Development of sensitizer peptide-fused endolysin Lys1S-L9P acting against multidrug-resistant gram-negative bacteria

The advent of multidrug-resistant (MDR) bacteria poses a major threat to public health, garnering attention to novel antibiotic replacements. Endolysin, a bacteriophage-derived cell wall-degrading enzyme, is a promising alternative to conventional antibiotics. However, it is challenging to control Gram-negative bacteria due to the presence of the outer membrane that shields the peptidoglycan layer from enzymatic degradation. To overcome this threshold, we constructed the fusion endolysin Lys1S-L9P by combining endolysin LysSPN1S with KL-L9P, a sensitizer peptide known to extend efficacy of antibiotics by perturbing the outer membrane of Gram-negative bacteria. In addition, we established a new endolysin purification procedure that increases solubility allowing a 4-fold increase in production yield of Lys1S-L9P. The sensitizer peptide-fused endolysin Lys1S-L9P exhibited high bactericidal effects against many MDR Gram-negative pathogens and was more effective in eradicating biofilms compared to LysSPN1S. Moreover, Lys1S-L9P showed potential for clinical use, maintaining stability at various storage temperatures without cytotoxicity against human cells. In the in vivo Galleria mellonella model, Lys1S-L9P demonstrated potent antibacterial activity against MDR Gram-negative bacteria without inducing any toxic activity. This study suggest that Lys1S-L9P could be a potential biocontrol agent to combat MDR Gram-negative bacteria

Transcriptomic Analysis of Shiga Toxin-Producing Escherichia coli FORC_035 Reveals the Essential Role of Iron Acquisition for Survival in Canola Sprouts and Water Dropwort

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that poses a serious threat to humans. Although EHEC is problematic mainly in food products containing meat, recent studies have revealed that many EHEC-associated foodborne outbreaks were attributable to spoiled produce such as sprouts and green leafy vegetables. To understand how EHEC adapts to the environment in fresh produce, we exposed the EHEC isolate FORC_035 to canola spouts (Brassica napus) and water dropwort (Oenanthe javanica) and profiled the transcriptome of this pathogen at 1 and 3 h after incubation with the plant materials. Transcriptome analysis revealed that the expression of genes associated with iron uptake were down-regulated during adaptation to plant tissues. A mutant strain lacking entB, presumably defective in enterobactin biosynthesis, had growth defects in co-culture with water dropwort, and the defective phenotype was complemented by the addition of ferric ion. Furthermore, gallium treatment to block iron uptake inhibited bacterial growth on water dropwort and also hampered biofilm formation. Taken together, these results indicate that iron uptake is essential for the fitness of EHEC in plants and that gallium can be used to prevent the growth of this pathogen in fresh produce

Roles of RpoN in the resistance of Campylobacter jejuni under various stress conditions

<p>Abstract</p> <p>Background</p> <p><it>Campylobacter jejuni </it>is a leading foodborne pathogen worldwide. Despite the fastidious nature of <it>C. jejuni </it>growth, increasing numbers of human campylobacteriosis suggest that <it>C. jejuni </it>may possess unique mechanisms to survive under various stress conditions. <it>C. jejuni </it>possesses only three sigma factors (FliA, RpoD, and RpoN) and lacks stress-defense sigma factors. Since FliA and RpoD are dedicated to flagella synthesis and housekeeping, respectively, in this study, we investigated the role of RpoN in <it>C. jejuni</it>'s defense against various stresses.</p> <p>Results</p> <p>Survivability of an <it>rpoN </it>mutant was compared with the wild-type <it>C. jejuni </it>under various stress conditions. While the growth of the <it>rpoN </it>mutant was as comparably as that of the wild type in shaking cultures, the <it>rpoN </it>mutant exhibited significant survival defects when cultured statically. The <it>rpoN </it>mutant was more sensitive to osmotic stress (0.8% NaCl) with abnormally-elongated cell morphology. Compared to the wile type, the <it>rpoN </it>mutant was more susceptible to acid stress (pH 5) and more resistant to hydrogen peroxide. However, the <it>rpoN </it>mutation had little effect on the resistance of <it>C. jejuni </it>to alkaline pH, heat, cold and antimicrobials.</p> <p>Conclusions</p> <p>The results demonstrate that RpoN plays an important role in <it>C. jejuni</it>'s defense against various stresses which this bacterial pathogen may encounter during transmission to and infection of humans.</p

Colanic Acid Is a Novel Phage Receptor of Pectobacterium carotovorum subsp. carotovorum Phage POP72

