Mobley H L, Green D M, Trifillis A L, Johnson D E, Chippendale G R, Lockatell C V, Jones B D, Warren J W
Department of Medicine, University of Maryland School of Medicine, Baltimore 21201.
Infect Immun. 1990 May;58(5):1281-9. doi: 10.1128/iai.58.5.1281-1289.1990.
Acute pyelonephritis, a complication of Escherichia coli bacteriuria, must represent a bacterial invasion through the kidney epithelium. To study this process, we overlaid bacterial suspensions onto monolayers of cultured human kidney proximal tubular epithelial cells and measured cytotoxicity by release of lactate dehydrogenase (LDH). Thirty-four isolates cultured from patients with acute pyelonephritis were screened for the ability to cause pyelonephritis in CBA mice by transurethral challenge. The eight most virulent strains (greater than or equal to 70% of mice challenged developed greater than or equal to 10(3) CFU/g of kidney after 48 h) were selected for study. Each strain displayed mannose-resistant hemagglutination of human O erythrocytes; three strains were phenotypically and genotypically hemolytic. Pyelonephritogenic strains were significantly more cytotoxic (30.1 +/- 9.5% LDH release after 18 h) than eight fecal control strains (13.5 +/- 11.5% LDH release; P = 0.0068). We selected the most cytotoxic strain, CFT073, for further study. Sterile filtrate from this hemolytic strain was significantly more cytotoxic than was the filtrate of the fecal control strain, FN414. Transposon mutagenesis of CFT073 with TnphoA abolished hemolytic activity and cytotoxicity by both whole cells and sterile filtrate. Southern blot analysis revealed that the Tnphoa insertion mapped to the E. coli chromosomal hly determinant within a 12-kilobase SalI restriction fragment. Transformation of a nonhemolytic strain, CPZ005 with plasmid pSF4000, which carries a cloned hemolysin determinant, resulted in highly elevated cytotoxicity. Light micrographs of proximal tubular epithelial cell cultures demonstrated cell damage by pyelonephritogenic strains that was not induced by a fecal strain or the hemolysin-deficient mutant. Results indicate that pyelonephritogenic E. coli strains are more frequently cytotoxic for a putative target, that is, human renal tubular epithelium, than are fecal isolates. Hemolysin, in some strains, is apparently responsible for this cytotoxicity.
急性肾盂肾炎是大肠杆菌菌尿的一种并发症，必定是细菌通过肾上皮的侵袭所致。为研究此过程，我们将细菌悬液覆盖于培养的人肾近端小管上皮细胞单层上，并通过乳酸脱氢酶（LDH）释放来测定细胞毒性。从急性肾盂肾炎患者分离培养的34株菌株，经尿道攻击筛选其在CBA小鼠中引起肾盂肾炎的能力。选择8株毒性最强的菌株（48小时后，大于或等于70%受攻击小鼠的肾脏中菌落形成单位大于或等于10³CFU/g）进行研究。每株菌株均表现出对人O型红细胞的甘露糖抗性血凝反应；3株菌株在表型和基因型上具有溶血活性。致肾盂肾炎菌株的细胞毒性（18小时后LDH释放30.1±9.5%）显著高于8株粪便对照菌株（LDH释放13.5±11.5%；P = 0.0068）。我们选择细胞毒性最强的菌株CFT073进行进一步研究。该溶血菌株的无菌滤液比粪便对照菌株FN414的滤液细胞毒性显著更强。用TnphoA对CFT073进行转座子诱变消除了全细胞和无菌滤液的溶血活性及细胞毒性。Southern印迹分析显示，Tnphoa插入定位于大肠杆菌染色体hly决定簇内一个12千碱基的SalI限制片段。用携带克隆溶血素决定簇的质粒pSF4000转化非溶血菌株CPZ005，导致细胞毒性显著升高。近端小管上皮细胞培养物的光学显微镜照片显示，致肾盂肾炎菌株可导致细胞损伤，而粪便菌株或溶血素缺陷突变体则不会。结果表明，与粪便分离株相比，致肾盂肾炎的大肠杆菌菌株对假定靶标即人肾小管上皮细胞的细胞毒性更常见。在某些菌株中，溶血素显然是这种细胞毒性的原因。
