Mini-Tn7 vectors as genetic tools for single copy gene cloning in Acinetobacter baumannii

Acinetobacter baumannii is an emerging pathogen that causes serious infections with high mortality rates in immunocompromised individuals. Genetic manipulations in this medically-relevant pathogen are limited by the paucity of molecular tools. In this study, we show the application of the mini-Tn7-based single copy insertion system in A. baumannii. Mini-Tn7 elements are known to integrate at a naturally evolved, therefore presumably neutral location (intergenic region) downstream of the glmS gene (glucosamine-fructose-6-phosphate aminotransferase) in Gram-negative bacteria. We identified the site of insertion of mini-Tn7 in A. baumannii and demonstrated application of this useful cloning tool by inserting the gfp gene into the chromosome. Our work shows that mini-Tn7 elements are useful tools for genetic studies in this important pathogen.     

Constitutive expression of fluorescent protein by Aspergillus var. niger and Aspergillus carbonarius to monitor fungal colonization in maize plants

Aspergillus niger and Aspergillus carbonarius are two species in the Aspergillus section Nigri (black-spored aspergilli) frequently associated with peanut (Arachis hypogea), maize (Zea mays), and other plants as pathogens. These infections are symptomless and as such are major concerns since some black aspergilli produce important mycotoxins, ochratoxins A, and the fumonisins. To facilitate the study of the black aspergilli–maize interactions with maize during the early stages of infections, we developed a method that used the enhanced yellow fluorescent protein (eYFP) and the monomeric red fluorescent protein (mRFP₁) to transform A. niger and A. carbonarius, respectively. The results were constitutive expressions of the fluorescent genes that were stable in the cytoplasms of hyphae and conidia under natural environmental conditions. The hyphal in planta distribution in 21-day-old seedlings of maize were similar wild type and transformants of A. niger and A. carbonarius. The in planta studies indicated that both wild type and transformants internally colonized leaf, stem and root tissues of maize seedlings, without any visible disease symptoms. Yellow and red fluorescent strains were capable of invading epidermal cells of maize roots intercellularly within the first 3 days after inoculation, but intracellular hyphal growth was more evident after 7 days of inoculation. We also tested the capacity of fluorescent transformants to produce ochratoxin A and the results with A. carbonarius showed that this transgenic strain produced similar concentrations of this secondary metabolite. This is the first report on the in planta expression of fluorescent proteins that should be useful to study the internal plant colonization patterns of two ochratoxigenic species in the Aspergillus section Nigri.     

Improvement of MALDI-TOF MS profiling for the differentiation of species within the Acinetobacter calcoaceticus—Acinetobacter baumannii complex

MALDI-TOF MS is currently becoming the method of choice for rapid identification of bacterial species in routine diagnostics. Yet, this method suffers from the inability to differentiate reliably between some closely related bacterial species including those of the Acinetobacter calcoaceticus–Acinetobacter baumannii (ACB) complex, namely A. baumannii and Acinetobacter nosocomialis. In the present study, we evaluated a protocol which was different from that used in the Bruker Daltonics identification system (MALDI BioTyper) to improve species identification using a taxonomically precisely defined set of 105 strains representing the four validly named species of the ACB complex. The novel protocol is based on the change in matrix composition from alpha-cyano-4-hydroxycinnamic acid (saturated solution in water:acetonitrile:trifluoroacetic acid, 47.5:50:2.5, v/v) to ferulic acid (12.5 mg ml⁻¹ solution in water:acetonitrile:formic acid 50:33:17, v/v), while the other steps of sample processing remain unchanged. Compared to the standard protocol, the novel one extended the range of detected compounds towards higher molecular weight, produced signals with better mass resolution, and allowed the detection of species-specific signals. As a result, differentiation of A. nosocomialis and A. baumannii strains by cluster analysis was improved and 13 A. nosocomialis strains, assigned erroneously or ambiguously by using the standard protocol, were correctly identified.     

