嗜肺军团菌致病性与效应蛋白的研究进展

嗜肺军团菌是一种可以引起军团菌肺炎和庞蒂亚克热的兼性胞内病原菌，主要侵染阿米巴原虫和人类巨噬细胞。该菌在宿主胞内能依靠Dot/Icm IVB型分泌系统产生的效应蛋白成功逃避溶酶体的降解。本文主要对嗜肺军团菌的致病物质、胞内存活与增殖机制及其效应蛋白的生物学功能进行综述，详细介绍嗜肺军团菌的毒力因子与致病机制，为军团病防治的研究提供新思路，也为其他胞内病原菌所致感染性疾病的研究提供重要的借鉴意义。     

Experimental infection with high‐ and low‐virulence strains of border disease virus (BDV) in Pyrenean chamois (Rupicapra p. pyrenaica) sheds light on the epidemiological diversity of the disease

Since 2001, Pyrenean chamois (Rupicapra pyrenaica pyrenaica) populations have been affected by border disease virus (BDV) causing mortalities of more than 80% in some areas. Field studies carried out in France, Andorra, and Spain have shown different epidemiological scenarios in chamois populations. This study was designed to confirm the presence of BDV strains of a high and low virulence in free‐ranging chamois populations from Pyrenees and to understand the implications of these findings to the diverse epidemiological scenarios. An experimental infection of Pyrenean chamois with a high‐virulence (Cadí‐6) and low‐virulence (Freser‐5) BDV strains was performed. Pregnant and non‐pregnant animals with and without antibodies against BDV were included in each group. Cadí‐6 BDV strain was confirmed to be of high virulence for seronegative adults and their foetuses. The antibody negative chamois infected with Freser‐5 BDV strain did not show symptoms, presented less viral distribution and RNA load in tissues than Cadí‐6 group, and cleared the virus from the serum. However, foetuses died before the end of the experiment and RNA virus was detected in sera and tissues although with lower RNA load than the Cadí‐6 group. Chamois from both groups presented lesions in brain but the ones infected with the low‐virulence Freser‐5 BDV strain were mild and most likely transient. In both groups, seropositive pregnant females and all but one of their foetuses did not present viraemia or viral RNA in tissues. The existence of a low‐virulence strain has been confirmed experimentally and related to chamois population infection dynamics in the area where it was isolated. Such strain may persist in the chamois population through PI animals and may induce cross‐protection in chamois against high‐virulence strains. This study demonstrates that viral strain diversity is a significant factor in the heterogeneity of epidemiological scenarios in Pyrenean chamois populations.     

Virulence factors impair epithelial junctions during bacterial infection

Epithelial cells are typically connected through different types of cell junctions that are localized from the apical membrane to the basal surface. In this way, epithelium cells form the first barrier against pathogenic microorganisms and prevent their entry into internal organs and the circulatory system. Recent studies demonstrate that bacterial pathogens disrupt epithelial cell junctions through targeting junctional proteins by secreted virulence factors. In this review, we discuss the diverse strategies used by common bacterial pathogens, including Pseudomonas aeruginosa, Helicobacter pylori, and enteropathogenic Escherichia coli, to disrupt epithelial cell junctions during infection. We also discuss the potential of targeting the pathogenic mechanisms in the treatment of pathogen‐associated diseases.     

Synergistic virulence of Porphyromonas gingivalis and Treponema denticola in a murine periodontitis model

Chronic periodontitis is characterized by the destruction of the tissues supporting the teeth and has been associated with the presence of a subgingival polymicrobial biofilm containing Porphyromonas gingivalis and Treponema denticola. We have investigated the potential synergistic virulence of P. gingivalis and T. denticola using a murine experimental model of periodontitis. An inoculation regime of four intra-oral doses of 1 × 10(10) P. gingivalis cells induced significant periodontal bone loss compared with loss in sham-inoculated mice, whereas doses of 1 × 10(9) cells or lower did not induce bone loss. Inoculation with T. denticola with up to eight doses of 1 × 10(10) cells failed to induce bone loss in this model. However, four doses of a co-inoculum of a 1 : 1 ratio of P. gingivalis and T. denticola at 5 × 10(8) or 1 × 10(9) total bacterial cells induced the same level of bone loss as four doses of 1 × 10(10) P. gingivalis cells. Co-inoculation induced strong P. gingivalis-specific T-cell proliferative and interferon-γ-dominant cytokine responses, and induced a strong T. denticola-specific interferon-γ dominant cytokine response. Only at the higher co-inoculum dose of 1 × 10(10) total cells was a T. denticola-specific T-cell proliferative response observed. These data show that P. gingivalis and T. denticola act synergistically to stimulate the host immune response and to induce alveolar bone loss in a murine experimental periodontitis model.     

