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

Proteus mirabillis

Model for Pathogenesis in the Urinary Tract

Infection of the urinary tract by P. mirabilis is governed by the general principles of bacterial pathogenesis. Proteins produced by this species during infection may contribute to a) colonization of the host; b) evasion of host defense; and c) damage of host tissue.

a. colonization of the urinary tract.

Colonization requires movement of P. mirabilis to its niche, attachment to specific receptors, and survival in the urinary tract. At least four fimbriae have been identified in this species [29-35] and 11 more are predicted by the newly completed genome sequence of our strain. MR/P (mannose-resistant Proteus-like) fimbriae and PMF (P. mirabilis fimbriae) appear to contribute to colonization by P. mirabilis. When separate groups of mice were transurethrally inoculated with the parent strain and an MR/P-deficient isogenic mutant (constructed in our laboratory), the mutant was recovered in lower numbers than the parent strain from the bladder and kidneys of mice. Separate mrp mutants (mrpA, mrpG, and mrpH) are all similarly attenuated [36-38]. When mice were coinfected with parent and the isogenic MrpH-deficient strains, the adhesin-negative mutant was severely out-competed [38]. That MR/P fimbriae are of paramount importance was illustrated by the ability to develop an intranasal vaccine containing the N-terminus of the MrpH tip adhesin. Vaccinated mice were protected from transurethral challenge with the homologous P. mirabilis HI4320 strain [39]. We also demonstrated that the entire bacterial population in the bladder expresses MR/P fimbriae when bacteria are present at high cfu (>10 5 cfu/ml). Indeed, bacteria expressing MR/P fimbriae are selected under the low oxygen conditions that prevail at high bacterial concentration (Li and Mobley, under revision). In other experiments, a PMF fimbrial mutant was found in significantly lower concentration in the bladder only [40]. The contribution of two other fimbriae, ATF (ambient temperature fimbriae) and NAF (non-agglutinating fimbriae), have not yet been tested but are of doubtful significance because of their synthesis at 23 oC (and not at 37 oC) and lack of identifiable adherence property, respectively.

To move to the site of colonization, P. mirabilis may undergo swarmer cell differentiation (i.e., development of an elongated form on solid surfaces) that is accompanied by production of hundreds of flagella per cell. We had postulated that the flagella-mediated motility is required to ascend the ureters to the kidney. Allison et al.[41] had shown that the genes encoding flagellin, urease, and hemolysin are coordinately expressed in these differentiated cells. Indeed, in our hands, flagella-deficient mutants are significantly attenuated in the CBA mouse model [42]. These data suggest that differentiation into swarmer cells is an adaptive response of P. mirabilis to the host environment. Surprisingly, however, we demonstrated that swarmer cells, visualized by examination of GFP-expressing bacteria by confocal microscopy in infected mouse tissue, were very rare [43]. Nevertheless, motility itself (which can be mediated by a single flagellum per bacterium) does appear to contribute to virulence.

Like other pathogenic microorganisms, P. mirabilis was found to possess a variety of iron acquisition mechanisms to overcome low free iron concentrations encountered in the host, which tend to be 10 8-fold lower than the concentration necessary for growth [44]. Mechanisms of iron acquisition include (i) direct utilization of the host iron compounds, including heme, hemoglobin, transferrin, and lactoferrin and (ii) synthesis of high affinity iron-chelating molecules, known as siderophores, that are secreted into the iron-limiting environment to scavenge extracellular iron [45]. Five mutants were identified by STM as having iron acquisition gene homologs, including nrpG (homologous to yersiniabactin), hasR, and genes encoding an iron transport permease and an iron-regulated outer membrane protein [46]. Other genes discovered by STM will be discussed in the Progress Report (Table 1).

b. evasion of host defense.

