Krishnamurthy P, Parlow M, Zitzer J B, Vakil N B, Mobley H L, Levy M, Phadnis S H, Dunn B E
Department of Pathology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
Infect Immun. 1998 Nov;66(11):5060-6. doi: 10.1128/IAI.66.11.5060-5066.1998.
Helicobacter pylori, an important etiologic agent in a variety of gastroduodenal diseases, produces large amounts of urease as an essential colonization factor. We have demonstrated previously that urease is located within the cytoplasm and on the surface of H. pylori both in vivo and in stationary-phase culture. The purpose of the present study was to assess the relative contributions of cytoplasmic and surface-localized urease to the ability of H. pylori to survive exposure to acid in the presence of urea. Toward this end, we compared the acid resistance in vitro of H. pylori cells which possessed only cytoplasmic urease to that of bacteria which possessed both cytoplasmic and surface-localized or extracellular urease. Bacteria with only cytoplasmic urease activity were generated by using freshly subcultured bacteria or by treating repeatedly subcultured H. pylori with flurofamide (1 microM), a potent, but poorly diffusible urease inhibitor. H. pylori with cytoplasmic and surface-located urease activity survived in an acid environment when 5 mM urea was present. In contrast, H. pylori with only cytoplasmic urease shows significantly reduced survival when exposed to acid in the presence of 5 mM urea. Similarly, Escherichia coli SE5000 expressing H. pylori urease and the Ni2+ transport protein NixA, which expresses cytoplasmic urease activity at levels similar to those in wild-type H. pylori, survived minimally when exposed to acid in the presence of 5 to 50 mM urea. We conclude that cytoplasmic urease activity alone is not sufficient (although cytoplasmic urease activity is likely to be necessary) to allow survival of H. pylori in acid; the activity of surface-localized urease is essential for resistance of H. pylori to acid under the assay conditions used. Therefore, the mechanism whereby urease becomes associated with the surface of H. pylori, which involves release of the enzyme from bacteria due to autolysis followed by adsorption of the enzyme to the surface of intact bacteria ("altruistic autolysis"), is essential for survival of H. pylori in an acid environment. The ability of H. pylori to survive exposure to low pH is likely to depend on a combination of both cytoplasmic and surface-associated urease activities.
幽门螺杆菌是多种胃十二指肠疾病的重要病原体，它产生大量尿素酶作为一种重要的定植因子。我们之前已经证明，在体内和稳定期培养中，尿素酶都存在于幽门螺杆菌的细胞质内和表面。本研究的目的是评估细胞质和表面定位的尿素酶对幽门螺杆菌在有尿素存在的情况下抵抗酸暴露能力的相对贡献。为此，我们比较了仅具有细胞质尿素酶的幽门螺杆菌细胞与同时具有细胞质和表面定位或细胞外尿素酶的细菌在体外的耐酸性。仅具有细胞质尿素酶活性的细菌是通过使用新鲜传代培养的细菌或用氟罗酰胺（1微摩尔）处理反复传代培养的幽门螺杆菌产生的，氟罗酰胺是一种强效但扩散性差的尿素酶抑制剂。当存在5毫摩尔尿素时，具有细胞质和表面定位尿素酶活性的幽门螺杆菌能在酸性环境中存活。相比之下，仅具有细胞质尿素酶的幽门螺杆菌在5毫摩尔尿素存在的情况下暴露于酸时，存活率显著降低。同样，表达幽门螺杆菌尿素酶和镍离子转运蛋白NixA的大肠杆菌SE5000，其细胞质尿素酶活性水平与野生型幽门螺杆菌相似，在5至50毫摩尔尿素存在的情况下暴露于酸时，存活率极低。我们得出结论，仅细胞质尿素酶活性不足以（尽管细胞质尿素酶活性可能是必需的）使幽门螺杆菌在酸性环境中存活；在所使用的检测条件下，表面定位的尿素酶活性对于幽门螺杆菌抵抗酸至关重要。因此，尿素酶与幽门螺杆菌表面结合的机制，即由于自溶使酶从细菌中释放出来，随后酶吸附到完整细菌的表面（“利他性自溶”），对于幽门螺杆菌在酸性环境中的存活至关重要。幽门螺杆菌在低pH环境中存活的能力可能取决于细胞质和表面相关尿素酶活性的共同作用。
