Inhibition of <i>Salmonella</i> Typhimurium adhesion, invasion, and intracellular survival via treatment with methyl gallate alone and in combination with marbofloxacin

Birhanu, Biruk Tesfaye,Park, Na-Hye,Lee, Seung-Jin,Hossain, Md Akil,Park, Seung-Chun BioMed Central 2018 VETERINARY RESEARCH Vol.49 No.-

<P><I>Salmonella enterica</I> serovar Typhimurium infects intestinal epithelia and macrophages, which is prevented by inhibiting adhesion and cell invasion. This study aimed to investigate the role of methyl gallate (MG) in adhesion, invasion, and intracellular survival of <I>Salmonella</I> Typhimurium in Caco-2 and RAW 264.7 cells via a gentamicin protection assay, confocal microscopy, and quantitative reverse-transcription polymerase chain reaction. MG (30 µg/mL) inhibited adhesion and invasion of <I>Salmonella</I> Typhimurium by 54.01% and 60.5% in RAW 264.7 cells, respectively. The combination of MG with sub-minimum inhibitory concentration (MIC) of marbofloxacin (MRB) inhibited the adhesion, invasion, and intracellular survival by 70.49%, 67.36%, and 74%, respectively. Confocal microscopy further revealed reductions in bacterial count in Caco-2 cells treated with MG alone or with sub-MIC of MRB. Furthermore, MG alone or in combination with sub-MIC of MRB decreased the motility of <I>Salmonella</I> Typhimurium. Quorum sensing genes including <I>sdiA</I>, <I>srgE</I>, and <I>rck</I> were downregulated by 52.8%, 61.7%, and 22.2%, respectively. Moreover, <I>rac</I>-<I>1</I> was downregulated by 56.9% and 71.9% for MG alone and combined with sub-MIC of MRB, respectively, in mammalian cells. Furthermore, MG downregulated virulence genes of <I>Salmonella</I> Typhimurium including <I>cheY, ompD</I>, <I>sipB</I>, <I>lexA</I>, and <I>ompF</I> by 59.6%, 60.2%, 20.5%, 31.4%, and 16.2%, respectively. Together, the present results indicate that MG alone or in combination with a sub-MIC of MRB effectively inhibited the adhesion, invasion, and intracellular survival of <I>Salmonella</I> Typhimurium in vitro by downregulating quorum sensing and virulence genes.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (10.1186/s13567-018-0597-8) contains supplementary material, which is available to authorized users.</P>

In silico analysis of putative drug and vaccine targets of the metabolic pathways of Actinobacillus pleuropneumoniae using a subtractive/comparative genomics approach

Birhanu Biruk Tesfaye,이승진,Na-Hye Park,송주범,Seung-Chun Park 대한수의학회 2018 Journal of Veterinary Science Vol.19 No.2

Actinobacillus pleuropneumoniae is a Gram-negative bacterium that resides in the respiratory tract of pigs and causes porcine respiratorydisease complex, which leads to significant losses in the pig industry worldwide. The incidence of drug resistance in this bacterium isincreasing; thus, identifying new protein/gene targets for drug and vaccine development is critical. In this study, we used an in silico approach,utilizing several databases including the Kyoto Encyclopedia of Genes and Genomes (KEGG), the Database of Essential Genes (DEG),DrugBank, and Swiss-Prot to identify non-homologous essential genes and prioritize these proteins for their druggability. The results showed20 metabolic pathways that were unique and contained 273 non-homologous proteins, of which 122 were essential. Of the 122 essentialproteins, there were 95 cytoplasmic proteins and 11 transmembrane proteins, which are potentially suitable for drug and vaccine targets,respectively. Among these, 25 had at least one hit in DrugBank, and three had similarity to metabolic proteins from Mycoplasmahyopneumoniae, another pathogen causing porcine respiratory disease complex; thus, they could serve as common therapeutic targets. Inconclusion, we identified glyoxylate and dicarboxylate pathways as potential targets for antimicrobial therapy and tetra-acyldisaccharide 4ʹ-kinaseand 3-deoxy-D-manno-octulosonic-acid transferase as vaccine candidates against A. pleuropneumoniae.

Biofilm formation and eradication in Mycoplasma hyopneumoniae

Abraham Mechesso,Biruk Birhanu,Seung Chun Park 대한수의학회 2016 대한수의학회 학술대회발표집 Vol.2016 No.-

BaeR protein acts as an activator of nuclear factor-kappa B and Janus kinase 2 to induce inflammation in murine cell lines

Lee, Seung-Jin,Birhanu, Biruk Tesfaye,Awji, Elias Gebru,Kim, Myung Hee,Park, Ji-Yong,Suh, Joo-Won,Park, Seung-Chun National Research Council 2016 Canadian journal of microbiology Vol.62 No.9

