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Streptococcus agalactiae npx Is Required for Survival in Human Placental Macrophages and Full Virulence in a Model of Ascending Vaginal Infection during Pregnancy

ABSTRACT Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a Gram-positive encapsulated bacterium that colonizes the gastrointestinal tract of 30 to 50% of humans. GBS causes invasive infection during pregnancy that can lead to chorioamnionitis, funisitis, preterm prelabor rupture of membranes (PPROM), preterm birth, neonatal sepsis, and maternal and fetal demise. Upon infecting the host, GBS encounters sentinel innate immune cells, such as macrophages, within reproductive tissues. Once phagocytosed by macrophages, GBS upregulates the expression of the gene npx, which encodes an NADH peroxidase. GBS mutants with an npx deletion (Δnpx) are exquisitely sensitive to reactive oxygen stress. Furthermore, we have shown that npx is required for GBS survival in both THP-1 and placental macrophages. In an in vivo murine model of ascending GBS vaginal infection during pregnancy, npx is required for invading reproductive tissues and is critical for inducing disease progression, including PPROM and preterm birth. Reproductive tissue cytokine production was also significantly diminished in Δnpx mutant-infected animals compared to that in animals infected with wild-type (WT) GBS. Complementation in trans reversed this phenotype, indicating that npx is critical for GBS survival and the initiation of proinflammatory signaling in the gravid host.

converts superoxide to H 2 O 2 and O 2 (33). GBS also produces glutathione and a carotenoid pigment, which protect against oxidative damage (34,35). A previous study revealed that GBS npx encodes an NADH peroxidase that is critical for the detoxification of and resistance to peroxide stress and survival within THP-1 macrophage-like cells (36). The deletion of the npx locus in a Dnpx mutant resulted in an attenuated ability to detoxify peroxide, a result that was reversed via genetic complementation in trans (36).
During ascending vaginal infections in pregnancy, some of the first immune cells that GBS encounters at the maternal-fetal interface are placental macrophages. We hypothesized that npx is also required for GBS to survive within PMs and to cause disease progression during pregnancy. To test this, we sought to characterize its role in pathogenesis in vivo using an established pregnant mouse model of ascending vaginal infection and ex vivo using primary human PMs. Here, we demonstrate that npx is required for GBS survival in PMs, full virulence, and invasion of gravid reproductive tissues in vivo. We have also demonstrated that npx is required for the induction of specific inflammatory cytokines expressed during GBS infection.

RESULTS
Transmission electron microscopy visualization of intracellular GBS in placental macrophages. Transmission electron microscopy (TEM) was used to visualize intracellular GB112 and isogenic mutants in PMs. This analysis revealed that few intracellular Dnpx cells were observed compared to wild-type (WT) GB112 and an isogenic complemented Dnpx derivative harboring a plasmid containing the npx locus (Dnpx::c) (Fig. 1A). Indeed, PM samples infected with WT GB112 averaged 17 bacterial cells per macrophage, while PMs infected with the Dnpx mutant averaged 5 bacterial cells per macrophage. This result was reversed in the strain with a genetic complementation of the npx allele in trans, which infected an average of 16 bacterial cells per macrophage (Fig. 1B). Quantitative culture analyses of living bacterial cells in PMs via gentamicin Streptococcus agalactiae npx Is Critical for Virulence mBio protection assays demonstrated that an average of 1.1 Â 10 7 WT GBS cells survived per 5 Â 10 5 to 1 Â 10 6 macrophages, compared to an average of 2.0 Â 10 5 cells for the Dnpx mutant (P , 0.05 by one-way analysis of variance [ANOVA] with Tukey's post hoc test) (Fig. 1C). Meanwhile, complementation with the Dnpx::c vector restored survival inside macrophages, with an average of 1.4 Â 10 7 cells surviving per 5 Â 10 5 to 1 Â 10 6 macrophages, a result that was comparable to those for WT GB112 (P , 0.01 by one-way ANOVA with Tukey's post hoc test). Taken