Dong Hyeok Kim

Toll-Like Receptor 4-Linked Janus Kinase 2 Signaling Contributes to Internalization of Brucella abortus by Macrophages

ABSTRACT Brucella abortus is an intracellular pathogen that uses a crafty strategy to invade and proliferate within host cells, but the distinct signaling pathways associated with phagocytic mechanisms of B. abortus remain unclear. The present study was performed to test the hypothesis that Toll-like receptor 4 (TLR4)-linked signaling interacting with Janus kinase 2 (JAK2) plays an essential role in B. abortus phagocytosis by macrophages. The effects of TLR4-JAK2 signaling on B. abortus phagocytosis in murine macrophage RAW 264.7 cells were observed through an infection assay and confocal microscopy. We determined that the uptake of B. abortus was negatively affected by the dysfunction of TLR4 and JAK2. F-actin polymerization detected by flow cytometry and F-actin assay was amplified for B. abortus entry, whereas that event was attenuated by the disruption of TLR4 and JAK2. Importantly, JAK2 phosphorylation and actin skeleton reorganization were suppressed immediately after B. abortus infection in bone marrow-derived macrophages (BMDMs) from TLR4−/− mice, showing the cooperation of JAK2 with TLR4. Furthermore, small GTPase Cdc42 participated in the intermediate pathway of TLR4-JAK2 signaling on B. abortus phagocytosis. Consequently, TLR4-associated JAK2 activation in the early cellular signaling events plays a pivotal role in B. abortus-induced phagocytic processes in macrophages, implying the pathogenic significance of JAK2-mediated entry. Here, we elucidate that this specific phagocytic mechanism of B. abortus might provide achievable strategies for inhibiting B. abortus invasion.

6-Acetonyl-5,6-dihydrosanguinarine (ADS) from Chelidonium majus L. triggers proinflammatory cytokine production via ROS-JNK/ERK-NFκB signaling pathway.

Chelidonium majus L. is an herbal plant that is commonly used in Western phytotherapy and traditional Chinese medicine for diuretic, antitussive, eye-regenerative, anti-osteoporotic, and radioprotective purposes. In this study, we purified 6-acetonyl-5,6-dihydrosanguinarine (ADS) from C. majus and investigated its immune-stimulatory effect. We found that ADS has the potential to induce the inflammatory cytokines TNF-α, IL-6, and IL-8 in macrophages and dendritic cells (DCs), that NFκB activation is a critical mediator of ADS-induced cytokine production, and that the activation of NFκB was dependent on reactive oxygen species (ROS). ADS induced phosphorylation of ERK and JNK, which was also associated with NFκB activation; phosphorylarion and cytokine production were inhibited by ROS scavenger and by specific MAPK inhibitors. Taken together, the results suggest that ADS from C. majus, as a positive immune modulator, induces inflammatory cytokines that might improve immunity, via the ROS-ERK/JNK-NFκB pathway.

Protective effects of recombinant Brucella abortus Omp28 against infection with a virulent strain of Brucella abortus 544 in mice.

The outer membrane proteins (OMPs) of Brucella (B.) abortus have been extensively studied, but their immunogenicity and protective ability against B. abortus infection are still unclear. In the present study, B. abortus Omp28, a group 3 antigen, was amplified by PCR and cloned into a maltose fusion protein expression system. Recombinant Omp28 (rOmp28) was expressed in Escherichia coli and was then purified. Immunogenicity of rOmp28 was confirmed by Western blot analysis with Brucella-positive mouse serum. Furthermore, humoral- or cell-mediated immune responses measured by the production of IgG1 or IgG2a in rOmp28-immunized mice and the ability of rOmp28 immunization to protect against B. abortus infection were evaluated in a mouse model. In the immunogenicity analysis, the mean titers of IgG1 and IgG2a produced by rOmp28-immunized mice were 20-fold higher than those of PBS-treated mice throughout the entire experimental period. Furthermore, spleen proliferation and bacterial burden in the spleen of rOmp28-immunized mice were approximately 1.5-fold lower than those of PBS-treated mice when challenged with virulent B. abortus. These findings suggest that rOmp28 from B. abortus is a good candidate for manufacturing an effective subunit vaccine against B. abortus infection in animals.

