具核梭杆菌促进结直肠癌发生发展机制研究进展

具核梭杆菌(Fusobacterium nucleatum)是一种人体共生菌,尤其富集于口腔,在特定情况下可导致机会感染.近年来,随着微生物群与健康或疾病相关性研究的深入,具核梭杆菌与结直肠癌(colorectal cancer,CRC)之间的关联性研究备受关注.大量临床研究表明,具核梭杆菌在CRC中更为富集,且进一步...     

Correct folding of the beta-barrel of the human membrane protein VDAC requires a lipid bilayer

Spontaneous membrane insertion and folding of beta-barrel membrane proteins from an unfolded state into lipid bilayers has been shown previously only for few outer membrane proteins of Gram-negative bacteria. Here we investigated membrane insertion and folding of a human membrane protein, the isoform 1 of the voltage-dependent anion-selective channel (hVDAC1) of mitochondrial outer membranes. Two classes of transmembrane proteins with either alpha-helical or beta-barrel membrane domains are known from the solved high-resolution structures. VDAC forms a transmembrane beta-barrel with an additional N-terminal alpha-helix. We demonstrate that similar to bacterial OmpA, urea-unfolded hVDAC1 spontaneously inserts and folds into lipid bilayers upon denaturant dilution in the absence of folding assistants or energy sources like ATP. Recordings of the voltage-dependence of the single channel conductance confirmed folding of hVDAC1 to its active form. hVDAC1 developed first beta-sheet secondary structure in aqueous solution, while the alpha-helical structure was formed in the presence of lipid or detergent. In stark contrast to bacterial beta-barrel membrane proteins, hVDAC1 formed different structures in detergent micelles and phospholipid bilayers, with higher content of beta-sheet and lower content of alpha-helix when inserted and folded into lipid bilayers. Experiments with mixtures of lipid and detergent indicated that the content of beta-sheet secondary structure in hVDAC1 decreased at increased detergent content. Unlike bacterial beta-barrel membrane proteins, hVDAC1 was not stable even in mild detergents such as LDAO or dodecylmaltoside. Spontaneous folding of outer membrane proteins into lipid bilayers indicates that in cells, the main purpose of membrane-inserted or associated assembly factors may be to select and target beta-barrel membrane proteins towards the outer membrane instead of actively assembling them under consumption of energy as described for the translocons of cytoplasmic membranes.     

具核梭杆菌协同Cdk5促进结直肠癌细胞迁移

目的以结直肠癌细胞SW480作为研究载体,分析具核梭杆菌(F.nucleatum)对Cdk5和STAT3途径相关基因及其炎症因子表达的影响,阐明F.nucleatum协同Cdk5促进结直肠癌形成和发展的分子机制。方法以结直肠癌细胞SW480作为研究载体,运用Western Blotting、qPCR、免疫组化和细胞划痕等实验研究F.nucleatum和Cdk5对结直肠癌形成的影响。结果免疫组化结果显示,癌组织Cdk5阳性表达率明显高于癌旁组织(t=8.218,P0.01)。细胞划痕实验结果表明,F.nucleatum菌液作用的结直肠癌细胞迁移率明显高于对照组(24 h:t=5.868,P0.01; 48 h:t=6.941,P0.01)。Western Blotting结果显示,F.nucleatum协同Cdk5可能通过STAT3通路调控结直肠癌细胞凋亡。qPCR结果显示,F.nucleatum菌液作用的结直肠癌细胞的炎症因子表达明显高于对照组(IL-6:t=5.542,P0.05; COX2:t=16.893,P0.01; TNF-α:t=16.963,P0.01; IL-8:t=3.733,P0.01)。结论 F.nucleatum协同Cdk5促进了结直肠癌细胞的迁移。     

Oxidative stress response in the opportunistic oral pathogen Fusobacterium nucleatum

