探討PrsA在A型鏈球菌所扮演的角色

Investigate the role of PrsA on M4 Streptococcus pyogenes

劉昭賢(Chao-Hsien Liu)

指導教授 ： 張永祺

國立臺灣大學/醫學院/微生物學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803233

摘要

PrsA是一種負責摺疊與穩定分泌蛋白的膜上脂蛋白，在多種不同的細菌如李斯特菌、枯草桿菌中都有所研究。在A型鏈球菌中(Streptococcus pyogenes, GAS)存在有prsA1、prsA2兩套prsA基因，其中prsA2被認為是主要具有功能的基因。實驗室先前的實驗結果觀察到ΔprsA2突變株對部分抗生素的耐受性存有差異，為了更加釐清PrsA蛋白在實驗用菌株M4血清型GAS中的角色，我們新建構出剔除prsA1的突變株及剔除prsA1與prsA2的雙重突變株以進行後續實驗。首先觀察野生株與各突變株的prsA1與prsA2在mRNA的表現量，實驗結果發現當prsA2剔除後prsA1的表現量上升，prsA1剔除後prsA2的表現量下降。接著檢查GAS在剔除PrsA後對各種抗菌物質如過氧化氫、溶菌酶、抗菌胜肽LL-37的耐受性。我們發現在面對過氧化氫所產生的氧化壓力逆境時野生株的耐受能力較三株突變掉prsA的突變株來得更差，與之相反的是在面對其他抗菌物質即脂蛋白抗生素daptomycin、抗菌胜肽LL-37、以及破壞細胞壁的溶菌酶時野生株都比突變株有更好的耐受性，顯示PrsA在應對這些抗菌物質時提供細菌生存上的優勢。為了找出可能造成上述表徵不同的蛋白，我們以SDS-PAGE以及質譜儀分析觀察野生株與ΔprsA突變株在蛋白組成上的差異。由於PrsA主述功能影響細菌分泌蛋白，我們收集GAS野生株與突變株的分泌蛋白(secreted proteins)以及膜囊泡(membrane vesicle)進行比較。實驗發現在分泌蛋白的部分ΔprsA2突變株和ΔprsA1/A2突變株的蛋白組成較為相似，而野生株與ΔprsA1突變株各有獨自的蛋白組成。另外在膜囊泡蛋白部分則是僅ΔprsA1突變株與另三者差異較大。此外從質譜儀的分析數據結合SDS-PAGE膠圖結果我們發現ΔprsA1突變株會有大量成熟SpeB(Streptopain)釋出，而ΔprsA2突變株與ΔprsA1/A2突變株則有許多SpeB的未成熟前驅物存在，此一結果與過往研究提到PrsA2影響SpeB成熟活化相似，且暗示PrsA1功能可能與成熟SpeB的調控有關。總體而言，我們確認了在M4 GAS中PrsA1確實有表現並發揮功能，其功能可能與分泌蛋白與膜囊泡分泌相關，並且prsA突變確實造成M4 GAS對多種抗菌物質耐受性差異，但具體透過何種機轉達成我們觀察到的現象尚待後續研究證實。

關鍵字

A型鏈球菌 ； PrsA ； 抗菌物質

並列摘要

PrsA is a member of membrane-anchored chaperone responsible for the folding and stablization of secreted proteins. Most of the published PrsA researches were conducted on Listeria monocytogenes, Bacillus subtilis, and many other Gram positive bacteria. Two prsA gene copies, prsA1 and prsA2, are found in Group A Streptococcus (GAS, also called Streptococcus pyogenes), and prsA2 was considered as the major functional prsA gene. We previously observed a moderate alteration of antibiotic susceptibility on GAS ΔprsA2 isogenic mutants. In order to clarify the role of PrsA on M4 serotype GAS, we generated GAS mutants lacking prsA1 and both prsA1 and prsA2. We found that expression of prsA1 and prsA2 were differentially regulated. Expression of prsA1 decreased in the absence of prsA2 while expression of prsA2 increased in the absence of prsA1. We next examined whether PrsA regulates the susceptibility of GAS to various antimicrobial substances. Compared to wild type GAS, GAS deficient in prsA1, prsA2 or both prsA1 and prsA2 were more resistant to oxidative stress upon hydrogen peroxide treatment. In contrast, PrsA conferred the survival advantage of GAS upon encountering cell wall destroying lysozyme, membrane targeted daptomycin and host antimicrobial peptides, LL-37. Our results demonstrated that deletion of prsA sensitizes GAS to most of the antimicrobial substance tested. To elucidate the protein candidates contributing to the sensitizing phenomenon to lysozyme, daptomycin and LL-37, we analyzed the protein composition of GAS wild type and mutants deficient in PrsA expression by SDS-PAGE electrophoresis and mass spectrometry. Since PrsA is a protein chaperone known to modulate a broad spectrum of bacterial secreted proteins, secreted proteins and proteins within membrane vesicles were analyzed. Similar protein composition on secreted proteins was observed between ΔprsA2 mutant and ΔprsA1/A2 mutant, while ΔprsA1 mutant and wild type exhibited unique protein patterns. For membrane vesicles, only ΔprsA1 mutant showed very distinct protein composition compare to others. From our preliminary data generated by mass spectrometry analysis, we found that PrsA1 and PrsA2 differentially regulated Streptopain (SpeB) maturation where PrsA2 facilitated and PrsA1 reduced SpeB maturation, respectively. In summary, we demonstrated that prsA1 is expressing and plays a regulatory role in protein secretion and membrane vesicle protein composition in M4 GAS. In addition, prsA deletion altered the sensitivity of GAS to various antimicrobial substances, although the detail mechanisms for this observation needs further investigation.

並列關鍵字

group A Streptococcus ； PrsA ； antimicrobial substances

參考文獻

1. Lancefield, R.C., A Serological Differentiation of Human and Other Groups of Hemolytic Streptococci. J Exp Med, 1933. 57(4): p. 571-95.

2. Carapetis, J.R., et al., The global burden of group A streptococcal diseases. Lancet Infect Dis, 2005. 5(11): p. 685-94.

3. Steer, A.C., et al., Status of research and development of vaccines for Streptococcus pyogenes. Vaccine, 2016. 34(26): p. 2953-2958.

4. Brook, I., Penicillin failure in the treatment of streptococcal pharyngo-tonsillitis. Curr Infect Dis Rep, 2013. 15(3): p. 232-5.

5. Michos, A., et al., Molecular analysis of Streptococcus pyogenes macrolide resistance of paediatric isolates during a 7 year period (2007-13). J Antimicrob Chemother, 2016. 71(8): p. 2113-7.