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Abstract Background Obstructive sleep apnea (OSA) affects 10–26% of adults in the United States with known sex differences in prevalence and severity. OSA is characterized by elevated inflammation, oxidative stress (OS), and cognitive dysfunction. However, there is a paucity of data regarding the role of sex in the OSA phenotype. Prior findings suggest women exhibit different OSA phenotypes than men, which could result in under-reported OSA prevalence in women. To examine the relationship between OSA and sex, we used chronic intermittent hypoxia (CIH) to model OSA in rats. We hypothesized that CIH would produce sex-dependent phenotypes of inflammation, OS, and cognitive dysfunction, and these sex differences would be dependent on mitochondrial oxidative stress (mtOS). Methods Adult male and female Sprague Dawley rats were exposed to CIH or normoxia for 14 days to examine the impact of sex on CIH-associated circulating inflammation (IL-1β, IL-6, IL-10, TNF-α), circulating steroid hormones, circulating OS, and behavior (recollective and spatial memory; gross and fine motor function; anxiety-like behaviors; and compulsive behaviors). Rats were implanted with osmotic minipumps containing either a mitochondria-targeting antioxidant (MitoTEMPOL) or saline vehicle 1 week prior to CIH initiation to examine how inhibiting mtOS would affect the CIH phenotype. Results Sex-specific differences in CIH-induced inflammation, OS, motor function, and compulsive behavior were observed. In female rats, CIH increased inflammation (plasma IL-6 and IL-6/IL-10 ratio) and impaired fine motor function. Conversely, CIH elevated circulating OS and compulsivity in males. These sex-dependent effects of CIH were blocked by inhibiting mtOS. Interestingly, CIH impaired recollective memory in both sexes but these effects were not mediated by mtOS. No effects of CIH were observed on spatial memory, gross motor function, or anxiety-like behavior, regardless of sex. Conclusions Our results indicate that the impact of CIH is dependent on sex, such as an inflammatory response and OS response in females and males, respectively, that are mediated by mtOS. Interestingly, there was no effect of sex or mtOS in CIH-induced impairment of recollective memory. These results indicate that mtOS is involved in the sex differences observed in CIH, but a different mechanism underlies CIH-induced memory impairments.

BackgroundSex differences in oxidative stress-associated cognitive decline are influenced by sex hormone levels. Notably, oxidative stress-associated neuronal cell death can be exacerbated through testosterone signaling via membrane androgen receptor AR45, which is complexed with G protein Gαq within plasma membrane-associated lipid rafts. The objective of this study was to elucidate the impact of sex on the expression of AR45 and Gαq in brain regions associated with cognitive function, specifically hippocampus subregions and entorhinal cortex. Additionally, we investigated whether chronic intermittent hypoxia (CIH), an oxidative stressor with sex-specific effects, would modulate AR45 and Gαq expression in these brain regions.MethodsAdult male and female Sprague-Dawley rats were exposed to CIH or normoxia (room air) during their sleep phase for 14 days. We quantified AR45 and Gαq protein expression in various cognition-associated brain regions [dorsal hippocampal CA1, CA3, dentate gyrus (DG), and entorhinal cortex (ETC)] via western blotting. For comparisons, AR45 and Gαq protein expression were also assessed in brain regions outside the hippocampal-ETC circuit [thalamus (TH) and striatum (STR)].ResultsThe highest AR45 levels were expressed in the hippocampal CA1 and DG while the lowest expression was observed in the extrahippocampal STR. The highest Gαq levels were expressed in the hippocampal-associated ETC while the lowest expression was observed in the extrahippocampal TH. Females expressed higher levels of AR45 in the hippocampal DG compared to males, while no sex differences in Gαq expression were observed regardless of brain region assessed. Moreover, there was no effect of CIH on AR45 or Gαq expression in any of the brain regions examined. AR45 expression was positively correlated with Gαq expression in the CA1, DG, ETC, TH, and STR in a sex-dependent manner.ConclusionOur findings reveal enrichment of AR45 and Gαq protein expression within the hippocampal-ETC circuit, which is vulnerable to oxidative stress and neurodegeneration during cognitive decline. Nonetheless, CIH does not modulate the expression of AR45 or Gαq. Importantly, there are sex differences in AR45 expression and its association with Gαq expression in various brain regions, which may underlie sex-specific differences in cognitive and motor function-associated declines with aging.

