Recombinant soluble human CD6 ectodomain and rshCD5 proteins were purified following the reported methods (28) using SURE CHO-M Cell line™ clones (Selexis SUREtechnology Platform™, Geneva, Switzerland) and size-exclusion chromatography protocols developed at PX’Therapeutics (Grenoble, France). Human and bovine seroalbumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Peptides (>80% purity) were manufactured by ProteoGenix (Schiltigheim, France) and stocked at 5 mg/mL with diluted (1:3) acetonitrile.

5 × 10⁸ colony-forming units (CFU)/mL diluted in TTC buffer (50 mM Tris pH 7.5 plus 150 mM NaCl, 0.1% Tween 20, and 1 mM Ca²⁺) were mixed (1:1) with different peptide concentrations (0–200 µg/mL) in 96 U-bottomed well microtiter plates (Biofil) (29). After overnight incubation at 37°C, bacterial agglutination was examined by light microscopy and scored from − (absent) to + + + (maximal).

Multidrug-resistant Acinetobacter baumannii clinical isolate, Enterobacter cloacae ATCC 23355, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Listeria monocytogenes ATCC 19111, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, and Methicillin-resistant S. aureus (MRSA) clinical isolate were provided by Dr. Jordi Vila (Microbiology Department, Hospital Clinic of Barcelona) and grown in Luria Bertani or agar with 5% sheep blood (Becton Dickinson) at 37°C, except for L. monocytogenes that was cultured in Brain Heart infusion broth (Pronadisa).

The hydrodynamic diameters of peptides (10 µg/mL in PBS) were measured at 25°C in a Zetasizer Nano S from Malvern Instruments (Worcestershire, UK) equipped with a 633 nm HeNe laser, as described (32). Four scans were recorded for each sample, and the samples were analyzed in triplicate.

Spleens from 6- to 8-week-old C57BL/6 mice (Charles River) were disaggregated by filtering through a cell strainer and, after erythrocyte lysis, the cells were resuspended in RPMI 1640 with l-glutamine (Lonza) plus 10% fetal calf serum (BioWest), 100 U/mL penicillin, 100 µg/mL streptomycin, and 50 µM 2-β-mercaptoethanol (Merck). Cells (2 × 10⁵) were stimulated for 48 h (at 37°C in a humidified atmosphere with 5% CO₂) in U-bottomed 96-well plates (Biofil) containing LPS (0.5 µg/mL; E. coli O111:B4), in the presence or absence of increasing peptides (0.5–20 µg/mL). Culture supernatants were harvested and mouse cytokines measured by ELISA following manufacturer’s instructions (BD Biosciences OptEIA sets).

Animal procedures were approved by the Animal Experimentation Ethical Committee, University of Barcelona. High-grade mortality (≥90% mortality within the first 48–72 h) CLP-induced septic shock was induced in 8- to 10-week-old C57BL/6J male mice (20–25 g; Charles River) as previously reported (24).

For the assessment of bacterial load, blood and spleen samples from CLP-treated mice were collected, homogenized, and diluted aseptically in sterile PBS. Serial dilutions were plated overnight on agar with 5% sheep blood (Becton Dickinson) at 37°C. Viable bacterial counts were expressed as CFU/mL (blood) or per mg (spleen).

Survival assays were analyzed by a log-rank χ² test using GraphPad Prism software. The significance of differences between experimental groups was determined by two-tailed paired t test with 95% confidence interval (CI). P values were considered significant when P < 0.05. Statistical analysis (mean ± SEM) was performed using a two-tailed Mann–Whitney test, with 95% CI.

To investigate the bacterial-binding properties of CD6, intradomain peptide sequences homologous to the consensus 11-mer DMBT-1/SAG.pbs1 peptide sequence (GRVEVLYRGSW) (10) were synthesized. The sequence and physicochemical properties of such CD6-derived peptides mapping at SRCR domains 1–3 (CD6.PD1, CD6.PD2, and CD6.PD3, respectively), as well as of the other peptides and proteins used in this study are compiled in Figure 1A. Structural analyses depicted in Figure 1B showed that all CD6 peptides are accessible at the surface of CD6, with CD6.PD1 (and CD6.PD3) being exposed at opposing sides from that of CD6.PD2. Interestingly, CD6.PD3 mapped at a distant position of the amino acids involved in CD6 binding to CD166/ALCAM (20) (Figure 1B). As illustrated in Figure 1C, the amino acid conservation of the CD6-derived peptides among different animal species was relatively high for CD6.PD2 and CD6.PD3 and lower for CD6.PD1.

None of the CD6-derived peptides matched the minimal 9-mer DMBT-1/SAG.pbs1 consensus motif (VEVLxxxxW)—a fact also shared by the CD163p2 (GRIEIKFQRRW) peptide (15)—and function was explored in bacterial agglutination assays. The DMBT-1/SAG.pbs1 peptide was used as positive control (10), and the analogous peptide sequence (CD5.PD2) present in the second SRCR domain of CD5—a highly homologous lymphocyte receptor for which no bacterial binding properties have been reported—used as negative control. As illustrated by Figures 2A,B, dose-dependent agglutination of different Gram-positive and Gram-negative bacterial suspensions (including MDR strains) was observed for CD6.PD1 and CD6.PD3, but not for CD6.PD2. These results indicate that some but not all CD6-derived sequences retain the bacterial agglutination properties of native CD6 (14) and DMBT-1/SAG proteins and of pbs1 (10, 29).

