克雷白氏肺炎桿菌CG43雙分子系統與多黏菌素B抗性及莢膜多醣體生合成之調控

Abstract

克雷白氏肺炎桿菌是臨床上一種重要的病原菌，常造成伺機性感染，包含尿道感染、肺炎、化膿性感染、原發性肝膿瘍及敗血症等。隨著多重抗藥菌株的產生與散佈，改善目前治療方式，並深入瞭解其致病機轉顯得刻不容緩。為適應環境改變，病原菌常藉由雙分子系統調控並適當表現其毒性因子。雙分子系統 (two-component system; 2CS)由位於細胞內膜的感應蛋白及細胞質內的反應調控蛋白組成。本論文著重探討克雷白氏菌中雙分子系統PhoP/PhoQ、PmrA/PmrB、RstA/RstB及RcsCDB於其毒性因子的調控。 多黏菌素為近年重新評估使用的藥物之一，沙門氏菌中PhoP/PhoQ, PmrA/PmrB及PmrD蛋白質共同調控多黏菌素B之抗性，為瞭解克雷白氏菌中多黏菌素B抗性的機轉，我們對可能參與的基因進行突變。結果顯示，相對於參與莢膜多醣體生合成基因，合成PmrF、PhoP、PmrA、PmrD等參與脂多醣修飾及調控蛋白之基因缺損，會使克雷白氏菌多黏菌素B抗性有較顯著的降低。藉由LacZ報導基因及DNA電泳遲滯實驗 (electrophoretic mobility shift assay)，我們發現pmrD基因表現受到PhoP的直接活化，細菌雙雜合實驗 (bacterial two-hybrid analysis) 也證明PmrD與PmrA在細菌體內的交互作用，另外藉由磷酸根傳遞分析 (phosphotransfer assay) 與激酶/去磷酸酶實驗 (kinase/phosphatase assay) 更指出，此交互作用可防止感應蛋白PmrB對PmrA的去磷酸化。這些結果顯示克雷白氏菌PmrD扮演了連結雙分子系統PhoP/PhoQ及PmrA/PmrB的角色。進一步藉由點突變方式，我們在15個所選擇的PmrD殘基中，發現9個改變為丙氨酸後對多黏菌素抗性有顯著影響，顯示這些氨基酸序列可能在PmrD與PmrA交互作用扮演重要角色。 為瞭解克雷白氏菌RstA/RstB之調控，我們利用LacZ報導基因證明PhoP及RstA可正向調控rstA基因表現。此外利用克雷白氏菌野生株與rstA基因缺損株cDNA進行刪除雜交法 (subtractive cDNA hybridization)，我們搜尋到11個可能受到RstA活化及19個可能受到RstA抑制的基因，結果顯示RstA/RstB可能參與調控鐵分子的運送、攝取以及重金屬鉛之抗性；然而針對野生株、rstA、rstB以及rstArstB基因缺損突變株進行表型比較，顯示rstA或rstB基因缺損對於細菌在含鐵或缺鐵環境的生長、螯鐵分子生合成 (siderophore biosynthesis)、重金屬鉛抗性、酸性環境適應性或膽鹽抗性均沒有顯著影響，顯示克雷白氏菌中RstA/RstB的功能仍待釐清。 除雙分子系統RcsCDB之外，克雷白氏菌K2莢膜多醣體生合成亦受到轉錄因子RmpA2的調控。在克雷白氏菌CG43大型毒性質體pLVPK序列中，我們發現另一個黏性分子基因rmpA。分析rmpA基因缺損株與rmpA回補菌株，我們發現RmpA可能與RcsB共同活化莢膜多醣體生合成。藉由細菌雙雜合實驗及免疫共沈澱分析，顯示RmpA與可與RcsB交互作用。此外藉由LacZ報導基因、DNA電泳遲滯實驗、有限稀釋反轉錄聚合酶連鎖反應 (limiting-dilution RT-PCR) 及fur基因缺損株表型的分析，我們發現rmpA基因表現受到鐵離子攝取調控蛋白Fur抑制，顯示莢膜多醣體生合成與鐵離子攝取在克雷白氏菌致病過程中可能存在交互作用。 綜合以上所述，本論文探討克雷白氏菌雙分子系統兩種調控方式：一種可藉由連結蛋白PmrD與反應調控蛋白PmrA相互作用，達成其轉譯後修飾來活化下游多黏菌素B抗性基因表現；另一種則包含反應調控蛋白RcsB與其協助分子RmpA之共同作用，藉以活化莢膜多醣體生合成。這些結果暗示在不同環境下，克雷白氏菌可藉由許多有彈性及效率的方式調控其致病因子表現。