together, these results show that npx plays an important role in persistence and survival within PMs. Placental macrophage cytokine responses to intracellular GBS infection. Because we observed a difference in intracellular survival among the GB112 (WT), Dnpx, and Dnpx::c strains, we hypothesized that there may be differences in cytokine production by PMs. Following infection of PMs with GBS and its isogenic mutants overnight, the cell supernatants were collected, and cytokine quantifications were performed. Intracellular infection with WT GB112 resulted in the increased secretion of a variety of proinflammatory cytokines, including CXC motif ligand 1 (CXCL1), interleukin 1 receptor antagonist (IL-1RA), interleukin 1a (IL-1a), interleukin 1b (IL-1b), interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1a (MIP-1a), macrophage inflammatory protein 1b (MIP-1b), and tumor necrosis factor alpha (TNF-a) (Fig. 2). Nonetheless, these cytokines were similarly increased in PMs infected with the Dnpx and FIG 2 GBS npx expression is dispensable for the initiation of proinflammatory cytokine production by human placental macrophages. (A) Heat map results (blue, low levels; red, high levels) of multiplex cytokine analyses of primary human placental macrophage-secreted fractions after coculture with wild-type GB112, the isogenic Dnpx mutant, or the isogenic complemented Dnpx derivative (Dnpx::c) reveal that GBS infection induces the production of multiple proinflammatory cytokines compared to the uninfected controls (UI). EGF, epidermal growth factor; FGF, fibroblast growth factor; TGFa, transforming growth factor a; IFNa2, interferon alpha 2; PDGF-AA, platelet-derived growth factor AA; sCD40L, soluble CD40 ligand; VEGF-A, vascular endothelial growth factor A. (B) Quantitation of cytokine levels reveals the significantly enhanced production of G-CSF, GM-CSF, GRO-a, IL-1RA, IL-1a, IL-1b, IL-6, IL-8, MCP-1, MIP-1a, MIP-1b, and TNF-a in macrophages cocultured with the GB112 (red), Dnpx (white), and Dnpx::c (blue) strains compared to the uninfected controls (black). *, P , 0.05; **, P , 0.01; ***, P , 0.001; ****, P , 0.0001 (by one-way ANOVA with Tukey's post hoc multiple-comparison test). #, P , 0.05 (by Student's t test with Welch's correction) (3 biological replicates).
Dnpx::c strains, suggesting that npx does not influence the production of proinflammatory cytokines by PMs ex vivo.
GBS npx is associated with preterm birth in a mouse model of ascending infection. To assess the role of GBS npx in vivo in the context of ascending infection during pregnancy, we utilized a pregnant mouse model initiated by intravaginal GBS infection (37). Previous work demonstrated that in this model, ascending vaginal GBS infection results in bacterial invasion, inflammation of reproductive tissues, preterm prelabor rupture of membranes (PPROM), preterm birth (PTB), as well as maternal and neonatal demise (37). Hence, we hypothesized that Npx could be important for GBS evasion of innate immune responses in the gravid reproductive tract. Our results (Fig. 3) demonstrate that mice infected with WT GB112 had higher incidences of PPROM and PTB than did uninfected and Dnpx mutant-infected animals (P = 0.0024 by a Mantel-Cox log rank test and P = 0.0067 by a Gehan-Breslow-Wilcoxon test). Notably, all GB112-infected animals experienced PPROM or PTB by 6 days postinfection. Complementation of the npx locus in trans resulted in 65% PTB and PPROM by 6 days postinfection; these results were statistically indistinguishable from those for cohorts infected with WT GB112 (P = 0.0766 by a Mantel-Cox log rank test and P = 0.1135 by a Gehan-Breslow-Wilcoxon test). By 7 days postinfection, GB112-infected animals had a 100% maternal mortality rate, which was significantly higher than that for the uninfected controls and Dnpx mutant-infected cohorts, which exhibited 0% maternal mortality by 8 days postinfection (P = 0.0164 by a Mantel-Cox log rank test and P = 0.0442 by a Gehan-Breslow-Wilcoxon test). Complementation in trans partially decreased the mortality rate (33% maternal death by 8 days postinfection), which was statistically indistinguishable from that for WT GB112 (P = 0.2205 by a Mantel-Cox log rank test and P = 0.4190 by a Gehan-Breslow-Wilcoxon test).
