A human monoclonal antibody that specifically binds and inhibits the staphylococcal complement inhibitor protein SCIN

Abstract

Staphylococcus aureus is a serious public health burden causing a wide variety of infections. Earlier detection of such infections could result in faster and more directed therapies that also prevent resistance development. Human monoclonal antibodies (humAbs) are promising tools for diagnosis and therapy owing to their relatively straightforward synthesis, long history of safe clinical use and high target specificity. Here we show that the humAb 6D4, which was obtained from a random screen of B-cells producing antibodies that bind to whole cells of S. aureus, targets the staphylococcal complement inhibitor (SCIN). The epitope recognized by 6D4 was localized to residues 26 to 36 in the N-terminus of SCIN, which overlap with the active site. Accordingly, 6D4 can inhibit SCIN activity as demonstrated through the analysis of C3b deposition on S. aureus cells and complement-induced lysis of rabbit erythrocytes. Importantly, while SCIN is generally regarded as a secreted virulence factor, 6D4 allowed detection of strongly increased SCIN binding to S. aureus cells upon exposure to human serum, relating to the known binding of SCIN to C3 convertases deposited on the staphylococcal cell surface. Lastly, we show that labeling of humAb 6D4 with a near-infrared fluorophore allows one-step detection of SCIN-producing S. aureus cells. Together, our findings show that the newly described humAb 6D4 specifically recognizes S. aureus SCIN, which can potentially be used for detection of human serum-incubated S. aureus strains expressing SCIN.

Exoproteomic profiling uncovers critical determinants for virulence of livestock-associated and human-originated Staphylococcus aureus ST398 strains

“…To investigate the cytotoxicity of the SCIN and CHIPS proteins of S. aureus , these proteins were expressed with a His 6 -tag in Lactococcus lactis as described before [ 27 ]. As a control, the LysM domain of the S. aureus protein Sle1 was also expressed with a His 6 -tag in L. lactis and purified.…”

There's no place like OM: Vesicular sorting and secretion of the peptidylarginine deiminase of Porphyromonas gingivalis

“…Antibody production – Rabbit polyclonal antibodies were raised at Eurogentec (Seraing, Liège, Belgium) for detection of PPAD, Omp41, Kgp, and RgpA + RgpB. To this end, the PPAD gene from reference strain W83 was cloned and expressed without its signal sequence in Lactococcus lactis PA1001[ 31,32 ], using plasmid pNG4210[ 33,34 ] and primers P1LF and P1LR (S2 Table). To raise Omp41-specific antibodies, the omp41 gene of strain W83 was amplified with primers P1F and P1R (S2 Table).…”

“…The resulting omp41, kgp, rgpA/B fragments were all cloned and expressed in E. coli BL21 using pET26B + . Expression of PPAD in L. lactis was induced overnight with nisin as described [ 33 ], while expression of Omp41 or the Kgp and RgpA catalytic domains in E. coli was induced with Isopropyl-β-D-thiogalactopyranoside for 2 h. After induction, cells were collected by centrifugation and disrupted by sonication ( L. lactis ) or bead-beating ( E. coli ) in binding buffer (S3 Table). Cell lysates were added to 500 µL of HisLink™ Protein Purification Resin (Promega, USA) and incubated at room temperature for 30–60 min.…”

Antibody-Based Agents in the Management of Antibiotic-Resistant Staphylococcus aureus Diseases

“…These include mAbs that block virulence factors such CHIPS and SCIN and that can consequently restore and revitalize host immunity. In this context, a human monoclonal antibody, named 6D4, that blocks the complement inhibitory activity of SCIN has recently been generated [ 127 ]. Of note, the immunodominant S. aureus antigen A (IsaA), an invariant, widely expressed cell wall-associated protein, suggested to be involved in growth, division, separation, and survival of S. aureus cells [ 128 ], may be another promising target for passive immunization in human population.…”