Virulence properties of a peptide hemolysin produced by &amp;lt;i&amp;gt;Enterococcus faecalis&amp;lt;/i&amp;gt;

M T Furumura,Valério Monteiro Neto,P M S Figueirêdo,Rosimary De Jesus Gomes Turri,G V Carbonell,Francyelle Costa Moraes,Hermínio Benitez Rabello Mendes,Maria Rosa Quaresma Bomfim,A L Darini,T Yano,Cristina Monteiro De Andrade

doi:10.4236/abb.2012.37112

Virulence properties of a peptide hemolysin produced by &lt;i&gt;Enterococcus faecalis&lt;/i&gt;

M T Furumura, Valério Monteiro Neto + Show 9 more

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https://doi.org/10.4236/abb.2012.37112

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Abstract

The characterization and prevalence of virulence factors associated with enterococcal invasiveness and severity of disease are important areas to be investigated. Recently, we described the production of a heat-stable hemolysin by clinical isolates of Enterococcus faecalis cultived in BHI-GA (BHI with glucose and L-arginine). Now, we purified the hemolysin from the culture supernatant by ultra-filtration (PM-10 membrane) and ethanol extraction followed by chromatography in a mBondapak C18 and Superdex Peptide columns. The hemolytic activity was not affected by the proteolytic enzymes. Cholesterol, phospholipids, EDTA and also bivalent ions did not inhibit the hemolytic activity. Among the various carbohydrates, only dextran 4 protected the erythrocytes against lyse. Scanning electron microscopy showed that lyse of erythrocytes occured at once after the exposure to the hemolysin. The mito-chondrial activity and the cell membrane integrity were significantly affected by the hemolysis, within 20 min of exposure and caused apoptosis after 12 h incubation, 51.92% in HeLa and 68% in HEp-2 cells, analyzed by flow cytometry. These results suggest that the heat-stable pore forming hemolysin might be a putative virulence factor in enterococci infections.

Highlights

IntroductionEnterococcus faecalis is an important nosocomial pathogen that causes urinary tract infections, bacteremia and endocarditis [1].Due to the importance of the Enterococcus species as nosocomial pathogens and the increasing prevalence of antimicrobial resistance among enterococci, the identification and characterization of virulence factors associated with enterococcal invasiveness and severity of disease are important areas to be investigated [2].Enterococcal virulence factors such as hemolysin (Hly), aggregation substance (Agg or AS) and an enterococcal protease commonly referred to as gelatinase (Gel) have been suggested as possible contributors to the virulence of E. faecalis; the mechanisms involved remain unclear [2,3].Hemolysin is commonly produced by Gram-positive and Gram-negative bacteria and, in most cases, is considered to be a virulence factor, its relative contribution to disease is variable among microbes and different host species [4]
The hemolytic activity was eluted as a sharp peak before the linear gradient of acetonitrile 66%, showing a hydrophilic character (Figure 1(a))
The hemolytic activity peak was concentrated by speed vac (Savant) and applied on a Superdex Peptide (GE Healthcare) column chromatography and the hemolytic activity was eluted at 47.95 min at a flow rate of 0.5 mL/min (Figure 1(b))

Summary

Introduction

Enterococcus faecalis is an important nosocomial pathogen that causes urinary tract infections, bacteremia and endocarditis [1].Due to the importance of the Enterococcus species as nosocomial pathogens and the increasing prevalence of antimicrobial resistance among enterococci, the identification and characterization of virulence factors associated with enterococcal invasiveness and severity of disease are important areas to be investigated [2].Enterococcal virulence factors such as hemolysin (Hly), aggregation substance (Agg or AS) and an enterococcal protease commonly referred to as gelatinase (Gel) have been suggested as possible contributors to the virulence of E. faecalis; the mechanisms involved remain unclear [2,3].Hemolysin is commonly produced by Gram-positive and Gram-negative bacteria and, in most cases, is considered to be a virulence factor, its relative contribution to disease is variable among microbes and different host species [4]. Due to the importance of the Enterococcus species as nosocomial pathogens and the increasing prevalence of antimicrobial resistance among enterococci, the identification and characterization of virulence factors associated with enterococcal invasiveness and severity of disease are important areas to be investigated [2]. Enterococcal virulence factors such as hemolysin (Hly), aggregation substance (Agg or AS) and an enterococcal protease commonly referred to as gelatinase (Gel) have been suggested as possible contributors to the virulence of E. faecalis; the mechanisms involved remain unclear [2,3]. The cytolysin lyses mouse erythrocytes, macrophages and polymorphonuclear neutrophils [7], which may enable the infecting bacteria to obtain nutrients and to escape from immune clearance [8]

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