Expression of urease does not affect the ability of Bordetella bronchiseptica to colonise and persist in the murine respiratory tract

Abstract

To investigate the role played by urease during the Bordetella bronchiseptica infection process, the ability to colonise and persist in the mouse respiratory tract of a urease-negative B. bronchiseptica BB7865 and a BB7865 derivative constitutively expressing urease was compared with that of the wild-type strain. The results obtained showed that neither constitutive expression nor abolishment of urease activity had any significant effect on the course of B. bronchiseptica infection. Therefore, under our experimental conditions, urease is not essential for B. bronchiseptica to colonise and persist within the murine host.

Urease, Respiratory challenge, Bordetella bronchiseptica

1 Introduction

Bordetella bronchiseptica is a Gram-negative respiratory pathogen able to infect both wild and domestic animals [1]. The expression of most virulence determinants, including adhesins and toxins, involved in the pathogenesis of B. bronchiseptica are positively regulated by the products of the bvg (Bordetella virulence gene) locus. This locus encodes a transmembrane environmental sensory protein (BvgS) and a transcriptional activator (BvgA) [2]. Activation of the bvg locus (e.g. temperatures of 37°C or MgSO₄ concentrations under 5 mM) results in the expression of both the bvg gene and bvg-activated gene products. The bvg locus is also a negative regulator of certain phenotypes such as motility and siderophore expression [3–5].

It has been suggested that bvg-activated genes are expressed during or immediately after the infection process and are the only genes required for infection. In this model, bvg-repressed genes are expressed when bacteria are outside the host and are only required for bacterial survival in the external environment [6]. However, the formulation of this model has neglected the capacity of B. bronchiseptica to survive in various eukaryotic cell lines [7–10]. The intracellular niche is not only rich in nutrients but also provides protection against specific and non-specific host clearance mechanisms. It has also been shown that constitutive bvg-negative mutant strains of B. bronchiseptica exhibit a significant long term survival advantage over the wild-type strains in in vitro models, suggesting that some bvg-activated gene products are not required and may even be detrimental for intracellular persistence of bacteria. In this regard, one study has demonstrated down-regulation of the bvg-activated adenylate cyclase upon invasion of eukaryotic cells [7]. Down-regulation of bvg-activated genes, which encode products toxic to eukaryotic cells, may constitute a mechanism to reduce cell damage, thereby reducing detection by the host immune system.

We have previously shown that urease expression in B. bronchiseptica is negatively regulated by the bvg locus [11]. In wild-type B. bronchiseptica BB7865, urease is expressed maximally at 37°C only in the presence of modulating concentrations of MgSO₄, whereas in the isogenic avirulent ΔbvgS derivative BB7866, it is constitutively expressed independently of the presence or absence of MgSO₄. The fact that expression of urease occurs at 37°C suggests that this protein may play a role during the infection process, possibly during the intracellular stage. Ammonia, a product of the urea catalysis, has been shown to inhibit phagosome-lysosome fusion in vitro [12]. In addition, the increase in pH that accompanies urea degradation may also facilitate bacterial survival within phagosomes. We have previously shown, using an in vitro infection model of B. bronchiseptica, that an increment in the urea concentration resulted in increased bacterial survival [11]. In a separate study using a mixed guinea pig infection model, urease deficient mutants were found to survive more readily than the parental wild-type strains [13]. To provide further insight into the role or urease in the pathogenesis of B. bronchiseptica, a urease-negative B. bronchiseptica BB7865 and a BB7865 derivative constitutively expressing urease was compared with that of the wild-type strain in an in vitro murine respiratory infection assay.

2 Materials and methods

2.1 Bacterial strains and manipulations

B. bronchiseptica BB7865 is a wild-type strain that undergoes phenotypic modulation between the bvg-positive and bvg-negative phases. BB7866 is a isogenic avirulent derivative of BB7865 that carries a deletion within BvgS [14]. BB7865 U5 is a urease-negative strain generated by transposon mutagenesis of BB7865 [11]. Unless otherwise specified, all Bordetella strains were grown on Bordet-Gengou agar supplemented with 15% defibrinated horse blood and 10 g l⁻¹ glycerol. Liquid cultures were grown in modified versions of Stainer and Scholte (SS) medium [15]. SS-X containing NaCl was used to grow B. bronchiseptica in the bvg-positive phase and SS-C medium modified by the addition of 5 g l⁻¹ magnesium sulfate and removal of sodium chloride was used to grow strains in the bvg-negative phase. Escherichia coli strains were grown on Z agar or in Luria Bertani broth. All genetic manipulations, conjugations and urease assays were performed as previously described [11].

2.2 In vitro murine infection assays

A mouse model was used to determine the ability of individual B. bronchiseptica strains to colonise and persist in the respiratory tract. Approximately 10⁵ bacteria were resuspended in 1% casamino acids in PBS and administered intranasally to 4-weeks old female BALB/c mice. Groups of animals (n= 4) were killed at 2 h and at 5, 12, 20 and 40 days after challenge. The lungs were homogenised using a Polytron PT 1200 homogeniser (Kinematica, Switzerland) and the number of viable microorganisms was determined by plating appropriate dilutions onto Bordet-Gengou agar supplemented with kanamycin and cephalexin.

