Proteus mirabilis UreR coordinates cellular functions required for urease activity.

A hallmark of Proteus mirabilis infection of the urinary tract is the formation of stones. The ability to induce urinary stone formation requires urease, a nickel metalloenzyme that hydrolyzes urea. This reaction produces ammonia as a byproduct, which can serve as a nitrogen source and weak base that raises the local pH. The resulting alkalinity induces the precipitation of ions to form stones. Transcriptional regulator UreR activates expression of urease genes in a urea-dependent manner. Thus, urease genes are highly expressed in the urinary tract where urea is abundant. Production of mature urease also requires the import of nickel into the cytoplasm and its incorporation into the urease apoenzyme. Urease accessory proteins primarily acquire nickel from one of two nickel transporters and facilitate incorporation of nickel to form mature urease. In this study, we performed a comprehensive RNA-seq to define the P. mirabilis urea-induced transcriptome as well as the UreR regulon. We identified UreR as the first defined regulator of nickel transport in P. mirabilis. We also offer evidence for the direct regulation of the Ynt nickel transporter by UreR. Using bioinformatics, we identified UreR-regulated urease loci in 15 Morganellaceae family species across three genera. Additionally, we located two mobilized UreR-regulated urease loci that also encode the ynt transporter, implying that UreR regulation of nickel transport is a conserved regulatory relationship. Our study demonstrates that UreR specifically regulates genes required to produce mature urease, an essential virulence factor for P. mirabilis uropathogenesis. IMPORTANCE: Catheter-associated urinary tract infections (CAUTIs) account for over 40% of acute nosocomial infections in the USA and generate $340 million in healthcare costs annually. A major causative agent of CAUTIs is Proteus mirabilis, an understudied Gram-negative pathogen noted for its ability to form urinary stones via the activity of urease. Urease mutants cannot induce stones and are attenuated in a murine UTI model, indicating this enzyme is essential to P. mirabilis pathogenesis. Transcriptional regulation of urease genes by UreR is well established; here, we expand the UreR regulon to include regulation of nickel import, a function required to produce mature urease. Furthermore, we reflect on the role of urea catalysis in P. mirabilis metabolism and provide evidence for its importance.

Organ agar serves as physiologically relevant alternative for in vivo bacterial colonization.

Animal models for host-microbial interactions have proven valuable, yielding physiologically relevant data that may be otherwise difficult to obtain. Unfortunately, such models are lacking or nonexistent for many microbes. Here, we introduce organ agar, a straightforward method to enable the screening of large mutant libraries while avoiding physiological bottlenecks. We demonstrate that growth defects on organ agar were translatable to bacterial colonization deficiencies in a murine model. Specifically, we present a urinary tract infection agar model to interrogate an ordered library of Proteus mirabilis transposon mutants, with accurate prediction of bacterial genes critical for host colonization. Thus, we demonstrate the ability of ex vivo organ agar to reproduce in vivo deficiencies. Organ agar was also useful for identifying previously unknown links between biosynthetic genes and swarming motility. This work provides a readily adoptable technique that is economical and uses substantially fewer animals. We anticipate this method will be useful for a wide variety of microorganisms, both pathogenic and commensal, in a diverse range of model host species.

MrpH, a new class of metal-binding adhesin, requires zinc to mediate biofilm formation.

Proteus mirabilis, a Gram-negative uropathogen, is a major causative agent in catheter-associated urinary tract infections (CAUTI). Mannose-resistant Proteus-like fimbriae (MR/P) are crucially important for P. mirabilis infectivity and are required for biofilm formation and auto-aggregation, as well as for bladder and kidney colonization. Here, the X-ray crystal structure of the MR/P tip adhesin, MrpH, is reported. The structure has a fold not previously described and contains a transition metal center with Zn2+ coordinated by three conserved histidine residues and a ligand. Using biofilm assays, chelation, metal complementation, and site-directed mutagenesis of the three histidines, we show that an intact metal binding site occupied by zinc is essential for MR/P fimbria-mediated biofilm formation, and furthermore, that P. mirabilis biofilm formation is reversible in a zinc-dependent manner. Zinc is also required for MR/P-dependent agglutination of erythrocytes, and mutation of the metal binding site renders P. mirabilis unfit in a mouse model of UTI. The studies presented here provide important clues as to the mechanism of MR/P-mediated biofilm formation and serve as a starting point for identifying the physiological MR/P fimbrial receptor.

