Characterization of Edwardsiella tarda waaL: roles in lipopolysaccharide biosynthesis, stress adaptation, and virulence toward fish

Edwardsiella tarda is the causative agent of edwardsiellosis in fish. The genome sequence of a virulent strain EIB202 has been determined. According to the genome sequence, the lipopolysaccharide (LPS) synthesis cluster containing a putative O-antigen ligase gene waaL was identified. Here, the in-frame deletion mutant ΔwaaL was constructed to analyze the function of WaaL in E. tarda EIB202. The ΔwaaL mutant displayed absence in O-antigen side chains in the LPS production. The ΔwaaL mutant exhibited an increased sensitivity to hydrogen peroxide indicating that the LPS was involved in the endurance to the oxidative stress in hosts during infection. In addition, the resistance of ΔwaaL to serum and polymyxin B decreased remarkably. The ΔwaaL mutant was also attenuated in virulence, showed an impaired ability in internalization of epithelioma papulosum cyprinid (EPC) cells and a comparatively poor ability of proliferation in vivo, which was in line with the increased LD₅₀ value. These results indicated that waaL gene was a functional member of the gene cluster involved in LPS synthesis and highlighted the importance of the O-antigen side chains to stress adaption and virulence in E. tarda, signifying the gene as a potential target for live attenuated vaccine against this bacterium.     

Identification of antigenic Edwardsiella tarda surface proteins and their role in pathogenesis

Edwardsiella tarda causes an infectious fish disease called edwardsiellosis. Several outer membrane proteins (OMPs) are associated with virulence factors and are attractive as vaccine candidates. In this study, 4 immuno-reactive OMPs of E. tarda were detected using anti-sera from flounder infected with E. tarda. Using matrix-assisted laser desorption/ionization mass spectrometry analyses, 2 of the 4 OMPs were identified as OmpA and murein lipoprotein (Lpp), which are highly conserved surface proteins in gram-negative bacteria. For further characterization of these surface proteins, we generated ompA- and lpp-inactivated mutants by insertion of a kanamycin cassette in the corresponding genes, and named these mutants E. tarda CK99 and CK164, respectively. As expected, immuno-reactive OmpA and Lpp proteins were absent in E. tarda CK99 and CK164, respectively, confirming that OmpA and Lpp are antigenic surface proteins. Interestingly, the LD₅₀ value of E. tarda CK164 in fish (2.0 × 10⁸ colony-forming unit [CFU]/fish) was greater than that of the parental strain (3.0 × 10⁷ CFU/fish). The LD₅₀ of E. tarda CK99 did not differ from that of its parental strain. After administering attenuated E. tarda CK164 to fish, we monitored the E. tarda-specific immune response profile. We observed that the E. tarda-specific serum IgM titer increased in a time-dependent manner, and was much higher than the value observed after the administration of a heat-killed E. tarda control. Moreover, fish vaccinated with E. tarda CK164 were 100% protected when challenged by CK41, a pathogenic strain. Our results suggest that E. tarda CK164 can potentially be used for developing an effective live attenuated vaccine for edwardsiellosis that can be applied in the aquaculture industry.     

CXCL8 Chemokines in Teleost Fish: Two Lineages with Distinct Expression Profiles during Early Phases of Inflammation

Background

During the inflammatory process, chemokine CXCL8 plays a pivotal role in recruitment of human neutrophilic granulocytes. A diversity of sequences similar to CXCL8 was reported in fish, but their evolutionary relationships and functional homology with their human homolog remain unclear.

Principal Findings

We screened fish genomes to seek for sequences related to CXCL8. A first lineage was retrieved in all teleosts, while a second CXCL8 lineage was found in zebrafish and carp only. An early inflammatory function for both lineages was indicated by several lines of evidence. The induction of carp CXCL8s, CXCb, and CXC receptor-1 and -2 was analyzed after in vitro stimulation of leukocyte subpopulations and in two in vivo inflammation models. Recombinant proteins of carp CXCL8 proteins were produced and showed significant chemotactic activity for carp leukocytes.