Analysis of bacterial burdens in reproductive tissues. To quantify the bacterial burden and classify host responses within reproductive tissues, intravaginal infections were performed using an infectious dose of 5 Â 10 2 to 1 Â 10 3 CFU. Mice were sacrificed at 48 h postinfection to collect reproductive tissues for bacteriological and immunological Wild-type GB112-infected animals (red) had higher incidences of PPROM and PTB than did uninfected (black) and Dnpx mutant-infected (white) animals, with 100% of the GB112-infected cohort experiencing PPROM or PTB by 6 days postinfection. Complementation of the npx locus in trans (Dnpx::c) (blue) resulted in 65% PTB and PPROM by 6 days postinfection. (B) Evaluation of maternal mortality over time (8 days postinfection and term for gestation). GB112-infected animals had a 100% maternal mortality rate by 7 days postinfection, a rate that was significantly higher than those for the uninfected control and Dnpx mutant-infected cohorts, which exhibited 0% maternal mortality by 8 days postinfection. Complementation in trans partially restored mortality (33% maternal death by 8 days postinfection) (n = 6 to 9 dams total from 3 separate experiments) (*, P , 0.05; **, P , 0.01 [by a Mantel-Cox log rank test and a Gehan-Breslow-Wilcoxon test]).
analyses. The bacterial burden was evaluated by quantitative culture methods for reproductive tissues, including uterine, decidual, placental, amnion, and fetal tissues (Fig. 4). The Dnpx mutant exhibited a 3.5-log decrease in the burden in uterine tissue compared to the parental strain (P , 0.01 by one-way ANOVA with Tukey's post hoc multiple-comparison test). Genetic complementation in trans resulted in a significant increase in the bacterial burden (P , 0.05 by one-way ANOVA with Tukey's post hoc multiple-comparison test) compared to the Dnpx mutant. The Dnpx mutant exhibited a 7.5-log decrease in the burden in decidual tissue compared to the parental strain (P , 0.0001 by one-way ANOVA with Tukey's post hoc multiple-comparison test). Genetic complementation in trans resulted in a significant increase in the bacterial burden (P , 0.0001 by one-way ANOVA with Tukey's post hoc multiple-comparison test) compared to the Dnpx mutant. The Dnpx mutant exhibited a 7.2-log decrease in the burden in placental tissue compared to the parental strain (P , 0.0001 by one-way ANOVA with Tukey's post hoc multiple-comparison test). Genetic complementation in trans resulted in a significant increase in the bacterial burden (P , 0.0001 by one-way ANOVA with Tukey's post hoc multiple-comparison test) compared to the Dnpx mutant. The Dnpx mutant exhibited a 5.7-log decrease in the burden in amnion tissue compared to the parental strain (P , 0.0001 by one-way ANOVA with Tukey's post hoc multiple-comparison test). Genetic complementation in trans resulted in a significant increase in the bacterial burden (P , 0.001 by one-way ANOVA with Tukey's post hoc multiple-comparison test) compared to the Dnpx mutant. The Dnpx mutant exhibited a 6.5-log decrease in the burden in fetal tissue compared to the parental strain (P , 0.0001 by one-way ANOVA with Tukey's post hoc multiple-comparison test). Genetic complementation in trans resulted in a significant increase in the bacterial burden (P , 0.0001 by one-way ANOVA with Tukey's post hoc multiple-comparison test) compared to the Dnpx mutant. This in vivo study revealed that Npx is required for GBS to achieve full bacterial burdens in reproductive tissues in a model of ascending infection during pregnancy.
Analysis of bacterial growth in amniotic fluid. Because the Dnpx mutant exhibited attenuated bacterial burdens within reproductive tissues compared to the WT or the complemented derivative, we hypothesized that the Dnpx mutant might have an attenuated ability to grow in certain environments such as amniotic fluid. To test this, we utilized human and mouse amniotic fluid and performed an inoculation with WT GBS, the Dnpx mutant, or the complemented derivative at a 1:100 dilution. Samples were incubated overnight, and viable bacteria were enumerated via serial dilution and quantitative culture techniques. The results indicate that the Dnpx mutant is attenuated in its ability to grow in human amniotic fluid and mouse amniotic fluid (P , 0.001 and P , 0.0001, respectively, by one-way ANOVA with Tukey's post hoc multiple-comparison test), results that were reversed by genetic complementation techniques (see Fig. S1 in the supplemental material).
