Journal of Microbiology

Review

Envelope‑Stress Sensing Mechanism of Rcs and Cpx Signaling Pathways in Gram‑Negative Bacteria: Seung-Hyun Cho , Kilian Dekoninck , Jean-Francois Collet; J. Microbiol. 2023;61(3):317-329. Published online March 9, 2023; DOI: https://doi.org/10.1007/s12275-023-00030-y

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9 Citations

Abstract: The global public health burden of bacterial antimicrobial resistance (AMR) is intensified by Gram-negative bacteria, which have an additional membrane, the outer membrane (OM), outside of the peptidoglycan (PG) cell wall. Bacterial twocomponent systems (TCSs) aid in maintaining envelope integrity through a phosphorylation cascade by controlling gene expression through sensor kinases and response regulators. In Escherichia coli, the major TCSs defending cells from envelope stress and adaptation are Rcs and Cpx, which are aided by OM lipoproteins RcsF and NlpE as sensors, respectively. In this review, we focus on these two OM sensors. β-Barrel assembly machinery (BAM) inserts transmembrane OM proteins (OMPs) into the OM. BAM co-assembles RcsF, the Rcs sensor, with OMPs, forming the RcsF-OMP complex. Researchers have presented two models for stress sensing in the Rcs pathway. The first model suggests that LPS perturbation stress disassembles the RcsF-OMP complex, freeing RcsF to activate Rcs. The second model proposes that BAM cannot assemble RcsF into OMPs when the OM or PG is under specific stresses, and thus, the unassembled RcsF activates Rcs. These two models may not be mutually exclusive. Here, we evaluate these two models critically in order to elucidate the stress sensing mechanism. NlpE, the Cpx sensor, has an N-terminal (NTD) and a C-terminal domain (CTD). A defect in lipoprotein trafficking
results
in NlpE retention in the inner membrane, provoking the Cpx response. Signaling requires the NlpE NTD, but not the NlpE CTD; however, OM-anchored NlpE senses adherence to a hydrophobic surface, with the NlpE CTD playing a key role in this function.

Journal Articles

Development of a neutralization assay based on the pseudotyped chikungunya virus of a Korean isolate: Woo-Chang Chung , Kwang Yeon Hwang , Suk-Jo Kang , Jae-Ouk Kim , Moon Jung Song; J. Microbiol. 2020;58(1):46-53. Published online November 25, 2019; DOI: https://doi.org/10.1007/s12275-020-9384-0

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Abstract: The Chikungunya virus (CHIKV) belongs to the Alphavirus genus of Togaviridae family and contains a positive-sense single stranded RNA genome. Infection by this virus mainly causes sudden high fever, rashes, headache, and severe joint pain that can last for several months or years. CHIKV, a mosquito- borne arbovirus, is considered a re-emerging pathogen that has become one of the most pressing global health concerns due to a rapid increase in epidemics. Because handling of CHIKV is restricted to Biosafety Level 3 (BSL-3) facilities, the evaluation of prophylactic vaccines or antivirals has been substantially hampered. In this study, we first identified the whole structural polyprotein sequence of a CHIKV strain isolated in South Korea (KNIH/2009/77). Phylogenetic analysis showed that this sequence clustered within the East/ Central/South African CHIKV genotype. Using this sequence information, we constructed a CHIKV-pseudotyped lentivirus expressing the structural polyprotein of the Korean CHIKV isolate (CHIKVpseudo) and dual reporter genes of green fluorescence protein and luciferase. We then developed a pseudovirus-based neutralization assay (PBNA) using CHIKVpseudo. Results from this assay compared to those from the conventional plaque reduction neutralization test showed that our PBNA was a reliable and rapid method to evaluate the efficacy of neutralizing antibodies. More importantly, the neutralizing activities of human sera from CHIKVinfected individuals were quantitated by PBNA using CHIKVpseudo. Taken together, these results suggest that our PBNA for CHIKV may serve as a useful and safe method for testing the neutralizing activity of antibodies against CHIKV in BSL-2 facilities.