P. mirabilis may avoid host defenses by at least four possible mechanisms. These include production of an IgA-degrading protease (ZapA) that cleaves secretory IgA, produced in response to infection [47-51]. As well, P. mirabilis has three distinct copies of flagellin genes and Belas and colleagues have demonstrated that these genes can recombine and express antigenically novel flagellins [52]. Bacteria expressing these recombinant flagellins could thrive under the selective pressure of the host immune response. Since flagella represent major surface antigens for this species during infection, synthesis of antigenically distinct flagella could represent an effective mechanism for avoidance of host defense. In a similar vein, expression of MR/P fimbriae can also be modulated by a mechanism of phase variation, resulting in expression of the fimbriae by some bacteria and no expression by other bacteria within the same population at a given time [53, 54]. Finally, urease-mediated stone formation imbeds bacteria within a stone matrix. These encased bacteria are protected from various assaults ranging from antibiotics to neutrophils.

c. damage to host tissue.

Hemolysin and urease appear to contribute to damage of host tissue [10, 11, 55, 56]. The HpmA hemolysin is a potent cytotoxin in vitro for renal epithelial cells, cytolyzing such primary human cell cultures after only brief contact [11]. Urease hydrolyzes urea in urine, present in high concentration [~0.4 M], releasing ammonia and raising the local pH, which also damages epithelium [10, 56]. Stones formed due to urease can also physically damage the epithelium and block urine flow.

d. current model of P. mirabilis ascending UTI.

P. mirabilis uses biofilm formation and swarming motility to colonize indwelling urinary catheters [57-60], and then migrates through the urethra and into the bladder. The high level of urea (~0.4 M) in urine most likely allows a sufficient urea level within colonizing bacteria, and the urea-induced transcriptional activator of urease, UreR, facilitates transcription of ureDABCEFG [61, 62]. Urease increases the local pH surrounding the bacteria and causes precipitation of calcium and magnesium [24, 63, 64]; these crystals form a matrix in which the bacteria are found in high numbers. The bacteria are the short swimmer morphotype within the stone and there are necrotic bits of host cells and neutrophils within the matrix of the stone. The environment within the bladder either selects or signals production of MR/P fimbriae, as >85% of the bacteria are expressing this surface structure two to four days after infection [65]. High levels of MR/P expression in the bladder continues in the majority of the bacteria at least until day seven [66], as detected by the orientation of the mrp promoter that resides on an invertible element. Numerous white blood cells, many of them neutrophils, are apparent in the bladder by two days post-infection but appear to diminish in number as the infection continues. At some signal, a very small percentage of bacteria elongate into swarmer cells. While their function in the bladder is unknown, they do exist, albeit in numbers <0.1% of the population [67]. The predominate morphotype, the swimmer cell [43], colonizes the bladder uroepithelium in a mosaic pattern during chronic infection (7 days post-inoculation); that is, selected bladder epithelial cells are colonized by high numbers of bacteria while an adjacent epithelial cell may remain uncolonized [68]. P. mirabilis produces many virulence factors during ascending infection, that when inactivated, attenuate the bacteria. These virulence proteins include the aforementioned urease, flagellin, and ZapA metalloprotease [56, 69, 70].

Bacteria ascend the ureters by swimming motility, and the majority of bacteria within the lumen of the ureters are producing MR/P fimbriae [65]. There are occasional areas of inflammation in ureter tissue, with influx of neutrophils and erythrocytes into the lumen of the ureter.

P. mirabilis is capable of infecting the kidney and causing pyelonephritis. Once in the kidney, expression levels of MR/P fimbriae are lower than in the lower urinary tract [65] . Occasional bacteria differentiate into elongated swarmer cells but primarily the short swimmer morphotype predominates [67] . There is massive neutrophil infiltration [67] . Bacteria within the kidney adhere to various surfaces, including mucus strands, and host epithelium. Once bacteria gain access to the proximal tubules within the kidney they are two cell layers removed from the bloodstream, and are capable of crossing the endothelium and entering the renal circulation [67] as evidenced by localization in the spleen (Pearson and Mobley, unpublished). Subsequently, organized structure within the renal parenchyma is lost due to necrosis likely triggered both by bacterial products and the inflammatory response.