New pneumococcal serotype 20C is a WciG O-acetyltransferase deficient variant of canonical serotype 20B

Abstract

The polysaccharide (PS) capsule of Streptococcus pneumoniae (pneumococcus) is the immunodominant surface structure that shields the bacteria from the host immune system. Since the capsule is the primary target of currently available pneumococcal vaccines, anti-capsular antibodies are highly protective but serotype-specific. Pneumococci may evade host or vaccine-induced immunity as a result of variation in capsule structure mediated via multiple mechanisms, such as the loss or gain of O-acetylation. Previous biochemical studies of serogroup 20 isolates have identified two subtypes-20A and 20B, whose capsule PS differs in the WhaF-mediated glucose side chain. Herein, we characterize a newly discovered capsule type, 20C, that differs from serotype 20B via the inactivation of capsule O-acetyltransferase gene, wciG. Structural analysis demonstrated that 20C and 20B share an identical repeat unit [→3)-α-D-GlcpNAc-[β-D-Galf-(1→4)][α-D-Glcp-(1→6)]-(1→P→6)-α-D-Glcp-(1→6)- β-D-Glcp-(1→3)-β-D-Galf 5,6Ac₂-(1→3)-β-D-Glcp-(1→], except for the absence of WciG-mediated O-acetyl group at terminal galactofuranose (β-D-Galf). We confirmed that deletion of the wciG gene in a 20B strain resulted in the expression of the 20C capsule. Serotype 20C is serologically indistinguishable from the canonical 20A and 20B using conventional serotyping antibodies, but serogroup 20 subtypes can be distinguished by sequencing of cps genes-whaF, wciG, and wcjE. While genetic screening suggests 20C to be globally less prevalent, a new variant was identified which appears to have both wciG and whaF genes inactive, potentially indicating it to be a new serotype. Consequently, genome-based serotyping/bioinformatic tools must scrutinize all cps genes for mutations that might inactivate/modify cps-encoded enzymes, ensuring effective tracking of emerging capsule variants in response to ongoing vaccination efforts.

Importance: Streptococcus pneumoniae (pneumococcus) is a significant human pathogen known for producing a wide array of antigenically and structurally diverse capsule types, a fact that poses a serious challenge to the effectiveness of vaccines targeting pneumococcal capsule polysaccharide (PS). Herein, we provide a comprehensive analysis-genetic, antigenic, and biochemical of a newly identified capsule type, 20C, which differs from the canonical serotype 20B due to the inactivation of the capsule O-acetyltransferase gene, wciG. Our findings highlight how pneumococci can alter their capsule PS structure and immunological characteristics through minor genetic modifications. Since the appearance of new capsule types can directly affect pneumococcal conjugate vaccine (PCV) implementation, a deeper understanding of capsule PS at the genetic, immunological, and biochemical levels is critical for the development of future diagnostic tools and vaccines.

Keywords: O-acetyltransferase; Streptococcus pneumoniae; capsule polysaccharide; serogroup 20; vaccine.

Fig 1

Capsule synthesis loci and capsule polysaccharide structures of serogroup 20 subtypes. (A) Representative cps loci of serotypes 20A and 20B. Highly conserved regulatory genes (light gray arrows), genes encoding glycosyltransferases (blue arrows), Wzy polymerases (green arrows), Wzx flippases (pink arrows), O-acetyltransferases (red arrows), carbohydrate synthetases (dark gray arrows), transposable elements (black arrows) are labeled at the top of 20A cps locus. Same gene annotation is followed for the 20B cps locus. White color cross on the whaF gene arrow in the 20A cps locus indicates that this gene is defective. cps, capsule synthesis locus. (B) Symbol nomenclature for glycans diagrams of elucidated serotypes 20A and 20B PS repeat unit structures. cps-encoded glycosyltransferase, O-acetyltransferases, and polymerase are listed in blue, red, and green text, respectively, on top of their putatively assigned linkages. (C) Alignment of WciG amino acid sequences showing substitutions and translational defects at different residues. The alignment shows WciG sequences of serotype 20A (ATCC6320), serotype 20B (CDC5931-06), and WciG variants (MNK0184 and MNK0952). The numbers on the top of the alignment refer to the amino acid position. Symbol (*) indicates the stop codon. Dots refer to conserved amino acids in reference to the ATCC6320 WciG sequence.