Klebsiella pneumoniae, an important opportunistic pathogen, causes urinary tract infections, pneumonia, purulent infections, primary liver abscess and septicemia. The emergence and wide-spread of multiresistant strains has urged the improvement of current therapeutic strategies and a deeper understanding of its pathogenesis. Pathogenic bacteria usually utilize two-component systems (2CSs), consisting of an inner membrane sensor kinase and a cytoplasmic response regulator, to modulate the expression of virulence factors in response to environmental stimuli. The objectives of this dissertation were to investigate the functional role and regulation of 2CSs PhoP/PhoQ, PmrA/PmrB, RstA/RstB and RcsCDB in K. pneumoniae virulence determinants. The 2CSs PhoP/PhoQ, PmrA/PmrB and a small protein PmrD regulated Salmonella enterica resistance to polymyxin B, one of the reevaluated antimicrobial agents. To investigate the regulation of polymyxin B resistance in K. pneumoniae, mutation effects of the genetic determinants likely involved in the resistance were investigated. Compared with the deletion of the genes involved in capsular polysaccharide (CPS) biosynthesis, the deletion of pmrF, phoP, pmrA or pmrD encoding the proteins participated in the lipopolysaccharide modifications or regulation resulted in a significantly reduced resistance. Through LacZ reporter assay and DNA electrophoretic mobility shift assay (EMSA), we have found that the expression of pmrD was directly activated by PhoP. Bacterial two-hybrid analysis has indicated an in vivo interaction between PmrD and PmrA. As illustrated by in vitro phosphotransfer assay and kinase/phosphatase assay, such an interaction could protect PmrA from the dephosphorylation by its cognate sensor protein PmrB. The results suggest a role of Klebsiella PmrD in connecting 2CSs PhoP/PhoQ and PmrA/PmrB. By point mutation strategy, 9 of the 15 selected residues on PmrD were shown to be critically involved in the polymyxin B resistance implying these residues are required for PmrD/PmrA interaction. To characterize the 2CS RstA/RstB in K. pneumoniae, LacZ reporter assay conducted indicated a positive regulatory role of PhoP and RstA on the expression of rstA. Subtractive hybridization of the cDNA from the parental strain and isogenic 岛rstA mutant has led to the identification of 11 RstA-activated genes and 19 RstA-repressed genes involved in various cellular functions, such as iron transport/uptake and lead resistance. Comparative phenotype analysis of the wild-type strain, 岛rstA, 岛rstB and 岛rstA岛rstB mutants, however, showed that the deletion of rstA or rstB had no apparent effects on the growth under iron-depletion or -repletion conditions, siderophore biosynthesis, lead resistance, acid stress response or the resistance to bile salts, indicating that the functional role of RstA/RstB in K. pneumoniae remained to be clarified. In addition to the 2CS RcsCDB, K. pneumoniae K2 CPS biosynthesis was also regulated by the transcription factor RmpA2. Sequence analysis of pLVPK in K. pneumoniae CG43 has revealed rmpA, another mucoid factor encoding gene. Phenotype analysis of the rmpA deletion mutant and the complemented strain has implied that RmpA activated K2 CPS biosynthesis through an cooperation with RcsB. Both bacterial two-hybrid analysis and co-immunoprecipitation have further confirmed RmpA/RcsB interaction. Besides, through LacZ reporter assay, DNA EMSA, limiting-dilution PCR and phenotype analysis of 岛fur mutant strain, we have shown that the expression of rmpA was negatively regulated by Fur, the global ferric ion uptake regulator. The results indicated the interplay between capsular polysaccharide biosynthesis and iron uptake in K. pneumoniae pathogenesis. In summary, two modes of 2CS regulation in K. pneumoniae were investigated in this dissertation: one regarding the interaction between the connector protein PmrD and PmrA, resulting in a post-translational modification to enhance the expression of downstream genes involved in polymyxin B resistance; the other comprising the cooperation between the response regulator RcsB and its accessory factor RmpA for the activation of the K2 CPS biosynthesis. The findings indicated that under different environmental conditions, K. pneumoniae could regulate the expression of its virulence determinants in a versatile and efficient manner.