Phylogenetic lineage and pilus protein Spb1/SAN1518 affect opsonin-independent phagocytosis and intracellular survival of Group B Streptococcus

Abstract

Opsonin-independent phagocytosis of Group B Streptococcus (GBS) is important in defense against neonatal GBS infections. A recent study indicated a role for GBS pilus in macrophage phagocytosis. We studied 163 isolates from different phylogenetic backgrounds and those possessing or lacking the gene encoding the pilus backbone protein, Spb1 (SAN1518, PI-2b) and spb1-deficient mutants of wild-type (WT) serotype III-3 GBS 874391 in non-opsonic phagocytosis assays using J774A.1 macrophages. Numbers of GBS phagocytosed differed up to 23-fold depending on phylogenetic background; isolates possessing spb1 were phagocytosed more than isolates lacking spb1. Comparing WT GBS and isogenic spb1-deficient mutants showed WT was phagocytosed better compared to mutants; Spb1 also enhanced intracellular survival as mutants were killed more efficiently. Complementation of mutants restored phagocytosis and resistance to killing in J774A.1 macrophages. Spb1 antiserum revealed surface expression in WT GBS and spatial distribution relative to capsular polysaccharide. spb1 did not affect macrophage nitric oxide and TNF-alpha responses; differences in phagocytosis did not correlate with N-acetyl D-glucosamine (from GBS cell wall) according to enzyme-linked lectin-sorbent assay. Together, these findings support a role for phylogenetic lineage and Spb1 in opsonin-independent phagocytosis and intracellular survival of GBS in J774A.1 macrophages.

Infection and Cellular Defense Dynamics in a Novel 17β-Estradiol Murine Model of Chronic Human Group B Streptococcus Genital Tract Colonization Reveal a Role for Hemolysin in Persistence and Neutrophil Accumulation

“…Finally, given that GBS survives phagocytosis (42,84,85) and can kill monocytes and macrophages by apoptosis (74,75), it will be crucial to investigate immune-subversive mechanisms in the genital tract in this model (86). Resident macrophages in the genital tract are important in antimicrobial defense (29), however, also may harbor microbes with intracellular lifestyle traits.…”

“…Prewet nitrocellulose was used to blot bacteria from the agar surface (5 min), which were fixed over steam for 5 min, and then placed in 1% Triton X-100/PBS for 20 min. Membranes were washed with PBS for 20 min and blocked in 2% BSA for 1 h. Membranes were incubated for 1 h in primary Ab (1/1000 dilution), specific for GBS surface binding protein 1 (42), washed, and incubated for 1 h in secondary Ab (goat anti-rat IgG, alkaline phosphotase conjugated; Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were washed and developed with NBT chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt (Sigma-Aldrich).…”

Differing mechanisms of surviving phagosomal stress among group BStreptococcusstrains of varying genotypes

“…demonstrated that GBS strains containing the pilus island 2b backbone protein, Spb1, had increased phagocytosis and intracellular survival compared to strains lacking Spb1. 32 Since Spb1 is specific to ST-17 strains, 33 this finding suggests that the enhanced phagocytosis and ability to survive intracellularly is ST-dependent. However, using our multiple stress medium, ST-17 strains did not survive significantly better than other STs.…”

Regulation of PI-2b Pilus Expression in Hypervirulent Streptococcus agalactiae ST-17 BM110

“…Two additional genes, orf and lep , are present at the beginning of the PI-2b operon encoding a conserved hypothetical protein and a putative signal peptidase, respectively. The backbone pilin Spb1 whose crystal structure has been solved recently [12] was proposed to enhance phagocytosis and survival in macrophages [13]. …”