Mobley Research Laboratory: Department of Microbiology & Immunology: University of Michigan Medical School
 

Proteus mirabillis

Specific Studies in the Mobley Laboratory

1. Proteus mirabilis genes that contribute to the pathogenesis of urinary tract infection: Identification of 25 signature-tagged mutants attenuated at least 100-fold.

To identify new virulence determinants that do not have obvious phenotypes (as do urease, hemolysin, fimbria, metalloproteases, and flagella), our study utilized the CBA mouse model of ascending urinary tract infection to evaluate the colonization of mutants of P. mirabilis HI4320 that were generated by signature-tagged mutagenesis. Primary screening of 2088 P. mirabilis transposon mutants identified 502 mutants ranging from slightly attenuated to unrecoverable. Secondary screening of these mutants defined 114 transposon mutants as reproducibly attenuated. Co-challenge of 84 of these single mutants with the parent strain in the mouse model identified 37 consistently out-competed P. mirabilis transposon mutants, 25 of which that were out-competed >100-fold for colonization of the bladder and/or kidneys by the parent strain (Table 1). 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 mutants localized to a 36 kb plasmid resident in this strain, suggesting its importance to 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, demonstrates the CBA mouse model and mutant pools to be a reliable source of attenuated mutants in virulence genes.

Burall*, Laurel S., Janette M. Harro*, Xin Li, C.Virginia Lockatell, Stephanie D. Himpsl, J. Richard Hebel, David E. Johnson, and Harry L.T. Mobley. 2004. Proteus mirabilis Genes That Contribute to the Pathogenesis of Urinary Tract Infection: Identification of 25 Signature-Tagged Mutants Attenuated at least 100-fold. Infect. Immun. 72:2922-2938. [*contributed equally]

Since the publication of these signature tagged mutagenesis results, we have screened another pool of 1880 mutants. When analyzed using all criteria described above, 24 additional mutants were found to be significantly attenuated. Nine of these mutants have been identified including fliF, exbD, cyaA, guaB, metN, yagY, edd, aceE, sdhC. Seven additional genes lack homologs.

Table 1. Homologs of disrupted genes in attenuated P. mirabilis STM mutants.

