In vitro activation of the hemolysin in Prevotella nigrescens ATCC 33563 and Prevotella intermedia ATCC 25611

Laboratório de Microbiologia Oral e Anaeróbios, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627 Pampulha, CEP 31 270-901, Belo Horizonte, MG, Brazil.
Research in Microbiology (Impact Factor: 2.71). 01/2004; 155(1):31-8. DOI: 10.1016/j.resmic.2003.09.010
Source: PubMed

ABSTRACT Hemolytic activity was evaluated in the putative periodontopathogens Prevotella intermedia and Prevotella nigrescens. Whole cells of both species present weak hemolytic activity evidenced only by solid media assays after 48 h of bacterial growth or after 5 h of interaction with erythrocytes at 37 degrees C in liquid assays. In this work we show that the use of crude extract allowed the detection of a higher hemolytic activity for P. intermedia, but surprisingly not for P. nigrescens. Incubation at 37 degrees C for 9 h, or treatment with trypsin or proteinase K, increased or exposed the hemolytic activity of P. intermedia and P. nigrescens crude extract, respectively. The activation process was inhibited by TLCK and PMSF but not by EDTA, E-64 or pepstatin A, indicating the serino-protease nature of the factor involved in activation of P. intermedia and P. nigrescens hemolysins. Both the buffer and the pH employed for cell fractionation influenced the activation of hemolysin, and the best results were obtained with Universal buffer at pH 8.0. The activated hemolysins acted optimally at pH 6.5 at 37 degrees C and the maximum hemolytic activity was detected at the early log phase of growth. The results of this study show for the first time a strong hemolytic activity for P. nigrescens and evidence of proteolytic activation of hemolysins produced by periodontopathogens.

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    • "The culture supernatant obtained from the early logarithmic growth phase had a much higher hemolytic activity, compared to the low activity of the culture supernatant obtained from the stationary phase. A similar pattern of hemolysin production has been reported for P. intermedia [22], P. nigrescens [25], Porphyromonas gingivalis [20], and Aggregatibacter actinomycetemcomitans [28]. However, the underlying reason for this phenomenon remains unknown. "
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    ABSTRACT: We observed hemolytic activity in culture supernatant of Prevotella oris. Results from growth-phase experiments show that hemolysin production increased during the logarithmic growth phase and decreased during the stationary phase. The hemolysin produced by P. oris was purified from the culture supernatant by ultrafiltration, diethylaminoethyl (DEAE) and carboxymethyl (CM) ion-exchange chromatography, and gel filtration chromatography; further, we investigated the purified hemolysin characteristics, including its ability to lyse human, horse, sheep, and rabbit erythrocytes. The purified hemolysin was observed as a single, 16-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The specific activity was obtained by concentrating the purified hemolysin by 9200 fold. Although hemolysin was inactivated by heat treatment, ethylenediaminetetraacetic acid (EDTA), l-cysteine, dithiothreitol (DTT), and 2-mercaptoethanol enhanced its activity. Further, treatments using trypsin, MgCl2, CaCl2, and cholesterol did not affect its hemolytic activity. A pH of 6.0 was optimal for inducing the hemolysin activity. To the best of our knowledge, this is the first report describing the purification and characterization of hemolysin produced by P. oris.
    Journal of Oral Biosciences 05/2012; 54(2):113–118. DOI:10.1016/j.job.2012.03.002
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    • "P. nigrescens produces protease that can degrade the host immunoglobin G [7], and transferrin-binding protein, which can inhibit the uptake of iron by host cells [4]. In addition, although the hemolysin of P. nigrescens generally does not have hemolytic activity in vivo [12], it can have hemolytic activity in terms of the treatment of trypsin or proteinase K in vitro [15]. This suggests that the non-active hemolysin of P. nigrescens can be transformed to the active form by degradation via the trypsin-like protease produced by oral bacteria, such as Porphyromonas gingivalis in peripical and periodontitis lesions. "
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    ABSTRACT: A previous study reported the cloning of a putative Prevotella nigrescens-specific DNA probe, Pn23, using random shotgun method. The present study evaluated the species-specificity of Pn23 for P. nigrescens using the clinical strains of Prevotella intermedia and P. nigrescens to develop P. nigrescens-specific polymerase chain reaction (PCR) primers. Southern blot analysis showed that the DNA probe, Pn23, detected only the genomic DNA of P. nigrescens strains. PCR showed that the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, had species-specificity for P. nigrescens. Interestingly, the two sets of PCR primers, Pn23-F6/Pn23-R6 and Pn23-F7/Pn23-R7, had strain-specificity for P. nigrescens ATCC 33563. The detection limits of the four primer sets were 40 or 4 pg of the purified genomic DNA of P. nigrescens ATCC 33563. These results suggest that the DNA probe, Pn23, and the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, can be useful for the detection of P. nigrescens in the molecular epidemiological studies of oral infectious diseases.
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