The anaerobic, Gram-negative bacillus Fusobacterium nucleatum plays a vital role in oral biofilm formation and the development of periodontal disease. The organism plays a central bridging role between early and late colonizers within dental plaque and plays a protective role against reactive oxygen species. Using a two-dimensional gel electrophoresis and mass spectrometry approach, we have annotated 78 proteins within the proteome of F. nucleatum subsp. nucleatum and identified those proteins whose apparent intracellular concentrations change in response to either O(2)- or H(2)O(2)-induced oxidative stress. Three major protein systems were altered in response to oxidative stress: (i) proteins of the alkyl hydroperoxide reductase/thioredoxin reductase system were increased in intracellular concentration; (ii) glycolytic enzymes were modified by oxidation (i.e. D-glyceraldehyde 3-phosphate dehydrogenase, and fructose 6-phosphate aldolase) or increased in intracellular concentration, with an accompanying decrease in ATP production; and (iii) the intracellular concentrations of molecular chaperone proteins and related proteins (i.e. ClpB, DnaK, HtpG, and HrcA) were increased.     

Biochemical and biological characterization of ruminal Fusobacterium necrophorum

Abstract Biochemical characteristics, biological activities, and antimicrobial susceptibilities of ruminal Fusobacterium necrophorum (eight subsp. necrophorum and eight subsp. funduliforme ) and of isolates (three of each subsp.) obtained from bovine hepatic abscesses were determined. F. necrophorum subsp. necrophorum strains had higher phosphatase and DNase activities, produced more leukotoxin, and were more pathogenic to mice than subsp. funduliforme strains. The leukotoxin titer for culture supernatants of ruminal subsp. necrophorum strains was approximately 15 times lower than that of hepatic subsp. necrophorum strains. Hemagglutination activity was present in all hepatic, but only in some ruminal, strains of subsp. necrophorum . The antimicrobial sensitivity profile of the ruminal isolates was similar to that of hepatic isolates.     

具核梭杆菌对结直肠癌诊疗作用的研究进展

口腔菌群作为人体的第二大微生物群,对人体健康具有非常重要的作用。具核梭杆菌是口腔中的常驻菌之一,与其他菌共同维持机体的稳态,现已被证实是结直肠癌最相关的菌属之一,参与结直肠癌的发生、发展以及预后。很多学者根据具核梭杆菌在结直肠癌中的致病机制,寻找以具核梭杆菌为靶点的对结直肠癌进行早期诊断和治疗的新方法。本文阐述了具核梭杆菌在结直肠癌中的致病机制,着重探讨其在结直肠癌的诊断和治疗中的潜在作用,以期为临床诊疗提供参考。

     

磁纳米散射探针暗场超灵敏检测具核梭杆菌

【背景】具核梭杆菌（Fusobacterium nucleatum）作为机会致病菌,能够引起许多感染性疾病,已被证实是促直肠癌发展的潜在重要危险因素,临床检测中亟需一种快速简单检测F. nucleatum的方法。【目的】通过建立一种磁纳米探针结合暗场显微镜直接观察计数的方法,可方便快速地检测样本中具核梭杆菌的数量。【方法】在磁纳米颗粒（magnetic nanoparticle,MNP）表面修饰制备的抗F. nucleatum抗体,构建一种特异性结合F. nucleatum的MNP探针。此外,比较MNP探针-暗场显微计数法与实时荧光定量PCR （qPCR）方法检测F. nucleatum的灵敏度。【结果】该方法的检测限可低至3.42×10¹ copies/μL,比qPCR的灵敏度高5倍左右。在实际样本的检测中,该方法与qPCR方法所检测F.nucleatum数量保持一致。【结论】本研究建立的方法用于检测F.nucleatum,操作简单、检测快速（约30 min）、灵敏且成本低,有应用于临床样本检测的前景。     

Studies on the possible role of cattle nuisance flies, especially Hydrotaea irritans, in the transmission of summer mastitis in Denmark