Abstract Background Gestational sleep apnea is a hypoxic sleep disorder that affects 8–26% of pregnancies and increases the risk for central nervous system dysfunction in offspring. Specifically, there are sex differences in the sensitivity of the fetal hippocampus to hypoxic insults, and hippocampal impairments are associated with social dysfunction, repetitive behaviors, anxiety, and cognitive impairment. Yet, it is unclear whether gestational sleep apnea impacts these hippocampal-associated functions and if sex and age modify these effects. To examine the relationship between gestational sleep apnea and hippocampal-associated behaviors, we used chronic intermittent hypoxia (CIH) to model late gestational sleep apnea in pregnant rats. We hypothesized that late gestational CIH would produce sex- and age-specific social, anxiety-like, repetitive, and cognitive impairments in offspring. Methods Timed pregnant Long-Evans rats were exposed to CIH or room air normoxia from GD 15–19. Behavioral testing of offspring occurred during either puberty or young adulthood. To examine gestational hypoxia-induced behavioral phenotypes, we quantified hippocampal-associated behaviors (social function, repetitive behaviors, anxiety-like behaviors, and spatial memory and learning), hippocampal neuronal activity (glutamatergic NMDA receptors, dopamine transporter, monoamine oxidase-A, early growth response protein 1, and doublecortin), and circulating hormones in offspring. Results Late gestational CIH induced sex- and age-specific differences in social, repetitive, and memory functions in offspring. In female pubertal offspring, CIH impaired social function, increased repetitive behaviors, and elevated circulating corticosterone levels but did not impact memory. In contrast, CIH transiently induced spatial memory dysfunction in pubertal male offspring but did not impact social or repetitive functions. Long-term effects of gestational CIH on social behaviors were only observed in female offspring, wherein CIH induced social disengagement and suppression of circulating corticosterone levels in young adulthood. No effects of gestational CIH were observed in anxiety-like behaviors, hippocampal neuronal activity, or circulating testosterone and estradiol levels, regardless of sex or age of offspring. Conclusions Our results indicate that hypoxia-associated pregnancy complications during late gestation can increase the risk for behavioral and physiological outcomes in offspring, such as social dysfunction, repetitive behaviors, and cognitive impairment, that are dependent on sex and age.

Obstructive sleep apnea (OSA) is a sleep disorder characterized by intermittent complete or partial occlusion of the airway. Despite a recognized association between OSA and glaucoma, the nature of the underlying link remains unclear. In this study, we investigated whether mild OSA induces morphological, inflammatory, and metabolic changes in the retina resembling those seen in glaucoma using a rat model of OSA known as chronic intermittent hypoxia (CIH). Rats were randomly assigned to either normoxic or CIH groups. The CIH group was exposed to periodic hypoxia during its sleep phase with oxygen reduction from 21% to 10% and reoxygenation in 6 min cycles over 8 h/day. The eyes were subsequently enucleated, and then the retinas were evaluated for retinal ganglion cell number, oxidative stress, inflammatory markers, metabolic changes, and hypoxic response modulation using immunohistochemistry, multiplex assays, and capillary electrophoresis. Statistically significant differences were observed between normoxic and CIH groups for oxidative stress and inflammation, with CIH resulting in increased HIF-1α protein levels, higher oxidative stress marker 8-OHdG, and increased TNF-α. Pyruvate dehydrogenase kinase-1 protein was significantly reduced with CIH. No significant differences were found in retinal ganglion cell number. Our findings suggest that CIH induces oxidative stress, inflammation, and upregulation of HIF-1α in the retina, akin to early-stage glaucoma.

