and are currently determining their function.
Lipid metabolism in Pseudomonas aeruginosa
Fatty acid synthesis occupies a central role in Pseudomonas aeruginosa cell envelope physiology and infection biology. In addition to supplying acyl chains for membrane phospholipids and the outermembrane saccharolipid lipopolysaccharide, FAS is required for acylation of quorum sensing signals, siderophore assembly, and lipoproteins. Our group recently discovered a new class of fatty acid biosynthesis initiation enzymes (PA5174, renamed FabY) which is only present in the aeruginosa sp. sub group of Pseudomonads 1. When overexpressed ectopically, virulence factor production and quorum sensing signaling was significantly enhanced. In addition, we also discovered a new medium chain fatty acid metabolic shunt pathway involving the previously unannotated gene PA3286, that directly funnels exogenous fatty acids back into de novo biosynthesis. We are interested in understanding how these unique
P. aeruginosa FAS elements are integrated into cell envelop biogenesis, environmental sensing, and lipid metabolism.