Identification of genes involved in PB susceptibility and virulence factor expression of Serratia marcescens

• Main Authors: Quen-Yen Lin, 林奎延
• Other Authors: Shwu-Jen Liaw
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/87040237768606687157

碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 100 === 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.