Highlights Brain maturation of the nigrostriatal pathway is sex- and age- dependent. Exposure to hypoxia in late pregnancy impacts brain maturation of the nigrostriatal pathway that can be observed during puberty and young adulthood. Gestational hypoxia impacted female offspring during puberty more than males, whereas it impacted male offspring during young adulthood more than females. These novel findings demonstrate that hypoxic insults during pregnancy mediate developmental programming of the cortical and subcortical pathways, in which male offspring exhibit long-term adverse effects compared to female offspring. Long-term adverse effects of gestational hypoxia in offspring can occur in the absence of pregnancy complications, especially if they occur within critical embryological developmental periods.

ABSTRACT Streptococcus pneumoniae colonizes the human nasopharynx and causes several diseases. Pneumococcal vaccines target the polysaccharide capsule and prevent most serious disease, but there has been an increase in the prevalence of nonencapsulated S. pneumoniae (NESp). Previously, it was thought that a capsule was necessary to cause invasive disease. NESp strains expressing the oligopeptide transporters AliC and AliD have been isolated from patients with invasive disease. The AliC and AliD oligopeptide transporters regulate the expression of several genes, including choline binding protein AC (CbpAC) (a homolog of PspA), which aids in reducing C3b deposition. It is hypothesized that by altering CbpAC expression, AliC and AliD provide protection from classical complement-mediated clearance by reducing C-reactive protein (CRP) binding. Our study demonstrates that AliC and AliD regulate CbpAC expression in NESp and that AliD found in certain serotypes of encapsulated strains regulates PspA expression. C3b deposition was increased in the NESp ΔaliD and encapsulated mutants in comparison to the wild type. NESp strains expressing AliC and AliD have a significant decrease in C1q and CRP deposition in comparison to the ΔaliC ΔaliD mutant. The complement protein C1q is required for NESp clearance in a murine model and increases opsonophagocytosis. By regulating CbpAC expression, NESp inhibits CRP binding to the bacterial surface and blocks classical complement activation, leading to greater systemic survival and virulence. Due to the increase in the prevalence of NESp, it is important to gain a better understanding of NESp virulence mechanisms that aid in establishing disease and persistence within a host by avoiding clearance by the immune system. IMPORTANCE Streptococcus pneumoniae (pneumococcus) can cause a range of diseases. Although there is a robust pneumococcal vaccination program that reduces invasive pneumococcal disease by targeting various polysaccharide capsules, there has been an increase in the isolation of nonvaccine serotypes and nonencapsulated S. pneumoniae (NESp) strains. While most studies of pneumococcal pathogenesis have focused on encapsulated strains, there is little understanding of how NESp causes disease. NESp lacks a protective capsule but contains novel genes, such as aliC and aliD, which have been shown to regulate the expression of numerous genes and to be required for NESp virulence and immune evasion. Furthermore, NESp strains have high transformation efficiencies and harbor resistance to multiple drugs. This could be deleterious to current treatment strategies employed for pneumococcal disease as NESp can be a reservoir of drug resistance genes. Therefore, deciphering how NESp survives within a host and facilitates disease is a necessity that will allow the fabrication of improved, broad-spectrum treatments and preventatives against pneumococcal disease. Our study provides a better understanding of NESp virulence mechanisms during host-pathogen interactions through the examination of genes directly regulated by the NESp proteins AliC and AliD.

Background Circulating cell‐free mitochondrial DNA (ccf‐mtDNA) is a damage‐associated molecular pattern that reflects cell stress responses and tissue damage, but little is known about ccf‐mtDNA in preeclampsia. The main objectives of this study were to determine (1) absolute concentrations of ccf‐mtDNA in plasma and mitochondrial DNA content in peripheral blood mononuclear cells and (2) forms of ccf‐mtDNA transport in blood from women with preeclampsia and healthy controls. In addition, we sought to establish the association between aberrance in circulating DNA‐related metrics, including ccf‐mtDNA and DNA clearance mechanisms, and the clinical diagnosis of preeclampsia using bootstrapped penalized logistic regression. Methods and Results Absolute concentrations of ccf‐mtDNA were reduced in plasma from women with preeclampsia compared with healthy controls (P≤0.02), while mtDNA copy number in peripheral blood mononuclear cells did not differ between groups (P>0.05). While the pattern of reduced ccf‐mtDNA in patients with preeclampsia remained, DNA isolation from plasma using membrane lysis buffer resulted in 1000‐fold higher ccf‐mtDNA concentrations in the preeclampsia group (P=0.0014) and 430‐fold higher ccf‐mtDNA concentrations in the control group (P0.05). Penalized regression analysis showed that women with preeclampsia were more likely to have lower concentrations of ccf‐mtDNA as well as higher concentrations of nuclear DNA and DNase I compared with their matched controls. Conclusions Women with preeclampsia have aberrant circulating DNA dynamics, including reduced ccf‐mtDNA concentrations and DNA clearance mechanisms, compared with gestational age–matched healthy pregnant women.

