Russo T A, Jodush S T, Brown J J, Johnson J R
Department of Medicine, SUNY at Buffalo 14214, USA.
Mol Microbiol. 1996 Oct;22(2):217-29. doi: 10.1046/j.1365-2958.1996.00096.x.
Our knowledge of the traits possessed by extraintestinal isolates of Escherichia coli, necessary for growth and survival in urine, is limited. To identify such determinants, transposon (TnphoA'1,4) mutant libraries of a clinical isolate (CP9) were generated and screened for derivatives exhibiting decreased growth in urine in vitro, and for mutants with active lacZ fusions that were induced in urine relative to laboratory medium. Using this approach we identified two genes, guaA (CPA24) and argC (CPI-1), which were previously unrecognized as being important for growth in human urine. Unexpectedly, not only does CPA24 (guaA) not grow in human urine in vitro, but it is sensitive to its effects, undergoing a 2-3 log loss of viability over 6 h. By contrast, CPA24 neither grows nor is killed in M9 minimal medium and artificial urine. Therefore, we postulate that lack of guanine or its derivatives in urine, and the inability of CPA24 to synthesize these compounds de novo, prevents CPA24 from synthesizing other guanine (or derivatives)-dependent products that are critical for growth and survival in urine. Although it seems logical that decreased growth in urine in vitro should correlate with diminished urovirulence, this concept was tested by challenging mice with CPA24 in vivo in a mouse model of urinary tract infection (UTI). Indeed, CPA24 was found to be significantly less virulent compared with its wild-type parent CP9. CPI-1(argC) was identified because of the significant induction of its argC::lacZ fusion in urine. Subsequent testing in urine demonstrated that its growth was significantly diminished in all urine samples tested (four females, three males). Polyamine synthesis is dependent upon, in part, the arginine biosynthetic pathway. Therefore, we tested whether the induction of argC in urine and/or the decreased growth of CPI-1 was a result of low levels of polyamines or arginine in urine. The results suggest that low levels of arginine, but not polyamines, in human urine are responsible. When tested in vivo in the mouse model of UTI, CPI-1 was also found to be significantly less virulent than CP9. In summary, we have established that guaA and argC are the first genes, which we are aware of, that have been shown to contribute to the growth of E. coli in urine in vitro and both have diminished urovirulence in vivo. These results support the concept that urine can be used in vitro as a screening tool to identify urovirulence traits.
我们对大肠杆菌肠外分离株在尿液中生长和存活所必需的特性的了解有限。为了鉴定这些决定因素，构建了临床分离株（CP9）的转座子（TnphoA'1,4）突变体文库，并筛选了在体外尿液中生长减少的衍生物，以及与实验室培养基相比在尿液中被诱导产生活性lacZ融合的突变体。通过这种方法，我们鉴定出两个基因，guaA（CPA24）和argC（CPI-1），它们以前未被认为对在人尿液中生长很重要。出乎意料的是，CPA24（guaA）不仅在体外人尿液中不生长，而且对其影响敏感，在6小时内活力损失2-3个对数。相比之下，CPA24在M9基本培养基和人工尿液中既不生长也不被杀死。因此，我们推测尿液中缺乏鸟嘌呤或其衍生物，以及CPA24无法从头合成这些化合物，阻止了CPA24合成其他对在尿液中生长和存活至关重要的依赖鸟嘌呤（或衍生物）的产物。虽然体外尿液中生长减少似乎与尿路致病性降低相关这一概念看似合理，但在尿路感染（UTI）小鼠模型中用CPA24对小鼠进行体内挑战对此概念进行了验证。事实上，发现CPA24的毒力与其野生型亲本CP9相比显著降低。CPI-1（argC）因其argC::lacZ融合在尿液中的显著诱导而被鉴定。随后在尿液中的测试表明，在所有测试的尿液样本（四名女性，三名男性）中其生长均显著减少。多胺合成部分依赖于精氨酸生物合成途径。因此，我们测试了尿液中argC的诱导和/或CPI-1生长减少是否是由于尿液中多胺或精氨酸水平低所致。结果表明，人尿液中精氨酸水平低而非多胺水平低是原因所在。在UTI小鼠模型中进行体内测试时，还发现CPI-1的毒力比CP9显著降低。总之，我们已经确定guaA和argC是我们所知的首批已被证明有助于大肠杆菌在体外尿液中生长且两者在体内尿路致病性均降低的基因。这些结果支持尿液可在体外用作筛选工具以鉴定尿路致病性状这一概念。
