• Genomics & Informatics
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• 제21권1호
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• pp.5.1-5.13
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• 2023

Neisseria gonorrhoeae is a Gram-negative aerobic diplococcus bacterium that primarily causes sexually transmitted infections through direct human sexual contact. It is a major public health threat due to its impact on reproductive health, the widespread presence of antimicrobial resistance, and the lack of a vaccine. In this study, we used a bioinformatics approach and performed subtractive genomic methods to identify potential drug targets against the core proteome of N. gonorrhoeae (12 strains). In total, 12,300 protein sequences were retrieved, and paralogous proteins were removed using CD-HIT. The remaining sequences were analyzed for non-homology against the human proteome and gut microbiota, and screened for broad-spectrum analysis, druggability, and anti-target analysis. The proteins were also characterized for unique interactions between the host and pathogen through metabolic pathway analysis. Based on the subtractive genomic approach and subcellular localization, we identified one cytoplasmic protein, 2Fe-2S iron-sulfur cluster binding domain-containing protein (NGFG RS03485), as a potential drug target. This protein could be further exploited for drug development to create new medications and therapeutic agents for the treatment of N. gonorrhoeae infections.

• Molecules and Cells
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• 제27권2호
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• pp.149-157
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• 2009

In both gram-positive and several gram-negative bacteria, the transcription of dnaK and groE operons is negatively regulated by HrcA; however, the mechanism modulating HrcA protein activity upon thermal stress remains elusive. Here, we demonstrate that HrcA is modulated via reduction and oligomerization in vitro. Native-PAGE analysis was used to reveal the oligomeric structure of HrcA. The oligomeric HrcA structure became monomeric following treatment with the reducing agent dithothreitol, and this process was reversed by treatment with hydrogen peroxide. Moreover, the mutant HrcA C118S exhibited reduced binding to CIRCE elements and became less oligomerized, suggesting that cysteine residue 118 is important for CIRCE element binding as well as oligomerization. Conversely, HrcA mutant C280S exhibited increased oligomerization. An HrcA double mutant (C118S, C280S) was monomeric and exhibited a level of oligomerization and CIRCE binding similar to wild type HrcA, suggesting that cysteine residues 118 and 280 may function as checks to one another during oligomer formation. Biochemical fractionation of E. coli cells overexpressing HrcA revealed the presence of HrcA in the membrane fraction. Together, these results suggest that the two HrcA cysteine residues at positions 118 and 280 function as reduction sensors in the membrane and mediate oligomerization upon stress.

• 한국미생물·생명공학회지
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• 제40권2호
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• pp.92-97
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• 2012

항균 펩타이드(Antimicrobial peptides(AMPs)는 그람 양성, 그람 음성 세균과 진균병원체에 대항하는 생명체에서 중요한 역할을 하는 물질이다. 인간의 Cathelicidin 항균 펩타이드는 임상학적으로 사용할 수 있는 여러 가지의 생물학적 활성을 가진다. 항균 펩타이드의 생산 비용은 재조합 방법으로 낮출 수가 있다. 대장균은 저렴하며 손쉬운 조작이 가능하기 때문에 다른 재조합 단백질처럼 항균 펩타이드의 발현에 훌륭한 숙주가 될 수 있다. 그러나 대장균에서의 항균 펩타이드의 과발현은 항균 펩타이드가 과발현 되었을 때 대장균에 독성을 보일 수 있으므로 어려움이 보고가 되어있다. 본 연구에서는 이러한 문제점을 극복하고자 항균 펩타이드를 Glutathione S-transferase(GST) 결합 단백질에 융합하여 항균펩타이드의 독성을 감소시키도록 설계하여 발현을 시도하였다. 이 때 발현한 항균 펩티드는 LL37과, gaegurin4과 LL37의 잡종 펩타이드 GGN4-LL37(GL32로 명명)를 GST에 융합되도록 벡터를 구축하고 설계하여 대장균에서 GST 융합단백질로 발현시켰다. 융합 단백질은 친화력 컬럼을 사용하여 분리하고 GST를 절단하여 항균펩타이드 만을 분리하였고 분리한 펩타이드는 웨스턴 블롯팅으로 확인하였고 그람 양성, 그람 음성 세균에 대하여 항균 활성을 나타내는 것을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.836-843
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• 2004

