Activation of mRNA translation by phage protein and low temperature: the case of Lactococcus lactis abortive infection system AbiD1

Background  

Abortive infection (Abi) mechanisms comprise numerous strategies developed by bacteria to avoid being killed by bacteriophage (phage). Escherichia coli Abis are considered as mediators of programmed cell death, which is induced by infecting phage. Abis were also proposed to be stress response elements, but no environmental activation signals have yet been identified. Abis are widespread in Lactococcus lactis, but regulation of their expression remains an open question. We previously showed that development of AbiD1 abortive infection against phage bIL66 depends on orf1, which is expressed in mid-infection. However, molecular basis for this activation remains unclear.     

Phage abortive infection in lactococci: variations on a theme

Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance.     

Growth Inhibition and Appearance of the Membranous Structure in Escherichia coli Infected with Bacteriophage fd

Thirty-six mutants of fd, a virus that infects but does not kill Escherichia coli, were isolated; 35 mutants were categorized into six complementation groups. Abortive infection with mutants in genes 1, 3, 4, 5, and 6, but not in gene 2, produced a cessation of host cell growth, generally linked to low burst size and to the formation of aberrant intracytoplasmic membranous structures. The membranous structure was studied during infection with various phage and hosts. Appearance of the membranous structure was linked specifically to incomplete phage maturation at the cell membrane, rather than solely to the inhibition of host cell growth or to infection with mutant phage, since (i) in one host, cell growth was inhibited, but no membranous structure developed; and (ii) when antibody against virus was added to cells infected with wild-type phage, phage extrusion was inhibited, cell growth stopped, and the membranous structure once again developed.     

Role of small t antigen in the acute transforming activity of SV40

A plasmid, pHR402, containing SV40 sequences that include a truncated early region bearing an intact t-coding sequence and a functionally intact late region, was introduced into thymidine kinase deficient (tk-) mouse L cells by cotransformation with a cloned tk gene. tk+ cotransformants synthesized SV40 t but not T antigen, and no truncated T-coding sequence products were detected. The viral sequences of pHR402 were reconstituted as a virus in COS1 cells, and acute infection of untransformed mouse cells with this viral stock (SV402) also led to the appearance of t but not T or a truncated T. Abortive transformation assays of such infected cells were negative, as were those performed on the same cells infected with either of two viral mutants (dl883 and dl884), each of which leads to T but not t synthesis. However, mixed infection with SV402 and either dl883 or dl884 led to a clear abortive and permanent transformation response. Thus, at least in part, t and T appear to function in a complementary fashion in eliciting transformation expression by SV40-infected cells.     

Viral regulation of RANTES expression during human cytomegalovirus infection of endothelial cells

Human cytomegalovirus (HCMV) evades healthy immune responses during infection, and this evasion may allow HCMV to establish latency in the host. The human vasculature has been recognized as a site of HCMV infection and may also be a site of latent HCMV infection. As the interface between circulating cells and underlying parenchymal cells, the vascular endothelium provides signals for local reaction of inflammatory cells. We propose that HCMV down-regulates expression of the proinflammatory chemokine RANTES from the infected endothelium, which may result in reduced recruitment of mononuclear cells to the site of infection. Abortive HCMV infection of primary endothelial cells with the clinical isolate HCMV 4010, under conditions in which viral gene expression could not occur, induced high levels of RANTES expression. Replicative HCMV infection, however, induced cells in parallel cultures to express significantly lower levels of RANTES. Expression of the chemokines interleukin 8 and MCP-1 by endothelial cells was found to be unaffected by replicative HCMV infection and thus may not play an important role during early HCMV infection of the endothelium. HCMV may regulate RANTES expression from endothelial cells as a mechanism to evade the local immune response to infection.     