Expression of GBS npx alters cytokine responses to GBS infection. Because the Dnpx mutant showed decreased bacterial burdens and disease progression in the pregnant mice, we hypothesized that these could be attributed to changes in proinflammatory cytokine production as a consequence of bacterial infection. To test this, we utilized multiplex cytokine assays to quantify the repertoire of cytokines and chemokines produced within reproductive tissues in response to GBS infection and compared them to those produced in uninfected animal tissues. Our results indicate that infection with WT GBS significantly enhanced the production of IL-1b, MIP-1a, and TNF-a in the uterus, decidua, placenta, amnion, and fetus ( Fig. 5 to 9 and Fig. S2 to S6) compared to the uninfected controls (*, P , 0.05 by one-way ANOVA; #, P , 0.05 by Student's t test). Importantly, the deletion of the npx gene resulted in the significantly reduced production of these cytokines and chemokines compared to the levels in WTinfected samples (*, P , 0.05 by one-way ANOVA; #, P , 0.05 by Student's t test). Similarly, granulocyte colony-stimulating factor (G-CSF), MCP-1, MIG, and MIP-2 were significantly upregulated in the uterus, decidua, placenta, and amnion of animals infected with WT GBS compared to the uninfected animals (*, P , 0.05 by one-way ANOVA; #, P , 0.05 by Student's t test), and the loss of the npx gene resulted in significant reductions in the production of these cytokines and chemokines compared to the levels in WT-infected samples (*, P , 0.05 by one-way ANOVA; #, P , 0.05 by Student's t test). IL-6 and MIP-1 were upregulated in the uterus, decidua, placenta, and fetus in response to WT GBS infection compared to the uninfected controls, and the deletion of the npx gene revealed significant reductions in the production of these cytokines and chemokines compared to the levels in WT-infected samples (*, P , 0.05 by oneway ANOVA; #, P , 0.05 by Student's t test). IP-10 was upregulated in the uterus, decidua, and placenta in response to WT GBS infection compared to the uninfected controls, and significant reductions in the production of these cytokines and chemokines were observed with the inactivation of the npx gene compared to the levels in WT-infected samples (*, P , 0.05 by one-way ANOVA; #, P , 0.05 by Student's t test). Complementation with the WT npx allele in trans restored cytokine and chemokine production to levels that were similar to those observed in WT GBS-infected animals or significantly higher than those measured in the isogenic Dnpx mutant-infected samples (*, P , 0.05 by one-way ANOVA; #, P , 0.05 by Student's t test; NS, not statistically significant [statistically indistinguishable from WT GBS]).
Infection with WT GBS resulted in the significantly enhanced production of select cytokines and chemokines, including eotaxin, G-CSF, granulocyte-macrophage colonystimulating factor ( Fig. S6), compared to the uninfected controls (*, P , 0.05 by oneway ANOVA; #, P , 0.05 by Student's t test). The inactivation of the npx gene, however, evoked significant reductions in the production of many of these cytokines and chemokines compared to the levels in WT-infected samples (*, P , 0.05 by one-way ANOVA; #, P , 0.05 by Student's t test).

DISCUSSION
While GBS has been identified as a perinatal pathogen since the 1930s, there are still major gaps in the knowledge of the pathophysiology of infection and disease outcomes by this bacterium. We previously observed that GBS interacts with gestational tissue macrophages and that GBS can invade the reproductive tract in a mouse model of ascending vaginal infection during pregnancy (17,29,38). We sought to understand the importance of individual GBS virulence factors that influence the outcome of GBSmacrophage interactions and might also have significance in clinical outcomes during pregnancy. We previously identified that a peroxide-detoxifying enzyme NADH peroxidase gene, npx, was upregulated when GBS was cultured with THP-1 macrophages (36). We further expanded that work in our current study by demonstrating that GBS npx aids in GBS survival within primary human placental macrophages.
Macrophages represent the second most common leukocytes within fetal membrane tissues, and these cells perform many roles, including regulating tissue remodeling during development and modulating maternal-fetal tolerance (39). Less is understood about the roles that macrophages may play during infection, as these cells are typically thought to be polarized to an anti-inflammatory M2 tolerogenic state (28). Some recent studies have noted that in response to bacteria, these cells change their polarization toward a more inflammatory M1 phenotype (40). GBS has evolved mechanisms such as a capsule to evade phagocytosis by placental macrophages (37), but once engulfed by innate immune cells, GBS deploys enhanced expression of the npx locus as a strategy to survive the peroxide stress encountered within the phagosomes of THP-1 macrophages (36).