Conclusions

While both carp CXCL8s appear to be functional homologs of mammalian CXCL8, their different induction requirements and kinetics evoke a gene-specific sub-functionalization.     

Search for live attenuated vaccine candidate against edwardsiellosis by mutating virulence-related genes of fish pathogen Edwardsiella tarda

Aims: The aims of this study were to construct and evaluate the live attenuated vaccine against edwardsiellosis on zebra fish model. Methods and Results: In this study, the deletion mutant of aroC gene for the biosynthesis of chorismic acid in Edwardsiella tarda EIB202 was firstly constructed by allelic exchange strategy. According to the genome information, 19 double mutants and one multiple mutant were successively constructed by deleting virulence‐associated genes based on the ΔaroC mutant. Zebra fish model was used to assay the virulence of the mutants by intramuscular (i.m.) injection. Fourteen mutants were significantly attenuated with accumulated mortality ranged from 0 to 63% (P < 0·05). The zebra fish vaccinated with ΔaroC, ΔaroCΔesrC, ΔaroCΔslyA and ΔaroCΔeseBCDΔesaC via i.m. injection showed ideal protection, resulting in relative per cent survival (RPS) of 68·3, 71·3, 80·1 and 81% against subsequent challenge with the wild‐type Edw. tarda EIB202. Conclusions: ΔaroCΔeseBCDΔesaC behaved a low virulence and the highest RPS on zebra fish model. When the zebra fish were vaccinated with ΔaroCΔeseBCDΔesaC via injection, the expression of immune‐related factors including IgM and MHC II was up‐regulated. Significance and Impact: The mutant ΔaroCΔeseBCDΔesaC might serve as an effective live attenuated vaccine against edwardsiellosis.     

Comparative genome analysis identifies few traits unique to the Escherichia coli ST131 H30Rx clade and extensive mosaicism at the capsule locus

Background

E.coli ST131 is a globally disseminated clone of multi-drug resistant E. coli responsible for that vast majority of global extra-intestinal E. coli infections. Recent global genomic epidemiological studies have highlighted the highly clonal nature of this group of bacteria, however there appears to be inconsistency in some phenotypes associated with the clone, in particular capsule types as determined by K-antigen testing both biochemically and by PCR.

Results

We performed improved quality assemblies on ten ST131 genomes previously sequenced by our group and compared them to a new reference genome sequence JJ1886 to identify the capsule loci across the drug-resistant clone H30Rx. Our data shows considerable genetic diversity within the capsule locus of H30Rx clone strains which is mirrored by classical K antigen testing. The varying capsule locus types appear to be randomly distributed across the H30Rx phylogeny suggesting multiple recombination events at this locus, but that this capsule heterogeneity has little to no effect on virulence associated phenotypes in vitro.

Conclusions

Our data provides a framework for determining the capsular genetics of E. coli ST131 and further beyond to ExPEC strains, and highlights how capsular mosaicism may be an important strategy in becoming a successful globally disseminated human pathogen.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-830) contains supplementary material, which is available to authorized users.     

Comparative genomics of first available bovine Anaplasma phagocytophilum genome obtained with targeted sequence capture

Background

Anaplasma phagocytophilum is a zoonotic and obligate intracellular bacterium transmitted by ticks. In domestic ruminants, it is the causative agent of tick-borne fever, which causes significant economic losses in Europe. As A. phagocytophilum is difficult to isolate and cultivate, only nine genome sequences have been published to date, none of which originate from a bovine strain.Our goals were to; 1/ develop a sequencing methodology which efficiently circumvents the difficulties associated with A. phagocytophilum isolation and culture; 2/ describe the first genome of a bovine strain; and 3/ compare it with available genomes, in order to both explore key genomic features at the species level, and to identify candidate genes that could be specific to bovine strains.