Proteus mirabilis fimbriae- and urease-dependent clusters assemble in an extracellular niche to initiate bladder stone formation.

The catheter-associated uropathogenProteus mirabilisfrequently causes urinary stones, but little has been known about the initial stages of bladder colonization and stone formation. We found thatP. mirabilisrapidly invades the bladder urothelium, but generally fails to establish an intracellular niche. Instead, it forms extracellular clusters in the bladder lumen, which form foci of mineral deposition consistent with development of urinary stones. These clusters elicit a robust neutrophil response, and we present evidence of neutrophil extracellular trap generation during experimental urinary tract infection. We identified two virulence factors required for cluster development: urease, which is required for urolithiasis, and mannose-resistantProteus-like fimbriae. The extracellular cluster formation byP. mirabilisstands in direct contrast to uropathogenicEscherichia coli, which readily formed intracellular bacterial communities but not luminal clusters or urinary stones. We propose that extracellular clusters are a key mechanism ofP. mirabilissurvival and virulence in the bladder.

MrpJ Directly Regulates Proteus mirabilis Virulence Factors, Including Fimbriae and Type VI Secretion, during Urinary Tract Infection.

Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs) and urolithiasis. The transcriptional regulator MrpJ inversely modulates two critical aspects of P. mirabilis UTI progression: fimbria-mediated attachment and flagellum-mediated motility. Transcriptome data indicated a network of virulence-associated genes under MrpJ's control. Here, we identify the direct gene regulon of MrpJ and its contribution to P. mirabilis pathogenesis, leading to the discovery of novel virulence targets. 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. Selected MrpJ-regulated genes were mutated and assessed for their contribution to UTI using a mouse model. ChIP-seq revealed a palindromic MrpJ binding sequence and 78 MrpJ-bound regions, including binding sites upstream of genes involved in motility, fimbriae, and a type VI secretion system (T6SS). A combinatorial mutation approach established the contribution of three fimbriae (fim8A, fim14A, and pmpA) to UTI and a new pathogenic role for the T6SS in UTI progression. In conclusion, this study (i) establishes the direct gene regulon and an MrpJ consensus binding site and (ii) led to the discovery of new virulence genes in P. mirabilis UTI, which could be targeted for therapeutic intervention of CAUTI.

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

UNLABELLED: Proteus mirabilis contributes to a significant number of catheter-associated urinary tract infections, where coordinated regulation of adherence and motility is critical for ascending disease progression. Previously, the mannose-resistant Proteus-like (MR/P) fimbria-associated transcriptional regulator MrpJ has been shown to both repress motility and directly induce the transcription of its own operon; in addition, it affects the expression of a wide range of cellular processes. Interestingly, 14 additional mrpJ paralogs are included in the P. mirabilis genome. Looking at a selection of MrpJ paralogs, we discovered that these proteins, which consistently repress motility, also have nonidentical functions that include cross-regulation of fimbrial operons. A subset of paralogs, including AtfJ (encoded by the ambient temperature fimbrial operon), Fim8J, and MrpJ, are capable of autoinduction. We identified an element of the atf promoter extending from 487 to 655 nucleotides upstream of the transcriptional start site that is responsive to AtfJ, and we found that AtfJ directly binds this fragment. Mutational analysis of AtfJ revealed that its two identified functions, autoregulation and motility repression, are not invariably linked. Residues within the DNA-binding helix-turn-helix domain are required for motility repression but not necessarily autoregulation. Likewise, the C-terminal domain is dispensable for motility repression but is essential for autoregulation. Supported by a three-dimensional (3D) structural model, we hypothesize that the C-terminal domain confers unique regulatory capacities on the AtfJ family of regulators. IMPORTANCE: Balancing adherence with motility is essential for uropathogens to successfully establish a foothold in their host. Proteus mirabilis uses a fimbria-associated transcriptional regulator to switch between these antagonistic processes by increasing fimbrial adherence while simultaneously downregulating flagella. The discovery of multiple related proteins, many of which also function as motility repressors, encoded in the P. mirabilis genome has raised considerable interest as to their functionality and potential redundancy in this organism. This study provides an important advance in this field by elucidating the nonidentical effects of these paralogs on a molecular level. Our mechanistic studies of one member of this group, AtfJ, shed light on how these differing functions may be conferred despite the limited sequence variety exhibited by the paralogous proteins.

