Structural and Functional Characterization of Acinetobacter baumannii Nucleoside Diphosphate Kinase

近年来,鲍曼不动杆菌(Acinetobacter baumannii)在医院里越来越受到人们的关注,尤其是在重症监护病房(ICUs)．它以强大的多重耐药性(multiresistance)而闻名．核苷二磷酸激酶(nucleoside diphosphate kinase,NDK)是一种进化上非常保守的酶,它能催化核苷之间磷酸基团的转移．我们解析了鲍曼不动杆菌NDK野生型和C端氨基酸残基Arg141-Thr142-Arg143(RTR)截短突变体的结构．通过和黄色黏菌(Myxococcus xanthus)NDK的三维结构进行比较,推断鲍曼不动杆菌NDK的催化机制和黄色黏菌类似．通过激酶活性实验和圆二色谱实验,发现鲍曼不动杆菌NDK E28A突变体二级结构发生了改变,从而导致蛋白催化活性降低,说明Glu28是鲍曼不动杆菌NDK结构中非常关键的氨基酸残基．鲍曼不动杆菌NDK C端RTR截短突变体显示出催化活性极大的降低,这可能与C端RTR残基介导的二体间相互作用有关．虽然RTR截短突变体中的Lys33伸向了和野生型中不同的方向,和Val15产生相互作用弥补了一部分因为RTR截短丢失的相互作用,维持了RTR截短突变体和野生型类似的结构．但是,Lys33产生的相互作用依然太弱,不足以维持蛋白在催化的动态过程中整体结构的高效转换．我们解析的鲍曼不动杆菌NDK晶体高分辨率结构将有助于科学家设计针对鲍曼不动杆菌的药物．     

Comparative genomic analysis of Acinetobacter baumannii clinical isolates reveals extensive genomic variation and diverse antibiotic resistance determinants

Background

Acinetobacter baumannii is an important nosocomial pathogen that poses a serious health threat to immune-compromised patients. Due to its rapid ability to develop multidrug resistance (MDR), A. baumannii has increasingly become a focus of attention worldwide. To better understand the genetic variation and antibiotic resistance mechanisms of this bacterium at the genomic level, we reported high-quality draft genome sequences of 8 clinical isolates with various sequence types and drug susceptibility profiles.

Results

We sequenced 7 MDR and 1 drug-sensitive clinical A. baumannii isolates and performed comparative genomic analysis of these draft genomes with 16 A. baumannii complete genomes from GenBank. We found a high degree of variation in A. baumannii, including single nucleotide polymorphisms (SNPs) and large DNA fragment variations in the AbaR-like resistance island (RI) regions, the prophage and the type VI secretion system (T6SS). In addition, we found several new AbaR-like RI regions with highly variable structures in our MDR strains. Interestingly, we found a novel genomic island (designated as GI_BJ4) in the drug-sensitive strain BJ4 carrying metal resistance genes instead of antibiotic resistance genes inserted into the position where AbaR-like RIs commonly reside in other A. baumannii strains. Furthermore, we showed that diverse antibiotic resistance determinants are present outside the RIs in A. baumannii, including antibiotic resistance-gene bearing integrons, the bla_OXA-23-containing transposon Tn2009, and chromosomal intrinsic antibiotic resistance genes.

Conclusions

Our comparative genomic analysis revealed that extensive genomic variation exists in the A. baumannii genome. Transposons, genomic islands and point mutations are the main contributors to the plasticity of the A. baumannii genome and play critical roles in facilitating the development of antibiotic resistance in the clinical isolates.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1163) contains supplementary material, which is available to authorized users.     

Structural and serological characterisation of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter baumannii strain 24

Extraction of dry bacteria of Acinetobacter baumannii strain 24 by phenol-water yielded a lipopolysaccharide (LPS) that was studied by serological methods and fatty acid analysis. After immunisation of BALB/c mice with this strain, monoclonal antibody S48-3-13 (IgG(3) isotype) was obtained, which reacted with the LPS in western blot and characterized it as S-form LPS. Degradation of the LPS in aqueous 1% acetic acid followed by GPC gave the O-antigenic polysaccharide, whose structure was determined by compositional analyses and NMR spectroscopy of the polysaccharide and O-deacylated polysaccharide as [carbohydrate structure: see text] where QuiN4N is 2,4-diamino-2,4,6-trideoxyglucose and GalNAcA 2-acetamido-2-deoxygalacturonic acid. The amino group at C-4 of the QuipN4N residues is acetylated in about 2/3 of LPS molecules and (S)-3-hydroxybutyrylated in the rest.     

