Interruption of the cydB locus in Brucella abortus attenuates intracellular survival and virulence in the mouse model of infection - PubMed (original) (raw)

Interruption of the cydB locus in Brucella abortus attenuates intracellular survival and virulence in the mouse model of infection

S Endley et al. J Bacteriol. 2001 Apr.

Abstract

Brucellosis is characterized by abortion in ruminants and a protracted undulant fever in humans, which often results in severe pathological manifestations. Scant information exists about the molecular mechanisms employed by Brucella abortus to combat host defenses or to persist and replicate within host cells. Transposon (Tn5) mutagenesis of B. abortus and the subsequent screening of mutants for sensitivity to killing in murine macrophages and in the mouse model led to the identification of mutants which were severely attenuated for intracellular survival. One group of mutants was interrupted in cydB, a gene that is part of the cydAB operon encoding cytochrome bd oxidase, which catalyzes an alternate terminal electron transport step in bacterial respiration. The elevated affinity for molecular oxygen of this enzyme in Escherichia coli has suggested that it is involved in the protection of sensitive enzymatic activities such as those of hydrogenases and nitrogenases from damage. B. abortus cydB::Tn5 strains exhibited heightened sensitivity to the respiratory inhibitors zinc and azide, highly reactive oxygen species such as hydrogen peroxide, low pH, and attenuated virulence in the mouse model of infection. Virulence was restored by an intact copy of cydAB or by B. abortus genes encoding the oxidative radical-scavenging enzyme Cu/Zn superoxide dismutase or catalase. These results suggest a bifunctional role for the products of the cydAB operon, both in preventing the buildup of oxidative free radicals and in detoxifying the intracellular compartment, thus indicating the importance of these products in preventing intracellular destruction. Intracellular conditions that favor expression of the cydAB operon are under investigation and may be linked to the acid sensitivity also observed in this strain.

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Figures

FIG. 1

Restriction map of the cydAB region. The restriction map was deduced from the partial sequence derived from the _Xba_I fragment obtained from the λ2001 recombinant. Outlined arrows represent the size and direction of transcription from the cydA and cydB structural genes. The cydA open reading frame is 526 amino acids, and the cydB open reading frame is 385 amino acids. There are two open reading frames upstream of the cydAB structural genes that share homology with the ATP binding cassette (ABC) transporters cydDC. The _Bam_HI site within cydA is 430 nucleotides downstream from the presumed translational start site. pSEK101, pSEK102, and pSEK103 carry the corresponding _Xba_I, _Hin_dIII, and _Bam_HI fragments, respectively, on pBBR1MCS, a B. abortus shuttle vector. The _Hin_dIII site downstream from cydB is 589 nucleotides from the putative stop codon of cydB.

FIG. 2

Sensitivity of S2308 and S2308 cydB::Tn_5_ to respiratory inhibitors. Liquid cultures were grown to mid-log phase, and portions were serially diluted and plated on TSA plates in the presence of 0.15 mM ZnSO4 and 0.15 mM NaN3 as described in Materials and Methods. Results are expressed as percentages of the cells growing on TSA plates. The limit of detection was 10 CFU/ml. Abbreviations of bacterial strains and plasmids are defined in Table 1.

FIG. 3

Hydrogen peroxide sensitivity assay. Bacteria from exponential-phase cultures grown in TSB were plated on TSA plates at 108 CFU/plate (as described in Materials and Methods). Oxidative killing was determined by measuring the diameter of the clear zone around a disk containing 5 μl of 30% hydrogen peroxide. The values are from three independent experiments and represent the averages of clearance zones from nine plates. Error bars represent the means of the three independent trials ± standard deviations. The asterisks denote values significantly different from those of the wild-type B. abortus S2308 as determined by analysis of variance using the Tukey test method. For a description of bacterial strains and plasmids, see Table 1.

FIG. 4

Acid tolerance response of S2308 and S2308 cydB::Tn_5_. Stationary-phase cells were exposed to TSB adjusted to various pHs for 2 h. After incubation, bacterial cells were serially diluted and plated in triplicate on TSA plates. The results expressed are averages of CFU per milliliter from triplicate plates, from two independent experiments. Error bars represent average values ± the standard deviations from the means. The asterisks denote values significantly different from those of wild-type S2308 (P < 0.05) as determined by analysis of variance using the Tukey test method. Abbreviations of bacterial strains and plasmids are defined in Table 1.

FIG. 5

Recovery of B. abortus from the spleens of mice. Mice were inoculated with 5 × 104 CFU (solid arrow) of either the wild-type strain S2308 (open triangles); the _cyd_-negative mutant BA582 (open circles); or the _cyd_-negative mutant with either the intact cydAB locus, BA582/pSEK102 (closed triangles), or a truncated version of the cydAB locus, BA582/pSEK103 (open triangles). Recovery of inoculated organisms from the spleens of mice is presented as the log10 CFU per spleen from serial dilutions plated in duplicate and averaged over five mice. Error bars represent standard deviations from the means. The limit of detection in these experiments is ≤25 CFU per spleen.

FIG. 6

Restoration of virulence of B. abortus cydB::Tn_5_ by Brucella sod and kat genes. Mice were inoculated with a 1:1 ratio of wild-type strain S2308 to cydB::Tn_5_ mutant strain BA582 or BA582 containing the plasmid pSEK101 (cyd+), pMEK21 (kat+), or pMEK15 (sod+). Brucellae were recovered from the spleens of mice, and the ratios of wild-type to mutant bacteria were calculated using the following formulas: CFUTSA = total number of Brucella bacteria recovered, CFUTSA/kan = the number of mutant bacteria recovered, and CFUTSA − CFUTSA/kan = the number of wild-type organisms recovered. Data are presented as the log10 of the ratios of wild-type to mutant CFU from serial dilutions plated in duplicate and averaged over four mice. Error bars represent standard deviations from the means. Asterisks indicate statistically significant differences. The limit of detection was ≤25 CFU/spleen.

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Interruption of the cydB locus in Brucella abortus attenuates intracellular survival and virulence in the mouse model of infection - PubMed (original) (raw)