Streptococcus pneumoniae remains a global health threat, particularly to young children, the elderly, and immunocompromised individuals. Pneumococcal vaccines targeting the bacterial capsule polysaccharide do not protect against all 100+ pneumococcal serotypes, contributing to non-vaccine serotype infections and antibiotic resistance. To address these limitations, we isolated human monoclonal antibodies (mAbs) targeting pneumococcal surface proteins and identified a first-in-class mAb, derived from a patient with prior pneumococcal infection, namely mAb 5995-40. mAb 5995-40 bound multiple pneumococcal proteins, including PcpA and PspA, through a conserved choline-binding domain shared across serotypes. Functionally, mAb 5995-40 provided complete protection in lethal pneumococcal challenge models and improved survival in influenza A, influenza B, and respiratory syncytial virus-associated bacterial coinfection models. Mechanistic studies showed enhanced opsonophagocytic killing, reduced bacterial dissemination, and blocked epithelial translocation. Cryo-electron microscopy identified a repeating motif within the choline-binding domain targeted by mAb 5995-40, highlighting its potential as a broadly protective pneumococcal therapeutic.

Serotype 3 (ST3) Streptococcus pneumoniae remains a major cause of invasive pneumococcal disease and pneumonia despite PCV13 introduction, in part due to potent immune evasion properties. The contribution of the pyruvate metabolic node (SpxB/LctO pathways) to ST3 pathogenesis is poorly defined. We investigated oxygen-dependent fitness, colonization efficiency, and lung pathology in the ST3-strain WU2 and isogenic ΔspxB, ΔlctO, and ΔspxBΔlctO mutants. In vitro growth was assessed under nasopharyngeal (21

Antimicrobial resistance (AMR) and virulence have traditionally been viewed as competing traits in bacterial evolution due to fitness costs. However, Streptococcus pneumoniae has emerged as a paradigm of successful coevolution, with multidrug-resistant clones simultaneously maintaining or enhancing pathogenic potential. This review examines the molecular mechanisms, epidemiological patterns, and clinical consequences of the convergence between AMR and virulence in Streptococcus pneumoniae. Resistance to β-lactams is driven by mosaic penicillin-binding protein genes (pbp1a, pbp2b, pbp2x), while macrolide resistance is mediated primarily by the erm(B) gene (MLS phenotype) and mef(A/E)-msr(D) genes encoding an efflux system. These determinants are frequently co-localized on integrative and conjugative elements, ICEs, (e.g., Tn916 family) within successful clonal complexes such as CC271/320 and lineages including ST320 and GPSC10. Contrary to the classical fitness cost hypothesis, compensatory epistasis, capsular recombination, metabolic adaptations, and intra-serotype phenotypic variation enable certain clones to combine high-level resistance to β-lactams, macrolides, and tetracyclines with enhanced colonization, biofilm formation, immune evasion, and invasive capacity. Post-pneumococcal conjugate vaccine (PCV) surveillance reveals the persistence and expansion of these high-risk lineages, contributing to treatment-refractory invasive pneumococcal disease (IPD), increased morbidity, and mortality. Although PCVs have reduced vaccine-type resistant strains in some settings, serotype replacement and emerging metabolic genotypes continue to drive adaptation. This review highlights the need for integrated genomic surveillance, novel therapeutics (e.g., omadacycline, lefamulin, endolysins), monoclonal antibodies, and next-generation vaccines targeting both resistance and conserved virulence determinants. A multifaceted strategy combining antimicrobial stewardship, strengthened surveillance, and innovative interventions is essential to curb the evolving threat of resistant and virulent S. pneumoniae.

