Gene expression profiles of wild-type Yersinia pestis CO92 and its Braun lipoprotein mutant at flea and human temp

Braun/murein lipoprotein (Lpp) is one of the major outer membrane components of gram-negative bacteria belonging to the enterobacteriaceae family that that is involved in inflammatory responses and septic shock. We previously characterized a delta-lpp isogenic mutant of Yersinia pestis CO92 and found that this mutant was defective in surviving in macrophages and was attenuated in a mouse inhalation model of plague when compared to the highly virulent wild-type (WT) bacterium. To better understand how deletion of the lpp gene might affect Yersinia virulence, we performed global transcriptional profiling of the genes in the WT Y. pestis CO92 and its delta-lpp mutant by using microarrays. The organisms were cultured at both 26 and 37 degrees Celsius to simulate the flea vector and mammalian host environments, respectively. Our data revealed vastly different effects of lpp mutation on the transcriptomes of Y. pestis grown at 37 versus 26°C. While the absence of Lpp resulted mainly in the down-regulation of metabolic genes at 26°C, the Y. pestis CO92 lpp mutant cultured at 37°C exhibited profound alterations in stress response and virulence genes, compared to the WT bacterium. We further investigated one of the stress-related genes (htrA) down-regulated in the lpp mutant relative to WT Y. pestis, as HtrA was previously shown to be important for intracellular survival of Y. enterocolitica in macrophages. Indeed, complementation of the delta-lpp mutant with the htrA gene in trans restored intracellular survival of the Y. pestis lpp mutant. Our results support a role for Lpp in Y. pestis CO92 adapatation to the host environment, possibly via transcriptional activation of htrA. WT Y. pestis CO92 was obtained from the Centers for Disease Control and Prevention (CDC, Atlanta, GA) and maintained in our restricted access biosafety level (BSL)-2 laboratory. The creation and characterization of the strain deficient in the expression of the lpp gene were described in detail previously. All Yersinia strains were grown in brain, heart, infusion broth (BHI, Difco, Voigt Global Distribution Inc, Lawrence, KS) at 26-28°C. Prior to RNA isolation, bacteria were grown in BHI broth overnight at 26°C. The overnight culture was diluted 1:20 in BHI broth and grown at 26°C for an additional 6 h. In another set of experiment, after 2 h of cultivation at 26°C, the temperature was shifted to 37°C for 4 h to facilitate activation of the T3SS and production of Yops. Bacteria were harvested and RNA isolated using RiboPure (Ambion/Applied Biosystems, Austin, TX). Microarray for Y. pestis are available to our laboratory through the NIAID's Pathogen Functional Genomics Resource Center at The Institute for Genomic Research (TIGR). RNA was processed and hybridized by the Molecular Genomics Core Facility at UTMB.