Gene Function STM Mutants Attenuated in Co-challenge Ratio of Mutant/Wild-type (P Value) Closest Homolog Description and Species Homolog present in E. coli CFT073
Urease B6-40 2.6x10 -5 (<0.001) UreG P. mirabilis urease accessory protein UreG -
Motility E6-43 7.3x10 -6 (<0.001) CheW Y. pestis positive regulator of CheA; chemotaxis +
Periplasmic Protein Folding F4-37 6.2x10 -6 (<0.001) DsbA Y. pestis thiol:disulfide interchange protein +
  G4-2 5.6x10 -1 (.038) HasR Pasteurella multocida membrane-bound heme receptor +
Iron A1-8 1.9x10 -2 (<0.005) NrpG P. mirabilis nrp operon; non-ribosomal peptide synthesis; phosphopantetheinyl transferase +
Acquisition H3-12 9.2x10 -3 (<0.0001)   Yersinia pestis putative iron transport permease -
  D6-33 1.5x10 -6 (<0.001)   Y. pestis putative outer membrane protein +
  C6-9 9.0x10 -2 (.024) SocE Myxococcusxanthus stringent response regulator +
  D5-9 3.0x10 -2 (.025) HdfR Y. pestis regulator of FlhDC (master flagellar operon) +
DNA-binding/ C1-18 1.4x10 -1 (.012) HdfR Y. pestis regulator of FlhDC (master flagellar operon) +
Regulatory Proteins E5-36 1.4x10 -5 (<0.001)   Y. pestis two-component system sensor kinase +
  E6-41 6.9x10 -6 (<0.001) AsnC Y. pestis regulatory protein for asnA and gidA +
  F1-29 1.9x10 -6 (<0.001) DusB conserved hypothetical protein +
Plasmid C6-15 6.9x10 -5 (<0.0001) ParE Nitrosomonas europaea plasmid stabilization factor -
Encoded C4-41 6.0x10 -4 (.019) PilX3 E. coli conjugative plasmid transfer -
  B6-23 8.7x10 -3 (<.0001) MrcA S. typhimurium peptidoglycan synthetase +
  B4-2 1.4x10 -2 (.006)   Y. pestis membrane heat shock protein +
Cell Envelope D6-26 2.8x10 -6 (.001)   Y. pestis putative glycosylase +
  F6-34 8.4x10 -6 (<.001)   Y. pestis putative exported protein +
  A3-39 2.0x10 -5 (<.001)   Y. pestis Putative exported protein +
Transport G1-43 1.4x10 -5 (.006) PstA Y. pestis phosphate transport system permease +
  H4-34 6.4x10 -6 (<.001) PstS Y. pestis phosphate-binding periplasmic protein +
  B2-9 7.7x10 -1 (.028) HemY E. coli late step of protoheme IX synthesis weak
  C4-2 1.5x10 -1 (.001)   P. falciparum putative chromosomal maintenance weak
Metabolic D4-8 1.1x10 -1 (.001) CarA E. coli carbamoyl phosphate synthetase +
  G4-12 6.1x10 -3 (.022) SerC Y. enterocolitica 3-phosphoserine aminotransferase +
  D6-39 4.5x10 -3 (.001)  NrdD Y. pestis anaerobic ribonucleoside triphosphate reductase +
  G1-38 4.6x10 -5 (<.001) AroG  Phospho-2-dehydro-3-deoxyheptonate aldolase +
Hypothetical B2-22 1.9x10 -1 (<.0001)   Y. pestis hypothetical gene -
  G5-30 5.5x10 -6 (<.001)l   Y. pestis hypothetical protein +
  A5-34 6.6x10 -6 (.005)   Limited Sequence- No Hits N/A
Unknown B4-38 1.5x10 -2 (.002)   Limited Sequence- No Hits N/A
  D1-30 1.0x10-5 (<.001)   Limited Sequence- No Hits N/A
  D2-41 9.6x10 -6 (.022)   Limited Sequence- No Hits N/A

2. Identification of MrpI as the sole recombinase that regulates the phase variation of MR/P fimbria, a bladder colonization factor of uropathogenic Proteus mirabilis.

The expression of mannose-resistant/Proteus-like (MR/P) fimbria is phase-variable due to the inversion of a 251-bp DNA fragment that carries the promoter for the mrp operon. Previous studies have shown that mrpI, which is transcribed divergently from the mrp operon, encodes a recombinase capable of switching the orientation of this invertible element (Fig. 1). In this study, we constructed isogenic mrpI null mutants from clinical isolate P. mirabilis HI4320. A PCR-based invertible element assay revealed that the isogenic mrpI null mutants were locked in one phase, either expressing (locked-on) or not expressing (locked-off) MR/P fimbriae, which indicated that MrpI was the sole recombinase that regulated the phase variation of MR/P fimbria. The locked-on and the locked-off mutants were evaluated for virulence in the CBA mouse model of ascending urinary tract infection by cochallenges with each other and with the wild type strain. Results from these experiments conclusively demonstrated that the MR/P fimbria was a critical bladder colonization factor of uropathogenic P. mirabilis and also suggested that the ability to switch off the expression of MR/P fimbria might be important for kidney colonization.

Fig. 1.  A schematic drawing of the invertible element assay. It illustrates the mrp gene cluster, which includes the invertible element (IE), the transcript mrpI and the mrp operon (mrpABCDEFGHJ), the location of primers P1, P2, P3 and P4 and the AflII site, and the sizes of DNA fragments after the AflII digestion.

Li, X., V. Lockatell, D.E. Johnson, and H.L.T. Mobley. 2002. Identification of MrpI as the sole recombinase that regulates the phase variation of MR/P fimbria, a bladder colonization factor of uropathogenic Proteus mirabilis. Molec. Microbiol. 45:865-874.

