Elsner Andrea, Kreikemeyer Bernd, Braun-Kiewnick Andrea, Spellerberg Barbara, Buttaro Bettina A, Podbielski Andreas
Department of Medical Microbiology and Hygiene, University Hospital, D-18057 Rostock, Germany.
Infect Immun. 2002 Sep;70(9):4859-69. doi: 10.1128/IAI.70.9.4859-4869.2002.
Three open reading frames (ORFs) were identified by a genome walking strategy in the genomes of serotype M49 group A streptococcal (GAS) strains CS101 and 591. These ORFs were located between the mga core regulon and the dipeptide permease operon. The deduced amino acid (aa) sequences contained signature sequences indicative of a lipoprotein (306 aa), an intracellular protein (823 aa), and a secreted peptide (66 aa), respectively. ORF1 (named Lsp for lipoprotein of Streptococcus pyogenes) and ORF2 exhibited a high degree of homology to the lmb/ORF2 genes of S. agalactiae (B. Spellerberg et al., Infect. Immun. 67:871-878, 1999). The three ORFs were found to be present in each of the 27 GAS serotype strains tested. Transcription analysis revealed a polycistronic lsp/ORF2 and a monocistronic ORF3 message that were detected primarily at the transition from exponential to stationary growth phase. lsp and ORF2 mutants, ORF2- and ORF3-luciferase reporter fusions, and antiserum against recombinant Lsp were produced to examine the biological role of these genes. Although high Zn(2+) and Cu(2+) ion concentrations decreased lsp operon expression, Lsp did not transport divalent cations as described for other LraI-type operons. The lsp mutant had reduced fibronectin binding. Although no direct binding of Lsp to fibronectin could be demonstrated, the lsp mutant showed decreased transcription of prtF2 encoding the fibronectin-binding protein F2. Both the lsp and ORF2 mutants showed decreased laminin binding. Adherence to and internalization into A549 epithelial cells of both mutants was reduced without a detectable effect on eukaryotic cell viability. The transcription of a number of virulence factors was altered in the lsp mutants and ORF2 mutants. The changes in laminin binding and eukaryotic cell internalization could be explained by changes in transcription of speB (cysteine protease) and/or the global regulators mga, csrRS, and nra.
通过基因组步移策略，在A群M49型链球菌（GAS）菌株CS101和591的基因组中鉴定出三个开放阅读框（ORF）。这些ORF位于mga核心调控子和二肽通透酶操纵子之间。推导的氨基酸（aa）序列分别包含指示脂蛋白（306个氨基酸）、细胞内蛋白（823个氨基酸）和分泌肽（66个氨基酸）的特征序列。ORF1（命名为Lsp，即化脓性链球菌的脂蛋白）和ORF2与无乳链球菌的lmb/ORF2基因具有高度同源性（B. Spellerberg等人，《感染与免疫》67:871 - 878，1999）。在所测试的27株GAS血清型菌株中，发现这三个ORF均存在。转录分析显示，一个多顺反子lsp/ORF2和一个单顺反子ORF3信息主要在从指数生长期向稳定期转变时被检测到。构建了lsp和ORF2突变体、ORF2 - 和ORF3 - 荧光素酶报告融合体以及针对重组Lsp的抗血清，以研究这些基因的生物学作用。尽管高浓度的Zn(2+)和Cu(2+)离子会降低lsp操纵子的表达，但Lsp并不像其他LraI型操纵子那样转运二价阳离子。lsp突变体的纤连蛋白结合能力降低。尽管无法证明Lsp与纤连蛋白有直接结合，但lsp突变体显示编码纤连蛋白结合蛋白F2的prtF2转录减少。lsp和ORF2突变体的层粘连蛋白结合能力均降低。两种突变体对A549上皮细胞的黏附和内化能力均降低，且对真核细胞活力无明显影响。lsp突变体和ORF2突变体中一些毒力因子的转录发生了改变。层粘连蛋白结合和真核细胞内化的变化可以通过speB（半胱氨酸蛋白酶）和/或全局调节因子mga、csrRS和nra转录的变化来解释。
