• Molecules and Cells
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• v.38 no.8
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• pp.715-722
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• 2015

In Gram-negative bacteria in the periplasmic space, the dimeric thioredoxin-fold protein DsbC isomerizes and reduces incorrect disulfide bonds of unfolded proteins, while the monomeric thioredoxin-fold protein DsbA introduces disulfide bonds in folding proteins. In the Gram-negative bacteria Salmonella enterica serovar Typhimurium, the reduced form of CueP scavenges the production of hydroxyl radicals in the copper-mediated Fenton reaction, and DsbC is responsible for keeping CueP in the reduced, active form. Some DsbA proteins fulfill the functions of DsbCs, which are not present in Gram-positive bacteria. In this study, we identified a DsbA homologous protein (CdDsbA) in the Corynebacterium diphtheriae genome and determined its crystal structure in the reduced condition at $1.5{\AA}$ resolution. CdDsbA consists of a monomeric thioredoxin-like fold with an inserted helical domain and unique N-terminal extended region. We confirmed that CdDsbA has disulfide bond somerase/reductase activity, and we present evidence that the N-terminal extended region is not required for this activity and folding of the core DsbA-like domain. Furthermore, we found that CdDsbA could reduce CueP from C. diphtheriae.

• Molecules and Cells
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• v.42 no.3
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• pp.262-269
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• 2019

The porcine myeloid antimicrobial peptide (PMAP), one of the cathelicidin family members, contains small cationic peptides with amphipathic properties. We used a putative lysozyme originated from the bacteriophage P22 (P22 lysozyme) as a fusion partner, which was connected to the N-terminus of the PMAP36 peptide, to markedly increase the expression levels of recombinant PMAP36. The PMAP36-P22 lysozyme fusion protein with high solubility was produced in Escherichia coli. The final purified yield was approximately 1.8 mg/L. The purified PMAP36-P22 lysozyme fusion protein exhibited antimicrobial activity against both Gram-negative and Grampositive bacteria (Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, and Bacillus subtilis). Furthermore, we estimated its hemolytic activity against pig erythrocytes as 6% at the high concentration ($128{\mu}M$) of the PMAP36-P22 lysozyme fusion protein. Compared with the PMAP36 peptide (12%), our fusion protein exhibited half of the hemolytic activity. Overall, our recombinant PMAP36-P22 lysozyme fusion protein sustained the antimicrobial activity with the lower hemolytic activity associated with the synthetic PMAP36 peptide. This study suggests that the PMAP36-P22 lysozyme fusion system could be a crucial addition to the plethora of novel antimicrobials.

• Journal of Microbiology and Biotechnology
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• v.29 no.3
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• pp.454-464
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• 2019

Salmonellosis is a highly contagious bacterial disease that threatens both human and poultry health. Tests that can detect Salmonella in the field are urgently required to facilitate disease control and for epidemiological investigations. Here, we combined loop-mediated isothermal amplification (LAMP) with a chromatographic lateral flow dipstick (LFD) to rapidly and accurately detect Salmonella. LAMP primers were designed to target the Salmonella invA gene. LAMP conditions were optimized by adjusting the ratio of inner to outer primers, $MgSO_4$ concentration, dNTP mix concentration, amplification temperature, and amplification time. We evaluated the specificity of our novel LAMP-LFD method using six Salmonella species and six related non-Salmonella strains. All six of the Salmonella strains, but none of the non-Salmonella strains, were amplified. LAMP-LFD was sensitive enough to detect concentrations of Salmonella enterica subsp. enterica serovar Pullorum genomic DNA as low as $89fg/{\mu}l$, which is 1,000 times more sensitive than conventional PCR. When artificially contaminated feed samples were analyzed, LAMP-LFD was also more sensitive than PCR. Finally, LAMP-LFD gave no false positives across 350 chicken anal swabs. Therefore, our novel LAMP-LFD assay was highly sensitive, specific, convenient, and fast, making it a valuable tool for the early diagnosis and monitoring of Salmonella infection in chickens.

