克雷白氏肺炎桿菌CG43中HdeA、HdeB、HdeD伴隨蛋白 在抗酸反應中所扮演的角色

Abstract

胃酸是對抗經口而入人體病原菌的第一道防線，在這極酸的環境下，腸道菌具有特定抗酸機制來抵禦胃酸的傷害。大腸桿菌HdeA、HdeB座落在抗酸島嶼上，當面臨酸壓力時HdeA、HdeB在膜間質執行伴隨蛋白功能保護受質蛋白質不受到酸傷害。克雷白氏肺炎桿菌CG43具有兩套抗酸島嶼AFI-I和AFI-II，有趣的是，AFI-II在NTUH-K2044和MGH78578並不存在。AFI-I上有hdeDB、hdeB1、yfdX和kvhAS；AFI-II上有hdeA、hdeD1B2、kvgAS和kvgA1S1。先前研究指出這些基因缺損株的抗酸能力依序為ΔhdeB1>ΔhdeD>ΔyfdX>ΔhdeB；重組蛋白HdeB與YfdX皆具伴隨蛋白活性；AFI-I上的基因會受到弱酸誘導，yfdX的表現量在靜置培養下會受RcsB調控。本研究建構了hdeA及hdeB2D1基因缺損株，其抗酸能力依序為ΔhdeB1>ΔhdeD>ΔhdeB>ΔhdeB2D1，hdeA基因缺損只有在穩定生長期對細菌的抗酸能力有明顯影響；此外，在yfdX、hdeB、hdeDhdeD1或rcsB基因缺損株中增加HdeA表現皆可回補其抗酸能力，但當HdeA與受質結合的44苯丙氨酸點突變後後即無法增加細菌抗酸能力，顯示HdeA具很強的伴隨蛋白活性，且第44個胺基酸對於其伴隨蛋白活性是很重要的。最後，PhdeA或PhdeB2D1活性不會受到弱酸誘導，在震盪培養或靜置培養下rcsB和rpoS的基因缺失皆不影響PhdeA和PhdeB2D1的活性。

Acid resistance or acid tolerance is an important adaptation for enterobacteria in order to evade the stomach acid before the intestinal tract colonization. Escherichia coli HdeA and HdeB, encoded on the acid fitness island (AFI) that is activated by acidic stress, function as chaperone in the periplasmic space to protect the proteins from damage. Analysis of the genome sequence of K. pneumoniae CG43 revealed two AFI homologs namely AFI-I and AFI-II. Interestingly, AFI-II is not found in the genome of NTUH-K2044 or MGH78578. The AFI-I contains hdeDB, hdeB1, yfdX and kvhAS, while AFI-II contains hdeA, hdeD1B2, kvgAS and kvgA1S1. The previous study of the specific gene deletion mutants revealed that the acid survival activity could be ranked as ΔhdeB1>ΔhdeD>ΔyfdX>ΔhdeB. Both the recombinant HdeB and YfdX exhibit chaperone activity, and the expression of the AFI-I gene is acid-inducible and positively regulated by RcsB under statically cultured condition. Here, the specific gene deletion mutants including ΔhdeA and ΔhdeB2 have been generated. The order of acid stress survivals is ΔhdeB1>ΔhdeD>ΔhdeB>ΔhdeB2, while ΔhdeA had no apparent change of the sensitivity when compared to the parental strain CG43S3. Nevertheless, the hdeA deletion exerts a profound reduction of the acid stress (pH2.5) survivals when the bacteria were grown to stationary phase. Increasing expression of hdeA could rescue the deletion defect of yfdX, hdeB, hdeDD1, or rcsB suggesting HdeA exhibits a strong chaperone activity. Moreover, phenylalanine 44 of HdeA was critical for the chaperone activity. Finally, the expression of hdeA and hdeB2D1 was not acid inducible and not affected by the deletion of rcsB or rpoS under shaking or static cultures.