3 Results and discussion

3.1 Construction and characterisation of B. bronchiseptica bbuR mutants

To construct a virulent B. bronchiseptica strain that constitutively expressed urease, single cross-over mutations were introduced into the bbuR, located directly upstream of the urease operon [16]. The open reading frame encoded by bbuR is homologous to the LysR family of transcriptional regulators. Within this family, BbuR is most homologous to the subset of OxyR proteins that protects bacteria from oxidative attack in the phagolysosomal compartment [17] and to NAC, a protein required for the expression of urease in K. aerogenes. A potential BbuR binding region, homologous to the NAC and LysR binding consensus sequences [18,19], is located upstream from the putative urease promoter region [16]. The location and orientation of bbuR in respect to the urease operon is also similar to that of ureR, a transcriptional activator of urease in Proteus mirabilis[20]. Together, these data led us to hypothesise that BbuR was involved in the regulation of urease expression in B. bronchiseptica.

Single cross-over mutations of bbuR in BB7865 and BB7866 were constructed by homologous recombination. Two primers, BB18 (5′-GGGAATTCCAACAGTATCGAGGGAGCCTTG-3′) and BB19 (5′-GGGAATTCTATGCCGCCTACTTCCTGCAAC-3′), designed with flanking EcoRI restriction sites, were used to amplify an internal 0.5-kb segment of bbuR from pMC3 [10], a plasmid carrying a partial bbuR open reading frame. The PCR product was cloned into the PCR cloning vector pCR2.1 (QIAGEN, USA) and subcloned into the EcoRI site of the kanamycin resistant suicide vector pJP5603 [21]. The resulting plasmid, pMC48, was then introduced into the donor strain E. coli S-17λpir. Conjugation between E. coli S-17λpir harbouring pMC48 and recipient BB7866 or BB7865 strains was carried out and transconjugants were selected on Bordet-Gengou agar supplemented with kanamycin and cephalexin. Southern hybridisation was then used to demonstrate the successful incorporation of pMC48 into the bbuR locus of a single kanamycin resistant mutant from each mating (Fig. 1). The two bbuR mutants, BB7865 B1 and BB7866 B8 were used in a further analysis.

Figure 1

Southern blot analysis of XhoI-restricted chromosomal DNA from BB7865, BB7866, BB7865 B1 and BB7866 B8 (lanes 1–4) probed with pMC48.

The urease activities of BB7865 B1 and BB7866 B8, when grown in media promoting either the bvg-positive and bvg-negative phenotypes, were compared to the parental strains. Growth favouring the bvg-positive phenotype was achieved by growing B. bronchiseptica in SS-X media at 37°C. Growth in SS-X at 30°C or growth in SS-C media at 37 or 30°C results in expression of the bvg-negative phenotype. In contrast to the regulated expression of urease in BB7865, urease activity was constitutive in BB7865 B1. As urease activity of BB7866 is also constitutive, BB7866 B8 showed no difference in comparison to the parental strain, regardless of the incubation temperature or growth media (Fig. 2). These results provide further evidence for a role for BbuR in the repression of urease activity in B. bronchiseptica. As abolition of bvg or bbuR activity results in the constitutive expression of urease, it is tempting to suggest that bvg may exert its regulatory influence through bbuR. However, no identifiable BvgA or BvgR binding site lies proximal to the putative bbuR promoter [22,23].

Figure 2

Comparison of the urease activities of (a) BB7865 and (b) BB7866 with their bbuR deficient derivatives BB7865 B1 and BB7865 B8. Bacteria were grown in SS-X or SS-C at both 30 and 37°C and urease was activity assayed. Data represent the means of at least three independent experiments±S.E.M.

3.2 Murine respiratory infection assays

We have previously shown, using an in vitro infection model of B. bronchiseptica, that an increment in the urea concentration resulted in increased bacterial survival [11]. In the present study, we have chosen a mouse model to determine the abilities of BB7865 B1 and BB7865 U5 mutants to colonise and persist in the respiratory tract in comparison with the wild-type strains. The results in Fig. 3 show that a similar progressive bacterial growth was observed during the first 5 days post-infection in all the strains tested. After this time, bacterial numbers declined gradually, but were still recoverable 40 days post-infection in all bacterial strains. No significant differences in the survival rate of mice infected with any of the strains was detected at any of the time points. As all strains showed similar growth kinetics in vivo, we can suggest that urease is not a critical factor during the infection processes.

Figure 3

Colonisation and persistence of BB7865, BB7865 B1 and BB7865 U5 after intranasal inoculation of mice. The number of viable B. bronchiseptica at 2 h and at 5, 12, 20 and 40 days post-inoculation was determined by serial dilution of homogenised murine lung tissue onto Bordet-Gengou agar. Data points represent the geometric means of four individual mice.

Both B. bronchiseptica and Bordetella pertussis produce oxidoreductases, including superoxide dismutase, SOD, [24,25] and catalase [26], which are presumed to be involved in intracellular survival. These enzymes detoxify reactive oxygen metabolites in the phagolysosomal compartment that are toxic to bacterial cells. However, abolition of sodB activity or deletion of the catalase gene katA does not affect the ability of B. pertussis to survive within polymorphonuclear leucocytes [25,26]. Furthermore, it was shown that sodA and sodB mutants of B. pertussis and B. bronchiseptica survive equally well as the parental strains in a mouse respiratory infection model [24]. This could be explained by the existence of redundant oxidoreductase activities which compensate for mutations in any of these genes. If urease is involved in intracellular survival, similar compensatory mechanisms may be employed by B. bronchiseptica to overcome the abolition of urease in vivo. To assess this possibility, however, a greater understanding of the general processes involved in the intracellular survival of B. bronchiseptica must first be developed.

Acknowledgments

This work was supported by grants from the Australian Research Council and the University of Wollongong.