Comparative Analysis of the Antimicrobial Activities of Plant Defensin-Like and Ultrashort Peptides against Food-Spoiling Bacteria.

UNLABELLED: Antimicrobial peptides offer potential as novel therapeutics to combat food spoilage and poisoning caused by pathogenic and nonpathogenic bacteria. Our previous studies identified the peptide human beta-defensin 3 (HBD3) as a potent antimicrobial agent against a wide range of beer-spoiling bacteria. Thus, HBD3 is an excellent candidate for development as an additive to prevent food and beverage spoilage. To expand the repertoire of peptides with antimicrobial activity against bacteria associated with food spoilage and/or food poisoning, we carried out an in silico discovery pipeline to identify peptides with structure and activity similar to those of HBD3, focusing on peptides of plant origin. Using a standardized assay, we compared the antimicrobial activities of nine defensin-like plant peptides to the activity of HBD3. Only two of the peptides, fabatin-2 and Cp-thionin-2, displayed antimicrobial activity; however, the peptides differed from HBD3 in being sensitive to salt and were thermostable. We also compared the activities of several ultrashort peptides to that of HBD3. One of the peptides, the synthetic tetrapeptide O3TR, displayed biphasic antimicrobial activity but had a narrower host range than HBD3. Finally, to determine if the peptides might act in concert to improve antimicrobial activity, we compared the activities of the peptides in pairwise combinations. The plant defensin-like peptides fabatin-2 and Cp-thionin-2 displayed a synergistic effect with HBD3, while O3TR was antagonistic. Thus, some plant defensin-like peptides are effective antimicrobials and may act in concert with HBD3 to control bacteria associated with food spoilage and food poisoning. IMPORTANCE: Food spoilage and food poisoning caused by bacteria can have major health and economic implications for human society. With the rise in resistance to conventional antibiotics, there is a need to identify new antimicrobials to combat these outbreaks in our food supply. Here we screened plant peptide databases to identify peptides that share structural similarity with the human defensin peptide HBD3, which has known antimicrobial activity against food-spoiling bacteria. We show that two of the plant peptides display antimicrobial activity against bacteria associated with food spoilage. When combined with HBD3, the peptides are highly effective. We also analyzed the activity of an easily made ultrashort synthetic peptide, O3TR. We show that this small peptide also displays antimicrobial activity against food-spoiling bacteria but is not as effective as HBD3 or the plant peptides. The plant peptides identified are good candidates for development as natural additives to prevent food spoilage.

Defect of Dpb2p, a noncatalytic subunit of DNA polymerase ɛ, promotes error prone replication of undamaged chromosomal DNA in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae DNA polymerase epsilon holoenzyme (Pol ɛ HE) is composed of four subunits: Pol2p, Dpb2p, Dpb3p and Dpb4p. The biological functions of Pol2p, the catalytic subunit of Pol ɛ, are subject of active investigation, while the role of the other three, noncatalytic subunits, is not well defined. We showed previously that mutations in Dpb2p, a noncatalytic but essential subunit of Pol ɛ HE, influence the fidelity of DNA replication in yeast cells. The strength of the mutator phenotype due to the different dpb2 alleles was inversely proportional to the strength of protein-protein interactions between Pol2p and the mutated forms of Dpb2p. To understand better the mechanisms of the contribution of Dpb2p to the controlling of the level of spontaneous mutagenesis we undertook here a further genetic analysis of the mutator phenotype observed in dpb2 mutants. We demonstrate that the presence of mutated forms of Dpb2p in the cell not only influences the intrinsic fidelity of Pol ɛ but also facilitates more frequent participation of error-prone DNA polymerase zeta (Pol ζ) in DNA replication. The obtained results suggest that the structural integrity of Pol ɛ HE is a crucial contributor to accurate chromosomal DNA replication and, when compromised, favors participation of error prone DNA Pol ζ in this process.

Defective interaction between Pol2p and Dpb2p, subunits of DNA polymerase epsilon, contributes to a mutator phenotype in Saccharomyces cerevisiae.

Most of the prokaryotic and eukaryotic replicative polymerases are multi-subunit complexes. There are several examples indicating that noncatalytic subunits of DNA polymerases may function as fidelity factors during replication process. In this work, we have further investigated the role of Dpb2p, a noncatalytic subunit of DNA polymerase epsilon holoenzyme from Saccharomyces cerevisiae in controlling the level of spontaneous mutagenesis. The data presented indicate that impaired interaction between catalytic Pol2p subunit and Dpb2p is responsible for the observed mutator phenotype in S. cerevisiae strains carrying different mutated alleles of the DPB2 gene. We observed a significant correlation between the decreased level of interaction between different mutated forms of Dpb2p towards a wild-type form of Pol2p and the strength of mutator phenotype that they confer. We propose that structural integrity of the Pol epsilon holoenzyme is essential for genetic stability in S. cerevisiae cells.

Dpb2p, a noncatalytic subunit of DNA polymerase epsilon, contributes to the fidelity of DNA replication in Saccharomyces cerevisiae.

Most replicases are multi-subunit complexes. DNA polymerase epsilon from Saccharomyces cerevisiae is composed of four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol2p and Dpb2p are essential. To investigate a possible role for the Dpb2p subunit in maintaining the fidelity of DNA replication, we isolated temperature-sensitive mutants in the DPB2 gene. Several of the newly isolated dpb2 alleles are strong mutators, exhibiting mutation rates equivalent to pol2 mutants defective in the 3' --> 5' proofreading exonuclease (pol2-4) or to mutants defective in mismatch repair (msh6). The dpb2 pol2-4 and dpb2 msh6 double mutants show a synergistic increase in mutation rate, indicating that the mutations arising in the dpb2 mutants are due to DNA replication errors normally corrected by mismatch repair. The dpb2 mutations decrease the affinity of Dpb2p for the Pol2p subunit as measured by two-hybrid analysis, providing a possible mechanistic explanation for the loss of high-fidelity synthesis. Our results show that DNA polymerase subunits other than those housing the DNA polymerase and 3' --> 5' exonuclease are essential in controlling the level of spontaneous mutagenesis and genetic stability in yeast cells.