Essential Staphylococcus aureus toxin export system

Abstract

Widespread antibiotic resistance among important bacterial pathogens such as Staphylococcus aureus calls for alternative routes of drug development. Interfering with crucial virulence determinants is considered a promising new approach to control bacterial infection. Phenol-soluble modulins (PSMs) are peptide toxins with multiple key roles in pathogenesis and have a major impact on the ability of highly virulent S. aureus to cause disease. However, targeting PSMs for therapeutic intervention is hampered by their multitude and diversity. Here we report that an ATP-binding cassette transporter with previously unknown function is responsible for the export of all PSMs, thus representing a single target for complete obstruction of PSM production. The transporter had a strong effect on virulence phenotypes, such as neutrophil lysis, and the extent of its effect on the development of S. aureus infection was similar to that of the sum of all PSMs. Notably, the transporter was essential for bacterial growth. Furthermore, it contributed to producer immunity toward secreted PSMs and defense against PSM-mediated bacterial interference. Our study reveals a noncanonical, dedicated secretion mechanism for an important class of toxins and identifies this mechanism as a comprehensive potential target for the development of drugs to efficiently inhibit the growth and virulence of pathogenic staphylococci.

Fig. 1

The Pmt PSM exporter. (a) Location and arrangement of the pmt operon in the S. aureus USA300 FPR3757 genome. aa, amino acids. (b) Dependence of PSM secretion on Pmt. (c) Dependence of growth on Pmt. (d,e) Energy dependence of Pmt function. PSMα3 secretion is shown in (d) and growth in (e). All tested strains are Δpmt with induced expression of PSMα peptides (PSMα1–4); control, without induction. The pmt genes were expressed in their natural form (wild-type) or with mutated Walker A or B boxes (“Pmt Walker A”, “Pmt Walker B”). (f,g) PSM secretion by Pmt in the heterologous host L. lactis. PSMα3 secretion is shown in (f) and growth in (g). Control, without induction. In all panels: WT, USA300; WT*, USA300 with all psm genes deleted; Δpmt, pmt deletion mutant, isogenic with WT*; PSMα1–4, induced expression of PSMα peptides (S. aureus, with xylose; L. lactis, with nisin). Pmt, constitutive expression of pmt. (b,c,f,g) WT*, L. lactis, and Δpmt controls harbor the respective PSMα1–4 expression vector, but samples were not induced. (d–g) Control and PSMα1–4 strains also harbor the empty Pmt expression vector. See Supplementary Table 1 for complete designations of used strains and plasmids. See Supplementary Figures 6–9 online for results with other PSMs and USA400. Statistical analyses are by 1-way ANOVA and Bonferroni post-tests, in (b) and (f) shown for the PSMα1–4-expressing WT* versus all other samples.

Fig. 2

Absence of Pmt leads to intracellular accumulation of PSMs and major cellular defects. (a) Intracellular accumulation of PSMs. Strain designations are as in Fig. 1. PSMα3 is shown as example. Statistical analysis is by 1-way ANOVA and Bonferroni post-test shown for the PSMα1–4-expressing Δpmt strain versus all other samples. See Supplementary Figure 10 online for other PSMs. (b–d) Effects on subcellular morphology, transmission electron microscopy (TEM) images. (b) control: strain WT*, PSMα1–4. (c) Δpmt, PSMα1–4. Note disruption of membrane integrity (white arrows) and abnormal cell division, for example the additional septum marked by a black arrow. (d) Δpmt, PSMα1–4, higher magnification. Note formation of abnormal macromolecular assemblies (white arrows) and higher electron density of the cytosolic compartment of control (b) versus Δpmt, PSMα1–4 samples (c,d).

Fig. 3

Pmt contributes to producer immunity and defense against PSM-based bacterial interference. Protective effects against S. aureus and S. epidermidis PSMs. Killing assays were performed with 3-h incubation of 10⁶ CFU of bacteria and addition of different concentration of the respective peptides in carbonate-containing buffer, which mimics in-vivo conditions and was used in previous studies on PSM antimicrobial activities^,. WT*, USA300 with all psm genes deleted; Δpmt, isogenic pmt deletion mutant; Statistical analysis is by t-tests versus the corresponding WT* sample.

Fig. 4

Pmt promotes virulence phenotypes and progression of S. aureus skin infection. (a) Neutrophil lysis after phagocytosis. Lysis of human neutrophils was determined microscopically after addition of equal numbers of live bacteria. Phagocytosis rates were measured under the same conditions. At > 60 min, virtually all bacteria were ingested (Supplementary Fig. 11 online). (b) Hemolysis. Culture filtrates were concentrated and butanol-extracted (to remove the overlaying hemolytic activity of α-toxin), and hemolysis was determined using incubation with human erythrocytes (left) or human blood agar plates (right). (c) Survival in human blood. Bacteria were incubated with heparinized blood (10⁶ CFU per ml) for 60 min. (d) Skin infection model. Groups of mice (n = 12) were subcutaneously injected at the dorsum with the indicated strains. Abscess sizes (lesions and surrounding inflamed areas) were measured daily. Representative images of formed abscesses are shown on the right. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; 2-way ANOVA with Bonferroni post-tests; only results comparing the two PSMα1–4-expressing strains are shown (see Supplementary Table 2 online for complete ANOVA results). (e) Bacterial numbers in abscesses at day 4. CFU are calculated from the performed measurement of bacterial genomic DNA by qRT-PCR of the gyrB gene. (f) Model depicting the multitude of Pmt functions. Pmt facilitates PSM export, provides producer immunity to PSMs, and protects from the antimicrobial activity of PSMs of non-self. (g) Model of consequences of Pmt absence. PSMs are not secreted, leading to strongly decreased virulence. In addition, PSMs accumulate in the cytosol, leading to the disruption of membrane integrity, disruption of vital macromolecular interactions, abnormal cell division and cell death. Finally, there is sensitivity to the antimicrobial activity of PSMs of non-self. (a–e) Strain designations are as in Fig. 1.