Two methionine aminopeptidases from Acinetobacter baumannii are functional enzymes

Drug resistance in Gram-negative bacteria, such as Acinetobacter baumannii, is emerging as a significant healthcare problem. New antibiotics with a novel mechanism of action are urgently needed to overcome the drug resistance. Methionine aminopeptidase (MetAP) carries out an essential cotranslational methionine excision in many bacteria and is a potential target to develop such novel antibiotics. Two putative MetAP genes were identified in A. baumannii genome, but whether they actually function as MetAP enzymes was not known. Therefore, we established an efficient E. coli expression system for their production as soluble and metal-free proteins for biochemical characterization. We demonstrated that both could carry out the metal-dependent catalysis and could be activated by divalent metal ions with the order Fe(II) ≈ Ni(II) > Co(II) > Mn(II) for both. By using a set of metalloform-selective inhibitors discovered on other MetAP enzymes, potency and metalloform selectivity on the A. baumannii MetAP proteins were observed. The similarity of their catalysis and inhibition to other MetAP enzymes confirmed that both may function as competent MetAP enzymes in A. baumannii and either or both may serve as the potential drug target.     

Development of a pyrosequencing assay for rapid assessment of quinolone resistance in Acinetobacter baumannii isolates

Rapid and reliable assessment of Acinetobacter baumannii resistance to quinolones was successfully achieved through pyrosequencing of the gyrA and parC quinolone-resistance determining regions. A strong correlation was found between quinolone resistance and mutations in gyrA codon 83 and/or in the parC gene (codons 80 or 84). Absence of QRDR mutations was associated with susceptibility to quinolones.     

Characterization and Fungal Inhibition Activity of Siderophore from Wheat Rhizosphere Associated Acinetobacter calcoaceticus Strain HIRFA32

Acinetobacter calcoaceticus HIRFA32 from wheat rhizosphere produced catecholate type of siderophore with optimum siderophore (ca. 92 % siderophore units) in succinic acid medium without FeSO₄ at 28 °C and 24 h of incubation. HPLC purified siderophore appeared as pale yellow crystals with molecular weight [M⁺¹] m/z 347.18 estimated by LCMS. The structure elucidated by ¹H NMR, ¹³C NMR, HMQC, HMBC, NOESY and decoupling studies, revealed that siderophore composed of 2,3-dihydroxybenzoic acid with hydroxyhistamine and threonine as amino acid subunits. In vitro study demonstrated siderophore mediated mycelium growth inhibition (ca. 46.87 ± 0.5 %) of Fusarium oxysporum. This study accounts to first report on biosynthesis of acinetobactin-like siderophore by the rhizospheric strain of A. calcoaceticus and its significance in inhibition of F. oxysporum.     

CarO, an Acinetobacter baumannii outer membrane protein involved in carbapenem resistance, is essential for L-ornithine uptake

We previously associated the emergence of carbapenem resistance in Acinetobacter baumannii with the loss of an outer membrane (OM) protein designated CarO. CarO was found essential for L-ornithine uptake: CarO-deficient strains were specifically impaired to grow only on L-ornithine, and failed to incorporate L-[(14)C] ornithine from the medium. L-arginine, and histidine and lysine to a lower extent, could effectively compete for L-[(14)C] ornithine uptake. L-ornithine also reduced A. baumannii sensitivity to imipenem, suggesting that both compounds compete for uptake. The overall results indicate that CarO participates in the selective uptake of L-ornithine, carbapenems, and other basic amino acids in A. baumannii.     