鲍曼不动杆菌VI型分泌系统功能蛋白的研究及应用新进展

细菌VI型分泌系统（type VI secretion system,T6SS）作为一个动态多蛋白复合体,各元件之间分工明确,转运各种效应蛋白作用于竞争细菌获得自我生长优势。鲍曼不动杆菌（Acinetobacter baumannii,Ab）通过T6SS介导细菌在微生物群落中的竞争能力,影响其耐药进化、宿主侵袭感染等过程。其中,缬氨酸-甘氨酸-精氨酸G蛋白三聚体（valine-glycine repeat protein G,VgrG）、脯氨酸-丙氨酸-丙氨酸-精氨酸重复序列蛋白（proline-alanine-alanine-arginine,PAAR）、溶血素共调节蛋白（hemolysin-coregulated protein,Hcp）和效应-免疫（effector-immunity,E-I）对发挥着关键作用。有关T6SS的研究总结虽然很多,但是鲜有文章系统概述其临床应用前景,因为这对T6SS功能蛋白的鉴定、特性、转运机制等基础研究的进展提出了挑战。本文通过综述鲍曼不动杆菌中T6SS的分布、主要功能蛋白的特性及转运机制的研究进展,结合T6SS的应用案例,提供其应用的可行性证据。以期进一步推动鲍曼不动杆菌VI型分泌系统基因和功能的研究,为开发新型抗感染疫苗、筛选合适的靶点抑制剂及生产工程化药物递送工具提供新的思路。     

鲍曼不动杆菌携带前噬菌体的生物信息学分析

【目的】预测并分析82株全基因组测序的鲍曼不动杆菌前噬菌体的携带情况,鉴定前噬菌体编码的抗生素耐药基因和毒力因子。【方法】利用PHASTER (Phage Search Tool Enhanced Release)软件预测鲍曼不动杆菌携带的前噬菌体,采用CARD (The Comprehensive Antibiotic Research Database)和VFDB(VirulenceFactorsDatabase)在线分析软件预测前噬菌体编码的抗生素耐药基因和毒力因子。【结果】预测到472条鲍曼不动杆菌前噬菌体,其中完整型前噬菌体201条,疑似型前噬菌体91条,缺陷型前噬菌体180条。平均每株鲍曼不动杆菌基因组中可携带至少2条完整型前噬菌体。每株鲍曼不动杆菌所携带的全部前噬菌体占其基因组比例约为4%–6%。29条前噬菌体携带77个耐药基因,耐药表型共有14种,分别来自15个不同的家族,涵盖6种抗生素耐药的作用机制。132条前噬菌体编码毒力基因,归类为38种毒力基因和34种毒力因子。不同类型的前噬菌体普遍携带1–2种毒力因子,少数前噬菌体携带3种及以上毒力因子。分析毒力因子可能的宿主...     

Microarray analysis of the human antibody response to synthetic Cryptosporidium glycopeptides

Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17 kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40 kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.     

KL2型鲍曼不动杆菌噬菌体解聚酶克隆及抗菌活性分析

【背景】噬菌体解聚酶是噬菌体在裂解细菌过程中产生的一种抗菌蛋白,关于鲍曼不动杆菌荚膜分型及常见型别噬菌体解聚酶的研究报道较少。【目的】以KL2型鲍曼不动杆菌为研究对象,从噬菌体IME-AB2中克隆解聚酶,在大肠杆菌中进行可溶性表达并研究其体外抗菌活性。【方法】应用二代测序及生物信息学方法鉴定鲍曼不动杆菌荚膜型,分析IME-AB2全基因组。应用分子克隆技术克隆ORF76假定的尾丝蛋白(Putative Tail Fiber)基因,构建重组表达载体pEASY-Blunt-E1-gp76,在大肠杆菌BL21(DE3)中诱导表达,通过Ni-NTA亲和层析纯化解聚酶,研究解聚酶体外抗菌活性。【结果】构建了pEASY-Blunt-E1-gp76解聚酶重组表达质粒,该重组质粒在大肠杆菌中得到可溶性表达;体外活性分析显示,该重组蛋白在体外能够对所有的KL2型鲍曼不动杆菌具有较好的抗菌活性,解聚酶联合人和狗的血清具有很好的杀菌活性。【结论】鉴定解聚酶并提高其抗菌谱具有重要意义,也是噬菌体及解聚酶用于治疗耐药菌研究领域急需解决的重要问题之一。     

Identification and evaluation of universal epitopes in Plasmodium vivax Duffy binding protein

Selected PvDBP-derived synthetic peptides were tested in competition assays with HLA molecules in order to identify and evaluate their binding to a wide range of MHC class II molecules. Binding was evaluated as the peptide’s ability to displace the biotinylated control peptide (HA_306-318) and was detected by a conventional ELISA. Thus, one epitope for the HLA-DR1 molecule, two epitopes for the HLA-DR4 molecule, six epitopes for the HLA-DR7 molecule and three epitopes for the HLA-DR11 molecule displaying a high binding percentage (above 50%) were experimentally obtained. The in vitro results were compared with the epitope prediction results. Two peptides behaved as universal epitopes since they bound to a larger number of HLA-DR molecules. Given that these peptides are located in the conserved PvDBP region II, they could be considered good candidates to be included in the design of a synthetic vaccine against Plasmodium vivax malaria.     

