Jeong-A Lim

Outer Membrane Proteins A (OmpA) and X (OmpX) Are Essential for Basolateral Invasion of Cronobacter sakazakii

ABSTRACT Cronobacter sakazakii is an opportunistic pathogen that actively invades host eukaryotic cells. To identify invasion factors responsible for the intestinal translocation of C. sakazakii, we constructed for the first time outer membrane protein X (OmpX) and A (OmpA) deletion mutants using the lambda Red recombination system. The ompX and ompA deletion mutants showed significantly reduced invasion of human enterocyte-like epithelial Caco-2 and human intestinal epithelial INT-407 cells, and significantly fewer mutant cells were recovered from the livers and spleens of rat pups. Furthermore, compared with intact target cells, the invasion and initial association potentials of the mutants increased at a rate similar to that of the wild type in tight-junction-disrupted target cells, suggesting that OmpX and OmpA are involved in basolateral invasion by C. sakazakii. This is the first report of C. sakazakii virulence determinants that are essential for basolateral invasion and that may be critical for the virulence of C. sakazakii.

Characterization of LysB4, an endolysin from the Bacillus cereus-infecting bacteriophage B4

BackgroundBacillus cereus is a foodborne pathogen that causes emetic or diarrheal types of food poisoning. The incidence of B. cereus food poisoning has been gradually increasing over the past few years, therefore, biocontrol agents effective against B. cereus need to be developed. Endolysins are phage-encoded bacterial peptidoglycan hydrolases and have received considerable attention as promising antibacterial agents.ResultsThe endolysin from B. cereus phage B4, designated LysB4, was identified and characterized. In silico analysis revealed that this endolysin had the VanY domain at the N terminus as the catalytic domain, and the SH3_5 domain at the C terminus that appears to be the cell wall binding domain. Biochemical characterization of LysB4 enzymatic activity showed that it had optimal peptidoglycan hydrolase activity at pH 8.0-10.0 and 50°C. The lytic activity was dependent on divalent metal ions, especially Zn2+. The antimicrobial spectrum was relatively broad because LysB4 lysed Gram-positive bacteria such as B. cereus, Bacillus subtilis and Listeria monocytogenes and some Gram-negative bacteria when treated with EDTA. LC-MS analysis of the cell wall cleavage products showed that LysB4 was an L-alanoyl-D-glutamate endopeptidase, making LysB4 the first characterized endopeptidase of this type to target B. cereus.ConclusionsLysB4 is believed to be the first reported L-alanoyl-D-glutamate endopeptidase from B. cereus-infecting bacteriophages. The properties of LysB4 showed that this endolysin has strong lytic activity against a broad range of pathogenic bacteria, which makes LysB4 a good candidate as a biocontrol agent against B. cereus and other pathogenic bacteria.

Anti-Tumoral Effect of the Mitochondrial Target Domain of Noxa Delivered by an Engineered Salmonella typhimurium

Bacterial cancer therapy relies on the fact that several bacterial species are capable of targeting tumor tissue and that bacteria can be genetically engineered to selectively deliver therapeutic proteins of interest to the targeted tumors. However, the challenge of bacterial cancer therapy is the release of the therapeutic proteins from the bacteria and entry of the proteins into tumor cells. This study employed an attenuated Salmonella typhimurium to selectively deliver the mitochondrial targeting domain of Noxa (MTD) as a potential therapeutic cargo protein, and examined its anti-cancer effect. To release MTD from the bacteria, a novel bacterial lysis system of phage origin was deployed. To facilitate the entry of MTD into the tumor cells, the MTD was fused to DS4.3, a novel cell-penetrating peptide (CPP) derived from a voltage-gated potassium channel (Kv2.1). The gene encoding DS4.3-MTD and the phage lysis genes were placed under the control of PBAD, a promoter activated by L-arabinose. We demonstrated that DS4.3-MTD chimeric molecules expressed by the Salmonellae were anti-tumoral in cultured tumor cells and in mice with CT26 colon carcinoma.