Macrophages are implicated as a replicative niche for a variety of bacteria, including  (45), and Legionella pneumophila (46). Recent work has demonstrated that S. pneumoniae can survive and replicate within splenic macrophages, which serve as a reservoir for septicemia (47), and that group A Streptococcus can survive and replicate within human macrophages (48). These results mirror what we observed with GBS in primary human PMs. Furthermore, macrophages have been implicated as a potential Trojan horse aiding in the dissemination of a variety of microbial pathogens, including Candida albicans (49), Mycobacterium tuberculosis (50), Toxoplasma gondii (51), Staphylococcus aureus (52), Cryptococcus neoformans (53), and Chlamydia trachomatis (54). The depletion of host macrophages impedes Chlamydia and GBS dissemination in the reproductive tract (38,55), demonstrating the important role that intracellular bacterial survival within macrophages plays in bacterial invasion of the reproductive tract. Our previous results indicate that GBS utilizes cadD, a metal resistance determinant, to circumnavigate metal stress within placental macrophages and to enhance bacterial ascension and invasion of the gravid reproductive tract (38). Similarly, our current work demonstrates that GBS utilizes npx, a peroxide resistance determinant, to circumnavigate peroxide and TNF-a levels revealed that wild-type GB112 infection (red bars) significantly enhances the production of these cytokines compared to the levels in the uninfected controls (black bars), but the isogenic Dnpx mutant (white bars) is significantly attenuated in its ability to induce these cytokines compared to the parental strain, except for MIP-2. Conversely, the complemented derivative (Dnpx::c) (blue bars) is often statistically indistinguishable from the parental strain (not statistically significant [NS]). Bars indicate mean values 6 standard errors of the means, with individual data points representing results from placental tissues from individual fetal-placental units from separate dams. #, P , 0.05 (by Student's t test). *, P , 0.05; **, P , 0.01; ***, P , 0.001; ****, P , 0.0001 (by one-way ANOVA with Tukey's post hoc multiple-comparison test). The results indicate that GBS npx is required for the initiation of numerous proinflammatory cytokine responses in placental tissues in a model of ascending infection during pregnancy.
Streptococcus agalactiae npx Is Critical for Virulence mBio stress within placental macrophages and to aid in ascending infection and disease progression during pregnancy. Taken together, these results support the hypothesis that GBS could exploit macrophages as a Trojan horse to aid in the promotion of invasive bacterial infections during pregnancy. Upon the recognition of pathogens by immune pattern recognition receptors (PRRs) such as Toll-like receptors, a cascade of responses by the macrophage is initiated. One mechanism of defense is the phagocytosis of the bacterial cell and chemical assault within the phagosome via the deployment of peroxides and reactive oxygen species (56). Highly reactive oxygen species can damage macromolecules, including lipids, proteins, and nucleic acids, ultimately leading to cell death (30). Within the reproductive tract, invading pathogens, including C. trachomatis (57) and N. gonorrhoeae (58), are assaulted with reactive oxygen species. There is an association between Chlamydia and spontaneous abortion, with oxidative stress being implicated, highlighting the oxidative response in the reproductive tract against invading pathogens (57). Additionally, the presence of ROS has been demonstrated in human amniotic fluid collected in the second and third trimesters of gestation (59). This likely presents an environmental challenge for bacteria, which are highly sensitive to oxidative stress, results that are supported by the survival and growth defects observed in the Dnpx mutant compared to the wild type and the complemented derivative grown in human or mouse amniotic fluid.