The emergence and widespread nature of pathogen resistance to antibiotics and chemicals has led to the re-consideration of bacteriophages as an alternative biocontrol agent in several fields, including agriculture. In this study, we isolated and characterized a novel bacteriophage, POP72, that specifically infects Pectobacterium carotovorum subsp. carotovorum (Pcc), which frequently macerates agricultural crops. POP72 contains a 44,760 bp double-stranded DNA genome and belongs to the family Podoviridae. To determine the phage receptor for POP72, a random mutant library of Pcc was constructed using a Tn5 transposon and screened for resistance against POP72 infection. Most of the resistant clones had a Tn5 insertion in various genes associated with colanic acid (CA) biosynthesis. The phage adsorption rate and CA production decreased dramatically in the resistant clones. Complementation of the clones with the pUHE21-2 lacIq vector harboring genes associated with CA biosynthesis restored their sensitivity to POP72, as well as their ability to produce CA. These results suggest that CA functions as a novel phage receptor for POP72. The application of POP72 protected Chinese cabbage from Pcc infection, suggesting that phage POP72 would be an effective alternative antimicrobial agent to protect agricultural products from Pcc

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

<p>Abstract</p> <p>Background</p> <p><it>Bacillus cereus </it>is a foodborne pathogen that causes emetic or diarrheal types of food poisoning. The incidence of <it>B. cereus </it>food poisoning has been gradually increasing over the past few years, therefore, biocontrol agents effective against <it>B. cereus </it>need to be developed. Endolysins are phage-encoded bacterial peptidoglycan hydrolases and have received considerable attention as promising antibacterial agents.</p> <p>Results</p> <p>The endolysin from <it>B. cereus </it>phage B4, designated LysB4, was identified and characterized. <it>In silico </it>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 <it>B. cereus, Bacillus subtilis </it>and <it>Listeria monocytogenes </it>and some Gram-negative bacteria when treated with EDTA. LC-MS analysis of the cell wall cleavage products showed that LysB4 was an <smcaps>L</smcaps>-alanoyl-<smcaps>D</smcaps>-glutamate endopeptidase, making LysB4 the first characterized endopeptidase of this type to target <it>B. cereus</it>.</p> <p>Conclusions</p> <p>LysB4 is believed to be the first reported <smcaps>L</smcaps>-alanoyl-<smcaps>D</smcaps>-glutamate endopeptidase from <it>B. cereus</it>-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 <it>B. cereus </it>and other pathogenic bacteria.</p

Expression of stm4467-encoded arginine deiminase controlled by the stm4463 regulator contributes to salmonella enterica serovar typhimurium virulence

Arginine deiminase (ADI), carbamate kinase (CK), and ornithine transcarbamoylase (OTC) constitute the ADI system. In addition to metabolic functions, the ADI system has been implicated in the virulence of certain pathogens. The pathogenic intracellular bacterium Salmonella enterica serovar Typhimurium possesses the STM4467, STM4466, and STM4465 genes, which are predicted to encode ADI, CK, and OTC, respectively. Here we report that the STM4467 gene encodes an ADI and that ADI activity plays a role in the successful infection of a mammalian host by S. Typhimurium. An STM4467 deletion mutant was defective for replication inside murine macrophages and was attenuated for virulence in mice. We determined that a regulatory protein encoded by the STM4463 gene functions as an activator for STM4467 expression. The expression of the ADI pathway genes was enhanced inside macrophages in a process that required STM4463. Lack of STM4463 impaired the ability of S. Typhimurium to replicate within macrophages. A mutant defective in STM4467-encoded ADI displayed normal production of nitric oxide by macrophages

Glucose repression of the Escherichia coli sdhCDAB operon, revisited: regulation by the CRP·cAMP complex

Expression of the Escherichia coli sdhCDAB operon encoding the succinate dehydrogenase complex is regulated in response to growth conditions, such as anaerobiosis and carbon sources. An anaerobic repression of sdhCDAB is known to be mediated by the ArcB/A two-component system and the global Fnr anaerobic regulator. While the cAMP receptor protein (CRP) and Cra (formerly FruR) are known as key mediators of catabolite repression, they have been excluded from the glucose repression of the sdhCDAB operon. Although the glucose repression of sdhCDAB was reported to involve a mechanism dependent on the ptsG expression, the molecular mechanism underlying the glucose repression has never been clarified. In this study, we re-examined the mechanism of the sdhCDAB repression by glucose and found that CRP directly regulates expression of the sdhCDAB operon and that the glucose repression of this operon occurs in a cAMP-dependent manner. The levels of phosphorylated enzyme IIA(Glc) and intracellular cAMP on various carbon sources were proportional to the expression levels of sdhC-lacZ. Disruption of crp or cya completely abolished the glucose repression of sdhC-lacZ expression. Together with data showing correlation between the intracellular cAMP concentrations and the sdhC-lacZ expression levels in several mutants and wild type, in vitro transcription assays suggest that the decrease in the CRP·cAMP level in the presence of glucose is the major determinant of the glucose repression of the sdhCDAB operon

Full sequence analysis and characterization of the South Korean Norovirus GII-4 variant CUK-3

