Staphylococcus aureus β-toxin production is common in strains with the β-toxin gene inactivated by bacteriophage

Abstract

Background: Staphylococcus aureus causes life-threatening infections, including infective endocarditis, sepsis, and pneumonia. β-toxin is a sphingomyelinase encoded for by virtually all S. aureus strains and exhibits human immune cell cytotoxicity. The toxin enhances S. aureus phenol-soluble modulin activity, and its activity is enhanced by superantigens. The bacteriophage φSa3 inserts into the β-toxin gene in human strains, inactivating it in the majority of S. aureus clonal groups. Hence, most strains are reported not to secrete β-toxin.

Methods: This dynamic was investigated by examining β-toxin production by multiple clonal groups of S. aureus, both in vitro and in vivo during infections in rabbit models of infective endocarditis, sepsis, and pneumonia.

Results: β-toxin phenotypic variants are common among strains containing φSa3. In vivo, φSa3 is differentially induced in heart vegetations, kidney abscesses, and ischemic liver compared to spleen and blood, and in vitro growth in liquid culture. Furthermore, in pneumonia, wild-type β-toxin production leads to development of large caseous lesions, and in infective endocarditis, increases the size of pathognomonic vegetations.

Conclusions: This study demonstrates the dynamic interaction between S. aureus and the infected host, where φSa3 serves as a regulator of virulence gene expression, and increased fitness and virulence in new environments.

Keywords: Staphylococcus aureus; bacteriophage; infective endocarditis; pneumonia; sepsis; β-toxin.

Figure 1.

Phage φSa3 excision restores β-toxin production. A, Schematic of φSa3 encoding for virulence factors, including SEl-K, SEl-Q, SEA, SAK, and SCIN. B, Hyperlytic patches produced by S. aureus MW2 plated on SBAPs at high density. C, MW2 isolates producing varying levels of β-toxin (left panel shows isolates with a narrow zone or ellipse zone of hemolysis, and right panel shows isolates with a bright zone of hemolysis surrounded by a large dark zone due to β-toxin). D, CAMP test comparing MW2 wild-type and MW2 hyper-β-toxin synergism with PSMs.

Figure 2.

Intact β-toxin structural gene is present among different S. aureus clonal groups. PCR screen for the full-length β-toxin gene in USA100–400 clonal groups and nontypeable strains. Abbreviation: PCR, polymerase chain reaction.

Figure 3.

Quantification of β-toxin production via antibody hemolysis-inhibition assays. A, hemolysis inhibition assays of strains MW2 (hyper-β), MN8, and MW2 wild-type showing culture concentrate hemolysis compared to culture hemolysis inhibited by anti-β toxin serum. B, Area of lysis measured in cm from hemolysis inhibition assays of USA200 and USA400 strains; black bar represents area of lysis from culture concentrates. Gray bar represents area of lysis when inhibited by anti-β toxin serum.

Figure 4.

Hemolysis inhibition assays of USA100 and USA300 strains. A, Image of hemolysis inhibition assay of S. aureus RN4220 culture concentrate alone, with anti-β toxin serum, with anti-α toxin serum, or with both anti-β and anti-α toxin serum to demonstrate the hemolysis due to each toxin; areas of lysis measured in centimeters from hemolysis inhibition assays of (B) S. aureus RN4220, USA100 strains, and (C) USA300 strains. Black bar represents area of lysis from culture concentrates, dark gray bar represents area of lysis when inhibited by anti-β toxin serum, gray bar represents area of lysis when inhibited by anti-α toxin serum, and light gray bar represents area of lysis when inhibited by both anti-β and anti-α serum.

Figure 5.

MW2 and MW2 (hyper-β) in a rabbit model of pneumonia. Lungs dissected from rabbits infected with (A) MW2 wild-type or (B) MW2 (hyper-β). Dotted lines highlight areas of granulomas.

Figure 6.

MW2 and MW2 (hyper-β) in a rabbit model of infective endocarditis and sepsis. A, The percent survival of rabbits from the MW2 and MW2 (hyper-β) infected groups each day postinfection until termination of the experiment at day 4. B, CFUs/mL recovered from the blood at death for each experimental group. C, Mean mass of vegetations in milligrams recovered from the heart valves of each experimental group. D, Mean total CFUs recovered per vegetation from each experimental group.

Figure 7.

Frequency of β-hemolytic colonies dependent on environmental niche. A, Ratio of β-hemolytic colonies to total number of colonies from cultures grown in TH or BH media at 30°C, 37°C, or 42°C and plated on sheep-blood agar plates; B, Ratio of β-hemolytic colonies to total number of recovered colonies from tissues of rabbits used in a model of infective endocarditis and sepsis when inoculated with S. aureus MW2. *P ≤ 0.05, **P ≤ 0.009, ***P ≤ 0.0009. Asterisks in panel A compare mean differences at 30°C, 37°C, and 42°C in TH or BH media. Asterisks in panel B compare mean differences of blood versus vegetations, liver, or kidneys.