McMillan D J, Shojaei M, Chhatwal G S, Guzmán C A, Walker M J
Department of Biological Sciences, University of Wollongong, N.S.W., Australia.
Microb Pathog. 1996 Nov;21(5):379-94. doi: 10.1006/mpat.1996.0069.
Bordetella bronchiseptica is a ureolytic mammalian respiratory pathogen. We have investigated the regulation of urease in B. bronchiseptica and the potential role of this enzyme in eukaryotic invasion and intracellular survival. Our results indicate urease is a bordetella virulence repressed gene. Urease activity in virulent B. bronchiseptica BB7865 is up-regulated from basal levels by 5 gl1 magnesium sulphate at 37 degrees C. At 30 degrees C, urease activity remained at basal levels, even in the presence on magnesium sulphate, suggesting a second temperature dependent mechanism of urease regulation was also operating. Urease was not inducible by 10 mM urea nor up-regulated in nitrogen limiting conditions. To evaluate the role of urease in intracellular invasion and survival urease-negative mutants of B. bronchiseptica BB7865 and B. bronchiseptica BB7866 were created by transposon mutagenesis, and compared to the urease-positive parental strains in a HeLa cell invasion assay. We demonstrate that increasing the concentration of urea in the assay increased survival of the urease-positive but not urease-negative strains after 24 h, suggesting that urease does have a role in intracellular survival. Partial DNA sequence analysis of an 11.0 kb EcoRI DNA fragment encoding urease activity revealed an open reading frame containing 50%, 45%, 45%, and 41% homology to the UreA urease subunit protein of Klebsiella aerogenes, Proteus vulgaris, Helicobacter pylori and Proteus mirabilis respectively. We also show Bordetella pertussis to contain sequences homologous with a DNA probe containing the gene encoding UreA of B. bronchiseptica indicating the possible presence of cryptic urease genes in this species.
支气管败血波氏杆菌是一种能分解尿素的哺乳动物呼吸道病原体。我们研究了支气管败血波氏杆菌中脲酶的调控以及该酶在真核细胞侵袭和细胞内存活中的潜在作用。我们的结果表明脲酶是波氏杆菌毒力抑制基因。在37℃时，毒力强的支气管败血波氏杆菌BB7865中的脲酶活性通过5 gl1硫酸镁从基础水平上调。在30℃时，即使存在硫酸镁，脲酶活性仍保持在基础水平，这表明还存在第二种依赖温度的脲酶调控机制。脲酶不能被10 mM尿素诱导，在氮限制条件下也不会上调。为了评估脲酶在细胞内侵袭和存活中的作用，通过转座子诱变创建了支气管败血波氏杆菌BB7865和支气管败血波氏杆菌BB7866的脲酶阴性突变体，并在HeLa细胞侵袭试验中与脲酶阳性亲本菌株进行比较。我们证明，在试验中增加尿素浓度可提高脲酶阳性菌株而非脲酶阴性菌株在24小时后的存活率这表明脲酶在细胞内存活中确实起作用。对一个编码脲酶活性的11.0 kb EcoRI DNA片段进行的部分DNA序列分析显示，一个开放阅读框分别与产气克雷伯菌、普通变形杆菌、幽门螺杆菌和奇异变形杆菌的脲酶亚基蛋白UreA具有50%、45%、45%和41%的同源性。我们还表明百日咳博德特氏菌含有与包含支气管败血波氏杆菌UreA编码基因的DNA探针同源的序列，这表明该物种中可能存在隐性脲酶基因。