• Molecules and Cells
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• v.43 no.12
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• pp.989-1001
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• 2020

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes salmonellosis and mortality worldwide. S. Typhimurium infects macrophages and survives within phagosomes by avoiding the phagosome-lysosome fusion system. Phagosomes sequentially acquire different Rab GTPases during maturation and eventually fuse with acidic lysosomes. Lysophosphatidylcholine (LPC) is a bioactive lipid that is associated with the generation of chemoattractants and reactive oxygen species (ROS). In our previous study, LPC controlled the intracellular growth of Mycobacterium tuberculosis by promoting phagosome maturation. In this study, to verify whether LPC enhances phagosome maturation and regulates the intracellular growth of S. Typhimurium, macrophages were infected with S. Typhimurium. LPC decreased the intracellular bacterial burden, but it did not induce cytotoxicity in S. Typhimurium-infected cells. In addition, combined administration of LPC and antibiotic significantly reduced the bacterial burden in the spleen and the liver. The ratios of the colocalization of intracellular S. Typhimurium with phagosome maturation markers, such as early endosome antigen 1 (EEA1) and lysosome-associated membrane protein 1 (LAMP-1), were significantly increased in LPC-treated cells. The expression level of cleaved cathepsin D was rapidly increased in LPC-treated cells during S. Typhimurium infection. Treatment with LPC enhanced ROS production, but it did not affect nitric oxide production in S. Typhimurium-infected cells. LPC also rapidly triggered the phosphorylation of IκBα during S. Typhimurium infection. These results suggest that LPC can improve phagosome maturation via ROS-induced activation of NF-κB pathway and thus may be developed as a therapeutic agent to control S. Typhimurium growth.

• Journal of Animal Science and Technology
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• v.64 no.6
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• pp.1144-1172
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• 2022

Salmonella enterica serovar Typhimurium isolate HJL777 is a virulent bacterial strain in pigs. The high rate of salmonella infection are at high risk of non-typhoidal salmonella gastroenteritis development. Salmonellosis is most common in young pigs. We investigated changes in gut microbiota and biological function in piglets infected with salmonella via analysis of rectal fecal metagenome and intestinal transcriptome using 16S rRNA and RNA sequencing. We identified a decrease in Bacteroides and increase in harmful bacteria such as Spirochaetes and Proteobacteria by microbial community analysis. We predicted that reduction of Bacteroides by salmonella infection causes proliferation of salmonella and harmful bacteria that can cause an intestinal inflammatory response. Functional profiling of microbial communities in piglets with salmonella infection showed increasing lipid metabolism associated with proliferation of harmful bacteria and inflammatory responses. Transcriptome analysis identified 31 differentially expressed genes. Using gene ontology and Innate Immune Database analysis, we identified that BGN, DCN, ZFPM2 and BPI genes were involved in extracellular and immune mechanisms, specifically salmonella adhesion to host cells and inflammatory responses during infection. We confirmed alterations in gut microbiota and biological function during salmonella infection in piglets. Our findings will help prevent disease and improve productivity in the swine industry.

• Journal of Animal Science and Technology
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• v.65 no.2
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• pp.441-458
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• 2023

The poultry industry, which produces excellent sources of protein, suffers enormous economic damage from diseases. To solve this problem, research is being conducted on the early detection of infection according to the behavioral characteristics of poultry. The purpose of this study was to evaluate the potential of a non-movement behavior observation method to detect sick chickens. Forty 1-day-old Ross 308 males were used in the experiments, and an isolator equipped with an Internet Protocol (IP) camera was fabricated for observation. The chickens were inoculated with Salmonella enterica serovar Gallinarum A18-GCVP-014, the causative agent of fowl typhoid (FT), at 14 days of age, which is a vulnerable period for FT infection. The chickens were continuously observed with an IP camera for 2 weeks after inoculation, chickens that did not move for more than 30 minutes were detected and marked according to the algorithm. FT infection was confirmed based on clinical symptoms, analysis of cardiac, spleen and liver lesion scores, pathogen re-isolation, and serological analysis. As a result, clinical symptoms were first observed four days after inoculation, and dead chickens were observed on day six. Eleven days after inoculation, the number of clinical symptoms gradually decreased, indicating a state of recovery. For lesion scores, dead chickens scored 3.57 and live chickens scored 2.38. Pathogens were re-isolated in 37 out of 40 chickens, and hemagglutination test was positive in seven out of 26 chickens. The IP camera applied with the algorithm detected about 83% of the chickens that died in advance through non-movement behavior observation. Therefore, observation of non-movement behavior is one of the ways to detect infected chickens in advance, and it appears to have potential for the development of remote broiler management system.

