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BACTERIAL PATHOGENICITY |
Departments of Microbiology & Immunology and Comparative Medicine, University of South Alabama College of Medicine, Mobile, AL 36688 and *Department of Microbiology & Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
Corresponding author: Dr D. J. McGee (e-mail: dmcgee{at}jaguar1.usouthal.edu).
Received 6 March 2002; revised version accepted 17 June 2002.
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Abstract |
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 TOP Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TOPAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Urease, which catalyses the hydrolysis of urea to CO2 and NH4+, is central to the pathogenesis of H. pylori infection and urease-negative mutants fail to colonise various animal models [5–7]. Because urease is a nickel-requiring enzyme, nickel transporters, such as NixA, are required for full urease activity in both H. pylori [8] and in an Escherichia coli model of H. pylori urease [9, 10]. H. pylori urease was thought initially to be constitutively expressed [11, 12], but mounting evidence suggests otherwise. Recently, a number of genes, including a flagellar biosynthesis and regulatory gene, flbA, was demonstrated to modulate urease activity [9]. Furthermore, urease protein levels [13] and activity [14, 15] are elevated under acidic conditions and nickel has been shown to activate H. pylori urease expression transcriptionally [16]. Thus, H. pylori may modulate urease activity in vivo in response to specific environmental cues.
Proteus mirabilis causes urinary tract infections including pyelonephritis and kidney stone formation, particularly in patients with indwelling catheters or structural abnormalities of the urinary tract [17–19]. Like H. pylori, the urease of P. mirabilis is required for virulence [20]. However, in contrast with H. pylori, P. mirabilis urease is activated transcriptionally by UreR [21] in the presence of urea [22]; no UreR homologues exist in the H. pylori genome [23, 24], and H. pylori urease is not urea-inducible [25]. UreR is transcribed from its own promoter and then activates the divergently transcribed ureDABCEFG urease operon [21]. E. coli carrying the P. mirabilis urease gene cluster also has urease activity that is inducible by urea [22] and requires UreR [26], but optimal urease activity does not require addition of a nickel transporter gene, as it does in E. coli containing the H. pylori urease gene cluster. Urea-induced expression of the P. mirabilis urease (ureD) promoter-lacZ transcriptional fusion is likewise dependent on a functionally intact UreR [26]. Clearly, P. mirabilis urease is regulated differently from that of H. pylori.
The flbA gene, which modulates H. pylori urease activity, is a cytoplasmic membrane protein of 80 kDa of the LcrD protein family that is thought to be a structural component of the flagellar secretion apparatus [9, 27, 28]. Although much in-vitro data suggest that FlbA homologues are involved in virulence [29–33], these have not been assessed in vivo for virulence. H. pylori motility is required for colonisation of gnotobiotic piglets [34, 35], mice [36] and gerbils [37]. However, in the gerbil study, undefined and non-isogenic non-motile variants of H. pylori were employed. Thus, no specific H. pylori flagellar biosynthesis gene has been tested for its role in virulence in the gerbil model.
A previously described isogenic flbA mutant of one strain of H. pylori had both loss of motility and elevated urease activity in a qualitative assay [27], and another study indicated that flbA significantly decreased urease activity and protein levels in E. coli containing the H. pylori urease gene cluster and the nixA nickel transporter gene (on pHP8080) [9]. New matters were generated by these studies. For example, urease activity and motility have not been quantified in flbA mutants of H. pylori, nor has the effect of flbA on urease of other motile bacterial species, such as P. mirabilis, been addressed. Furthermore, the in-vivo relevance of flbA homologues has not been demonstrated. The present study examined these matters.
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Materials and methods |
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TOPAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Oligonucleotide primers used for PCR and sequence analyses are listed in Table 1. Plasmids (detailed in Table 1) were constructed by standard molecular biology techniques [39, 40]. Constructs were verified by restriction endonuclease digestions or PCR analyses, or both, and subsequently confirmed by sequence analysis of restriction endonuclease site junctions. Plasmids pBS-flbA, pBR322-flbA and their corresponding kanamycin disruption constructs are represented diagrammatically in Fig. 1.
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Motility assay
Motility was assessed on F-12 soft agar. F-12 powder mix (Life Technologies) was reconstituted to a 2x stock, filter sterilised and mixed with an equal volume of Bacto agar (0.7%). Fetal bovine serum was added to a final concentration of 4%. Strains were inoculated by stabbing the agar and the plates were incubated at 37°C for several days in a CO2 5% incubator with 100% humidity. Strains were considered motile if they moved away from the initial stab within 2–3 days, whereas non-motile strains remained at the inoculation site.
