• The Journal of Natural Sciences
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• v.14 no.1
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• pp.7-24
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• 2004

We found new type of thiol peroxidase, fused with GRX.(TPx-GRX) The TPx-GRX exists in pathogenic bacteria including -. This protein was homogeneously purified from the E.coli recombinant overexpressing TPx-GRX. In the presence of a thiol-containing electron donor such as DTT, the purified TPx-GRX has potent the antioxidant to prevent the inactivation of GS by the MCO system, which is comprised of DTT, $Fe^{3+}$, and $O^2$. The antioxidant activity is much higher that other thiol peroxidase. The investigate the peroxidase activity of TPx-GRX, we directly measured the peroxidase activity of TPx-GRX toward peroxides in terms of the removal of peroxides in the presence of GSH. This result demonstrates that the peroxidase activity of TPx-GRX. These taken together results suggest that TPx-GRX is a new member of thiol peroxidase. These observations also suggest that in the pathogenic bacteria, TPx-GRX plays an important antioxidative role as a multiple array defence mechanism against oxidative stress.

• Clinical and Experimental Pediatrics
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• v.50 no.2
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• pp.143-150
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• 2007

Purpose : This study was conducted to validate enzyme immunoassay (EIA) for the quantitative measurement of human IgG antibodies specific for Haemophilus influenzae type b (Hib) capsular polysaccharide. Method : We evaluated specificity, repeatability, intermediate precision, accuracy, lower limit of quantification (LLOQ), and stability to validate standardized EIA for the quantitative measurement of human anti-polyribosylribitol phosphate (PRP) IgG antibodies. Results : The results indicated that this EIA showed specificity to HbO-HA antigen and repeatability and intermediate precision were within acceptance criteria (repeatability: $CV{\leq}15%$, intermediate precision: $CV{\leq}20%$). The EIA-derived results from this laboratory were equivalent to those obtained by the standard radioactive antigen binding assay (RABA) for quantitation of anti-PRP antibodies in the 28 sera. Spiking recovery result was within acceptance criteria ($100{\pm}20%$). The precision and accuracy of samples in LLOQ were from -14.7 to -4.7% in nominal values, which were within acceptance criteria (precision: $CV{\leq}25%$, accuracy: ${\pm}25%$). Freeze-thaw stability and short term temperature stability were within ${\pm}20%$ of acceptance criteria. Conclusions : The EIA which is performed at the Center for Vaccine Evaluation and Study Ewha Medical Research Institute, is an appropriate serologic assay which can be used for quantitation of anti-PRP IgG antibodies in human sera.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.103-110
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• 2003

The regulation of pyrimidine nucleotide synthesis has been proved to be controlled by a regulatory protein PyrR-mediated attenuation in the Gram-positive bacteria. After several bacterial genome sequencing projects, we have discovered the PyrR orthologues in the databases for Haemophilus influenzae and Synechocystis and sp. PCC6803 genome sequences. To investigate whether these PyrR orthologue proteins regulate pyrimidine nucleotide synthesis as well as the cases of Bacillus, the PyrR regions of each strains were amplified by PCR and cloned with pUC19 or T-vector in Escherichia coli and with a shuttle vector pHPS9 for E. coli and B. subtilis. For the regulation test of the PyrR orthologues, the aspartate-transcarbamylase (ATCase) assay was carried out. From the results of the ATCase assay, it was confirmed that Synechocystis sp. PCC6803 could not restore by pyrimidines to a B. subtilis, PyrR but H. influenzae PyrR could. For Purification of PyrR orthologue proteins, PyrR orthologue genes were cloned into the expression vector (pET14b). Over-expressed product of PyrR orthologue genes was purified and analyzed by the SDS-PACE. The purified PyrR orthologue proteins from H. influenzae and Synechocystis sp. PCC6803 turned out to be molecular mass of 18 kDa and 21 kDa, respectively. The result of uracil phosphoribosyl transferase (UPRTase) assay with purified PyrR orthologue proteins showed that H. influenzae PyrR protein only has UPRTase activity. In addition, we could predict several regulatory mechanisms that PyrR orthologue proteins regulate pyrimidine de novo synthesis in bacteria, through phylogenetic analysis for PyrR orthologue protein sequences.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.147-148
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• 2002

Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis are encapsulated bacteria which encounter the respiratory mucosa and cause nasopharyngeal carriage that may lead to mild mucosal infections or severe invasive disease. (omitted)

• Journal of Microbiology and Biotechnology
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• v.13 no.3
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• pp.469-472
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• 2003

A method that effectively precipitates capsular polysaccharide of Haemophilus influenzae type b (polyribosylribitol phosphate, PRP) conjugated to tetanus toxoid (TT), PRP TT in a liquid vaccine has been developed to measure free PRP present in TT-conjugate vaccine. The method involves adding anti-TT antibody and ammonium sulfate to precipitate PRP-TT conjugate and measuring free PRP in tile supernatant. This new method provides a complete precipitation of the total PRP-TT, and provides an accurate and reproducible measurements of free PRP. The accuracy of the assay was confirmed by spiking known amounts of unconjugated PRP to PRP-TT conjugate, and the new method was found to have no effect on free PRP while precipitating PRP-TT. The published acid precipitation method did not produce reproducible results due to incomplete precipitation of PRP-TT, especially when the vaccine is formulated in a salt-buffered solution.

