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The type III secretion system of Proteus mirabilis HI4320 does not contribute to virulence in the mouse model of ascending urinary tract infection

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-0620, USA

Correspondence
Harry L. T. Mobley
hmobley{at}umich.edu

Received 27 March 2007
Accepted 25 June 2007

Abbreviations: TTSS, type III secretion system; UTI, urinary tract infection.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Proteus mirabilis, a major cause of complicated urinary tract infection (UTI) in humans, primarily infects patients with long-term indwelling urinary catheters (>30 days) or individuals with anatomical abnormalities of the urinary tract (Coker et al., 2000). P. mirabilis-induced UTI may be complicated by acute pyelonephritis, fever, kidney or bladder stone formation, catheter encrustation or blockage, and bacteraemia (Coker et al., 2000).

Once this species has gained a foothold in the urinary tract, it raises the local pH by the action of urease (Griffith et al., 1976). Urease catalyses the formation of bladder and kidney stones, which may also encrust or block a urinary catheter. Flagella also play a role in P. mirabilis pathogenesis (Mobley et al., 1996); this highly motile member of the Enterobacteriaceae is able to convert between short, vegetative swimmer cells and very long, hyperflagellated swarmer cells (Mobley & Belas, 1995). Several fimbriae have been identified in P. mirabilis, including MR/P (Adegbola et al., 1983; Bahrani et al., 1994), UCA (Cook et al., 1995; Wray et al., 1986), MR/K (Adegbola et al., 1983; Bahrani et al., 1993) and ATF (Massad et al., 1994). Other virulence factors include a haemolysin (Mobley et al., 1991; Swihart & Welch, 1990), an IgA protease (Senior et al., 1987; Walker et al., 1999) and capsular polysaccharide (Dumanski et al., 1994).

Type III secretion systems (TTSSs) are involved in both plant and animal pathogenesis or symbiosis by several Gram-negative bacterial species (reviewed by Cornelis, 2006). TTSSs consist of a needle-like structure extending from a channel that spans both the inner and outer bacterial membranes. This needle inserts into the membrane of a target eukaryotic cell and injects effector proteins directly into the cytoplasm. The role of these proteins varies by pathogen, but effects may include host cell attachment (McDaniel & Kaper, 1997), invasion (Menard et al., 1993), antiphagocytosis (Rosqvist et al., 1988), apoptosis (Mills et al., 1997) and modulation of inflammation (Espinosa & Alfano, 2004; Schulte et al., 1996).

Sequencing of the genome of P. mirabilis HI4320, a strain isolated from a patient with a long-term urinary catheter, was recently completed. Annotation of the genome facilitated the identification of new potential virulence factors. Several genes appeared to encode components of a previously unknown TTSS. This report details experiments conducted to characterize the nature of this potential P. mirabilis TTSS.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Bacterial strains and culture conditions. Proteus mirabilis HI4320 is a wild-type strain that was isolated from a patient with a long-term indwelling urinary catheter (Mobley & Chippendale, 1990). Escherichia coli DH5 (Sambrook et al., 1989) was used as the host for plasmid construction. All strains and mutants were cultured at 37 °C in non-swarming Luria–Bertani (LB) broth (10 g tryptone l^–1, 5 g yeast extract l^–1, 0.5 g NaCl l^–1) or on LB solidified with 1.5 % agar. Antibiotic supplementation with chloramphenicol (20 µg ml^–1), ampicillin (100 µg ml^–1) or kanamycin (25 µg ml^–1) was provided as necessary.

Identification of TTSS genes. Conserved TTSS genes from E. coli, Shigella flexneri and Salmonella typhimurium were submitted for BLAST analysis (Altschul et al., 1990) against the P. mirabilis genome (http://www.sanger.ac.uk/Projects/P_mirabilis/). The extent of the TTSS pathogenicity island was determined by both BLAST analysis and mapping the percentage GC content of the TTSS region.

Construction of a spa47 mutant. A kanamycin-resistance gene was inserted into the spa47 gene using the TargeTron method (Sigma) according to the manufacturer's directions. Briefly, a group II intron was reprogrammed to specifically insert into spa47 by mutagenic PCR using primers spa47-IBS, spa47-EBS2 and spa47-EBS1d (Table 1). The retargeted intron was ligated into plasmid pACD4K-C to create plasmid pMP122 and introduced to E. coli DH5 by electroporation. Correct retargeting of the intron was confirmed by nucleotide sequence analysis using plasmid-based primers pACD4K-C5' and pACD4K-C3' (Table 1). P. mirabilis HI4320 was then electroporated with plasmid pMP122 and the T4 helper plasmid pAR1219 (Davanloo et al., 1984). Addition of 0.5 mM IPTG to a chloramphenicol- and ampicillin-resistant isolate induced the intron to jump from pMP122 into the spa47 gene in the P. mirabilis chromosome, an event that was detectable by selection with kanamycin. This mutant, HI4320spa4 kan, was confirmed by PCR using primers spa47-L5 and spa47-R3 (Table 1). The single insertion of the kanamycin gene into the spa47 locus was verified by Southern blotting.

