福氏痢疾菌膜成分与细菌抗原性和毒力的关系

应用免疫转移技术,用从感染福氏痢疾菌的病人获取的恢复期血清分析了福氏痢疾菌膜成分与细菌抗原性和毒力的关一。发现福氏痢疾菌的膜蛋白67kD和63kD均含有两种成分,一种和膜蛋白60kD都可能是保护性抗原,而另一种与膜蛋白78kD和35kD一样与福氏痢疾菌的毒力有关。采用微细胞同位素掺入示踪显示这些与病人恢复期血清反应的膜蛋白由质粒编码。     

Development of quinolone resistance and prevalence of different virulence genes among Shigella flexneri and Shigella dysenteriae in environmental water samples

The purpose of this study was to find out the mechanism of quinolone resistance in Shigella sp. isolated from environmental water samples from various parts of Kolkata, India. Out of 196 Shigella sp. isolated from 2014 to 2017, we selected 32 Shigella isolates for antimicrobial susceptibility tests. The minimum inhibitory concentrations (MIC) for quinolones ranged from 30 to 50 μg ml⁻¹ for ofloxacin, 5–20 μg ml⁻¹ for ciprofloxacin and 20–30 μg ml⁻¹ for norfloxacin. A few amino acid changes were found in quinolone resistance determining region (QRDR) of gyrA. Mutations in gyrA lead to a higher increment of MIC of quinolones. Among the plasmid-mediated (PMQR) quinolone resistance genes investigated, qnrB and aac(6')-lb-cr genes were found in all isolates. qnrA and qnrS were found in 25% and 62% of the isolates, respectively. ipaH gene was found in all of the isolates followed by the presence of other virulence genes ial, sen and stx1. Almost all the isolates having high MICs showed efflux pump activity in drug accumulation assay. All the mechanisms may or may not be present in a single strain. Several types of efflux pumps, presence of PMQR genes and mutations in drug target site of QRDR region may play the crucial role for resistance in our isolates.     

Detection of protease specifically splitting actin in revertants of Shigella flexneri L-forms

Proteolytic activity of cell extracts from revertants of Shigella flexneri L-forms as well as biochemical properties of these strains and their sensitivity to antibiotics were studied. The protease found earlier in cells of strain E. coli A2 was shown to be synthesized by one of 8 revertants under study. This protease split actin and did not split some other proteins, its activity was inhibited by inhibitors of metalloproteases. Strain 5a2c which produced the protease was similar to the strain E. coli A2 and differed from other revertants in some biochemical properties, resistance to ampicillin and sensitivity to furazolidone. Thus the protease activity can be a marker of structural and functional transformation of Sh. flexneri under the influence of furazolidone.     

肺炎链球菌体内诱导基因的筛选及初步鉴定

为筛选鉴定肺炎链球菌宿主体内诱导的基因,寻找潜在的抗生素作用靶点和疫苗候选者,应用体内表达技术,以肺炎链球菌荚膜合成的关键基因galU作为体内报告基因,利用其缺陷体不能合成荚膜多糖,从而不能在宿主体内存活的特点,筛选鉴定肺炎链球菌体内诱导基因。首先,把肺炎链球菌基因组DNA的随机酶切片段(200~500bp)克隆到含有体内、体外双重报告基因(galU-lacZ)的报告载体pEVP3-galU的BglⅡ位点,将获得的质粒库转化肺炎链球菌galU缺陷菌株,得到肺炎链球菌体内启动子诱捕文库,将此文库去感染BALB/c小鼠,经过两轮体内筛选,在涂布有X-gal的TSA血清平板上得到了165个白色菌落,对插入的随机片段进行测序及生物信息学分析,共证实15个不同的体内诱导基因片段,8个为单独的ORF,7个为含有多个ORFs的操纵子结构,它们分别参与细菌在宿主体内的定植与粘附、能量代谢、物质转运、转录调节、DNA复制与重组、细胞壁合成等,另外还包括功能不明的假想蛋白。其中部分ORFs可能与细菌毒力相关,可以作为候选疫苗和药物的靶标。     

Biological properties of bacteria of the species Shigella flexneri

Some biological properties of S. flexneri have been studied. The strains of this bacterial species have been shown to produce DNAase in 98.8 +/- 0.77% of cases and RNAase in 97.4 +/- 1.5% of cases. The capacity for the positive reaction with Congo red as early as after 24-hour incubation in the thermostat has proved to be characteristic of S. flexneri (91.1 +/- 3.6%). If stored at 4 degrees C in semiliquid agar, S. flexneri cultures have been found to retain their capacity for producing the above-mentioned enzymes as long as 10-13 years. All S. flexneri serovars under study, with the exception of serovar 6, have shown high activity in the manifestation of their properties.     

