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

Jiang, Wangshu; Ubhayasekera, Wimal; Breed, Michael C; Norsworthy, Allison N; Serr, Nina; Mobley, Harry L T; Pearson, Melanie M; Knight, Stefan D
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.
PMID: 32780778
ISSN: 1553-7374
CID: 4563672

Structures of two fimbrial adhesins, AtfE and UcaD, from the uropathogen Proteus mirabilis

Jiang, Wangshu; Ubhayasekera, Wimal; Pearson, Melanie M; Knight, Stefan D
The important uropathogen Proteus mirabilis encodes a record number of chaperone/usher-pathway adhesive fimbriae. Such fimbriae, which are used for adhesion to cell surfaces/tissues and for biofilm formation, are typically important virulence factors in bacterial pathogenesis. Here, the structures of the receptor-binding domains of the tip-located two-domain adhesins UcaD (1.5 Å resolution) and AtfE (1.58 Å resolution) from two P. mirabilis fimbriae (UCA/NAF and ATF) are presented. The structures of UcaD and AtfE are both similar to the F17G type of tip-located fimbrial receptor-binding domains, and the structures are very similar despite having only limited sequence similarity. These structures represent an important step towards a molecular-level understanding of P. mirabilis fimbrial adhesins and their roles in the complex pathogenesis of urinary-tract infections.
PMID: 30387764
ISSN: 2059-7983
CID: 3400452

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

Debnath, Irina; Stringer, Anne M; Smith, Sara N; Bae, Emily; Mobley, Harry L T; Wade, Joseph T; Pearson, Melanie M
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 flagella-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.
PMID: 30082479
ISSN: 1098-5522
CID: 3226542

From Catheter to Kidney Stone: The Uropathogenic Lifestyle of Proteus mirabilis

Norsworthy, Allison N; Pearson, Melanie M
Proteus mirabilis is a model organism for urease-producing uropathogens. These diverse bacteria cause infection stones in the urinary tract and form crystalline biofilms on indwelling urinary catheters, frequently leading to polymicrobial infection. Recent work has elucidated how P. mirabilis causes all of these disease states. Particularly exciting is the discovery that this bacterium forms large clusters in the bladder lumen that are sites for stone formation. These clusters, and other steps of infection, require two virulence factors in particular: urease and MR/P fimbriae. Highlighting the importance of MR/P fimbriae is the cotranscribed regulator, MrpJ, which globally controls virulence. Overall, P. mirabilis exhibits an extraordinary lifestyle, and further probing will answer exciting basic microbiological and clinically relevant questions.
PMCID:5365347
PMID: 28017513
ISSN: 1878-4380
CID: 2383442

Distinct residues contribute to motility repression and autoregulation in the Proteus mirabilis fimbria-associated transcriptional regulator AtfJ

Bode, Nadine J; Chan, Kun-Wei; Kong, Xiang-Peng; Pearson, Melanie M
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 to affecting the expression of a wide range of cellular processes. Interestingly, 14 additional mrpJ paralogs are encoded by the P. mirabilis genome. Looking at a selection of MrpJ paralogs, we discovered that these proteins, which consistently repress motility, also have non-identical functions that include cross-regulation of fimbrial operons. A subset of paralogs, including AtfJ (encoded by the ambient temperature fimbrial operon), Fim8J, and MrpJ, is capable of auto-induction. 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 3D-structural model, we hypothesize the C-terminal domain confers unique regulatory capacities to 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, 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 by elucidating the non-identical 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.
PMCID:4944230
PMID: 27246571
ISSN: 1098-5530
CID: 2124252

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

Schaffer, Jessica N; Norsworthy, Allison N; Sun, Tung-Tien; Pearson, Melanie M
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.
PMCID:4843424
PMID: 27044107
ISSN: 1091-6490
CID: 2066032

Proteus mirabilis and Urinary Tract Infections

Schaffer, Jessica N; Pearson, Melanie M
Proteus mirabilis is a Gram-negative bacterium and is well known for its ability to robustly swarm across surfaces in a striking bulls'-eye pattern. Clinically, this organism is most frequently a pathogen of the urinary tract, particularly in patients undergoing long-term catheterization. This review covers P. mirabilis with a focus on urinary tract infections (UTI), including disease models, vaccine development efforts, and clinical perspectives. Flagella-mediated motility, both swimming and swarming, is a central facet of this organism. The regulation of this complex process and its contribution to virulence is discussed, along with the type VI-secretion system-dependent intra-strain competition, which occurs during swarming. P. mirabilis uses a diverse set of virulence factors to access and colonize the host urinary tract, including urease and stone formation, fimbriae and other adhesins, iron and zinc acquisition, proteases and toxins, biofilm formation, and regulation of pathogenesis. While significant advances in this field have been made, challenges remain to combatting complicated UTI and deciphering P. mirabilis pathogenesis.
PMCID:4638163
PMID: 26542036
ISSN: 2165-0497
CID: 2482952