Background:Serotype 3 (ST3) Streptococcus pneumoniae remains a major cause of invasive pneumococcal disease and pneumonia despite PCV13 introduction, in part due to potent immune evasion properties. The contribution of the pyruvate metabolic node (SpxB/LctO pathways) to ST3 pathogenesis is poorly defined. Methods:We investigated oxygen-dependent fitness, virulence, and lung pathology in the ST3 strain WU2 and isogenic ΔspxB, ΔlctO, and ΔspxBΔlctO mutants. In vitro growth was assessed under nasopharyngeal (21% O2) and alveolar (14% O2) conditions. Murine pneumonia models evaluated survival, bacterial burdens, histopathology (H&E, confocal microscopy), and lung transcriptomics (RNA-seq). Results:ST3-strain exhibited a unique oxygen-sensitive growth defect at 21% O2, alleviated by spxB deletion, indicating metabolic burden from pyruvate flux. In mice, wild-type WU2 caused high mortality with severe suppurative bronchopneumonia, alveolar consolidation, hemorrhage, and perivascular inflammation. The ΔspxB mutant accelerated lethality with enhanced distal lung damage, uncontrolled dissemination, and amplified inflammation. Wild-type infection uniquely induced targeted reorganization of bronchial epithelial membranes, forming prominent bacterium-laden blebs-host-derived membrane protrusions encapsulating intact pneumococci. These novel structures facilitated organized bacterial translocation into tissue without overt cytotoxicity and were largely absent in spxB-deficient mutants despite comparable lung burdens. RNA-seq analysis revealed SpxB-dependent suppression of T cell activation (e.g., Rag1 and Themis) and acute inflammatory pathways, consistent with immune sequestration via blebs. Conclusions:The SpxB-dependent pathway orchestrates ST3 virulence by enabling metabolic adaptation and driving bleb-mediated epithelial invasion and immune evasion in the lung. These bacterium-laden blebs represent a novel hallmark mechanism in pneumococcal pathogenesis, offering new insights and potential therapeutic targets.

ABSTRACT Infections caused by Streptococcus pneumoniae (Spn, pneumococcus), manifesting as pneumonia, meningitis, and otitis media, represent a leading cause of severe morbidity and mortality globally. Escalating antibiotic resistance and serotype replacement by conjugate vaccines underscore the need for serotype-independent therapies. Hypothiocyanous acid (HOSCN), a reactive oxidant naturally produced by the host innate immune system, is formed through the interaction of hydrogen peroxide, thiocyanate anions (SCN−), and peroxidase enzymes. HOSCN is produced at mucosal surfaces, including the respiratory epithelium, where it functions as a broad-spectrum oxidative agent. HOSCN has been reported to kill Spn in vitro, but tolerance mechanisms of Spn against HOSCN have also been described. This review synthesizes recent advances in understanding HOSCN’s molecular mechanisms of action, tolerance pathways of Spn, and the effects of HOSCN on host-Spn interactions. The article also discusses current limitations and key challenges that must be addressed to translate HOSCN from preclinical promise to clinical application in the management of Spn lung infections. By leveraging an innate microbicidal pathway already active in the healthy lung, HOSCN-based therapeutics could offer rapid, resistance-refractory control of pneumococcal disease.

Pneumococcal pneumonia continues to be a significant global health burden, affecting both children and adults. Traditional diagnostic methods for sputum analysis remain challenging. The objective of this study was twofold: to develop a rapid and easy-to-perform assay for the identification of Streptococcus pneumoniae (Spn) directly in sputum specimens using fluorescence microscopy, and to characterize with high-resolution confocal microscopy the ultrastructure of pneumococci residing in human sputum. We fluorescently labeled antibodies against the pneumococcal capsule (Spn-FLUO). The specificity and sensitivity of Spn-FLUO for detecting Spn was evaluated in vitro and in vivo using mouse models of carriage and disease, human nasopharyngeal specimens, and sputum from patients with pneumococcal pneumonia. Spn was confirmed in the specimens using culture and a species-specific qPCR assay. Spn strains were serotyped by Quellung. Confocal microscopy and Imaris software analysis were utilized to resolve the ultrastructure of pneumococci in human sputum. Compared with cultures and qPCR, Spn-FLUO demonstrated high sensitivity (78–96

