Vaccination with Proteus Toxic Agglutinin, a Hemolysin-Independent Cytotoxin In Vivo, Protects against Proteus mirabilis Urinary Tract Infection

ABSTRACT Complicated urinary tract infections (UTI) caused by Proteus mirabilis are associated with severe pathology in the bladder and kidney. To investigate the roles of two established cytotoxins, the HpmA hemolysin, a secreted cytotoxin, and proteus toxic agglutinin (Pta), a surface-associated cytotoxin, mutant analysis was used in conjunction with a mouse model of ascending UTI. Inactivation of pta, but not inactivation of hpmA, resulted in significant decreases in the bacterial loads of the mutant in kidneys (P < 0.01) and spleens (P < 0.05) compared to the bacterial loads of the wild type; the 50% infective dose (ID50) of an isogenic pta mutant or hpmA pta double mutant was 100-fold higher (5 × 108 CFU) than the ID50 of parent strain HI4320 (5 × 106 CFU). Colonization by the parent strain caused severe cystitis and interstitial nephritis as determined by histopathological examination. Mice infected with the same bacterial load of the hpmA pta double mutant showed significantly reduced pathology (P < 0.01), suggesting that the additive effect of these two cytotoxins is critical during Proteus infection. Since Pta is surface associated and important for the persistence of P. mirabilis in the host, it was selected as a vaccine candidate. Mice intranasally vaccinated with a site-directed (indicated by an asterisk) (S366A) mutant purified intact toxin (Pta*) or the passenger domain Pta-α*, each independently conjugated with cholera toxin (CT), had significantly lower bacterial counts in their kidneys ( P = 0.001) and spleens (P = 0.002) than mice that received CT alone. The serum immunoglobulin G levels correlated with protection (P = 0.03). This is the first report describing the in vivo cytotoxicity and antigenicity of an autotransporter in P. mirabilis and its use in vaccine development.

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Transcriptome of Proteus mirabilis in the Murine Urinary Tract: Virulence and Nitrogen Assimilation Gene Expression

Infection and Immunity ◽

10.1128/iai.05152-11 ◽

2011 ◽

Vol 79 (7) ◽

pp. 2619-2631 ◽

Cited By ~ 46

Author(s):

Melanie M. Pearson ◽

Alejandra Yep ◽

Sara N. Smith ◽

Harry L. T. Mobley

Keyword(s):

Gene Expression ◽

Urinary Tract ◽

Glutamate Dehydrogenase ◽

Proteus Mirabilis ◽

Transcriptional Analysis ◽

Broth Culture ◽

Pathogenic Potential ◽

Content Type ◽

Tract Infections

ABSTRACTThe enteric bacteriumProteus mirabilisis a common cause of complicated urinary tract infections. In this study, microarrays were used to analyzeP. mirabilisgene expressionin vivofrom 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 downregulatedin vivocompared toin vitrobroth culture. Genes upregulatedin vivoencoded mannose-resistantProteus-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 geneglnA(glutamine synthetase) was repressedin vivo, whilegdhA(glutamate dehydrogenase) was upregulatedin vivo. Contrary to our expectations, ammonia availability due to urease activity inP. mirabilisdid not drive this gene expression. AgdhAmutant was growth deficient in minimal medium with citrate as the sole carbon source, and loss ofgdhAresulted in a significant fitness defect in the mouse model of urinary tract infection. UnlikeEscherichia coli, which repressesgdhAand upregulatesglnAin vivoand cannot utilize citrate, the data suggest thatP. mirabilisuses glutamate dehydrogenase to monitor carbon-nitrogen balance, and this ability contributes to the pathogenic potential ofP. mirabilisin the urinary tract.

