Analysis of a Spontaneous Non-Motile and Avirulent Mutant Shows That FliM Is Required for Full Endoflagella Assembly in Leptospira interrogans

Pathogenic Leptospira strains are responsible for leptospirosis, a worldwide emerging zoonotic disease. These spirochetes are unique amongst bacteria because of their corkscrew-like cell morphology and their periplasmic flagella. Motility is reported as an important virulence determinant, probably favoring entry and dissemination of pathogenic Leptospira in the host. However, proteins constituting the periplasmic flagella and their role in cell shape, motility and virulence remain poorly described. In this study, we characterized a spontaneous L. interrogans mutant strain lacking motility, correlated with the loss of the characteristic hook-shaped ends, and virulence in the animal model. Whole genome sequencing allowed the identification of one nucleotide deletion in the fliM gene resulting in a premature stop codon, thereby preventing the production of flagellar motor switch protein FliM. Genetic complementation restored cell morphology, motility and virulence comparable to those of wild type cells. Analyses of purified periplasmic flagella revealed a defect in flagella assembly, resulting in shortened flagella compared to the wild type strain. This also correlated with a lower amount of major filament proteins FlaA and FlaB. Altogether, these findings demonstrate that FliM is required for full and correct assembly of the flagella which is essential for motility and virulence.     

Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum

Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold. Together, these results indicate that chemotaxis is an important trait for virulence and pathogenic fitness in this plant pathogen.     

Relationship between cell coiling and motility of spirochetes in viscous environments.

The lowest viscosity that stops translational motility of cells (minimum immobilizing viscosity [MIV] was determined for various spirochetes. The viscous agent used was polyvinylpyrrolidone, The MIV for either Spirochaeta halophila P1 or Spirochaeta aurantia J4T was approximately 1,000 centipoise (cp), and for Leptospira interrogans (biflexa) B16 the MIV was greater than 500 cp. In comparison, the MIV for the flagellated bacteria Escherichia coli and Spirillum serpens was 60 cp. MIV values for two S. halophila mutant strains lacking the characteristic cell coiling (Hel-mutants) were 70 and 120 cp, approximately one-tenth the MIV for the wild-type strain. MIV values for cells of S. aurantia strains with fewer coils than comparably long cells of S. aurantia J4T were 300 to 600 cp. The average velocity of strains of S. aurantia and S. halophila decreased at viscosities higher than 2 to 3 cp. At 2 cp the average velocity of S. halophila P1 was 16 micron/s, whereas the average velocities of Hel-mutant strains were 7 to 9 micron/s. This study indicates that the coiling of spirochetes plays a role in their ability to move through environments of realtively high viscosity. Among the spirochetes we investigated, this ability is greater in the more extensively coiled strains.     

Whole Genome Sequence of Treponema pallidum ssp. pallidum,Strain Mexico A,Suggests Recombination between Yaws and Syphilis Strains

Background

Treponema pallidum ssp. pallidum (TPA), the causative agent of syphilis, and Treponema pallidum ssp. pertenue (TPE), the causative agent of yaws, are closely related spirochetes causing diseases with distinct clinical manifestations. The TPA Mexico A strain was isolated in 1953 from male, with primary syphilis, living in Mexico. Attempts to cultivate TPA Mexico A strain under in vitro conditions have revealed lower growth potential compared to other tested TPA strains.

Methodology/Principal Findings

The complete genome sequence of the TPA Mexico A strain was determined using the Illumina sequencing technique. The genome sequence assembly was verified using the whole genome fingerprinting technique and the final sequence was annotated. The genome size of the Mexico A strain was determined to be 1,140,038 bp with 1,035 predicted ORFs. The Mexico A genome sequence was compared to the whole genome sequences of three TPA (Nichols, SS14 and Chicago) and three TPE (CDC-2, Samoa D and Gauthier) strains. No large rearrangements in the Mexico A genome were found and the identified nucleotide changes occurred most frequently in genes encoding putative virulence factors. Nevertheless, the genome of the Mexico A strain, revealed two genes (TPAMA_0326 (tp92) and TPAMA_0488 (mcp2-1)) which combine TPA- and TPE- specific nucleotide sequences. Both genes were found to be under positive selection within TPA strains and also between TPA and TPE strains.

