Bladder invasion and histopathology

Internalization of P. mirabilis by bladder epithelial cells has been directly demonstrated in vitro using a hemolysin (hpmA) mutant to avoid confounding from the effects of the cytolytic toxin. In these experiments, it was determined that P. mirabilis utilizes the AipA autotransporter for internalization into bladder cells (46, 55). In addition to internalization, P. mirabilis is capable of lysing bladder epithelial cells using a combination of the Proteus toxic agglutinin (Pta) and hemolysin (55).

In the murine model of ascending UTI, P. mirabilis invades bladder epithelial cells as early as 30 minutes post-inoculation, which may provide transient protection from the immune response and an intracellular niche for initial replication and survival (44, 50, 52, 56–58). However, intracellular bacteria are uncommonly observed at later times post-inoculation, and P. mirabilis appears to instead form large, extracellular clusters within the bladder lumen and adjacent to the urothelium after this initial invasion phase rather than establishing the intracellular communities that are characteristic of uropathogenic E. coli (44) (Fig. 6). Formation of these clusters requires urease activity and the mannose-resistant Proteus-like (MR/P) fimbriae, and provides protection from infiltrating neutrophils (44).

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Figure 6

P. mirabilis invades the urothelium, but infrequently forms intracellular bacterial communities (IBCs)

(A–D) Representative images of P. mirabilis (A and B) and uropathogenic E. coli (UPEC) (C and D) attachment and invasion. Bacteria (green), UPIIIa (red), and DNA (blue) show localization of the bacteria relative to the apical surface of the urothelium. Scale bars, 10 μm. (B and D) A regional view of the bladder section containing the 10 hpi IBC shown in A and C, respectively. Scale bars, 100 μm. L, bladder lumen. (E and F) Quantification of P. mirabilis (E) and UPEC (F) bladder invasion at 0.5 hpi following either ex vivo gentamicin treatment (Gent) or mock treatment (Mock) (n = 6–8). *P < 0.05. Figure adapted, with permission, from (44).

Bladder colonization by P. mirabilis most often results in mild to moderate cystitis in murine models of UTI, including transmural neutrophilic inflammation, epithelial transcytosis of neutrophils, and submucosal edema (25, 55). The pathology of P. mirabilis bladder colonization during ascending UTI is largely due to a combination of urease activity, Pta, and hemolysin (25, 55). In the murine model of CAUTI, infection with P. mirabilis results in more severe cystitis, and this appears to be largely due to urease activity and encrustation of the catheter (25).

ICEPm1

Comparative genomic hybridization of Providencia stuartii and Morganella morganii with a P. mirabilis HI4320 microarray revealed an integrative and conjugative element (ICE) common to all three species (107) (link ICE section). Further analysis of this 94 kb element, called ICEPm1, revealed genes encoding two previously-identified virulence factors: Proteus toxic agglutinin (Pta) and Nrp siderophore (108–110). Carried within ICEPm1 is the Yersinia spp. high pathogenicity island (HPI), which includes the nrp operon. Consistent with a virulence role, ICEPm1 is found more often in urinary isolates than colonizing isolates (100% vs. 65%) (107). Furthermore, ICEPm1 has been observed to transmit between clinical isolates in a laboratory setting (111). Examination of sequenced genomes reveals that ICEPm1, when present, is not always inserted at the same genetic locus.

P. mirabilis strains may encode other conjugative elements, but they do not appear to be as prevalent as ICEPm1. For example, HI4320 encodes another conjugative transposon, named as either ICEPmiUSA1 or ICEPmi HI4320, that is a member of the SXT/R391 family of ICEs (112–114). No genes in ICEPmiUSA1 have thus far been identified as virulence-associated. However, other ICEs in the SXT/R391 family carry multiple antibiotic resistance genes, and some of these ICEs are found in P. mirabilis (115–117).

T6SS

Because the type VI secretion system (T6SS) of P. mirabilis is involved in strain self-recognition and competition between strains (link secretion section), it is not surprising that there is a high degree of variability in these genes. Specifically, the genes encoding the T6SS secretion apparatus are highly conserved, but the secreted effectors vary in sequence, type, and number. For example, P. mirabilis HI4320 and BB2000 encode T6SS effector operons adjacent to, and in the reverse orientation from, the T6SS secretion apparatus genes (118, 119). In both cases, the effector operons begin with hcp and vgrG homologs, which are required for T6SS function (120). After that, however, the operons diverge in both sequence and number of genes; for example, BB2000’s idr operon comprises five genes while HI4320’s pef operon has nine (118, 119) (Fig. 12). Furthermore, HI4320 encodes at least two more operons identified as likely T6SS effectors than BB2000 is predicted to encode (112, 121). Examination of other P. mirabilis sequenced genomes indicates that this variability continues throughout the species (Fig. 10A).

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Figure 12

T6SS effector operons are highly variable

The T6SS effector operon immediately adjacent to the conserved T6SS secretion apparatus operon is depicted for P. mirabilis HI4320 (top) and BB2000 (bottom). The HI4320 locus, called pef for primary effector operon, comprises PMI0750-0758, and the BB2000 locus, called idr for identity recognition, comprises BB2000_0822-0826. The proteins encoded by the first gene (red) and the first 596 amino acids of the second gene (orange) are 99% identical, but the remaining ~130 amino acids of the encoded proteins share no homology. The rest of the operons are not homologous (black). A portion of IdrD shares identity with PMI0761, which is not part of the pef operon. The scale at the top is in nucleotides.

O-antigen

Like other gram-negative bacteria, P. mirabilis strains have diverse O-polysaccharides as part of their LPS; 76 O-serogroups have been described thus far for Proteus spp. (122). In fact, the O and H antigen designations for LPS and flagellar typing were originally named by Weil and Felix’s observation that swarming P. mirabilis had a breath-like appearance (German Hauch, breath) while non-swarming colonies did not (ohne Hauch, without breath) (95). In a survey of P. mirabilis strains with different O-serotypes, virulence factors were widespread (urease, Zap protease, swarming), but strains differed in the levels of these activities. Specifically, strains with negatively-charged O-polysaccharides displayed higher urease, protease, and swarming activities compared with positive or neutral O-polysaccharides (104). LPS is abundant on the cell surface of gram-negative bacteria, and its properties can affect bacterial interaction with their environment. Thus, some aspects of strain variability may be determined by LPS biochemistry.

Flagellin

Flagella (link flagella section) are targets of the innate immune system and also energetically expensive to produce (123). Correspondingly, although most flagellar genes are conserved in P. mirabilis, the gene encoding the whip itself is highly variable, as in other bacterial species. P. mirabilis also encodes a second, silent flagellar gene flaB, which can recombine with flaA and yield hybrid FlaAB flagella (124). The properties of FlaAB hybrids have been extensively reviewed elsewhere (19).

Phage

Like other bacteria, P. mirabilis genomes are peppered with integrated phage. HI4320 encodes three apparently complete and three degenerate prophages (112). CRISPR-Cas systems are encoded in approximately one-third of sequenced P. mirabilis genomes as determined by the Bacterial Bioinformatics Resource Center PATRIC (125); notably, CRISPR is not present in two strains commonly used for experimentation: HI4320 and BB2000 (112, 121).

Differential expression of phage genes has been observed in P. mirabilis during swarming (126, 127); specifically, one phage is inserted adjacent to the rcs operon, which encodes a regulator of flagella and swarming. Furthermore, phage have been used to transduce swarming ability into non-swarming strains (128). Thus, it is likely that phage account for some of the observed differences in swarming behavior between strains.

Microarrays

The four following studies (109, 126, 131, 132) were conducted using a microarray with 70-mer oligonucleotide probes designed to hybridize with each of the 3,719 predicted open reading frames from an early draft version of the P. mirabilis HI4320 genome (112).