Nontypeable H. influenzae (NTHi), a Gram-negative obligate human pathogen, causes pneumonia, chronic bronchitis, and otitis media, and the respiratory epithelium is the first line of defense that copes with the pathogen. In an effort to identify transcriptional responses of human respiratory epithelial cells to infection with NTHi, we examined its differential gene expression using high density cDNA microarrays. BEAS-2B human bronchial epithelial cells were exposed to NTHi for 3 hand 24 h, and the alteration of mRNA expression was analyzed using microarrays consisting of 8,170 human cDNA clones. The results indicated that approximately 2.6% of the genes present on the microarrays increased in expression over 2-fold and 3.8% of the genes decreased during the 24-h infection period. Upregulated genes included cytokines (granulocyte-macrophage colony stimulating factor 2, granulocyte chemotactic protein 2, IL-6, IL-10, IL-8), transcription factors (Kruppel-like factor 7, CCAAT/enhancer binding protein $\beta$, E2F-1, NF-$\kappa$B, cell surface molecules (CD74, ICAM-1, ICAM-2, HLA class I), as well as those involved in signal transduction and cellular transport. Selected genes were further confirmed by reverse-transcription-PCR. These data expand our knowledge of host cellular responses during NTHi infection and should provide a molecular basis for the study of host-NTHi interaction.

• 한국수산과학회지
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• 제54권6호
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• pp.927-937
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• 2021

This study was performed to screen the antimicrobial activities of the extract from the Pacific oyster Crassostrea gigas against skin pathogens and to purify the relevant antibacterial peptide. The acidified extract showed potent antibacterial activities against gram-positive and gram-negative bacteria but showed no activity against Candida albicans and no significant cell toxicity. Among acne-causing pathogens, the acidified extract showed potent antibacterial activity only against Staphylococcus aureus, and its antibacterial activity was completely abolished by treatment with trypsin or chymotrypsin, and was inhibited by salt treatment. The acidified extract showed strong DNA-binding ability but did not show bacterial membrane permeabilizing ability. Based on antimicrobial activity screening and cytotoxic effects, a novel antibacterial peptide was purified from the acidified gill extract using solid-phase extraction, cation-exchange, and reversed-phase HPLC. The resulting peptide had a molecular weight of 4800.8 Da and showed partial sequence homology with the carbonic anhydrase 4 (CA4) protein in the hard-shelled mussel. Overall, we purified a novel antibacterial peptide, named cgCAFLP, which is related to carbonic anhydrase 4 (CA4) protein, against skin pathogens. Our results suggest that the Pacific oyster extract could be used as an additive to control some acne-related skin pathogens (S. aureus).

• Journal of Plant Biotechnology
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• 제36권3호
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• pp.268-274
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• 2009

Porphyromonas gingivalis, the gram-negative anaerobic oral bacterium, initiates periodontal disease by binding to saliva-coated oral surface. The cholera toxin B subunit (CTB) genetically linked to FimA1 (1-200 aa) or FimA2 (201-337 aa) of the P. gingivalis fimbrial antigen were introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation method. The integration of CTB-FimA1 or CTB-FimA2 fusion genes were confirmed in the chromosome of transformed leaves by genomic DNA PCR amplification method. Synthesis and assembly of the CTB-FimA fusion proteins into oligomeric structures with pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding activities of CTB-FimA fusion proteins to intestinal epithelial cell membrane receptors were confirmed by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). The ELISA showed that the expression levels of the CTB-FimA1 or CTB-FimA2 fusion proteins were 0.0019, 0.002% of the total soluble protein in transgenic tuber tissues, respectively The synthesis of CTB-FimA monomers and their assembly into biologically active oligomers in transformed potato tuber tissues demonstrates the feasibility of using edible plants for the production of enterocyte targeted fimbrial antigens that could elicit mucosal immune responses.

• Molecules and Cells
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• 제38권2호
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• pp.180-186
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• 2015

Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation of how the three components assemble in the functional state. Here, we created fusion proteins composed of AcrB, a transmembrane linker, and two copies of AcrA. The fusion protein exhibited acridine pumping activity, suggesting that the protein reflects the functional structure in vivo. To discern the assembling mode with TolC, the AcrBA fusion protein was incubated with TolC or a chimeric protein containing the TolC aperture tip region. Three-dimensional structures of the complex proteins were determined through transmission electron microscopy. The overall structure exemplifies the adaptor bridging model, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel interaction with the ${\alpha}$-barrel tip region of TolC, and a direct interaction between AcrB and TolC is not allowed. These observations provide a structural blueprint for understanding multidrug resistance in pathogenic Gram-negative bacteria.

