Robertson Gregory T, Ng Wai-Leung, Foley Joseph, Gilmour Raymond, Winkler Malcolm E
Infectious Diseases Research, Lilly Research Laboratories, Indianapolis, Indiana 46285, USA.
J Bacteriol. 2002 Jul;184(13):3508-20. doi: 10.1128/JB.184.13.3508-3520.2002.
Streptococcus pneumoniae is an important human pathogen that contains single copies of genes encoding the ClpP and FtsH ATP-dependent proteases but lacks the Lon and HslV proteases. We constructed and characterized the phenotypes of clpP, clpC, and clpX deletion replacement mutants, which lack the ClpP protease subunit or the putative ClpC or ClpX ATPase specificity factor. A DeltaclpP mutant, but not a DeltaclpC or DeltaclpX mutant, of the virulent D39 type 2 strain of S. pneumoniae grew poorly at 30 degrees C and failed to grow at 40 degrees C. Despite this temperature sensitivity, transcription of the heat shock regulon determined by microarray analysis was induced in a DeltaclpP mutant, which was also more sensitive to oxidative stress by H2O2 and to puromycin than its clpP+ parent strain. A DeltaclpP mutant, but not a DeltaclpC mutant, was strongly attenuated for virulence in the murine lung and sepsis infection models. All of these phenotypes were complemented in a DeltaclpP/clpP+ merodiploid strain. Consistent with these complementation patterns, clpP was found to be in a monocistronic operon, whose transcription was induced about fivefold by heat shock in S. pneumoniae as determined by Northern and real-time reverse transcription-PCR analyses. Besides clpP, transcription of clpC, clpE, and clpL, but not clpX or ftsH, was induced by heat shock or entry into late exponential growth phase. Microarray analysis of DeltaclpP mutants showed a limited change in transcription pattern (approximately 80 genes) consistent with these phenotypes, including repression of genes involved in oxidative stress, metal ion transport, and virulence. In addition, transcription of the early and late competence regulon was induced in the DeltaclpP mutant, and competence gene expression and DNA uptake seemed to be constitutively induced throughout growth. Together, these results indicate that ClpP-mediated proteolysis plays a complex and central role in numerous pneumococcal stress responses, development of competence, and virulence.
肺炎链球菌是一种重要的人类病原体，它含有编码ClpP和FtsH ATP依赖性蛋白酶的单拷贝基因，但缺乏Lon和HslV蛋白酶。我们构建并表征了clpP、clpC和clpX缺失替代突变体的表型，这些突变体缺乏ClpP蛋白酶亚基或假定的ClpC或ClpX ATP酶特异性因子。肺炎链球菌2型强毒株D39的DeltaclpP突变体在30℃下生长不良，在40℃下无法生长，而DeltaclpC或DeltaclpX突变体则不然。尽管有这种温度敏感性，但通过微阵列分析确定的热休克调节子的转录在DeltaclpP突变体中被诱导，该突变体对H2O2引起的氧化应激和嘌呤霉素也比其clpP+亲本菌株更敏感。DeltaclpP突变体在小鼠肺部和败血症感染模型中对毒力有强烈的减弱作用，而DeltaclpC突变体则没有。所有这些表型在DeltaclpP/clpP+部分二倍体菌株中得到互补。与这些互补模式一致，发现clpP存在于一个单顺反子操纵子中，通过Northern和实时逆转录-PCR分析确定，其转录在肺炎链球菌中受热休克诱导约五倍。除了clpP，clpC、clpE和clpL的转录受热休克或进入指数生长后期阶段诱导，但clpX或ftsH不受诱导。对DeltaclpP突变体的微阵列分析显示转录模式的变化有限（约80个基因），与这些表型一致，包括参与氧化应激、金属离子转运和毒力的基因的抑制。此外，在DeltaclpP突变体中诱导了早期和晚期感受态调节子的转录，并且感受态基因表达和DNA摄取似乎在整个生长过程中都被组成性诱导。总之，这些结果表明ClpP介导的蛋白水解在众多肺炎链球菌应激反应、感受态发育和毒力中发挥着复杂而核心的作用。
