Project description:This series of microarrays compares gene expression by the bacterial pathogen Proteus mirabilis when the transcriptional regulator mrpJ is deleted or induced to levels found during experimental urinary tract infection. The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections. Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for successful disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects a wide array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking expression levels that occur during UTI leads to differential expression of 217 genes related to, among others, bacterial virulence, type VI secretion and metabolism. We probed the molecular mechanism of transcriptional regulation through MrpJ using reporter assays and chromatin immunoprecipitation (ChIP). Two virulence-associated target genes, the flagellar master regulator flhDC and mrp itself, appear to be regulated through a binding site proximal to the transcriptional start, complemented by a more distantly situated enhancer site. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observe that mrpJ is widely conserved in a collection of recent clinical isolates, leading us to conclude that our results elucidate an unanticipated role of MrpJ as a global regulator of P. mirabilis virulence.
Project description:This series of microarrays compares gene expression by the bacterial pathogen Proteus mirabilis when the transcriptional regulator mrpJ is deleted or induced to levels found during experimental urinary tract infection. The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections. Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for successful disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects a wide array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking expression levels that occur during UTI leads to differential expression of 217 genes related to, among others, bacterial virulence, type VI secretion and metabolism. We probed the molecular mechanism of transcriptional regulation through MrpJ using reporter assays and chromatin immunoprecipitation (ChIP). Two virulence-associated target genes, the flagellar master regulator flhDC and mrp itself, appear to be regulated through a binding site proximal to the transcriptional start, complemented by a more distantly situated enhancer site. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observe that mrpJ is widely conserved in a collection of recent clinical isolates, leading us to conclude that our results elucidate an unanticipated role of MrpJ as a global regulator of P. mirabilis virulence. Four biological replicates were analyzed for each set of arrays (P. mirabilis HI4320 wild type vs. ΔmrpJ, and vector pLX3607 vs. mrpJ plasmid pLX3805).
Project description:Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (UTIs) and urolithiasis. The transcriptional regulator MrpJ inversely modulates two critical aspects of P. mirabilis UTI progression: fimbria-mediated attachment to the urinary tract, and flagella-mediated motility. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) was used for the first time in a CAUTI pathogen to probe for in vivo direct targets of MrpJ. ChIP-seq revealed 81 78 direct MrpJ targets, including genes for motility, fimbriae and a type VI secretion system (T6SS), and the putative MrpJ binding sequence ACnCnnnnnnnGnGT.
Project description:The enteric bacterium Proteus mirabilis is a common cause of complicated urinary tract infections. In the study, microrarrays were used to analyze P. mirabilis gene expression in vivo from experimentally infected mice. Urine was collected at 1, 3, and 7d postinfection, and RNA was isolated from bacteria in the urine for transcriptional analysis. Across 9 microarrays, 471 genes were upregulated and 82 were downregulated in vivo compared to in vitro broth culture. Genes upregulated in vivo encoded MR/P fimbriae, urease, iron uptake systems, amino acid and peptide transporters, pyruvate metabolism, and portions of the TCA cycle. Flagella were downregulated. Ammonia assimilation gene glnA (glutamine synthetase) was repressed in vivo while gdhA (glutamate dehydrogenase) was upregulated in vivo. Contrary to our expectations, ammonia availability due to urease activity in P. mirabilis did not drive this gene expression. A gdhA mutant was growth-deficient in minimal medium with citrate as the sole carbon source, and loss of gdhA resulted in a significant fitness defect in the mouse model of urinary tract infection. Unlike Escherichia coli, which represses gdhA and upregulates glnA in vivo and cannot utilize citrate, the data suggest that P. mirabilis uses glutamate dehydrogenase to monitor carbon-nitrogen balance, and this ability contributes to the pathogenic potential of P. mirabilis in the urinary tract.
Project description:The enteric bacterium Proteus mirabilis is a common cause of complicated urinary tract infections. In the study, microrarrays were used to analyze P. mirabilis gene expression in vivo from experimentally infected mice. Urine was collected at 1, 3, and 7d postinfection, and RNA was isolated from bacteria in the urine for transcriptional analysis. Across 9 microarrays, 471 genes were upregulated and 82 were downregulated in vivo compared to in vitro broth culture. Genes upregulated in vivo encoded MR/P fimbriae, urease, iron uptake systems, amino acid and peptide transporters, pyruvate metabolism, and portions of the TCA cycle. Flagella were downregulated. Ammonia assimilation gene glnA (glutamine synthetase) was repressed in vivo while gdhA (glutamate dehydrogenase) was upregulated in vivo. Contrary to our expectations, ammonia availability due to urease activity in P. mirabilis did not drive this gene expression. A gdhA mutant was growth-deficient in minimal medium with citrate as the sole carbon source, and loss of gdhA resulted in a significant fitness defect in the mouse model of urinary tract infection. Unlike Escherichia coli, which represses gdhA and upregulates glnA in vivo and cannot utilize citrate, the data suggest that P. mirabilis uses glutamate dehydrogenase to monitor carbon-nitrogen balance, and this ability contributes to the pathogenic potential of P. mirabilis in the urinary tract. Voided urine from female CBA/J mice infected with Proteus mirabilis was collected and pooled in RNA stabilizing reagent (RNAprotect). Urine was collected at 1, 3, and 7 d postinfection. RNA was isolated from urine and log-phase LB cultures, converted to cDNA, and labeled with CyDye. Three arrays were completed per time point (9 arrays total). Slides were scanned with a ScanArray Express microarray scanner (Perkin Elmer) at 10 μm resolution and quantified using ScanArray Express software. Resulting data were normalized by total intensity and median spot intensities were identified using MIDAS (v. 2.22) software.
Project description:Swarming motility by the urinary tract pathogen Proteus mirabilis has been a long-studied but little understood phenomenon. On agar, a P. mirabilis colony grows outward in a bull's-eye pattern formed by consecutive waves of rapid swarming followed by consolidation into shorter cells. To examine differential gene expression in these growth phases, a microarray was constructed based on the completed genome sequence and annotation. RNA was extracted from broth-cultured, swarming, and consolidation-phase cells to assess transcription during each of these growth states. A total of 587 genes were differentially expressed in broth-cultured cells versus swarming cells, and 527 genes were differentially expressed in broth-cultured cells versus consolidation-phase cells (consolidate). Flagellar genes were highly upregulated in both swarming cells and consolidation-phase cells. Fimbriae were downregulated in swarming cells, while genes involved in cell division and anaerobic growth were upregulated in broth-cultured cells. Direct comparison of swarming cells to consolidation-phase cells found that 541 genes were upregulated in consolidate, but only nine genes were upregulated in swarm cells. Genes involved in flagellar biosynthesis, oligopeptide transport, amino acid import and metabolism, cell division, and phage were upregulated in consolidate. Mutation of dppA, oppB, and cysJ, upregulated during consolidation compared to during swarming, revealed that although these genes play a minor role in swarming, dppA and cysJ are required during ascending urinary tract infection. Swarming on agar to which chloramphenicol had been added suggested that protein synthesis is not required for swarming. These data suggest that the consolidation phase is a state in which P. mirabilis prepares for the next wave of swarming.