Mechanistic studies of MrpJ paralogs in Proteus mirabilis [Meeting Abstract]

Bode, N J; Pearson, M M
The enteric Gram-negative bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections. P. mirabilis exhibits robust swimming and swarming motility, yet it produces a variety of adherence proteins. Both adherence and motility, mediated by fimbriae and flagella, respectively, contribute to UTI. Hence, strict regulation of these antagonistic processes is essential for successful disease progression. Objectives: The transcriptional regulator MrpJ, which is encoded by the mrp (mannose-resistant Proteus-like) fimbrial operon, has been shown to repress both swimming and swarming motility. Recent work in our laboratory elucidated MrpJ as a global regulator of virulence-associated genes. The P. mirabilis genome encodes an additional 14 mrpJ paralogs, the majority of which also negatively affect motility. Our study aims to elucidate the differences in target genes or mechanism of action between these transcriptional regulators, necessitating their coexistence in this important uropathogen. Materials and Methods: In order to study these regulatory proteins, we tested expression of potential target genes by qRTPCR. Mechanistic studies focused on promoter deletion analyses of transcriptional LacZ reporters, as well as the production of chimeric fusion proteins in order to assess domain functionality. Results: Transcriptional analysis of strains overexpressing individual paralogs revealed differences in regulation of target genes. Notably, several MrpJ paralogs autoregulate their own operons. We identified an MrpJ-responsive region in the mrp promoter, resulting in positive autoregulation of the fimbrial operon. We narrowed the MrpJ binding site to a 100 bp region located about 150-250 nucleotides upstream of the transcriptional start. Current studies focus on AtfJ, an MrpJ paralog associated with ambient temperature fimbriae (ATF). As with MrpJ, we observed positive autoregulation, albeit at a much greater magnitude. Interestingly, the atfJ-responsive element is placed at a greater distance (-487 to -655 nt) from the start of transcription of the atf operon. Chimeric fusion proteins between AtfJ and two other MrpJ-type proteins demonstrated that the C-terminal domain of AtfJ is necessary but not sufficient for transcriptional activation of the atf operon. Interestingly, several chimeric proteins eliciting negligible LacZ responses retained the ability to repress motility, suggesting that these two protein functions are not invariably linked. Conclusion: Our findings support the hypothesis that mrpJ paralogs fulfill non-identical functions. In addition to the possible use of different mechanisms of regulation, these results strengthen the importance of MrpJ-type regulators in P. mirabilis biology
EMBASE:72056667
ISSN: 1438-4221
CID: 1840092

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

Bode, Nadine J; Debnath, Irina; Kuan, Lisa; Schulfer, Anjelique; Ty, Maureen; Pearson, Melanie M
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 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 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 by MrpJ using transcriptional reporters and chromatin immunoprecipitation (ChIP). Binding of MrpJ to two virulence-associated target gene promoters, 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.
PMCID:4432738
PMID: 25847961
ISSN: 1098-5522
CID: 1528382

Characterization of 17 Chaperone-Usher Fimbriae Encoded by Proteus mirabilis Reveals Strong Conservation

Kuan, Lisa; Schaffer, Jessica; Zouzias, Christos D; Pearson, Melanie M
Proteus mirabilis is a Gram-negative enteric bacterium that causes complicated urinary tract infections, particularly in patients with indwelling catheters. Sequencing of clinical isolate P. mirabilis HI4320 revealed the presence of 17 predicted chaperone-usher fimbrial operons. We classified these fimbriae into three groups by their genetic relationship to other chaperone-usher fimbriae. Sixteen of these fimbriae are encoded by all six currently sequenced P. mirabilis genomes. The predicted protein sequence of the major structural subunit for 14 of these fimbriae was highly conserved (>/=95% identity), whereas three other structural subunits (Fim3A, UcaA, and Fim6A) were highly variable. Further examination of 58 clinical isolates showed that 12 of the 17 predicted major structural subunit genes of the fimbriae were present in most strains (>85%). Transcription of the predicted major structural subunit genes for all 17 fimbriae was measured under different culture conditions designed to mimic conditions in the urinary tract. The majority of the fimbrial genes were induced during stationary phase, static culture, or colony growth compared to logarithmic-phase aerated culture. Major structural subunit proteins for six of these fimbriae were detected using mass spectrometry of proteins sheared from the surface of broth-cultured P. mirabilis, demonstrating that this organism may produce multiple fimbriae within a single culture. The high degree of conservation of P. mirabilis fimbriae stands in contrast to uropathogenic Escherichia coli and Salmonella enterica, which exhibit greater variability in their fimbrial repertoires. These findings suggest there may be evolutionary pressure for P. mirabilis to maintain a large fimbrial arsenal.
PMCID:4064351
PMID: 24809384
ISSN: 0022-2615
CID: 988132