Adaptive immune response to lipoproteins of Staphylococcus aureus in healthy subjects

Abstract

Staphylococcus aureus is a frequent commensal but also a dangerous pathogen, causing many forms of infection ranging from mild to life-threatening conditions. Among its virulence factors are lipoproteins, which are anchored in the bacterial cell membrane. Lipoproteins perform various functions in colonization, immune evasion, and immunomodulation. These proteins are potent activators of innate immune receptors termed Toll-like receptors 2 and 6. This study addressed the specific B-cell and T-cell responses directed to lipoproteins in human S. aureus carriers and non-carriers. 2D immune proteomics and ELISA approaches revealed that titers of antibodies (IgG) binding to S. aureus lipoproteins were very low. Proliferation assays and cytokine profiling data showed only subtle responses of T cells; some lipoproteins did not elicit proliferation. Hence, the robust activation of the innate immune system by S. aureus lipoproteins does not translate into a strong adaptive immune response. Reasons for this may include inaccessibility of lipoproteins for B cells as well as ineffective processing and presentation of the antigens to T cells.

Keywords: Antibody; Human; Lipoprotein; Microbiology; S. aureus; T cell.

Figure 1

Human IgG binding to S. aureus lipoproteins using immunoproteomics. S. aureus extracellular proteins were separated on 2D gels. Proteins were identified by MS (MALDI‐TOF). A human serum pool was used as source of IgG for staining immunoblots. The superimposed images were created with the software Delta2D version 4.4. (A) Superimposed 2D gel images of extracellular proteins derived from an lgt‐competent S. aureus strain RN4220 (orange) and its isogenic lgt mutant (blue). The blue spots represent presumptive lipoproteins, which are released into the cell culture supernatant. MS analysis identified these proteins as lipoproteins. Superimposed 2D immunoblots and 2D gel images show the human serum IgG binding to S. aureus extracellular proteins of the WT (B) and the lgt mutant (C); blue: proteins from S. aureus, orange: IgG‐bound proteins. IgG binding to S. aureus lipoproteins was not observed (blue spots in panels A and C). Similar probing in the pH range 4–7 revealed no secreted lipoproteins (see Supporting Information Fig. S3). Experiments were carried out in duplicate with similar results.

Figure 2

Low serum IgG binding to recombinant bacterial lipoproteins. Recombinant lipoproteins (100 ng per cavity) were coated on ELISA plates and incubated with human serum samples—16 samples from S. aureus non‐carriers and 16 samples from S. aureus carriers—each at a dilution of 1:100. After incubation with the goat anti‐human‐IgG‐POD secondary antibody, IgG binding was measured using TMB substrate. (A) The absorbance at OD_{450 nm} showed at best weak IgG binding to eight recombinant lipoproteins. IsdB and Plc, known immunodominant proteins, were included as controls. (B) In addition, 20 putative S. aureus lipoproteins (according to LocateP) were recombinantly expressed and tested for binding of human serum antibodies as described above. Binding of human serum antibodies to these proteins was very low as well. Each experiment was repeated twice. Box‐and‐whisker plots visualize median, upper and lower quartiles, maximum, minimum and outliers for each dataset.

Figure 3

A moderate proliferative response in human PBMCs. Proliferation assays were conducted to investigate how PBMCs respond to S. aureus lipoproteins. Fresh human blood samples from ten individuals were used to isolate PBMCs. Antigens were applied in serial dilution from 0.006 to 25 μg/mL. The recall antigen tetanus toxoid (TT) and the immunodominant protein S. aureus phospholipase C (Plc) were included as controls. Proliferation was measured by 3H‐thymidine incorporation. Experiments were performed in triplicate. Means of the average T‐cell responses of the 10 individuals are depicted. Lipoproteins stimulated PBMCs only at high antigen concentrations (≥6.25 μg/mL). Three of the five tested lipoproteins (Opp1A, Opp4A and SirA) did not trigger proliferation at all.

Figure 4

Th1/Th17 cytokine secretion. Cytokine profiling was conducted on five proliferation‐stimulated lipoprotein assays. Cytokine secretion during the proliferative response was analyzed by cytokine bead assay (BD Biosciences, Heidelberg, Germany). The three lipoproteins IsdE, Opp1A, and PstS induced the secretion of the Th1/Th17 cytokines INF‐γ and IL‐17A. Other cytokines, including IL‐1β, IL‐10, and IL‐12p70, were also induced by these lipoproteins. In contrast, MntC and SstD weakly stimulated the secretion of cytokines belonging to the Th1/Th17 group (for details see Supporting Information Fig. S6). Kruskal–Wallis tests were conducted using the GraphPad Prism v6.02 software to calculate p values. Asterisks at the top of each panel correspond to significant differences to no antigen control as following *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.