Anticoccidial effect of supplemental dietary Galla Rhois against infection with Eimeria tenella in chickens

The anticoccidial effects of Galla Rhois (GR) powder, which contains a major tannin-derived component of 52.7%, were evaluated in chickens following oral infection with Eimeria tenella. One-day-old chickens were assigned to five groups (control, unsupplemented, GR 0.5% supplemented [GRS 0.5%], GRS 1.0% [GRS 1.0%] and salinomycin supplemented [SS]). The chickens were fed a standard diet supplemented or not supplemented with GR or salinomycin for 10 days prior to infection. The birds received the supplemented diets continuously until 10 days post infection. The effects of GR on a E. tenella infection were evaluated by several parameters, including body weight gain, feed intake, oocyst excretion, bloody diarrhoea, and lesion scores. Infected chickens on the GRS and SS diets had a relatively moderate body weight loss (reduction ratio < 15%) and improved feed conversion. GRS and SS chickens produced significantly fewer faecal oocysts (P<0.05) and showed milder bloody diarrhoea compared with the E. tenella-infected control group. Furthermore, the lesion scores of both the GRS 0.5% and GRS 1.0% groups were significantly lower than the scores of the unsupplemented group on day 5 post infection. The lesion scores for the GR groups were similar to the scores for the SS group. In conclusion, this study suggests that GR appears to be as efficacious as salinomycin against E. tenella infection. GR supplementation leads to a reduction in infected chickens, although infected chickens are still affected compared with the uninfected control group. GR-based diets may be beneficial in preventing or treating coccidial infections in poultry.

Characterization of betaine aldehyde dehydrogenase (BetB) as an essential virulence factor of Brucella abortus

The pathogenic mechanisms of Brucellosis used to adapt to the harsh intracellular environment of the host cell are not fully understood. The present study investigated the in vitro and in vivo characteristics of B. abortus betaine aldehyde dehydrogenase (BetB) (Gene Bank ID: 006932) using a betB deletion mutant constructed from virulent B. abortus 544. In test under stress conditions, including osmotic- and acid stress-resistance, the betB mutant had a lower osmotic-resistance than B. abortus wild-type. In addition, the betB mutant showed higher internalization rates compared to the wild-type strain; however, it also displayed replication failures in HeLa cells and RAW 264.7 macrophages. During internalization, compared to the wild-type strain, the betB mutant was more adherent to the host surface and showed enhanced phosphorylation of protein kinases, two processes that promote phagocytic activity, in host cells. During intracellular trafficking, colocalization of B. abortus-containing phagosomes with LAMP-1 was elevated in betB mutant-infected cells compared to the wild-type cells. In mice, the betB mutant was predominantly cleared from spleens compared to the wild-type strain after 2 weeks post-infection, and the vaccination test with the live betB mutant showed effective protection against challenge infection with the virulent wild-type strain. These findings suggested that the B. abortus betB gene substantially affects the phagocytic pathway in human phagocytes and in host cells in mice. Furthermore, this study highlights the potential use of the B. abortus betB mutant as a live vaccine for the control of brucellosis.

Interplay between Clathrin and Rab5 Controls the Early Phagocytic Trafficking and Intracellular Survival of Brucella abortus within HeLa cells

Background: Phagocytic mechanisms are important for understanding the host-pathogen interactions of intracellular parasitic pathogens. Results: The entry and early intracellular trafficking of B. abortus are dependent on clathrin cooperation with Rab5. Conclusion: Clathrin influences phagocytic pathways of B. abortus associating with Rab5 at a host subcellular site allowing replication. Significance: This study promotes the early control of B. abortus infection. Lipid raft-associated clathrin is essential for host-pathogen interactions during infection. Brucella abortus is an intracellular pathogen that circumvents host defenses, but little is known about the precise infection mechanisms that involve interaction with lipid raft-associated mediators. The aim of this study was to elucidate the clathrin-mediated phagocytic mechanisms of B. abortus. The clathrin dependence of B. abortus infection in HeLa cells was investigated using an infection assay and immunofluorescence microscopy. The redistribution of clathrin in the membrane and in phagosomes was investigated using sucrose gradient fractionation of lipid rafts and the isolation of B. abortus-containing vacuoles, respectively. Clathrin and dynamin were concentrated into lipid rafts during B. abortus infection, and the entry and intracellular survival of B. abortus within HeLa cells were abrogated by clathrin inhibition. Clathrin disruption decreased actin polymerization and the colocalization of B. abortus-containing vacuoles with clathrin and Rab5 but not lysosome-associated membrane protein 1 (LAMP-1). Thus, our data demonstrate that clathrin plays a fundamental role in the entry and intracellular survival of B. abortus via interaction with lipid rafts and actin rearrangement. This process facilitates the early intracellular trafficking of B. abortus to safe replicative vacuoles.