In response to this, bacterial pathogens have evolved a range of mechanisms to   (33). Our study demonstrates that the full repertoire of antioxidant defenses is required for invasive infection of the gravid reproductive tract and that the npx-encoded NADH peroxidase aids in GBS survival within reproductive tissue macrophages and is critical for full virulence in a pregnant animal model. Interestingly, a dye-neutralizing peroxidase (DyP) has been identified in M. tuberculosis, which is critical for bacterial survival within host macrophages as well (68). Similarly, in Listeria monocytogenes, peroxidases encoded within the fri and ahpA loci were each required for L. monocytogenes to survive acute peroxide stress, and the fri locus was essential for cytosolic growth within host macrophages (69). These studies further underscore the critical role that bacterial peroxidases play in the host-pathogen dialogue, specifically with respect to intracellular survival. In addition to aiding in GBS intracellular survival within host immune cells, the npx locus is critical for virulence in vivo. In our mouse model of ascending vaginal infection during pregnancy, we observed that mutants lacking npx showed impaired invasion of gestational tissues, cognate inflammatory responses, and disease compared to the parental strain or the complemented mutant, demonstrating the importance of npx for pathogenesis (Fig. 10). We observed drastic reductions in the bacterial burdens in the uterus, decidua, placenta, amnion, and fetal tissue compartments derived from animals infected with the Dnpx mutant compared to the burdens in animals infected with the parental strain or the complemented isogenic derivative. These reductions in burdens correlated with the decreased production of proinflammatory cytokines such as IL-1b, MIP-1a, and TNF-a in all of the examined tissue compartments of the gravid reproductive tract. Interestingly, the diminutions of proinflammatory cytokine production and bacterial burdens were associated with cognate decreases in adverse pregnancy outcomes such as PPROM, preterm birth, and maternal demise. Previous work has linked high levels of IL-1b, MIP-1a, and TNF-a with an enhanced risk of preterm birth (70). It is likely that the expression of proinflammatory cytokines perturbs maternal tolerance of the semiallogeneic fetus, leading to an enhanced risk of adverse pregnancy outcomes (71). Recently, interest has been piqued in exploiting the NLRP3 inflammasome pathway as a potential chemotherapeutic strategy to ameliorate the risk associated with perinatal disease outcomes (72). Because the expression of the npx locus is critical for GBS ascension of the reproductive tract and the initiation of these signaling pathways that promote inflammation, a dually targeted approach of the inhibition of the GBS NADH peroxidase and the NLRP3 inflammasome pathway could prove useful in combating GBS perinatal infections.
Intracellular infection of PMs resulted in the production of proinflammatory cytokines such as G-CSF, GM-CSF, growth-regulated protein alpha (GRO-a), IL-1RA, IL-1a, IL-1b, IL-6, IL-8, MCP-1, MIP-1a, MIP-1b, and TNF-a. However, npx was dispensable for FIG 10 Conceptual model of npx-dependent invasion and proinflammatory signal initiation in ascending GBS infections during pregnancy. GBS ascends the reproductive tract by surviving within placental macrophages, in part by encoding an NADH peroxidase to aid in detoxifying oxidative stress (such as peroxides [H 2 O 2 ]). GBS invasion and full burdens within the gravid reproductive tract (uterus, decidua, placenta, amnion, and fetus) require npx expression and trigger the production of tissue compartment-specific proinflammatory cytokines, resulting in inflammation and disease progression, including rupture of membranes, preterm birth, and maternal demise. (Image created with BioRender.com.) this induction. This was a surprising result considering the significant differences in bacterial loads within these macrophages, underscoring that even low levels of GBS intracellular infection are sufficient to induce the production of these cytokines. The production of proinflammatory cytokines by macrophages instigates the recruitment of neutrophils in mice upon GBS infection (15,73). A similar response has also been observed in humans (73). Neutrophils aid in the clearance of bacteria by phagocytosis and subsequent killing in internal vacuoles called phagosomes (74). Finally, neutrophils excrete neutrophil extracellular traps (NETs) loaded with various antimicrobial peptides (75). These extracellular DNA traps have been exhibited in response to GBS infection (15). Collectively, placental macrophages and neutrophils predominate the innate immune response against GBS infection.
Our studies indicate that NADH peroxidase plays an important role in the full virulence of GBS in a mouse model of ascending vaginal infection during pregnancy. Other reactive oxygen species-detoxifying enzymes such as catalase and superoxide dismutase are also important for the virulence of many bacterial pathogens, especially those that survive and establish replicative niches within macrophages (76,77). The introduction of bacteriumspecific inhibitors of oxidative stress response pathways may target bacteria without impacting the host, providing exciting new avenues for drug development. As such, developing small molecules or other chemotherapeutic strategies to inhibit these enzymes may be a viable option to defend against these bacterial infections. The identification of these bacterium-specific inhibitors is being researched currently and includes small molecules that inhibit M. tuberculosis catalase (78). A plausible future direction of our work may include screening preexisting banks of small molecules against purified GBS NADH peroxidase protein or creating a crystal structure to identify regions for targeted drug design.