Growth factor and bcl-2 mediated survival during abortive proliferation of hybridoma cell line

Cultures of the CRL-1606 hybridoma (ATCC) have been reported to undergo continuous proliferation with simultaneous death during nutrient limited fed-batch fermentations. The bcl-2 proto-oncogene has been shown to prevent cell death under a variety of otherwise death inducing conditions. We were interested in elucidating the nature of the massive death observed in cultures of CRL-1606, specifically with respect to the possible environmental causes, and the ability of overexpressed human bcl-2 (hbcl-2) to mitigate cell death. Abortive proliferation, or continuous proliferation in the presence of continuous death, could be induced in serum free cultures of CRL-1606 through the withdrawal of insulin provided the culture was competent for cell proliferation. Culture competency for proliferation was found to be solely determined by the presence of cell culture nutrients. Abortive proliferation was defective in cultures transfected with hbcl-2 and the enhanced viability observed resulted from an increased viable cell population and at the expense of the nonviable cell population normally found in untransfected cultures. Abortive proliferation was also observed in serum containing cultures upon serum shiftdowns. Like the insulin-supplemented serum free culture system, hbcl-2 transfected cultures exhibited defects in the abortive proliferation process. These results suggest that the massive death observed during nutrient-limited fed-batch fermentation originate, in part, from growth or survival factor limitations. Hence, approaches to design cell culture media that account for the cell's proliferation requirements without accounting for the cell's survival requirements may represent a cell death sentence. Given the transformed nature of the hybridomas, we conclude that the abortive proliferation of CRL-1606 is a consequence of inappropriate cell cycle entry in a survival factor limited environment.     

红莲型细胞质雄性不育水稻线粒体DNA的RFLP分析

应用RFLP技术,研究了红莲型不育系、保持系、杂种一代及野败型、马协型不育系的线粒体基因组。结果表明,红莲型不育系与保持系线粒体基因组之间在多个基因区域存在明显差异,为红莲型细胞质雄性不育分子机理研究提供了线索;红莲型不育系与野败型不育系的线粒体基因组之间存在显著差异,马协型不育系与野败型不育系的线粒体基因组之间存在一定差异,在分子水平揭示了不育胞质的多样性。 Abstract:Restriction fragment length polymorphism (RFLP) was used to analyze rice mitochondrial genome.The results indicated obvious differences between mt genome of rice cytoplasmic male sterile line and its maintain line of Honglian type,which further clued CMS mechanism research.Mitochondrial genome difference between sterile line of Honglian type and Wild Abortive type was obvious,some difference between sterile Maxie and Wild Abortive was also detected,it offered molecular evidence for cytoplasmic heterogeneity.     

Polyoma virus-human cell interactions: persistence of T-antigen in two cell lines with and without transformation.

The interaction of polyoma virus and human cells was investigated. Abortive infection as evidenced by the synthesis of T-antigen was observed in normal fibroblast and abnormal (transformed) cells but not in normal epithelial cells. A high percentage of simian virus 40-transformed WI-18 Va2 and spontaneously transformed BE skin cells produced T-antigen after high-multiplicity infection, but most of the cells rapidly lost antigen-producing capacity upon cell passage, and the cultures became negative by passage 3. All fibroblast cells displayed varying degrees of susceptibility to infection, but most of the cell lines became negative for T-antigen except for two. In one, T-antigen persisted in a small percentage of the cells throughout the lifetime of the culture, without cellular transformation occurring. In the other, the entire culture became morphologically transformed and eventually consisted of 100% T-antigen-positive cells. This is the first time that normal diploid human fibroblast cells have been transformed by polyoma virus.     