Construction and confirmation of H. pylori isogenic mutants of flbA
H. pylori strains were electroporated (800 ohms, 2.5 kV, 25 µF; Gene Pulser II, BioRad, Hercules, CA, USA) with insertionally inactivated flbA to generate flbA1 mutants (derived from pBS-flbA::aphA3) or flbA2 mutants (derived from pBR322-flbA::aphA3) (Fig. 1 and Table 1). Two different flbA mutant constructs were employed to ensure reproducibility of the data. The kanamycin cassette was non-polar, thereby minimising effects on adjacent downstream genes. Chromosomal DNA was isolated [41] from kanamycin-resistant transformants and was used to re-transform wild-type strains to remove potential background mutations. Because H. pylori urease activity decreases significantly upon in-vitro passage (D. J. McGee and H. L. T. Mobley, unpublished observations), first-passage transformants were used for urease extracts and transformants were passaged a second time for isolation of chromosomal DNA and subsequent PCR-based confirmation of mutants. The following PCR primer pairs were used (Table 1 and Fig. 1): FlhA-F1 and KanDM-R2 (1.2-kb product only in mutants), FlhA-R1 and KanDM-F2 (1.8-kb product only in mutants), FlhA-F1 and FlhA-R1 (2.4-kb product in wild-type, 3.6-kb product in mutants). PCR conditions were: 94°C for 5 min (first cycle only), 30 cycles of 94°C for 60 s, 60°C for 90 s and 72°C for 90 s, followed by extension for 5 min at 72°C. The expected size product (or lack of product) was obtained in all cases (data not shown).
Urease extract preparations, protein determinations and urease activity determinations
For H. pylori [42] or for E. coli containing the H. pylori urease gene cluster [9], extracts were prepared and protein concentration was determined as described. The phenol hypochlorite assay for urease activity was as described previously [9, 42]. For P. mirabilis or for E. coli containing the P. mirabilis urease gene cluster, extracts were prepared and measured for protein concentration and for urease activity by the phenol red urease assay as described previously [25].
ß-galactosidase activity determinations
E. coli cells were grown to mid-exponential phase with appropriate antibiotics as described above. ß-Galactosidase activity was determined by the method of Miller [43].
Inoculation of gerbils, tissue processing and recovery of H. pylori
Animal experiments were performed at the University of Maryland, with the approval of the Institute Animal Care and Use Committee. Male Mongolian gerbils (Meriones unguiculatus; Charles River) were inoculated twice orally (2 days apart) with 50 µl of F12-broth-grown H. pylori strains (SS1 background) suspended in sterile PBS (pH 7.4) to 109 viable cfu/ml. Control animals received PBS. At 4 weeks after infection, animals were anaesthetised (Avertin, 125 mg/kg), exsanguinated by cardiac puncture and euthanased by cervical dislocation. Stomachs were removed, dissected longitudinally along the greater curvature and washed several times in sterile PBS. The antrum was dissected into two halves. One was weighed and then homogenised (Ultra-Turrax T25, IKA Works.) in 1 ml of sterile PBS. The other half was fixed in formalin 10% for histology. Antrum homogenates and dilutions of them in PBS were plated for viable counts in triplicate on CBA containing nalidixic acid 10 µg/ml, vancomycin 10 µg/ml, amphotericin B 2 µg/ml, bacitracin 30 µg/ml, polymyxin B 10 U/ml and trimethoprim 10 µg/ml to suppress normal flora.
Histology
Antrum sections were embedded in paraffin, sectioned (5 µm), stained with haematoxylin and eosin and evaluated in a blind fashion.
Statistical analysis of data
Statistical analyses of urease and ß-galactosidase activities were calculated by the alternative Welch's t test; statistical analysis of colonisation data was made by the Mann-Whitney t test. Instat 2.03 software (GraphPad Software, San Diego, CA, USA) was employed. p <0.05 was considered statistically significant.