Swarming microarray

Perhaps the most dramatic change P. mirabilis undergoes is the cyclic switch from elongated, hypermotile swarmer cells to short, nonmotile consolidated cells during culture on solid surfaces (19). This transformation was the target of the first P. mirabilis transcriptomic study, where (i) swarm and consolidate transcripts were shown to be more similar to each other than to RNA derived from broth culture, and (ii) while many genes are induced during consolidation, very few are upregulated during active swarming (126). The data were consistent with earlier reports of increased expression of certain virulence genes during swarming (133, 134), and swarmer cells being less metabolically active than consolidates (135). Curiously, although P. mirabilis is largely nonmotile during consolidation, flagellar transcripts were among the most highly-transcribed messages in both swarm and consolidate (126). Based on the accumulated transcription and metabolic data, the consolidation phase has been proposed to be a time of preparation for the next round of swarming, by increasing nutrient uptake systems, central metabolism, and respiration. As P. mirabilis swarming is very robust and occurs under conditions not permissive for other species of swarming bacteria, the preparation that occurs during consolidation phase may contribute to how P. mirabilis is able to swarm under a wider range of conditions that other species, including on solid surfaces such as hard agar and catheters.

Iron limitation microarray

Iron is sequestered by the host to combat bacterial infection, and pathogens activate iron acquisition systems during UTI (link metals section). P. mirabilis was previously thought to lack common iron scavenging systems in earlier studies. However, microarray analysis conducted on iron-restricted P. mirabilis revealed 21 iron-regulated systems, including two new acquisition systems which are detailed later in this chapter (109).

Ascending UTI microarray

A critical point for studying how P. mirabilis causes disease requires knowledge regarding the physiological state of this pathogen during UTI. To answer this question, urine was collected from experimentally infected mice at 1, 3, and 7 days post-inoculation (dpi) for RNA extraction and microarray hybridization (131). Iron acquisition, urease, and peptide transporters were induced during UTI, consistent with an iron-restricted, urea- and peptide-rich urine environment. The genes encoding MR/P fimbriae (link fimbriae section), which were previously shown to contribute to infection, were the most-highly upregulated of the transcriptome compared with broth culture (Fig. 13). However, some known virulence factors were either poorly expressed or downregulated during UTI, including genes encoding PMF fimbriae, Zap metalloprotease, hemolysin, and Proteus toxic agglutinin (Pta). This suggests that voided urine provides an important, but incomplete, snapshot of bacterial gene expression during UTI.

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Figure 13

P. mirabilis gene expression during experimental UTI

(A) Heat map of expression data for specific virulence-associated genes, depicting the ratio of expression in LB broth versus in vivo. The legend at the left indicates the color associated with log₂ fold change: red, upregulated in vivo; green, downregulated in vivo; black, not differentially regulated. (B) Adherence and motility genes are inversely regulated during UTI. Each line represents fold-change of a specific flagellar (left panel) or fimbrial (right panel) gene in vivo relative to mid-logarithmic phase culture in vitro. Genes in the mrp operon are highly induced early during infection, but expression falls by 7 days post infection. Flagellar genes are initially repressed, but expression increases late in infection. Figure adapted, with permission, from (131).

Interestingly, central metabolic pathways that were required during infection differed in several ways compared with a previous study of uropathogenic E. coli (136). Nitrogen assimilation gene expression suggested that P. mirabilis has greater access to nitrogen compared with E. coli, but follow-up experiments indicated this was not simply due to ammonia derived from urease activity (131). In contrast with E. coli, P. mirabilis induced glucose uptake and glycolysis genes, suggesting that P. mirabilis is able to access nutrient sources not available to E. coli (perhaps due to the activity of toxins on bladder epithelium). Gene expression at 1, 3, and 7 days post-inoculation was also compared. Flagellar genes were initially repressed, but this repression was relieved by day 7. In contrast, mrp genes were highly-transcribed throughout the infection but their degree of induction was lower at day 7; this topic is further detailed in the fimbrial section (link fimbriae section).

The transcriptional regulator MrpJ is encoded by the mrp fimbrial operon (link fimbriae section). Originally proposed as a switch between adherent and motile states (137), MrpJ was found to contribute to other phenotypes as well (138). Microarray experiments comparing an mrpJ in vivo mimic, where mrpJ is expressed in trans at levels comparable to those detected in mice during experimental UTI, with an empty vector control showed that MrpJ’s regulatory network extends considerably beyond repression of flagella to include, among others, regulation of additional fimbriae and the type VI secretion system (132). MrpJ and MrpJ paralogs are discussed later in this chapter (link).

Proteases

In an early study, it was found that 90–94% of 48 P. mirabilis strains secreted proteolytic enzymes, detected at pH 8 using gelatin as substrate. A correlation between the ability to swarm and protease secretion was noted. These proteases were all EDTA-sensitive metalloproteinases (204). However, the specific secretion systems involved in secretion of these proteases were not determined in this study.

Type III Secretion

Nucleotide sequencing of the genome of P. mirabilis HI4320 (112) revealed genes appearing to encode a type III secretion system on a low GC-content pathogenicity island (148). A cluster of 24 genes clearly have the potential to encode an intact T3SS with genes for an intact needle complex and at least two effector proteins and their chaperones. The genetic organization of the T3SS is similar to that of Shigella flexneri, there was no evidence of mutation of the genes resulting in inactivation of the system, and RT-PCR analysis demonstrated that these genes were expressed in P. mirabilis HI4320. However, mutation of two genes including a putative ATPase and negative regulator of T3S had no effect on the secreted protein profile when compared to the wild-type strain (148). Furthermore, in the murine model of ascending UTI, there was no difference in CFUs between the mutant and wild-type strain in urine, bladder, or kidneys during either independent infection or cochallenge. Thus, no pathogenicity phenotype for the T3SS was identified for P. mirabilis HI4320 during ascending UTI. However, two components of the T3SS (PMI2688 and PMI2696) were identified as significant fitness factors during polymicrobial CAUTI by Tn-Seq (129), indicating that the T3SS may be involved in mediating host-microbe interactions during polymicrobial infection.

Type V Secretion

There are three subclasses of type V secretion systems (Va, Vb, and Vc), and the nucleotide sequence of P. mirabilis HI4320 predicts members of all three (112, 205, 206).

Classical autotransporters (Va)

Classical autotransporters are a family of virulence proteins in Gram-negative pathogens that contain three domains: an amino-terminal leader peptide for export across the inner membrane via the Sec pathway, a surface-localizing passenger domain, and a carboxy-terminal domain for translocation across the outer membrane (Henderson 2004). One of the three predicted classical autotransporters encoded by P. mirabilis HI4320 has been studied in detail: Proteus toxic agglutinin (pta), encodes a serine protease that was previously identified as an immunogenic outer membrane protein (161) (link Toxins section). The 117-kDa protein has a 58-amino acid long signal peptide, a 75-kDa long N-terminal passenger domain, and a 30-kDa C-terminal translocator (110). The autotransported protease, either cell-associated or secreted, has cytotoxic effects on cultured bladder and kidney epithelial cells. Catalytic residues Ser366, His147, and Asp533, when mutated, abolish protease activity. The protein also has autoaggregation properties not associated with the proteolytic activity. Virulence of a protease mutant, as tested in the murine model of ascending UTI, was significantly reduced.