Surprising dependence on postsegregational killing of host cells for maintenance of the large virulence plasmid of Shigella flexneri

Low-copy-number plasmids all encode multiple systems to ensure their propagation, including replication, partition (active segregation), and postsegregational killing (PSK) systems. PSK systems kill those rare cells that lose the plasmid due to replication or segregation errors. PSK systems should not be used as the principle means of maintaining the plasmid. The metabolic cost of killing the many cured cells that would arise from random plasmid segregation is far too high. Here we describe an interesting exception to this rule. Maintenance of the large virulence plasmid of Shigella flexneri is highly dependent on one of its PSK systems, mvp, at 37 degrees C, the temperature experienced during pathogenesis. At 37 degrees C, the plasmid is very unstable and mvp efficiently kills the resulting cured bacterial cells. This imposes a major growth disadvantage on the virulent bacterial population. The systems that normally ensure accurate plasmid replication and segregation are attenuated or overridden at 37 degrees C. At 30 degrees C, a temperature encountered by Shigella in the outside environment, the maintenance systems function normally and the plasmid is no longer dependent on mvp. We discuss why the virulent pathogen tolerates this self-destructive method of propagation at the temperature of infection.     

痢疾杆菌体内诱导基因筛选方法的建立

分别用氯霉素乙酰转移酶基因(cat)和天冬氨酸半醛脱氢酶基因(asd)作为报告基因,用小鼠肺感染模型和HeLa细胞侵袭模型来试验筛选痢疾杆菌体内诱导基因的可行性。结果表明,以asd为报告基因,用HeLa细胞侵袭试验作为筛选模型,可成功地用于筛选痢疾杆菌的体内诱导基因。     

Regulation of O-antigen chain length is required for Shigella flexneri virulence

It is shown that Shigella flexneri maintains genetic control over the modal chain length of the O-antigen polysaccharide chains of its lipopolysaccharide (LPS) molecules because such a distribution is required for virulence. The effect of altering O-antigen chain length on S. flexneri virulence was investigated by inserting a kanamycin (Km)-resistance cassette into the rol gene (controlling the modal O-antigen chain length distribution), and into the rfbD gene, whose product is needed for synthesis of dTDP-rhamnose (the precursor of rhamnose in the O-antigen). The mutations had the expected effect on LPS structure. The rol ::Km mutation was impaired in the ability to elicit keratoconjunctivitis, as determined by the Serény test. The rol ::Km and rfbD ::Km mutations prevented plaque formation on HeLa cells, but neither mutation affected the ability of S. flexneri to invade and replicate in HeLa cells. Microscopy of bacteria-infected HeLa cells stained with fluorescein isothiocyanate (FITC)-phalloidin demonstrated that both the rol ::Km and rfbD ::Km mutants were defective in F-actin tail formation: the latter mutant showed distorted F-actin tails. Plasma-membrane protrusions were occasionally observed. Investigation of the location of IcsA (required for F-actin tail formation) on the cell surface by immunofluorescence and immunogold electron microscopy showed that while most rol mutant bacteria produced little or no cell-surface IcsA, 10% resembled the parental bacterial cell (which had IcsA at one cell pole; the rfbD mutant had IcsA located over its entire cell surface although it was more concentrated at one end of the cell). That the O-antigen chains of the rol ::Km mutant did not mask the IcsA protein was demonstrated by using the endorhamnosidase activity of Sf6c phage to digest the O-antigen chains, and comparing untreated and Sf6c-treated cells by immunofluorescence with anti-IcsA serum.     

Comparative characteristics of cell-free filtrates of Shigella flexneri of different virulence]

It was shown for the first time that the virulent Sh. flexneri strain grown on Luria broth differed from the avirulent one by the yield of readily released surface-located complexes--lipopolysaccharide (determined by rhamnose) and protein into the filtrate. There was no distinct correlation between the strain virulence and the content of rhamnose-determined lipopolysaccharide in the filtrate; growing bacteria in the presence of Ca and Mg ions had no significant influence on the lipopolysaccharide release into the filtrate. Protein release into the cell-free filtrate was thrice that in the virulent shigella strain than in the avirulent one. When bacteria were grown in the presence of Ca ions protein release from the virulent strain increased 1.5-fold and changed but little in the avirulent culture. Cell-free filtrates of the virulent strain produced toxic action on L tissue culture cells; in conjunctival infection of guinea pigs they caused some reduction of the LD50 of the virulent strain and sharply aggravated the course of the infectious process. Heating of the filtrate at 100 degrees C for 15 min decreased their toxic action on L cells. The data obtained indicated that the active biological factor revealed in the virulent strain of Sh. flexneri was protein or its derivative.     

Molecular cloning and characterization of chromosomal virulence region kcpA of Shigella flexneri

In Shigella flexneri, in addition to several well-recognized plasmid-borne virulence loci, at least three genetic loci implicated in pathogenesis have been recognized on the chromosome. To understand more about the pathogenesis of bacillary dysentery at a molecular level, the genetically recognized but previously unidentified KcpA region (one of the chromosomal regions near purE) was cloned and sequenced. A single translatable open reading frame encoding a 12310 Dalton protein corresponding to the minicell product was found. Immunofluorescence microscopy, as well as optical and electron microscopic comparison of tissue-cultured cells and guinea-pigs' eyes infected with wild-type or kcpA mutant bacteria, revealed that the kcpA product is required by invading bacteria for spread into adjacent cells.