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Isolation of Listeria monocytogenes mutants with high-level in vitro expression of host cytosol-induced gene products 总被引:1,自引:0,他引:1
Shetron-Rama LM Mueller K Bravo JM Bouwer HG Way SS Freitag NE 《Molecular microbiology》2003,48(6):1537-1551
The facultative intracellular bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors upon entry into the host cell cytosol. actA, the protein product of which is required for cell-to-cell spread of the bacterium, is expressed at low to undetectable levels in vitro and increases in expression more than 200-fold after L. monocytogenes escape from the phagosome. To identify bacterial factors that participate in the intracellular induction of actA expression, L. monocytogenes mutants expressing high levels of actA during in vitro growth were selected after chemical mutagenesis. The resulting mutant isolates displayed a wide range of actA expression levels, and many were less sensitive to environmental signals that normally mediate repression of virulence gene expression. Several isolates contained mutations affecting actA gene expression that mapped at least 40 kb outside the PrfA regulon, supporting the existence of additional regulatory factors that contribute to virulence gene expression. Two actA in vitro expression mutants contained novel mutations within PrfA, a key regulator of L. monocytogenes virulence gene expression. PrfA E77K and PrfA G155S mutations resulted in high-level expression of PrfA-dependent genes, increased bacterial invasion of epithelial cells and increased virulence in mice. Both prfA mutant strains were significantly less motile than wild-type L. monocytogenes. These results suggest that, although constitutive activation of PrfA and PrfA-dependent gene expression may enhance L. monocytogenes virulence, it may conversely hamper the bacterium's ability to compete in environments outside host cells. 相似文献
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Christian U. Riedel Ian R. Monk Pat G. Casey Mark S. Waidmann Cormac G. M. Gahan Colin Hill 《Molecular microbiology》2009,71(5):1177-1189
The Listeria monocytogenes Agr peptide-sensing system has been analysed by creating a deletion mutant in agrD , the structural gene for the putative quorum-sensing peptide. The Δ agrD mutant displayed significantly reduced biofilm formation, a defect which could be restored by genetic or physical complementation. A reduced invasion of Caco-2 intestinal epithelial cells was observed for the Δ agrD mutant while phagocytosis by THP-1 macrophages was unaffected. Additionally, the level of internalin A (InlA) in the cell wall was decreased in the Δ agrD mutant. Expression profiling of virulence genes ( hlyA , actA , plcA , prfA and inlA ) identified a finely tuned regulation which resulted in an impaired virulence response in the Δ agrD mutant. The mutant is also significantly attenuated for virulence in mice, as revealed by bioluminescent in vivo imaging. On day 3 post infection, systemic dissemination to livers and spleens had occurred for the wild type, whereas the Δ agrD mutant remained localized to the liver. Microarray analysis identified 126 and 670 genes as significantly regulated in exponential and stationary phase respectively. The results presented here suggest that peptide sensing plays an important role in the biology of L. monocytogenes , with relevant phenotypes in both the saprophytic and parasitic lifecycles. 相似文献
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PrfA是单核细胞增生李斯特菌(LM)中迄今为止发现的惟一个调控绝大多数毒力基因转录表达的蛋白因子.为了研究PrfA转录调控毒力基因表达的分子机制,将无启动子的绿色荧光蛋白(GFP)基因与毒力基因actA的启动子融合,连接到穿梭载体pLSV16质粒上,构建成表达融合载体pLSV16-PactA-gfp,然后将其电转化入LM野生株P14、PrfA高表达突变株P14a和prfA基因等位缺失突变株A42中表达.利用荧光显微镜和荧光酶标仪检测上述3株细菌中绿色荧光蛋白的不同表达强度,从而评价actA基因依赖于PrfA的转录活性强弱.结果显示,绿色荧光蛋白在P14a中发出的荧光强度最高,P14次之,A42最弱,两两比较均有显著差异(P<0.01),表明毒力基因actA的转录水平高低与PrfA的活性成正相关,其转录表达依赖于PrfA的调控;该试验同时也显示GFP能方便、有效地用于研究PrfA调控LM不同毒力基因的转录表达水平. 相似文献
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The InlB protein of Listeria monocytogenes is sufficient to promote entry into mammalian cells 总被引:4,自引:1,他引:3