Absence of bacterial agglutination does not fully exclude direct binding to bacterial PAMPs, so binding of biotin-labeled CD6-derived peptides to solid-phase bound LPS or LTA was tested by ELISA. As shown by Figures 3A,B, all CD6-derived peptides showed dose-dependent binding to LPS and LTA, similar to the pbs1 peptide and rshCD6 protein used as positive controls. As expected, no significant binding was observed for the CD5.PD1 peptide and the rshCD5 protein. These results confirm that CD6-derived peptides retain binding properties to LPS and LTA as reported for native CD6 but not CD5 proteins (14, 23).

To further characterize the interaction of CD6-derived peptides with LPS and LTA, the corresponding dissociation constants (K_d) were determined by tryptophan fluorescence emission. As summarized in Figure 3C (Figure S1 in Supplementary Material), all CD6-derived peptides displayed high affinities for both LPS and LTA, being higher for CD6.PD1 and/or CD6.PD2 compared to CD6.PD3 (PD1 ≥ PD2 > PD3). K_d values for CD6.PD1 and CD6.PD2, but not CD6.PD3, are lower than for the prototypical DMBT-1/SAG.pbs1 peptide or the rshCD6 protein itself. The greater affinity of CD6.PD1 and CD6.PD2 for LPS, and LTA is not correlated with their bacterial agglutination properties, which are absent in CD6.PD2.

To determine whether self-aggregation properties of CD6-derived peptides are related to their agglutination properties, the hydrodynamic size of CD6-derived peptides in solution was analyzed by DLS. Results indicate that CD6-derived peptides formed particles of different hydrodynamic sizes according to their self-aggregation properties (Figure 3D). CD6.PD3 particles exhibited two peaks, at 630 ± 3 and 5.151 ± 7 nm, indicative of self-aggregation. In contrast, CD6.PD2 showed a single peak centered at 379 ± 5 nm, whereas CD6.PD1 showed two peaks at 321 ± 5 and 1.484 ± 8 nm, in line with the greater bacterial agglutination properties of CD6.PD3 (and CD6.PD1) with respect to CD6.PD2. Higher CD6.PD3 binding to immobilized LPS or LTA (Figures 3A,B) may respond to the fact that self-association of biotinylated peptide would produce an enhancement of the chromogenic signal. Whatever the case, the binding results univocally support the direct and substantial interaction of CD6-derived peptides with essential cell wall components from Gram-negative and Gram-positive bacterial strains.

Next, the functional relevance of CD6-derived peptides interaction with key pathogenic bacterial products was explored ex vivo. To this end, the modulatory effects of increasing concentrations of CD6-peptides on cytokine release by mouse splenocytes exposed to LPS were tested. As illustrated by Figure 4, only CD6.PD3 showed significant dose-dependent inhibitory effects on pro-inflammatory IL-6 and IL-1β cytokine release, which reached statistical significance in the former case. The same CD6.PD3 peptide also induced a non-statistically significant increased release of the anti-inflammatory cytokine IL-10, as reported for rshCD6 (23). No significant effect was observed regarding TNF-α release for any of the CD6-derived peptides tested (data not shown).

The effects of CD6-derived peptides in vivo were tested in mice undergoing CLP-induced septic shock (27). To this end, a single intravenous (i.v.) dose (6 mg/kg) of the different CD6-derived peptides was infused to C57BL/6 mice 1 h post CLP-induction, and survival monitored thereafter. As shown in Figure 5, increased survival was observed among mice infused with CD6.PD2 (12.5%, P = 0.0005) and CD6.PD3 (36.36%, P < 0.0001) compared to the saline-treated group, a fact also observed in mice infused with the DMBT-1/SAG.pbs1 (23.07%, P = 0.0025). In contrast, no effects were evidenced for CD6.PD1.

Since the in vivo protective properties of CD6.PD3 against septic shock excelled CD6.PD1 and CD6.PD2, additional experiments exploring its time-, dose-, and systemic via-dependent effects were performed. As shown in Figures 6A–C, maximal survival rates post CLP were obtained following CD6.PD3 infusion at 6 or 12 mg/kg doses (37.5 and 40%, respectively, vs 23.08% at 3 mg/kg), and 1 h after CLP induction (40% at + 1 h vs 20% at + 3 h), irrespective of the i.v. or intraperitoneal (i.p.) infusion pathway used.

Next, the effect of the CD6.PD3 peptide on serum cytokine levels and bacterial load post CLP were further monitored. To this end, C57BL/6 mice undergoing CLP-induced septic shock were treated with saline or CD6.PD3 peptide under the previously stated optimal conditions (single i.v. infusion of 6 mg/kg at 1 h post CLP) and thereafter bled and sacrificed at 4 and 20 h later, respectively.

As shown in Figure 7A, CD6.PD3-treated mice exhibited lower levels (P < 0.05) of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α at 20 h post CLP, compared to the saline-treated group. Similarly, the same CD6.PD3-treated mice also produced lower CFU isolated from blood and spleen when sacrificed at 20 h post CLP, compared to the control group (Figure 7B). These results indicate that the CD6.PD3 peptide retains the therapeutic properties reported for the rshCD6 protein in experimental models of septic shock (24). This also holds for septic mice simultaneously treated with CD6.PD3 and the broad-spectrum bactericidal antibiotic Imipenem/Cilastatin. As illustrated in Figure 8, the combined administration of CD6.PD3 (6 mg/kg i.v.) and Imipenem/Cilastatin (50 mg/kg/12 h i.p.) 1 h post CLP-induced septic shock resulted in additive/synergistic effects on mice survival (90.9%), compared to either treatment individually (30.8 and 44.4%, respectively).