研究rmpA 在克雷伯氏肺炎桿菌中的功能

Abstract

近二十年來，在台灣出現了一種新型的侵襲性克雷伯氏肺炎桿菌，會引起典型的臨床表現－原發性肝膿瘍 (primary liver abscess, PLA) 合併菌血症，已知克雷伯氏肺炎桿菌的致病因子之中包含莢膜與黏性型態，莢膜能夠保護細菌不受吞噬細胞 (phagocytes) 的吞噬作用及血清中補體 (complement) 的殺菌作用所摧毀。 本研究發現rmpA基因的分布在PLA菌株較non-PLA菌株為高 (100% vs. 22%)。rmpA-突變株已失去了野生株的高黏性特徵，且相較於野生株降低對小鼠的致死能力，而rmpA異位互補株 (trans-complementation) 則使得黏性回復。利用克雷伯氏肺炎桿菌基因微陣列，分析野生株與rmpA-突變株基因表現之差異來尋找RmpA直接或間接調控的致病因子，經過RT-qPCR的確認，找到在rmpA-突變株表現量下降，並且在rmpA異位互補株表現量上升的基因：莢膜生合成基因組 (cps gene cluster) 與nirB、nirD (nitrite reductase) 基因。已知莢膜生合成基因組為重要致病因子，且會受到RmpA之相似蛋白質RmpA2的活化。而nirB、nirD基因的表現並不受莢膜合成的影響，可能為RmpA影響莢膜以外的致病因子。然而小鼠實驗顯示nirBD-突變株對小鼠的致死能力與野生株無差異。因此只有莢膜生合成基因組為RmpA所調控的致病因子。

In the past 20 years, a new type of invasive Klebsiella pneumoniae disease has emerged in Taiwan that typically presents a community-acquired primary liver abscess (PLA) with sepsis and bacteremia. It has been known that two of the virulent factors of K. pneumoniae are capsule and mucoid phenotype. The capsular polysaccharide protect against the bactericidal effect of serum complement and against ingestion and killing by professional phagocytes. In this study, we found rmpA was significantly more prevalent in PLA strains. Furthermore, the rmpA- mutants lost the hypermucoviscosity and resulted in an increase of LD50 compared to wild-type strains in a mouse intraperitoneal infection model. The mucoviscosity was restored by complementation using a rmpA-containing plasmid. The above data suggested that rmpA was correlated to virulence resulted from K. pneumoniae. In order to find RmpA-regulated virulent factors, we compared RNA expression profiles between wild-type strains and rmpA- mutants by using microarray analysis. The microarray data were confirmed by RT-qPCR, and we found cps (capsular polysaccharide) gene cluster and nir operon (nitrite reductase) expression were decreased in rmpA- mutants but increased in rmpA-complementation strains. It has been well documented that CPS is an important virulent factor activated by RmpA homolog, RmpA2. The expression of nir operon is not correlated to CPS but regulated by RmpA. The nir operon may be involved in CPS-independent virulence mechanism regulated by RmpA. However, there was no significant difference in LD50 between wild-type strains and nirBD- mutants. Therefore, only cps gene clusters are found to be virulent genes regulated by RmpA.