Streptococcus pneumoniae (Spn), a primary cause of pneumonia, induces acute lung parenchymal damage through a unique metabolic pathway generating hydrogen peroxide (H₂O₂) as a byproduct. This study demonstrates that Spn-derived H₂O₂, primarily produced by pyruvate oxidase (SpxB), inhibits key tricarboxylic acid (TCA) cycle enzymes (aconitase, glutamate dehydrogenase, and α-ketoglutarate dehydrogenase) in lung epithelial cells, leading to citrate accumulation and diminished NADH production for oxidative phosphorylation. RNA sequencing reveals SpxB-dependent upregulation of glycolytic genes (HIF1A, IER3, HK2, PFKP), restricting pyruvate entry into the TCA cycle and increasing glucose consumption and lactate/acetate production, indicative of a Warburg-like metabolic shift that may enhance bacterial survival. Notably, mitochondrial membrane potential remains largely preserved, with minimal apoptosis despite Spn-induced stress. These findings uncover a novel mechanism of Spn-driven host metabolic reprogramming, highlighting potential therapeutic targets for pneumococcal diseases.IMPORTANCEStreptococcus pneumoniae (Spn) remains a leading cause of community-acquired pneumonia worldwide, yet the mechanisms by which it manipulates host metabolism to promote its survival and pathogenesis are not fully understood. This study reveals a novel metabolic strategy whereby pneumococcus-derived hydrogen peroxide, generated by pyruvate oxidase (SpxB), disrupts the host tricarboxylic acid (TCA) cycle and drives a Warburg-like metabolic shift in lung epithelial cells. By inhibiting key TCA cycle enzymes and rewiring glycolytic gene expression, Spn effectively reprograms host cell metabolism to favor its persistence while minimizing host cell apoptosis and maintaining mitochondrial function. These insights expand our understanding of host-pathogen metabolic interactions and identify potential metabolic vulnerabilities that could be targeted to mitigate tissue damage and improve treatment outcomes in pneumococcal pneumonia.

BACKGROUND:In the United States, the invasive pneumococcal disease incidence in Indigenous populations is higher than in the general population. Molecular detection and/or specimen sampling from multiple body sites could enhance our understanding of pneumococcal carriage, a prerequisite for disease. METHODS:Persons aged <5 and ≥18 years from the Navajo Nation and White Mountain Apache Tribal lands were enrolled in an observational carriage study from October 2015 through September 2017. Swabs from the nasopharynx (all participants) and oropharynx (adults only) were collected and tested by enriched culture or molecular methods (lytA and piaB polymerase chain reaction [PCR]). Cultured Streptococcus pneumoniae was serotyped by sequencing. PCR-positive samples were serotyped by 13-valent pneumococcal conjugate vaccine (PCV13)-type PCR and TaqMan array card PCR. RESULTS:An overall 1503 participants were enrolled (age <5 years, n = 600; ≥18 years, n = 903). Among children, pneumococcal positivity was similar by culture (49.5%) and PCR (50.8%); PCV13-type carriage was 8.0% by any method. Among adults, oropharyngeal swab positivity by PCR was 18.5%, an increase when compared with cultured oropharyngeal swabs (0.6%) and nasopharyngeal swabs by culture (7.9%) or PCR (5.3%); PCV13-type carriage by any sample or method was 8.0%. CONCLUSIONS:PCV13-type carriage persists in Indigenous populations. Use of molecular methods and oropharyngeal swabs for adults increased carriage prevalence estimates.

Pneumococcal conjugate vaccines (PCVs) have influenced population dynamics of Streptococcus pneumoniae in the nasopharynx and may have contributed to increased Staphylococcus aureus colonization. This study assessed the prevalence of colonization, antibiotic resistance patterns, and associated risk factors for colonization and co-colonization of S. aureus and S. pneumoniae in healthy Peruvian children post-PCV introduction. Nasopharyngeal swabs from children <24 months were collected in five hospitals in Lima (2018-2019). Microbiological identification and antibiotic susceptibility tests were performed, and multinomial regression evaluated factors influencing colonization. Among 894 children, 19.7% were colonized with S. aureus, 20.3% with S. pneumoniae, and 2.9% co-colonized. Of the 176 S. aureus strains isolated, 1.7% were methicillin resistant and 20.5% were clindamycin resistant; no resistance to trimethoprim-sulfamethoxazole (SXT) was found. Among 182 S. pneumoniae strains isolated, 48.9% were resistant to macrolides, 74.7% to SXT; no resistance to penicillin was found. Breastfeeding and vaccination with PCV13 were associated with a reduced prevalence of S. aureus colonization, while vaccination with PCV13 increased the prevalence of S. pneumoniae colonization, mainly by non-vaccine serotypes. This study highlights the need to continue monitoring the changes in colonization dynamics and antimicrobial resistance patterns after vaccine introduction, to guide empirical therapy and future vaccine strategies.