Two-Partner Secretion (Vb)

P. mirabilis hemolysin, encoded by hpmA, is exported by the protein product of hmpB via the type Vb two-partner secretion pathway. This requires sequential unfolding of the HpmA protein (207), for which a partial crystal structure has been solved (208). Two polypeptides, HpmB and HpmA, synthesized in that transcriptional order, are responsible for hemolysin activity of P. mirabilis (link Toxins section). A Fur-binding site upstream of hmpB overlapping the −35 site of the promoter indicates that expression is governed by iron concentration. HpmB and HpmA have 17- and 29-amino acid leader peptides, confirmed for purified HpmA. HpmB is necessary for secretion of HpmA. HpmB and HpmA are most similar with respect to amino acid sequence to the Serratia marcescens hemolysin proteins ShlB and ShlA, respectively (209).

Trimeric autotransporters (Vc)

P. mirabilis HI4320 encodes three putative trimeric autotransporter proteins (Pearson 2008). Two of these, AipA and TaaP, are annotated as “adhesin-like” and “agglutinating adhesin-like”. Based on their homology with other trimeric autotransporters, the two autotransporters would contain four antiparallel β-sheets and form homotrimers. Recombinant AipA and TaaP bind extracellular matrix proteins, produce polypeptides of 28-kDa and 78-kDa, respectively, and form high molecular weight homotrimers. A 51-amino acid invasin-like motif of AipA is necessary for function. Gly-247 in AipA and Gly708 in TaaP are required for trimerization and activity. AipA and TaaP confer an advantage at 7 dpi during cochallenge in the murine model of ascending UTI.

Hemolysin

Secreted pore-forming toxins are a common feature of pathogenic bacteria. In particular, hemolysins are secreted pore-forming toxins that insert into eukaryotic cell membranes, causing efflux of sodium ions and cell damage (213). Two hemolysins have been described for members of the Proteus genus (214), one that is calcium-dependent and similar to the α-hemolysin of E. coli (hlyA), and another that is calcium-independent. The calcium-independent hemolysin is encoded by two genes comprising a two-partner secretion system: hpmA, which produces a 166 kDa secreted exoprotein, and hpmB, which produces a 63 kDa translocase protein that is required for both the activation and secretion of HpmA through cleavage of an N-terminal peptide (209). HpmA is the predominant hemolysin in Proteus species and appears to be the only hemolysin encoded by P. mirabilis, present in 268/274 (98%) of P. mirabilis clinical and fecal isolates from Brazil and the United States while none of these isolates encoded hlyA (102, 103). Similarly, hpmA is present in all 7 complete P. mirabilis genome sequences available through NCBI as of May 2017. An N-terminal fragment of HpmA, called HpmA265, has been crystallized and its structure solved (Fig. 20) (208). This fragment lacks the C-terminal pore-forming domain, but can still activate full-length HpmA in the absence of HpmB when the proteins are mixed with erythrocytes. The formation of HpmA265 dimers suggests that full-length HpmA may also function as a dimer.

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Figure 20

HpmA265 crystallographic dry dimer interface leads to a filamentous appearance

Solid lines represent hydrogen bonds shared between β23 strands of both subunits. Reproduced from (208), with permission.

HpmA mediates lysis of a broad range of cell types from numerous host species and appears to be the primary P. mirabilis virulence factor responsible for cytotoxicity to human renal proximal tubular epithelial cells (HRPTECs) (47, 48, 50, 56). Deletion of hpmA dramatically decreases cytotoxicity, and allows for internalization of P. mirabilis by HRPTECs in vitro (47, 56). As HRPTECs form a protective barrier for the kidney parenchyma, it was hypothesized the hemolysin may be a critical virulence factor that mediates spread of P. mirabilis into the kidneys and development of pyelonephritis (28). However, deletion of hpmA does not appear to impact tissue colonization or damage during independent challenge in the murine model of ascending UTI (45, 48, 55), indicating that either hemolysin has less of an impact during experimental infection than the in vitro cell culture studies suggest, or the activity of other virulence factors mask its contribution in vivo. In agreement with this finding, hpmBA was not identified as a fitness factor for UTI or CAUTI in any of the genome-wide transposon mutagenesis studies (108, 129, 179, 180), likely due to a combination of complementation in trans by the other hemolysin-producing transposon mutants present during infection and possibly production of other cytolysins with similar functions.

Proteus toxic agglutinin

The genome of P. mirabilis strain HI4320 encodes six putative autotransporters (112) (link secretion section), of which only the Proteus toxic agglutinin (Pta) has been fully characterized. Pta was initially identified as an outer membrane surface-expressed protein recognized by the murine immune system (161), and determined to be encoded within the integrative and conjugative element ICEPm1 in P. mirabilis strain HI4320 (107) (link to ICEPm1).

Pta is a 120 kDa protein that is catalytically processed to become a ~110 kDa active protein within the outer membrane (110). Expression of this protein in E. coli promotes autoaggregation of the bacteria, as well as lysis of bladder epithelial cells in vitro. Both properties of Pta require translocation to the outer membrane and cytotoxicity requires serine protease activity from the passenger domain, but protease activity is not required for autoaggregation. Pta is also classified as a subtilisin-like alkaline protease owing to the observation that expression of pta is induced by alkaline pH and protease activity is maximal at pH 8.5–9.0, conditions that would be encountered by P. mirabilis during urinary tract infection due to its urease activity.

Pta contributes to colonization of the bladder and kidneys in a murine cochallenge model of ascending UTI, as well as dissemination to the spleen (110). Interestingly, HpmA and Pta have an additive effect on cytotoxicity both in vitro and during experimental UTI, particularly with respect to cystitis and possibly interstitial nephritis (55). However, Pta appears to be the more potent toxin during experimental infection, as disruption of pta has a much greater impact on infectivity than loss of hpmA. Similar to hpmA, pta is present in all seven of the complete P. mirabilis genome sequences in NCBI as of May 2017, and was also encoded in eight P. mirabilis infection and fecal isolates (55).

Mannose-resistant Proteus-like fimbriae (MR/P)

These are the most-extensively studied of the fimbriae encoded by P. mirabilis. MR/P fimbriae were originally named for their ability to agglutinate untreated erythrocytes in the presence of mannose, a trait which was frequently found in P. mirabilis isolates (229) (Fig. 23). This was in contrast to mannose-sensitive agglutination, e.g., type 1 fimbriae from E. coli, or mannose-resistant Klebsiella-like (MR/K) agglutination, which occurred in the presence of tannic acid-treated bovine erythrocytes (254). MrpA is the major structural subunit of the fimbria, and MrpH is a two-domain adhesin which is located at the tip of MR/P fimbriae. Extensive details on MR/P structure and assembly have been recently reviewed elsewhere (19).

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Figure 23

Mannose-resistant hemagglutination (MRHA) patterns of MR/P fimbriae expressed in E. coli

E. coli DH5α expressing the entire mrp operon under its native promoter (mrpA-J) (pXL4206), mrp minus mrpH and mrpJ (“ΔmrpH”; pXL4401), ΔmrpH plus empty vector (pON-184), or ΔmrpH plus complemented mrpH (pXL8906) were cultured in Luria broth at 37°C and mixed with chicken erythrocytes. MRHA only occurs when MR/P tip adhesin MrpH is present. Figure adapted, with permission, from (156).

Expression

The mrp gene cluster consists of two transcripts. Genes required for fimbrial structure and assembly are encoded by mrpABCDEFGHJ (156). The last gene, mrpJ, encodes a transcriptional regulator and is discussed later. The second transcript comprises a single gene, mrpI, which is divergently transcribed from the rest of the mrp genes (241). MrpI is a recombinase that flips a 252 bp invertible element containing the mrpABCDEFGHJ promoter (241). The transcriptional start site lies within the invertible element (132), and thus the orientation of this element dictates whether or not mrp fimbrial genes are transcribed. MrpI is the sole recombinase for the invertible element, and therefore mutations in mrpI result in bacteria that either constitutively produce MR/P fimbriae (“locked on”) or are devoid of MR/P fimbriae (“locked off”) (158). The orientation of the element may be easily detected using PCR-based assays (Fig. 24A), and phase variation of MR/P fimbriae has been observed at the single cell level (Fig. 24B).