The summer mastitis pathogens Actinomyces pyogenes, Peptococcus indolicus, Bacteroides melaninogenicus ss. levii, Fusobacterium necrophorum and Streptococcus dysgalactiae were isolated from the polyphagous symbovine dipterans Hydrotaea irritans (Fallén) and Morellia sp. caught around dairy heifers on pasture, but not from the haematophagous species Haematobia irritans (L.), Haematobosca stimulans (Meigen), Culicoides sp. and Simulium sp. Secretions from clinical cases of summer mastitis proved to be sources of summer mastitis bacteria for more than 3 weeks despite antibiotic treatment and teat amputation. Taking into account the seasonal activity pattern of Hydrotaea irritans and its topographical distribution on grazing cattle, it appears evident that this fly may play a central role in the establishment and maintenance of the bacterial contamination with summer mastitis pathogens on the teats of healthy cattle. In the present study the survival of A.pyogenes and P.indolicus for 7 days in experimentally infected Hydrotaea irritans, as demonstrated by the recovery of these microorganisms from agar plates exposed to live infected flies, is described. However, experimental transmission of summer mastitis from sick to healthy heifers by Hydrotaea irritans proved unsuccessful.     

具核梭杆菌对结直肠癌小鼠化疗敏感性的影响

目的研究具核梭杆菌对结直肠癌小鼠化疗敏感性的影响。方法建立结直肠癌荷瘤小鼠,分为空白对照组、5-氟尿嘧啶对照组和实验组、奥沙利铂实验组和对照组、伊立替康实验组和对照组、阿霉素实验组和对照组、丝裂霉素实验组和对照组,每组5只;各实验组荷瘤鼠灌胃给予具核梭杆菌菌液(10~9CFU),0.2 mL/d,1次/周。连续灌胃4周后,5-氟尿嘧啶实验组、阿霉素实验组、丝裂霉素实验组荷瘤鼠分别腹腔注射给予30.00 mg/(kg·d)、1.75 mg/(kg·d)、2.00 mg/(kg·d)对应药物,1次/d,连用7 d;奥沙利铂实验组、伊立替康实验组荷瘤鼠分别第1天腹腔注射给予29 mg/kg、66 mg/kg对应药物,其余6 d用生理盐水0.1 mL代替。各对照组荷瘤鼠除不灌胃具核梭杆菌菌液外,其余操作均与实验组相同。对比各实验组和对照组的瘤重和抑瘤率(IR%)。结果各组成瘤裸鼠一般情况均正常,肿瘤呈膨胀性生长,未见明显浸润或转移发生。各实验组荷瘤鼠的瘤重(g)均显著大于对照组荷瘤鼠[(1.42±0.15)vs(0.97±0.12),(1.76±0.16)vs(1.45±0.13),(1.50±0.09)vs(1.03±0.08),(1.38±0.07)vs(0.87±0.05),(1.26±0.08)vs(0.79±0.05);均P<0.05],IR%显著小于对照组荷瘤鼠[(27.55±2.83)vs(50.51±5.02),(10.20±1.78)vs(26.02±2.36),(23.47±2.76)vs(47.45±4.86),(29.59±3.02)vs(55.61±5.35),(35.71±3.47)vs(59.69±5.45);均P<0.05]。结论具核梭杆菌降低抗结直肠癌药物的敏感性。     

Isolation and characterization of a human neutrophil aggregation defective mutant of Fusobacterium nucleatum

Fusobacterium nucleatum is known to adhere to human polymorphonuclear neutrophils (PMNs) and cause them to aggregate. In this study, we isolated a spontaneously occurring aggregation defective (AGG(-)) mutant and this mutant will be used for future study of the interactions between this bacterium and human PMN. Genomic DNA fingerprinting by random-primed polymerase chain reaction method revealed a difference between the parent strain and the AGG(-) mutant. This mutant also showed an altered phenotype in both microbicidal and phagocytic assays, suggesting that the bacterial factor involved in the aggregation may also be very important for the phagocytosis and, subsequently, the killing by human PMNs. Further study of this mutant may help to clarify the molecular mechanisms of the interaction between this pathogen and human PMNs.