ABSTRACT Pneumonia is a pulmonary disease affecting people of all ages and is consistently a leading cause of childhood mortality and adult hospitalizations. Streptococcus pneumoniae and Pseudomonas aeruginosa are major lung pathogens commonly associated with community-acquired and nosocomial pneumonia. Additionally, mixed lung infections involving these bacterial pathogens are increasing in prevalence and are frequently more severe than single infections. The cooperative interactions of these two pathogens that impact pulmonary disease severity are understudied. A major secreted virulence factor of P. aeruginosa, protease IV (PIV), cleaves interleukin 22 (IL-22), a cytokine essential for maintaining innate mucosal defenses against extracellular pathogens. Here, we investigate the ability of PIV to augment the virulence of a pneumococcal strain with limited virulence, S. pneumoniae EF3030, in a C57BL/6 murine model of pneumonia. We demonstrate that pulmonary coinfection involving P. aeruginosa 103-29 and S. pneumoniae EF3030 results in pneumococcal bacteremia that is abrogated during pneumococcal coinfection with a PIV-deficient strain. Furthermore, intratracheal administration of exogenous PIV and EF3030 resulted in abundant immune cell infiltration into the lung with large abscess formation, as well as severe bacteremia leading to 100% mortality. Heat-inactivated PIV did not worsen pneumonia or reliably induce bacteremia, suggesting that the specific activity of PIV is required. Our studies also show that PIV depletes IL-22 in vivo. Moreover, PIV-mediated enhancement of pneumonia and disease severity was dependent on the expression of pneumolysin (Ply), a prominent virulence factor of S. pneumoniae. Altogether, we reveal that PIV and Ply additively potentiate pneumonia in a murine model of lung infection. IMPORTANCE S. pneumoniae remains the leading cause of bacterial pneumonia despite widespread use of pneumococcal vaccines, forcing the necessity for appropriate treatment to control pneumococcal infections. Coinfections involving S. pneumoniae with other bacterial pathogens threaten antibiotic treatment strategies and disease outcomes. Currently, there is not an effective treatment for alveolar-capillary barrier dysfunction that precedes bacteremia. An understanding of the dynamics of host-pathogen interactions during single and mixed pulmonary infections could elucidate proper treatment strategies needed to prevent or reduce invasive disease. Antibiotic treatment decreases bacterial burden in the lung but also increases acute pathology due to cytotoxins released via antibiotic-induced bacterial lysis. Therefore, targeted therapeutics that inhibit or counteract the effects of bacterial proteases and toxins are needed in order to limit pathology and disease progression. This study identifies the cooperative effect of PIV and Ply, products of separate lung pathogens that additively alter the lung environment and facilitate invasive disease.