Scaling of immune responses against intracellular bacterial infection

Macrophages detect bacterial infection through pattern recognition receptors (PRRs) localized at the cell surface, in intracellular vesicles or in the cytosol. Discrimination of viable and virulent bacteria from non-virulent bacteria (dead or viable) is necessary to appropriately scale the anti-bacterial immune response. Such scaling of anti-bacterial immunity is necessary to control the infection, but also to avoid immunopathology or bacterial persistence. PRR-mediated detection of bacterial constituents in the cytosol rather than at the cell surface along with cytosolic recognition of secreted bacterial nucleic acids indicates viability and virulence of infecting bacteria. The effector responses triggered by activation of cytosolic PRRs, in particular the RIG-I-induced simultaneous rapid type I IFN induction and inflammasome activation, are crucial for timely control of bacterial infection by innate and adaptive immunity. The knowledge on the PRRs and the effector responses relevant for control of infection with intracellular bacteria will help to develop strategies to overcome chronic infection.     

DNA微阵列技术在细菌感染后宿主反应研究中的应用

感染性疾病是病原微生物和宿主紧密相互作用的结果。深入理解宿主对病原微生物感染发生反应的分子基础是预防感染性疾病发生和组织损伤的必要条件。本文通过介绍体内、体外2种感染模型中宿主对细胞内和细胞外致病菌感染后的基因表达谱变化,简述了DNA微阵列技术在病原菌一宿主相互作用中宿主反应研究中的应用。     

Experimental study of the antibiotic activity of cefuroxime

The antimicrobial spectrum of cefuroxime, an antibiotic of the cephalosporin family was studied in vitro with respect to 11 species (16 strains) of gram-positive and gram-negative bacteria and in vivo on albino mice with experimental salmonellosis or pneumococcal infections. The bacteria were either test cultures or isolates from patients. The studies showed that cefuroxime had a wide antibacterial spectrum in vitro. It inhibited the growth of Staph. aureus, Str. pneumoniae, E. coli, Salm. typhimurium, Kl. pneumoniae, Bac. subtilis and had no effect on Ps. aeruginosa, Pr. vulgaris, M. tuberculosis and M. fortuitum. Cefuroxime had also a high bacteriostatic effect with respect to the experimental pneumococcal infection and a lower bacteriostatic effect with respect to the experimental salmonellosis infection.     

口腔细菌对铜绿假单胞菌黏附及侵入肺上皮细胞能力的影响

目的 通过比较铜绿假单胞菌和口腔细菌单独或共同作用于肺上皮细胞时,细菌黏附和侵入细胞的能力,探讨细菌间相互作用在呼吸道感染的最初阶段的作用机制.方法 应用培养法和抗生素保护法检测铜绿假单胞菌和口腔细菌单独或共同作用于肺上皮细胞时,细菌黏附和侵入肺上皮细胞的能力.结果 铜绿假单胞菌与口腔细菌共同作用于肺上皮细胞,牙龈卟啉单胞菌和伴放线放线杆菌降低了铜绿假单胞菌的黏附能力,却增强了其侵入能力;而铜绿假单胞菌能够影响口腔细菌对肺上皮细胞的黏附,同时增强口腔细菌侵入肺上皮细胞的能力.结论 口腔细菌,尤其是牙周可疑致病菌主要通过增强铜绿假单胞菌对肺上皮细胞的侵入而影响呼吸道感染过程.     

A Modified CDC Biofilm Reactor to Produce Mature Biofilms on the Surface of PEEK Membranes for an In Vivo Animal Model Application

Biofilm-related infections have become a major clinical concern. Typically, animal models that involve inoculation with planktonic bacteria have been used to create positive infection signals and examine antimicrobial strategies for eradicating or preventing biofilm-related infection. However, it is estimated that 99.9% of bacteria in nature dwell in established biofilms. As such, open wounds have significant potential to become contaminated with bacteria that reside in a well-established biofilm. In this study, a modified CDC biofilm reactor was developed to repeatably grow mature biofilms of Staphylococcus aureus on the surface of polyetheretherketone (PEEK) membranes for inoculation in a future animal model of orthopaedic implant biofilm-related infection. Results indicated that uniform, mature biofilms repeatably grew on the surface of the PEEK membranes.     