• Journal of Veterinary Science
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• v.24 no.6
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• pp.82.1-82.12
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• 2023

Background: The current conventional serotyping based on antigen-antisera agglutination could not provide a better understanding of the potential pathogenicity of Salmonella enterica subsp. enterica serovar Brancaster. Surveillance data from Malaysian poultry farms indicated an increase in its presence over the years. Objective: This study aims to investigate the virulence determinants and antimicrobial resistance in S. Brancaster isolated from chickens in Malaysia. Methods: One hundred strains of archived S. Brancaster isolated from chicken cloacal swabs and raw chicken meat from 2017 to 2022 were studied. Two sets of multiplex polymerase chain reaction (PCR) were conducted to identify eight virulence genes associated with pathogenicity in Salmonella (invasion protein gene [invA], Salmonella invasion protein gene [sipB], Salmonella-induced filament gene [sifA], cytolethal-distending toxin B gene [cdtB], Salmonella iron transporter gene [sitC], Salmonella pathogenicity islands gene [spiA], Salmonella plasmid virulence gene [spvB], and inositol phosphate phosphatase gene [sopB]). Antimicrobial susceptibility assessment was conducted by disc diffusion method on nine selected antibiotics for the S. Brancaster isolates. S. Brancaster, with the phenotypic ACSSuT-resistance pattern (ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracycline), was subjected to PCR to detect the corresponding resistance gene(s). Results: Virulence genes detected in S. Brancaster in this study were invA, sitC, spiA, sipB, sopB, sifA, cdtB, and spvB. A total of 36 antibiogram patterns of S. Brancaster with a high level of multidrug resistance were observed, with ampicillin exhibiting the highest resistance. Over a third of the isolates displayed ACSSuT-resistance, and seven resistance genes (β-lactamase temoneira [bla_TEM], florfenicol/chloramphenicol resistance gene [floR], streptomycin resistance gene [strA], aminoglycoside nucleotidyltransferase gene [ant(3")-Ia], sulfonamides resistance gene [sul-1, sul-2], and tetracycline resistance gene [tetA]) were detected. Conclusion: Multidrug-resistant S. Brancaster from chickens harbored an array of virulence-associated genes similar to other clinically significant and invasive non-typhoidal Salmonella serovars, placing it as another significant foodborne zoonosis.

• Journal of Microbiology and Biotechnology
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• v.33 no.9
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• pp.1130-1140
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• 2023

Among the AAA+ proteases in bacteria, FtsH is a membrane-bound ATP-dependent metalloprotease, which is known to degrade many membrane proteins as well as some cytoplasmic proteins. In the intracellular pathogen Salmonella enterica serovar Typhimurium, FtsH is responsible for the proteolysis of several proteins including MgtC virulence factor and MgtA/MgtB Mg²⁺ transporters, the transcription of which is controlled by the PhoP/PhoQ two-component regulatory system. Given that PhoP response regulator itself is a cytoplasmic protein and also degraded by the cytoplasmic ClpAP protease, it seems unlikely that FtsH affects PhoP protein levels. Here we report an unexpected role of the FtsH protease protecting PhoP proteolysis from cytoplasmic ClpAP protease. In FtsH-depleted condition, PhoP protein levels decrease by ClpAP proteolysis, lowering protein levels of PhoP-controlled genes. This suggests that FtsH is required for normal activation of PhoP transcription factor. FtsH does not degrade PhoP protein but directly binds to PhoP, thus sequestering PhoP from ClpAP-mediated proteolysis. FtsH's protective effect on PhoP can be overcome by providing excess ClpP. Because PhoP is required for Salmonella's survival inside macrophages and mouse virulence, these data implicate that FtsH's sequestration of PhoP from ClpAP-mediated proteolysis is a mechanism ensuring the amount of PhoP protein during Salmonella infection.