3. Coordinated control of bacterial motility and adhesion by a novel fimbrial gene.

The mannose-resistant, Proteus-like (MR/P) fimbria and flagellum are among virulence factors of Proteus mirabilis that contribute to its colonization in a murine model of ascending urinary tract infection. mrpJ, the last of nine genes of the mrp operon, encodes a 107-amino acid residue protein that contains a putative helix-turn-helix domain and inhibits flagellar synthesis and swarming (Fig. 2). Using transcriptional lacZ fusions integrated into the chromosome and mutagenesis studies, we demonstrate that MrpJ actively represses transcription of the flagellar regulon and thus reduces flagella synthesis when MR/P fimbriae are produced. This coordinated control of bacterial motility and adhesion by MrpJ is required for a successful colonization by P. mirabilis in the murine model. [Our data also indicate that PapX encoded by a pap (pyelonephritis-associated pilus) operon of uropathogenic Escherichia coli is a functional homolog of MrpJ, suggesting that such a negative feedback on flagella synthesis may be conserved among fimbriae assembled through the chaperone-usher pathway.]

Figure 2. Elevated expression of MrpJ in P.mirabilis inhibits motility due to reduced flagella production. The three strains assayed here are P.mirabilis HI4320 transformed with pLX3607 (vector), pLX3805 (+MrpJ) and pLX5401 (DeltamrpJ). (A) The three strains were assayed for swarming on 1.5% Luria agar and for swimming in 0.35% Luria agar. (B) Three overnight Luria broth cultures of each of the three strains were adjusted to the same optical density, and equal volumes processed for SDS−PAGE and subsequent western blot analyses with antiserum against MrpJ or P.mirabilis flagella (FlaA). A Coomassie Blue-stained gel confirmed that equivalent amounts of protein were loaded to each lane (data not shown). (C) Two-fold serial dilutions of protein samples were assayed by western blot analysis to assess the difference in flagella production between the two strains.

Li, X., D.A. Rasko, V. Lockatell, D.E. Johnson, and H.L.T. Mobley. 2001. Repression of bacterial motility by a novel fimbrial gene. EMBO J. 20:4854-4862.

4. Visualization of Proteus mirabilis within the matrix of urease-induced bladder stones during experimental urinary tract infection.

Urinary stone formation (urolithiasis) is the hallmark of infection by P. mirabilis (Fig. 3). When tested in the CBA mouse model of ascending urinary tract infection, the urease-negative mutant was unable to colonize bladder or kidney tissue, or to cause urolithiasis or death. In this study, the urease-negative mutant was tested in long-term catheterized CBA mice, a clinically more relevant animal model. The long-term catheterized mice were more susceptible to infection by the wild type P. mirabilis than the uncatheterized mice. The urease-negative mutant, at an inoculum size of 10 9 CFU/mouse, was able to colonize bladder to a level similar to the wild type at an inoculum size of 10 6CFU/mouse. Despite the successful colonization in bladder, the urease-negative mutant did not cause urolithiasis or ascend to colonize the kidneys. Using confocal laser scanning microscopy and scanning electron microscopy, we have demonstrated the presence of P. mirabilis in urease-induced stone matrix. An alizarin red S-based stain method was developed to detect calcium-containing deposits in bladder and kidney tissues of P. mirabilis infected mice.

Fig. 3. Scanning electron micrographs of P. mirabilis urease-induced bladder stone. (A) One-quarter of the bladder viewed at a low magnification (bar, 500 µm). The orientation of the bladder is indicated by an arrow pointing to the inferior end of the bladder (the end leading to the urethra). (B) Higher magnification (bar, 100 µm) of the area enclosed in a box in panel A. (C) Higher magnification (bar, 5 µm) of the area enclosed in a box in panel B. (D and E) Representative views of the bladder stone (bars, 2 µm).