Conclusions/Significance

The observed mosaic character of the TPAMA_0326 and TPAMA_0488 loci is likely a result of inter-strain recombination between TPA and TPE strains during simultaneous infection of a single host suggesting horizontal gene transfer between treponemal subspecies.     

Detection of genes encoding cholera toxin (CT), zonula occludens toxin (ZOT), accessory cholera enterotoxin (ACE) and heat-stable enterotoxin (ST) in Vibrio mimicus clinical strains

A total of 51 clinical strains of Vibrio mimicus were searched for the presence of virulence-associated genes, like ctx, zot or ace genes which locate in "cholera virulence cassette," and the st gene by polymerase chain reaction. Moreover, the pathological potential of each clinical strain was also examined by rabbit ileal loop (RIL). Three strains showed to have the ctx gene, of which only one strain was zot gene-positive. Meanwhile, one other strain was zot+ but ctx-. All of these four strains were found to have the ace gene and to belong to serogroup O115. Nine strains showed to carry the st gene. However, none of these ST-gene-positive strains was indicated to contain the genes located in the "cholera virulence cassette." It is of interest to note that all of the RIL-positive and/or virulence gene-positive strains were restricted to three serogroups, O20, O41 and O115. These results suggest a significant association between O antigens and enterotoxic activities in V. mimicus clinical strains, and clearly demonstrate multifactorial virulence potentials of this human pathogen.     

DNA characterization of Lyme disease spirochetes.

Lyme disease spirochetes (LDS) have phenotypic characteristics of both treponemes and borreliae. To ascertain whether one or more species of LDS exist, as well as their taxonomic status, we determined the DNA base (G + C) content for three strains of LDS, the DNA relatedness of ten strains isolated in the United States or Europe, and the DNA relatedness of LDS to other spirochetes. The G + C content of the three LDS strains was 28.1-29.0 mol%, most similar to those of Borellia hermsii (30.6 mol %) and Treponema hyodysenteriae (25.6 mol %) among the other spirochetes tested. DNA hybridization studies of nine LDS strains to a reference strain isolated from human blood revealed divergence (unpaired bases) within related nucleotide sequences of only 0.0-1.0 percent, indicating the strains were one species. Similarly, relatedness values of seven strains to the reference strain were high: 58-98 percent (mean, 71 percent) in 50 degrees C reactions and 50-93 percent (mean, 69 percent) in 65 degrees C reactions. Labeled DNA from B. hermsii was 30-40 percent related to three Lyme disease spirochete strains in 50 degrees C reactions and 8-10 percent related in 65 degrees C reactions. In contrast, DNA from the reference LDS strain showed relatedness of only 1 percent to DNAs of two leptospires and only 16 percent to DNA from T. hyodysenteriae. We conclude that LDS are a single species, genetically unlike treponemes or leptospires, which belong in the genus Borrelia.     

Effect of osmolarity and viscosity on the motility of pathogenic and saprophytic Leptospira

The motility of bacteria is an important factor in their infectivity. In this study, the motility of Leptospira, a member of the spirochete family that causes a zoonotic disease known as leptospirosis, was analyzed in different viscous or osmotic conditions. Motility assays revealed that both pathogenic and saprophytic strains increase their swimming speeds with increasing viscosity. However, only pathogenic Leptospira interrogans maintained vigorous motility near physiological osmotic conditions. This suggests that active motility in physiological conditions is advantageous when Leptospira enters hosts and when it migrates toward target tissues.     