Fig 2

Biochemical analysis of capsule polysaccharide produced by serogroup 20 WciG variants. (A) ¹H NMR spectra of native capsule PS purified from strains of serotype 20A (ATCC6320), 20B (CDC5931-06), 20C (MNK0184 and MNK0952). Acetylation peaks in the O-acetyl methyl region are labeled (chemical shift: 2.05–2.25 ppm), and O-acetylation of terminal galactofuranose (tGalf) is labeled in blue letters. Asterisk denotes a signal arising from the cell wall polysaccharide (CWPS). Strain name is provided on the left of the spectra and the corresponding serotype is in parenthesis. (B) cps locus of serotypes 20C. The gene annotation is followed as described for 20A and 20B cps loci in Fig. 1A. White color cross on the wciG gene arrow in the 20C cps locus indicates that this gene is defective. (C) Symbol nomenclature for glycans diagrams of elucidated serotype 20C PS repeat unit structure. cps-encoded glycosyltransferase, O-acetyltransferases, and polymerase are listed in blue, red, and green text, respectively, on top of their putatively assigned linkages. The symbol key is found in Fig. 1B.

Fig 3

Deletion of wciG results in the loss of O-acetylation at the terminal galactofuranose (tGalf) branch of serotype 20B PS. (A) SJC strategy to create the wciG knock-out mutant strain, JY21, from CDC5931-06 (serotype 20B). YNL001 is a pneumococcal strain containing SJC (genes—sacB, kan, rpsL) (8, 34). The five-digit numbers indicate the PCR primers, which are described in Table S4. The black arrows (short and thin) underneath the primers indicate the direction of the primers. (B) ¹H NMR spectra of native capsule PS purified from CDC5931-06 (serotype 20B, black spectra), JY21 (wciG knock-out, red spectra), and MNK0184 (serotype 20C, blue spectra). The ¹H NMR overlay shows the signals arising in the anomeric (4.9 to 5.6 ppm) and acetyl-methyl (2.05 to 2.25 ppm) regions. Acetylation peaks are labeled, and the O-acetylation of tGalf is labeled in blue.

Fig 4

Serological properties of serogroup 20 subtypes. (A) FCSA histograms depicting antibody deposition on strains ATCC6320 (20A), CDC5931-06 (20B), MNK0184 (20C), JY21 (20C), and SPEC6B (serotype 6B, control strain). Black curves represent the fluorescence of bacteria incubated in different antisera (bottom labels), while gray-filled curves represent negative-control preparations incubated with secondary antibodies only. Hyp6BG13 and Hyp20G5 represent monoclonal antibodies (Mabs) specific to serotype 6B and serogroup 20, respectively. Type-20 antiserum and factor serum 20b are the polyclonal rabbit antisera. (B) Minimized molecular models for 6RU of 20A (left), 20B (middle), and 20C (right), shown in space-filling representation and colored according to residue type. The models show the same extended helical conformation with 2RU per helical turn for the three serotypes. (C) Inhibition enzyme-linked immunosorbent assay (ELISA) illustrates the ability of purified 20A, 20B, and 20C capsule PS to inhibit the binding of Hyp20G5. 20A capsule PS was commercially obtained from ATCC. 20B and 20C capsule PS were purified from CDC5931-06 and MNK0184 strains, respectively. The Y-axis shows the antibody (Hyp20G5) bound to ELISA plates coated with 20A PS, while the X-axis displays different inhibitory concentrations of purified capsule PS. (D) Reactivity of purified 20A, 20B, and 20C capsule PS with the anthrone reagent. Y-axis represents the absorbance at OD₆₃₀, indicating the strength of reactivity, and X-axis shows different PS dilutions (twofold serial dilutions) used to react with the anthrone reagent. Average OD values from quadruplets are plotted. (E) Opsonic titers against target strains ATCC6320 (20A), CDC5931-06 (20B), MNK0184 (20C) using postimmunization PPSV23 sera samples from four human adults (P007B, P008B, P014B, and P016B) and a reference sample (007sp). Data were analyzed by one-way analysis of variance with Tukey’s multiple-comparison test. OPK, opsonophagocytosis killing.