MATERIALS AND METHODS
Bacterial strains and culture conditions. S. agalactiae strain GB00112 (GB112), which represents the wild-type (WT) or parental strain, was utilized in this study. GB112 is a clinical, sequence type 12 (ST-12), capsular polysaccharide (CPS) serotype III strain isolated from a rectovaginal swab of a postpartum patient (79). We previously examined GB112 for interactions with host macrophages and fetal membranes (36,80). Isogenic GB112 mutants were constructed previously. These mutants include an npx deletion mutant (Dnpx) and a complemented Dnpx mutant harboring a plasmid containing the npx locus (Dnpx::c), as previously described (36). Bacterial strains were grown on tryptic soy agar plates supplemented with 5% sheep blood or in Todd-Hewitt broth (THB) at 37°C. Derivatives harboring the pLZ12 plasmid were grown in medium supplemented with 3 mg/mL chloramphenicol. E. coli DH5a strains used for the mutation and complementation processes were grown in LB broth or agar supplemented with either 150 mg/mL erythromycin or 20 mg/mL chloramphenicol when necessary.
Purification of placental macrophages. Deidentified placental tissue was collected from nonlaboring women who delivered healthy, full-term infants by Caesarian section at the Vanderbilt University Medical Center with approval from the Vanderbilt University Medical Center Institutional Review Board (VUMC IRB) (approval number 181998). Placental macrophages (PMs) were isolated according to our previously described methods (80,81). Briefly, villous core tissue was macerated and enzymatically digested with hyaluronidase, collagenase, and DNase (Sigma-Aldrich) before being strained through a stainless steel filter and suspended in RPMI 1640 with HEPES, L-glutamine, and fetal bovine serum supplemented with antibiotic and antifungal factors. Cells were filtered and centrifuged, and CD14 1 cells were isolated using the magnetic activated cell sorting (MACS) cell separation system with CD14 microbeads (Miltenyi Biotec). Cells were incubated in RPMI 1640 medium (Thermo Fisher) with 10% charcoal-stripped fetal bovine serum (FBS) (Thermo Fisher) and a 1% antibiotic-antimycotic solution (Thermo Fisher) overnight at 37°C in 5% carbon dioxide. The following day, PMs were suspended in RPMI 1640 medium without antibiotic-antimycotic and distributed into polystyrene plates. Cells were seeded at a density of 200,000 cells per well in a polystyrene, 24-well culture plate in RPMI 1640 with a 1% antibiotic-antimycotic solution and 10% charcoal dextran FBS (RPMI 1/1 ) and then incubated for 24 h in a humidified atmosphere at 37°C with 5% CO 2 .
Coculture of GBS with placental macrophages and survival assays. PMs were cultured in RPMI 1640 medium (Thermo Fisher) with 10% charcoal-stripped fetal bovine serum (Thermo Fisher) and a 1% antibiotic-antimycotic solution (Thermo Fisher) overnight at 37°C in room air supplemented with 5% carbon dioxide. Cocultured cells were incubated at 37°C in air supplemented with 5% carbon dioxide for 1 to 24 h with medium free of the antibiotic-antimycotic solution. Macrophages were inoculated at a multiplicity of infection (MOI) of 10:1 bacteria to host cells for 1 h. Cocultures were washed with sterile medium, resuspended in fresh medium containing 100 mg/mL of gentamicin (Sigma) to kill extracellular bacteria, and further incubated for 4 h at 37°C. Gentamicin kills extracellularly located GBS but is limited in its ability to gain access to intracellular organisms. Subsequently, the samples were extensively washed with sterile phosphate-buffered saline (PBS) and dislodged with trypsin (1Â 0.05% trypsin-EDTA; Gibco). Following the collection of the PMs, the cells were lysed by the addition of 1 mL of distilled water (dH 2 O). Cellular cytoplasmic contents were serially diluted in PBS and plated onto blood agar plates to determine the number of viable intracellular bacteria. Samples containing only bacteria were used to estimate the efficacy of antibiotic killing as a control experiment (82).
Transmission electron microscopy analyses. Cocultures of GBS and macrophages were subjected to primary fixation with 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.05 M sodium cacodylate buffer at room temperature for 24 h. Subsequently, samples were washed three times with 0.05 M sodium cacodylate buffer and subjected to a secondary fixation step with 0.1% osmium tetroxide for 15 min. Samples were washed three times with 0.05 M sodium cacodylate buffer before being sequentially dehydrated with increasing concentrations of ethanol. After dehydration, samples were embedded in resin, polymerized, and sectioned into 70-to 90-nm sections via ultramicrotomy. Sections were lifted onto nickel or copper 100-mesh grids (Electron Microscopy Sciences) and secondarily stained with 1% phosphotungstic acid. Grids were imaged with a Philips/FEI T12 transmission electron microscope to visualize intracellular bacteria. Bacteria were enumerated in a blind manner using the ImageJ software package.
