Group B Streptococcus cpsE Is Required for Serotype V Capsule Production and Aids in Biofilm Formation and Ascending Infection of the Reproductive Tract during Pregnancy

Abstract

Group B Streptococcus (GBS) is an encapsulated Gram-positive pathogen that causes ascending infections of the reproductive tract during pregnancy. The capsule of this organism is a critical virulence factor that has been implicated in a variety of cellular processes to promote pathogenesis. Primarily comprised of carbohydrates, the GBS capsule and its synthesis is driven by the capsule polysaccharide synthesis (cps) operon. The cpsE gene within this operon encodes a putative glycosyltransferase that is responsible for the transfer of a Glc-1-P from UDP-Glc to an undecaprenyl lipid molecule. We hypothesized that the cpsE gene product is important for GBS virulence and ascending infection during pregnancy. Our work demonstrates that a GBS cpsE mutant secretes fewer carbohydrates, has a reduced capsule, and forms less biofilm than the wild-type parental strain. We show that, compared to the parental strain, the ΔcpsE deletion mutant is more readily taken up by human placental macrophages and has a significantly attenuated ability to invade and proliferate in the mouse reproductive tract. Taken together, these results demonstrate that the cpsE gene product is an important virulence factor that aids in GBS colonization and invasion of the gravid reproductive tract.

Keywords: Streptococcus; capsule; carbohydrate; innate immunity; pathogenesis.

Figure 1.. Genetic architecture and putative activity of CpsE.

A) Genetic arrangement of the cps operon in S. agalactiae GB37. B) Putative activity of CpsE (as determined in S. pneumoniae). CpsE cleaves a glucose-1-phosphate from uridine diphosphate glucose and transfers this to a polyprenyl phosphate acceptor (such as undecaprenyl phosphate).

Figure 2.. Transmission electron microscopy analysis of GBS capsule biosynthesis.

Ammonium molybdate staining and TEM of whole bacterial cells reveals A & C) wild-type GB37 produces a thick capsule visualized by negative stain as a white region surrounding the cell (arrows). B & D) An isogenic ΔcpsE mutant exhibits diminished capsule associated with the cell surface. Magnification is 6500X (A & B) and 11,000X (C & D). Micrographs are representative of three biological replicates.

Figure 3.. Analysis of the role of cpsE in GBS biofilm formation.

A) Bacterial biofilm was visualized with crystal violet stain (purple) which revealed that GB37 forms robust biofilm when cultured in THB supplemented with glucose. The isogenic ΔcpsE mutant has diminished capacity to form biofilms on polystyrene. B) Quantification of biofilm by spectrophotometric measurement, (bars indicate mean optical density of 560 nm (OD₅₆₀) of solubilized crystal violet in ratio to cellular density (OD₆₀₀) prior to staining) indicates wild-type GB37 forms significantly more quantifiable biofilm than the isogenic ΔcpsE mutant. High-resolution scanning electron microscopy imaging reveals wild-type GB37 forms larger, tertiary architectural structures of cells with well-formed channels between cellular clusters (C & D). Conversely, the isogenic ΔcpsE mutant adheres sparsely to the abiotic surface and rarely forms tertiary cellular structures. C & E) 5,000X magnification. D & F) 10,000X magnification. Bars indicate mean +/− SEM. N=3-6 biological replicates. **P<0.01, Student’s t test with Welch’s correction.

Figure 4.. Analyses of carbohydrates within GBS biofilm.

Confocal laser scanning microscopy of GBS (stained green with Syto9, panels A and B) and cell-associated carbohydrates (stained blue with calcofluor white stain, panels C and D) reveals GB37 (A, C, E) secretes more carbohydrate matrix in its biofilm than a ΔcpsE mutant (B, D, F). Merged images (Panels E and F) include ortho stack analyses which confirm that GB37 (Panel E) forms thicker biofilms than the ΔcpsE mutant (Panel F). G) Monomeric carbohydrate assay of adherent bacterial cells indicates the ΔcpsE mutant secretes less carbohydrates compared to the wild-type strain. *P<0.05, Student’s t test.

Figure 5.. Evaluation of placental macrophage phagocytosis of wild-type vs. cpsE mutant.

Bacterial cells were labeled with fluorescent dye (FITC) and co-cultured with primary human placental macrophages for 3 hours before extracellular bacteria were washed away or quenched with trypan blue stain. Mean fluorescence was measured in placental macrophages co-cultured with fluorescently labeled GB37 or isogenic ΔcpsE mutant (+/− SEM). An isogenic isogenic ΔcpsE mutant is more readily taken up by placental macrophages than the parental strain, indicating cpsE aids in evasion of phagocytosis. **P<0.01, Student’s t test with Welch’s correction (N=4).

Figure 6.. Analysis of bacterial burden within reproductive tissues from the mouse model of ascending GBS infection of the vagina during pregnancy.

Pregnant mice received a vaginal inoculation of bacterial cells (either GB37 or isogenic ΔcpsE mutant) at embryonic day 13.5. Two days post-infection, mice were sacrificed and reproductive tissues were collected, homogenized, and analyzed by quantitative culture to determine bacterial burden (colony forming units per gram of tissue; CFU/g). GB37 was able to ascend and invade the reproductive tract more effectively and had enhanced burden in the vagina, uterus, decidua, placenta, fetal membranes, and fetus compared to the isogenic ΔcpsE mutant. N=3, *P<0.05, **P<0.01, Student’s t test with Welch’s correction. Dotted line indicates the limit of detection.

Figure 7.. Histopathological evaluation of gravid reproductive tissues in response to GBS infection.

Microscopical analysis of hematoxylin and eosin-stained placental tissue sections shows that, A) uninfected tissues have intact tissue architecture with few detectable polymorphonuclear cells. B) Infection with wild-type GB37 results in a disruption of tissue integrity and polymorphonuclear cell infiltrates (inset panel) indicative of inflammation. C) Infection with the isogenic ΔcpsE mutant results in less disruption of tissue integrity, and fewer polymorphonuclear cell infiltrates, indicating less tissue damage in these animals. Micrographs were collected at 400X magnification and are representative of three separate experiments. Tissue compartments are labeled as follows: decidua (D), junctional zone (JZ), labyrinthine placenta (LP).

Figure 8.. Conceptual model of the role of cpsE during GBS infection of the reproductive tract.

GBS CpsE catalyzes the transfer of glucose-1-phosphate (Glc-1-P) to an undecaprenyl phosphate acceptor, initiating capsule synthesis. CpsJ is a predicted glycosyltransferase which transports the molecule to the opposite side of the cellular membrane where CpsK facilitates sialylation of the capsular polysaccharide. CpsA is a LytR-domain protein that acts as a transcriptional regulator and assists in insertion of the capsular polysaccharide into the peptidoglycan (PG) cell wall. Capsular biogenesis promotes biofilm formation, evasion of phagocytosis by placental macrophages, invasion and ascending infection during pregnancy.