Staphylococcus aureus Alpha-Toxin in Deep Tracheal Aspirates-Preliminary Evidence for Its Presence in the Lungs of Sepsis Patients

Abstract

The pore forming alpha-toxin (hemolysin A, Hla) of Staphylococcus aureus (S. aureus) is a major virulence factor with relevance for the pathogenicity of this bacterium, which is involved in many cases of pneumonia and sepsis in humans. Until now, the presence of Hla in the body fluids of potentially infected humans could only be shown indirectly, e.g., by the presence of antibodies against Hla in serum samples or by hemolysis testing on blood agar plates of bacterial culture supernatants of the clinical isolates. In addition, nothing was known about the concentrations of Hla actually reached in the body fluids of the infected hosts. Western blot analyses on 36 samples of deep tracheal aspirates (DTA) isolated from 22 hospitalized sepsis patients using primary antibodies against different epitopes of the Hla molecule resulted in the identification of six samples from five patients containing monomeric Hla (approx. 33 kDa). Two of these samples showed also signals at the molecular mass of heptameric Hla (232 kDa). Semiquantitative analyses of the samples revealed that the concentrations of monomeric Hla ranged from 16 to 3200 ng/mL. This is, to our knowledge, the first study directly showing the presence of S. aureus Hla in samples of airway surface liquid in human patients.

Keywords: Staphylococcus aureus; alpha-toxin; deep tracheal aspirate; sepsis patients.

Figure 1

Detection and semi-quantification of Hla in DTA samples from patients (P) with sepsis by semiquantitative Western blotting. Shown is an example blot that was incubated with the anti-Hla polyclonal antibody from Sigma (image on the left) and, upon stripping, with the anti-Hla monoclonal antibody from Abcam (image on the right). Five lanes of this blot were loaded with different amounts (1 to 20 ng) of recombinant Hla (rHla) to compare their signal intensities with those of the DTA samples for semi-quantification of the Hla content of each sample. Note that the Sigma antibody is slightly more sensitive compared with the Abcam antibody (different signal intensities in the rHla lanes). m_Hla—monomeric Hla; h_Hla—heptameric Hla.

Figure 2

Detection of monomeric (m_Hla) as well as heptameric Hla (h_Hla) in DTA samples from patients with sepsis by Western blotting. Shown is an example blot that was incubated with the anti-Hla polyclonal antibody from Sigma. Five lanes of this blot were loaded with different amounts (0.05 to 5 ng) of rHla to compare their signal intensities with those of the DTA sample of Patient 21 (P21) for semi-quantification of the monomeric Hla content of each sample. The presence of a band at approximately 230 kDa indicates that the sample of Patient 21 contains heptameric Hla in addition to the monomeric form of the toxin. No attempt was made to quantify the heptameric Hla as this depends on the amount of cellular material in the sample. *—Bands of monomeric and multimeric Hla in the lane containing the sample obtained from Patient 21.