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Figure 24

Phase variation of P. mirabilis MR/P fimbriae

(A) Urine, bladder and kidney samples were collected at 7 dpi from mice infected with wild-type P. mirabilis HI4320, an MR/P locked-off mutant or an MR/P locked-on mutant and subjected to the invertible element assay. (B) Electron micrograph showing the phase variation of MR/P fimbrial expression in a broth culture of P. mirabilis. MrpH, the tip adhesin of MR/P fimbriae, is immunogold labeled. Note that the top left bacterium is gold labeled, while the top right bacterium is unlabeled. Scale bar, 500 nm. (C) Correlation between MR/P fimbrial expression and bacterial colonization in the bladder. Bladders from mice challenged with P. mirabilis HI4320 were collected at 7 dpi and bacteria were both quantitatively cultured and subjected to the IE assay. A positive correlation was found between MR/P fimbrial expression (percentage of IE in the ON orientation; y axis) and bacterial colonization in the bladder (log₁₀ CFU/g tissue; x-axis): y = 17× − 30, r² = 0.9, n = 18, P < 0.0001. Figure adapted, with permission, from (158) (A) and (152) (B and C).

The nine genes in the mrp operon are the most highly induced genes by bacteria in urine collected from experimentally-infected mice compared with in vitro broth culture (131). Correspondingly, even when mice are infected with P. mirabilis cultured so the population is almost completely phase off, after seven days, the majority of the bacteria have the invertible element in the on orientation (241). MR/P fimbriae are produced at a relatively low level in vitro, with the highest amounts produced during 48 h static culture in broth or culture under 5% oxygenation, followed by aerated broth, and finally, growth on an agar surface (152, 249, 255). Expression of mrp genes is both induced and subject to positive selection in a 5% oxygen atmosphere, which is logical for a virulence factor given reduced oxygen availability in the bladder (152). That is, an mrp locked on mutant outcompetes locked off in broth, and, at the same time, mrpA expression increases during oxygen limitation (152). Transcription of the recombinase mrpI may also be regulated by oxygen levels (152). Furthermore, orientation of the invertible element is influenced by the bacterium’s ability to assemble fimbriae. Assembly requires disulfide bond formation, and mutation of the gene encoding thiol:disulfide interchange protein DsbA leads to an increase in element-off bacteria (152).

In vitro function

In addition to hemagglutination, MR/P fimbriae mediate in vitro phenotypes that give clues to their function during infection. Biofilm formation (link biofilm section), measured in plastic multi-well plates, on glass coverslips, as pellicles in culture tubes, or in static cultures in glass tubing, is dependent on MR/P fimbriae (156, 244, 256). Interestingly, phase variation contributes to biofilm development. MR/P locked on bacteria establish biofilms more rapidly than wild-type, but over time the wild-type biofilm is much thicker (65 μm vs 12 μm) and has a classic structured channel appearance (244). In contrast, the locked on biofilm is short, very dense, and lacks channels. MR/P locked on bacteria autoaggregate (152, 244), which is reminiscent of the bacterial clustering that happens prior to stone formation in the bladder (44) (Fig. 25A–D). Furthermore, MR/P fimbriae allow P. mirabilis to adhere to some cultured cell lines, including T24 and Vero (150, 245). There are conflicting reports about MR/P-mediated binding to shed bladder epithelial cells, where purified MR/P and MR/P-positive P. mirabilis bound cells (240), but there was no difference in binding between a different wild-type strain and its isogenic mrpA mutant (149). It is possible that these two studies were in fact not examining the same fimbria (19).

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Figure 25

P. mirabilis mrpA mutant is defective in cluster formation

(A–D) Representative sections of (A and B) P. mirabilis wild-type and (C and D) mrpA-infected murine bladders at 24 hpi. (A and C) The wild-type–infected bladder shows a regional view of clusters, whereas the mutant-infected bladder shows a close-up of the urothelial surface. Bacteria are in green, UPIIIa in red, and DAPI in blue. (Scale bars, in micrometers, are as marked.) (B and D) Alizarin Red staining of bladder sections. Only wild-type P. mirabilis-infected bladders contain significant mineral deposition. L, bladder lumen; an asterisk indicates an extracellular cluster. Arrows indicate regions with increased DAPI signal. (E and F) SEM micrographs of MR/P ON and MR/P OFF cells colonizing the murine bladder at 4 dpi. MR/P ON colonized the bladder uroepithelium (E), while MR/P OFF colonized the lamina propria where bladder cells had sloughed off (F). Bars = 10 μm (E), 2 μm (F). Figure adapted, with permission, from (44) (A–D) and (244) (E and F).

Contributions to infection

MR/P fimbriae contribute to UTI as described in multiple studies (Table 6). The magnitude of deficiency of an mrp mutant in mouse model independent challenges varies from study to study, from the initial mrpA experiment, where a 6–18-fold decrease was observed in urine, bladder, and kidneys, to a four log defect in the bladder for an mrpG mutant (146, 149, 151). A mutation in mrpH, which encodes the fimbrial adhesin, does not decrease fitness in independent challenge, although the mutant was unrecoverable following a one-week cochallenge experiment (156). Subsequent cochallenge infections with an mrpA or an mrpA-D mutant likewise resulted in the mutant being significantly out-competed in both the bladder and kidneys (150, 151).

Newer evidence may explain the observed variability in the requirement for MR/P expression. Two studies have reported equal numbers of recovered bacteria from the bladders of mice infected with wild-type compared with an mrpA mutant (44) or mrp locked-off (244). However, in both studies, the mrp mutant was not found in the same location as wild-type, suggesting that MR/P fimbriae not only facilitate adherence to urinary tract tissue but also dictate the localization of P. mirabilis within the bladder. Notably, both of these studies used a relatively early endpoint (24 hpi) (44, 244) compared with other mouse independent challenges lasting 7 days (146, 149, 151, 156). Thus, the location of P. mirabilis in the urinary tract, whether extracellular, intracellular, or bound to regions where uroepithelial cells have sloughed off, may be important for its long-term survival (Fig. 25E and F).

Although prior studies suggested that MR/P fimbriae bind a receptor in renal cells and contribute to pyelonephritis (149, 240), recent data point to their importance in cystitis. First, when bacteria are isolated from the bladder or urine of infected mice, the invertible element of the mrp promoter is overwhelmingly (95–100%) in the on orientation, allowing transcription of mrp genes. In contrast, the invertible element orientation is highly variable in the kidneys, ranging from mostly on to 85% off (158, 241). Second, the percentage of bacteria with the mrp invertible element in the on orientation directly correlates with the amount of bladder colonization (Fig. 24C) (152). Third, when a locked-on mrpI mutant is competed against wild-type bacteria in a 7 d mouse cochallenge experiment, locked-on bacteria outcompete wild-type in the bladder but not the kidneys (158). Fourth, P. mirabilis locked-on mrpI mutants adhere to the luminal surface of the bladder when examined 24 hpi, while locked-off bacteria adhere to regions where umbrella cells have been shed, revealing the lamina propria (244). Fifth, wild-type P. mirabilis forms large clusters in the bladder lumen within 24 hpi, but an mrpA mutant does not form clusters and is primarily intracellular (44) (Fig. 25A–D). The precise function of MR/P fimbriae and their contributions to cystitis and/or pyelonephritis will be difficult to address until their binding target(s) are identified.