Ascending vaginal infection model. GBS infection of pregnant mice and subsequent analyses were performed as previously described (15,37). Briefly, C57BL/6J mice were purchased from Jackson Laboratories and mated in harem breeding strategies (1 male to 3 to 4 females) overnight. Pregnancy was confirmed by the presence of a vaginal mucus plug establishing the embryonic date (embryonic day 0.5 [E0.5]) the following day. On E13.5, pregnant dams were anesthetized via the inhalation of isoflurane and vaginally infected with 5 Â 10 2 to 10 4 CFU in 0.05 mL of THB plus 10% gelatin. Uninfected controls were also maintained. Infections were allowed to progress until term (E21.5) for disease progression studies or until E15.5 for bacterial and immunological assays. Animals were euthanized by carbon dioxide asphyxiation, and necropsy was performed to harvest reproductive tissues, including the uterus, placenta, decidua, amnion, and fetus tissues.
Quantitative culture evaluation of bacterial burdens. To determine the bacterial burden in reproductive tissues, quantitative culture methods were employed, as previously described (37). Briefly, reproductive tissues were weighed and placed into sterile THB. Tissues were homogenized, subjected to serial dilution, and plated onto blood agar to quantify bacteria (CFU per milligram) in host tissue. Plates were incubated at 37°C in air supplemented with 5% carbon dioxide overnight, and individual colonies were counted to quantify CFU.
Evaluation of bacterial growth in amniotic fluid. To determine bacterial growth in human or mouse amniotic fluid, quantitative culture methods were employed, as described above. Briefly, human amniotic fluid was pipetted off the surface of the placentas from term, nonlaboring C-section deliveries. Mouse amniotic fluid was collected after necropsy on embryonic day 15.5. All amniotic fluid samples were stored at 280°C until utilization for growth assays. Amniotic fluid samples (100 L) were placed into 96-well plates, and a 1:100 dilution of a bacterial culture grown overnight (either WT GBS, the Dnpx mutant, or the complemented derivative) was inoculated into the amniotic fluid. Samples were incubated overnight (16 to 24 h) at 37°C in room air supplemented with 5% CO 2 . The following day, viable bacteria were enumerated via serial dilution and plating onto bacteriological medium (blood agar plates). Plates were incubated at 37°C in room air supplemented with 5% CO 2 overnight, and the following day, bacterial colonies were counted to enumerate viable bacterial cells.
Evaluation of cytokine responses to GBS infection. Mouse reproductive tissues, maternal sera, and amniotic fluid were analyzed by multiplex cytokine assays. Mouse tissues were placed into 0.5 mL of sterile PBS or THB plus 10 mg/mL penicillin, homogenized, and passed through a 0.22-mm filter. Samples were frozen at 280°C or on dry ice until analyses were performed. Samples were analyzed by Eve Technologies (Alberta, Canada) via a multiplex cytokine array, as previously described (83). Validation of host targets for specific cytokines (IL-1b, IL-6, KC, and TNF-a) was performed by a sandwich enzyme-linked immunosorbent assay (ELISA) (Abcam), as indicated in our previous study (84).
Ethics statement. This study was carried out in accordance with the recommendations of the Vanderbilt University Medical Center Institutional Review Board. This protocol was approved by the IRB (approval numbers 181998 and 00005756). All animal experiments were performed in accordance with the Animal Welfare Act, U.S. federal law, and NIH guidelines. All experiments were carried out under a protocol approved by the Vanderbilt University Institutional Animal Care and Use Committee (IACUC) (approvals M/14/034, M/17/002, and V2000089), a body that has been accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
Statistical analyses. Statistical analysis of parametric data with more than two groups was performed using one-way ANOVA with either Tukey's or Dunnett's post hoc correction for multiple comparisons; all reported P values were adjusted to account for multiple comparisons. For parametric data with two groups, Student's t test or one-way ANOVA was used. P values of #0.05 were considered significant. Nonparametric data (such as log-transformed CFU data) were analyzed by Mann-Whitney U or Kruskal-Wallis tests. Disease outcome data, including survival curves, were analyzed using the Mantel-Cox log rank test and the Gehan-Breslow-Wilcoxon test. All data analyzed in this work were derived from at least three biological replicates (representing different human or mouse samples). Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software Inc.).
Data availability. Data are available upon reasonable request to the authors.  We declare no competing interests.