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.

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.

Organ agar serves as physiologically relevant alternative for in vivo colonization.

Animal models for host-microbial interactions have proven valuable, yielding physiologically relevant data that may be otherwise difficult to obtain. Unfortunately, such models are lacking or nonexistent for many microbes. Here, we introduce organ agar, a straightforward method to enable the screening of large mutant libraries while avoiding physiological bottlenecks. We demonstrate that growth defects on organ agar were translatable to colonization deficiencies in a murine model. Specifically, we present a urinary tract infection agar model to interrogate an ordered library of Proteus mirabilis transposon mutants, with accurate prediction of bacterial genes critical for host colonization. Thus, we demonstrate the ability of ex vivo organ agar to reproduce in vivo deficiencies. This work provides a readily adoptable technique that is economical and uses substantially fewer animals. We anticipate this method will be useful for a wide variety of microorganisms, both pathogenic and commensal, in a diverse range of model host species.

Construction of an Ordered Transposon Library for Uropathogenic Proteus mirabilis HI4320.

Ordered transposon libraries are a valuable resource for many bacterial species, especially those with difficult methods for generating targeted genetic mutations. Here, we present the construction of an ordered transposon library for the bacterial urinary tract pathogen Proteus mirabilis strain HI4320. This library will facilitate future studies into P. mirabilis biology. For large experimental screens, it may be used to overcome bottleneck constraints and avoid biased outcomes resulting from gene length. For smaller studies, the library allows sidestepping the laborious construction of single targeted mutants. This library, containing 18,432 wells, was condensed into a smaller library containing 1,728 mutants. Each selected mutant had a single transposon insertion in an open reading frame, covering 45% of predicted genes encoded by P. mirabilis HI4320. This coverage was lower than expected and was due both to library wells with no mapped insertions and a surprisingly high proportion of mixed clones and multiple transposon insertion events. We offer recommendations for improving future library construction and suggestions for how to use this P. mirabilis library resource. IMPORTANCE Ordered libraries facilitate large genetic screens by guaranteeing high genomic coverage with a minimal number of mutants, and they can save time and effort by reducing the need to construct targeted mutations. This resource is now available for P. mirabilis, a common and complicating agent of catheter-associated urinary tract infection. We also present obstacles encountered during library construction with the goal to aid others who would like to construct ordered transposon libraries in other species.

Transcriptome of Proteus mirabilis in the murine urinary tract: virulence and nitrogen assimilation gene expression.

The enteric bacterium Proteus mirabilis is a common cause of complicated urinary tract infections. In this study, microarrays were used to analyze P. mirabilis gene expression in vivo from experimentally infected mice. Urine was collected at 1, 3, and 7 days postinfection, and RNA was isolated from bacteria in the urine for transcriptional analysis. Across nine microarrays, 471 genes were upregulated and 82 were downregulated in vivo compared to in vitro broth culture. Genes upregulated in vivo encoded mannose-resistant Proteus-like (MR/P) fimbriae, urease, iron uptake systems, amino acid and peptide transporters, pyruvate metabolism enzymes, and a portion of the tricarboxylic acid (TCA) cycle enzymes. 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.

Proteobactin and a yersiniabactin-related siderophore mediate iron acquisition in Proteus mirabilis.

