The Role Of Proline-rich Region In ActA Protein Of Listeria Monocytogenes

Listeria monocytogenes (Lm) is an important food-borne pathogen, widely spread in environments such as soil, water, food products, could infect both human being and animals through unpasteurized dairy products or undercooked meat and other contaminated foods. Listeriosis caused by this bacterium, manifests as gastroenteritis, meningitis, encephalitis, mother-to-fetus infections and septicaemia, immunocompromised individuals, pregnant women and newborns are populations at high risk. Lm is able to cross three host barriers:intestinal barrier, blood brain barrier and materno-fetal barrier. The bacterial surface protein ActA is one of the most critical and best characterized virulence factors of Lm. It fulfills various essential functions within host cells, enabled Lm escaping from autophagy and recruiting an actin polymerization complex that promotes Lm actin-based motility, cell-to-cell spread and dissemination within host tissues. ActA protein consists with N region, P region and C region. The P region binds Ena/VASP, contributing to the length of actin tails and the velocity of bacterial intracellular movement with four PRRs. In this study, the ActA protein of serotype 4b Lm strain NTSN naturally absent one PRR motif was investigated in non-phagocytic cells and mice model as well as its potential function on pathogenesis, to provide a new understanding of the pathogenic mechanism of ActA.1. The transcriptional level analysis of Lm actA and construction of its mutant strainsIn this study, the relative transcriptional level of virulence-related genes (actA、hly、mpl、 prfA、plcA、ami、dltA、fbpA、hfq、inlC、inlH、inlK、sigB、virR、virS、lntA、oppA、prsA2、 aut、pdgA、srtA、mprF、murA、secA2、sod、vip) in Lm was assessed by real-time PCR in vitro infection of human intestinal epithelial cell Caco-2. Results showed that the expression of actA genes up-regulated 320-fold, and several virulence-related genes (virS、vip、mpl、plcA、lntA、 inlK、 inlC) up-regulated significantly after in vitro infection of Caco-2 cell.The amino acid sequences of ActA in 58 Lm strains from NCBI database were analyzed with BioEdit.22 strains exist 35 aa deletion in the proline-rich region, including the serotype 4b Lm NTSN isolated from sheep, while the reference strain Lm EGDe has full-length protein of ActA. Further analysis of their origin and serotype,7 strains are 4b serotype, all of them were isolated from people or animals of listeriosis. Interestingly, among strains belonging to Lineage I (37.9%) and Lineage II (32.7%), the Lineage I strains (59%) have higher tendency with the PRR deletetion than Lineage II strains (9%). It suggests that the PRR deletion has unique specificity with lineages.Thus, in this study, a set of the recombinant Lm NTSN, including Lm NTSN△actA designated for act A gene-deleted NTSN, NTSN(actA-105) for recovered the absent PRR of ActA and NTSN(actA-FL) for replacing actA from the reference strain Lm EGDe were successfully constructed with homology recombination system to investigate the function of proline-rich region deleted.2. The role of proline-rich region in Lm ActA proteinMeasurement of the Lm biofilm formation ability in BHI medium showed that, the biofilm formation ability of Lm NTSN △actA decreased significantly (P< 0.01), suggesting actA gene could promote the biofilm formation in vitro.Adhesion and invasion of Lm in the human cervical carcinoma cell line Hela were evaluated in vitro. Results demonstrated that the mutant Lm NTSN (actA-105) has a lower adhesion and invasion ability to non-phagocytic cells compared to the WT strain (P<0.05). Lm NTSN△actA lacked the plaque formation ability, and WT strains, Lm NTSN (actA-105), Lm NTSN (actA-FL) could effectively form the plaques in Hela cell cultures, but the number of Lm NTSN (actA-105) plaques were lower amount and their sizes were smaller compared th those of the WT strain(P<0.001). Furthermore, the actin-tail formation of bacteria in infected Hela cells was observed. Results showed that Lm NTSN△actA strain cannot gather actin; while the accumulation actin ability of Lm NTSN (actA-105) strain decreased compared with wild strains and Lm NTSN (actA-FL), and the length of actin-tails are shorter. In conclusion, Lm NTSN with naturally deletion of one PRR region has more powerful adhesion or invasion ability, and the capability of formation actin tails is stronger.In addition, the pathogenicity of mutant strains was further evaluated in mice model via three infection routes. Results showed that NTSN△actA strain displayed 247-fold increase in LD50 compared to WT strain. Interestingly, the bacteria load of Lm NTSN (actA-105) in mice spleen via oral, intraperitoneal, intravenous routes were significantly higher than that of wild type strain (i.g.: P<0.05; i.p.:P<0.05; i.v.:P<0.01), and the bacteria load in mice liver via oral route were also significantly higher than that of WT strain(P<0.01). Additionally, the bacteria load of Lm NTSN (actA-FL) in mice spleen via oral, intraperitoneal, intravenous routes were significantly higher than that of WT strain (i.g.:P<0.05; i.p.:P<0.01; i.v.:P<0.01), and the bacteria load in mice liver via oral and intravenous routes were also significantly higher than that of WT strain (i.g.: P<0.05, i.p.:P<0.01). In conclusion, the colonization of Lm-NTSN(actA-105) and Lm NTSN(act4-FL) in livers and spleens of mice showed significantly increase compared to the WT strain. It suggests that the full-length PRRs playing important role in colonization in mice.In conclusion, all results demonstrated that the PRR deletion of Lm NTSN influenced its adhesion and invasion in non-phagocytic cells and cell-to-cell spread, but in the process of infection in mice, a full-length ActA protein is more beneficial to bacterial colonization in the host. It needs further expand the mechanism of ActA protein role during the infection process of Lm.