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Results |
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Urease activity of H. pylori flbA mutants
Eleven of 14 flbA mutants of strain UMAB41 [45] and two of three flbA mutants of strain 43504 had elevated urease activity compared with the corresponding wild-type strain (Fig. 2a and b, respectively; representatives shown). In contrast, flbA mutants of strains 26695 (21 of 31 mutants tested) and HPDJM17 (4 of 4 mutants tested), showed reduced urease activity (Fig. 2c and d, respectively; representatives shown), whereas flbA mutants of the fresh clinical isolate J75 had no detectable urease activity (2 of 2 mutants tested; Fig. 2e). Other strains of H. pylori containing the flbA mutation exhibited no effect on urease activity: strain SS1 (3 of 3), fresh clinical isolates J68 (2 of 2), B194A (3 of 3), and J166 (3 of 3). The urease data were consistent regardless of the construct (flbA1 or flbA2) used to obtain the flbA mutants. Two-thirds (42 of 65) of the flbA mutants and five of nine H. pylori strain backgrounds showed changed urease activity, but whether this was an increase or decrease was clearly strain-dependent. Consistent data were obtained for wild-type and mutants of a single strain. The second observation from these experiments was that urease activity among wild-type H. pylori strains varied widely, ranging from 7000 units for strain 26695 to 55 000 units for strain HPDJM17 (Fig. 2a–e).
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H. pylori flbA effect on urease activity in E. coli containing genes for H. pylori urease and NixA nickel transporter
Because flbA caused a strain-dependent modulation of urease activity in H. pylori, it would be difficult to decipher the reasons for the strain differences without extensive genetic characterisation of them. Therefore, an E. coli model of H. pylori urease activity was used, in which genetic variability could be minimised. In this model, the H. pylori urease gene cluster and the nixA nickel transporter gene were on plasmid pHP8080 and this permitted urease activity in E. coli [9]. The model allowed exploration of genes that affect urease or nixA expression without the excessive variability observed with H. pylori. To investigate further the role of flbA in modulating H. pylori urease activity, the flbA gene was subcloned and the resultant plasmid (pBS-flbA) DNA was transformed into E. coli (pHP8080). Urease activity was reduced 15-fold compared with the vector control strain (Fig. 3). Disruption of flbA with a kanamycin resistance cassette restored urease activity in E. coli (pHP8080/pBS-flbA::aphA3) to levels of the vector control strain, E. coli (pHP8080/pBS) (Fig. 3). This suggested that flbA functioned as a urease-decreasing factor in the E. coli model.
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Effect of flbA on the H. pylori urease promoter and on expression of the nixA promoter
The flbA gene may decrease urease activity by decreasing promoter activity of urease or nixA within pHP8080, or by increasing turnover of urease subunits. It was shown previously that flbA decreased expression of the urease subunits UreA and UreB, supporting the increased turnover model [9]. To address the other two possibilities, flbA was transformed into E. coli containing nixA promoter- or urease promoter-lacZ transcriptional fusion plasmids (Table 1) and the resultant strains were assayed for ß-galactosidase activity. H. pylori flbA had no effect on the H. pylori urease promoter in E. coli strain MC1061 containing pRS415-ureAP (urease promoter-lacZ fusion) and pCC038 (Table 1; low copy plasmid harbouring flbA); the mean ß-galactosidase activity was 1275 Miller Units in MC1061 (pRS415-ureAP/pCC038) versus 1529 Miller Units in the vector control strain, MC1061 (pRS415-ureAP/pKHKS303) (p >0.05). Similarly, no differences were observed when the same constructs were transformed into E. coli strain DH5
. No ß-galactosidase activity was observed when the H. pylori urease promoter was omitted from the construct (as plasmid pRS415). In contrast, the flbA gene significantly decreased expression of the nixA promoter in E. coli MC1061 (pLX2106-nixAP) by about three-fold (p <0.0001) (Fig. 4). No ß-galactosidase activity was observed when the H. pylori nixA promoter was omitted from the construct (as plasmid pLX2106).
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Effect of flbA on P. mirabilis urease activity in E. coli
Because flbA decreased urease activity of some H. pylori strains, it was of interest to investigate the effect of flbA on other bacterial ureases. The P. mirabilis urease was chosen as a model because urease regulation is well understood in this system [7]. The P. mirabilis urease gene cluster has a positive transcriptional activator of urease gene expression, UreR, which is divergently transcribed from the rest of the ureDABCEFG urease gene cluster and activates transcription of itself and the ureDABCEFG operon only in the presence of urea. Plasmid pCC038, containing flbA in low copy, was transformed into E. coli DH5 harbouring pMID1010, which encodes the entire P. mirabilis urease gene cluster. Transformants were grown in the presence of 100 mM urea and assayed for urease activity. Urease activity was decreased seven-fold when compared with the vector control-containing strain, DH5 (pKHKS303/pMID1010) (p <0.001) (Fig. 5a). Only very low basal levels of urease activity were observed for both strains cultured in the absence of urea.