Results

DNA was extracted from a bovine blood sample infected by A. phagocytophilum. Following a whole genome capture approach, A. phagocytophilum DNA was enriched 197-fold in the sample and then sequenced using Illumina technology. In total, 58.9% of obtained reads corresponded to the A. phagocytophilum genome, covering 85.3% of the HZ genome. Then by performing comparisons with nine previously-sequenced A. phagocytophilum genomes, we determined the core genome of these ten strains. Following analysis, 1281 coding DNA sequences, including 1001 complete sequences, were detected in the A. phagocytophilum bovine genome, of which four appeared to be unique to the bovine isolate. These four coding DNA sequences coded for "hypothetical proteins of unknown function” and require further analysis. We also identified nine proteins common to both European domestic ruminants tested.

Conclusion

Using a whole genome capture approach, we have sequenced the first A. phagocytophilum genome isolated from a cow. To the best of our knowledge, this is the first time that this method has been used to selectively enrich pathogenic bacterial DNA from samples also containing host DNA. The four proteins unique to the A. phagocytophilum bovine genome could be involved in host tropism, therefore their functions need to be explored.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-973) contains supplementary material, which is available to authorized users.     

What Makes a Difference in Exercise-Induced Bronchoconstriction: An 8 Year Retrospective Analysis

Background

Exercise-induced bronchoconstriction (EIB) was recently classified into EIB alone and EIB with asthma, based on the presence of concurrent asthma.

Objective

Differences between EIB alone and EIB with asthma have not been fully described.

Methods

We retrospectively reviewed who visited an allergy clinic for respiratory symptoms after exercise and underwent exercise bronchial provocation testing. More than a 15% decrease of forced expiratory volume in 1 second (FEV1) from baseline to the end of a 6 min free-running challenge test was interpreted as positive EIB.

Results

EIB was observed in 66.9% of the study subjects (89/133). EIB-positive subjects showed higher positivity to methacholine provocation testing (61.4% vs. 18.9%, p<0.001) compared with EIB-negative subjects. In addition, sputum eosinophilia was more frequently observed in EIB-positive subjects than in EIB-negative subjects (56% vs. 23.5%, p = 0.037). The temperature and relative humidity on exercise test day were significantly related with the EIB-positive rate. Positive EIB status was correlated with both temperature (p = 0.001) and relative humidity (p = 0.038) in the methacholine-negative EIB group while such a correlation was not observed in the methacholine-positive EIB group. In the methacholine-positive EIB group the time to reach a 15% decrease in FEV1 during exercise was significantly shorter than that in the methacholine-negative EIB group (3.2±0.7 min vs. 8.6±1.6 min, p = 0.004).

Conclusions

EIB alone may be a distinct clinical entity from EIB with asthma. Conditions such as temperature and humidity should be considered when performing exercise tests, especially in subjects with EIB alone.     

High correlation between genotypes and phenotypes of environmental bacteria Comamonas testosteroni strains

Background

Members of Comamonas testosteroni are environmental microorganisms that are usually found in polluted environment samples. They utilize steroids and aromatic compounds but rarely sugars, and show resistance to multiple heavy metals and multiple drugs. However, comprehensive genomic analysis among the C. testosteroni strains is lacked.

Results

To understand the genome bases of the features of C. testosteroni, we sequenced 10 strains of this species and analyzed them together with other related published genome sequences. The results revealed that: 1) the strains of C. testosteroni have genome sizes ranging from 5.1 to 6.0 Mb and G + C contents ranging from 61.1% to 61.8%. The pan-genome contained 10,165 gene families and the core genome contained 3,599 gene families. Heap’s law analysis indicated that the pan-genome of C. testosteroni may be open (α = 0.639); 2) by analyzing 31 phenotypes of 11 available C. testosteroni strains, 99.4% of the genotypes (putative genes) were found to be correlated to the phenotypes, indicating a high correlation between phenotypes and genotypes; 3) gene clusters for nitrate reduction, steroids degradation and metal and multi-drug resistance were found and were highly conserved among all the genomes of this species; 4) the genome similarity of C. testosteroni may be related to the geographical distances.

Conclusions

This work provided an overview on the genomes of C. testosteroni and new genome resources that would accelerate the further investigations of this species. Importantly, this work focused on the analysis of potential genetic determinants for the typical characters and found high correlation between the phenotypes and their corresponding genotypes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1314-x) contains supplementary material, which is available to authorized users.     

