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 Original Articles |
| Unique Mutation in Pneumolisin (ply) Gene in Streptococcus pneumoniae with Decreased Hemolytic Activity |
| Won Sup Oh1, Hyun Ha Chang1, Sook-In Jung1 , Yeon-Sook Kim1, Hyuck Lee2, Shin Woo Kim3, Sungmin Kim1, Kyong Ran Peck1, Nam Yong Lee4 and Jae-Hoon Song1.5 |
| Division of Infectious Diseases1 and Department of Clinical Pathology4, Samsung Medical Center, Sungkyunkwan University School of Medicine ; Division of Infectious Disease2, Dong-A University College of Medicine ; Division of Infectious Diseases, Kyungbuk University School of Medicine ; and the Asian-Pacific Research Foundation for Infectious Diseases5 |
Vol.20 Num.3 (p233~241)
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Background : Pneumolysin (Ply) is one of the most important virulence factors of Streptococcus pneumoniae. To investigate the functional and structural variation of Ply, hemolytic activity (HA) and gene sequences of pneumolysin were determined.
Methods : A total of 40 invasive pneumococcal isolates (20 penicillin-susceptible & 20 -resistant strains) were analyzed. HA of each isolate was measured in crude extracts (1x108 CFU/mL hourly from the time of inoculation to 9h. Crude extracts (50 μL) and DTT buffer (100μL) were serially diluted in a 96-well plate and mixed with 1% sheep blood (50μL). HA was represented as the reciprocal of the greatest dilution, which resulted in the complete lysis. Two sets of oligonucleotide primers were used to amplify a 1,474-bp fragment containing the coding region and a 578-bp fragment of upstream region of ply gene, respectively. Amplified products were directly sequenced.
Results : HA showed a wide variation from 0 to 87,480 regardless of penicillin MICs, serotypes or specimen sources. Two bacteremic strains showed completely no hemolytic activity. No significant differences in HA between penicillin-susceptible (Mean±SE: 15,468±4,693) and penicillin-resistant strains (21,384±4,087) were found (p > 0.05). Sequencing of the coding region showed unique alterations in amino acids in strains with markedly reduced hemolytic activity (≤ 40): Thr-172→lie and Lys-224→Arg. However, upstream region of ply gene was highly conserved.
Conclusions : Ply activity was not related with antibiotic resistance. Absence of HA in some strains was associated with unique alterations in amino acid sequences in ply gene. Relationnship between genomic alteration and functional changes in pneumolysin should be further investigated. |
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Keywords : Pneumolysin, Strepococcus pneumoniae, Virulence, Hemolytic activity, DNA sequence |
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