Li, X, H. Zhao, C.V. Lockatell, C.B. Drachenberg, D.E. Johnson, and H.L.T. Mobley. 2002. Visualization of Proteus mirabilis within the matrix of urease-induced bladder stones during experimental urinary tract infection. Infect. Immun. 70:389-394.

5. Swarmer cells are rarely observed in ascending urinary tract infection.

Proteus mirabilis colonizes the bladder and ascends the ureters to the proximal tubules of the kidneys, leading to the development of acute pyelonephritis. P. mirabilis is capable of swarming, a form of multicellular behavior in which bacteria differentiate from the short rod, typical of the Enterobacteriaceae, into hyperflagellated elongated bacteria capable of rapid and coordinated population migration across surfaces. In vitro evidence shows a correlation between the swarming morphotype and upregulation of certain virulence factors. There has been considerable debate as to which morphotype predominates during urinary tract infection. P. mirabilis (pBAC001), which expresses green fluorescent protein in both swimming and swarming morphotypes, was constructed to quantify the prevalence of each morphotype in ascending urinary tract infection (Fig. 4). Transurethral inoculation of P. mirabilis (pBAC001) resulted in ascending urinary tract infection and kidney pathology in mice examined both two and four days post-inoculation. Using confocal microscopy we were able to investigate the morphotypes of the bacteria in the urinary tract. Of 5087 bacteria measured in bladder, ureters, and kidneys only 7 (0.08%) were identified as swarmers. MR/P fimbriae expression, which correlates with the swimmer phenotype, was prevalent in the ureters and bladder. We conclude that, by far, the predominant morphotype present in the urinary tract during ascending infection is the short rod – the swimmer cell.

Fig. 4. GFP-expressing bacteria colonize the urinary tract. Confocal micrographs showing typical fields (seen with a 63x lens objective) of bladder (A) and kidney parenchyma (B and D) sections and pus in kidney (C). Host cells and actin are red, and host cell nuclei are blue. GFP-expressing bacteria are green. e, epithelium; l, lumen; c, capillary; t, tubule; p, polymorphonuclear cell; a, actin. Bars, 10 µm.

Jansen, A.M., C. Virginia Lockatell, D.E. Johnson, and H.L.T. Mobley. 2003. Visualization of Proteus mirabilis morphotypes in the urinary tract: The elongated swarmer cell is rarely observed in ascending urinary tract infection. Infect Immun. 71:3607-3613.

Jansen, A.M., V. Lockatell, D.E. Johnson, and H.L.T. Mobley. 2004. 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. Infect. Immun. 72:7294-7305. (Appendix 6)

6. Development of an intranasal vaccine to prevent urinary tract Infection by Proteus mirabilis.

Proteus mirabilis commonly infects the complicated urinary tract and is associated with urolithiasis. Stone formation is caused by bacterial urease, which hydrolyzes urea to ammonia, causing local pH to rise with subsequent precipitation of magnesium ammonium phosphate (struvite) and calcium phosphate (apatite) crystals.

To prevent these infections, we vaccinated CBA/J mice with formalin-killed bacteria or purified MR/P fimbriae, a surface antigen expressed by P. mirabilis during experimental urinary tract infection, via four routes of immunization: subcutaneous, intranasal, transurethral, and oral. We assessed the efficacy of vaccination using the CBA/J mouse model of ascending urinary tract infection. Subcutaneous or intranasal immunization with formalin-killed bacteria and intranasal or transurethral immunization with purified MR/P fimbriae significantly protected CBA mice from ascending urinary tract infection by P. mirabilis (p < 0.05). To investigate the potential of MrpH, the MR/P fimbrial tip adhesin, as a vaccine, the mature MrpH peptide (H: residues 23-275, excluding the signal peptide) and the N-terminal receptor-binding domain of MrpH (H’:residues 23-157) were overexpressed as C-terminal fusions to maltose-binding protein (MBP), and purified on amylose resins. Intranasal immunization of CBA mice with MBP-MrpH (23-157) conferred effective protection against urinary tract infection by P. mirabilis (p<.002) when compared to naïve (N) unvaccinated controls (Fig.).