Intracellular replication inhibitory effects of Galla Rhois ethanol extract for Brucella abortus infection

ETHNOPHARMACOLOGICAL RELEVANCE Galla Rhois (GR) has long been applied in traditional Korean and Oriental medicine. Although GR has an anti-bacterial effect, the anti-bacterial mechanism and therapeutic efficiency of GR for intracellular parasitic Brucella infection are still unclear. AIM OF THE STUDY The objective of this study was to investigate the antibacterial and therapeutic effects of GR ethanol extract (GRE), which is a natural antibacterial component for the treatment of Brucella abortus infection. MATERIALS AND METHODS The antibacterial activity of GRE towards Brucella abortus was evaluated by incubating Brucella abortus with GRE. Following treatment with GRE, Brucella abortus adherence, uptake, intracellular growth, and intracellular trafficking in macrophages were monitored. Mice were infected intraperitoneally with Brucella abortus and treated orally with GRE for 14 days, and then the weight and CFUs from each spleen were monitored. RESULTS The viability of Brucella abortus was markedly decreased in a dose-dependent manner. Moreover, Brucella abortus internalization and intracellular growth within macrophages were reduced in GRE-treated cells. The number of bacteria that adhered to GRE-pretreated cells was significantly lower than that of untreated cells. With regards to intracellular trafficking, treatment with GRE augmented the colocalization of Brucella abortus-containing phagosomes with LAMP-1. GRE-treated mice showed considerably decreased weight and bacterial burdens in the spleen compared to untreated mice. CONCLUSION GRE exhibits antibacterial and protective effects on Brucella abortus in vitro and in vivo. These results highlight the beneficial effects of GRE in the prevention and treatment of brucellosis.

Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis

In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host-pathogen interaction.

Identification of genes contributing to the intracellular replication of Brucella abortus within HeLa and RAW 264.7 cells

Brucella abortus, the causative agent of brucellosis, can survive and replicate within host cells. Understanding bacterial virulence factors and bacteria-host cell interactions is critical for controlling brucellosis, yet very little is known about the virulence strategies and signaling pathways activated in phagocytes during infection to ensure their growth and survival. B. abortus was mutagenized by mini-Tn5Km2 transposon mutagenesis to identify virulence genes related to the internalization and intracellular replication of the bacteria. Of the total 2300 mutants used to infect HeLa cells, 23 mutants defective for intercellular growth and the mutated genes were identified. Sequence analysis of DNA flanking the transposon showed various insertion sites in bacterial genes that might be associated with virulence, including genes associated with lipoproteins, amino acid metabolism, translation, transcription, carbohydrate transport, coenzyme transport, inorganic ion transport, energy metabolism, membrane transport, and cell wall/membrane biogenesis. Moreover, mutants were classified into class I, class II and class III as higher, similar, and lower internalization, respectively, into HeLa cells. Furthermore, defective mutants for intracellular growth in HeLa cells were found to be defective in RAW 264.7 cells. Taken together, we suggest that the identified virulence associated genes might contribute to the intracellular growth and survival of B. abortus in phagocytes.

Redundant effects of ketamine on the pathogenesis and severity of Brucella abortus infection

Brucella abortus is an intracellular bacterium and leading to a serious debilitating disease known as brucellosis. Ketamine is an anesthetic and a sedative that affects the immunomodulatory activities of various immune cells. The current study was to elucidate the role of ketamine in B. abortus infection, focusing on the phagocytic activity and immune response of macrophages. Following incubation of murine macrophages with ketamine, the phagocytosis of B. abortus was markedly reduced compared with the unincubated control. Interestingly, ketamine-incubated cells displayed a decreased intensity of F-actin fluorescence compared with the B. abortus-induced amplification of intensity. Conversely, the intracellular replication of B. abortus within macrophages was notably enhanced by ketamine. Furthermore, the in vivo assessment using a mouse model revealed that continual injections with ketamine led to augmented bacterial burdens in the spleen, which was accompanied by decreased levels of mRNA expression of cytokines in the spleen. The elevations of serum cytokines such as IFN-γ, IL-12 and IL-6, as well as the chemokine MCP-1, were also reduced by ketamine. These findings verify that ketamine suppresses the phagocytic activity and immune response during B. abortus infection. Therefore, the current study might provide novel insights into the potential influences of ketamine on infectious diseases caused by B. abortus, considering the host-pathogen interaction.