Uroepithelial cell adhesin (UCA)

A fimbrial preparation from P. mirabilis cultured to stationary phase in a minimal medium at 37 °C was used to isolate a long, 4 nm-thin protein that adhered to human uroepithelial cells obtained from voided urine (246). The importance of this protein, UCA, in uroepithelial cell adhesion was subsequently confirmed when a mutation of the gene encoding the major structural subunit of UCA (ucaA) was tested with desquamated cells (163).

A second group working on UCA renamed the protein “non-agglutinating fimbriae” (NAF) and found the protein in shear preparations obtained from diverse P. mirabilis isolates (248). UCA/NAF are readily expressed under several laboratory conditions at 37°C, including Luria broth or agar; production may be enhanced by addition of serum to Luria broth or during culture on urine agar (248, 249).

Expressing cloned uca genes in E. coli conferred the ability to bind uroepithelial cells (247). However, no fimbriae were observed on the recombinant E. coli; only ucaA was sequenced and it is not clear whether all genes in the uca operon were present in the expression construct (247). A UCA/NAF preparation purified from P. mirabilis binds to cultured cells, including EJ/28 (urinary carcinoma), HEp-2 (laryngeal carcinoma), and MDCK (canine kidney epithelium) (249, 251), although these results have not been confirmed using other methods, such as by comparing adhesion of a uca mutant to that of wild-type.

Although UCA fimbriae are encoded by most P. mirabilis isolates, their sequence is more variable compared with other fimbriae (106). Antiserum raised against purified fimbriae further indicated that UCA is widely present but variable in size (248). It is possible that variations in UCA may explain distinct phenotypes reported by different groups, who are typically using locally-obtained isolates in their experiments. Notably, the uca operon is part of a mobile element and flanked by phage genes; in the first two fully-sequenced P. mirabilis genomes, HI4320 and BB2000, the uca operon is not in the same chromosomal locus (106).

In vitro receptor experiments

A possible receptor for UCA/NAF has been identified using fimbrial protein purified from a shear preparation using differential centrifugation (248, 250). Purified UCA/NAF bound in vitro to glycolipids, including asialo-GM₁, asialo-GM₂, and lactosyl ceramide (250). A follow-up study suggested another molecule, galectin-3, was also a receptor for purified UCA/NAF on MDCK cells (251). The possible receptors have not been identified in healthy bladder tissue, although galectin-3 is a marker for bladder cancer (257). Thus, it is not apparent whether these molecules are true in vivo targets for UCA.

Infection studies

Given that bladder cell adherence is a defining feature of UCA, it is perhaps paradoxical that a ucaA mutant is significantly attenuated in the kidneys but not the bladder during either independent or cochallenge of the murine urinary tract (163). When this ucaA mutant was injected into the tail vein of mice, bacteria were only recoverable from 7/16 mice 7 days post-inoculation, while the wild-type parent was recovered from 15/16 mice (163). Transcription of ucaA has been detected in urine from infected mice, although the level was 2.5-fold decreased relative to in vitro broth culture (131). UCA also contribute to CAUTI, where ucaA and ucaB were both detected in a Tn-Seq study (129).

Proteus mirabilis fimbriae (PMF)

PMF fimbriae were initially identified when a P. mirabilis cosmid gene bank was screened using antiserum generated against UCA (255). These fimbriae are encoded by the pmfACDEF operon (226, 258). Despite initial speculation that PMF was the unidentified MR/K agglutinin (255), a pmfA mutant had no effect on either MR/P or MR/K-type agglutination (153). Nevertheless, PMF fimbriae contribute to UTI in mouse models, with an 83-fold decrease in recoverable CFU for a pmfA mutant during independent challenge in the bladder (153). The defect was more profound in cochallenge, where the mutant was significantly attenuated in both bladder and kidneys (154). In a hematogenous model, where P. mirabilis was introduced through the tail vein of mice, the pmfA mutant was unrecoverable from the kidneys of 55% of mice 7 days post-inoculation, while the wild-type parent was still recovered from 94% (154). There are conflicting data on whether PMF fimbriae mediate adherence to voided human uroepithelial cells (153, 154); however, a pmfA mutant had diminished adherence to cultured T24/83 bladder carcinoma cells (154). In summary, evidence exists for PMF to contribute to infection in the bladder, kidneys, or both, and the receptor(s) have not yet been identified. PMF also contribute to fitness during CAUTI (129).

Interestingly, although PMF are expressed during UTI and mice infected with P. mirabilis generate an immune response against these fimbriae, pmf genes are repressed in urine relative to in vitro culture (131, 243). This suggests that PMF fimbriae may be transiently produced during infection, or that PMF-producing bacteria are not readily voided in urine. A pmfA/mrpA-D double mutant is more attenuated during a triple challenge in mice than either single mutation or wild-type, but interestingly, the double mutant is still infective in independent challenge (albeit less than the wild-type parent strain) (151). This is consistent with overlapping roles for the P. mirabilis “pan-fimbriome” and suggests that other fimbriae also contribute to adherence in the urinary tract (Fig. 26).

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Figure 26

MR/P and PMF fimbriae have additive roles in urinary tract infection

Virulence of wild‐type P. mirabilis Pr2921 (wt), single fimbrial mutants (mrpA-D or pmfA), and double mrpA-D pmfA mutant was assessed in an ascending UTI model in mice at 7 dpi. (A and B) Independent challenge. The double mutant is significantly less fit compared with either single mutant. (A) CFU recovered per kidney, (B) CFU recovered per bladder. (C and D) Mice were challenged with a 1:1:1 mixture of three strains (tri-challenge). (C) Tri-challenge with wt, mrpA-D, and mrpA-D pmfA double mutant. (D) Tri-challenge with wt, pmfA, and mrpA-D pmfA double mutant. Each dot represents the log₁₀ CFU recovered from each organ. The median (horizontal bar) is indicated for each group. The range of detection in this assay is 10² to 10⁹ CFU per organ. Figure adapted, with permission from (151).

Ambient temperature fimbriae (ATF)

ATF fimbriae were first identified in a preparation of sheared surface proteins derived from an mrpA mutant (234). The atf operon was originally reported to consist of three genes, atfABC (259) as isolated from a cosmid clone, but subsequent genome sequencing revealed three additional genes (atfDEF) including an mrpJ paralog (112). Although ATF are produced at 37°C (106, 234), they are optimally expressed following static culture at 23°C (234). In a similar vein, an atfA mutant is recovered at the same rate as wild-type from either independent or cochallenge mouse studies (157). However, when an mrpA mutant was examined by immunofluorescence microscopy in infected mice, AtfA was readily detected (Fig. 27) (158, 244). This suggests that ATF may play a role in infection that is masked by other fimbriae, such as MR/P, and also there may be a fimbrial hierarchy that determines gene expression. ATF may contribute to biofilm formation under some conditions (256).

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Figure 27

MR/P and ATF expression during ascending UTI

(A and B) Phase-locked mutants express MR/P fimbriae (MR/P ON) (A) or do not express MR/P fimbriae (MR/P OFF) (B) during experimental UTI at 2 dpi. Green, GFP-expressing P. mirabilis; blue, DAPI-stained bladder cell nuclei; red, rabbit anti-MrpA reacted with goat anti-rabbit IgG conjugated to Alexa Fluor 568; yellow, colocalization of bacteria and MR/P. (C and D) MR/P ON does not express ATF (C), but MR/P OFF does (D). Green, GFP-expressing P. mirabilis; blue, DAPI-stained bladder cell nuclei, red, rabbit anti-ATF serum reacted with goat anti-rabbit IgG conjugated to Alexa Fluor 568; yellow, colocalization of bacteria and ATF. Bars = 10 μm. Reproduced from (244), with permission.