Li, Xin, C. Virginia, Lockatell, David E. Johnson, M. Chelsea Lane, John W. Warren, and Harry L.T. Mobley. 2004. Development of an intranasal vaccine to prevent urinary tract infection by Proteus mirabilis. Infect. Immun. 72:66-75 .

Li, X. and H.L.T. Mobley. 2002. Vaccines for Proteus mirabilis in urinary tract infection. Int. J. Antimicrob. Ag. 19:461-465.

7. Use of a translational fusion of the mrpH fimbrial adhesin binding domain with cholera toxin A2 domain, coexpressed with cholera toxin B subunit, as an intranasal vaccine to prevent experimental urinary tract infection by Proteus mirabilis.

MrpH is the tip adhesin of MR/P fimbriae, which are expressed by P. mirabilis during experimental urinary tract infections. A previous study indicated that intranasal immunization with a recombinant maltose-binding protein fusion of the N-terminal receptor-binding domain of MrpH (residues 23-157) (MHT), covalently conjugated to cholera toxin (CT), protected mice from urinary tract infection by P. mirabilis. Here, we test whether chemical- or biological (translational fusion to cholera toxin A2 domain)-conjugation of the MrpH-adhesin-based vaccine to nontoxic cholera toxin B subunit is sufficient to induce a protective immune response in mice immunized via the intranasal route.

We demonstrate that MrpH 23-157-holotoxin-like chimera (HA2-B) induces protective immunity against transurethral challenge with P. mirabilis.

Li, Xin*, Jarrod Erbe*, C. Virginia Lockatell, David E. Johnson, Michael G. Jobling, Randall K. Holmes, and Harry L.T. Mobley. 2004. Use of a translational fusion of the MrpH fimbrial adhesin binding domain with cholera toxin A2 domain, coexpressed with cholera toxin B subunit, as an intranasal vaccine to prevent experimental urinary tract infection by Proteus mirabilis. Infect. Immun. 72:7306-7310. [*contributed equally]


8. Completion of P. mirabilis HI4320 genome sequence.
Sequencing of the genome of our type strain, P. mirabilis HI4320.

The strain that we isolated from a patient with catheter-associated bacteriuria in 1986 [64] and have used for 43 published studies was selected for genomic sequencing by the Sanger Institute. As of November 1, 2005, the genome sequence of P. mirabilis HI4320 (cultured by our laboratory from the urine of a nursing home patient with a long-term (>30 days) indwelling urinary catheter [64]) is complete and has been closed. The genome is 4.063 Mb with a G+C content of 38.88%. There is a single plasmid of 36,289 nucleotides. Preliminary annotation of the genome identified 3719 coding sequences and seven rRNA loci. Analysis of the sequence confirmed the presence of previously identified virulence determinants, including a 54 kb contiguous flagellar regulon and up to 17 types of fimbriae. Surprisingly, genes encoding a potential type III secretion system (TTSS) have been found on a low GC-content pathogenicity island (30.7% vs. 38.9% for the entire genome). This island contains 24 intact genes that appear to encode all the necessary components to assemble a TTSS needle complex, plus at least two putative secreted effector proteins and their chaperones. Furthermore, two genes with homologies to known TTSS regulators (the positive regulator virF from Shigella flexneri and the negative regulator exsD from Pseudomonas aeruginosa) have been identified in the HI4320 genome. The genomic organization and homology of this pathogenicity island is most similar to the TTSS regulon from S. flexneri. In addition to the virulence determinants mentioned above, the P. mirabilis HI4320 genome also possesses four tandem copies of the zapE metalloprotease gene, genes encoding six putative autotransporters, at least five iron uptake mechanisms, and two possible type IV secretion systems.