Thiophenyl oxime-derived phosphonates as nano-molar class C beta-lactamase inhibitors reducing MIC of imipenem against Pseudomonas aeruginosa and Acinetobacter baumannii

The preparation and characterization of a series of thiophenyl oxime phosphonate beta-lactamase inhibitors is described. A number of these analogs were potent and selective inhibitors of class C beta-lactamases from Pseudomonas aeruginosa and Enterobacter cloacae. Compounds 3b and 7 reduced the MIC of imipenem against an AmpC expressing strain of imipenem-resistant P. aeruginosa. A number of the title compounds retained micromolar potency against the class D OXA-40 beta-lactamase from Acinetobacter baumannii and at high concentrations compound 3b was shown to reduce the MIC of imipenem against a highly imipenem-resistant strain of A. baumanii expressing the OXA-40 beta-lactamase. In mice compound 3b exhibited phamacokinetics similar to imipenem.     

分离自老年病房的鲍曼不动杆菌耐药机制初步研究*

目的:检测老年住院患者分离的鲍曼不动杆菌的主要耐药基因,并研究不同耐药基因型与耐药表型之间的对应关系。方法:用PCR方法检测分离自老年住院患者的不同标本来源的170例非重复鲍曼不动杆菌的耐药基因。检测的耐药基因包括D类碳青霉烯酶:bla_(OXA-51),bla_(OXA-23),bla_(OXA-24),bla_(OXA-58),B类金属碳青霉烯酶:bla_(VIM),bla_(IMP),bla_(SIM),bla_(GIM),bla_(DIM),bla_(NDM-1),以及A类超广谱β-内酰胺酶:blaKPC,共计11种。根据检测结果对菌株进行基因分型,并研究不同基因型与CRAB和CSAB这两种耐药表型之间的对应关系。结果:170株鲍曼不动杆菌的固有基因bla_(OXA-51)均为阳性,此外,主要检出基因为bla_(OXA-23),共124株。另外检测出blaKPC12株,bla OXA-58 6株,bla_(NDM-1)3株,bla_(SIM)2株,bla_(OXA-24)、bla_(VIM)和bla_(DIM)各1株,IMP和GIM未检出。根据检出耐药基因的不同组合,分为bla OXA-51+bla_(OXA-23)阳性为基础的A型(124株)及bla_(OXA-23)阴性为基础的B型(bla_(OXA-51),39株)、C型(bla_(OXA-51)+bla_(OXA-58),6株)、D型(bla_(OXA-51)+bla_(OXA-24),1株)共计四类基因型。从耐药表型来看,128株碳青霉烯耐药菌中有122株bla_(OXA-23)为阳性,在CRAB中占95.3%(122/128),42株碳青霉烯敏感株中,有40株bla_(OXA-23)为阴性,在CSAB中占95.2%(40/42)。结论:老年病房流行的耐碳青霉烯鲍曼不动杆菌的耐药基因型以bla_(OXA-23)阳性为主。其与鲍曼不动杆菌CRAB耐药表型、bla_(OXA-23)阴性与CSAB耐药表型之间有良好的对应关系。     

鲍曼不动杆菌裂解性噬菌体LZ35的分离及全基因组分析

目的 通过分离鉴定鲍曼不动杆菌噬菌体并进行遗传信息分析,为今后噬菌体用于治疗鲍曼不动杆菌引起的感染提供依据。方法 以鲍曼不动杆菌临床分离株为宿主菌,从医院污水中分离鲍曼不动杆菌噬菌体并进行纯化、电镜观察形态特征、提取噬菌体DNA,进行全基因组测序,分析全基因组的结构特征,比较基因组分析其进化关系。结果 分离到鲍曼不动杆菌裂解性噬菌体LZ35,电镜观察显示,该噬菌体属于有尾噬菌体目肌尾病毒科。基因组全长44 885 bp,G＋C含量为37.95%,含有83个开放阅读框,其中22个编码序列可预测其功能,61个编码序列为未知基因。噬菌体LZ35的基因组与鲍曼不动杆菌噬菌体IME-AB2和YMC-13-01-C62具有很高的同源性（分别为97%和99%）,与鲍曼不动杆菌噬菌体YMC11/12/R1215的进化关系最近。结论 以鲍曼不动杆菌临床分离株为宿主菌,分离到鲍曼不动杆菌裂解性噬菌体LZ35,明确了其形态和基因组特征,为防治噬菌体疗法奠定基础。     