• International Journal of Oral Biology
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• 제47권1호
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• pp.1-8
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• 2022

Inflammation is a protective mechanism against pathogens, but if maintained continuously, it destroys tissue structures. Aggregatibacter actinomycetemcomitans is a gram-negative, facultative anaerobic bacterium often found in severe periodontitis. A. actinomycetemcomitans invades epithelial cells and triggers inflammatory response in the immune cells. In this study, we investigated the effect of water-soluble rosehip extract on A. actinomycetemcomitans-induced inflammatory responses. A human monocytic cell line (THP-1) was differentiated to macrophages by phorbol 12-mystristate 13-acetate treatment. The cytotoxic effect of extract was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effects of extract on bacterial growth were examined by measuring the optical densities using a spectrophotometer. THP-1-derived macrophages were infected A. actinomycetemcomitans after extract treatment, and culture supernatants were analyzed for cytokine production using enzyme-linked immunosorbent assay. Protein expression was measured by western blotting. Extract was not toxic to THP-1-derived macrophages. A. actinomycetemcomitans growth was inhibited by 1% extract. The extract suppressed A. actinomycetemcomitans-induced tumor necrosis factor-α, interleukin (IL)-1β, and IL-8 production. It also decreased mitogen-activated protein kinase (MAP kinase) and nuclear factor-κB (NF-κB) phosphorylation. Moreover, the extract inhibited the expression of inflammasome components, including nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3, Absent in Melanoma 2, and apoptosis associated speck-like protein containing a CARD. And cysteine-aspartic proteases-1 and IL-1β expression were decreased by the extract. In summary, extract suppressed A. actinomycetemcomitans growth and decreased inflammatory cytokine production by inhibiting activation of MAP kinase, NF-κB, and inflammasome signaling. Rosehip extract could be effective in the treatment of periodontal inflammation induced by A. actinomycetemcomitans infection.

• 약학회지
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• 제40권1호
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• pp.102-111
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• 1996

LB10522 is a new parenteral broad spectrum cephalosporin with a catechol moiety at C-7 position of beta-lactam ring. This compound can utilize tonB-dependent iron transp ort system in addition to porin proteins to enter bacterial periplasmic space and access to penicillin-binding proteins (PBPs) which are the lethal targets of ${\beta}$-lactam antibiotics. The chelating activity of LB10522 to metal iron was measured by spectrophotometrically scanning the absorbance from 200 to 900nm. When $FeCl_3$ was added, optical density was increased between 450 and 800nm. LB10522 was more active against gram-negative strains in iron-depleted media than in iron-replete media. This is due to the increased expression of iron transport channels in iron-depleted condition. LB10522 showed a similar activity against E. coli DC2 (permeability mutant) and E. coli DCO (wild type strain) in both iron-depleted and iron-replete media, indicating a minimal permeaility barrier for LB10522 uptake. LB10522 had high affinities to PBP 3 and PBP 1A, 1B of E. coli. By blocking these proteins, LB10522 caused inhibition of cell division and the eventual death of cells. This result was correlated well with the morphological changes in E. coli exposed to LB10522. Although the in vitro MIC of LB10522 against P. aeruginosa 1912E mutant (tonB) was 8-times higher than that of the P. aeruginosa 1912E parent strain, LB10522 showed a similar in vivo protection efficacy against both strains in the mouse systemic infection model. This result suggested that tonB mutant, which requires a high level of iron for normal growth, might have a difficulty in surviving in their host with an iron-limited environment.

• Asian-Australasian Journal of Animal Sciences
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• 제21권8호
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• pp.1080-1087
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• 2008

Lipopolysaccharide binding protein (LBP) and CD14 protein play important roles in the defense against infection of Gram-negative bacteria. In the present study, tissue distribution and polymorphism of porcine LBP and CD14 genes were analyzed. Real-time PCR results showed that the porcine LBP gene was especially highly expressed in liver, while CD14 gene was highly expressed in liver and spleen tissues. A 1,732 bp cDNA fragment of porcine LBP gene and a 1,682 bp genomic DNA fragment of CD14 gene were isolated. Polymorphisms were identified in these two fragments and showed that there were 14 potential SNPs in the porcine LBP gene and 3 potential SNPs in the porcine CD14 gene. Three SNPs, 292G/A (Gly/Ser), 1168G/A (Ala/Thr) of the LBP gene and -61G/A of the CD14 gene, were genotyped using restriction fragment length polymorphism (RFLP) method. Association analyses indicated that polymorphism of the 292G/A locus was significantly associated with porcine immune traits hematocrit (HCT), IgG and delayed-type hypersensitivity (DTH) (p<0.01), and the 1168G/A locus was significantly associated with HCT and mean corpuscular volume (MCV) traits (p<0.05). No significant association was found between the -61G/A locus and immune traits of the pig. Our data indicated that the LBP gene was significantly associated with immune traits of pig. Also, we identified some SNPs which may be useful markers for disease-resistant breeding of pigs.