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Cloning, expression, and purification of a new antibacterial substance gene from larvae of Musca domestica (Diptera: Muscidae).

ABSTRACT: Musca domestica L. (Diptera: Muscidae), the housefly, exhibits unique immune defenses and can produce antibacterial substances upon stimulation with bacteria. On the basis of the cDNA library constructed using the suppression subtractive hybridization method, a 1188-bp antibacterial substance gene, which we named AS566, was amplified by rapid amplification of cDNA ends from M. domestica larva stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In this study, the full-length AS566 gene was cloned and inserted into a His-tagged Escherichia coli (Enterobacteriaceae: Escherichia) prokaryotic expression system to enable production of the recombinant protein. The recombinant AS566 protein was purified in denatured form from inclusion bodies and renatured to obtain functionally active AS566 protein. The bacteriostatic activity of the recombinant purified AS566 protein was assessed using the Oxford plate assay system and the results indicated that AS566 had antibacterial activity against six bacteria, including an E. coli clinical isolate, S. pullorum, Streptococcus bovis (Streptococcaceae: Streptococcus), Streptococcus suis, and Staphylococcus aureus (Staphylococcaceae: Staphylococcus) in vitro. The antibacterial activity of AS566 toward Gram- bacteria was two times greater than that against Gram+ bacteria. The sequencing results and BLAST analysis showed that the antibacterial substance gene AS566 was not homologous to any other antibacterial substance genes in GenBank. The antibacterial mechanisms of the newly discovered AS566 protein warrant further study.

SUBMITTER: Pei Z

PROVIDER: S-EPMC5634066 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Cloning, expression, and purification of a new antibacterial substance gene from larvae of Musca domestica (Diptera: Muscidae).

Pei Zhihua Z Bian Lu L Zhang Hui H Gao Yunhang Y Ma Hongxia H

Journal of insect science (Online) 20140101

Musca domestica L. (Diptera: Muscidae), the housefly, exhibits unique immune defenses and can produce antibacterial substances upon stimulation with bacteria. On the basis of the cDNA library constructed using the suppression subtractive hybridization method, a 1188-bp antibacterial substance gene, which we named AS566, was amplified by rapid amplification of cDNA ends from M. domestica larva stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In this study, the full-length AS5 ...[more]

PMID: 25434038

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Project description:BACKGROUND:It has been reported that there are more than ten antimicrobial peptides (AMPs) belonging to the cecropin family in Musca domestica; however, few of them have been identified, and the functions of the other molecules are poorly understood. METHODS:Sequences of the M. domestica cecropin family of genes were cloned from cDNA template, which was reverse-transcribed from total mRNA isolated from third-instar larvae of M. domestica that were challenged with pathogens. Sequence analysis was performed using DNAMAN comprehensive analysis software, and a molecular phylogenetic tree of the cecropin family was constructed using the Neighbour-Joining method in MEGA v.5.0 according to the mature peptide sequences. Antibacterial activity of the synthetic M. domestica cecropin protein was detected and the minimum inhibitory concentration (MIC) values were determined using broth microdilution techniques. Time-killing assays were performed on the Gram-negative bacteria, Acinetobacter baumannii, at the logarithmic or stabilizing stages of growth, and its morphological changes when treated with Cec4 were assessed by scanning electron microscopy (SEM) and detection of leakage of 260 nm absorbing material. RESULTS:Eleven cecropin family genes, namely Cec01, Cec02 and Cec1-9, show homology to the Cec form in a multigene family on the Scaffold18749 of M. domestica. In comparing the encoded cecropin protein sequences, most of them have the basic characteristics of the cecropin family, containing 19 conservative amino acid residues. To our knowledge, this is the first experimental demonstration that most genes in the Cec family are functional. Cec02, Cec1, Cec2, Cec5 and Cec7 have similar antibacterial spectra and antibacterial effects against Gram-negative bacteria, while Cec4 displays a more broad-spectrum of antimicrobial activity and has a very strong effect on A. baumannii. Cec4 eliminated A. baumannii in a rapid and concentration-dependent manner, with antibacterial effects within 24 h at 1× MIC and 2× MIC. Furthermore, SEM analysis and the leakage of 260 nm absorbing material detection indicated that Cec4 sterilized the bacteria through the disruption of cell membrane integrity. CONCLUSIONS:Although there are more than ten cecropin genes related to M. domestica, some of them have no preferred antibacterial activity other than Cec4 against A. baumannii.

Project description:Beneficial symbiotic bacteria have positive effects on some insects' (such as mosquitoes, cockroaches, and flies) biological activities. However, the effects of a lack of one specific symbiotic bacterium on the life activities of some insects and their natural gut microbiota composition remain unclear. Bacteriophages are viruses that specifically target and kill bacteria and have the potential to shape gut bacterial communities. In previous work, Pseudomonas aeruginosa that naturally colonized the intestines of housefly larvae was shown to be essential to protect housefly larvae from entomopathogenic fungal infections, leading us to test whether a deficiency in Pseudomonas aeruginosa strains in housefly larvae that was specifically caused using bacteriophages could remold the composition of the intestinal bacteria and affect the development of housefly larvae. Our research revealed that the phage, with a titer of 108 PFU/ml, can remove 90% of Pseudomonas aeruginosa in the gut. A single feeding of low-dose phage had no effect on the health of housefly larvae. However, the health of housefly larvae was affected by treatment with phage every 24 h. Additionally, treating housefly larvae with bacteriophages every 24 h led to bacterial composition changes in the gut. Collectively, the results revealed that deficiency in one symbiotic gut bacteria mediated by precise targeting using bacteriophages indirectly influences the intestinal microbial composition of housefly larvae and has negative effects on the development of the host insect. Our results indicated the important role of symbiotic gut bacteria in shaping the normal gut microbiota composition in insects. IMPORTANCE The well-balanced gut microbiota ensures appropriate development of the host insect, such as mosquitoes, cockroaches, and flies. Various intestinal symbiotic bacteria have different influences on the host gut community structure and thus exert different effects on host health. Therefore, it is of great importance to understand the contributions of one specific bacterial symbiont to the gut microbiota community structure and insect health. Bacteriophages that target certain bacteria are effective tools that can be used to analyze gut bacterial symbionts. However, experimental evidence for phage efficacy in regulating insect intestinal bacteria has been little reported. In this study, we used phages as precision tools to regulate a bacterial community and analyzed the influence on host health after certain bacteria were inhibited by bacteriophage. The ability of phages to target intestinal-specific bacteria in housefly larvae and reduce the levels of target bacteria makes them an effective tool for studying the function of gut bacteria.

Dataset Information

Cloning, expression, and purification of a new antibacterial substance gene from larvae of Musca domestica (Diptera: Muscidae).

Publications

Cloning, expression, and purification of a new antibacterial substance gene from larvae of Musca domestica (Diptera: Muscidae).

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