Specific and Selective Bacteriophages in the Fight against Multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii causes serious infections especially in immunocompromised and/or hospitalized patients. Several A. baumannii strains are multidrug resistant and infect wounds, bones, and the respiratory tract. Current studies are focused on finding new effective agents against A. baumannii. Phage therapy is a promising means to fight this bacterium and many studies on procuring and applying new phages against A. baumannii are currently being conducted. As shown in animal models, phages against multidrug-resistant A. baumannii may control bacterial infections caused by this pathogen and may be a real hope to solve this dangerous health problem.     

Endosymbionts of Acanthamoeba Isolated from Domestic Tap Water in Korea

In a previous study, we reported our discovery of Acanthamoeba contamination in domestic tap water; in that study, we determined that some Acanthamoeba strains harbor endosymbiotic bacteria, via our molecular characterization by mitochondrial DNA restriction fragment length polymorphism (Mt DNA RFLP). Five (29.4%) among 17 Acanthamoeba isolates contained endosymbionts in their cytoplasm, as demonstrated via orcein staining. In order to estimate their pathogenicity, we conducted a genetic characterization of the endosymbionts in Acanthamoeba isolated from domestic tap water via 16S rDNA sequencing. The endosymbionts of Acanthamoeba sp. KA/WP3 and KA/WP4 evidenced the highest level of similarity, at 97% of the recently published 16S rDNA sequence of the bacterium, Candidatus Amoebophilus asiaticus. The endosymbionts of Acanthamoeba sp. KA/WP8 and KA/WP12 shared a 97% sequence similarity with each other, and were also highly similar to Candidatus Odyssella thessalonicensis, a member of the α-proteobacteria. The endosymbiont of Acanthamoeba sp. KA/WP9 exhibits a high degree of similarity (85-95%) with genus Methylophilus, which is not yet known to harbor any endosymbionts. This is the first report, to the best of our knowledge, to show that Methylophilus spp. can live in the cytoplasm of Acanthamoeba.     

Characterization of Insecticidal Genes of Bacillus thuringiensis Strains Isolated from Arid Environments

This study aimed at characterizing the insecticidal genes of eight Bacillus thuringiensis isolates that were recovered from the local environment of western Saudi Arabia. The screening for the presence of lepidopteran-specific cry1A family and vip3A genes, dipteran-specific cry4 family and coleopteran-specific cry3A, vip1A and vip2A genes, was carried out by PCR. All eight isolates produced PCR products that confirmed the presence of cry1Aa, cry1Ab, cry1Ac, cry4A, cry4B genes, but not cry3A, vip1A and vip2A genes. However, three isolates only were found to carry vip3A genes as revealed by PCR. The observation of cry1 and cry4 genes suggests that these eight isolates may have dual activity against Lepidoptera and Diptera species, while three isolates possessed vip3 genes in addition to cry1 and cry4 which suggests that these three isolates have toxic crystals and vegetative proteins. The results of this study are interesting in the sense that they may help developing new strategies for controlling insects of economic and medical importance in Saudi Arabia, using B. thuringiensis strains that naturally exist in the local environment instead of the current control strategies that are based solely on chemical insecticides.     

耐碳青霉烯类鲍曼不动杆菌OXA-23-like基因的克隆和表达

扩增耐碳青霉烯类鲍曼不动杆菌(Acinetobacter baumannii) OXA-23-like基因,并表达纯化该蛋白,为深入研究鲍曼不动杆菌亚单位蛋白疫苗提供理论基础。从60份样品中分离并扩增出OXA-23-like基因,构建于p GEX-6p-1表达载体中,用BL21表达宿主细胞诱导表达并纯化蛋白;免疫印迹试验(Western blotting)验证OXA-23-like蛋白保守性。结果显示,成功构建pGEX-6p-1-OXA-23-like质粒,表达并纯化蛋白;Western blotting实验表明临床菌株OXA-23-like蛋白表达阳性。OXA-23-like基因和蛋白表达保守性高,具有免疫原性,是鲍曼不动杆菌疫苗良好的抗原靶点。     