Proteus mirabilis causes complicated urinary tract infections (UTIs). While the urinary tract is an iron-limiting environment, iron acquisition remains poorly characterized for this uropathogen. Microarray analysis of P. mirabilis HI4320 cultured under iron limitation identified 45 significantly upregulated genes (P ≤ 0.05) that represent 21 putative iron-regulated systems. Two gene clusters, PMI0229-0239 and PMI2596-2605, encode putative siderophore systems. PMI0229-0239 encodes a non-ribosomal peptide synthetase-independent siderophore system for producing a novel siderophore, proteobactin. PMI2596-2605 are contained within the high-pathogenicity island, originally described in Yersinia pestis, and encodes proteins with apparent homology and organization to those involved in yersiniabactin production and uptake. Cross-feeding and biochemical analysis shows that P. mirabilis is unable to utilize or produce yersiniabactin, suggesting that this yersiniabactin-related locus is functionally distinct. Only disruption of both systems resulted in an in vitro iron-chelating defect; demonstrating production and iron-chelating activity for both siderophores. These findings clearly show that proteobactin and the yersiniabactin-related siderophore function as iron acquisition systems. Despite the activity of both siderophores, only mutants lacking the yersiniabactin-related siderophore have reduced fitness in vivo. The fitness requirement for the yersiniabactin-related siderophore during UTI shows, for the first time, the importance of siderophore production in vivo for P. mirabilis.

Transcriptome of swarming Proteus mirabilis.

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.

Oxygen-limiting conditions enrich for fimbriate cells of uropathogenic Proteus mirabilis and Escherichia coli.

MR/P fimbriae of uropathogenic Proteus mirabilis undergo invertible element-mediated phase variation whereby an individual bacterium switches between expressing fimbriae (phase ON) and not expressing fimbriae (phase OFF). Under different conditions, the percentage of fimbriate bacteria within a population varies and could be dictated by either selection (growth advantage of one phase) or signaling (preferentially converting one phase to the other in response to external signals). Expression of MR/P fimbriae increases in a cell-density dependent manner in vitro and in vivo. However, rather than the increased cell density itself, this increase in fimbrial expression is due to an enrichment of fimbriate bacteria under oxygen limitation resulting from increased cell density. Our data also indicate that the persistence of MR/P fimbriate bacteria under oxygen-limiting conditions is a result of both selection (of MR/P fimbrial phase variants) and signaling (via modulation of expression of the MrpI recombinase). Furthermore, the mrpJ transcriptional regulator encoded within the mrp operon contributes to phase switching. Type 1 fimbriae of Escherichia coli, which are likewise subject to phase variation via an invertible element, also increase in expression during reduced oxygenation. These findings provide evidence to support a mechanism for persistence of fimbriate bacteria under oxygen limitation, which is relevant to disease progression within the oxygen-restricted urinary tract.

Repression of motility during fimbrial expression: identification of 14 mrpJ gene paralogues in Proteus mirabilis.

Proteus mirabilis alternates between motile and adherent forms. MrpJ, a transcriptional regulator previously reported to repress motility, is encoded at the 3' end of the mrp fimbrial operon in P. mirabilis. Sequencing of the P. mirabilis genome revealed 14 additional paralogues of mrpJ, 10 of which are associated with fimbrial operons. Twelve of these genes, when overexpressed, repressed motility; several distinct patterns of swarming motility were noted. Expression of 10 of the 14 mrpJ paralogues repressed flagellin (FlaA) synthesis. Alignment of the predicted amino acid sequences of MrpJ and its 14 paralogues revealed a conserved consensus motif (SQQQFSRYE) within the helix-turn-helix domain. Site-directed mutagenesis of these residues coupled with linker insertion mutagenesis of MrpJ confirmed the importance of this domain for repression of motility. Gel shift assays demonstrated that MrpJ and another paralogue UcaJ bind directly to the promoter region of the flagellar master regulator flhDC. Thus, P. mirabilis appears to use a related mechanism to inhibit motility during the production of at least 10 of its predicted fimbriae.

Culture Methods for Proteus mirabilis.

