Brucella suis urease encoded by ure1 but not ure2 is necessary for intestinal infection of BALB/c mice

BACKGROUND: In prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. The Brucella genomes contain two urease operons designated as ure1 and ure2. We investigated the role of the two Brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-pH killing. RESULTS: The deduced amino acid sequence of urease-α subunits of operons-1 and -2 exhibited substantial identity with the structural ureases of alpha- and beta-proteobacteria, Gram-positive and Gram-negative bacteria, fungi, and higher plants. Four ure deficient strains were generated by deleting one or more of the genes encoding urease subunits of B. suis strain 1330 by allelic exchange: strain 1330Δure1K (generated by deleting ureD and ureA in ure1 operon), strain 1330Δure2K (ureB and ureC in ure2 operon), strain 1330Δure2C (ureA, ureB, and ureC in ure2 operon), and strain 1330Δure1KΔure2C (ureD and ureA in ure1 operon and ureA, ureB, and ureC in ure2 operon). When grown in urease test broth, strains 1330, 1330Δure2K and 1330Δure2C displayed maximal urease enzyme activity within 24 hours, whereas, strains 1330Δure1K and 1330Δure1KΔure2C exhibited zero urease activity even 96 h after inoculation. Strains 1330Δure1K and 1330Δure1KΔure2C exhibited slower growth rates in tryptic soy broth relative to the wild type strain 1330. When the BALB/c mice were infected intraperitoneally with the strains, six weeks after inoculation, the splenic recovery of the ure deficient strains did not differ from the wild type. In contrast, when the mice were inoculated by gavage, one week after inoculation, strain 1330Δure1KΔure2C was cleared from livers and spleens while the wild type strain 1330 was still present. All B. suis strains were killed when they were incubated in-vitro at pH 2.0. When the strains were incubated at pH 2.0 supplemented with 10 mM urea, strain 1330Δure1K was completely killed, strain 1330Δure2C was partially killed, but strains 1330 and 1330Δure2K were not killed. CONCLUSION: These findings suggest that the ure1 operon is necessary for optimal growth in culture, urease activity, resistance against low-pH killing, and in vivo persistence of B. suis when inoculated by gavage. The ure2 operon apparently enhances the resistance to low-pH killing in-vitro

<it>Brucella suis </it>urease encoded by <it>ure</it>1 but not <it>ure</it>2 is necessary for intestinal infection of BALB/c mice

Abstract Background In prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. The Brucella genomes contain two urease operons designated as ure1 and ure2. We investigated the role of the two Brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-pH killing. Results The deduced amino acid sequence of urease-α subunits of operons-1 and -2 exhibited substantial identity with the structural ureases of alpha- and beta-proteobacteria, Gram-positive and Gram-negative bacteria, fungi, and higher plants. Four ure deficient strains were generated by deleting one or more of the genes encoding urease subunits of B. suis strain 1330 by allelic exchange: strain 1330Δure1K (generated by deleting ureD and ureA in ure1 operon), strain 1330Δure2K (ureB and ureC in ure2 operon), strain 1330Δure2C (ureA, ureB, and ureC in ure2 operon), and strain 1330Δure1KΔure2C (ureD and ureA in ure1 operon and ureA, ureB, and ureC in ure2 operon). When grown in urease test broth, strains 1330, 1330Δure2K and 1330Δure2C displayed maximal urease enzyme activity within 24 hours, whereas, strains 1330Δure1K and 1330Δure1KΔure2C exhibited zero urease activity even 96 h after inoculation. Strains 1330Δure1K and 1330Δure1KΔure2C exhibited slower growth rates in tryptic soy broth relative to the wild type strain 1330. When the BALB/c mice were infected intraperitoneally with the strains, six weeks after inoculation, the splenic recovery of the ure deficient strains did not differ from the wild type. In contrast, when the mice were inoculated by gavage, one week after inoculation, strain 1330Δure1KΔure2C was cleared from livers and spleens while the wild type strain 1330 was still present. All B. suis strains were killed when they were incubated in-vitro at pH 2.0. When the strains were incubated at pH 2.0 supplemented with 10 mM urea, strain 1330Δure1K was completely killed, strain 1330Δure2C was partially killed, but strains 1330 and 1330Δure2K were not killed. Conclusion These findings suggest that the ure1 operon is necessary for optimal growth in culture, urease activity, resistance against low-pH killing, and in vivo persistence of B. suis when inoculated by gavage. The ure2 operon apparently enhances the resistance to low-pH killing in-vitro.</p

Survival of strains 1330, 1330Δ1K, 1330Δ2K, and 1330Δ2C after incubation at pH 2

<p><b>Copyright information:</b></p><p>Taken from "urease encoded by 1 but not 2 is necessary for intestinal infection of BALB/c mice"</p><p>http://www.biomedcentral.com/1471-2180/7/57</p><p>BMC Microbiology 2007;7():57-57.</p><p>Published online 19 Jun 2007</p><p>PMCID:PMC1983905.</p><p></p>0 with or without urea. At each urea concentration, the value for the difference among mean cfu wa

The schematic representations of the operons with corresponding Ure subunits, and deletion sites of mutant strains 1330Δ1K, 1330Δ2K and 1330Δ2C

<p><b>Copyright information:</b></p><p>Taken from "urease encoded by 1 but not 2 is necessary for intestinal infection of BALB/c mice"</p><p>http://www.biomedcentral.com/1471-2180/7/57</p><p>BMC Microbiology 2007;7():57-57.</p><p>Published online 19 Jun 2007</p><p>PMCID:PMC1983905.</p><p></p> A: 1 operon. B: 2 operon. The numbers represent the location of the genes in the chromosome I

Recovery of cfu from livers one week after BALB/c mice were inoculated by gavage with the wild type strain 1330 or strain 1330Δ1KΔ2C with or without urea supplementation

<p><b>Copyright information:</b></p><p>Taken from "urease encoded by 1 but not 2 is necessary for intestinal infection of BALB/c mice"</p><p>http://www.biomedcentral.com/1471-2180/7/57</p><p>BMC Microbiology 2007;7():57-57.</p><p>Published online 19 Jun 2007</p><p>PMCID:PMC1983905.</p><p></p> value for the difference among mean values wa

Recovery of cfu from spleens one week after BALB/c mice were inoculated by gavage with wild type strain 1330 or strain 1330Δ1KΔ2C with or without urea supplementation

<p><b>Copyright information:</b></p><p>Taken from "urease encoded by 1 but not 2 is necessary for intestinal infection of BALB/c mice"</p><p>http://www.biomedcentral.com/1471-2180/7/57</p><p>BMC Microbiology 2007;7():57-57.</p><p>Published online 19 Jun 2007</p><p>PMCID:PMC1983905.</p><p></p> value for the difference among mean values wa