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Identification of virulence determinants in uropathogenic Proteus mirabilis using signature-tagged mutagenesis

Journal of Medical Microbiology ◽

10.1099/jmm.0.2008/002071-0 ◽

2008 ◽

Vol 57 (9) ◽

pp. 1068-1078 ◽

Cited By ~ 38

Author(s):

Stephanie D. Himpsl ◽

C. Virginia Lockatell ◽

J. Richard Hebel ◽

David E. Johnson ◽

Harry L. T. Mobley

Keyword(s):

Urinary Tract ◽

Mouse Model ◽

Proteus Mirabilis ◽

Virulence Determinants ◽

Indwelling Catheters ◽

Cba Mice ◽

Transposon Mutants ◽

Tract Infections

The Gram-negative bacterium Proteus mirabilis causes urinary tract infections (UTIs) in individuals with long-term indwelling catheters or those with functional or structural abnormalities of the urinary tract. Known virulence factors include urease, haemolysin, fimbriae, flagella, DsbA, a phosphate transporter and genes involved in cell-wall synthesis and metabolism, many of which have been identified using the technique of signature-tagged mutagenesis (STM). To identify additional virulence determinants and to increase the theoretical coverage of the genome, this study generated and assessed 1880 P. mirabilis strain HI4320 mutants using this method. Mutants with disruptions in genes vital for colonization of the CBA mouse model of ascending UTI were identified after performing primary and secondary in vivo screens in approximately 315 CBA mice, primary and secondary in vitro screens in both Luria broth and minimal A medium to eliminate mutants with minor growth deficiencies, and co-challenge competition experiments in approximately 500 CBA mice. After completion of in vivo screening, a total of 217 transposon mutants were attenuated in the CBA mouse model of ascending UTI. Following in vitro screening, this number was reduced to 196 transposon mutants with a probable role in virulence. Co-challenge competition experiments confirmed significant attenuation for 37 of the 93 transposon mutants tested, being outcompeted by wild-type HI4320. Following sequence analysis of the 37 mutants, transposon insertions were identified in genes including the peptidyl-prolyl isomerases surA and ppiA, glycosyltransferase cpsF, biopolymer transport protein exbD, transcriptional regulator nhaR, one putative fimbrial protein, flagellar M-ring protein fliF and hook protein flgE, and multiple metabolic genes.

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Mannose-Resistant Proteus-Like Fimbriae Are Produced by Most Proteus mirabilis Strains Infecting the Urinary Tract, Dictate the In Vivo Localization of Bacteria, and Contribute to Biofilm Formation

Infection and Immunity ◽

10.1128/iai.72.12.7294-7305.2004 ◽

2004 ◽

Vol 72 (12) ◽

pp. 7294-7305 ◽

Cited By ~ 50

Author(s):

Angela M. Jansen ◽

Virginia Lockatell ◽

David E. Johnson ◽

Harry L. T. Mobley

Keyword(s):

Urinary Tract ◽

Proteus Mirabilis ◽

Biofilm Formation ◽

Constitutive Expression ◽

Etiologic Agent ◽

Phase Variable ◽

Wild Type ◽

Bladder Tissue ◽

Tract Infections

ABSTRACT Proteus mirabilis, an etiologic agent of complicated urinary tract infections, expresses mannose-resistant Proteus-like (MR/P) fimbriae whose expression is phase variable. Here we examine the role of these fimbriae in biofilm formation and colonization of the urinary tract. The majority of wild-type P. mirabilis cells in transurethrally infected mice produced MR/P fimbriae. Mutants that were phase-locked for either constitutive expression (MR/P ON) or the inability to express MR/P fimbriae (MR/P OFF) were phenotypically distinct and swarmed at different rates. The number of P. mirabilis cells adhering to bladder tissue did not appear to be affected by MR/P fimbriation. However, the pattern of adherence to the bladder surface was strikingly different. MR/P OFF colonized the lamina propria underlying exfoliated uroepithelium, while MR/P ON colonized the luminal surfaces of bladder umbrella cells and not the exfoliated regions. Wild-type P. mirabilis was usually found colonizing intact uroepithelium, but it occasionally adhered to exfoliated areas. MR/P ON formed significantly more biofilm than either P. mirabilis HI4320 (P = 0.03) or MR/P OFF (P = 0.05). MR/P OFF was able to form a biofilm similar to that of the wild type. MR/P ON formed a three-dimensional biofilm structure as early as 18 h after the initiation of the biofilm, while MR/P OFF and the wild type did not. After 7 days, however, P. mirabilis HI4320 formed a 65-μm-thick biofilm, while the thickest MR/P ON and MR/P OFF biofilms were only 12 μm thick. We concluded that MR/P fimbriae are expressed by most P. mirabilis cells infecting the urinary tract, dictate the localization of bacteria in the bladder, and contribute to biofilm formation.