• Pediatric Infection and Vaccine
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• v.24 no.3
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• pp.125-133
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• 2017

Purpose: After the introduction of Haemophilus influenzae type b (Hib) vaccine in 1995 in Korea, it was included in the national immunization program in 2013. In the post-Hib vaccine era, some studies in other countries reported that invasive Hib disease affects adults, especially the elderly and immunocompromised persons, more often than it affects children. To evaluate disease susceptibility, quantitative and qualitative analysis of anti-polyribosylribitol phosphate (PRP) antibodies were carried out in Korean adults aged 20 to 85 years. Methods: Sera were collected from 39 healthy adults (20 to 50 years of age) and from 30 elderly adults (75 to 85 years of age) who did not have immune-compromising conditions. The concentration of anti-PRP immunoglobulin G (IgG) and serum bactericidal indices (SBIs) were measured by enzyme-linked immunosorbent assay and serum bactericidal assay. Results: Geometric mean concentrations of anti-PRP IgG and geometric mean SBIs were $0.88{\mu}g/mL$ (95% confidence interval [CI], 0.17 to 3.85) and 354 (95% CI, 50 to 2,499) in young adults and $1.67{\mu}g/mL$ (95% CI, 0.53 to 5.24) and 449 (95% CI, 146 to 1,376) in elderly adults, respectively. When the threshold of seropositivity for anti-PRP IgG was applied as 0.15 or $1.0{\mu}g/mL$, which is the protective antibody level in children, seropositive rates were 87.2% or 53.8% in young adults and 100% or 60% in elderly adults. The seropositivity rates of the SBI ($SBI{\geq}4$) were 82.1% and 100% in the groups, respectively. Conclusions: Most subjects in the adult and elderly adult groups display immunity to Hib based on quantitative and qualitative antibody levels, but not all. Because high immunization and low Hib circulation rates may reduce the natural Hib immunity in the population, monitoring Hib immunity as well as disease are needed continuously.

• Bulletin of the Korean Chemical Society
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• v.32 no.8
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• pp.2549-2552
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• 2011

In this study, the kinetic properties of wild-type and C117D mutant H. influenzae MurA (Hi MurA), which catalyzes the first reaction in the biosynthetic pathway of the cell wall, were characterized. Purified recombinant Hi MurA was active at pH values ranging from pH 5.5 to pH 10, and its $K_m$ (UNAG), $K_m$ (PEP), and $k_{cat}$ values were measured to be 31 ${\mu}M$, 24 ${\mu}M$, and 210 $min^{-1}$, respectively. Hi MurA activity was effectively inhibited by fosfomycin with an $IC_{50}$ value of 60 ${\mu}M$. Hi MurA contains a cysteine residue (C117) at the loop region near the PEP binding, whereas MurA from fosfomycin resistant Mycobaterium tuberculosis or Chlamydia trachomatis contain an aspartate residue instead of the cysteine at the corresponding site. Aspartate substitution of Cys117 in Hi MurA shifted its optimum pH from 7.8 to 6.0. In addition, the $K_m$ values for UNAG and PEP were increased to 160 ${\mu}M$ and 150 ${\mu}M$, respectively, and the $k_{cat}$ value was significantly reduced to 41 $min^{-1}$. Furthermore, the C117D mutant form of Hi MurA was not inhibited by 1 mM fosfomycin. These results indicate that the Cys117 of Hi MurA is the binding site of fosfomycin and plays an important role in the fast turnover of the catalytic reaction.