Introduction:Nonencapsulated Streptococcus pneumoniae (NESp) are isolated worldwide. Due to the lack of capsule in NESp strains the current vaccines, that target the pneumococcal capsule are ineffective. Some NESp contain the oligopeptide transporters AliC and AliD which are required for virulence through unknown mechanisms. AliC and AliD have been previously shown to reduce rates of phagocytosis and alter the transcriptome and proteome of MNZ41. We hypothesize that oligopeptide regulated genes are responsible for reduced phagocytosis and increased survival through resistance to reactive oxygen species (ROS). Methods:To test this a mutant library of AliC and AliD regulated genes was used in in vitro and in vivo models. ROS resistance was tested through quantifying bacterial counts after exposure to hydrogen peroxide (H2O2). A modified surface killing assay was also used to calculate resistance to phagocytosis of our mutant library. A Galleria mellonella larvae model of infection was used to determine survival curve analyses. Results:Two mutant genes in our library, ∆lytFN1 (CDT04) and ∆mgtC (CDT05), displayed greater sensitivity to H2O2 killing and phagocytosis compared to wildtype MNZ41. Deletion of AliD in an AliD-expressing encapsulated strain reduced virulence. Conclusion:This research demonstrates that proteins encoded by genes regulated by AliC and AliD alter susceptibility to host-derived mechanisms for bacterial clearance and increases bacterial survival in response to ROS.

Streptococcus pneumoniae (the pneumococcus) causes cytotoxicity and encapsulates within the lung parenchyma, leading to pneumococcal pneumonia. However, the underlying mechanisms remain unclear and likely involve multiple bacterial and host factors.We investigated the selection process of encapsulated pneumococci, a critical factor in lung damage during pneumococcal pneumonia.Our study revealed that pneumococci initially lack capsules but re-encapsulate upon reaching the alveoli. This process is driven by S. pneumoniae-derived hydrogen peroxide (Spn-H₂O₂), which oxidizes lung hemoglobin, leading to heme release and polymerized hemoglobin formation. Physiologically relevant levels of heme were found to promote the selection of encapsulated bacteria. Furthermore, encapsulation protects bacteria from intracellular heme toxicity, a defense absent in non-encapsulated strains. Ultrastructural analysis demonstrated interactions between hemoglobin and both encapsulated and non-encapsulated pneumococci in human sputum.These findings reveal a critical connection between oxidative stress-mediated lung damage and the selection of encapsulated pneumococci, suggesting potential therapeutic avenues by targeting these oxidative processes.

AIMS:To investigate the molecular events associated with acquiring macrolide resistance genes [mefE/mel (Mega) or ermB] in Streptococcus pneumoniae (Spn) during nasopharyngeal colonization. METHODS AND RESULTS:Genomic analysis of 128 macrolide-resistant Spn isolates revealed recombination events in genes of the conjugation apparatus, or the competence system, in strains carrying Tn916-related elements. Studies using confocal and electron microscopy demonstrated that during the transfer of Tn916-related elements in nasopharyngeal cell biofilms, pneumococcal strains formed clusters facilitating their acquisition of resistance determinants at a high recombination frequency (rF). Remarkably, these aggregates comprise both encapsulated and nonencapsulated pneumococci that span extracellular and intracellular compartments. rF assessments showed similar rates regardless Mega was associated with large integrative and conjugative elements (ICEs) (>23 kb) or not (∼5.4 kb). The rF for Mega Class IV(c) insertion region (∼53 kb) was three orders of magnitude higher than the transformation of the capsule locus. Metabolomics studies of the microenvironment created by colonization of human nasopharyngeal cells revealed a link between the acquisition of ICEs and the pathways involving nicotinic acid and sucrose. CONCLUSIONS:Pneumococcal clusters, both extracellular and intracellular, facilitate macrolide resistance acquisition, and ICEs were acquired at a higher frequency than the capsule locus. Metabolic changes could serve as intervention targets.

