Transcriptional Analysis of the MrpJ Network: Modulation of Diverse Virulence-Associated Genes and Direct Regulation of mrp Fimbrial and flhDC Flagellar Operons in Proteus mirabilis

Abstract

The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections (UTIs). Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for 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 an array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking levels observed during UTIs leads to differential expression of 217 genes related to, among other functions, bacterial virulence, type VI secretion, and metabolism. We probed the molecular mechanism of transcriptional regulation by MrpJ using transcriptional reporters and chromatin immunoprecipitation (ChIP). Binding of MrpJ to two virulence-associated target gene promoters, the promoters of the flagellar master regulator flhDC and mrp itself, appears to be affected by the condensation state of the native chromosome, although both targets share a direct MrpJ binding site proximal to the transcriptional start. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observed that mrpJ is widely conserved in a collection of recent clinical isolates. Altogether, these findings support a role of MrpJ as a global regulator of P. mirabilis virulence. Bacterial pathogens utilize a myriad of complex regulatory networks to adapt gene expression in response to environmental cues encountered in the host. Trying to walk a fine balance of avoiding detection by the host immune system, while ensuring acquisition of all essential nutrients and promoting growth, bacteria employ transcriptional and posttranscriptional strategies to combat the host's defenses. A prominent example is the specific induction of iron and zinc uptake systems during infection, regulated by Fur and Zur, respectively, which allows pathogens to gain access to these essential transition metals in the restricted host environment (1, 2).Urinary tract infections (UTIs) are among the most common bacterial infections (3), presenting a significant public health burden amounting to annual costs of about 3.5 billion dollars in the United States alone (4). Although Proteus mirabilis causes a relatively small proportion of UTIs in healthy individuals, it is a common cause of cystitis and pyelonephritis in individuals with indwelling catheters or anatomical abnormalities of the urinary tract (4, 5).UTIs occur almost exclusively in an ascending route, meaning that bacteria of fecal origin gain entry to the bladder via the urethra and then spread to the kidneys via the ureters (4). Initially, bacteria adhere to host epithelial cells via proteinaceous supramolecular structures referred to as fimbriae (or pili) (6), which allow pathogens to withstand the mechanical flow of urine (5)...

Distinct Residues Contribute to Motility Repression and Autoregulation in the Proteus mirabilis Fimbria-Associated Transcriptional Regulator AtfJ

“…We recently reported the transcriptomic microarray analysis of a strain expressing MrpJ in trans at levels mimicking those observed in vivo. Production of MrpJ in this physiologically relevant amount resulted in altered expression of several fimbrial genes (pmfA, fim8A, and fim14A), besides positively affecting the transcription of its own mrp operon (24). To expand our limited knowledge of the interplay and regulation of the 17 potential P. mirabilis fimbrial operons, we tested whether select MrpJ paralogs could (i) autoregulate their operons and (ii) cross-regulate other fimbrial operons or fimbria-related genes.…”

“…We first assessed whether the selected MrpJ paralogs showed an autoregulatory phenotype similar to that of MrpJ (24). To do so, we utilized a set of strains expressing individual MrpJ paralogs from the same IPTG-inducible vector used in our microarray studies (9).…”

“…All cloned inserts were sequenced using primers NB52 and NB60. Operon fusions were integrated at the HI4320 attTn7 locus downstream of glmS in single copy, as previously described (24). Successful integration events were confirmed by PCR.…”

“…Pellets were used directly in enzymatic assays or after overnight storage at Ϫ20°C. ␤-Galactosidase activity was determined at room temperature, exactly as described previously (24). Activities are expressed in arbitrary Miller units (27) or as percentages of activity after all values were normalized to that measured for the atfAp-lacZ operon fusion strain containing nucleotides Ϫ655 to ϩ220 (where ϩ1 is the transcription start site of atfA), herein called ⌽(Ϫ655 to ϩ220), when atfJ (pMP129) was expressed in trans.…”

“…This fimbria-associated transcription factor directly binds the promoters of the flagellar master regulator flhDC and its own operon, thereby repressing motility while simultaneously upregulating the production of MR/P fimbriae (10,24). Recent microarray studies utilizing a strain that mimics levels of MrpJ observed during UTIs further illustrated that MrpJ has many nonmotility targets and implicated this protein as a master regulator of virulence (24). A previous report studying the functions of 14 additional P. mirabilis mrpJ paralogs found that the majority repressed both swimming and swarming motility when provided individually in trans, like MrpJ.…”