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Effect of flbA on urease activity in P. mirabilis HI4320
When grown in the presence of 100 mM urea, P. mirabilis HI4320 (pCC038), which contains flbA, produced 20% less urease activity than P. mirabilis containing the vector control, pKHKS303 (Fig. 5b). This suggested that the flbA gene product of H. pylori repressed the expression of urease produced by P. mirabilis. Only very low basal levels of urease activity were observed for both strains when cultured in the absence of urea.
Effect of flbA on P. mirabilis ureD promoter expression in E. coli
To determine whether flbA repressed the P. mirabilis urease promoter, plasmid pCC038 containing flbA or the vector control, pKHKS303, was transformed into E. coli DH5a harbouring plasmid p
R10bureD-lacZ, which encodes the P. mirabilis ureD promoter transcriptionally fused to lacZYA [26]. This plasmid also has the functional ureR gene, which is required for expression of the ureD promoter. ß-Galactosidase activity from urea-induced cultures of DH5 (pR10bureD-lacZ/pCC038) was decreased 4–5-fold (p <0.001), as compared with the vector control strain, DH5 (pR10bureD-lacZ/pKHKS303) (Fig. 5c). Only nominal basal levels of ß-galactosidase activity were detected in the uninduced controls grown in the absence of urea. This suggested that the flbA-mediated decrease of urease activity was dependent on urea and a functional UreR.
Requirement for flbA for colonisation of gerbils by H. pylori
Previous work based solely on in-vitro data has speculated that flbA homologues are important in virulence [29–33]. To determine whether flbA was important for virulence in vivo, gerbils were inoculated with either wild-type H. pylori strain SS1, the isogenic flbA mutant or sterile buffer. Of six animals inoculated with SS1, five were colonised with a mean of 5.4x106cfu/g of antrum (Table 2). In contrast, only one of six animals inoculated with the flbA mutant was colonised and this one animal had a mean of only 5.5x103cfu/g of antrum, barely above the detection limit of 9x102cfu/g. Lack of colonisation by the flbA mutant was not due to loss of urease activity, because the flbA mutant of strain SS1 had wild-type urease activity. No H. pylori or Helicobacter-like organisms were recovered from animals inoculated with buffer alone. These results indicated that flbA was required for H. pylori to colonise gerbils.
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Occurrence of chronic gastritis and ulcers in antral tissue from gerbils infected with wild-type H. pylori and the flbA mutant
The antrum from gerbils inoculated with wild-type H. pylori strain SS1 exhibited micro-ulcer formation (three of six animals) (Fig. 6a), lymphoid follicle formation and lymphocytic infiltration (one of six animals) (Fig. 6b), disruption of the ordered gastric pit and glandular architecture (six of six animals) and small foci of necrosis. In contrast, the antra from gerbils inoculated with the flbA mutant (six of six animals) (Fig. 6c) or sterile buffer (Fig. 6d) exhibited no lesions.
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Discussion |
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The contrasting mechanisms of flbA-mediated modulation of urease in H. pylori and P. mirabilis may reflect the differences in the importance of a high affinity nickel transporter (Table 3) and the distinct niches that these two organisms occupy in vivo. H. pylori, which has very few regulatory genes [23, 24], may exert regulatory control of gene expression through more subtle mechanisms than observed for organisms with larger genomes and more regulatory genes such as P. mirabilis. H. pylori is found in a very low nickel environment (0.1–0.5 µg/L in serum and presumably in similar amounts in the gastric milieu [46–48]) and thus has evolved the high affinity nickel transporter nixA for optimal delivery of nickel to apo-urease. In contrast, P. mirabilis resides in the urinary tract, where nickel concentrations are about 10-fold higher (1–3 µg/L [47, 49]) and thus a high affinity nickel transporter is unnecessary.
Although both H. pylori and P. mirabilis ureases were examined in E. coli models that were optimised for urease activity, urease activities in both models were significantly lower (10–30-fold for H. pylori urease, 100-fold for P. mirabilis urease) than those observed in the native organisms, suggesting that additional loci and compounds are necessary to achieve peak urease activity. In support of this hypothesis, Soriano and Hausinger have shown that bicarbonate and GTP are needed to achieve high urease activities in an E. coli model of Klebsiella urease [50]. Furthermore, a previous study uncovered a number of genes, including flbA and a putative DNA helicase, which influenced urease activity [9].