Response of Bacillus spores to combinations of germinative compounds

Foerster, Harold F. (University of Texas, Austin), and J. W. Foster. Response of Bacillus spores to combinations of germinative compounds. J. Bacteriol. 91:1168-1177. 1966.-Spores of 21 strains of Bacillus megaterium and 25 other strains representing 13 species of Bacillus were produced under standardized conditions. The germination of a washed spore suspension of each strain was measured as a response to various combinations of 30 different germinative compounds. The strains were first typed with respect to their response to "primary" germination compounds, i.e., glucose, l-alanine, inosine, and l-alanine-inosine mixture, and also Na(+) and K(+). The second stage was the determination of the response to various organic and inorganic anions and cations, each strain being supplied with the "primary" compounds best for it. Marked differences in germination patterns were observed among species and strains of the same species. No relation to established taxonomic lines was evident. A nonspecific requirement for ions was found for all strains, but not all ions were effective. A striking degree of interchangeability of germinative chemicals was found. "Fractional germination" was very common. A mixture of l-alanine and inosine and various ions was the best germinative solution for most strains. Some anomalous germination patterns were encountered. Those studied included a strain whose cells lysed spontaneously upon germination and other strains for which l-leucine had striking germinative powers.     

Gac系统中gacS基因的缺失对东湖假单胞菌HYS毒性的影响

细菌致病因子是细菌毒性的重要部分。Gac系统是细菌的一种全局性调控系统,调控着细菌多种毒性因子的产生。在前期研究中,首次从武汉东湖水域分离得到一株具备高产铁载体能力的革兰氏阴性菌--东湖假单胞菌（Pseudomonas donghuensis）HYS菌株。为了探索Gac系统对于HYS毒性的影响,首先通过氨基酸序列BlastP比对以定位HYS中Gac系统GacS蛋白的位置,经生物信息学分析,确定了HYS中存在Gac系统;再将来源于P. donghuensis HYS和铜绿假单胞菌（P. aeruginosa）PAO1的gacS片段分别通过基因克隆回补至HYS的gacS基因敲除株ΔgacS中,并将获得的菌株喂食秀丽隐杆线虫,通过比较线虫的生存数量和生存天数来判断菌株毒性的强弱。结果表明,ΔgacS菌株毒性明显减弱,而2株回补菌株对线虫的毒性均有不同程度的恢复,说明HYS中的Gac系统参与了毒性调控途径,且P. aeruginosa PAO1的gacS片段也能在HYS中发挥一定的毒性调控作用。为了进一步探索ΔgacS菌株的减毒特性是否与HYS中毒力因子的丧失有关,利用脱脂牛奶平板检测菌株胞外蛋白酶的活性,并利用泳动运动平板、群集运动平板、蹭行运动平板检测菌株的运动能力,同时还对脂多糖、氢氰酸等代谢产物进行了检测。结果表明,相比于HYS,ΔgacS菌株的蛋白酶分泌完全消失,且3种运动能力相比HYS均显著下降（P<0.05）。此外,ΔgacS菌株的胞外脂多糖与氢氰酸的产量相比野生型HYS菌株也有不同程度的下降。而这些毒力因子表型在回补菌株ΔgacS/pBBR2-gacSHYS中均可恢复至野生型水平,且回补PAO1的同源gacS基因甚至可在一定程度上回补gacS的正调控毒性物质的功能。研究表明,潜在的动物致病菌--P. donghuensis HYS通过Gac系统正调控产生毒性因子,从而发挥对秀丽隐杆线虫的致死效应,这为将来生物体抵御HYS感染提供了理论基础。     

Complement inhibitor factor H binding to Lyme disease spirochetes is mediated by inducible expression of multiple plasmid-encoded outer surface protein E paralogs

Borrelia burgdorferi spirochetes can circumvent the vertebrate host's immune system for long periods of time. B. burgdorferi sensu stricto and B. afzelii, but not B. garinii, bind the complement inhibitor factor H to protect themselves against complement-mediated opsonophagocytosis and killing. We found that factor H binding and complement resistance are due to inducible expression of a wide repertoire of outer surface protein E (OspE) lipoproteins variably called OspE, p21, ErpA, and ErpP. Individual Borrelia strains carry multiple plasmid-encoded OspE paralogs. Together the OspE homologs were found to constitute an array of proteins that bind factor H via multiple C-terminal domains that are exposed outwards from the Borrelial surface. Charged residue substitutions in the key binding regions account for variations between OspE family members in the optimal binding pH, temperature, and ionic strength. This may help the spirochetes to adapt into various host environments. Our finding that multiple plasmid-encoded OspE proteins act as virulence factors of Borrelia can provide new tools for the prevention and treatment of borreliosis.     

