Diaz, O. R. | Search bioRxiv

bioRxiv2023

Temporal profiling of Salmonella transcriptional dynamics during macrophage infection using a comprehensive reporter library

The transcriptome of Salmonella enterica serovar Typhimurium (S. Tm) dynamically responds to the rapid environmental shifts intrinsic to S. Tm lifestyle, exemplified by entry into the Salmonella-containing vacuole (SCV) within macrophages. Intracellular S. Tm must respond to the acidity of the SCV, accumulation of reactive oxygen/nitrogen species, and fluctuations in nutrient availability. Despite thorough RNA-seq-based investigations, the precise transcriptional timing of the expression of many secretion systems, metabolic pathways, and virulence effectors involved in infection has yet to be elucidated. Here, we construct a comprehensive library of GFP-reporter strains representing [~]3,000 computationally identified S. Tm promoter regions to study the dynamics of transcriptional regulation. We quantified promoter activity during in vitro growth in defined and complex media and throughout the timeline of intracellular infection of RAW 246.7 macrophages. Using bulk measurements and single-cell imaging, we uncovered condition-specific transcriptional regulation and population-level heterogeneity in the activity of virulence-related promoters, including SPI2 genes such as ssaR and ssaG. We discovered previously unidentified transcriptional activity from 234 genes, including ones with novel activity during infection that are associated with pathogenecity islands and are involved in metabolism and metal homeostasis. Our library and data sets should provide powerful resources for systems-level interrogation of Salmonella transcriptional dynamics.

microbiology↗

bioRxiv2021

Allosteric activation of CwlD amidase activity by the GerS lipoprotein during Clostridioides difficile spore formation

Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. Spore germination depends on the degradation of the protective spore peptidoglycan layer known as the spore cortex. Cortex degradation is mediated by enzymes that recognize the spore-specific peptidoglycan modification, muramic-{partial}-lactam (MAL). In C. difficile, MAL synthesis depends on the activity of the CwlD amidase and the GerS lipoprotein, which directly binds CwlD. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind zinc stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to zinc, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of zinc co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.

microbiology↗