Characterization of an endolysin, LysBPS13, from a Bacillus cereus bacteriophage

Use of bacteriophages as biocontrol agents is a promising tool for controlling pathogenic bacteria including antibiotic-resistant bacteria. Not only bacteriophages but also endolysins, the peptidoglycan hydrolyzing enzymes encoded by bacteriophages, have high potential for applications as biocontrol agents against food-borne pathogens. In this study, a putative endolysin gene was identified in the genome of the bacteriophage BPS13, which infects Bacillus cereus. In silico analysis of this endolysin, designated LysBPS13, showed that it consists of an N-terminal catalytic domain (PGRP domain) and a C-terminal cell wall binding domain (SH3_5 domain). Further characterization of the purified LysBPS13 revealed that this endolysin is an N-acetylmuramyl-l-alanine amidase, the activity of which was not influenced by addition of EDTA. In addition, LysBPS13 demonstrated remarkable thermostability in the presence of glycerol, and it retained its lytic activity even after incubation at 100 °C for 30 min. Taken together, these results indicate that LysBPS13 can be considered a favorable candidate for a new antimicrobial agent to control B. cereus.

Genetic Diversity of Pectobacterium carotovorum subsp. brasiliensis Isolated in Korea.

The plant pathogenic bacterial genus Pectobacteirum consists of heterogeneous strains. The P. carotovorum species is a complex strain showing divergent characteristics, and a new subspecies named P. carotovorum subsp. brasiliensis has been identified recently. In this paper, we re-identified the P. carotovorum subsp. brasiliensis isolates from those classified under the subspecies carotovorum and newly isolated P. carotovorum subsp. brasiliensis strains. All isolates were able to produce plant cell-wall degrading enzymes such as pectate lyase, polygalacturonase, cellulase and protease. We used genetic and biochemical methods to examine the diversity of P. carotovorum subsp. brasiliensis isolates, and found genetic diversity within the brasiliensis subsp. isolates in Korea. The restriction fragment length polymorphism analysis based on the recA gene revealed a unique pattern for the brasiliensis subspecies. The Korean brasiliensis subsp. isolates were divided into four clades based on pulsed-field gel electrophoresis. However, correlations between clades and isolated hosts or year could not be found, suggesting that diverse brasiliensis subsp. isolates existed.

The Interaction of Human Enteric Pathogens with Plants

There are an increasing number of outbreaks of human pathogens related to fresh produce. Thus, the growth of human pathogens on plants should be explored. Human pathogens can survive under the harsh environments in plants, and can adhere and actively invade plants. Plant-associated microbiota or insects contribute to the survival and transmission of enteric pathogens in plants. Human enteric pathogens also trigger plant innate immunity, but some pathogens-such as Salmonella-can overcome this defense mechanism.

Survival of Salmonella enterica and Listeria monocytogenes in Chicken and Pig Manure Compost

Livestock manure is a valuable source of nutrients and organic matter for plant. Thus, livestock manure compost is commonly used fertilizer in organic vegetable and fruit production in many countries. However, contaminated or inadequate manure compost can give negative effect to soil microorganisms. This study was conducted to investigate the survival difference of Salmonella enterica and Listeria monocytogenes in chicken and pig manure compost under the selected environmental conditions. Commercially available manure compost (pig, chicken) was inoculated with S. enterica and L. monocytogenes. Manure compost was incubated at 25℃ and consistent moisture content. Samples had been collected during 200 days depending on the given conditions. S. enterica survived for 130 days in pig manure compost and over 200 days in chicken manure compost, respectively. L. monocytogenes persisted for 120 days in pig manure compost and over 200 days in chicken manure compost, respectively. It is noted that the number of S. enterica and L. monocytogenes gradually decreased over time. The results indicate that S. enterica survived longer than L. monocytogenes in manure compost at 25℃. S. enterica and L. monocytogenes survived longer in chicken manure compost than in pig manure compost. Increased knowledge of pathogen behavior in agricultural environments is a valuable part of future work on improving risk evaluations and, in a longer perspective, in providing data for guidelines regarding safe handling of pathogen-contaminated manure compost and soil.