Sensitive and Rapid Detection of Edwardsiellosis in Fish by a Loop-Mediated Isothermal Amplification Method

Here we report a rapid and sensitive method (using loop-mediated isothermal amplification [LAMP]) for the diagnosis of edwardsiellosis, a fish disease caused by Edwardsiella tarda, in Japanese flounder. A set of four primers was designed, and conditions for the detection were optimized for the detection of E. tarda in 45 min at 65°C. No amplification of the target hemolysin gene was detected in other related bacteria. When the LAMP primers were used, detection of edwardsiellosis in infected Japanese flounder kidney, and spleen and seawater cultures was possible. We have developed a rapid and sensitive diagnostic protocol for edwardsiellosis detection in fish. This is the first report of the application of LAMP for the diagnosis of a fish pathogen.     

Human Apolipoprotein E Resequencing by Proteomic Analysis and Its Application to Serotyping

Background

Apolipoprotein E (ApoE) typing is considered important because of the association between ApoE and Alzheimer’s disease and familial dyslipidemia and is currently performed by genetic testing (APOE genotyping). ApoE levels in plasma and serum are clinically determined by immunoassay.

Methods

Combining an ApoE immunoassay reagent with proteomic analysis using an Orbitrap mass spectrometer, we attempted to resequence ApoE from trace amounts of serum for typing (serotyping). Most (24 of 33) ApoE mutant proteins registered to date with Online Mendelian Inheritance in Man, such as ApoE2 and ApoE4, involve lysine and arginine mutations. Digestion of mutant ApoE with trypsin will thus result in fragments that differ substantially from wild-type ApoE3 in terms of mass, making serotyping ideally suited to mass spectrometry analysis.

Results

The mean coverage of the amino acid sequence of full-length ApoE was 91.6% in the protein resequence. Residues 112 and 158 (which are mutated in ApoE2 and ApoE4) were covered in all samples, and the protein sequences were used for serotyping. Serotypes including all heterozygous combinations (ApoE2/E3, E2/E4, E3/E4) corresponded exactly to the APOE genotyping results in each of the subjects.

Conclusion

Our novel ApoE serotyping method with protein resequencing requires no synthesis of stable isotope-labeled peptides or genome analysis. The method can use residual blood from samples collected for routine clinical tests, thus enabling retrospective studies with preserved body fluids. The test could be applied to samples from subjects whose DNA is unavailable. In future studies, we hope to demonstrate the capability of our method to detect rare ApoE mutations.     

Complete genome determination and analysis of Acholeplasma oculi strain 19L,highlighting the loss of basic genetic features in the Acholeplasmataceae

Background

Acholeplasma oculi belongs to the Acholeplasmataceae family, comprising the genera Acholeplasma and ‘Candidatus Phytoplasma’. Acholeplasmas are ubiquitous saprophytic bacteria. Several isolates are derived from plants or animals, whereas phytoplasmas are characterised as intracellular parasitic pathogens of plant phloem and depend on insect vectors for their spread. The complete genome sequences for eight strains of this family have been resolved so far, all of which were determined depending on clone-based sequencing.

Results

The A. oculi strain 19L chromosome was sequenced using two independent approaches. The first approach comprised sequencing by synthesis (Illumina) in combination with Sanger sequencing, while single molecule real time sequencing (PacBio) was used in the second. The genome was determined to be 1,587,120 bp in size. Sequencing by synthesis resulted in six large genome fragments, while the single molecule real time sequencing approach yielded one circular chromosome sequence. High-quality sequences were obtained by both strategies differing in six positions, which are interpreted as reliable variations present in the culture population. Our genome analysis revealed 1,471 protein-coding genes and highlighted the absence of the F₁F_O-type Na⁺ ATPase system and GroEL/ES chaperone. Comparison of the four available Acholeplasma sequences revealed a core-genome encoding 703 proteins and a pan-genome of 2,867 proteins.