Secondary metabolites from Eurotium species, Aspergillus calidoustus and A. insuetus common in Canadian homes with a review of their chemistry and biological activities

As part of studies of metabolites from fungi common in the built environment in Canadian homes, we investigated metabolites from strains of three Eurotium species, namely E. herbariorum, E. amstelodami, and E. rubrum as well as a number of isolates provisionally identified as Aspergillus ustus. The latter have been recently assigned as the new species A. insuetus and A. calidoustus. E. amstelodami produced neoechinulin A and neoechinulin B, epiheveadride, flavoglaucin, auroglaucin, and isotetrahydroauroglaucin as major metabolites. Minor metabolites included echinulin, preechinulin and neoechinulin E. E. rubrum produced all of these metabolites, but epiheveadride was detected as a minor metabolite. E. herbariorum produced cladosporin as a major metabolite, in addition to those found in E. amstelodami. This species also produced questin and neoechinulin E as minor metabolites. This is the first report of epiheveadride occurring as a natural product, and the first nonadride isolated from Eurotium species. Unlike strains from mainly infection-related samples, largely from Europe, neither ophiobolins G and H nor austins were detected in the Canadian strains of A. insuetus and A. calidoustus tested, all of which had been reported from the latter species. TMC-120 A, B, C and a sesquiterpene drimane are reported with certainty for the first time from indoor isolates, as well as two novel related methyl isoquinoline alkaloids.     

In silico structural homology modeling of nif A protein of rhizobial strains in selective legume plants

Symbiosis is a complex genetic regulatory biological evolution which is highly specific pertaining to plant species and microbial strains. Biological nitrogen fixation in legumes is a functional combination of nodulation by nod genes and regulation by nif, fix genes. Three rhizobial strains (Rhizobium leguminosarum, Bradyrhizobium japonicum, and Mesorhizobium ciceri) that we considered for in silico analysis of nif A are proved to be the best isolates with respect to N₂ fixing for ground nut, chick pea and soya bean (in vitro) out of 47 forest soil samples. An attempt has been made to understand the structural characteristics and variations of nif genes that may reveal the factors influencing the nitrogen fixation. The primary, secondary and tertiary structure of nif A protein was analyzed by using multiple bioinformatics tools such as chou-Fasman, GOR, ExPasy ProtParam tools, Prosa -web. Literature shows that the homology modeling of nif A protein have not been explored yet which insisted the immediate development for better understanding of nif A structure and its influence on biological nitrogen fixation. In the present predicted 3D structure, the nif A protein was analyzed by three different software tools (Phyre2, Swiss model, Modeller) and validated accordingly which can be considered as an acceptable model. However further in silico studies are suggested to determine the specific factors responsible for nitrogen fixing in the present three rhizobial strains.     

Homo floresiensis: a cladistic analysis

The announcement of a new species, Homo floresiensis, a primitive hominin that survived until relatively recent times is an enormous challenge to paradigms of human evolution. Until this announcement, the dominant paradigm stipulated that: 1) only more derived hominins had emerged from Africa, and 2) H. sapiens was the only hominin since the demise of Homo erectus and Homo neanderthalensis. Resistance to H. floresiensis has been intense, and debate centers on two sets of competing hypotheses: 1) that it is a primitive hominin, and 2) that it is a modern human, either a pygmoid form or a pathological individual. Despite a range of analytical techniques having been applied to the question, no resolution has been reached. Here, we use cladistic analysis, a tool that has not, until now, been applied to the problem, to establish the phylogenetic position of the species. Our results produce two equally parsimonious phylogenetic trees. The first suggests that H. floresiensis is an early hominin that emerged after Homo rudolfensis (1.86 Ma) but before H. habilis (1.66 Ma, or after 1.9 Ma if the earlier chronology for H. habilis is retained). The second tree indicates H. floresiensis branched after Homo habilis.     

Mutational analysis of Arabidopsis thaliana plant uncoupling mitochondrial protein

In this study, point mutations were introduced in plant uncoupling mitochondrial protein AtUCP1, a typical member of the plant uncoupling protein (UCP) gene subfamily, in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and in two more residues, Cys28 and His83, specific for plant UCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted proteoliposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to linoleic acid (LA) whereas His83 plays an important role in AtUCP1 transport activity. Residues Cys28, Lys147, and also Tyr269 are probably essential for correct protein function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to inhibitors (purine nucleotides). Interestingly, Cys28 substitution reduced ATP inhibitory effect on AtUCP1, while Tyr269Phe mutant exhibited 2.8-fold increase in sensitivity to ATP, in accordance with the reverse mutation Phe267Tyr of mammalian UCP1.     

Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing

In a previous report (Luyo-Acero et al., 2004), we demonstrated that cytochrome b (Cyt b) gene analysis is an effective method for classifying several isolates of the genus Leishmania; hence, we have further applied this method to other Leishmania species in an effort to enhance the accuracy of the procedure and to construct a new phylogenic tree. In this study, a total of 30 Leishmania and Endotrypanum WHO reference strains, clinical isolates from our patients assigned to 28 strains (human and non-human pathogenic species) and two species of the genus Endotrypanum were analyzed. The Cyt b gene in each sample was amplified by PCR, and was then sequenced by several primers, as reported previously. The phylogenic tree was constructed based on the results obtained by the computer software MEGA v3.1 and PAUP* v4.0 Beta. The present phylogenic tree was almost identical to the traditional method of classification proposed by Lainson and Shaw (1987). However, it produces the following suggestions: (1) exclusion of L. (Leishmania) major from the L. (L.) tropica complex; (2) placement of L.tarentolae in the genus Sauroleishmania; (3) L. (L.) hertigi complex and L. (V.) equatorensis close to the genus Endotrypanum; (4) L. (L.) enrietti, defined as L. (L.) mexicana complex, placed in another position; and (5) L. (L.) turanica and L. (L.) arabica are located in an area far from human pathogenic Leishmania strains. Cyt b gene analysis is thus applicable to the analyzing phylogeny of the genus Leishmania and may be useful for separating non-human pathogenic species from human pathogenic species.     

Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime^®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.     

The morphology of the larval stages of three species (Arrenurus mediorotundatus Thor, 1898; Arrenurus conicus Piers 1894; and Arrenurus cylindratus Piers, 1896) of the Megaluracarus subgenus (Acari: Hydrachnidia: Arrenuridae)

The morphology of the larval stages of three species (Arrenurus mediorotundatus, Arrenurus conicus and Arrenurus cylindratus) belonging to the Megaluracarus subgenus is described. Particular attention is paid to the common characters of the three species in which they differ from the other species belonging to the other subgenera. These are: the shape of the excretory pore plate, the shape of the dorsal plate, the length of coxal plate II medial margin and the C1-CpI, C4-CpIII distances. Other characters differ between the three species also. These are: the shape of the dorsal plate, the shape of coxal plate III, the shape of excretory pore plate and the presence or absence of secondary setae of the IFe3 setae.     

五爪金龙中香豆素类物质含量及其对福寿螺、水稻和稗草的影响

在不同季节采集并测定了入侵植物五爪金龙不同器官中伞形花内酯(Umbelliferone(UMB))和东莨菪亭内酯(Scopoletin(SCO))的含量、同时室内分析了这两种物质的杀螺效果及其对水稻和稗草生长的影响、并研究了这两种物质在土壤中的降解情况。结果表明:五爪金龙植株中SCO含量高于UMB含量,两物质均是夏季含量最高,春季次之,秋冬季低,SCO夏季在茎中含量最大,秋冬季幼茎和幼叶中含量较高;UMB对福寿螺的毒杀力强于SCO,UMB浓度为C2(200μg/mL)时处理24 h对福寿螺的致死率达100%,而SCO浓度为C2时处理24 h对福寿螺的致死率仅为8%;SCO对水稻和稗草苗生长的影响较UMB强,在C0(50μg/mL),C1(100μg/mL)、C2浓度下均促进水稻及稗草苗的生长,而C3(400μg/mL)浓度下则抑制水稻和稗草苗的生长。UMB和SCO混合溶液比单一物质在相同浓度的杀螺效果及对水稻和稗草的生长影响要强;UMB和SCO在土壤中10 d内完全降解。入侵植物五爪金龙中的次生物质UMB和SCO在稻田中的生态效应值得进一步研究。     

Molecular characterization and expression analysis of Acmago and AcY14 in Antrodia cinnamomea

Mago nashi (Mago) and Y14 proteins, highly conserved among eukaryotes, participate in mRNA localization and splicing, and as such play important roles in oogenesis, embryogenesis and germ-line sex determination during animal development. Here we identified mago (Acmago) and Y14 (AcY14) homologues derived from Antrodia cinnamomea. Acmago encodes 149 amino acids and AcY14 encodes 168 amino acids. Multiple amino acid sequence alignment as well as secondary and tertiary structure prediction showed that AcMago and AcY14 have similar protein structure to the reported crystal structures of other Mago and Y14 proteins. During fungal development both Acmago and AcY14 genes were abundantly expressed in natural basidiomes. This is the first report of the molecular characterization and expression analysis of the mago and Y14 genes from fungi.