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doi:10.1128/AAC.01706-13. the controls, 1E2 significantly altered the expression of over 50 genes. It increased the expression of the operon, which encodes an iron uptake system, and decreased the expression of required divalent binding, as Fab fragments did not reduce NP colonization or alter PF 4981517 ST3 gene expression. and 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 gene expression (12). The current 13-valent PCV (PCV13) is usually less effective against ST3 than against other STs (1, 13,C16), highlighting the need for a better understanding of vaccine-mediated protection against ST3. Our group is usually interested in how pneumococcal capsular polysaccharide (PPS) antibodies protect against pneumococcal disease, particularly ST3 disease (17,C21). We previously identified an ST3 PPS (PPS3)-binding mouse IgG1 monoclonal antibody (MAb), 1E2. 1E2 does not promote opsonic killing of ST3 (23). In this study, we determined the effect of 1E2 on ST3 gene expression during NP colonization in mice. We found that 1E2 altered the expression of more than 50 genes compared to the expression achieved with a control MAb. All genes in the operon, encoding the iron import complex, were upregulated, and the gene encoding an oxidoprotective iron-binding protein, and insight into how these effects may translate into decreased bacterial viability during NP colonization. RESULTS 1E2 alters ST3 gene expression during NP colonization. We first tested the effects of 1E2 (a nonopsonic PPS3 IgG1 MAb) on NP colonization relative to those of 5F6 (an opsonic PPS3 IgG1 MAb), 31B12 (a PPS8-specific IgG1 MAb, here referred to as the control MAb), or phosphate-buffered saline (PBS). 1E2 reduced the level of colonization and prevented dissemination relative to the control MAb, as previously described (22), whereas 5F6 had no effect on colonization or dissemination (see Fig. S1 in the supplemental material). We also tested the ability of 1E2 fragments to reduce the number of NP CFU and found that F(ab)2 fragments retained the ability to reduce colonization, as previously described (22), whereas Fab fragments did not (Fig. S2A). We then analyzed by transcriptome sequencing (RNA-seq) bacterial gene expression in NP lavage samples obtained 24 h after ST3 colonization of mice treated with 1E2, the control MAb, or PBS. In a single experiment, we also treated mice with 7A9, an opsonic PPS3 IgG1 MAb that does not reduce NP colonization (22). Unfortunately, further studies with this MAb were not possible as the cell line was lost. After filtering out genes PF 4981517 with low expression (see Materials and Methods), we examined the expression of the remaining 1,884 ST3 genes (Data Set S1). Since there were no significant differences in ST3 gene expression between mice that received the control MAb and mice that received PBS (Data Set S2, control versus PBS), results obtained under control MAb and PBS treatment conditions were combined to increase the statistical power (here referred to as the control condition). A stringent cutoff of a Ofold change (FC)O in expression of 3 and an adjusted value (= 0.0017; Fig. S3). Functional classification of genes with increased expression in 1E2-treated mice revealed numerous genes involved PF 4981517 in carbohydrate transport/metabolism and inorganic ion transport (Table 1), a pattern that was also observed when using a less stringent cutoff for differential expression (FC 2, iron import operon was upregulated 4-fold in 1E2-treated mice (Table 1). Notable downregulated genes included two genes required for fatty acid biosynthesis, and values for the difference in expression relative to that PF 4981517 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. *, 0.05; **, 0.01; ***, 0.001; ****, 0.0001. Data are from two impartial experiments using two replicates per group per experiment. CoA, coenzyme A; ACP, acyl carrier protein. TABLE 1 Functional grouping of ST3 genes upregulated in the NP of 1E2-treated mice transporter subunit IIBCMembrane3.21.0E?02????RS09035biogenesis proteinMembrane3.89.0E?03????RS08830(reverse transcription-quantitative PCR Prox1 (RT-qPCR) experiments, which were performed with RNA prepared from NP lavage fluid obtained from 1E2-, 5F6-, or control MAb-treated mice at 1.