Conclusions

The application of two state-of-the-art sequencing technologies highlights the potential of single molecule real time sequencing for complete genome determination. Comparative genome analyses revealed that the process of losing particular basic genetic features during genome reduction occurs in both genera, as indicated for several phytoplasma strains and at least A. oculi. The loss of the F₁F_O-type Na⁺ ATPase system may separate Acholeplasmataceae from other Mollicutes, while the loss of those genes encoding the chaperone GroEL/ES is not a rare exception in this bacterial class.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-931) contains supplementary material, which is available to authorized users.     

Rapid MALDI-TOF Mass Spectrometry Strain Typing during a Large Outbreak of Shiga-Toxigenic Escherichia coli

Background

In 2011 northern Germany experienced a large outbreak of Shiga-Toxigenic Escherichia coli O104:H4. The large amount of samples sent to microbiology laboratories for epidemiological assessment highlighted the importance of fast and inexpensive typing procedures. We have therefore evaluated the applicability of a MALDI-TOF mass spectrometry based strategy for outbreak strain identification.

Methods

Specific peaks in the outbreak strain’s spectrum were identified by comparative analysis of archived pre-outbreak spectra that had been acquired for routine species-level identification. Proteins underlying these discriminatory peaks were identified by liquid chromatography tandem mass spectrometry and validated against publicly available databases. The resulting typing scheme was evaluated against PCR genotyping with 294 E. coli isolates from clinical samples collected during the outbreak.

Results

Comparative spectrum analysis revealed two characteristic peaks at m/z 6711 and m/z 10883. The underlying proteins were found to be of low prevalence among genome sequenced E. coli strains. Marker peak detection correctly classified 292 of 293 study isolates, including all 104 outbreak isolates.

Conclusions

MALDI-TOF mass spectrometry allowed for reliable outbreak strain identification during a large outbreak of Shiga-Toxigenic E. coli. The applied typing strategy could probably be adapted to other typing tasks and might facilitate epidemiological surveys as part of the routine pathogen identification workflow.     

The Mitochondrial Genome of an Aquatic Plant,Spirodela polyrhiza

Background

Spirodela polyrhiza is a species of the order Alismatales, which represent the basal lineage of monocots with more ancestral features than the Poales. Its complete sequence of the mitochondrial (mt) genome could provide clues for the understanding of the evolution of mt genomes in plant.

Methods

Spirodela polyrhiza mt genome was sequenced from total genomic DNA without physical separation of chloroplast and nuclear DNA using the SOLiD platform. Using a genome copy number sensitive assembly algorithm, the mt genome was successfully assembled. Gap closure and accuracy was determined with PCR products sequenced with the dideoxy method.

Conclusions

This is the most compact monocot mitochondrial genome with 228,493 bp. A total of 57 genes encode 35 known proteins, 3 ribosomal RNAs, and 19 tRNAs that recognize 15 amino acids. There are about 600 RNA editing sites predicted and three lineage specific protein-coding-gene losses. The mitochondrial genes, pseudogenes, and other hypothetical genes (ORFs) cover 71,783 bp (31.0%) of the genome. Imported plastid DNA accounts for an additional 9,295 bp (4.1%) of the mitochondrial DNA. Absence of transposable element sequences suggests that very few nuclear sequences have migrated into Spirodela mtDNA. Phylogenetic analysis of conserved protein-coding genes suggests that Spirodela shares the common ancestor with other monocots, but there is no obvious synteny between Spirodela and rice mtDNAs. After eliminating genes, introns, ORFs, and plastid-derived DNA, nearly four-fifths of the Spirodela mitochondrial genome is of unknown origin and function. Although it contains a similar chloroplast DNA content and range of RNA editing as other monocots, it is void of nuclear insertions, active gene loss, and comprises large regions of sequences of unknown origin in non-coding regions. Moreover, the lack of synteny with known mitochondrial genomic sequences shed new light on the early evolution of monocot mitochondrial genomes.     