Comparison of virulence between matt and mucoid colonies of Klebsiella pneumoniae coproducing NDM-1 and OXA-232 isolated from a single patient: Haejeong Lee , Jin Yang Baek , So Yeon Kim , HyunJi Jo , KyeongJin Kang , Jae-Hoon Ko , Sun Young Cho , Doo Ryeon Chung , Kyong Ran Peck , Jae-Hoon Song , Kwan Soo Ko; J. Microbiol. 2018;56(9):665-672. Published online August 23, 2018; DOI: https://doi.org/10.1007/s12275-018-8130-3

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21 Citations

Abstract: Nine Klebsiella pneumoniae isolates coproducing NDM-1 and OXA-232 carbapenemases were successively isolated from a single patient. Although they were isolated simultaneously and were isogenic, they presented different colony phenotypes (matt and mucoid). All nine isolates were resistant to most antibiotics except colistin and fosfomycin. In addition, matt-type isolates were resistant to tigecycline. No differences were detected in the cps cluster sequences, except for the insertion of IS5 in the wzb gene of two matt-type isolates. In vitro virulence assays based on production of capsular polysaccharide, biofilm formation, and resistance to human serum indicated that the mucoid-type isolates were significantly more virulent than the matt-type. In addition, mucoid-type isolates showed higher survival rates than the matt-type ones in infection experiments in the fruit fly, suggesting a higher virulence of K. pneumoniae isolates with a mucoid phenotype. To our knowledge, this is the first report of K. pneumoniae colonies with different phenotypes being isolated from the same sample. In addition, we show that virulence varies with colony phenotype. Dissemination of K. pneumoniae isolates expressing both antibiotic resistance and high virulence would constitute a great threat.

Research Support, Non-U.S. Gov't

Characterization of Streptococcus pneumoniae N-Acetylglucosamine-6-Phosphate Deacetylase as a Novel Diagnostic Marker: Chi-Won Choi , Hee-Young An , Yong Ju Lee , Yeol Gyun Lee , Sung Ho Yun , Edmond Changkyun Park , Yeonhee Hong , Gun-Hwa Kim , Jae-Eun Park , Sun Jong Baek , Hyun Sik Kim , Seung Il Kim; J. Microbiol. 2013;51(5):659-664. Published online October 31, 2013; DOI: https://doi.org/10.1007/s12275-013-3451-8

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5 Citations

Abstract: The identification of novel diagnostic markers of pathogenic bacteria is essential for improving the accuracy of diagnoses and for developing targeted vaccines. Streptococcus pneumoniae is a significant human pathogenic bacterium that causes pneumonia. N-acetylglucosamine-6-phosphate deacetylase (NagA) was identified in a protein mixture secreted by S. pneumoniae and its strong immunogenicity was confirmed in an immuno-proteomic assay against the anti-serum of the secreted protein mixture. In this study, recombinant S. pneumoniae NagA protein was expressed and purified to analyze its protein characteristics, immunospecificity, and immunogenicity, thereby facilitating its evaluation as a novel diagnostic marker for S. pneumoniae. Mass spectrometry analysis showed that S. pneumoniae NagA contains four internal disulfide bonds and that it does not undergo posttranslational modification. S. pneumoniae NagA antibodies successfully detected NagA from different S. pneumoniae strains, whereas NagA from other pathogenic bacteria species was not detected. In addition, mice infected with S. pneumoniae generated NagA antibodies in an effective manner. These results suggest that NagA has potential as a novel diagnostic marker for S. pneumoniae because of its high immunogenicity and immunospecificity.

Journal Article

Study on Persistent Infection of Japanese Encephalitis Virus Beijing-1 Strain in Serum-free Sf9 Cell Cultures: Hun Kim , Su Jeen Lee , Jin Yong Park , Yong Wook Park , Hyun Sung Kim , Heui-Yun Kang , Byung-Ki Hur , Yeon-Woo Ryu , Sang In Han , Jong Su Kim; J. Microbiol. 2004;42(1):25-31.; DOI: https://doi.org/2005 [pii]

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Abstract: Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8×10^6 cells/ml in a 500ml spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-1 strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15% of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-1 strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-1 stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-1 strain rose from 1.0×10^5 to 1.5×10^6 pfu/ml. The infected Sf9 cells could be subcultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-1 strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-1 strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.

Cloning and characterization of the multiprotein bridging factor 1 (YlMBF1) gene from the dimorphic yeast Yarrowia lipolytica: Janghwan Kim , Seon Ah Cheon , Yunkyoung Song , Jeong-Yoon Kim; J. Microbiol. 2002;40(2):173-177.

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Abstract: In order to identify Yarrowia lipolytica genes induced by serum, cDNA representational difference analysis was performed using a PCR-select cDNA subtraction method. One of the genes cloned from the subtraction was a gene (YlMBF1) homologous to Saccharomyces cerevisiae MBF1 encoding the coactivator multiprotein bridging factor 1. Disruption of YlMBF1 revealed that the gene was not essential for viability, and the Ylmbf1[delta] strain did not show any distinct phenotypic change on solid serum medium. In liquid medium, however, a difference was found in the ability to maintain hyphae induced by serum. This result suggests that the YlMbf1 protein may mediate transcriptional activation of certain genes involved in the hypha formation of Y. lipolytica.

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