P. mirabilis P-like fimbriae (PMP)

This fimbria was first discovered in a sheared protein preparation from a canine UTI isolate of P. mirabilis, where PMP was found along with UCA (253). PMP fimbriae have also been implicated in both single-species and polymicrobial CAUTI, where pmpG was identified by Tn-Seq (129), and a pmpA mutant was found to be attenuated in a diabetic mouse UTI model (260). The pmp operon was also reported to be directly regulated by cyclic AMP receptor protein (Crp) (260).

Fimbria 14

Fimbria 14, so named because it is encoded by the fourteenth of the 17 chaperone-usher fimbrial operons in HI4320, seems like it should be non-functional because the fim14 operon completely lacks a chaperone and has a frameshift mutation in usher gene fim14C (112). However, putative minor fimbrial subunit fim14B was detected in an STM screen for genes that contribute to UTI (179), and putative major structural subunit Fim14A was identified in a preparation of sheared surface proteins (106). The last gene in the operon, fim14D, is induced during UTI (131), and genes from the fim14 operon were found to contribute to polymicrobial CAUTI (129). Although chaperone-usher secretion apparatuses are usually specific for a single fimbrial type, it is likely that Fimbria 14 is assembled using one or more of the other 16 chaperone-usher systems encoded by P. mirabilis.

Other fimbriae

The remaining fimbriae encoded by P. mirabilis are poorly characterized (Table 6). P. mirabilis have been reported to employ an unknown fimbria to agglutinate tannic acid-treated erythrocytes, a pattern that is called mannose-resistant Klebsiella-like (MR/K) (100, 229, 230, 261). This ability is variable for P. mirabilis, with some studies suggesting MR/K agglutination by P. mirabilis is widespread (229, 230) but others suggesting MR/K is more often seen for Proteus vulgaris or Proteus penneri (100, 252, 261). MR/K fimbriae produced by Providencia stuartii and P. penneri mediate attachment to catheter surfaces, and therefore may contribute to catheter colonization by P. mirabilis strains that possess MR/K fimbriae (261, 262). They have also been shown to mediate adherence to Bowman’s capsules in kidney glomeruli (240). Some of the 17 fimbriae are more variable in sequence than others. For example, one operon, fim3, is present in less than half the isolates in a diverse collection (106). Likewise, UCA is widespread in P. mirabilis, but its size and sequence are variable, so UCA binding properties might not be constant from strain to strain (106, 248). Thus, MR/K agglutination could be mediated by one or more of these fimbriae.

Transcriptomic and high throughput sequencing methods are beginning to shed light on the complex collection of the 17 fimbriae. Transcripts from three additional fimbriae, fim5, fim8, and fim15, were detected by microarray analysis of urine from infected mice, although both fim5 and fim8 were decreased compared with in vitro culture (131). In addition, Fim8A, E, and F protein were detected by mass spectrometry from sheared surface proteins produced during in vitro culture (106). Southern blot analysis suggested P. mirabilis strains encoded two copies of mrp genes (150). Subsequent genome sequencing revealed the mrp′ operon, which is an apparent duplication of the mrp operon; the two operons are encoded next to each other in the chromosome (112). Tn-Seq analysis of genes that contribute to single-species CAUTI suggest a role for mrp′ but not mrp, which is unexpected given the well-documented contributions of MR/P fimbriae to uncomplicated UTI (129). Most of the remaining fimbriae were also detected in polymicrobial CAUTI Tn-Seq with P. stuartii but not single-species CAUTI (mrp, atf, fim3, fim5, fim12, fim14, fim15, fim16, fim17, plus orphan fimbrial genes PMI1812, PMI1920, and PMI3023), indicating once again that the contributions of these multiple fimbriae may be subtle and much work remains to discern their functions (129).

Non-fimbrial adhesins

P. mirabilis produces other, non-fimbrial adhesins that are described elsewhere in this chapter, including autotransporter proteins Pta, AipA, and TaaP (link toxins and autotransporter sections). P. mirabilis also encodes two putative type IV pili (Pearson 2008).

Transcriptional regulation by MrpJ

MrpJ is a transcriptional regulator encoded at the end of the mrp fimbrial operon (137). Like other mrp genes, it is not expressed well in vitro, but is among the most highly-induced genes in urine collected from infected mice (131). MrpJ was originally proposed to serve as a balance between adherent and motile states (reciprocal regulation); when MR/P fimbriae are produced (adherent state), MrpJ is also produced and switches off flagella (motility) (137). That is, expression of mrpJ on a plasmid represses FlaA flagellin, in turn inhibiting both swimming and swarming motility (137) (Fig. 28). Similarly, an mrpI locked-on mutant constitutively produces MR/P fimbriae but not flagella, while disruption of mrpJ in the locked-on background results in a bacterium that produces both fimbriae and flagella (137).

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Figure 28

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 (ΔmrpJ). (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). Reproduced from (137), with permission.

MrpJ is predicted to be a 110 amino acid protein consisting of a helix-turn-helix (HTH) DNA-binding domain and a unique C-terminal tail (137, 138). It is a member of the xenobiotic response element (XRE) superfamily of transcriptional regulators. Consistent with other HTH proteins, mutation of residues predicted to form DNA contacts interferes with MrpJ’s ability to repress swimming motility (138). However, deletion of the C-terminal domain has almost no effect on motility repression, suggesting that this region is disposable for reciprocal regulation (138). Consistent with its role as a repressor of motility, MrpJ directly binds the promoter of the master flagellar regulatory genes flhDC (132, 138).

In addition to influencing motility, MrpJ also appears to be involved in autoregulation of the mrp operon. Expression of mrpJ on a plasmid results in elevated mrpA expression (132), and an mrpJ mutant produces less MrpA protein (137). The mrpJ mutant also has the mrp invertible element overwhelmingly in the off orientation, even under mrp inducing conditions (152).

Deletion of mrpJ has little effect in vitro, which may be due to either low expression of mrp genes in most laboratory conditions or masking of MrpJ function by redundancy (see MrpJ paralogs below) (137). However, an mrpJ mutant is significantly outcompeted during mouse independent and cochallenge experiments (132, 137), indication a strong contribution to fitness and pathogenesis within the urinary tract. The exact role of MrpJ during infection is complicated considering that it regulates other virulence factors, such as production of MR/P fimbriae and flagella; MrpJ could be acting by altering either of these proteins, or eliminating coordination of motility and adherence, or by acting on other gene targets.

Transcriptomic analysis confirms that MrpJ has other virulence-associated targets, in addition to MR/P fimbriae and flagella (132). Because mrp genes are transcribed at a low level in vitro, an in vivo mimic has been used to study MrpJ targets, where mrpJ is expressed in trans at levels comparable to those detected in mice during experimental UTI (132). In a comparison of the in vivo mimic with a vector control, 217 genes were differentially regulated. Classes of MrpJ target genes include flagella, MR/P and other fimbriae, type VI secretion, metabolism, LPS modifications, virulence factors such as Zap and Pta, and transporters (132). Because many MrpJ-regulated genes have known or predicted roles in disease, MrpJ has been proposed as a master regulator of virulence.

Chromatin immunoprecipitation followed by PCR (ChIP-PCR) indicated that MrpJ binds within the invertible element of the mrp operon, in agreement with detection of an MrpJ-responsive element 156–256 bp upstream of the transcriptional start site of the mrp promoter (132). This is unusual, given that bacterial regulators typically bind in the vicinity of the −35 (activator) or +0 (repressor) promoter sequences so they can interact or interfere with RNA polymerase, respectively (263). However, a distant site of action for MrpJ, relative to other transcriptional regulators, is consistent with the flhDC promoter binding data mentioned above (132, 138).