InlB is one of the two Listeria monocytogenes invasion proteins required for bacterial entry into mammalian cells. Entry into human epithelial cells such as Caco-2 requires InlA, whereas InlB is needed for entry into cultured hepatocytes and some epithelial or fibroblast cell lines such as Vero, HEp-2 and HeLa cells. InlB-mediated entry requires tyrosine phosphorylation, cytoskeletal rearrangements and activation of the host protein phosphoinositide (PI) 3-kinase, probably in response to engagement of a receptor. In this study, we demonstrate for the first time that InlB is sufficient to promote internalization. Indeed, coating of normally non-invasive bacteria or inert latex beads with InlB leads to internalization into mammalian cells. In addition, a soluble form of InlB also appears to promote uptake of non-invasive bacteria, albeit at a very low level. Similar to entry of L. monocytogenes , uptake of InlB-coated beads required tyrosine phosphorylation in the host cell, PI 3-kinase activity and cytoskeletal reorganization. Taken together, these data indicate that InlB is sufficient for entry of L. monocytogenes into host cells and suggest that this protein is an effector of host cell signalling pathways. 相似文献
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To test the hypothesis that actA allelic variation contributes to virulence differences among Listeria monocytogenes strains, cell-to-cell spread and intracellular ActA phosphorylation patterns were characterized for 14 wild-type isolates and selected isogenic mutants. Our data show that (i) while actA allelic variation is not responsible for enhanced cell-to-cell spread observed in epidemic clone I strains, actA allelic variation may contribute to reduced plaque size observed in some isolates, (ii) actA sequence alone determines phosphorylation-dependent ActA banding patterns, and (iii) sequence variation at the positively selected ActA residue 498 does not contribute to ActA phosphorylation patterns or to differences in cell-to-cell spread. 相似文献
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PrfA is a key regulator of Listeria monocytogenes pathogenesis and induces the expression of multiple virulence factors within the infected host. PrfA is post-translationally regulated such that the protein becomes activated upon bacterial entry into the cell cytosol. The signal that triggers PrfA activation remains unknown, however mutations have been identified (prfA* mutations) that lock the protein into a high activity state. In this report we examine the consequences of constitutive PrfA activation on L. monocytogenes fitness both in vitro and in vivo. Whereas prfA* mutants were hyper-virulent during animal infection, the mutants were compromised for fitness in broth culture and under conditions of stress. Broth culture prfA*-associated fitness defects were alleviated when glycerol was provided as the principal carbon source; under these conditions prfA* mutants exhibited a competitive advantage over wild type strains. Glycerol and other three carbon sugars have been reported to serve as primary carbon sources for L. monocytogenes during cytosolic growth, thus prfA* mutants are metabolically-primed for replication within eukaryotic cells. These results indicate the critical need for environment-appropriate regulation of PrfA activity to enable L. monocytogenes to optimize bacterial fitness inside and outside of host cells. 相似文献
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A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin. 总被引:35,自引:6,他引:29
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E Domann J Wehland M Rohde S Pistor M Hartl W Goebel M Leimeister-Wchter M Wuenscher T Chakraborty 《The EMBO journal》1992,11(5):1981-1990
The ability of Listeria monocytogenes to move within the cytosol of infected cells and their ability to infect adjacent cells is important in the development of infection foci leading to systemic disease. Interaction with the host cell microfilament system, particularly actin, appears to be the basis for propelling the bacteria through the host cell cytoplasm to generate the membraneous protrusions whereby cell-to-cell spread occurs. The actA locus of L.monocytogenes encodes a 90 kDa polypeptide that is a key component of bacterium-host cell microfilament interactions. Cloning of the actA gene allowed the identification of its gene product and permitted construction of an isogenic mutant strain defective in the production of the ActA polypeptide. Sequencing of the region encoding the actA gene revealed that it was located region encoding the actA gene revealed that it was located between the metalloprotease (mpl) and