Chundan Zhang

Rapid detection of Enterobacter cloacae by immunomagnetic separation and a colloidal gold-based immunochromatographic assay

To establish the rapid detection of Enterobacter cloacae in food, immunomagnetic beads were prepared by coupling an anti-E. cloacae polyclonal antibody with magnetic beads. An immunochromatographic test strip was composited with an anti-E. cloacae monoclonal antibody marked by colloidal gold as the detection antibody, anti-E. cloacae polyclonal antibody as the test line, and donkey anti-mouse IgG secondary antibody as the control line. Immunomagnetic separation was combined with a colloidal gold-based immunochromatographic assay for the rapid detection of E. cloacae. The results showed that 102 CFU mL−1 E. cloacae could be detected using the immunochromatographic test strip after immunomagnetic separation. The sensitivity was 10 times higher than direct detection with an immunochromatographic test strip. With the immunochromatographic test strip, water samples spiked with E. cloacae yielded a negative result, whereas E. cloacae yielded a positive result with immunomagnetic separation. The proposed method enhances the rapid detection of E. cloacae in food.

A ferritin from Dendrorhynchus zhejiangensis with heavy metals detoxification activity.

Ferritin, an iron homeostasis protein, has important functions in transition and storage of toxic metal ions. In this study, the full-length cDNA of ferritin was isolated from Dendrorhynchus zhejiangensis by cDNA library and RACE approaches. The higher similarity and conserved motifs for ferritin were also identified in worm counterparts, indicating that it belonged to a new member of ferritin family. The temporal expression of worm ferritin in haemocytes was analyzed by RT-PCR, and revealed the ferritin could be induced by Cd2+, Pb2+ and Fe2+. The heavy metal binding activity of recombinant ferritin was further elucidated by atomic force microscopy (AFM). It was observed that the ferritin protein could form a chain of beads with different size against three metals exposure, and the largest one with 35∼40 nm in height was identified in the Cd2+ challenge group. Our results indicated that worm ferritin was a promising candidate for heavy metals detoxification.

iTRAQ-based proteomic profiling of Vibrio parahaemolyticus under various culture conditions

BackgroundVibrio parahaemolyticus is a common pathogen infecting humans and marine animals; this pathogen has become a major concern of marine food products and trade. In this study, V. parahaemolyticus isolated from sewage was exposed to different culture conditions and analyzed by isobaric tag for relative and absolute quantitation (iTRAQ) based reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique. Our goal is to gain further insights into the proteomics of V. parahaemolyticus, particularly differentially expressed proteins closely correlated with growth conditions and pathogenicity associated proteins.ResultsIn this study, a total of 2,717 proteins including numerous membrane proteins were significantly identified, and 616 proteins displayed significant differential expression under different conditions. Of them, 12 proteins mainly participating in metabolism showed the most elastic expression differentiation between different culture conditions. Some membrane proteins such as type I secretion outer membrane protein, TolC, lipoprotein, efflux system proteins iron-regulated protein A and putaive Fe-regulated protein B, ferric siderophore receptor homolog and several V. parahaemolyticus virulence-associated proteins were differentially regulated under different conditions. Some differentially regulated proteins were analyzed and confirmed at gene expression level by quantitative real time polymerase chain reaction (qRT-PCR).ConclusionsProteomics analysis results revealed the characteristics of V. parahaemolyticus proteome expression, provided some promising biomarkers related with growth conditions, the results likely advance insights into the mechanism involved in the response of V. parahaemolyticus to different conditions. Some virulence-associated proteins were discovered to be differentially expressed under different conditions.

Proteomics and 1 H NMR-based metabolomics analysis of pathogenic Vibrio vulnificus aquacultures isolated from sewage drains

Vibrio bacteria live in both marine and freshwater habitats and are associated with aquatic animals. Vibrio vulnificus is a pathogenic bacterium that infects people and livestock. It is usually found in offshore waters or within fish and shellfish. This study presents a comparative proteomic analysis of the outer membrane protein (OMP) changes in V. vulnificus proteins after stimulation with sewage from sewage drains. Using two-dimensional electrophoresis followed by MALDI-TOF MS/MS, 32 protein spots with significant differences in abundance were identified and characterized. These identified proteins were found to be involved in various functional categories, including catalysis, transport, membrane proteins progresses, receptor activity, energy metabolism, cytokine activity, and protein metabolism. The mRNA expression levels of 12 differential proteins were further assessed by qRT-PCR. Seven genes including carboxypeptidase, hemoglobin receptor, succinate dehydrogenase iron-sulfur subunit, ATP synthase subunit alpha, thioredoxin, succinyl-CoA synthetase subunit, and alanine dehydrogenase were downregulated upon stimulation, whereas the protein expression levels HupA receptor, type I secretion outer membrane protein, glutamine synthetase, superoxide dismutase, OmpU, and VuuA were upregulated. 1H NMR spectra showed 18 dysregulated metabolites from V. vulnificus after the sewage stimulation and the pathogenicity was enhanced after that.

Selective colonization mechanism of Shewanella putrefaciens in dyeing wastewater outlets

Large amounts of dyeing wastewater are discharged to rivers and oceans without appropriate treatment, especially in developing countries. It is imperative to control these wastewater discharges to prevent ecological contamination of rivers and oceans. However, most of the mechanisms of bacterial colonization in contaminated rivers and oceans are unknown, especially in dyeing wastewater outlets. We found Shewanella putrefaciens to be the primary bacteria in the dyeing wastewater outlets around Ningbo City, China. Therefore, in this study, we utilized a combination of differential proteomics, metabolomics, and real-time fluorescent quantitative PCR techniques to investigate the selective colonization mechanism of S. putrefaciens and to excavate related functional proteins and metabolites to provide a theoretical basis for the biological treatment of dyeing wastewater. We found 26 different proteins were filtrated by 2-DE, referring to the synthesis of fatty acids and amino acids, sulfur metabolism, RNA degradation, energy metabolism, two-component signal transduction, oxidative stress, siderophore transport, ABC transport, etc. Furthermore, 14 candidate genes in mRNA expression levels were researched by qRT-PCR, and the results showed that 9 genes were up-regulated, 3 genes were down-regulated, and 8 genes presented consistency in protein and gene expression levels. Additionally, 57 different metabolites of 8 classes were detected. Most metabolites were up-regulated with the highest up-regulated ratios being thymine (61.13), then ethanol (28.61) and putrescine (20.74). Arginine, AMP and malate were down-regulated though. These metabolites are involved in twin-arginine transduction systems and two-component signal transduction systems, which may be related to the adaptability of S. putrefaciens to dyeing wastewater. This work can help researchers understand the biological mechanism pathway of S. putrefaciens in dyeing wastewater. In our future research, we will look to apply this strain for dyeing wastewater treatment.