Chemotaxis in pathogenic spirochetes: directed movement toward targeting tissues?

Chemotaxis is an important feature of motile organisms that allows navigation through various environments. It enables them to detect nutrients and to avoid unfavorable or dangerous conditions. Motility and chemotaxis are widely acknowledged as important virulence factors for pathogenic bacteria. In this review, we try to explore the role of chemotaxis in the pathogenesis of spirochetes. Chemotaxis might be involved in tissue identification and penetration, and represents a possible mechanism for evasion of the host's immune defense. The recent development of genetic tools for pathogenic spirochetes and "tracking" techniques, employing fluorescent in situ hybridization (FISH), could revolutionize our understanding of the importance of chemotaxis for infection and persistence of these bacteria in their host.     

Absence of membrane phosphatidylcholine does not affect virulence and stress tolerance phenotypes in the opportunistic pathogen Pseudomonas aeruginosa

During growth in presence of choline, both laboratory and clinical Pseudomonas aeruginosa strains synthesize phosphatidylcholine (PC), and PC makes up ～4% of the total membrane phospholipid content. In all the strains tested, PC synthesis occurred only when choline is provided exogenously. Mutants defective in synthesis of PC were generated in the strain backgrounds PAO1 and PA14. Minimum inhibitory concentration studies testing sensitivity of PC-deficient strains towards various antibiotics and cationic antimicrobial peptides revealed no differences as compared to wild-type strains. Mutants incapable of synthesizing PC were also found to be unaffected in motility and biofilm formation on abiotic surfaces, colonization of biotic surfaces and virulence in a mouse infection model. A global phenotypic microarray was further used to identify conditions wherein membrane PC may play a role of in P. aeruginosa. No culture conditions were identified wherein wild-type and PC-deficient mutants showed phenotypic differences. Membrane PC may serve a highly specific role during P. aeruginosa interactions with its eukaryotic hosts based on all the clinical strains tested retaining the ability to synthesize it during availability of choline.     

一株北里孢菌株的分离鉴定及其对松材线虫的致病性

【目的】筛选、鉴定出对松材线虫杀灭活性较高的放线菌菌株,并确定生防菌株的毒力因子。【方法】采用平板活性测试及代谢杀虫活性检测方法进行筛选,采用形态学及16S rDNA序列分析等进行鉴定。对发酵液中的活性物质稳定性分析后,利用醇沉、萃取、层析、气相色谱/质谱分析等方法分离纯化出杀虫毒力因子。【结果】从河南南阳宝天曼的腐木及枯枝落叶样品中共分离获得了79株放线菌,从中筛选出对松材线虫有灭活作用的放线菌6株,其中分离株C620菌株对松材线虫的灭活性最高:该菌株的发酵液处理松材线虫48、60 h后线虫的死亡率分别达到60.0%、81.5%。结合该菌株的形态学、生理学特征及16S rDNA序列分析等结果将其归为北里孢菌属中的一个种,菌株编号Kitasatospora sp.strain C620。该菌株的发酵液中杀线虫活性物质的热稳定性、光稳定性及耐储藏性均较强,在中性偏碱性环境较稳定;经pH纸电泳层析初步确定该物质属于碱性水溶性物质。对菌株C620发酵液分离纯化,得到活性化合物为1-苯基-3-(2-吡啶)-5-吡唑啉酮。【结论】获得一株松材线虫高效生防菌Kitasatospora sp.strain C620,其活性物质为1-苯基-3-(2-吡啶)-5-吡唑啉酮。     

Early evolution of microtubules and undulipodia

A critique of both autogeneous and symbiotic hypotheses for the origin of microtubules and cilia and eukaryotic flagella (undulipodia) is presented. It is proposed that spirochetes provided the ancient eukaryotic cell with microtubules twice; cytoplasmic microtubules originated from phagocytosed spirochetes whereas axopodial tubules of undulipodia were transformed from ectosymbiotic spirochetes. A role in transport for microtubules in spirochetes together with a detailed scenario by which free-living spirochetes attached as ectosymbionts and subsequently differentiated into undulipodia is outlined. A mechanism for the continuity of motility in the form of "training" of the novel microtubular axoneme by the ancient spirochete motility apparatus is proposed. Transitional states (missing links) are unlikely to have survived. Constraints regarding the nature of the host cell are discussed. A corresponding flowchart of the early evolution of eukaryotes is presented in which plastids and mitochondria are polyphyletic in their origins.     

