抑制Stenotrophomonas maltophilia mrcA基因之功能可誘發L1和L2 β-lactamases 基本表現

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Title:	抑制Stenotrophomonas maltophilia mrcA基因之功能可誘發L1和L2 β-lactamases 基本表現 Inactivation of mrcA Gene Derepresses the Basal-level Expression of L1 and L2 β-lactamases in Stenotrophomonas maltophilia
Authors:	林昕潔;Hsin-Chieh Lin
Contributors:	健康照護學院醫學檢驗生物技術學系碩士班
Keywords:	嗜麥芽糖黃單胞菌;盤尼西林結合蛋白;Stenotrophomonas maltophilia;Penicillin-binding protein;β-lactamase;mrcA
Date:	2010
Issue Date:	2010-09-29 12:10:36 (UTC+8)
Abstract:	細菌的外型通常是受細胞壁所影響，革蘭氏陰性菌構成細胞壁的主要成份是 peptidoglycan 。細菌通常有一套各別的 penicillin binding proteins ( PBPs )，擁有 DD-transpeptidase activity，此活性和 glycan 的組成有關。在擁有 anpR-β-lactamase 調控系統的革蘭氏陰性菌，如 Enterobacteriaceae 、 Pseudomonas 和Stenotrophomonas ，其抵抗 β-lactams 類抗生素的主要機制為誘發 β-lactamase 。 PBPs 除了在 peptidoglycan 生合成中有扮演的角色之外，PBPs 和 β-lactams 的親合性也會影響 chromosomal β-lactamases 的誘發能力。本論文的研究分為兩部份。第一部份是將 S. maltophilia KJ 菌株中，除了 PBP3 之外，每個PBP個別突變，所得之突變株進行細菌生長之觀察、β-lactamases 定量分析及β-lactams 感受性測試。結果顯示， mrcA、mrcB、pbpC、dacB 和dacC 基因缺陷對於菌株的生長幾乎沒有明顯的影響。然而，mrcB 基因突變菌株在 stationary phase 的飽和 OD450nm 之數值比原生菌株之數值低。此外，不同的 PBPs 失去功能在 β-lactamases 活性表現和 β-lactams 抗性上有不同的表型。其中，mrcA 基因缺陷導致β-lactamase 誘發有一特殊的表型，即在沒有添加誘發物的情況下與原生菌株相比，其β-lactamase 活性表現明顯增加；當加入誘發物後，其β-lactamase 活性表現亦有誘發再上升的現象。本論文第二部份是針對 KJΔmrcA 突變菌株做進一步分析。 KJΔmrcA 突變菌株與 Pseudomonas aeruginosa dacB 基因缺陷菌株 (PAOΔdacB) 具有相似的表型。然而，KJΔmrcA 的表型與creBCD two-component 調控系統無關，此與影響 PAOΔdacB 表型的機制不同。為了探討 mrcA 基因與 S. maltophilia ampNG-ampDI-ampR 之 β-lactamase 調控系統的關係，構築了KJΔRΔmrcA、KJΔDIΔmrcA 及 KJΔNGΔmrcA 。並對此系列 double mutants 與其相對應的原生菌株 (wild-type) 進行 β-lactamase 活性測試與感受性試驗。結果顯示，mrcA 基因突變株所造成的 β-lactamase 活性表現上升的現象是 AmpR-, AmpN-, AmpG-dependent 。而 mrcA 和 ampDI 基因同時發生突變，會使得 β-lactamase 活性表現上升有加乘的作用。 The cell wall of gram-negative bacteria is composed of a rigid murein layer, peptidoglycan, which determines cell shape. Bacteria generally own a set of unique penicillin binding proteins (PBPs) which have the DD-transpeptidase activity involved in the cross-linking of the glycan strands. In the ampR-β-lactamase-bearing gram-negative bacteria, such as Enterobacteriaceae, Pseudomonas, and Stenotrophomonas, the inducible expression of the chromosomal β-lactamase gene is the major mechanism of the β-lactam resistance. The role of PBPs in the peptidoglycan turnover and the binding affinity between PBPs and β-lactams are responsible for the induction of the chromosomal β-lactamases. This study includes two major sections. The first section points on the each PBP role of S. maltophilia KJ, except PBP3, in the bacterial growth, β-lactamase activity, and β-lactam susceptibility. Inactivation of mrcA, mrcB, pbpC, dacB, and dacC genes made little effect on the bacterial growth. However, the KJΔmrcB displayed a lower saturated optical density at 450nm than wild-type KJ strain. In addition, inactivation of different PBPs conferred to various phenotypes in β-lactamase activity and β-lactam resistance. Among them, KJΔmrcA exhibited a noteworthy phenotype of basal derepressed β-lactamase activity and further inducibility. Therefore, in the second section of this study, we focus on characterization of the KJΔmrcA mutant. The phenotype of KJΔmrcA is similar to that of Pseudomonas aeruginosa dacB mutant (PAOΔdacB). Nevertheless, the creBCD two-component regulation system is not involved in the phenotype of KJΔmrcA, unlike its role in the phenotype of PAOΔdacB. To elucidate the relationship between mrcA and the known ampNG-ampDI-ampR regulon of S. maltophilia β-lactamase induction, mutants KJΔRΔmrcA, KJΔDIΔmrcA, and KJΔNGΔmrcA were constructed. The β-lactamase activity assay and susceptibility test were comparatively determined between the mutant and its respective parent strain. The results demonstrate that the basal derepressed β-lactamase activity of KJΔmrcA is ampN-ampG- and ampR-dependent. The introduction of an ΔmrcA allele into the ΔampDI background augments the induced β-lactamase expression, signifying that ΔampDI and ΔmrcA have an addition effect on the β-lactam resistance.
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