Function and Characterization of an Alanine Dehydrogenase Homolog From Nocardia seriolae

Fish nocardiosis is a chronic, systemic, granulomatous disease in aquaculture. Nocardia seriolae has been reported to be one of the main pathogenic bacteria of fish nocardiosis. There are few studies on the associated virulence factors and pathogenesis of N. seriolae. Alanine dehydrogenase (ALD), which may be a secreted protein, was discovered by analysis using bioinformatics methods throughout the whole genomic sequence of N. seriolae. Nevertheless, the roles of ALD and its homologs in the pathogenesis of N. seriolae are not demonstrated. In this study, the function of N. seriolae ALD (NsALD) was preliminarily investigated by gene cloning, host cell subcellular localization, secreted protein identification, and cell apoptosis detection. Identification of the extracellular products of N. seriolae via mass spectrometry (MS) analysis revealed that NsALD is a secreted protein. In addition, subcellular localization of NsALD-GFP recombinant protein in fathead minnow (FHM) cells showed that the strong green fluorescence co-localized with the mitochondria. Moreover, apoptosis assays demonstrated that the overexpression of NsALD induces apoptosis in FHM cells. This study may lay the foundation for further exploration of the function of NsALD and facilitate further understanding of the pathogenic mechanism and the associated virulence factors of N. seriolae.

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Identification of a Nocardia seriolae secreted protein targeting host cell mitochondria and inducing apoptosis in fathead minnow (FHM) cells

10.1101/341651 ◽

2018 ◽

Author(s):

Jianlin Chen ◽

Wenji Wang ◽

Liqun Xia ◽

Zhiwen Wang ◽

Yishan Lu ◽

...

Keyword(s):

Fusion Protein ◽

Subcellular Localization ◽

Host Cell ◽

Virulence Factor ◽

Signal Peptide ◽

Cell Apoptosis ◽

Fathead Minnow ◽

Secreted Protein ◽

Mitochondrial Targeting ◽

Nocardia Seriolae

AbstractNocardia seriolae, is a Gram-positive, partially acid-fast, aerobic, and filamentous bacterium. This bacterium is the main pathogen of fish nocardiosis. A bioinformatic analysis based on the genomic sequence of the N. seriolae strain ZJ0503 showed that ORF3141 encoded a secreted protein with a signal peptide at the N-terminate which may target the mitochondria in the host cell. However, the functions of this protein and its homologs remain unknown. In this study, we experimentally tested the bioinformatic prediction on this protein. Mass spectrometry analysis of the extracellular products from N. seriolae showed that ORF3141 was a secreted protein. Subcellular localization of the ORF3141-GFP fusion protein revealed that the green fluorescence protein co-localized with the mitochondria, while ORF3141Δsig-GFP (with the signal peptide deleted) fusion protein was evenly distributed in the whole cell of fathead minnow (FHM) cells. Thus, the N-terminate signal peptide had a significant impact on mitochondrial targeting. Notably, the expression of ORF3141 protein changed the distribution of mitochondria from perinuclear halo into lumps in the transfected FHM cells. In addition, apoptotic features were found in the transfected FHM cells by overexpression of ORF3141 and ORF3141Δsig proteins, respectively. Quantitative assays of mitochondrial membrane potential value, caspase-3 activity and apoptosis-related gene mRNA expression suggested that cell apoptosis was induced in the transfected FHM cells. In conclusion, the ORF3141 was a secreted protein of N. seriolae that targeted host cell mitochondria and induced apoptosis in FHM cells. This protein may participate in the cell apoptosis regulation and plays an important role in the pathogenesis of N. seriolae.Author summaryNocardia seriolae is the causative pathogen responsible for fish nocardiosis. This facultative intercellular bacterium, adapts to survive and colonize by evading intracellular killing after being engulfed with macrophages in the host. Despite considerable economic losses caused by N. seriolae in fish infection, the pathogenic mechanism and specific virulence factor of this bacterium remain ambiguous. In this study, the characteristic of ORF3141 protein function was investigated by subcellular localization and its possible contributions on the ability of N. seriolae to induce apoptosis in transfected fathead minnow (FHM) cells was investigated. Here, we confirmed that ORF3141 was a secreted protein that targeted host cell mitochondria and induced cell apoptosis in FHM cells. Interestingly, after deleting the signal peptide, ORF3141Δsig protein was evenly distributed in the whole host cell and did not co-localize with the mitochondria which could also induce cell apoptosis. Thus, the N-terminate signal peptide played an important role in mitochondrial targeting, and the domain part without the signal peptide had a critical relationship with cell apoptosis. These results demonstrated that ORF3141 mays act as a potential virulence factor that induces apoptosis in fish cells. This protein is significant to elucidate the pathogenic mechanism of N. seriolae and this study mays provide beneficial insight to prevent and treat fish nocardiosis.