Metabolic characterization of the genus Brucella. VI. Growth stimulation by i-erythritol compared with strain virulence for guinea pigs

Strains of Brucella abortus isolated 20 to 38 years ago and strains recently isolated were assessed for their virulence to guinea pigs and for their ability to grow in half-strength Tryptose Broth with and without i-erythritol. The recently isolated strains were virulent and i-erythritol enhanced their growth. The aged strains were avirulent and grew equally well in both media. Three of the recently isolated strains were subcultured serially every 24 hr alternately on Tryptose Agar slants and in half-strength Tryptose Broth without i-erythritol. After 8 to 13 such transfers, the growth of each strain was equivalent in both media. The subculture on which growth equivalence occurred was retested for virulence. None of the three strains had decreased in its virulence for guinea pigs. The conclusion was drawn that strain virulence for guinea pigs and growth enhancement by i-erythritol are independent characteristics.     

Determinants of bluetongue virus virulence in murine models of disease

Bluetongue is a major infectious disease of ruminants that is caused by bluetongue virus (BTV). In this study, we analyzed virulence and genetic differences of (i) three BTV field strains from Italy maintained at either a low (L strains) or high (H strains) passage number in cell culture and (ii) three South African "reference" wild-type strains and their corresponding live attenuated vaccine strains. The Italian BTV L strains, in general, were lethal for both newborn NIH-Swiss mice inoculated intracerebrally and adult type I interferon receptor-deficient (IFNAR(-/-)) mice, while the virulence of the H strains was attenuated significantly in both experimental models. Similarly, the South African vaccine strains were not pathogenic for IFNAR(-/-) mice, while the corresponding wild-type strains were virulent. Thus, attenuation of the virulence of the BTV strains used in this study is not mediated by the presence of an intact interferon system. No clear distinction in virulence was observed for the South African BTV strains in newborn NIH-Swiss mice. Full genomic sequencing revealed relatively few amino acid substitutions, scattered in several different viral proteins, for the strains found to be attenuated in mice compared to the pathogenic related strains. However, only the genome segments encoding VP1, VP2, and NS2 consistently showed nonsynonymous changes between all virulent and attenuated strain pairs. This study established an experimental platform for investigating the determinants of BTV virulence. Future studies using reverse genetics will allow researchers to precisely map and "weight" the relative influences of the various genome segments and viral proteins on BTV virulence.     

Temperature-sensitive clones of herpes simplex virus type 1 from laboratory and clinical strains. I. Cloning and basic pathogenetic and immunogenic properties]

Two HSV-1 strains were used in the study: McIntyre laboratory strain and "eye" strain isolated from a patient. Temperature-sensitive clone of HSV-1 was isolated from McIntyre strain as a consequence of virus replication carried out at lowered temperature (28 degrees C). Temperature-resistant clones were obtained from both strains through passages at 39 degrees C and through heating for four times at 45 degrees C. Pathogenic properties of the temperature clones obtained were determined in inbred mice Balb/c and CFw/Pzh. A loss of pathogenicity for mice of temperature-sensitive clone and an increase of pathogenicity of temperature-resistant clones were noted as compared to parental strains. It was found that an introduction of temperature-sensitive clone, with lowered virulence immunizes against highly virulent temperature-resistant clone.     

Avirulence of Candida albicans auxotrophic mutants in a rat model of oropharyngeal candidiasis

Abstract The virulence of Candida albicans strain SC5413 and two isogenic derivatives have been investigated in a rat model of oropharyngeal candidiasis. The results demonstrate that both mutant strains are avirulent in this animal model while the parental strain readily initiates infection. Avirulence is not related to altered growth characteristics or the inability of the strains to undergo yeast-to-hyphal morphogenesis. The potential importance of nutritional sufficiency as a virulence factor as well as the possibility of utilizing such strains in the development of an in vitro expression technology system for Candida albicans is discussed.