ABSTRACT Nonencapsulated Streptococcus pneumoniae (NESp) is an emerging human pathogen that colonizes the nasopharynx and is associated with noninvasive diseases such as otitis media (OM), conjunctivitis, and nonbacteremic pneumonia. Since capsule expression was previously thought to be necessary for establishment of invasive pneumococcal disease (IPD), serotype-specific polysaccharide capsules are targeted by currently licensed pneumococcal vaccines. Yet, NESp expressing oligopeptide binding proteins AliC and AliD have been isolated during IPD. Thus, we hypothesize AliC and AliD are major NESp virulence determinants that facilitate persistence and development of IPD. Our study reveals that NESp expressing AliC and AliD have intensified virulence compared to isogenic mutants. Specifically, we demonstrate AliC and AliD enhance murine nasopharyngeal colonization and pulmonary infection and are required for OM in a chinchilla model. Furthermore, AliC and AliD increase pneumococcal survival in chinchilla whole blood and aid in resistance to killing by human leukocytes. Comparative proteome analysis revealed significant alterations in protein levels when AliC and AliD were absent. Virulence-associated proteins, including a pneumococcal surface protein C variant (CbpAC), were significantly downregulated, while starvation response indicators were upregulated in the double mutant relative to wild-type levels. We also reveal that differentially expressed CbpAC was essential for NESp adherence to epithelial cells, virulence during OM, reduction of C3b deposition on the NESp surface, and binding to nonspecific IgA. Altogether, the rise in NESp prevalence urges the need to understand how NESp establishes disease and persists in a host. This study highlights the roles of AliC, AliD, and CbpAC in the pathogenesis of NESp. IMPORTANCE Despite the effective, widespread use of licensed pneumococcal vaccines over many decades, pneumococcal infections remain a worldwide burden resulting in high morbidity and mortality. NESp subpopulations are rapidly rising in the wake of capsule-targeted vaccine strategies, yet there is very little knowledge on NESp pathogenic potential and virulence mechanisms. Although NESp lacks a protective capsule, NESp lineages expressing AliC and AliD have been associated with systemic infections. Furthermore, higher antibiotic resistance rates and transformation efficiencies associated with emerging NESp threaten treatment strategies needed to control pneumococcal infections and transmission. Elucidating how NESp survives within a host and establishes disease is necessary for development of broadened pneumococcal prevention methods. Our study identifies virulence determinants and host survival mechanisms employed by NESp with a high pathogenic potential. Moreover, our study also identifies virulence determinants shared by NESp and encapsulated strains that may serve as broad prevention and therapeutic targets.

Streptococcus pneumoniae is an important human pathogen. To cause disease, it must first colonize the nasopharynx. The widespread use of pneumococcal-conjugate vaccines which target the capsular polysaccharide has led to decreased nasopharyngeal carriage of vaccine serotypes, but a concomitant increase in carriage of non-vaccine serotypes and nonencapsulated S. pneumoniae (NESp). Some NESp express pneumococcal surface protein K (PspK), a virulence factor shown to contribute to nasopharyngeal colonization. We present the case of a child with chronic adenoiditis caused by a PspK+ NESp. We tested the pneumococcal isolate, designated C144.66, for antimicrobial resistance, the presence of the pspK gene and the expression of PspK. Sequence typing and genome sequencing were performed. C144.66 was found to be resistant to erythromycin and displayed intermediate resistance to penicillin and trimethoprim/sulfamethoxazole. C144.66 has the pspK gene in place of the capsule locus. Additionally, PspK expression was confirmed by flow cytometry. NESp are a growing concern as an emerging human pathogen, as current pneumococcal vaccines do not confer immunity against them. An inability to vaccinate against NESp may result in increased carriage and associated pathology.

