A Capsular Polysaccharide-Specific Antibody Alters Streptococcus pneumoniae Gene Expression during Nasopharyngeal Colonization of Mice

Abstract

Pneumococcal conjugate vaccines (PCV) elicit opsonophagocytic (opsonic) antibodies to pneumococcal capsular polysaccharides (PPS) and reduce nasopharyngeal (NP) colonization by vaccine-included Streptococcus pneumoniae serotypes. However, nonopsonic antibodies may also be important for protection against pneumococcal disease. For example, 1E2, a mouse IgG1 monoclonal antibody (MAb) to the serotype 3 (ST3) PPS (PPS3), reduced ST3 NP colonization in mice and altered ST3 gene expression in vitro Here, we determined whether 1E2 affects ST3 gene expression in vivo during colonization of mice by performing RNA sequencing on NP lavage fluid from ST3-infected mice treated with 1E2, a control MAb, or phosphate-buffered saline. Compared to the results for the controls, 1E2 significantly altered the expression of over 50 genes. It increased the expression of the piuBCDA operon, which encodes an iron uptake system, and decreased the expression of dpr, which encodes a protein critical for resistance to oxidative stress. 1E2-mediated effects on ST3 in vivo required divalent binding, as Fab fragments did not reduce NP colonization or alter ST3 gene expression. In vitro, 1E2 induced dose-dependent ST3 growth arrest and altered piuB and dpr expression, whereas an opsonic PPS3 MAb, 5F6, did not. 1E2-treated bacteria were more sensitive to hydrogen peroxide and the iron-requiring antibiotic streptonigrin, suggesting that 1E2 may increase iron import and enhance sensitivity to oxidative stress. Finally, 1E2 also induced rapid capsule shedding in vitro, suggesting that this may initiate 1E2-induced changes in sensitivity to oxidative stress and gene expression. Our data reveal a novel mechanism of direct, antibody-mediated antibacterial activity that could inform new directions in antipneumococcal therapy and vaccine development.

Keywords: Streptococcus pneumoniae; antibody function; capsule; iron acquisition; monoclonal antibodies; nasopharyngeal colonization; pneumococcus; serotype 3.

FIG 1

1E2 alters ST3 gene expression during nasopharyngeal colonization of mice. (A) Mice were treated with 1E2, 7A9, a control MAb, or PBS for 2 h before intranasal infection with ST3 in 10 μl PBS. ST3 gene expression at 24 h postinfection was determined by RNA-seq in two independent experiments (experiments A and B) using RNA purified from pooled lavage fluid from 6 to 10 mice per treatment. A heatmap and a hierarchical clustering of log₂-transformed fold changes in expression of differentially expressed genes in 1E2-treated mice compared to their expression under the combined control conditions (treatment with control MAb and PBS) are shown. The color scale is representative of relative expression, as follows: red, upregulated; blue, downregulated. Asterisks indicate the genes tested by RT-qPCR, the results of which are presented in panels C to J. (B) Principal component (PC) analysis of gene expression data from all samples in panel A. (C to J) Mice were treated with 1E2, 5F6, or the control MAb for 2 h before ST3 infection, as described in the legend to panel A. Expression of the indicated genes in 1E2- or 5F6-treated mice (red and blue bars, respectively) relative to that in the control MAb-treated mice at 1 or 24 h postinfection was determined by RT-qPCR. For relative expression values of >2 or <−2, P values for the difference in expression relative to that in control MAb-treated mice were determined by one-way analysis of variance and Tukey's multiple-comparison test on nontransformed FC values at the same time points. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data are from two independent experiments using two replicates per group per experiment. CoA, coenzyme A; ACP, acyl carrier protein.

FIG 2

1E2 induces ST3 growth arrest and alters piuB and dpr expression in vitro. (A) ST3 bacteria were incubated with the indicated amounts of 1E2, 5F6, or the control MAb. Growth was monitored over 18 h and is shown as OD₆₀₀ values. The results of a representative experiment from three independent experiments with similar results are shown. Arrows indicate the times at which samples were collected for RT-qPCR analysis of gene expression and for which the results are shown in panels B to E. (B to E) The fold change in expression of the indicated genes in 1E2- or 5F6-treated bacteria relative to that in the control MAb-treated bacteria was determined by RT-qPCR at the indicated times post-MAb addition. For relative expression values of >2 or <−2, P values for the differences in expression relative to that in control MAb-treated mice were determined by one-way analysis of variance and Tukey's multiple-comparison test of nontransformed FC values at the same time points. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data are from two independent experiments using three samples per condition per experiment.

FIG 3

1E2 enhances ST3 sensitivity to iron-induced oxidative stress. ST3 bacteria were mixed with 1E2, 5F6, or the control MAb in C+Y medium. (A) MAb-mediated sensitivity to H₂O₂ was determined by mixing ST3 and the MAbs with 2 to 16 mM H₂O₂. (B) MAb-mediated sensitivity to iron was determined by mixing ST3 and the MAbs with 1 to 20 mM Fe₂SO₄. (C) MAb-mediated sensitivity to the combination of iron and H₂O₂ was determined by mixing ST3 and the MAbs with 1 mM Fe₂SO₄ and 2 mM H₂O₂. (D) The ability of catalase to rescue 1E2-mediated sensitivity to iron and H₂O₂ was determined by adding 0.1 to 10 units of catalase to mixtures of 1E2, iron, and H₂O₂, as described in the legend to panel C. (E) MAb-mediated sensitivity to streptonigrin was determined by incubating ST3 and MAbs with 19 μM streptonigrin for the indicated times before plating for determination of the number of viable CFU. For panels A to D, mixtures were incubated for 3 h before plating for determination of the number of viable CFU. P values for the differences compared to the results obtained with the control MAb under the same treatment conditions (A to C and E) or the 0 mM catalase condition (D) were determined by one-way analysis of variance and Tukey's multiple-comparison test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. All results are expressed as percent viability calculated relative to the viability under the MAb-only condition within each MAb group. All results are from two to three independent experiments with three samples per condition per experiment.

FIG 4

1E2 triggers ST3 capsule shedding in vitro. (A) Electron micrographs of ST3 following 3 h of incubation with the indicated amounts of 1E2 or control MAb in PBS. Representative images from a single experiment are shown. (B and C) A total of 10⁴ bacteria were incubated with 100 μg/ml of the indicated MAbs for 30 min or 3 h in PBS. Shed (C) and cell-associated (B) capsules were quantified by ELISA. (D) Bacteria were incubated with MAbs in the presence or absence of H₂O₂ for 3 h, and capsule shedding was quantified by ELISA. (E) Expression of the ST3 capsule synthase gene (cps3S) was determined by RT-qPCR after 1 h of incubation with 1E2, 5F6, or the control MAb in PBS. Results are shown as the level of expression relative to that achieved with control MAb treatment. For panels B to D, data are presented as the number of nanograms of capsule (PPS3) per microgram of protein in the corresponding cell pellets and are from two to three independent experiments using three samples per condition per experiment. *, P < 0.05 by one-way analysis of variance and Tukey's multiple-comparison test.