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Hop, Huynh Tan,Arayan, Lauren Togonon,Reyes, Alisha Wehdnesday Bernardo,Huy, Tran Xuan Ngoc,Min, WonGi,Lee, Hu Jang,Son, Jee Soo,Kim, Suk Elsevier 2017 Microbial pathogenesis Vol.113 No.-
<P><B>Abstract</B></P> <P> <I>Brucella</I> is a zoonotic pathogen that survives within macrophages; however the replicative mechanisms involved are not fully understood. We describe the isolation of sufficient <I>Brucella abortus</I> RNA from primary host cell environment using modified reported methods for RNA-seq analysis, and simultaneously characterize the transcriptional profiles of intracellular <I>B. abortus</I> and bone marrow-derived macrophages (BMM) from BALB/c mice at 24 h (replicative phase) post-infection. Our results revealed that 25.12% (801/3190) and 16.16% (515/3190) of the total <I>B. abortus</I> genes were up-regulated and down-regulated at >2-fold, respectively as compared to the free-living <I>B. abortus</I>. Among >5-fold differentially expressed genes, the up-regulated genes are mostly involved in DNA, RNA manipulations as well as protein biosynthesis and secretion while the down-regulated genes are mainly involved in energy production and metabolism. On the other hand, the host responses during <I>B. abortus</I> infection revealed that 14.01% (6071/43,346) of BMM genes were reproducibly transcribed at >5-fold during infection. Transcription of cytokines, chemokines and transcriptional factors, such as tumor necrosis factor (<I>Tnf</I>), interleukin-1α (<I>Il1α</I>), interleukin-1β (<I>Il1β</I>), interleukin-6 (<I>Il6</I>), interleukin-12 (<I>Il12</I>), chemokine C-X-C motif (<I>CXCL</I>) family, nuclear factor kappa B (<I>Nf-κb</I>), signal transducer and activator of transcription 1 (<I>Stat1</I>), that may contribute to host defense were markedly induced while transcription of various genes involved in cell proliferation and metabolism were suppressed upon <I>B. abortus</I> infection. In conclusion, these data suggest that <I>Brucella</I> modulates gene expression in hostile intracellular environment while simultaneously alters the host pathways that may lead to the pathogen's intracellular survival and infection.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The transcriptional profiles of <I>B. abortus</I> and BMM were analyzed by RNA-seq approach. </LI> <LI> The up-regulated <I>B. abortus</I> genes are involved in DNA, RNA manipulations and protein biosynthesis. </LI> <LI> The down-regulated <I>B. abortus</I> genes are involved in energy production and metabolism. </LI> <LI> The host defense genes are induced but cell proliferation and metabolism genes are suppressed in <I>B. abortus</I> infected BMM. </LI> </UL> </P>
Hop, Huynh Tan,Arayan, Lauren Togonon,Huy, Tran Xuan Ngoc,Reyes, Alisha Wehdnesday Bernardo,Min, WonGi,Lee, Hu Jang,Park, Soo Jong,Chang, Hong Hee,Kim, Suk Elsevier 2018 Vaccine Vol.36 No.21
<P><B>Abstract</B></P> <P>In this study, we assessed the protective efficacy of single subunit vaccines, encoded by the <I>B. abortus</I> 544 genes <I>aspC</I>, <I>dps</I>, <I>yaeC</I> and <I>inpB</I>, against <I>B. abortus</I> infection in mice. First, immunization with these antigens, with the exception of the YaeC protein, was found to elicit both humoral and cellular immune responses with IgG2a being dominant over IgG1. In addition, a massive production of IFN-γ but lower degree of IL-10 was observed, suggesting that all three antigens were able to induce predominantly cell-mediated immunity in response to <I>B. abortus</I> infection. Further investigation of a combined subunit vaccine (CSV) consisting of purified AspC, Dps, InpB and Ndk proteins showed a superior protective effect in mice against brucellosis. The intraperitoneal injection of this combination was shown to induce a remarkable production of IFN-γ and IL-2, which occurred in conjunction with an increase of blood CD4<SUP>+</SUP> and CD8<SUP>+</SUP> T cell proportions. In addition, the higher titer of IgG2a compared to IgG1 elicited by this CSV was obtained, suggesting that this CSV induced a typical T-helper-1-dominated immune response <I>in vivo</I>. Furthermore, the protection level induced by this combination was significantly higher than that induced by single antigens and was not significantly different compared to a group immunized with a live attenuated vaccine (RB51). Altogether, our findings suggest that the combination of different immunogenic antigens could be a useful approach for the development of a new, effective and safe brucellosis vaccine that can replace current vaccine strains.</P>