Co-culture of HI4320 and HI4320spa4 kan.

Construction of an exsD mutant. The exsD gene was inactivated using the TargeTron protocol, as described above. Primers exsD-IBS, exsD-EBS2 and exsD-EBS1d (Table 1) were used to retarget the intron. The intron was ligated into pACD4K-C to create plasmid pMP166 and was confirmed by nucleotide sequence analysis. HI4320 was electroporated as described above. The kanamycin-resistant mutant HI4320exs kan was confirmed by PCR using primers virF-RT5' and exsD-RT3' (Table 1).

RNA isolation and RT-PCR. To measure expression of TTSS genes, P. mirabilis strains were cultured to mid-exponential phase (OD₆₀₀ 0.8). RNAprotect (2 ml) (Qiagen) was added to 1 ml culture, and RNA was isolated using the RNeasy kit (Qiagen) according to the manufacturer's instructions. DNA was digested using TURBO DNA-free DNase (Ambion). RNA was used as the template for cDNA synthesis using the Superscript First-Strand Synthesis System (Invitrogen) according to the manufacturer's protocol. Three genes, located in diverse regions of the TTSS locus, were examined for expression by PCR. Primers used to amplify cDNA are shown in Table 1.

Analysis of concentrated culture supernatants. A 1 ml portion of an overnight P. mirabilis culture was collected by centrifugation and resuspended in 1 ml DMEM culture medium (Gibco). This suspension was used to inoculate 100 ml prewarmed DMEM, and the culture was cultured at 37 °C without aeration in an atmosphere of 95 % air/5 % CO₂. At mid-exponential phase (OD₆₀₀ 0.5) or late-exponential phase (OD₆₀₀ 1.0), the culture was centrifuged at 9000 g for 15 min at 4 °C. The supernatant was filtered (0.2 µm pore size) and concentrated to less than 1 ml using Centriprep YM-10 centrifugal filter units (Millipore). Protein was precipitated overnight using 10 % TCA. Samples were separated by SDS-PAGE and stained with silver (Silver Stain Plus, Bio-Rad) according to the manufacturer's directions. The same protocol was used to examine the culture supernatant of P. mirabilis cultured in Minimal A medium (Belas et al., 1991) to mid-exponential phase (OD₆₀₀ 0.5). Alternatively, to attempt to induce secretion of intracellular stores of TTSS effector proteins (Bahrani et al., 1997), P. mirabilis was cultured in LB to late-exponential phase (OD₆₀₀ 1.0), collected by centrifugation (9000 g for 15 min at 4 °C), and resuspended in PBS. Congo red was added to a final concentration of 20 µM, and the bacteria were incubated at 37 °C with aeration for 30 min (Bahrani et al., 1997). The suspension was then centrifuged (9000 g for 15 min at 4 °C), and the supernatant was concentrated and analysed by silver staining, as above.

Mouse model of ascending UTI. The CBA/J mouse model of ascending UTI has been described previously (Hagberg et al., 1983; Johnson et al., 1987). Briefly, 6-week-old female CBA/J mice were inoculated transurethrally with a 50 µl suspension of 1x10⁸ c.f.u. P. mirabilis HI4320 or HI4320spa4 kan. At 3 or 7 days post-infection, the urine, bladders and kidneys of the mice were examined for the presence of P. mirabilis by plating on LB using a spiral plater (Autoplate 4000, Spiral Biotech). At 7 days, the spleen was also analysed for infection (c.f.u. g^–1). Colony counts were enumerated using a QCount (Spiral Biotech).

The co-challenge experiment was conducted by mixing an estimated 2x10⁹ c.f.u. ml^–1 culture of HI4320 with an estimated 2x10⁹ c.f.u. ml^–1 culture of HI4320spa4 kan according to the method of Li et al. (1999). Quantification of the inoculum showed the actual amount of HI4320 to be 1.04x10⁹ c.f.u. ml^–1 and that of HI4320spa4 kan to be 1.43x10⁹ c.f.u. ml^–1. Mice were inoculated as above with a 50 µl portion (1x10⁸ c.f.u. per mouse) of this suspension. After 3 days, the urine, bladders and kidneys of the mice were assessed for bacterial load by plating equal portions onto LB or LB supplemented with kanamycin. Wild-type HI4320 infection was determined by subtracting the number of colonies on the kanamycin plate from the number of colonies on the plain LB plate.