Structure of bacteriophage SPN1S endolysin reveals an unusual two-module fold for the peptidoglycan lytic and binding activity

Bacteriophage SPN1S infects the pathogenic Gram‐negative bacterium Salmonella typhimurium and expresses endolysin for the release of phage progeny by degrading peptidoglycan of the host cell walls. Bacteriophage SPN1S endolysin exhibits high glycosidase activity against peptidoglycans, resulting in antimicrobial activity against a broad range of outer membrane‐permeabilized Gram‐negative bacteria. Here, we report a crystal structure of SPN1S endolysin, indicating that unlike most endolysins from Gram‐negative bacteria background, the α‐helical protein consists of two modular domains, a large and a small domain, with a concave groove between them. Comparison with other structurally homologous glycoside hydrolases indicated a possible peptidoglycan binding site in the groove, and the presence of a catalytic dyad in the vicinity of the groove, one residue in a large domain and the other in a junction between the two domains. The catalytic dyad was further validated by antimicrobial activity assay against outer membrane‐permeabilized Escherichia coli. The three‐helix bundle in the small domain containing a novel class of sequence motif exhibited binding affinity against outer membrane‐permeabilized E. coli and was therefore proposed as the peptidoglycan‐binding domain. These structural and functional features suggest that endolysin from a Gram‐negative bacterial background has peptidoglycan‐binding activity and performs glycoside hydrolase activity through the catalytic dyad.

Isolation and Genomic Characterization of the T4-Like Bacteriophage PM2 Infecting Pectobacterium carotovorum subsp. carotovorum.

In order to control Pectobacterium carotovorum subsp. carotovorum, a novel virulent bacteriophage PM2 was isolated. Bacteriophage PM2 can infect 48% of P. carotovorum subsp. carotovorum and 78% of P. carotovorum subsp. brasilliensis but none of atrosepticum, betavasculorum, odoriferum and wasabiae isolates had been infected with PM2. PM2 phage belongs to the family Myoviridae, and contains a large head and contractile tail. It has a 170,286 base pair genome that encodes 291 open reading frames (ORFs) and 12 tRNAs. Most ORFs in bacteriophage PM2 share a high level of homology with T4-like phages including IME08, RB69, and JS98. Phylogenetic analysis based on the amino acid sequence of terminase large subunits confirmed that PM2 is classified as a T4-like phage. It contains no integrase- or no repressor-coding genes related to the lysogenic cycle, and lifestyle prediction using PHACT software suggested that PM2 is a virulent bacteriophage.

Persistence of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in Soil, Liquid Manure Amended Soil, and Liquid Manure

While searching for healthier diets, people became more attentive to agricultural organic products. However, organic foods may be more susceptible to microbiological contamination because of the use of livestock manure compost and liquid manure, potential sources of pathogenic bacteria. This study was undertaken to investigate the persistence of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in soil, liquid manure amended soil, and liquid manure. Loamy soil, liquid manure amended soil, and liquid manure were inoculated with S. enterica, E. coli O157:H7, and L. monocytogenes. Samples were incubated in consistent moisture content at 25°C. Samples had been periodically collected during 120 days depending on the given conditions. S. enterica and E. coli O157:H7 survived over 120 days in loamy soil and over 60 days in liquid manure amended soil, respectively. L. monocytogenes decreased faster than other pathogens in soil. S. enterica, E. coli O157:H7, and L. monocytogenes survived for up to 5 days in liquid manure. S. enterica and E. coli O157:H7 in soil decreased by 2 to 2.5 log CFU g -1 for 120 days. S. enterica and E. coli O157:H7 in liquid manure amended soil decreased slowly for 21 days. However, S. enterica, E. coli O157:H7, and L. monocytogenes sharply decreased after 21 days. S. enterica, E. coli O157:H7, and L. monocytogenes in soil increased by 0.5 to 1.0 log CFU g -1 for 7 days. Foodborne pathogens in soil and liquid manure amended soil gradually decreased over time.