Streptococcus pneumoniae is a major cause of invasive disease of young children in low- and middle-income countries. In southern India, pneumococcal conjugate vaccines (PCVs) that can prevent invasive pneumococcal disease began to be used more frequently after 2015. To characterize pneumococcal evolution during the early time period of PCV uptake in southern India, genomes were sequenced and selected characteristics were determined for 402 invasive isolates collected from children <5 years of age during routine surveillance from 1991 to 2020. Overall, the prevalence and diversity of vaccine type (VT) and non-vaccine type (NVT) isolates did not significantly change post-uptake of PCV. Individually, serotype 1 and global pneumococcal sequence cluster (GPSC or strain lineage) 2 significantly decreased, whereas serotypes 6B, 9V and 19A and GPSCs 1, 6, 10 and 23 significantly increased in proportion post-uptake of PCV. Resistance determinants to penicillin, erythromycin, co-trimoxazole, fluoroquinolones and tetracycline, and multidrug resistance significantly increased in proportion post-uptake of PCV and especially among VT isolates. Co-trimoxazole resistance determinants were common pre- and post-uptake of PCV (85 and 93 %, respectively) and experienced the highest rates of recombination in the genome. Accessory gene frequencies were seen to be changing by small amounts across the frequency spectrum specifically among VT isolates, with the largest changes linked to antimicrobial resistance determinants. In summary, these results indicate that as of 2020 this pneumococcal population was not yet approaching a PCV-induced equilibrium and they highlight changes related to antimicrobial resistance. Augmenting PCV coverage and prudent use of antimicrobials are needed to counter invasive pneumococcal disease in this region.

Macrolide antibiotics are recommended for the treatment of pneumococcal pneumonia and invasive pneumococcal disease (IPD). Prior to 2000, ∼10% of Streptococcus pneumoniae strains isolated from IPD cases in Latin American countries were resistant to macrolides. The mechanism of resistance to macrolides was associated mainly with the efflux pump known as the macrolide efflux genetic assembly, since most pneumococcal strains carried the mef(A/E) gene, whereas <6% strains carried both the methylase gene ermB and mef(A/E). In the first decade of this century, a significant increase in the prevalence of macrolide resistance was observed in pneumococcal strains in both Mexico and Peru. Approximately 30% of S. pneumoniae strains in these countries were already resistant to erythromycin, while the prevalence in Colombia, Argentina, and Brazil remained below 10%. During the last decade, we have been experiencing a worrisome increase in pneumococcal strains carrying resistance to macrolides, with a prevalence of up to 80% for resistance to erythromycin. The mechanism for disseminating macrolide resistance has evolved. Currently, more than 55% of invasive S. pneumoniae macrolide-resistant strains carry both the ermB and the mef(A/E)/mel genes. Lessons learned from the current macrolide resistance crisis in Latin America can inform interventions in other regions.

Air pollution is a major global health issue and a significant risk factor for respiratory infections. Air pollution containing inhalable particulate matter (PM), including Diesel Exhaust Particles (DEP), Urban Particles (UP), tobacco smoke particles, dust particles, ambient Black Carbon (BC), household smoke, etc., emitted from vehicles, industry, construction, agriculture waste burning, cooking, etc., exerts a negative effect on human health. The exposure of inhalable PM to the upper airways, often colonized by opportunistic microbes, represents a unique risk for respiratory infections. Several epidemiological studies reported that PM exposure increases susceptibility to, and severity of, lower respiratory infections like pneumonia or other important diseases such as otitis media, asthma, lung cancer, cardiovascular disease, and Chronic Obstructive Pulmonary disease (COPD). It has been suggested that inhalable PM exposure damages airway epithelial cells, alters the immune response, affects the microbiota, and, as a result, opportunist or pathogenic bacteria (Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, or Staphylococcus aureus) establishes respiratory infections. PM and bacteria interaction alters bacteria physiology and enhances bacterial proliferation and biofilm mode of growth. However, the exact mechanism pertaining to how the PM reverts the opportunistic bacteria of the nasopharynx to a pathogenic state is not well understood. In the present review, we have focused on understanding the airborne PM and bacteria interaction that makes humans more susceptible to otherwise harmless bacteria, especially those in the upper airways. Further, we have provided an overview of potential mechanisms triggered by air pollutants to induce bacterial infectious diseases.