The statistical significance of data obtained in both independent challenge experiments was assessed using the Mann–Whitney test. The co-challenge experiment was analysed using the Wilcoxon matched-pairs test.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Identification of a TTSS locus in P. mirabilis HI4320

Annotation of the P. mirabilis HI4230 genome sequence is currently under way in collaboration with the Sanger Institute (H. L. T. Mobley and others, unpublished results). A routine BLAST (Altschul et al., 1990) search of draft genome sequences for putative pathogenicity genes identified homologues of several TTSS genes. Beginning with the initial TTSS genes, neighbouring ORFs were submitted for BLAST analysis to find other TTSS homologues. Genes encoding a potential TTSS were found on a low GC-content (30.7 vs 38.9 % for the entire genome) 22 kb pathogenicity island. This island contains 24 genes that appear to encode all components necessary to assemble a TTSS needle complex, plus at least two putative secreted effector proteins and their chaperones (Fig. 1a). The ORFs within the pathogenicity island are free of apparent premature termination mutations, deletions or other disruptions. The genes in the island encompass ORFs PMI2681 to PMI2704 in the annotated genome (H. L. T. Mobley and others, unpublished results). The genomic organization and homology of this pathogenicity island are most similar to those of the TTSS regulon from S. flexneri (Fig. 1b); thus, the S. flexneri gene designations have been adopted for simplicity. An additional gene (PMI2709), encoding a product homologous to the TTSS effector YopH from Yersinia enterocolitica, was found nearby (5 kb) the other TTSS genes.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Schematic representation of the P. mirabilis HI4320 TTSS. (a) The TTSS locus of P. mirabilis HI4320. Genes are named after their counterparts in the S. flexneri TTSS (Buchrieser et al., 2000). The filled arrow indicates the spa47 gene, which was mutated by insertion of a kanamycin-resistance gene. The predicted percentage similarity between translated P. mirabilis genes and S. flexneri proteins is listed above each gene. (b) The TTSS locus of S. flexneri. Shaded arrows indicate genes that are missing from the P. mirabilis TTSS. (c) Potential regulators of the P. mirabilis TTSS are found at a chromosomal site distant from the main TTSS locus.

Expression of TTSS genes

To examine whether the P. mirabilis TTSS genes are transcribed, RT-PCR was conducted using RNA isolated from HI4320 cultured to exponential phase. Selected TTSS genes were examined for message (Fig. 2a). Although the TTSS genes mxiA, ipaB, virF (Fig. 2a), mxiG and yopH (data not shown) were expressed in exponential-phase culture, the small amount of amplified message compared to the constitutively expressed rpoA gene suggested that the TTSS genes are expressed at a very low level under this growth condition.

View larger version (22K): [in this window] [in a new window]   Fig. 2. RT-PCR showing expression of selected TTSS genes. RNA was isolated from exponential-phase cultures of either wild-type HI4320 (WT) or the spa47 mutant. (a) RT-PCR of selected TTSS genes. The gene rpoA, encoding RNA polymerase A, was used as a positive control for gene expression. (b) RT-PCR of the spa47 gene in both HI4320 and HI4320spa4 kan. G, HI4320 genomic DNA template; –, no reverse transcriptase added; +, reverse transcriptase added.

RT-PCR on selected TTSS genes was conducted to determine whether this mutant had a perturbation in TTSS gene expression. However, transcript was detected for all TTSS genes tested: mxiA, ipaB, virF (Fig. 2a), mxiG and yopH (data not shown). RT-PCR analysis of the spa47 gene confirmed the disruption in this gene in HI4320spa4 kan (Fig. 2b), as no message was detected.

TTSS effector proteins have been detected in the culture supernatants of other bacterial species as an index of a functional TTSS (Demers et al., 1998; Gruenheid et al., 2004). A spa47 ATPase mutant should be unable to secrete TTSS effectors through the needle complex. Therefore, supernatants from wild-type HI4320 and HI4320spa4 kan cultures in DMEM or Minimal A medium were collected and concentrated over 100-fold. These concentrated culture supernatants were separated by SDS-PAGE and stained with silver (Fig. 3a, b, lanes 1 and 2). No differences were observed between the wild-type and mutant secreted protein profiles. The indicator dye Congo red has been used to induce secretion of intracellular stores of TTSS effector proteins in S. flexneri (Bahrani et al., 1997); therefore, P. mirabilis cultures were exposed to Congo red, and the culture supernatants examined. No differences were detected in the wild-type and mutant protein profiles (Fig. 3c, lanes 1 and 2).