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Identification of Catechin as One of the Flavonoids from Combretum albiflorum Bark Extract That Reduces the Production of Quorum-Sensing-Controlled Virulence Factors in Pseudomonas aeruginosa PAO1

Applied and Environmental Microbiology ◽

10.1128/aem.01059-09 ◽

2009 ◽

Vol 76 (1) ◽

pp. 243-253 ◽

Cited By ~ 175

Author(s):

Olivier M. Vandeputte ◽

Martin Kiendrebeogo ◽

Sanda Rajaonson ◽

Billo Diallo ◽

Adeline Mol ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Virulence Factors ◽

Pathogenic Bacteria ◽

Higher Plants ◽

Homoserine Lactone ◽

Bark Extract ◽

Negative Effect ◽

Virulence Factor Production

ABSTRACT Quorum-sensing (QS) regulates the production of key virulence factors in Pseudomonas aeruginosa and other important pathogenic bacteria. In this report, extracts of leaves and bark of Combretum albiflorum (Tul.) Jongkind (Combretaceae) were found to quench the production of QS-dependent factors in P. aeruginosa PAO1. Chromatographic fractionation of the crude active extract generated several active fractions containing flavonoids, as shown by their typical spectral features. Purification and structural characterization of one of the active compounds led to the identification of the flavan-3-ol catechin [(2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol]. The identity of catechin as one of the active molecules was confirmed by comparing the high-pressure liquid chromatography profiles and the mass spectrometry spectra obtained for a catechin standard and for the active C. albiflorum fraction. Moreover, standard catechin had a significant negative effect on pyocyanin and elastase productions and biofilm formation, as well as on the expression of the QS-regulated genes lasB and rhlA and of the key QS regulatory genes lasI, lasR, rhlI, and rhlR. The use of RhlR- and LasR-based biosensors indicated that catechin might interfere with the perception of the QS signal N-butanoyl-l-homoserine lactone by RhlR, thereby leading to a reduction of the production of QS factors. Hence, catechin, along with other flavonoids produced by higher plants, might constitute a first line of defense against pathogenic attacks by affecting QS mechanisms and thereby virulence factor production.

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Computational Screening and Characterization of Novel Bacteriocins through Peptidome Mining of Pathogenic Bacteria

SSRN Electronic Journal ◽

10.2139/ssrn.3574482 ◽

2020 ◽

Author(s):

Jinal Naik ◽

Dhwani Solanki ◽

John J. Georrge

Keyword(s):

Pathogenic Bacteria ◽

Computational Screening

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PoGB-Pred: Prediction of Antifreeze Proteins Sequences using Amino Acid Composition with Feature Selection followed by a Sequential based Ensemble Approach

Current Bioinformatics ◽

10.2174/1574893615999200707141926 ◽

2020 ◽

Vol 15 ◽

Author(s):

Affan Alim ◽

Abdul Rafay ◽

Imran Naseem

Keyword(s):

Amino Acid ◽

Dimension Reduction ◽

Protein Identification ◽

Cold Water ◽

Genomic Sequence ◽

Sequence Data ◽

Antifreeze Proteins ◽

Building Blocks ◽

Gradient Boosting ◽

Proposed Model

Background: Proteins contribute significantly in every task of cellular life. Their functions encompass the building and repairing of tissues in human bodies and other organisms. Hence they are the building blocks of bones, muscles, cartilage, skin, and blood. Similarly, antifreeze proteins are of prime significance for organisms that live in very cold areas. With the help of these proteins, the cold water organisms can survive below zero temperature and resist the water crystallization process which may cause the rupture in the internal cells and tissues. AFP’s have attracted attention and interest in food industries and cryopreservation. Objective: With the increase in the availability of genomic sequence data of protein, an automated and sophisticated tool for AFP recognition and identification is in dire need. The sequence and structures of AFP are highly distinct, therefore, most of the proposed methods fail to show promising results on different structures. A consolidated method is proposed to produce the competitive performance on highly distinct AFP structure. Methods: In this study, we propose to use machine learning-based algorithms Principal Component Analysis (PCA) followed by Gradient Boosting (GB) for antifreeze protein identification. To analyze the performance and validation of the proposed model, various combinations of two segments composition of amino acid and dipeptide are used. PCA, in particular, is proposed to dimension reduction and high variance retaining of data which is followed by an ensemble method named gradient boosting for modelling and classification. Results: The proposed method obtained the superfluous performance on PDB, Pfam and Uniprot dataset as compared with the RAFP-Pred method. In experiment-3, by utilizing only 150 PCA components a high accuracy of 89.63 was achieved which is superior to the 87.41 utilizing 300 significant features reported for the RAFP-Pred method. Experiment-2 is conducted using two different dataset such that non-AFP from the PISCES server and AFPs from Protein data bank. In this experiment-2, our proposed method attained high sensitivity of 79.16 which is 12.50 better than state-of-the-art the RAFP-pred method. Conclusion: AFPs have a common function with distinct structure. Therefore, the development of a single model for different sequences often fails to AFPs. A robust results have been shown by our proposed model on the diversity of training and testing dataset. The results of the proposed model outperformed compared to the previous AFPs prediction method such as RAFP-Pred. Our model consists of PCA for dimension reduction followed by gradient boosting for classification. Due to simplicity, scalability properties and high performance result our model can be easily extended for analyzing the proteomic and genomic dataset.

