A novel C5a-derived immunobiotic peptide reduces Streptococcus agalactiae colonization through targeted bacterial killing

Abstract

Streptococcus agalactiae (group B Streptococcus [GBS]) is a Gram-positive bacterium that colonizes the cervicovaginal tract in approximately 25% of healthy women. Although colonization is asymptomatic, GBS can be vertically transmitted to newborns peripartum, causing severe disease such as pneumonia and meningitis. Current prophylaxis, consisting of late gestation screening and intrapartum antibiotics, has failed to completely prevent transmission, and GBS remains a leading cause of neonatal sepsis and meningitis in the United States. Lack of an effective vaccine and emerging antibiotic resistance necessitate exploring novel therapeutic strategies. We have employed a host-directed immunomodulatory therapy using a novel peptide, known as EP67, derived from the C-terminal region of human complement component C5a. Previously, we have demonstrated in vivo that EP67 engagement of the C5a receptor (CD88) effectively limits staphylococcal infection by promoting cytokine release and neutrophil infiltration. Here, using our established mouse model of GBS vaginal colonization, we observed that EP67 treatment results in rapid clearance of GBS from the murine vagina. However, this was not dependent on functional neutrophil recruitment or CD88 signaling, as EP67 treatment reduced the vaginal bacterial load in mice lacking CD88 or the major neutrophil receptor CXCr2. Interestingly, we found that EP67 inhibits GBS growth in vitro and in vivo and that antibacterial activity was specific to Streptococcus species. Our work establishes that EP67-mediated clearance of GBS is likely due to direct bacterial killing rather than to enhanced immune stimulation. We conclude that EP67 may have potential as a therapeutic to control GBS vaginal colonization.

Fig 1

EP67 treatment reduces GBS vaginal colonization. GBS, 1 × 10⁷ CFU, was inoculated into the vaginal lumen of 8-week-old CD1 mice (n = 7 to 8 per group). GBS was allowed to establish colonization for 24 h prior to initial treatment with EP67 or sEP67. GBS persistence was measured by swabbing the vagina and enumerating recovered bacteria. Lines represent median CFU for each treatment group (the median was 0 CFU for the EP67 group on days 2, 3, and 5). In vivo experiments were conducted independently at least three times, and data from one representative experiment are shown. EP67 significantly decreased GBS persistence compared to sEP67 (P = 0.0124) as calculated using a log rank test.

Fig 2

GBS vaginal clearance by EP67 does not require signaling through the CXCL2 receptor. (A and B) Approximately 1 × 10⁷ CFU GBS was inoculated into the vaginal lumen of 16-week-old BALB/c mice (n = 5 to 10 per group) (A) or CXCr2 KO mice (n = 3 to 9 per group) (B). GBS was allowed to colonize for 9 days to ensure colonization following antibiotic removal (see maintenance of CXCr2 KO mice in Materials and Methods) prior to initial treatment with EP67 or sEP67. GBS persistence was measured by swabbing the vagina and enumerating recovered bacteria; horizontal lines represent median CFU recovered (A and B). (C) The percentages of mice that were colonized over time were calculated following EP67 or sEP67 treatment. (D) Chemokine MIP-2 levels were quantified by ELISA in vaginal lavage fluid collected from mice at the indicated time points.

Fig 3

Increased GBS vaginal clearance during EP67 treatment does not require signaling through the CD88 receptor. (A and B) Approximately 1 × 10⁷ CFU GBS was inoculated into the vaginal lumen of 8-week-old BALB/c mice (n = 7 per group) (A) or CD88 KO mice (n = 10 per group) (B). GBS was allowed to colonize for 2 days prior to initial treatment with EP67 or sEP67. GBS persistence was measured by swabbing the vagina and enumerating recovered bacteria. Lines represent median CFU recovered (A and B). (C) The percentages of mice that were colonized over time were calculated following EP67 or sEP67 treatment. (D) Neutrophils (Ly6G⁺ CD11b⁺) present in vaginal lavage fluid collected from mice at the indicated time points were quantified by flow cytometry as described in Materials and Methods.

Fig 4

EP67 exhibits antibacterial activity against GBS. (A) GBS strain A909 was grown to the logarithmic phase and subjected to increasing levels of EP67 or sEP67. After 24 h, GBS growth was measured by absorbance (OD₆₀₀). (B and C) Killing kinetics of WT GBS strains A909 and COH1 upon exposure to 200 μM EP67 or sEP67 control as measured by CFU (B) or Live-Dead staining as described in Materials and Methods (C). GBS with intact membrane fluoresces green, whereas GBS with damaged membranes fluoresces red. Representative images are shown. Magnification, ×1,000. All experiments were conducted independently at least twice, and representative data from one experiment are shown.

Fig 5

EP67 treatment inhibits bacterial load in vivo but not immune cell influx and acts independently of CXCr2 and CD88 signaling pathways. (A) Approximately 1 × 10⁷ CFU GBS was injected into the peritoneal cavity of 20-week-old BALB/c mice (n = 5 per group). Immediately following GBS injection, mice were treated with EP67 or sEP67 in the peritoneal cavity. Peritoneal lavage fluid was conducted 2 h posttreatment, and GBS survival was quantified. Peritoneal infection was conducted independently at least twice, and data from one representative experiment are shown. (B) Lavage fluid samples from the mice described in the panel A legend were analyzed for neutrophil (Ly6G⁺ CD11b⁺), macrophage (B220⁻ CD11c⁺), and dendritic cell (B220⁻ CD11c⁺, B220⁻ CD11c⁺ CD11b⁺) infiltration by flow cytometry. Data are expressed as percentages of 20,000 cells analyzed per sample. (C) Peritoneal infection and peptide treatment were performed as in described in the panel A legend using 8- to 16-week-old BALB/c (n = 5 to 8 per group), CXCr2 KO (n = 4 to 8 per group), and CD88 KO (n = 7 per group) mice. Bacterial CFU were recovered from peritoneal lavage fluid 2 h posttreatment. Data were analyzed by the use of an unpaired Student's t test. *, P < 0.05; **, P < 0.005; ***, P < 0.001.