View larger version (55K): [in this window] [in a new window]   Fig. 3. Silver staining of concentrated culture supernatants. HI4320, the spa47 mutant and the exsD mutant were grown to late-exponential phase in DMEM medium (a) or mid-exponential phase in Minimal A medium (b). Mid-exponential cultures in DMEM were also examined (data not shown). (c) P. mirabilis strains were cultured in LB before exposure to Congo red. The culture supernatants were collected and concentrated before separation by SDS-PAGE on a 10 % gel. Lanes: 1, wild-type HI4320; 2, spa47 mutant HI4320spa4 kan; 3, exsD mutant HI4320exs kan.

Virulence of the spa47 mutant in the mouse model of ascending UTI

To determine whether the P. mirabilis TTSS contributes to pathogenesis in the urinary tract, CBA/J mice were infected transurethrally with either wild-type HI4320 or the spa47 mutant HI4320spa4 kan. After 3 days, the bacterial load in the bladder and kidneys was enumerated (Fig. 4a). There was no significant difference between the c.f.u. (g tissue)^–1 values for HI4320 and the spa47 mutant. To determine whether the TTSS played a role in a more advanced stage of UTI, CBA/J mice were infected with HI4320 or HI4320spa4 kan as before. The infection was allowed to progress for 7 days before examination of the urine, bladder, kidneys and spleen of the mice. One mouse infected with HI4320spa4 kan died on day 6, and one mouse infected with HI4320 died on day 7; these were not included in the data analysis. However, there were no significant differences in bacterial numbers between wild-type HI4320 and the spa47 mutant (Fig. 4b). The presence of bacteria in the spleens of some mice indicated that P. mirabilis had crossed from the kidneys into the bloodstream of these mice.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Independent and co-challenges of CBA/J mice comparing P. mirabilis HI4320 with the spa47 mutant. For independent challenges at (a) 3 days and (b) 7 days, mice were inoculated with either wild-type HI4320 or the spa47 mutant. Each data point represents log10[c.f.u. (ml urine)–1] or log10[c.f.u. (g tissue)–1] collected from one mouse. For the 3 day co-challenge (c), each mouse was infected with a 1 : 1 mixture of HI4230 and the spa47 mutant. For both independent and co-challenge experiments, horizontal bars denote the median value of a population. The limit of detection of this assay was 102 c.f.u. per millilitre or per gram. , Wild-type HI4320 (WT); , HI4320spa4 kan mutant. No statistically significant differences were found between groups.

Conclusions

TTSS have been consistently implicated in direct interactions with eukaryotic hosts (Alfano & Collmer, 2004; Mota & Cornelis, 2005). However, TTSS have not yet been found to play a role for any species in urinary tract pathogenesis. P. mirabilis appears to possess all the machinery necessary to produce a functional TTSS, but if this structure is made, it does not contribute to virulence in the mouse model of ascending UTI. This bacterium is believed to gain access to the urinary tract following contamination of the periurethral area, and has been isolated from the intestinal microbiota of humans (Senior, 1983). Since several intestinal pathogens employ a TTSS, it is possible that P. mirabilis uses its TTSS while living in the gastrointestinal tract but not in the urinary tract. Another possibility is that the P. mirabilis TTSS does not play any role in human disease, but rather is used while the bacterium is living in soil or water (C. W. Penn, personal communication).

Alternatively, the P. mirabilis TTSS may not be functional. Although a functional TTSS has been well described in enterohaemorrhagic and enteropathogenic E. coli (EHEC and EPEC), an additional cryptic TTSS has also been reported in EHEC O157 : H7 (Hayashi et al., 2001; Perna et al., 2001). This TTSS, called ETT2, is present to varying degrees in diverse E. coli strains (Ren et al., 2004). However, in contrast to the P. mirabilis TTSS (which appears to be intact), the cryptic E. coli TTSS is marked by multiple deletions and frameshift mutations (Ren et al., 2004). Notably, TTSS regulators encoded within ETT2 have been shown to affect the expression of genes within the functional TTSS of EHEC (Zhang et al., 2004). Thus, even if P. mirabilis does not produce a TTSS needle complex, the genes within the TTSS cluster may have other functions.

	ACKNOWLEDGEMENTS

 
This study was supported in part by Public Health Service grant AI059722 to H. L. T. M. M. M. P. was supported in part by the Molecular Mechanisms of Microbial Pathogenesis training grant (T32 AI7528). The authors would like to thank M. Chelsea Lane and Amy Simms for substantial assistance with animal challenges.

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