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An iron-regulated outer-membrane protein of Proteus mirabilis is a haem receptor that plays an important role in urinary tract infection and in in vivo growth

Journal of Medical Microbiology ◽

10.1099/jmm.0.47320-0 ◽

2007 ◽

Vol 56 (12) ◽

pp. 1600-1607 ◽

Cited By ~ 15

Author(s):

Analía Lima ◽

Pablo Zunino ◽

Bruno D'Alessandro ◽

Claudia Piccini

Keyword(s):

Urinary Tract Infection ◽

Urinary Tract ◽

Tract Infection ◽

Outer Membrane ◽

Proteus Mirabilis ◽

Outer Membrane Proteins ◽

Mammalian Host ◽

Tract Infections

Proteus mirabilis, a common cause of urinary tract infections, expresses iron-regulated outer-membrane proteins (OMPs) in response to iron restriction. It has been suggested that a 64 kDa OMP is involved in haemoprotein uptake and that this might have a role in pathogenesis. In order to confirm this hypothesis, this study generated a P. mirabilis mutant strain (P7) that did not express the 64 kDa OMP, by insertion of the TnphoA transposon. The nucleotide sequence of the interrupted gene revealed that it corresponded to a haemin receptor precursor. Moreover, in vitro growth assays showed that the mutant was unable to grow using haemoglobin and haemin as unique iron sources. The authors also carried out in vivo growth and infectivity assays and demonstrated that P7 was not able to survive in an in vivo model and was less efficient than wild-type strain Pr 6515 in colonizing the urinary tract. These results confirmed that the P. mirabilis 64 kDa iron-regulated OMP is a haem receptor that has an important role for survival and multiplication of these bacteria in the mammalian host and in the development of urinary tract infection.

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Proteus mirabilis isolates of different origins do not show correlation with virulence attributes and can colonize the urinary tract of mice

Microbiology ◽

10.1099/mic.0.28846-0 ◽

2006 ◽

Vol 152 (7) ◽

pp. 2149-2157 ◽

Cited By ~ 19

Author(s):

Vanessa Sosa ◽

Geraldine Schlapp ◽

Pablo Zunino

Keyword(s):

Urinary Tract ◽

Virulence Factors ◽

Proteus Mirabilis ◽

Protein Patterns ◽

Wide Range ◽

Urease Production ◽

Tract Infections ◽

Different Origins

Proteus mirabilis has been described as an aetiological agent in a wide range of infections, playing an important role in urinary tract infections (UTIs). In this study, a collection of P. mirabilis isolates obtained from clinical and non-clinical sources was analysed in order to determine a possible correlation between origin, virulence factors and in vivo infectivity. Isolates were characterized in vitro, assessing several virulence properties that had been previously associated with P. mirabilis uropathogenicity. Swarming motility, urease production, growth in urine, outer-membrane protein patterns, ability to grow in the presence of different iron sources, haemolysin and haemagglutinin production, and the presence and expression of diverse fimbrial genes, were analysed. In order to evaluate the infectivity of the different isolates, the experimental ascending UTI model in mice was used. Additionally, the Dienes test and the enterobacterial repetitive intergenic consensus (ERIC)-PCR assay were performed to assess the genetic diversity of the isolates. The results of the present study did not show any correlation between distribution of the diverse potential urovirulence factors and isolate source. No significant correlation was observed between infectivity and the origin of the isolates, since they all similarly colonized the urinary tract of the challenged mice. Finally, all isolates showed unique ERIC-PCR patterns, indicating that the isolates were genetically diverse. The results obtained in this study suggest that the source of P. mirabilis strains cannot be correlated with pathogenic attributes, and that the distribution of virulence factors between isolates of different origins may correspond to the opportunistic nature of the organism.

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Proteus mirabilis Genes That Contribute to Pathogenesis of Urinary Tract Infection: Identification of 25 Signature-Tagged Mutants Attenuated at Least 100-Fold

Infection and Immunity ◽

10.1128/iai.72.5.2922-2938.2004 ◽

2004 ◽

Vol 72 (5) ◽

pp. 2922-2938 ◽

Cited By ~ 129

Author(s):

Laurel S. Burall ◽

Janette M. Harro ◽

Xin Li ◽

C.Virginia Lockatell ◽

Stephanie D. Himpsl ◽

...