phosphatidylcholine-specific phospholipase C (plcB) genes. Within the cytoplasm of the infected cells, the mutant strain grew as microcolonies, was unable to accumulate actin following escape from the phagocytic compartment and was incapable of infecting adjacent cells. It was also dramatically less virulent, demonstrating that the capacity to move intracellularly and spread intercellularly is a key determinant of L.monocytogenes virulence. Like all other virulence factors described for this microorganism, expression of the ActA polypeptide is controlled by the PrfA regulator protein. The primary sequence of this protein appeared to be unique with no extended homology to known protein sequences. However, an internal repeat sequence showed strong regional homology to a sequence from within the hinge region of the cytoskeletal protein vinculin. 相似文献
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产单核细胞李斯特菌actA基因在大肠杆菌中的表达及其单克隆抗体的研制 总被引:3,自引:0,他引:3
利用PCR技术从血清型1/2a的产单核细胞李斯特菌Lm-4株中扩增出actA基因,经克隆筛选和测序鉴定后,构建成该基因的原核表达载体pGEX-6P-1-actA及pET-actA,转入E·coli后,IPTG诱导目的蛋白的表达。SDS-PAGE结果表明,actA基因在两种载体中均获得表达,融合蛋白的大小分别约为120kDa和97kDa。以纯化蛋白为材料进行了ActA单抗的研制,获得4株抗ActA的单克隆抗体杂交瘤细胞株,腹水单抗ELISA效价为1∶5×104~1∶1×105。选取单抗1A5进行Westernblot分析,结果表明单抗1A5能和表达产物进行特异性反应,且与Lm-4多抗血清的Westernblot结果一致。actA基因的原核表达及单抗的研制为研究ActA蛋白的生物学活性及其致病作用奠定了基础。 相似文献
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Evidence implicating the 5' untranslated region of Listeria monocytogenes actA in the regulation of bacterial actin-based motility 总被引:4,自引:0,他引:4
The ActA protein of Listeria monocytogenes is a major virulence factor, essential for the recruitment and polymerization of host actin filaments that lead to intracellular motility and cell-to-cell spread of bacteria within the infected host. The expression of actA is tightly regulated and is strongly induced only when L. monocytogenes is within the host cytosol. Intracellular induction of actA expression is mediated through a single promoter element that directs the expression of a messenger RNA with a long (150 bp) 5' untranslated region (UTR). Deletion of the actA+3 to +130 upstream region was found to result in bacterial mutants that were no longer capable of intracellular actin recruitment or cell-to-cell spread, thus indicating that this region is important for actA expression. L. monocytogenes strains that contained smaller deletions (21-23 bp) within the actA upstream region demonstrated a range of actA expression levels that coincided with the amount of bacterial cell-to-cell spread observed within infected monolayers. A correlation appeared to exist between levels of actA expression and the ability of L. monocytogenes to transition from uniform actin accumulation surrounding individual bacteria (actin clouds) to directional assembly and the formation of actin tails. Bacterial mutants containing deletions that most significantly altered the predicted secondary structure of the actA mRNA 5' UTR had the largest reductions in actA expression. These results suggest that the actA 5' UTR is required for maximal ActA synthesis and that a threshold level of ActA synthesis must be achieved to promote the transition from bacteria-associated actin clouds to directional actin assembly and movement. 相似文献
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A novel mutation within the central Listeria monocytogenes regulator PrfA that results in constitutive expression of virulence gene products 总被引:7,自引:0,他引:7
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The PrfA protein of Listeria monocytogenes functions as a key regulatory factor for the coordinated expression of many virulence genes during bacterial infection of host cells. PrfA activity is controlled by multiple regulatory mechanisms, including an apparent requirement for either the presence of a cofactor or some form of posttranslational modification that regulates the activation of PrfA. In this study, we describe the identification and characterization of a novel PrfA mutation that results in constitutive activation of the PrfA protein. The PrfA L140F mutation was found to confer high-level expression of PrfA-regulated genes and to be functionally dominant over the wild-type allele. The presence of the PrfA L140F mutation resulted in the aggregation of L. monocytogenes in broth culture and, unlike previously described prfA mutations, appeared to be slightly toxic to the