<p>Abstract</p> <p>Background</p> <p>Many of researchers have focused on the emerging pathogen, Norovirus, since its first identification as the causing agent of nonbacterial acute gastroenteritis in humans. One of the virulence factors of norovirus, the great genetic diversity attributed to point mutations and recombinations, has brought forth the result of significant changes in the circulating norovirus genotype patterns.</p> <p>Findings</p> <p>In recognition of the necessity for tracking and monitoring of genetic diversity, a norovirus variant among the most prevalent genotype GII-4, Norovirus Hu/GII-4/CUK-3/2008/KR (CUK-3), was isolated from stool samples and analyzed on the level of whole genome sequence. Whole genome sequence analysis revealed three ORF composites of the whole genome, ORF1 (5100 bp), ORF2 (1623 bp), and ORF3 (807 bp). Each genetic relationship of CUK-3 variant analysis located the ORF1 (5,100 bp) in Cluster I, ORF2 (1623 bp) in Cluster I (2006b), ORF3 (807 bp) in Cluster I, and the whole genome sequence (about 5.1 kb) in Cluster I in the phylogenetic tree. And the phylogenetic analyses showed the same location of CUK-3 strain with the GII-4/2006b cluster in the phylogenetic tree.</p> <p>Conclusions</p> <p>In This study, a first concerning the full-length sequence of a NoV variant in South Korea is meaningful in that it can be used not only as a full-length NoV variant sequence standard for future comparison studies, but also as useful material for the public health field by enabling the diagnosis, vaccine development, and prediction of new emerging variants.</p

Transducer-Like Protein in Campylobacter jejuni With a Role in Mediating Chemotaxis to Iron and Phosphate

Chemotaxis-mediated motility enables Campylobacter jejuni to navigate through complex environmental gradients and colonize diverse niches. C. jejuni is known to possess several methyl accepting chemotaxis proteins (MCPs), also called transducer-like proteins (Tlps). While the role of some of the Tlps in chemotaxis has been identified, their regulation and role in virulence is still not very clear. Here, we investigated the contribution of Tlp2 to C. jejuni chemotaxis, stress survival and colonization of the chicken gastrointestinal tract. The Δtlp2 deletion mutant showed decreased chemotaxis toward aspartate, pyruvate, inorganic phosphate (Pi), and iron (FeSO4). Transcriptional analysis of tlp2 with a promoter fusion reporter assay revealed that the tlp2 promoter (Ptlp2) was induced by Pi and iron, both in the ferrous (Fe2+) and ferric form (Fe3+). RT-PCR analysis using overlapping primers indicated that the phoX gene, located immediately downstream of tlp2, is co-transcribed with tlp2. A transcription start site was identified at 53 bp upstream of the tlp2 start codon. The Δtlp2 mutant showed decreased colonization of the chicken gastrointestinal tract. Collectively, our findings revealed that the tlp2 plays a role in C. jejuni pathogenesis and colonization in the chicken host and its expression is regulated by iron

Presence of plasmid-mediated quinolone resistance (PMQR) genes in non-typhoidal Salmonella strains with reduced susceptibility to fluoroquinolones isolated from human salmonellosis in Gyeonggi-do, South Korea from 2016 to 2019

Non-typhoidal salmonellosis remains a pressing public health problem worldwide. Quinolones, particularly fluoroquinolones, are widely used to treat various infections, including non-typhoidal salmonellosis, which can be a serious illness. The emergence of fluoroquinolone-resistant Salmonella has resulted in treatment failure and high mortality rates. In this study, we estimated the presence of plasmid-mediated quinolone resistance (PMQR) genes in Salmonella enterica isolated from human salmonellosis patients in South Korea from 2016 to 2019. We evaluated the association of these genes with fluoroquinolone susceptibility. Antimicrobial susceptibility tests for Salmonella isolates were performed using the Vitek II system, and the minimum inhibitory concentrations (MIC) of ciprofloxacin and levofloxacin were determined using the E-test method. Plasmid-mediated quinolone resistance (PMQR) genes were detected by PCR amplification and quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were analyzed following Sanger sequencing of the PCR products. Thirty-four Salmonella strains with reduced susceptibility to fluoroquinolones (ciprofloxacin MIC ≥ 0.125 µg/mL and levofloxacin MIC ≥ 0.25 µg/mL) were selected from 208 human clinical Salmonella isolates. Among them, 22 Salmonella strains harbored one PMQR gene (qnrA, qnrB, or qnrS), and three Salmonella strains carried two PMQR genes (qnrS and aac(6′)-Ib-cr or qnrA and qnrB). qnrS was the most common PMQR gene. Serotyping revealed that Salmonella 4,[5]12:i:- (32.4%, 11/34) and Salmonella Typhimurium (29.4%, 10/34) were the two most predominant serovars, and Multi-locus sequence typing (MLST) showed that ST19 and ST34 were the most frequent sequence types. In conclusion, qnr gene-positive Salmonella 4,[5],12:i:- and Salmonella Typhimurium were the main serovars responsible for reduced susceptibility to fluoroquinolones. Therefore, our findings suggest that PMQR-positive Salmonella strains, which can be isolated from various samples including human, food, and the environment, should be carefully monitored.This research was supported by a Grant (19162MFDS037) from the Ministry of Food and Drug Safety in 2020