Cloning, expression, and enzymatic activity of Acinetobacter baumannii and Klebsiella pneumoniae acetyl-coenzyme A carboxylases

Pathogenic Gram-negative bacteria are a major public health concern because they are causative agents of life-threatening hospital-acquired infections. Due to the increasing rates of resistance to available antibiotics, there is an urgent need to develop new drugs. Acetyl-coenzyme A carboxylase (ACCase) is a promising target for the development of novel antibiotics. We describe here the expression, purification, and enzymatic activity of recombinant ACCases from two clinically relevant Gram-negative pathogens, Acinetobacter baumannii and Klebsiella pneumoniae. Recombinant ACCase subunits (AccAD, AccB, and AccC) were expressed and purified, and the holoenzymes were reconstituted. ACCase enzyme activity was monitored by direct detection of malonyl-coenzyme A (malonyl-CoA) formation by liquid chromatography tandem mass spectrometry (LC–MS/MS). Steady-state kinetics experiments showed similar k_cat and K_M values for both enzymes. In addition, similar IC₅₀ values were observed for inhibition of both enzymes by a previously reported ACCase inhibitor. To provide a higher throughput assay suitable for inhibitor screening, we developed and validated a luminescence-based ACCase assay that monitors ATP depletion. Finally, we established an enzyme activity assay for the isolated AccAD (carboxyltransferase) subunit, which is useful for determining whether novel ACCase inhibitors inhibit the biotin carboxylase or carboxyltransferase site of ACCase. The methods described here could be applied toward the identification and characterization of novel inhibitors.     

Biocontrol and Plant Growth Promotion Characterization of Bacillus Species Isolated from Calendula officinalis Rhizosphere

The phenotypic and genotypic diversity of the plant growth promoting Bacillus genus have been widely investigated in the rhizosphere of various agricultural crops. However, to our knowledge this is the first report on the Bacillus species isolated from the rhizosphere of Calendula officinalis. 15 % of the isolated bacteria were screened for their important antifungal activity against Fusarium oxysporum, Botrytis cinerea, Aspergillus niger, Cladosporium cucumerinium and Alternaria alternata. The bacteria identification based on 16S r-RNA and gyrase-A genes analysis, revealed strains closely related to Bacillus amyloliquefaciens, B. velezensis, B. subtilis sub sp spizezenii and Paenibacillus polymyxa species. The electro-spray mass spectrometry coupled to liquid chromatography (ESI-LC MS) analysis showed that most of the Bacillus isolates produced the three lipopeptides families. However, the P. polymyxa (18SRTS) didn’t produce any type of lipopeptides. All the tested Bacillus isolates produced cellulase but the protease activity was observed only in the B. amyloliquefaciens species (9SRTS). The Salkowsky colorimetric test showed that the screened bacteria synthesized 6–52 μg/ml of indole 3 acetic acid. These bacteria produced siderophores with more than 10 mm wide orange zones on chromazurol S. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. amyloliquefaciens (9SRTS) had no significant (P > 0.05) effect on the pre-germination of the chickpea seeds. However, it increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05).These results suggested that the Bacillus strains isolated in this work may be further used as bioinoculants to improve the production of C. officinalis and other crop systems.     

Detection and Partial Characterization of Egg Polypeptides from Heterodera glycines

The presence of two major egg polypeptides was demonstrated in the plant-parasitic nematode Heterodera glycines. The polypeptides were present in equal amounts in, and were most abundant in, eggs from yellow females. They were also present in brown females but were not detected in second-stage juveniles (J2). The two major egg polypeptides, MEP-I and MEP-II, accounted for more than 50% of the total protein in egg extracts evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. During development of females from the yellow stage to the brown stage, the levels of MEP-I and MEP-II declined at twice the rate as total protein. MEP-I and MEP-II had estimated molecular masses of 190 kD and 180 kD, respectively, similar to those reported for female-specific proteins, vitellins, from free-living nematodes.