• Journal of Microbiology and Biotechnology
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• v.28 no.10
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• pp.1730-1735
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• 2018

Bacterial pneumonia is one of the most common causes of mortality in Korea. In 2016, the mortality rate from pneumonia was 16,476 deaths per 100,000, which was an 11% increase from the previous year. The aim of our study was to determine the distribution of the bacterial pathogens causing respiratory symptoms in different age groups over a 10-year period. Between January 2008 and September 2017, 1,861 specimens from 1,664 patients admitted to Dankook University Hospital with respiratory symptoms were examined. We used multiplex polymerase chain reaction (PCR) to detect six bacterial pneumonia pathogens: Bordetella pertussis, Chlamydophila pneumoniae, Haemophilus influenzae, Legionella pneumophila, Mycoplasma pneumoniae, and Streptococcus pneumoniae. We detected bacterial pneumonia pathogens in 1,281 (68.83%) specimens. Of the 1,709 pathogens detected, S. pneumoniae was the most common (48.57%; n = 830) followed by H. influenzae (40.08%; n = 685). Most infections were found among children younger than 10 years (92.69%; n = 1,584). Although S. pneumoniae was the most common pathogen detected in all age groups, M. pneumoniae infection increased in prevalence with age (p < 0.05). The rate of co-infection was also high among these patients (31.1%; n = 399), which peaked in 2015 (54.55%; n = 42/77). The prevalence of bacterial pneumonia in Cheonan, along with the proportion of co-infections among patients increased over the 10-year study period. The findings will aid the development of treatment and prevention guidelines.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.11a
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• pp.65-73
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• 1996

LPS/LOS, the compound found only in gram-negative bacterial outer membrane, plays important roles in bacterial maintenance as well as its pathogenesis. We isolated and characterized several genes required for NTHi 2019 LOS biosynthesis, which encode enzymes required for sugar substrate synthesis or the transfer of substrates to receptor molecules. The htrB gene, however, appears to have more complex role. It has acryltransferase activity as well as various other activity, which may control regulation of LOS biosynthesis as well as its pathogenicity. Evidences supporting the latter come from the observations that the lipid A of the B29 induced significantly less TNF ${\alpha}$ from macrophages than that of the wild type LOS (unpublished data). H. influenzae A2-htrB mutant strain was also significantly less invasive than the wild type strain. The structural similarities of the enterobacterial LPS and the Haemophilus LOS enabled us to isolate the NTHi 2019 genes involved in LOS biosynthesis genes by using the S. typhimurium LPS deep core mutants. While a similar approach has been used for E. coli, this technique for selection of an LPS phenotype has not been applied to nonenterobacterial species. The difficulties inherent in the molecular manipulation of organism such as Neisseria and Haemophilus species make this approach particularly attractive in the identification and cloning LOS genes. Studies on genetic features of LPS/LOS biosynthesis would be useful for understanding bacterial pathogenesis as well as for developing vaccines for these gram-negative pathogenic bacteria.

• Pediatric Infection and Vaccine
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• v.7 no.2
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• pp.233-239
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• 2000

Purpose : A great deal of youngsters suffer from otitis media, for which antimicrobials are frequently prescribed. Increased antimicrobial resistance forces physicians to judiciously use antimicrobial agents in treating patients with acute otitis media. There have however been few references with regard to otitis media in Korean children, and authors proceeded investigation to look for the causative agents of otitis media in Korean children and their antimicrobial susceptibility. Methods : The study included 65 patients younger than 15 years old who had been cared at the department of pediatrics and otolaryngology in Hanyang University Hospital from July 1994 to June 1999, and diagnosed of otitis media with otorrhea which contained microorganisms isolated in otorrhea culture. The medical records were reviewed for demographic data, isolated organisms and their antimicrobial susceptibility. Results : Among 65 patients, 37(57%) were boys and 28(43%) girls. Distribution of the patients was reciprocal to the age of the patients; 27 patients(41.5%) were younger than 1 year old, 24(36.9%) were 1 to 3 years old with the average of 2.9 years of age. Staphylococus aureus was isolated in 32 patients(49.2%), Streptococcus pneumoniae in 19 patients(29.2%) Haemophilus influenzae in 9 patients(13.8%), Streptococcus oralis in 3 patients(4.6%), Moraxella catarrhalis in 1 patient(1.5%). The isolated microorganisms were not different whether patients had cleft lip/palate or not. The antibiotic resistance rates of S. aureus were ${\geq}90%$ to erythromycin, imipenem, cephalothin, and clindamycin, 86.2% to oxacillin, 25% to chloramphenicol, 12.5% to trimethoprim/sulfamethoxazole(TMP/SMX), and 0% to vancomycin and teicoplanin. The antibiotic resistance rates of S. pneumoniae were 71.4% to penicillin and greater than 60% to erythromycin, tetracycline, TMP/SMX, 7.1% to chloramphenicol, and 0% to vancomycin and teicoplanin. The antibiotic resistance rates of H. influenzae were 55% to ampicillin and TMP/SMX, and 0% to chloramphenicol, ceftriaxone, aztreonam, imipenem and ciprofloxacin. Conclusion : With otorrhea culture, the causative organisms of otitis media appear to be S. aureus, S. pneumoniae and H. influenzae. The high antibiotic resistance rates of the isolated organisms should affect the choice of antibiotics in treating patients with otitis media. Prospective investigations utilizing tympanocentesis in microbiologic studies are needed.