<P> BaeR, a response regulator protein, takes part in multidrug efflux, bacterial virulence activity, and other biological functions. Recently, BaeR was shown to induce inflammatory responses by activating the mitogen-activated protein kinases (MAPKs). In this study, we investigated additional pathways used by BaeR to induce an inflammatory response. BaeR protein was purified from Salmonella enterica Paratyphi A and subcloned into a pPosKJ expression vector. RAW 264.7 cells were treated with BaeR, and RNA was extracted by TRIzol reagent for RT-PCR. Cytokine gene expression was analyzed by using the comparative cycle threshold method, while western blotting and ELISA were used to assess protein expression. We confirmed that BaeR activates nuclear factor-kappa B (NF-κB), thereby inducing an inflammatory response and increases the production of interleukins (IL-)1β and IL-6. During this process, the Janus kinase 2 (JAK2)-STAT1 signaling pathway was activated, resulting in an increase in the release of interferons I and II. Additionally, COX-2 was activated and its expression increased with time. In conclusion, BaeR induced an inflammatory response through activation of NF-κB in addition to the MAPKs. Furthermore, activation of the JAK2-STAT1 pathway and COX-2 facilitated the cytokine binding activity, suggesting an additional role for BaeR in the modulation of the immune system of the host and the virulence activity of the pathogen. </P>

Sonicated Protein Fractions of Mycoplasma hyopneumoniae Induce Inflammatory Responses and Differential Gene Expression in a Murine Alveolar Macrophage Cell Line

Damte, Dereje,Lee, Seung-Jin,Birhanu, Biruk Tesfaye,Suh, Joo-Won,Park, Seung-Chun The Korean Society for Microbiology and Biotechnol 2015 Journal of microbiology and biotechnology Vol.25 No.12

Mycoplasma hyopneumoniae is known to cause porcine enzootic pneumonia (EP), an important disease in swine production. The objective of this study was to examine the effects of sonicated protein fractions of M. hyopneumoniae on inflammatory response and gene expression in the murine alveolar macrophage MH-S cell line. The effects of sonicated protein fractions and intact M. hyopneumoniae on the gene expression of cytokines and iNOS were assessed using RT-PCR. The Annealing Control Primer (ACP)-based PCR method was used to screen differentially expressed genes. Increased transcription of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, COX-2, and iNOS mRNA was observed after exposure to the supernatant (SPT), precipitant (PPT), and intact M. hyopneumoniae protein. A time-dependent analysis of the mRNA expression revealed an upregulation after 4 h for IL-6 and iNOS and after 12 h for IL-1β and TNF-α, for both SPT and PPT; the fold change in COX-2 expression was less. A dose- and time-dependent correlation was observed in nitrite (NO) production for both protein fractions; however, there was no significant difference between the effects of the two protein fractions. In a differential gene analysis, PCR revealed differential expression for nine gene bands after 3 h of stimulation — only one gene was downregulated, while the remaining eight were upregulated. The results of this study provide insights that help improve our understanding of the mechanisms underlying the pathogenesis of and macrophage defenses against M. hyopneumoniae assault, and suggest targets for future studies on therapeutic interventions for M. hyopneumoniae infections.

Enhancement of Lipid Metabolism and Hepatic Stability in Fat-Induced Obese Mice by Fermented <i> Cucurbita moschata</i> Extract

Hossain, Md. Akil,Lee, Seung-Jin,Park, Na-Hye,Birhanu, Biruk Tesfaye,Mechesso, Abraham Fikru,Park, Ji-Yong,Park, Eun-Jin,Lee, Sam-Pin,Youn, Sun-Joo,Park, Seung-Chun Hindawi 2018 Evidence-based Complementary and Alternative Medic Vol.2018 No.-

<P>The aim of this study was to evaluate the potentials of fermented<I> Cucurbita moschata</I> extract (FCME) in the treatment of obesity and nonalcoholic fatty liver disease (NAFLD). Five-week-old male C57BL/6 mice were assigned to 6 groups and treated for 8 weeks by feeding the normal diet (ND) and high fat diet (HFD) with and without FCME. Changes in body weight gain and consumption of feed and water were recorded. Major organs, adipose tissues, and blood samples were collected after the experimental period. The serum lipid profile, histological features of liver and adipose tissues, and mRNA expression of different adipogenic/lipogenic genes from liver tissue were evaluated. The supplementation of FCME in HFD significantly prevented HFD-induced increment of bodyweight. The adipose tissue mass, liver enzymes, and plasma lipids were also reduced significantly (<I>p</I> < 0.05) by the consumption of FCME. The mRNA expressions of adipogenic/lipogenic genes (PPAR<I>γ</I>, C/EBP<I>α</I>, C/EBP<SUB><I>β</I></SUB>, C/EBP<I>γ</I>, and SREBP-1C) in FCME-treated obese mice were considerably (<I>p</I> < 0.05) suppressed. FCME showed its antiobesity potential by suppressing the body weight gain and by modulating the plasma lipids and liver enzymes through the regulation of adipogenic/lipogenic transcriptional factors. Fermented<I> Cucurbita moschata</I> could be an opportunistic agent in controlling obesity and fatty liver changes.</P>