• Korean Journal of Clinical Laboratory Science
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• v.48 no.2
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• pp.74-81
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• 2016

Ureaplasma species can normally colonize in the bodies of healthy individuals. Their colonization is associated with various diseases including non-gonococcal urethritis, chorioamnionitis, neonatal meningitis, and prematurity. In 2012, the sum of the resistant and intermediate resistant rates of Ureaplasma spp. to ofloxacin and ciprofloxacin was 66.08% and 92.69%, respectively. DNA point mutations in the genes encoding DNA gyrase (topoisomerase II) and topoisomerase IV are commonly responsible for fluoroquinolone resistance. Each enzyme is composed of two subunits encoded by gyrA and gyrB genes for DNA gyrase and parC and parE genes for topoisomerase IV. In the current study, these genes were sequenced in order to determine the role of amino acid substitutions in Ureaplasma spp. clinical isolates. From December 2012 to May 2013, we examined mutation patterns of the quinolone resistance-determining region (QRDR) in Ureaplasma spp. DNA sequences in the QRDR region of Ureaplasma clinical isolates were compared with those of reference strains including U. urealyticum serovar 8 (ATCC 27618) and U. parvum serovar 3 (ATCC 27815). Mutations were detected in all ofloxacin- and ciprofloxacin-resistant isolates, however no mutations were detected in drug-susceptible isolates. Most of the mutations related to fluoroquinolone resistance occurred in the parC gene, causing amino acid substitutions. Newly found amino acid substitutions in this study were Asn481Ser in GyrB; Phe149Leu, Asp150Met, Asp151Ile, and Ser152Val in ParC; and Pro446Ser and Arg448Lys in ParE. Continuous monitoring and accumulation of mutation data in fluoroquinolone-resistant Ureaplasma clinical isolates are essential to determining the tendency and to understanding the mechanisms underlying antimicrobial resistance.

• Food Science and Preservation
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• v.15 no.3
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• pp.469-476
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• 2008

Lactic acid fermentation of peach juice was carried out by using Lactobacillus plantarum KLAB21, a strain with a high level of antimutagenic activity, When the fermentation was carried out at 25, 30, 37 and $40^{\circ}C$, the highest level in the viable counts and acid production was obtained at $37^{\circ}C$. The sterilized peach juice showed a higher level of viable counts and acid production than the non-sterilized juice. And more viable counts and acid production were observed in the juice fermented by L. plantarum KLAB21 only than that obtained by a mixed culture of L. plantarum KLAB21 and Leuconostoc mesenteroides cells. When the lactic acid fermentation was performed for 5 days, the first 3 days of fermentation resulted in an increase of the viable counts from 8.2 to of 9.2 of log cfu/mL which is the highest level, as well as a decrease of the residual reducing sugar content from 5.6 to 0.1 % Decrease in the viable counts and m significant changes in the residual reducing sugar content were observed for further fermentation up to 5 days. However, the titratable acid content increased and the pH value decreased during the fermentation for 5 days to reach the highest titratable acid content (1,98%) and the lowest pH value (3.14) after 5 days of fermentation. HPLC analysis of the organic acids showed 1,236 mg% of lactic acid and 841 mg% of galacturonic acid contents in the fermented juice which were not detected in the fresh juice before fermentation. Antimutagenic effects of $100\;{\mu}L$ of the fermented peach juice supernatant were shown to be 97.7% against MNNG(N-methyl-N'-nitro-N-nitrosoguanidine), and 58.3% against NPD(4-nitro-O-phenylenediamine) in Salmonella enterica serovar Typhimurium TA100.