Prevention of catheter blockage

Currently, the typical approaches to avoid urinary catheter biofilms are to 1) limit the duration of catheterization and 2) replace blocked/encrusted catheters. Multiple groups are developing new approaches to prevent biofilm establishment, including alternative catheter materials, antimicrobial coatings or instillation of anti-biofilm chemicals, mechanical or electrical biofilm dispersal, bacteriophage, and control of urinary pH (278, 285–287). Because P. mirabilis is the major agent of crystalline biofilm formation and catheter blockage during CAUTI, many of these efforts focus on this species. Thus far, no catheter type has been immune to P. mirabilis biofilm (270), although some materials and coatings delay blockage. In some cases, P. mirabilis colonizes antimicrobial surfaces after crystalline material has coated the catheter (288). Filling the retention balloon of urinary catheters with 10 g/L triclosan is able to inhibit P. mirabilis biofilm in an in vitro model (289, 290), although exposure of P. mirabilis to triclosan may rapidly select for triclosan resistance (291). Ultimately, a combination of these strategies may be necessary to successfully combat P. mirabilis crystalline biofilm formation in a clinical setting.

Methods for studying P. mirabilis biofilms

An important caveat for biofilm studies in general is that biofilms can be generated using many techniques, and the chosen method has a strong influence on the outcome of the study (292). For example, P. mirabilis mutants with altered biofilm in a high-throughput crystal violet screen had no correlation with time to blockage in a catheter/artificial bladder model (177). Thus, there is a need to use a combination of simple and/or high-throughput in vitro methods, and more complex in vitro models and in vivo methods to study the contributions of P. mirabilis biofilms to infection.

A common, simple method for surveying biofilm formation involves culturing bacteria in multi-well plates, and visualizing biofilm by staining with crystal violet. The major advantage is that this assay allows for simultaneous screening of many samples; it is also easily quantifiable by dissolving the crystal violet and measuring the dye with a spectrophotometer. For this reason, the assay has been used to screen P. mirabilis clinical isolates and transposon mutants, as well as screening of targeted mutants (53, 177, 256, 275, 280, 281, 293, 294). Other variations on this assay include the addition of short catheter segments to multiwall plates and culture in static, closed tubes (283, 295). It is, however, important to note that these methods have their limitations. While urinary catheter biofilms are subject to the flow of urine, bacteria in multi-well plates are in a closed system where nutrients are depleted. Furthermore, if urine (artificial or real) is used as the medium, P. mirabilis urease activity will cause a rapid increase in alkalinity to approximately pH9. This lethal effect in static systems can be seen in a comparison of P. mirabilis biofilms cultured in the presence or absence of urea (296). Therefore, while the crystal violet method is useful, results may be less likely to lead to translational applications.

Continuous flow systems, where fresh medium is supplied at a constant rate, allow study of P. mirabilis biofilm development in catheters (30, 273, 297). The flow can be adjusted to match physiological urine rates in humans, and a flow cell can be inserted to facilitate microscopy. Urease activity still leads to crystalline biofilm formation if urine is used as the medium, but the pH increase is not as extreme as in the multi-well plate biofilm assay and may be maintained within a physiological range. A modification of continuous flow is an artificial bladder (298). Flow is maintained into a glass chamber that serves as a bladder analog, which is maintained at 37°C via a water jacket. A catheter is inserted into the base of the bladder chamber, which may be further connected to tubing and a drainage bag. The advantage of this bladder model is that it captures several aspects of the urinary tract: continuous introduction of urine from above, insertion of a catheter from below (including inflation of the retention balloon), and retention of a residual volume of urine similar to what occurs in a catheterized patient. However, this model requires specialized equipment and is limited in how many samples can be analyzed at one time.

P. mirabilis adheres to cultured cells in an aggregative pattern that has been likened to biofilms (275). This method may be used to assess aspects of biofilm development that are host-influenced, or require a biotic substrate for the developing biofilm. A drawback is that P. mirabilis produces toxins, such as hemolysin, which lead to cell death within a few hours and pose a challenge for studying longer-term biofilm development.

Animal models are also used to study biofilm development. Stones and clusters form in the mouse ascending UTI model (40, 42, 44), and P. mirabilis biofilms may be studied in vivo using the CAUTI mouse model (link animal models section) (25, 129). These models are essential for investigating the interaction of P. mirabilis biofilms with urinary tissues, the innate immune response, and the catheter-bladder interface.

Culture medium also greatly affects biofilm development. Using continuous flow coupled with confocal scanning laser microscopy, P. mirabilis biofilms were found to produce towers of biomass separated by channels in a rich medium (LB). However, in artificial urine (299), biofilms were flat, lacked channels, and had swarmer cells protruding from the surface (273). Crystalline biofilms will only develop if urine or artificial urine is used. Catheters in patients will be coated with cellular material and other material not present in artificial urine, and these coatings may further facilitate biofilm formation (268, 270).

Flagella

In contrast to other motile bacteria, all of the flagellar components and chemotaxis proteins of P. mirabilis are encoded within a single locus in the chromosome that spans approximately 54 kb (112). Within this region, P. mirabilis encodes two flagellin genes, flaA and flaB (312). FlaA appears to be the predominant flagellin produced by P. mirabilis, but recombination can occur between flaA and flaB, resulting in occasional hybrids (124, 313, 314). As the flagella produced by P. mirabilis are recognized by the host immune response and can elicit a pro-inflammatory response, antigenic variation and flaAB hybrids may play a role in immune evasion during infection (77, 161).

Flagella are not an absolute requirement for establishment of UTI, as naturally-occurring nonmotile strains and flagellar mutants of P. mirabilis are still capable of colonizing the urinary tract in experimental models (155, 315). However, there is clear evidence for the contribution of flagella to pathogenesis. The importance of flagella to host cell invasion and ascending UTI was directly assessed by disrupting the gene encoding the flagellar filament (flaD) in a P. mirabilis mutant lacking hemolysin activity to avoid confounding by expression of the toxin. Production of the flagellar filament was found to facilitate invasion of human renal proximal tubular epithelial cells in vitro, partly by allowing the bacterial cells to come into close proximity to the host cells (45). Flagella may also promote internalization through other mechanisms, as the flaD mutant still exhibited reduced internalization compared to the parental strain when the bacteria were centrifuged onto the cell monolayer. This mutant also exhibited reduced bladder and kidney colonization in the murine model of ascending UTI, while loss of hemolysin alone did not significantly impact pathogenesis.

A role for chemotaxis and components of the flagellar apparatus in ascending UTI was also directly indicated by signature-tagged mutagenesis (STM) studies as a positive regulator of chemotaxis (cheW), the flagellar M-ring (fliF) and flagellar hook protein (flgE) were all identified as fitness factors for ascending UTI (108, 179). Transposon insertion in any of these genes resulted in a defect in kidney colonization, while fliF and cheW also affected bladder colonization. Transposon insertion-site sequencing (Tn-Seq) similarly indicated a role for chemotaxis and flagella during catheter-associated CAUTI (129). Specifically, a regulator of chemotaxis (cheR), the flagellar master regulator (flhDC), and a gene involved in regulation of flhDC (umoA) were identified as fitness factors for kidney colonization, while another gene involved in regulation of flhDC (umoB) and the flagellar basal-body rod protein (flgC) were fitness factors for colonization of the kidneys and the catheterized bladder, and the flagellar M-ring (fliF) and ATP synthase (fliI) were fitness factors for bladder colonization (129). Thus, the flagellar M-ring has been identified as having a significant contribution to fitness through two separate mutagenesis screens in two distinct models of infection. It is also noteworthy that flagellar genes are temporally regulated during ascending UTI, being poorly expressed in vivo relative to growth in rich medium at early time points post-inoculation but increasing in expression by 7 dpi, further indicating a role for flagella in pathogenicity (131).