In addition to the evidence for decreased urease activity by flbA in the E. coli models described above, evidence was also obtained that flbA played a significant role in urease modulation in H. pylori itself. Some H. pylori flbA mutants in some strain backgrounds had elevated urease levels, whereas flbA mutants of other strain backgrounds had a decrease or loss in urease activity (Fig. 2) [27]. This suggested that urease regulation differs among H. pylori isolates. This observation was complicated by the finding that H. pylori urease activity decreased (50–90%, depending on the strain) by the tenth in-vitro passage in nearly all strains, regardless of whether flbA was present or not (D. J. McGee and H. L. T. Mobley, unpublished observations). This problem was minimised by transforming DNA from flbA mutants back into the wild-type strain to remove background mutations, and by measuring urease activity from first-passage transformants. Differences in urease activity between wild-type and flbA mutants of various H. pylori strains may be explained by the observations of high mutation frequency leading to genetic variability [51–54] of urease, flbA or nixA or to numerous strain-specific genes [23]. For example, recent studies have found: two different cag(+) strains [55] exhibiting different effects on interleukin-8 production by gastric epithelial cells; strain differences with respect to urease activity in nixA mutants [8, 56]; and strain differences in arginase and urease activity (Fig. 2a–e) [25, 42, 57]. Clearly, phenotypic variation of H. pylori strains can make it difficult for investigators to make generalisations. Therefore, it is recommended that researchers use multiple H. pylori strains to investigate phenotypes of wild-type strains and their corresponding isogenic mutants because misleading or premature conclusions might be reached with only one strain.
This study confirmed the original observation of Schmitz et al. [27] that flbA is required for H. pylori motility and extended it by using more strains and by employing a transparent soft agar containing F-12, a chemically defined medium that supports the growth of H. pylori [38]. The homologous gene flhA is likewise required for motility in P. mirabilis [58]. Because inhibition of H. pylori urease activity by urease inhibitors abolishes motility and chemotaxis through a viscous medium, the proton motive force required for flagellar movement may be generated by the hydrolysis of urea [15, 59–61]. Indeed, urease-negative H. pylori mutants fail to swarm on motility agar [61]. These data, taken together with those of the present study, suggest that flbA alters urease activity in both H. pylori and P. mirabilis and provides a crucial link between two virulence attributes in both human pathogens – urease and motility.
The flbA gene was required for H. pylori to colonise gerbils. This is the first demonstration of a specific flagellar biosynthesis gene being required for H. pylori colonisation of gerbils. One other study suggested that motility is important for colonisation, but the aflagellate variant was of an undefined mutation, was not isogenic with the wild-type strain, and the mutation could potentially be reversible [37]. Notably, no lesions were observed in the antrum of gerbils inoculated with the flbA mutant, whereas lesions of gastritis were common in gerbils infected with wild-type H. pylori.
The H. pylori strains used for the gerbil study were SS1 and the isogenic flbA mutant, which have identical urease activities. Thus, the lack of colonisation by the flbA mutant was not due to altered urease activity. Attempts to complement the mutant have so far been unsuccessful. Other H. pylori mutants that affect flagellar biosynthesis are likewise severely attenuated in other animal models [34–36], emphasising the important role of motility in enabling H. pylori to penetrate the viscous mucous layer to adhere to the gastric epithelial cell surface and avoid the harsh gastric acidity through urease activity.
In summary, the flagellar biosynthesis and regulatory gene flbA was shown to modulate urease of both H. pylori and P. mirabilis, but this modulation was by distinct mechanisms. flbA was required for motility and for virulence in H. pylori.
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Acknowledgments |
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ko K. Potential virulence factors of Proteus bacilli. Microbiol Mol Biol Rev 1997; 61: 65–89.[Abstract]This article has been cited by other articles:
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average 35 000 nmol NH4+/min/mg of protein) was set to 100%. The percent decrease from vector-free HI4320 was calculated by the following formula: 100* [(urease activity of vector-free HI4320) – (urease activity of vector-containing or flbA-containing HI4320)] /urease activity of vector-free HI4320. The average and 2 SD are shown. *p <0.001 compared with HI4320 and with HI4320 (pKHKS303). (c) Effect of flbA on the P. mirabilis urease promoter. E. coli strain DH5