Polymorphism of mitochondrial DNA and distribution of cytoplasmic symbionts in the populations of two-spot ladybird beetle Adalia bipunctata

In geographically distant populations of ladybird beetle Adalia bipunctata from Eurasia mitotypes and infection with symbiotic bacteria Spiroplasma and Rickettsia were determined. All populations examined demonstrated mtDNA polymorphism and striking differences in prevalence of bacteria (from about 50% of individuals infected with Spiroplasma in St.-Petersburg population and 50% of the Rickettsia prevalence in Kem' population to complete absence of bacteria in the population from Archangelsk). In the populations studied a total of 14 mitotypes were discovered, including two mitotypes that were remarkably different from the others in nucleotide composition. Mitotype 10, which was the most different from all the others, was found in all populations from Germany to Transbaikalia, excluding the population from Tashkent. Linkage disequilibrium between mitotype 10 and the Rickettsia infection was confirmed. Infection with the Spiroplasma bacteria was typical of the individuals with haplotype 1 and relative to it. The results obtained supported the conclusion on the association between infection with Spiroplasma and Rickettsia and certain mitotype of A. bipunctata, which was the consequence of either absence or rare horizontal transfer of symbionts and ancientness of the first contact between the bacteria and A. bipunctata ladybird beetles.     

Microbiological study of the body wall lesions of the echinoid Tripneustes gratilla

The microbiota of the body wall lesions of the echinoid Tripneustes gratilla, initiated by the grazing action of the parasitic gastropod Vexilla vexillum, was investigated with a pluridisciplinary approach. Parasitised sea urchins showed body wall lesions strongly infected by bacteria that progressed through the test and reached the coelomic cavity after ca. 1 mo. We report here on the bacterial community observed in lesions of echinoids collected in situ and on the bacteria that successively appeared during laboratory experiments. Two Alphaproteobacteria, a CFB (Cytophaga-Flavobacterium-Bacteroides) bacterium, 3 Vibrio species and Exiguobacterium aestuarii were identified in the field-collected lesions by 16S rDNA sequencing. The last 4 bacteria were cultured and each induced the disease when inoculated on scalpel-made wounds, with 100% of the individuals infected within 2 d. Scalpel-induced scarifications tended to heal within 3 wk, while gastropod-induced lesions evolved into disease, suggesting a role of Vexilla vexillum in the development of the infection. Denaturing gradient gel electrophoresis (DGGE) and sequencing suggest that (1) bacteria associated with healthy integument were not present in the lesions and were thus not responsible for their infection, (2) Alphaproteobacteria with close phylogenetic affiliation with other bacteria involved in several diseases affecting marine invertebrates were present, and (3) these Alphaproteobacteria were present from the beginning of the infection and appeared earlier in the infection than other bacteria such as CFB bacteria.     

Bacteria-infected fibroblasts have enhanced susceptibility to the cytotoxic action of tumor necrosis factor.

The susceptibility of bacteria-infected fibroblasts to the cytotoxic action of tumor necrosis factor was investigated. L cells infected with Shigella flexneri, Salmonella typhimurium, or Listeria monocytogenes, had an enhanced susceptibility to the cytotoxic activity of TNF-alpha. This enhanced susceptibility was dependent upon the challenge dose of bacteria, the concentration of TNF, and upon the exposure time of bacteria-infected cells to TNF. L cells infected with S. flexneri were susceptible to the cytotoxic action of TNF at 2 to 6 h after bacterial infection. In contrast, L cells infected with S. typhimurium or L. monocytogenes did not show enhanced susceptibility to TNF until 14 h postbacterial infection and exposure to TNF. Enhanced susceptibility to TNF was dependent on bacterial invasion because fibroblasts pretreated with a noninvasive isogenic variant of S. flexneri, UV-treated invasive bacteria, bacterial cultural supernatant, or bacteria LPS were no more susceptible to TNF than untreated cells. Enhanced susceptibility to TNF by bacteria-infected cells was not unique to L cells. Mouse embryo fibroblasts and HeLa cells also showed similar reactivities after bacteria infection. Bacteria-infected cells were greatly suppressed in host cell protein synthesis that may play an important role in their enhanced susceptibility to TNF. These results suggest that an important role of TNF in host defense against bacterial infections is its cytotoxic activity against bacteria-infected cells.     