Polymerase chain reaction for the identification of Porphyromonas gingivalis collagenase genes

Porphyromonas gingivalis has been shown to exhibit genetic diversity possibly resulting in variation of virulence. In the present study a potential virulence factor was targeted for the detection of P. gingivalis. A 548 bp fragment of the collagenase gene ( prtC ) from Porphyromonas gingivalis ATCC 33277 was amplified by polymerase chain reaction (PCR) using oligonucleotides derived from the middle portion of prtC. From 16 of 21 clinical P. gingivalis strains, a PCR product of similar size to the prtC could be obtained. These 16 P. gingivalis strains were confirmed as positive for prtC using DNA hybridization with a digoxigenin-labeled prtC PCR product as a probe. In 12 of the 16 prtC positive strains, the restriction analysis of the PCR products revealed fragment patterns identical to the known sequence. In the other 4 prtC positive strains, 4 distinct patterns were found. Of these strains, nucleotide sequence analysis of a 400 bp PCR product stretch revealed 79.1%, 83.0%, 84.8 and 89.5% homology with the known nucleotide sequence for this specific region. Sequence analysis of the PCR products from the ATCC 33277 strain demonstrated 93.7% homology. The limit of detection for the PCR was about 100 organisms. None of the other 48 tested strains of 16 bacterial species derived from oral and extraoral infections yielded a PCR product. The PCR was also used for the detection of prtC sequences in dental plaque. Our data indicate that not all P. gingivalis strains have prtC. Nucleotide heterogeneity exists among P. gingivalis with prtC. Using a potential virulence factor for the detection of putative periodontal pathogens such as P. gingivalis may be valuable for the epidemiology of infection and clinical diagnosis of periodontal diseases.     

Virulence of a polymicrobic complex,Treponema denticola and Porphyromonas gingivalis,in a murine model

The effect of a polymierobic infection employing Treponema denticola and Porphyromonas gingivalis in the murine lesion model was used to determine the synergistic virulence of these two periodontopathic bacteria. At high doses of P. gingivalis W50, addition of T. denticola in the infection mixture had no effect on the formation and size of the spreading lesion caused by this microorganism. However, at low P. gingivalis challenge doses, T. denticola significantly enhanced the virulence of P. gingivalis compared with monoinfection of this microorganism. A potential role of the trypsin-like protease enzyme activity of P. gingivalis in this synergistic virulence was tested using P. gingivalis mutants deficient (i.e., BEI) or devoid (i.e., NG4B19) of this protease activity. These findings demonstrated that T. denticola—P. gingivalis complexes exhibit enhanced virulence in this model and that even using a polymicrobic challenge infection, the trypsin-like protease activity was important to P. gingivalis virulence expression.     

Modulation of host matrix metalloproteinases by bacterial virulence factors relevant in human periodontal diseases

OBJECTIVE: Bacterial pathogens involved in periodontal diseases exert a part of their destructive effect by triggering and inducing host cells to elevate their secretion of matrix metalloproteinases (MMPs). Pathogen-secreted phospholipase (PLC) is one bacterial product that may trigger this host response. The roles of exogenous PLC leading to the release, secretion and expression of MMPs by peripheral blood neutrophils (PMNs), cultured epithelial cells of human gingiva and porcine periodontal ligament were investigated. Also the activities of PLC in the diseased and healthy gingival sulcular fluid (crevicular fluid, GCF) and molecular forms of gelatin-ases present in dental plaque were investigated. MATERIALS AND METHODS GCF, salivary and dental plaque samples were analyzed for PLC and proteinase activities. The abilities of PLC to induce PMNs and oral epithelial cells to release and express their MMPs were examined by specific functional, immunological and molecular biology means. RESULTS: PMN-derived MMPs were found to predominate in periodontitis GCF and plaque, and PLC activities were higher in GCF of adult periodontitis patients than in healthy controls. Purified bacterial PLC (1 mU ml^-I) efficiently induced PMN degranulation. PLC also induced MMP expression in the cultured epithelial cells. The strongest response was seen in MMP-9 and less in MMP-2. The induction was dose-dependent in the range of 0.I-1.0 U ml^-1 PI-PLC, and quiescent cultures were more responsive than proliferating ones. PLC induction of MMPs was polar, with increased levels of MMP-9 in the apical region and increased MMP-2 levels secreted in the basal direction. Northern analysis showed a strong increase in mRNA levels of MMP-9 and a smaller increase for MMP-2 and MMP-I. In the second part of the study we investigated the molecular forms of the released MMPs during periodontitis. In bacterial plaque of periodontitis patients the MMP-9 were found to be converted into lower molecular weight forms. Isolated proteinase from Porphyromonas gingivalis (ATCC 33277) was able to convert human proMMPs to their active forms. CONCLUSION: Bacterial PLC may induce degranulation of PMN MMPs and increase MMP expression in oral epithelial cells. The released proteases can be converted into active form by the proteases of plaque bacteria. Thereby, the pathogenic oral bacteria may indirectly participate in the destruction of periodontal tissues.