Refolding process of cysteine-rich proteins:Chitinase as a model

Background:

Recombinant proteins overexpressed in E. coli are usually deposited in inclusion bodies. Cysteines in the protein contribute to this process. Inter- and intra- molecular disulfide bonds in chitinase, a cysteine-rich protein, cause aggregation when the recombinant protein is overexpressed in E. coli. Hence, aggregated proteins should be solubilized and allowed to refold to obtain native- or correctly- folded recombinant proteins.

Methods:

Dilution method that allows refolding of recombinant proteins, especially at high protein concentrations, is to slowly add the soluble protein to refolding buffer. For this purpose: first, the inclusion bodies containing insoluble proteins were purified; second, the aggregated proteins were solubilized; finally, the soluble proteins were refolded using glutathione redox system, guanidinium chloride, dithiothreitol, sucrose, and glycerol, simultaneously.

Results:

After protein solubilization and refolding, SDS-PAGE showed a 32 kDa band that was recognized by an anti-chitin antibody on western blots.

Conclusions:

By this method, cysteine-rich proteins from E. coli inclusion bodies can be solubilized and correctly folded into active proteins.Key Words: Chitinase, Cysteine-rich proteins, Protein refolding, Protein solubilization     

An evolutionary relationship between Stearoyl-CoA Desaturase (SCD) protein sequences involved in fatty acid metabolism

Background:

Stearoyl-CoA desaturase (SCD) is a key enzyme that converts saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs) in fat biosynthesis. Despite being crucial for interpreting SCDs’ roles across species, the evolutionary relationship of SCD proteins across species has yet to be elucidated. This study aims to present this evolutionary relationship based on amino acid sequences.

Methods:

Using Multiple Sequence Alignment (MSA) and phylogenetic construction methods, a hypothetical evolutionary relationship was generated between the stearoyl-CoA desaturase (SCD) protein sequences between 18 different species.

Results:

SCD protein sequences from Homo sapiens, Pan troglodytes (chimpanzee), and Pongo abelii (orangutan) have the lowest genetic distances of 0.006 of the 18 species studied. Capra hircus (goat) and Ovis aries (Sheep) had the next lowest genetic distance of 0.023. These farm animals are 99.987% identical at the amino acid level.

Conclusions:

The SCD proteins are conserved in these 18 species, and their evolutionary relationships are similar. Key Words: Phylogenetic analysis, Stearoyl-CoA desaturase (SCD) proteins, Multiple sequence alignment     

Proteomic analysis of the cyst stage of Entamoeba histolytica

Background

The category B agent of bioterrorism, Entamoeba histolytica has a two-stage life cycle: an infective cyst stage, and an invasive trophozoite stage. Due to our inability to effectively induce encystation in vitro, our knowledge about the cyst form remains limited. This also hampers our ability to develop cyst-specific diagnostic tools.

Aims

Three main aims were (i) to identify E. histolytica proteins in cyst samples, (ii) to enrich our knowledge about the cyst stage, and (iii) to identify candidate proteins to develop cyst-specific diagnostic tools.

Methods

Cysts were purified from the stool of infected individuals using Percoll (gradient) purification. A highly sensitive LC-MS/MS mass spectrometer (Orbitrap) was used to identify cyst proteins.

Results

A total of 417 non-redundant E. histolytica proteins were identified including 195 proteins that were never detected in trophozoite-derived proteomes or expressed sequence tag (EST) datasets, consistent with cyst specificity. Cyst-wall specific glycoproteins Jacob, Jessie and chitinase were positively identified. Antibodies produced against Jacob identified cysts in fecal specimens and have potential utility as a diagnostic reagent. Several protein kinases, small GTPase signaling molecules, DNA repair proteins, epigenetic regulators, and surface associated proteins were also identified. Proteins we identified are likely to be among the most abundant in excreted cysts, and therefore show promise as diagnostic targets.

Major Conclusions

The proteome data generated here are a first for naturally-occurring E. histolytica cysts, and they provide important insights into the infectious cyst form. Additionally, numerous unique candidate proteins were identified which will aid the development of new diagnostic tools for identification of E. histolytica cysts.