Sublethal β-lactam antibiotics induce PhpP phosphatase expression and StkP kinase phosphorylation in PBP-independent β-lactam antibiotic resistance of Streptococcus pneumoniae
Sublethal β-lactam antibiotics induce PhpP phosphatase expression and StkP kinase phosphorylation in PBP-independent β-lactam antibiotic resistance of Streptococcus pneumoniae
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亚致死β-内酰胺抗生素诱导PhpP 磷酸酶表达和StkP 激酶磷酸化肺炎链球菌的PBP 独立β-内酰胺抗生素耐药性
DOI：
10.1101/342188
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发表时间：
2018-06
期刊：
影响因子：
--
通讯作者：
Aihua Sun
中科院分区：
文献类型：
--
作者：
Yanying Huang;Yanhong Sun;Nan Huang;Xiaoxiang Liu;Jie Yan;Aihua Sun
已证实肺炎链球菌的StkP和PhpP组成一个信号对，其中前者是丝氨酸/苏氨酸（Ser/Thr）激酶，后者被注释为磷酸酶。据报道，StkP与肺炎链球菌青霉素结合蛋白（PBP）不依赖的青霉素耐药有关。然而，关于PhpP的酶学特性和PhpP在StkP-PhpP偶联中的作用仍然知之甚少。本研究发现，以β-内酰胺类抗生素为代表的青霉素（PCN）或头孢噻肟（CTX）的1/4最低抑菌浓度（MIC）可诱导PCN/CTX敏感菌株ATCC6306和3株肺炎链球菌（MIC: 0.02 ~ 0.5 μg/ml）中stkP和phpP基因的表达和stkP的磷酸化。phpP基因产物水解PP2C型丝氨酸/苏氨酸磷酸酶特异性RRA(pT)VA磷酸化肽底物，Km和Kcat值分别为277.35 μmol/L和0.71 S−1，水解活性被PP2C型丝氨酸/苏氨酸磷酸酶抑制剂氟化钠阻断。4个phpP基因敲除（ΔphpP）突变体的StkP磷酸化水平显著高于野生型菌株。特别是PCN和CTX对ΔphpP突变体的mic均显著升高，为4 ~ 16 μg/ml。因此，我们的研究结果证实，亚致死PCN和CTX作为环境诱导剂，导致phpP和stkP基因表达增加和stkP磷酸化。PhpP是一种PP2C型丝氨酸/苏氨酸蛋白磷酸酶，负责StkP的去磷酸化。敲除phpP基因可导致肺炎链球菌StkP高水平磷酸化和pbp无关的PCN/CTX耐药性。由于β-内酰胺耐药肺炎球菌菌株的流行，肺炎链球菌是许多国家和地区人群中的常见病原体。β-内酰胺酶的产生和青霉素结合蛋白（PBP）的突变被认为是细菌对β-内酰胺类抗生素耐药的主要机制，但尚未证实肺炎链球菌能产生任何β-内酰胺酶，许多肺炎球菌菌株表现出与PBP突变无关的β-内酰胺类抗生素耐药性。StkP是肺炎链球菌的丝氨酸/苏氨酸激酶，与PhpP蛋白组成信号偶联。本研究表明，PhpP是一种pp2c型磷酸酶，用于StkP的去磷酸化，亚致死青霉素（PCN）或头孢噻肟（CTX）作为环境信号分子诱导了PhpP的表达。敲除phpp编码基因导致PCN/CTX敏感肺炎球菌产生PCN/CTX耐药。这些数据表明，肺炎链球菌的StkP- phpp偶对通过StkP的磷酸化参与了PBP突变不依赖的β-内酰胺类抗生素耐药性。
StkP and PhpP of Streptococcus pneumoniae have been confirmed to compose a signaling couple, in which the former is a serine/threonine (Ser/Thr) kinase while the latter was annotated as a phosphotase. StkP has been reported to be involved in penicillin-binding protein (PBP)-independent penicillin resistance of S. pneumoniae. However, the enzymatic characterization of PhpP and the role of PhpP in StkP-PhpP couple remain poorly understood. Here we showed that 1/4 minimal inhibitory concentration (MIC) of penicillin (PCN) or cefotaxime (CTX), the representatives of β-lactam antibiotics, could induce the expression of stkP and phpP genes and phosphorylation of StkP in PCN/CTX-sensitive strain ATCC6306 and three isolates of S. pneumoniae (MICs: 0.02-0.5 μg/ml). The product of phpP gene hydrolyzed PP2C type Ser/Thr phosphotase-specific RRA(pT)VA phosphopeptide substrate with the Km and Kcat values of 277.35 μmol/L and 0.71 S−1, and the hydrolytic activity was blocked by sodium fluoride, a PP2C type Ser/Thr phosphatase inhibitor. The phosphorylation levels of StkP in the four phpP gene-knockout (ΔphpP) mutants were significantly higher than that in the wild-type strains. In particular, the MICs of PCN and CTX against the ΔphpP mutants were significantly elevated as 4-16 μg/ml. Therefore, our findings confirmed that sublethal PCN and CTX act as environmental inducers to cause the increase of phpP and stkP gene expression and StkP phosphorylation. PhpP is a PP2C type Ser/Thr protein phosphatase responsible for dephosphorylation of StkP. Knockout of the phpP gene results in a high level of StkP phosphorylation and PBP-independent PCN/CTX resistance of S. pneumoniae. Importance Streptococcus pneumoniae is a common pathogen in human populations in many countries and areas due to the prevalence of β-lactam antibiotic-resistant pneumococcal strains. Production of β-lactamases and mutation of penicillin-binding proteins (PBP) have been considered as the major β-lactam antibiotic-resistant mechanisms in bacteria, but S. pneumoniae has not been confirmed to produce any β-lactamases and many pneumococcal strains present PBP mutation-independent β-lactam antibiotic resistance. StkP is a Ser/Thr kinase of S. pneumoniae to compose a signal-couple with PhpP protein. The present study demonstrated that the PhpP is a PP2C-type phosphotase for dephosphorylation of StkP and the sublethal penicillin (PCN) or cefotaxime (CTX) acted as environmental signal molecules to induce the expression of PhpP. The knockout of PhpP-encoding gene caused the PCN/CTX resistance generation of PCN/CTX-sensitive pneumococcal strains. All the data indicate that StkP-PhpP couple of S. pneumoniae is involved in PBP mutation-independent β-lactam antibiotic resistance by phosphorylation of StkP.
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影响因子：
3.7
作者：
Chen Y;Deng W;Wang SM;Mo QM;Jia H;Wang Q;Li SG;Li X;Yao BD;Liu CJ;Zhan YQ;Ji C;Lopez AL;Wang XY
通讯作者：
Wang XY
2011-2013年中国某医院肺炎链球菌分离株血清型分布及耐药性分析
影响因子：
3.7
作者：
Huang S;Liu X;Lao W;Zeng S;Liang H;Zhong R;Dai X;Wu X;Li H;Yao Y
通讯作者：
Yao Y
影响因子：
3.7
作者：
Wang H;Wu Y;Ojcius DM;Yang XF;Zhang C;Ding S;Lin X;Yan J
通讯作者：
Yan J
影响因子：
13.8
作者：
Yeats, C;Finn, RD;Bateman, A
通讯作者：
Bateman, A
DOI：
10.1016/j.ijantimicag.2008.05.004
发表时间：
2008-11-01
影响因子：
10.8
作者：
Yang, Fan;Xu, Xiao-Gang;McGee, Lesley
通讯作者：
McGee, Lesley