( Huynh Tan Hop ),( Alisha Wehdnesday Bernardo Reyes ),( Hannah Leah Tadeja Simborio ),( Lauren Togonon Arayan ),( Won Gi Min ),( Hu Jang Lee ),( Jin Ju Lee ),( Hong Hee Chang ),( Suk Kim ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.1
In this study, the Brucella abortus ohr gene coding for an organic hydroperoxide resistance protein (Ohr) was cloned into a maltose fusion protein expression system (pMAL), inserted into Escherichia coli, and purified, and its immunogenicity was evaluated by western blot analysis using Brucella-positive mouse sera. The purified recombinant Ohr (rOhr) was treated with adjuvant and injected intraperitoneally into BALB/c mice. A protective immune response analysis revealed that rOhr induced a significant increase in both the IgG1 and IgG2a titers, and IgG2a reached a higher level than IgG1 after the second and third immunizations. Additionally, immunization with rOhr induced high production of IFN-γ as well as proinflammatory cytokines such as TNF, MCP-1, IL-12p70, and IL-6, but a lesser amount of IL-10, suggesting that rOhr predominantly elicited a cell-mediated immune response. In addition, immunization with rOhr caused a significantly higher degree of protection against a virulent B. abortus infection compared with a positive control group consisting of mice immunized with maltose-binding protein. These findings showed that B. abortus rOhr was able to induce both humoral and cell-mediated immunity in mice, which suggested that this recombinant protein could be a potential vaccine candidate for animal brucellosis.
Hop, Huynh Tan,Reyes, Alisha Wehdnesday Bernardo,Huy, Tran Xuan Ngoc,Arayan, Lauren Togonon,Min, WonGi,Lee, Hu Jang,Rhee, Man Hee,Chang, Hong Hee,Kim, Suk American Society for Biochemistry and Molecular Bi 2018 The Journal of biological chemistry Vol.293 No.9
<P>Brucella abortus is a Gram-negative zoonotic pathogen for which there is no 100% effective vaccine. Phagosomes in B. abortus infected cells fail to mature, allowing the pathogen to survive and proliferate. Interleukin 10 (IL10) promotes B. abortus persistence in macrophages by mechanisms that are not fully understood. In this study, we investigated the regulatory role of IL10 in the immune response to B. abortus infection. B. abortus infected macrophages were treated with either IL10 s1RNA or recombinant IL10 (rIL10), and the expression of phagolysosome- or inflammation-related genes was evaluated by qRT-PCR and Western blotting. Phagolysosome fusion was monitored by fluorescence microscopy. We found that the synthesis of several membrane-trafficking regulators and lysosomal enzymes was suppressed by IL10 during infection, resulting in a significant increase in the recruitment of hydrolytic enzymes by Brucella-containing phagosomes (BCPs) when IL10 signaling was blocked. Moreover, blocking IL10 signaling also enhanced proinflammatory cytokine production. Finally, concomitant treatment with STAT3 siRNA significantly reduced the suppression of proinflammatory brucellacidal activity but not phagolysosome fusion by rIL10. Thus, our data provide the first evidence that clearly indicates the suppressive role of IL10 on phagolysosome fusion and inflammation in response to B. abortus infection through two distinct mechanisms, STAT3-independent and-dependent pathways, respectively, in murine macrophages.</P>
( Huynh Tan Hop ),( Lauren Togonon Arayan ),( Alisha Wehdnesday Bernardo Reyes ),( Tran Xuan Ngoc Huy ),( Eun Jin Baek ),( Wongi Min ),( Hu Jang Lee ),( Chun Hee Lee ),( Man Hee Rhee ),( Suk Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.10
In this study, we evaluated the inhibitory effect of a rice bran mixture extract (RBE) on Brucella abortus pathogenesis in professional (RAW 264.7) and nonprofessional (HeLa) phagocytes. We fermented the rice bran mixture and then extracted it with 50% ethanol followed by gas chromatography-mass spectrometry to identify the components in RBE. Our results clearly showed that RBE caused a significant reduction in the adherence of B. abortus in both cell lines. Furthermore, analysis of phagocytic signaling proteins by western blot assay revealed that RBE pretreatment resulted in inhibition of phosphorylation of JNK, ERK, and p38, leading to decline of internalization compared with the controls. Additionally, the intensity of F-actin observed by fluorescence microscopy and FACS was remarkably reduced in RBE-pretreated cells compared with control cells. However, the intracellular replication of B. abortus within phagocytes was not affected by RBE. Taken together, these findings suggest that the phagocytic receptor blocking and suppressive effects of RBE on the MAPK-linked phagocytic signaling pathway could negatively affect the invasion of B. abortus into phagocytes.