Background: Because of the rise in antibiotic resistance and the control of pathogenicity, polymicrobial bacterial biofilms exacerbate wound infections. Since bacterial quorum sensing (QS) signals can dysregulate biofilm development, they are interesting therapeutic treatments. In this study, Pseudomonas Quinolone Signal (PQS) was used to treat an animal model of a wound that had both Staphylococcus aureus and Pseudomonas aeruginosa co-infection. Methods: S. aureus and P. aeruginosa mono- and co-infection models were developed in vitro on the L-929 cell line and in an animal model of wound infection. Moreover, PQS was extracted and purified using liquid chromatography. Then, the mono- and co-infection models were treated by PQS in vitro and in vivo. RT-PCR analysis was used to look into changes in biofilm, QS, tissue regeneration, and apoptosis genes after the treatment. Results: PQS significantly disrupted established biofilm up to 90% in both in vitro and in vivo models. Moreover, a 93% reduction in the viability of S. aureus and P. aeruginosa was detected during the 10 days of treatment in comparison to control groups. In addition, the biofilm-encoding and QS-regulating genes were down-regulated to 75% in both microorganisms. Also, fewer epithelial cells died when treated with PQS compared to control groups in both mono- and co-infection groups. Conclusion: According to this study, PQS may facilitate wound healing by stimulating the immune system and reducing apoptosis. It seems to be a potential medication to use in conjunction with antibiotics to treat infections that are difficult to treat.

Purpose Streptococcus pneumoniae (Spn) is a major cause of child death. We investigated the epidemiology of S. pneumoniae in a pediatric fever clinic and explored the genomics basis of the limited vaccine response of serotype 14 strains worldwide. Methods Febrile disease and pneumonia were diagnosed following criteria from the WHO at the end of 2019 at a tertiary children’s hospital. Spn was isolated by culture from nasopharyngeal (NP) swabs. The density was determined by lytA -base qPCR. Isolates were serotyped by Quellung and underwent antimicrobial susceptibility testing. Whole-genome sequencing was employed for molecular serotyping, MLST, antibiotic gene determination, SNP calling, recombination prediction, and phylogenetic analysis. Results The presence of pneumococcus in the nasopharynx (87.5%, 7/8, p = 0.0227) and a high carriage (100%, 7/7, p = 0.0123) were significantly associated with pneumonia development. Living with siblings (73.7%, 14/19, p = 0.0125) and non-vaccination (56.0%, 28/50, p = 0.0377) contributed significantly to the Spn carriage. Serotype 14 was the most prevalent strain (16.67%, 5/30). The genome analysis of 1497 serotype 14 strains indicated S14/ST876 strains were only prevalent in China, presented limited vaccine responses with higher recombination activities within its cps locus, and unique variation patterns in the genes wzg and lrp . Conclusion With the lifting of the one-child policy, it will be crucial for families with multiple children to get PCV vaccinations in China. Due to the highly variant cps locus and distinctive variation patterns in capsule shedding and binding proteins genes, the prevalent S14/ST876 strains have shown poor response to current vaccines. It is necessary to continue monitoring the molecular epidemiology of this vaccine escape clone.

Pneumococcal pneumonia causes cytotoxicity in the lung parenchyma but the underlying mechanism involves multiple factors contributing to cell death. Here, we discovered that hydrogen peroxide produced by Streptococcus pneumoniae (Spn-H2O2) plays a pivotal role by oxidizing hemoglobin, leading to its polymerization and subsequent release of labile heme. At physiologically relevant levels, heme selected a population of encapsulated pneumococci. In the absence of capsule and Spn-H2O2, host intracellular heme exhibited toxicity towards pneumococci, thus acting as an antibacterial mechanism. Further investigation revealed that heme-mediated toxicity required the ABC transporter GlnPQ. In vivo experiments demonstrated that pneumococci release H2O2 to cause cytotoxicity in bronchi and alveoli through the non-proteolytic degradation of intracellular proteins such as actin, tubulin and GAPDH. Overall, our findings uncover a mechanism of lung toxicity mediated by oxidative stress that favor the growth of encapsulated pneumococci suggesting a therapeutic potential by targeting oxidative reactions. Graphical abstract Highlights Oxidation of hemoglobin by Streptococcus pneumoniae facilitates differentiation to encapsulated pneumococci in vivo Differentiated S. pneumoniae produces capsule and hydrogen peroxide (Spn-H2O2) as defense mechanism against host heme-mediated toxicity. Spn-H2O2-induced lung toxicity causes the oxidation and non-proteolytic degradation of intracellular proteins tubulin, actin, and GAPDH. The ABC transporter GlnPQ is a heme-binding complex that makes Spn susceptible to heme toxicity.