bacteria. High-level PrfA-dependent gene expression showed no additional increase in L. monocytogenes strains containing an additional copy of prfA L140F despite a >4-fold increase in PrfA protein levels. In contrast, the introduction of multiple copies of the wild-type prfA allele to L. monocytogenes resulted in a corresponding increase in PrfA-dependent gene expression, although overall expression levels remained far below those observed for PrfA L140F strains. These results suggest a hierarchy of PrfA regulation, such that the relative levels of PrfA protein present within the cell correlate with the levels of PrfA-dependent gene expression when the protein is not in its fully activated state; however, saturating levels of the protein are then quickly reached when PrfA is converted to its active form. Regulation of the PrfA activation status must be an important facet of L. monocytogenes survival, as mutations that result in constitutive PrfA activation may have deleterious consequences for bacterial physiology. 相似文献
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Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion 总被引:10,自引:0,他引:10
Marie-Hélène Coconnier Marie-Françoise Bernet Sophie Kernéis Gilles Chauvière Jacky Fourniat Alain L. Servin 《FEMS microbiology letters》1993,110(3):299-305
Abstract Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC) were found to adhere to the brush border of differentiated human intestinal epithelial Caco-2 cells in culture, whereas Yersinia pseudotuberculosis and Listeria monocytogenes adhered to the periphery of undifferentiated Caco-2 cells. All these enterovirulent strains invaded the Caco-2 cells. Using a heat-killed human Lactobacillus acidophilus (strain LB) which strongly adheres both to undifferentiated and differentiated Caco-2 cells, we have studied inhibition of cell association with and invasion within Caco-2 cells by enterovirulent bacteria. Living and heat-killed Lactobacillus acidophilus strain LB inhibited both cell association and invasion of Caco-2 cells by enterovirulent bacteria in a concentration-dependent manner. The mechanism of inhibition of both adhesion and invasion appears to be due to steric hindrance of human enterocytic pathogen receptors by whole-cell lactobacilli rather than to a specific blockade of receptors. 相似文献
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依赖PrfA转录调控的单核细胞增生李斯特菌毒力基因inlC启动子结构特点的初步研究 总被引:1,自引:0,他引:1
为了研究单核细胞增生李斯特菌毒力基因启动子的结构特点与转录调控因子PrfA蛋白之间的关系,应用PCR定点突变和重组PCR技术缺失了该菌毒力基因inlC启动子上可能与PrfA蛋白结合以及诱发转录起始相关的碱基序列,构建了一系列突变启动子与lacZ报告基因融合表达质粒, 使lacZ基因的表达置于inlC突变启动子下,并分别电转化单核细胞增生李斯特菌野生株P14、PrfA蛋白高表达突变株P14a 和prfA基因等位缺失突变株A42中,检测相应的β-半乳糖苷酶活性。结果表明:位于inlC启动子转录起始点下游22bp 处的一段17bp的类似PrfA蛋白结合序列TTAACAGCGTTTGTTAA并没有增强和抑制PrfA转录调控活性的功能;甚至将其改造成“完美的” PrfA蛋白结合序列TTAACATTTGTTAA后,也不影响inlC依赖于PrfA的转录活性地表达;但是,如果缺失inlC启动子上原始的PrfA蛋白结合序列,则使inlC依赖于PrfA的转录活性完全丧失;另外,单核细胞增生李斯特菌毒力基因inlC和plcA 依赖于PrfA的转录活性的表达也与启动子上PrfA蛋白结合区(PrfA-box)距离-10区的碱基个数有关:最适为22或23bp,长于23bp或短于22bp的突变启动子的依赖PrfA的转录活性大大降低,甚至没有活性。说明除PrfA蛋白结合序列外,受PrfA调控的毒力基因启动子上还可能存在其它尚未阐明的结构和序列影响PrfA蛋白的结合以及启动转录表达。 相似文献
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AIMS: The mechanism of the host cell invasion of Plesiomonas shigelloides and its capability to induce apoptosis were investigated. METHODS AND RESULTS: We performed a time course experiment on the bacterial adherence and invasion of the P. shigelloides P-1 strain into Caco-2 cells using an invasion assay and flow cytometry. The adherence of P. shigelloides to the Caco-2 cells was almost completed within 10 min after the infection. Thereafter, P. shigelloides starts internalization within the Caco-2 cells, which was completed within 60 min after the infection. Based on the invasion assay using nocodazole, cytochalasin D, and genistein, it became clear that the mechanism of the internalization depended on the signal transduction followed by the rearrangement of the cytoskeletal protein. Based on the DNA laddering and TUNEL methods, the cytotoxicity of the Caco-2 cells by the invasion of P. shigelloides occurred through the induction of apoptosis. CONCLUSIONS: This work demonstrated that the mechanism of invasion of P. shigelloides into Caco-2 cells and the invasion of P. shigelloides induces apoptotic cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the virulence factor, which may be important for understanding of the pathogenesis of P. shigelloides. 相似文献