社区老年人上呼吸道定植菌调查及药敏分析

调查社区老年人上呼吸道的定植菌情况,能够了解引起社区老年人呼吸道感染的危险因素,分析主要定植菌的药敏结果,指导社区老年人呼吸道感染的经验治疗。取社区老年人咽喉部拭子进行细菌分离培养及鉴定,调查呼吸道定植菌情况,并对主要定植菌进行药敏试验,细菌鉴定应用法国生物梅里埃公司的细菌鉴定仪VITEK2和API系统,药敏试验采用K-B纸片琼脂扩散法,结果统计应用WHONET5.4软件。对706例社区老年人上呼吸道标本进行分离培养,有274份标本生长了致病菌或条件致病菌,其中248份标本有一种细菌生长,26份标本生长2种细菌,总体阳性率为38.8%。共分离9种细菌,居首位的细菌是肺炎克雷伯菌151株,占21.4%,其次为副流感嗜血杆菌74株,占10.5%,第3位的是β-溶血链球菌31株,占4.4%。75株肺炎克雷伯菌的药敏结果显示全部为敏感株,从药敏表型分析具有较高的同源性。老年人群上呼吸道定植菌以革兰阴性杆菌尤其以肺炎克雷伯杆菌为主,并对常用抗菌药物有高度敏感性,一旦发生定植菌感染应选择合适的抗菌药物并给予恰当的治疗方案,防止细菌产生耐药性。     

Genes in the Salmonella pathogenicity island 2 and the Salmonella virulence plasmid are essential for Salmonella-induced apoptosis in intestinal epithelial cells

Intestinal epithelial cells are an important site of the host's interaction with enteroinvasive bacteria. Genes in the chromosomally encoded Salmonella pathogenicity island 2 (SPI 2) that encodes a type III secretion system and genes on the virulence plasmid pSDL2 of Salmonella enteritica serovar Dublin (spv genes) are thought to be important for Salmonella dublin survival in host cells. We hypothesized that genes in those loci may be important also for prolonged Salmonella growth and the induction of apoptosis induced by Salmonella in human intestinal epithelial cells. HT-29 human intestinal epithelial cells were infected with wild-type S. dublin or isogenic mutants deficient in the expression of spv genes or with SPI 2 locus mutations. Neither the spv nor the SPI 2 mutations affected bacterial entry into epithelial cells or intracellular proliferation of Salmonella during the initial 8 h after infection. However, at later periods, bacteria with mutations in the SPI 2 locus or in the spv locus compared to wild-type bacteria, manifested a marked decrease in intracellular proliferation and a different distribution pattern of bacteria within infected cells. Epithelial cell apoptosis was markedly increased in response to infection with wild-type, but not the mutant Salmonella. However, apoptosis of epithelial cells infected with wild-type S. dublin was delayed for approximately 28 h after bacterial entry. Apoptosis was preceded by caspase 3 activation, which was also delayed for approximately 24 h after infection. Despite its late onset, the cellular commitment to apoptosis was determined in the early period after infection as inhibition of bacterial protein synthesis during the first 6 h after epithelial cell infection with wild-type S. dublin, but not at later times, inhibited the induction of apoptosis. These studies indicate that genes in the SPI 2 and the spv loci are crucial for prolonged bacterial growth in intestinal epithelial cells. In addition to their influence on intracellular proliferation of Salmonella, genes in those loci determine the ultimate fate of infected epithelial cells with respect to caspase 3 activation and undergoing death by apoptosis.