蛋白酶ClpQY在沙門氏菌入侵細胞時所扮演的角色之探討

Abstract

沙門氏桿菌利用沙門氏菌致病基因群1(SPI1)轉譯形成的第三型分泌系統(T3SS)入侵腸道上皮細胞，此第三型分泌系統將作用蛋白運輸至寄主細胞的細胞質內，促進腸道上皮細胞骨架重組及沒入細菌作用。細菌在惡劣的環境下，一連串的機制會被啟動，使細菌能夠克服所遭遇的困境。其中，蛋白酶扮演著重要的角色。之前研究發現沙門氏菌clpXP突變株導致持續性感染老鼠；而clpP單突變株則是容易受到外在壓力（例如：高溫，酸性環境與高滲透壓）的影響。另外，當沙門氏菌入侵上皮細胞時，Lon蛋白酶可降低其感染率，並藉由分解HilC與HilD調節蛋白以負調控SPI1基因表現。當沙門氏菌進入宿主上皮細胞後，Lon會抑制沙門氏菌在宿主細胞內的增生與存活。在大腸桿菌中，ClpQY蛋白酶與ClpXP及Lon有重複的分解蛋白活性。本實驗研究藉由改變氧氣濃度、滲透壓以及溫度分析ClpQY蛋白對於沙門氏菌在不同生長逆境下入侵腸道上皮細胞的影響。我們發現在lon缺失突變下大量表現ClpQY蛋白導致沙門氏菌胞外分泌蛋白量整體性明顯減少且抑制入侵上皮細胞的能力。於clpQ-lon-雙突變株中，於1% NaCl可觀察到與lon-相當或稍高的胞外蛋白分泌與入侵細胞能力。而clpY-lon-雙突變株在不同逆境下皆比lon單突變株表現較差的胞外蛋白分泌與入侵細胞能力，在高滲透壓時最為顯著。這些結果暗示著ClpY於沙門氏菌中扮演著伴隨蛋白（chaperone）的角色，以利於細菌的入侵。此外，在高溫逆境下可觀察到ClpQ與ClpY影響沙門氏菌入侵細胞能力，並且不受Lon蛋白酶的干擾。同樣的；clpY-突變株於1% NaCl厭氧環境下，與野生株相較有較差的入侵細胞能力。因此，在不同逆境下當沙門氏菌缺乏Lon蛋白酶入侵宿主上皮細胞時，ClpY蛋白可能扮演著伴隨蛋白的角色。另外，ClpQ與ClpY於lon突變株下亦會影響沙門氏菌存活於腸道上皮細胞，惟其間的機制需更進一步研究。

Salmonella enterica serotype Typhimurium is a Gram-negative pathogen, which cause a variety of diseases ranging from mild gastroenteritis to life threatening systemic infections. During the course of intestinal epithelial cells infection, two main type III secretion systems (T3SS, pathogenicity Island 1 (SPI1) and SPI2) encoded in Salmonella, were found out important for their fuction. The SPI1 T3SS mediates invasion of epithelial cells and involved in evoking enterocolitis. SPI1 encoded proteins form a needle-like structure that injects effector proteins directly into the cytosol of host cells. The effector proteins also alter signaling mechanisms and mediate cytoskeletal rearrangements, leading to the formation of large membrane ruffles that engulf the bacteria in vacuoles (Salmonella-containing vacuole). Once inside host cell, genes from SPI2 are expressed for intracellular survival and replication of Salmonella. Proteolysis in bacteria is a critical strategy for the control of many cellular processes: it limits an availability of regulatory proteins and rids the cell of irreversibly damaged proteins under stress. Most intracellular proteolysis in bacteria is initiated by ATP-dependent proteases, including Lon, FtsH, ClpXP, ClpAP and ClpQY (HslVU). They have a common structure comprising of ATPase and peptidase domain. Disruption of the genes for ClpXP protease in S. Typhimurium results in persistent infection in mice. The lon mutant revealed a high efficiency of an invasiveness. Recent studies show that HilC and HilD are degraded by Lon protease, resulting depletion of Lon dramatically enhances SPI1 gene transcription, including hilA. Thus, Lon presumably allows down-regulation of the system after invasion of host cells. Lon and ClpQY share a redundant fuction in E. coli. Therefore here we investigate the role of Salmonella ClpQY in a lon mutant background while bacteria infect host cells and examine the patterns of secreted proteins under an SPI1-induced or -repressed condition. We found that an overproduced ClpQY represses extracellular protein secretions. Alongside, this also leads to an inability of its invasiveness into epithelial cells. In combination of a lon mutation, a clpQlon mutant has similar SipA/SipC secreted proteins as the lon mutant does. In contrast, a clpYlon mutant showed a decreased SipA/SipC protein secretion as well as a less invasiveness. These results indicate that ClpY might play a role for SipA/SipC secretion. Followed this rationality, we reexamine the role of ClpQ and ClpY under a heat induction (a favorable condition for ClpQ and ClpY expression). We found that under a heat induction, clpY mutant alone has a defective SipA/SipC secretion as well as a ten-fold reduction of invasiveness as compared to the wild-type. Again, these results indicate that ClpY is likely playing a role ancillary to the SipA/SipC secretion under certain conditions.