Keyword(s):

Urinary Tract ◽

Mouse Model ◽

Virulence Factors ◽

Proteus Mirabilis ◽

Parent Strain ◽

Iron Acquisition ◽

Phosphate Transport ◽

Transposon Mutants ◽

Cell Surface Structure ◽

Tract Infections

ABSTRACT Proteus mirabilis, a common cause of urinary tract infections (UTI) in individuals with functional or structural abnormalities or with long-term catheterization, forms bladder and kidney stones as a consequence of urease-mediated urea hydrolysis. Known virulence factors, besides urease, are hemolysin, fimbriae, metalloproteases, and flagella. In this study we utilized the CBA mouse model of ascending UTI to evaluate the colonization of mutants of P. mirabilis HI4320 that were generated by signature-tagged mutagenesis. By performing primary screening of 2,088 P. mirabilis transposon mutants, we identified 502 mutants that ranged from slightly attenuated to unrecoverable. Secondary screening of these mutants revealed that 114 transposon mutants were reproducibly attenuated. Cochallenge of 84 of these single mutants with the parent strain in the mouse model resulted in identification of 37 consistently out-competed P. mirabilis transposon mutants, 25 of which were out-competed >100-fold for colonization of the bladder and/or kidneys by the parent strain. We determined the sequence flanking the site of transposon insertion in 29 attenuated mutants and identified genes affecting motility, iron acquisition, transcriptional regulation, phosphate transport, urease activity, cell surface structure, and key metabolic pathways as requirements for P. mirabilis infection of the urinary tract. Two mutations localized to a ∼42-kb plasmid present in the parent strain, suggesting that the plasmid is important for colonization. Isolation of disrupted genes encoding proteins with homologies to known bacterial virulence factors, especially the urease accessory protein UreF and the disulfide formation protein DsbA, showed that the CBA mouse model and mutant pools are a reliable source of attenuated mutants with mutations in virulence genes.

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1675. Epidemiology of Urinary Tract Infections in the United States, 2009 - 2018

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1853 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S823-S823

Author(s):

Kendra Foster ◽

Linnea A Polgreen ◽

Brett Faine ◽

Philip M Polgreen

Keyword(s):

United States ◽

Urinary Tract ◽

Klebsiella Pneumoniae ◽

Urinary Tract Infections ◽

Proteus Mirabilis ◽

Antibiotic Use ◽

The United States ◽

Drug Resistant ◽

E Coli ◽

Tract Infections

Abstract Background Urinary tract infections (UTIs) are one of the most common bacterial infections. There is a lack of large epidemiologic studies evaluating the etiologies of UTIs in the United States. This study aimed to determine the prevalence of different UTI-causing organisms and their antimicrobial susceptibility profiles among patients being treated in a hospital setting. Methods We used the Premier Healthcare Database. Patients with a primary diagnosis code of cystitis, pyelonephritis, or urinary tract infection and had a urine culture from 2009- 2018 were included in the study. Both inpatients and patients who were only treated in the emergency department (ED) were included. We calculated descriptive statistics for uropathogens and their susceptibilities. Multi-drug-resistant pathogens are defined as pathogens resistant to 3 or more antibiotics. Resistance patterns are also described for specific drug classes, like resistance to fluoroquinolones. We also evaluated antibiotic use in this patient population and how antibiotic use varied during the hospitalization. Results There were 640,285 individuals who met the inclusion criteria. Females make up 82% of the study population and 45% were age 65 or older. The most common uropathogen was Escherichia Coli (64.9%) followed by Klebsiella pneumoniae (8.3%), and Proteus mirabilis (5.7%). 22.2% of patients were infected with a multi-drug-resistant pathogen. We found that E. Coli was multi-drug resistant 23.8% of the time; Klebsiella pneumoniae was multi-drug resistant 7.4%; and Proteus mirabilis was multi-drug resistant 2.8%. The most common antibiotics prescribed were ceftriaxone, levofloxacin, and ciprofloxacin. Among patients that were prescribed ceftriaxone, 31.7% of them switched to a different antibiotic during their hospitalization. Patients that were prescribed levofloxacin and ciprofloxacin switched to a different antibiotic 42.8% and 41.5% of the time, respectively. Conclusion E. Coli showed significant multidrug resistance in this population of UTI patients that were hospitalized or treated within the ED, and antibiotic switching is common. Disclosures All Authors: No reported disclosures