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Tumor-specific overexpression of a novel keratinocyte lipid-binding protein. Identification and characterization of a cloned sequence activated during multistage carcinogenesis in mouse skin

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)85343-7 ◽

1993 ◽

Vol 268 (23) ◽

pp. 17362-17369

Author(s):

P. Krieg ◽

S. Feil ◽

G. Fürstenberger ◽

G.T. Bowden

Keyword(s):

Protein Identification ◽

Binding Protein ◽

Mouse Skin ◽

Lipid Binding ◽

Lipid Binding Protein ◽

Multistage Carcinogenesis ◽

Identification And Characterization

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Characterization of Saccharomyces cerevisiae thymidylate kinase, the CDC8 gene product. General properties, kinetic analysis, and subcellular localization.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)42612-3 ◽

1984 ◽

Vol 259 (23) ◽

pp. 14394-14398 ◽

Cited By ~ 5

Author(s):

A Y Jong ◽

J L Campbell

Keyword(s):

Saccharomyces Cerevisiae ◽

Subcellular Localization ◽

Kinetic Analysis ◽

Gene Product ◽

Thymidylate Kinase ◽

General Properties

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Incidence and characterisation of Bacillus cereus bacteriophages from Thua Nao, a Thai fermented soybean product

BioMolecular Concepts ◽

10.1515/bmc-2021-0009 ◽

2021 ◽

Vol 12 (1) ◽

pp. 85-93

Author(s):

Wallapat Phongtang ◽

Ekachai Chukeatirote

Keyword(s):

Bacillus Cereus ◽

Food Industry ◽

Pathogenic Bacteria ◽

Morphological Analysis ◽

Food Poisoning ◽

Fermented Soybean ◽

Foodborne Pathogenic Bacteria ◽

Ph Range ◽

Potential Use

Abstract Bacillus cereus is considered to be an important food poisoning agent causing diarrhea and vomiting. In this study, the occurrence of B. cereus bacteriophages in Thai fermented soybean products (Thua Nao) was studied using five B. cereus sensu lato indicator strains (four B. cereus strains and one B. thuringiensis strain). In a total of 26 Thua Nao samples, there were only two bacteriophages namely BaceFT01 and BaceCM02 exhibiting lytic activity against B. cereus. Morphological analysis revealed that these two bacteriophages belonged to the Myoviridae. Both phages were specific to B. cereus and not able to lyse other tested bacteria including B. licheniformis and B. subtilis. The two phages were able to survive in a pH range between 5 and 12. However, both phages were inactive either by treatment of 50°C for 2 h or exposure of UV for 2 h. It should be noted that both phages were chloroform-insensitive, however. This is the first report describing the presence of bacteriophages in Thua Nao products. The characterization of these two phages is expected to be useful in the food industry for an alternative strategy including the potential use of the phages as a biocontrol candidate against foodborne pathogenic bacteria.

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Isolation and Characterization of Bacteriophage ZCSE6 against Salmonella spp.: Phage Application in Milk

Biologics ◽

10.3390/biologics1020010 ◽

2021 ◽

Vol 1 (2) ◽

pp. 164-176

Author(s):

Abdallah S. Abdelsattar ◽

Anan Safwat ◽

Rana Nofal ◽

Amera Elsayed ◽

Salsabil Makky ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Raw Milk ◽

Salmonella Spp ◽

Isolation And Characterization ◽

Food Borne ◽

Wide Range ◽

Almost All ◽

And Control ◽

Milk And Dairy Products

Food safety is very important in the food industry as most pathogenic bacteria can cause food-borne diseases and negatively affect public health. In the milk industry, contamination with Salmonella has always been a challenge, but the risks have dramatically increased as almost all bacteria now show resistance to a wide range of commercial antibiotics. This study aimed to isolate a bacteriophage to be used as a bactericidal agent against Salmonella in milk and dairy products. Here, phage ZCSE6 has been isolated from raw milk sample sand molecularly and chemically characterized. At different multiplicities of infection (MOIs) of 0.1, 0.01, and 0.001, the phage–Salmonella interaction was studied for 6 h at 37 °C and 24 h at 8 °C. In addition, ZCSE6 was tested against Salmonella contamination in milk to examine its lytic activity for 3 h at 37 °C. The results showed that ZCSE6 has a small genome size (<48.5 kbp) and belongs to the Siphovirus family. Phage ZCSE6 revealed a high thermal and pH stability at various conditions that mimic milk manufacturing and supply chain conditions. It also demonstrated a significant reduction in Salmonella concentration in media at various MOIs, with higher bacterial eradication at higher MOI. Moreover, it significantly reduced Salmonella growth (MOI 1) in milk, manifesting a 1000-fold decrease in bacteria concentration following 3 h incubation at 37 °C. The results highlighted the strong ability of ZCSE6 to kill Salmonella and control its growth in milk. Thus, ZCSE6 is recommended as a biocontrol agent in milk to limit bacterial growth and increase the milk shelf-life.

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