Enzymes Catalyzing the TCA- and Urea Cycle Influence the Matrix Composition of Biofilms Formed by Methicillin-Resistant Staphylococcus aureus USA300.
Enzymes Catalyzing the TCA- and Urea Cycle Influence the Matrix Composition of Biofilms Formed by Methicillin-Resistant Staphylococcus aureus USA300.
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DOI：
10.3390/microorganisms6040113
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发表时间：
2018-10-29
期刊：
影响因子：
4.5
通讯作者：
Malhotra-Kumar S
中科院分区：
文献类型：
--
作者：
De Backer S;Sabirova J;De Pauw I;De Greve H;Hernalsteens JP;Goossens H;Malhotra-Kumar S
在甲氧西林敏感的金黄色葡萄球菌（MSSA）中，已知三羧酸（TCA）循环负调控主要生物膜基质胞外多糖PIA/PNAG的产生。然而，耐甲氧西林金黄色葡萄球菌（MRSA）产生主要的蛋白生物膜基质，tca循环在其中的作用尚不清楚。利用编码TCA-和尿素循环酶基因的USA300- je2 tn -突变体（NARSA），研究了TCA-和尿素循环以及eDNA和PIA/PNAG蛋白对基质的贡献。尿素循环中靶向的基因编码精氨酸琥珀酸裂解酶和精氨酸酶（argH::Tn和rocF::Tn）， tca循环中靶向的基因编码琥珀酰辅酶a合成酶、琥珀酸脱氢酶、乌头酸酶、异柠檬酸脱氢酶、富马酸水合酶II类和柠檬酸合成酶II （sucC::Tn、sdhA/B::Tn、acnA::Tn、icd::Tn、fumC::Tn和gltA::Tn）。与UAS391-EryS （OD492 0.814，综合密度10.676）相比，无流量和有流量条件下，argH::Tn、fumC::Tn和sdhA/B::Tn （OD492 0.374 ~ 0.667，综合密度2.065 ~ 4.875）显著降低了生物膜的形成（p≤0.008）。细胞和基质染色、酶处理（蛋白酶K、dna酶I）和基于逆转录酶pcr的纤维连接蛋白结合蛋白（fnbA/B）和葡萄球菌附属调节剂（sarA）在预形成的UAS391-Erys和n-突变体生物膜上的基因表达分析显示：(I)蛋白/eDNA基质中PIA/PNAG < 1%；（ii）与UAS391-Erys（平均为22和25（±4）%）相比，Tn突变体生物膜基质中无流动和流动条件下蛋白质含量分别增加了50和51（±11）% (p < 0.001)；（iii）与UAS391-EryS相比，n-突变体中fnbA/B和sarA分别下调和上调（平均为0.62-，0.57-和2.23倍）。综上所述，我们发现MRSA-USA300及其相应的Tn突变体的生物膜基质不依赖于PIA/ pnag，主要由蛋白质和eDNA组成。tca循环失活的主要影响是MRSA-USA300生物膜基质的蛋白质成分。
In methicillin-sensitive Staphylococcus aureus (MSSA), the tricarboxylic acid (TCA) cycle is known to negatively regulate production of the major biofilm-matrix exopolysaccharide, PIA/PNAG. However, methicillin-resistant S. aureus (MRSA) produce a primarily proteinaceous biofilm matrix, and contribution of the TCA-cycle therein remains unclear. Utilizing USA300-JE2 Tn-mutants (NARSA) in genes encoding TCA- and urea cycle enzymes for transduction into a prolific biofilm-forming USA300 strain (UAS391-Erys), we studied the contribution of the TCA- and urea cycle and of proteins, eDNA and PIA/PNAG, to the matrix. Genes targeted in the urea cycle encoded argininosuccinate lyase and arginase (argH::Tn and rocF::Tn), and in the TCA-cycle encoded succinyl-CoA synthetase, succinate dehydrogenase, aconitase, isocitrate dehydrogenase, fumarate hydratase class II, and citrate synthase II (sucC::Tn, sdhA/B::Tn, acnA::Tn, icd::Tn, fumC::Tn and gltA::Tn). Biofilm formation was significantly decreased under no flow and flow conditions by argH::Tn, fumC::Tn, and sdhA/B::Tn (range OD492 0.374−0.667; integrated densities 2.065−4.875) compared to UAS391-EryS (OD492 0.814; integrated density 10.676) (p ≤ 0.008). Cellular and matrix stains, enzymatic treatment (Proteinase K, DNase I), and reverse-transcriptase PCR-based gene-expression analysis of fibronectin-binding proteins (fnbA/B) and the staphylococcal accessory regulator (sarA) on pre-formed UAS391-Erys and Tn-mutant biofilms showed: (i) < 1% PIA/PNAG in the proteinaceous/eDNA matrix; (ii) increased proteins under no flow and flow in the matrix of Tn mutant biofilms (on average 50 and 51 (±11)%) compared to UAS391-Erys (on average 22 and 25 (±4)%) (p < 0.001); and (iii) down- and up-regulation of fnbA/B and sarA, respectively, in Tn-mutants compared to UAS391-EryS (0.62-, 0.57-, and 2.23-fold on average). In conclusion, we show that the biofilm matrix of MRSA-USA300 and the corresponding Tn mutants is PIA/PNAG-independent and are mainly composed of proteins and eDNA. The primary impact of TCA-cycle inactivation was on the protein component of the biofilm matrix of MRSA-USA300.
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影响因子：
3.7
作者：
Mrak LN;Zielinska AK;Beenken KE;Mrak IN;Atwood DN;Griffin LM;Lee CY;Smeltzer MS
通讯作者：
Smeltzer MS
影响因子：
3.1
作者：
Houston, Patrick;Rowe, Sarah E.;O'Gara, James P.
通讯作者：
O'Gara, James P.
影响因子：
4.4
作者：
Izano, Era A.;Amarante, Matthew A.;Kaplan, Jeffrey B.
通讯作者：
Kaplan, Jeffrey B.
影响因子：
4.4
作者：
Sabirova, J. S.;Hernalsteens, J-P;Malhotra-Kumar, S.
通讯作者：
Malhotra-Kumar, S.
影响因子：
3.2
作者：
Sadykov, Marat R.;Olson, Michael E.;Somerville, Greg A.
通讯作者：
Somerville, Greg A.