靈菌中影響多黏菌素B的感受性及毒力因子表現之基因研究

Abstract

Serratia marcescens屬於腸內桿菌科 (Enterobacteriaceae)，為革蘭氏陰性菌，除了感染尿道、呼吸道外，也可能造成菌血症、腦膜炎等疾病。多黏菌素B (Polymyxin B，後續簡稱為PB) 屬cationic antimicrobial peptide的一種，常用於治療multidrug-resistant革蘭氏陰性菌的感染。PB結構具有polycationic peptide ring，故可利用它所帶的正電與陰性菌lipidA上的負電做結合，進而造成膜的瓦解。在臨床中部分S. marcescens對於PB具有抗藥性，但目前為止對其抗PB的機制及所調控的路徑了解並不多。因此本篇論文主要在尋找哪些基因會影響PB的感受性，並探討這些基因可能的抗藥機制；此外也分析PB感受性及virulence之間的關聯。 首先利用transposon mutagenesis篩選到3株對PB感受性有差異的菌株，3株mutants的生長皆不受到影響，且經MIC確認皆為PB-susceptible (WT=2500μg/ml，mutants=2μg/ml)。其中包括參與lipid A 4-amino-4-deoxy-L-arabinose (L-Ara4N) 修飾的ΔarnB、ΔarnC，以及isopreniod biosynthesis的ΔispH。在先前的研究中發現，E. coli及Salmonella可透過PhoP-PhoQ及PmrA-PmrB (E. coli為BasSR) 兩套雙組成系統調控下游基因pmr operon (E. coli為arn) 的表現，pmr operon負責lipid A上L-Ara4N的修飾，此修飾會降低LPS所帶的負電荷，進而減少PB的結合。從LPS binding assay發現3株突變株的LPS皆較容易與PB結合 (ispH因為影響isopreniod的合成造成L-Ara4N的修飾受到阻礙)。在reporter assay中，野生株在低濃度Mg2+、PB、酸性 (三者皆為PhoP的訊號) 環境下，arn operon會被誘導而有較高的activity，phoP突變株則無此現象，另外在有PB的情況下，野生株經過低濃度Mg2+、酸性處理後有較高的存活率 (phoP突變株則無)，而從EMSA (electrophoretic mobility shift assay) 的結果得到PhoP可以直接結合至arn operon 的promoter上進而調控arn基因的表現，因此推測Serratia可能透過雙組成系統造成其PB的抗藥性增加。在phenotype的分析中，ΔarnB、ΔarnC在cell invasion的能力降低，ΔispH在外膜蛋白部分則呈現與WT不同的profile，其餘的phenotype如biofilm、SDS感受性則與WT無明顯差異。 總結上述，在Serratia marcescens中arnB、arnC，及ispH皆會影響到LPS的修飾，造成Serratia對於PB具有抗性。而PhoP-PhoQ這個雙組成系統則是做為arnB、arnC基因的regulator，當有訊號因子 (低濃度Mg2+、PB、酸性) 存在時，PhoP被活化爾後結合至arn operon 的promoter，造成arn基因的表現，這也是細菌在面對外在壓力時藉由基因調控所行使的反應。

Serratia marcescens is a Gram-negative，facultative anaerobe bacterium and a member of the family Enterobacteriaceae. It can cause infection in several sites，including the urinary tract、respiratory tract，besides it’s a rare cause of bacteremia and meningitis. Polymyxin B (PB) is a kind of cationic antimicrobial peptides (CAMPs)，which could be used for multidrug-resistant Gram-negative infections. PB is composed of a polycationic peptide ring which have positive charge，it can bind negative charged portion of LPS such as lipid A and core and then alter the membrane integrity by solubilization or pore formation. S. marcescens isolates were resistant to PB in clinical reports，but the mechanisms resistance to PB in S. marcescens still unknown. As a result，the purpose of this thesis is to identify which genes involved in PB susceptibility and explore the mechanisms of these genes. Moreover，analyzing the association between PB susceptibility and virulence factors. Three unique PB-sensitive S. marcescens mutants were identified (ΔarnB、ΔarnC、ΔispH) and they had the same growth curve with wild-type. These mutants were over 1000-fold more sensitive to PB than wild-type (WT=2500μg/ml，mutants=2μg/ml). Analysis of the amino acid sequence indicated that arnB、arnC were necessary for the biosynthesis and addition of 4-aminoarabinose (Ara4N) to the 4’ phosphate of lipid A. Besides，ispH participated in isopreniod biosynthesis. Previous studies indicated that Salmonella and E. coli can positively control the pmr (E. coli named arn) operon at the transcriptional level by PhoP-PhoQ and PmrA-PmrB (E. coli named BasSR) two-component systems. The pmr operon is involved in LPS modification，a modification which contributes to a reduction in the net negative charge of LPS and consequently decreases attraction of PB. According to LPS binding assay，LPS from all mutants bound more PB than wild-type (IspH defect block the synthesis of L-Ara4N ). Reporter assay indicated that low Mg2+、PB and acidic pH (PhoPQ could sense those signals) could enhance the transcription of arn operon in wild-type (ΔphoP failed to do so )，moreover，wild-type (ΔphoP failed to do so either ) grown at acidic pH (or low Mg2+ ) was more resistant to PB than organisms grown at neutral pH (or high Mg2+ ). Here we showed that PhoP could mediate the induction of arn operon，an electrophoretic mobility shift assay (EMSA) demonstrated that PhoP could bind directly to the putative arn promoter. As a result，we assumed that S. marcescens resistant to polymyxin B might controlled by PhoP-PhoQ two-component system. Finally，we assayed the virulence factors of these mutants，wild-type had greater cell invasion ability than ΔarnB、ΔarnC，ΔispH present a different profile of outermembrane protein compared with wild-type. Other phenotype (biofilm、SDS susceptibility etc.) had no significant difference between wild-type and mutants. In conclusion，ΔarnB、ΔarnC、ΔispH affected LPS modification，causing PB resistance in S. marcescens. Depend on our finding，PhoP-PhoQ two-component system acted as a regulator of arnB、arnC，on sensing activating signals (low Mg2+、PB and acidic pH)，PhoPQ directly enhanced the transcription of arn operon and finally triggered LPS modification . This is a instance that microbes are able to sense and respond to their environment primarily through the use of two-component regulatory systems.