Streptococcus pneumoniae persists as a leading cause of bacterial pneumonia despite the widespread use of polysaccharide-based vaccines. The limited serotype coverage of current vaccines has led to increased incidence of nonvaccine serotypes, as well as an increase in antibiotic resistance among these serotypes. Pneumococcal infection often follows a primary viral infection such as influenza virus, which hinders host defense and results in bacterial spread to the lungs. We previously isolated human monoclonal Abs (mAbs) against the conserved surface Ag pneumococcal histidine triad protein D (PhtD), and we demonstrated that mAbs to this Ag are protective against lethal pneumococcal challenge prophylactically and therapeutically. In this study, we elucidated the mechanism of protection of a protective anti-pneumococcal human mAb, PhtD3, which is mediated by the presence of complement and macrophages in a mouse model of pneumococcal infection. Treatment with mAb PhtD3 reduced blood and lung bacterial burden in mice, and mAb PhtD3 is able to bind to bacteria in the presence of the capsular polysaccharide, indicating exposure of surface PhtD on encapsulated bacteria. In a mouse model of secondary pneumococcal infection, protection mediated by mAb PhtD3 and another mAb targeting a different epitope, PhtD7, was reduced; however, robust protection was restored by combining mAb PhtD3 with mAb PhtD7, indicating a synergistic effect. Overall, these studies provide new insights into anti-pneumococcal mAb protection and demonstrate, to our knowledge, for the first time, that mAbs to pneumococcal surface proteins can protect against secondary pneumococcal infection in the mouse model.

ABSTRACT In the presence of molecular oxygen, the human pathogen Streptococcus pneumoniae produces and secretes large amounts of hydrogen peroxide (H2O2), which can readily interact with free and heme-bound iron. Here, we investigated the role of the endogenously produced H2O2 in iron acquisition. The data revealed that S. pneumoniae uses H2O2 to liberate iron from met-hemoglobin (Hb-Fe3+) extracellularly, allowing the bacterium to import and grow on free iron even when cultivated on met-hemoglobin as the only iron source. The loss of H2O2 production leads to a dramatic pneumococcal intake of heme and is associated with a robust upregulation of most iron uptake machinery (indicating an iron starvation signal). These and other data reveal a close and previously unexplored relation between H2O2 production and iron metabolism in S. pneumoniae. The data also show that, in addition to extracellular degradation, pneumococci are armed with H2O2-independent mechanisms for intracellular heme catabolism. IMPORTANCE Heme degradation provides pathogens with growth essential iron, leveraging on the host heme reservoir. Bacteria typically import and degrade heme enzymatically, and here, we demonstrated a significant deviation from this dogma. We found that Streptococcus pneumoniae liberates iron from met-hemoglobin extracellularly, in a hydrogen peroxide (H2O2)- and cell-dependent manner; this activity serves as a major iron acquisition mechanism for S. pneumoniae. Inhabiting oxygen-rich environments is a major part of pneumococcal biology, and hence, H2O2-mediated heme degradation likely supplies iron during infection. Moreover, H2O2 reaction with ferrous hemoglobin but not with met-hemoglobin is known to result in heme breakdown. Therefore, the ability of pneumococci to degrade heme from met-hemoglobin is a new paradigm. Lastly, this study will inform other research as it demonstrates that extracellular degradation must be considered in the interpretations of experiments in which H2O2-producing bacteria are given heme or hemoproteins as an iron source.