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Urinary Tract Infections Caused by Multi-Drug Resistant Proteus mirabilis: Risk Factors and Clinical Outcomes

Infection ◽

10.1007/s15010-009-8460-5 ◽

2009 ◽

Vol 38 (1) ◽

pp. 41-46 ◽

Cited By ~ 38

Author(s):

K. Cohen-Nahum ◽

L. Saidel-Odes ◽

K. Riesenberg ◽

F. Schlaeffer ◽

A. Borer

Keyword(s):

Risk Factors ◽

Urinary Tract ◽

Clinical Outcomes ◽

Urinary Tract Infections ◽

Proteus Mirabilis ◽

Drug Resistant ◽

Tract Infections

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New Aspects of RpoE in Uropathogenic Proteus mirabilis

Infection and Immunity ◽

10.1128/iai.02232-14 ◽

2014 ◽

Vol 83 (3) ◽

pp. 966-977 ◽

Cited By ~ 10

Author(s):

Ming-Che Liu ◽

Kuan-Ting Kuo ◽

Hsiung-Fei Chien ◽

Yi-Lin Tsai ◽

Shwu-Jen Liaw

Keyword(s):

Urinary Tract ◽

Virulence Factors ◽

Stress Responses ◽

Proteus Mirabilis ◽

Immune Cell ◽

Polymyxin B ◽

Urothelial Cells ◽

Cationic Antimicrobial Peptide ◽

Content Type ◽

Tract Infections

Proteus mirabilisis a common human pathogen causing recurrent or persistent urinary tract infections (UTIs). The underlying mechanisms forP. mirabilisto establish UTIs are not fully elucidated. In this study, we showed that loss of the sigma factor E (RpoE), mediating extracytoplasmic stress responses, decreased fimbria expression, survival in macrophages, cell invasion, and colonization in mice but increased the interleukin-8 (IL-8) expression of urothelial cells and swarming motility. This is the first study to demonstrate that RpoE modulated expression of MR/P fimbriae by regulatingmrpI, a gene encoding a recombinase controlling the orientation of MR/P fimbria promoter. By real-time reverse transcription-PCR, we found that the IL-8 mRNA amount of urothelial cells was induced significantly by lipopolysaccharides extracted fromrpoEmutant but not from the wild type. These RpoE-associated virulence factors should be coordinately expressed to enhance the fitness ofP. mirabilisin the host, including the avoidance of immune attacks. Accordingly,rpoEmutant-infected mice displayed more immune cell infiltration in bladders and kidneys during early stages of infection, and therpoEmutant had a dramatically impaired ability of colonization. Moreover, it is noteworthy that urea (the major component in urine) and polymyxin B (a cationic antimicrobial peptide) can induce expression ofrpoEby the reporter assay, suggesting that RpoE might be activated in the urinary tract. Altogether, our results indicate that RpoE is important in sensing environmental cues of the urinary tract and subsequently triggering the expression of virulence factors, which are associated with the fitness ofP. mirabilis, to build up a UTI.

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Interaction of Proteus mirabilis Urease Apoenzyme and Accessory Proteins Identified with Yeast Two-Hybrid Technology

Journal of Bacteriology ◽

10.1128/jb.183.4.1423-1433.2001 ◽

2001 ◽

Vol 183 (4) ◽

pp. 1423-1433 ◽

Cited By ~ 28

Author(s):

Susan R. Heimer ◽

Harry L. T. Mobley

Keyword(s):

Proteus Mirabilis ◽

Accessory Proteins ◽

Structural Protein ◽

Yeast Two Hybrid ◽

Nickel Ions ◽

Catalytically Active ◽

Tract Infections ◽

Two Hybrid

ABSTRACT Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)3. To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.

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