Project description:Genome-wide transcriptional responses of Yersinia entomophaga to shifts in temperature (25 - 37 degrees C) and in vitro and in vivo growth conditions were investigated using RNA-seq.
Project description:Identification the proteins co-secreted with YenTc and compare them to those remaining in yersinia entomophaga cells by bottom-up proteomics.
Project description:Tripartite Tc toxins are virulence factors of bacterial pathogens. Although their structure and mechanism of action are well understood, it remains elusive where this large macromolecular complex is assembled and how it is released. Here we show by an integrative multiscale imaging approach that Yersinia entomophaga Tc (YenTc) toxin components are expressed only in a subpopulation of cells that are “primed” with several other potential virulence factors, including filaments of the protease M66/StcE. A phage-like lysis cassette (LC) is required for YenTc release; however, before resulting in complete cell lysis, the LC generates intermediate “ghost” cells, which may serve as assembly compartments and become densely packed with assembled YenTc holotoxins. We hypothesize that this stepwise mechanism evolved to minimize the number of cells that need to be sacrificed. The occurrence of similar lysis cassettes in diverse organisms indicates a conserved mechanism for Tc toxin release that may apply to other extracellular macromolecular machines.
Project description:Yersinia entomophaga MH96 is a highly virulent entomopathogen that infects pest insects of economic importance. We discovered a new regulator, Yen6, which influences the virulence of MH96 in Galleria mellonella at 37 ˚C. Yen6 is directly upstream of the yen7 gene, which encodes for a RoeA-like PhoB-containing regulator and that is directly upstream of the Yen Toxin component (Yen-Tc) genes. We found that yen6 is the most temperature-responsive gene in MH96, producing both messenger RNA and non-coding RNA in its 3' untranslated region (UTR). Yen6 encodes a unique LytTR-containing transcription factor that targets sugar uptake genes and an RNA-binding protein. Of interest, the non-coding RNA from the yen6 3’ UTR overlaps with yen7 in the opposite orientation. Deleting yen6 did not significantly affect the transcriptional activity of yen7 or downstream Yen-Tc genes during infection at 37 ˚C. However, when we deleted the yen6 locus or induced yen6 plus a 478-bp region of its 3’ UTR at 25 ˚C, we observed a significant increase in the transcription/ translation rates of the Yen-Tc gene chi1 using a chi1:lacZ fusion. Our findings reveal a novel mode of virulence regulation in MH96, where the yen6 gene exerts control over multiple genes involved in G. mellonella infection and exhibits potential for dual functionality at both transcriptional and translational levels.