克雷白氏肺炎桿菌CG43 PerA及PerC的功能性探討

Abstract

克雷白氏肺炎桿菌CG43為一分離自糖尿病患的肝膿瘍菌株，除了其厚重的多醣莢膜外，線毛的表現與生物膜的形成能力、對小鼠的高毒性都可能與其肝膿瘍的致病機轉相關。已知，二級信使c-di-GMP可以促進克雷白氏肺炎桿菌第三型線毛的表現，為了進一步了解c-di-GMP的調控角色，先前研究以mRNA定序分析轉錄體，結果發現相對於CG43S3[pRK415]，在提高c-di-GMP濃度的CG43S3[pRK415-ydeH]中有94個基因被大量誘導、154個基因被抑制；除了mrkABCDF 和mrkHIJ基因組的表現提高外，另外有兩個轉錄因子基因KP1_0384及KP1_0385也大量表現，序列分析顯示與病原性大腸桿菌EPEC毒性相關的PerA與PerC為同源蛋白、perA與PNAG胞外多醣生合成基因組pgaABCD相鄰且轉錄方向相反。為了了解克雷白氏肺炎桿菌PerA與PerC的功能，首先建構perA及perC基因缺失株及perA及perC增量表現菌株，然後，分析perA及perC基因缺損及增量表現的影響，結果顯示perA或perC基因缺損會稍微降低第三型線毛單位蛋白MrkA表現，而perA基因缺失對MrkA生成的影響在CG43S3[pRK415-ydeH]更為明顯；然而，任一基因缺失對生物膜生成、胞外多醣、多醣莢膜與抗酸能力都沒有明顯影響；進一步大量表現perA及perC，結果發現perA表現可明顯促進胞外多醣生成卻降低對酸的抗性；最後，我們以LacZ報告系統分析其啟動子活性，結果發現perA、perC，及pgaA的啟動子活性在hns基因缺失的情況下都會增加，而在弱酸環境(pH 5)，三者的啟動子活性皆降低。

Klebsiella pneumoniae CG43 is a liver abscess isolate obtained from a diabetic patient. Besides its thick polysaccharide capsule, the fimbriae expression, biofilm forming ability, high virulence to mice may be also contributed to the pathogenesis of liver abscess. It is known that the second messenger c-di-GMP could stimulate the expression of K. pneumoniae type 3 fimbriae. In order to explore further the regulatory role of c-di-GMP, a comparative transcriptomic analysis via RNA-seq has been employed. There are 94 and 154 genes increased and reduced expression respectively in CG43S3[pRK415-ydeH] with increased c-di-GMP level compared to CG43S3[pRK415]. In addition to mrkABCDF and mrkHIJ with significantly elated expression levels, two transcription factor encoding gene KP1_0384 and KP1_0385 are also overexpressed. Sequence analysis revealed that they are related to the virulence regulator PerA and PerC of the enteropathogenic E. coli (EPEC), and the poly--1,6-N-acetylglucosamine (PNAG) exopolysaccharide (EPS) encoding operon pgaABCD is divergently transcribed and located next to perA. Toward understanding the function of PerA and PerC, the specific gene deletion mutants and the gene overexpression constructs were firstly generated and then the effect of gene deletion or overexpression analyzed. The results showed that deletion of perA or perC slightly reduced the type three fimbriae major unit protein MrkA production in CG43S3 and the perA deletion effect on MrkA production was more profound in CG43S3[pRK415-ydeH]. However, neither gene deletion had apparent influence on EPS or CPS production, or acid stress response. Nevertheless, overexpression of perA or perC in CG43S3 increased the EPS production but decreased acid stress resistance. Finally, the promoter activity measurement via LacZ reporter system revealed that the expression of perA, perC and pgaA were increased by the deletion of hns and all three promoter activity were decreased under the culture of weak acid (pH5).