Iron

The most highly studied of the nutrient competition systems is iron, and much like the rest of the human body, the urinary tract is iron-restricted (324). Similar to other pathogens, P. mirabilis encodes iron scavenging and uptake systems that are induced during infection (109, 112, 131).

Bacteria typically produce iron-scavenging molecules called siderophores, which bind iron with higher affinity than host iron sequestration systems (322). Early evidence suggested that P. mirabilis lacked siderophores because it fares poorly relative to other bacteria in iron chelation experiments and tested negative in siderophore detection assays (325, 326). In addition, P. mirabilis is inhibited in its ability to establish infection in an iron-deficient rat pyelonephritis model (327). However, we now know that P. mirabilis has at least three ways to scavenge iron: proteobactin (Pbt), a non-ribosomal peptide synthesis system (Nrp), and α-keto acids. It can also use enterobactin, a siderophore produced by other enteric bacteria, although this species is not able to synthesize enterobactin on its own (109).

The nrp system was initially identified as a possible iron-regulated swarming signaling system (328). Genome annotation, microarray analysis, and a more sensitive siderophore detection method revealed that nrp and a second system, proteobactin (pbt), are siderophore biosynthesis and transport operons (109, 112). While the nrp genes are homologous to yersiniabactin genes and are encoded within a high pathogenicity island derived from Yersinia spp. (107), the products are not interchangeable (109). Although both siderophore systems must be mutated to produce a negative iron scavenging result in the laboratory (chrome azurol S, or CAS assay), only the nrp system has a significant contribution to infection (109).

P. mirabilis produces α-keto acids from amino acids, likely via two amino acid deaminases (PMI2834 aad and PMI2149) (326, 329, 330). These α-keto acids act as non-canonical siderophores and can restore growth of P. mirabilis on iron-chelated medium when supplied externally (326, 329). Amino acid deaminase activity is restricted to genera closely related to P. mirabilis (that is, Proteus, Providencia, and Morganella), as most enteric bacteria do not have Aad activity and cannot use externally-provided α-keto or α-hydroxy acids to overcome iron chelation (326, 329).

P. mirabilis encodes ferric, ferrous, and heme uptake systems that have been discovered by analysis of genes and proteins differentially regulated during iron restriction, genome annotation, and transposon mutagenesis (109, 112, 331, 332). In vivo analyses have demonstrated that iron uptake proteins are immunogenic (161, 331) and produced in a rat intraperitoneal chamber model (333); likewise, iron-related genes are induced during UTI (131), and several of these have directly been shown to contribute to UTI (108, 161, 179, 332). Iron acquisition and storage systems are even more strongly required during polymicrobial CAUTI, suggesting that competition with other species increases pressure on P. mirabilis iron-scavenging capabilities (129). Details on iron-associated genes in P. mirabilis and their contributions to infection are listed in Table 8.

Table 8

Iron-related genes in P. mirabilis

Iron-related genes from P. mirabilis were identified by homology to other iron-related genes (334, 335). Genes identified as iron-related by homology but not identified using one of the four conditions shown were excluded. A checkmark indicates that one or more of the genes in the row were identified using the condition specified. Adapted from (19).

Gene Designation(s)	Proposed function	PMI number (s)	Upregulated in iron limitation	Upregulated in vivo	Implicated in vivo	Antigenic in vivo	CAUTI		Citations
							Single	Poly
Heme uptake
	TonB-dependent receptor	PMI0409							(336) (335) (129)
	hemin uptake protein	PMI1424							(335) (337)
hmuR1R2STUV	hemin uptake system	PMI1425–1430							(336) (335) (337) (338) (331) (129)
Ferrous Iron Uptake
sitDCBA	Iron ABC transporter	PMI1024–1027							(335) (337) (129)
feoAB	ferrus iron transport	PMI2920–2921							(337) (129)
Ferric citrate transport
	exported protease	PMI3704							(335) (129)
	iron-related ABC transporter	PMI3705							(335) (339) (129)
	iron receptor	PMI3706–3707							(335) (129)
	extracytoplasmic function family σ factor	PMI3708							(335) (129)
	TonB-like protein	PMI3709							(335) (129)
Siderophore production
	TonB-dependent receptor	PMI2596							(336) (335) (333) (129)
nrpXYRSUTABG	nrp siderophore	PMI2597–2605							(335) (337) (340) (129)
pbtIABCDEFGH	proteobact in	PMI0231–0239							(339) (129)
Other TonB-dependent receptors
irgA	TonB-dependent enterobact in receptor	PMI0842							(336) (335) (337) (333) (129)
ireA	ferric siderophore receptor	PMI1945							(336) (335) (337) (333) (129)
	TonB-dependent receptor	PMI0363							(335) (337) (129)
	TonB-dependent receptor	PMI1548–1551							(335) (337) (129)
hasR	TonB-dependent receptor	PMI3120–3121							(340) (129)
ABC-transport system
	iron-related ABC transporter	PMI0331							(335) (337) (129)
	iron-related ABC transporter	PMI2957–2960							(335) (337) (340) (129)
	Iron-related	PMI0229							(335) (129)
	ABC transporter	−0230
Iron Metabolism
	iron utilization protein	PMI1437							(335) (337) (129)
Iron Sulfur Cluster Formation/Uptake
	iron sulfur cluster	PMI1411–1416							(335) (337) (129)
	iron sulfur cluster	PMI3253							(335) (337) (129)
	iron sulfur cluster	PMI0176–0172							(335) (337) (129)

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Zinc

The high-affinity zinc importer genes znuACB are induced during UTI compared with laboratory culture (131), and ZnuB is recognized by the humoral immune response (161). Furthermore, a znuC mutant is outcompeted in mouse cochallenge experiments (162), and znuACB contributes to fitness in a CAUTI model (129). However, these systems are not an absolute requirement for infection, as the znuC mutant remains able to colonize during independent infection (162), and the requirement for znuACB in single-species CAUTI disappears during polymicrobial CAUTI (129). Zinc export may also contribute to infection, as exporter ppaA contributes to single-species CAUTI (129).

At least two virulence factors are linked to zinc: flagella and ZapA. ZapA is a member of the serralysin metalloprotease family of zinc metalloproteases, which have a conserved zinc-binding motif (219, 341). While this clearly suggests a zinc requirement for ZapA, it has not been directly tested. Work in E. coli showed that zinc is bound by the flagellar master regulator FlhC (342), so it is not surprising that a znuC mutant exhibits defects in flagella-mediated motility (both swimming and swarming) (162). Swarming is also inhibited in the presence of the transitional metal chelator TPEN, further supporting a role for zinc in motility (162). Interestingly, a zinc exporter ppaA mutant also exhibits aberrant swarming (343).

Nickel

Nickel is an essential component of catalytically active urease, in addition to other bacterial enzymes. However, there are few studies where P. mirabilis nickel homeostasis has been examined. Likewise, nickel sequestration by the host as a pathogen defense strategy has been proposed, but not yet conclusively demonstrated (344). Yet, there are some indications that nickel balance contributes to P. mirabilis fitness in the urinary tract. Genes from two predicted nickel import systems are induced in experimentally-infected mice compared with laboratory culture: nikAB and yntABCD (131). Curiously, in a CAUTI model, these import systems were not found to be essential, but nickel export (PMI1518) was (129). Thus, nickel homeostasis appears to be important during infection, but the balance between enzymatic requirements and metal toxicity may be affected by the presence of a catheter and/or competing nutritional requirements of other bacteria.