Proteus mirabilis is generally easy to culture, but its tendency to swarm on a wide variety of media can interfere with isolation of single colonies or identification of other species in a sample. Therefore, specialized media may be needed to control swarming or to study the bacteria under chemically defined conditions. Here, methods are described for routine culture of P. mirabilis, isolation of P. mirabilis from mixed cultures, and culture of P. mirabilis on physiologically relevant media.

Methods for Studying Swarming and Swimming Motility.

Proteus mirabilis is well known for using its flagella to swim through liquids or swarm across solid surfaces. Both phenomena are easy to observe. Described here are two agar-based assays for studying both swimming and swarming behavior, and considerations that affect the outcome.

Phase Variation of the mrp Fimbrial Promoter.

Mannose-resistant Proteus-like (MR/P) fimbriae, produced by the uropathogen Proteus mirabilis, are major contributors to urinary tract infection. Expression of mrp genes is controlled by an invertible element in the mrp operon promoter, and promoter orientation is controlled by a single recombinase, MrpI, which determines whether the element is in the on or off orientation. Detailed here is a simple assay to determine the orientation of the invertible element in a population of P. mirabilis, a rapid screen to detect element-locked mrpI mutants, and quantification of mixtures of on and off bacteria.

Insertional Mutagenesis Protocol for Constructing Single or Sequential Mutations.

Genetic mutation enables the study of the function of specific genes, particularly when a mutant is compared against its isogenic parent. In Proteus mirabilis bacteria, traditional allelic exchange mutation is labor-intensive and has a high failure rate in some strains. Likewise, there is no working protocol for lambda red recombinase-based mutation in P. mirabilis. Here we describe an alternative method of insertional mutagenesis based on retargeting of group II introns. The protocol includes steps to generate single or multiple mutations, with the possibility to delete intervening sequences of DNA.

Using Hemagglutination, Surface Shearing, and Acid Treatment to Study Fimbriae in Proteus mirabilis.

A critical first step in bacterial virulence and colonization is adherence to mucosal surfaces, often mediated by fimbriae and other protein adhesins. Here are described three short methods for studying these surface proteins and their behaviors, using protocols developed for the opportunistic pathogen Proteus mirabilis. Unlike the mannose-binding type 1 fimbriae produced by Escherichia coli, most P. mirabilis strains produce mannose-resistant/Proteus-like (MR/P) fimbriae. Both types of fimbrial production and adhesion can be easily demonstrated by a simple and economical hemagglutination assay which uses a model system of erythrocytes. The second and third fimbrial methods presented here show how to shear surface-exposed proteins and use acid treatment to separate interlocked fimbrial subunits into monomers.

Complete genome sequence of uropathogenic Proteus mirabilis, a master of both adherence and motility.

The gram-negative enteric bacterium Proteus mirabilis is a frequent cause of urinary tract infections in individuals with long-term indwelling catheters or with complicated urinary tracts (e.g., due to spinal cord injury or anatomic abnormality). P. mirabilis bacteriuria may lead to acute pyelonephritis, fever, and bacteremia. Most notoriously, this pathogen uses urease to catalyze the formation of kidney and bladder stones or to encrust or obstruct indwelling urinary catheters. Here we report the complete genome sequence of P. mirabilis HI4320, a representative strain cultured in our laboratory from the urine of a nursing home patient with a long-term (> or =30 days) indwelling urinary catheter. The genome is 4.063 Mb long and has a G+C content of 38.88%. There is a single plasmid consisting of 36,289 nucleotides. Annotation of the genome identified 3,685 coding sequences and seven rRNA loci. Analysis of the sequence confirmed the presence of previously identified virulence determinants, as well as a contiguous 54-kb flagellar regulon and 17 types of fimbriae. Genes encoding a potential type III secretion system were identified on a low-G+C-content genomic island containing 24 intact genes that appear to encode all components necessary to assemble a type III secretion system needle complex. In addition, the P. mirabilis HI4320 genome possesses four tandem copies of the zapE metalloprotease gene, genes encoding six putative autotransporters, an extension of the atf fimbrial operon to six genes, including an mrpJ homolog, and genes encoding at least five iron uptake mechanisms, two potential type IV secretion systems, and 16 two-component regulators.