Streptococcus pneumoniae infections result in a range of human diseases and are responsible for almost one million deaths annually. Pneumococcal disease is mediated in part through surface structures and an anti-phagocytic capsule. Recent studies have shown that nonencapsulated Streptococcus pneumoniae (NESp) make up a significant portion of the pneumococcal population and are able to cause disease. NESp lack some common surface proteins expressed by encapsulated pneumococci, but express surface proteins unique to NESp. A chinchilla model of otitis media (OM) was used to determine the effect various pneumococcal mutations have on pathogenesis in both NESp and encapsulated pneumococci. Epithelial cell adhesion and invasion assays were used to examine the effects in relation to deletion of intrinsic genes or expression of novel genes. A mouse model of colonization was also utilized for comparison of various pneumococcal mutants. It was determined that pneumococcal surface protein K (PspK) and pneumolysin (Ply) affect NESp middle ear pathogenesis, but only PspK affected epithelial cell adhesion. Experiments in an OM model were done with encapsulated strains testing the importance of native virulence factors and treatment of OM. First, a triple deletion of the common virulence factors PspA, PspC, and Ply, (ΔPAC), from an encapsulated background abolished virulence in an OM model while a PspC mutant had detectable, but reduced amounts of recoverable bacteria compared to wildtype. Next, treatment of OM was effective when starting antibiotic treatment within 24 hrs with resolution by 48 hrs post treatment. Expression of NESp-specific virulence factor PspK in an encapsulated strain has not been previously studied, and we showed significantly increased adhesion and invasion of human epithelial cells by pneumococci. Murine colonization was not significantly increased when an encapsulated strain expressed PspK, but colonization was increased when a capsule mutant expressed PspK. The ability of PspK expression to increase colonization in a capsule mutant despite no increase in adhesion can be attributed to other functions of PspK, such as sIgA binding or immune modulation. OM is a substantial economic burden, thus a better understanding of both encapsulated pneumococcal pathogenesis and the emerging pathogen NESp is necessary for effective prevention and treatment.

ATP-binding cassette (ABC) transport systems are crucial for bacteria to ensure sufficient uptake of nutrients that are not produced de novo or improve the energy balance. The cell surface of the pathobiont Streptococcus pneumoniae (pneumococcus) is decorated with a substantial array of ABC transporters, critically influencing nasopharyngeal colonization and invasive infections. Given the auxotrophic nature of pneumococci for certain amino acids, the Ami ABC transporter system, orchestrating oligopeptide uptake, becomes indispensable in host compartments lacking amino acids. The system comprises five exposed Oligopeptide Binding Proteins (OBPs) and four proteins building the ABC transporter channel. Here, we present a structural analysis of all the OBPs in this system. Multiple crystallographic structures, capturing both open and closed conformations along with complexes involving chemically synthesized peptides, have been solved at high resolution providing insights into the molecular basis of their diverse peptide specificities. Mass spectrometry analysis of oligopeptides demonstrates the unexpected remarkable promiscuity of some of these proteins when expressed in Escherichia coli, displaying affinity for a wide range of peptides. Finally, a model is proposed for the complete Ami transport system in complex with its various OBPs. We further disclosed, through in silico modelling, some essential structural changes facilitating oligopeptide transport into the cellular cytoplasm. Thus, the structural analysis of the Ami system provides valuable insights into the mechanism and specificity of oligopeptide binding by the different OBPs, shedding light on the intricacies of the uptake mechanism and the in vivo implications for this human pathogen.

Streptococcus pneumoniae (Pneumococcus) infections affect millions of people worldwide, cause serious mortality and represent a major economic burden. Despite recent successes due to pneumococcal vaccination and antibiotic use, Pneumococcus remains a significant medical problem. Airway epithelial cells, the primary responders to pneumococcal infection, orchestrate an extracellular antimicrobial system consisting of lactoperoxidase (LPO), thiocyanate anion and hydrogen peroxide (H2O2). LPO oxidizes thiocyanate using H2O2 into the final product hypothiocyanite that has antimicrobial effects against a wide range of microorganisms. However, hypothiocyanite's effect on Pneumococcus has never been studied. Our aim was to determine whether hypothiocyanite can kill S. pneumoniae. Bactericidal activity was measured in a cell-free in vitro system by determining the number of surviving pneumococci via colony forming units on agar plates, while bacteriostatic activity was assessed by measuring optical density of bacteria in liquid cultures. Our results indicate that hypothiocyanite generated by LPO exerted robust killing of both encapsulated and nonencapsulated pneumococcal strains. Killing of S. pneumoniae by a commercially available hypothiocyanite-generating product was even more pronounced than that achieved with laboratory reagents. Catalase, an H2O2 scavenger, inhibited killing of pneumococcal by hypothiocyanite under all circumstances. Furthermore, the presence of the bacterial capsule or lytA-dependent autolysis had no effect on hypothiocyanite-mediated killing of pneumococci. On the contrary, a pneumococcal mutant deficient in pyruvate oxidase (main bacterial H2O2 source) had enhanced susceptibility to hypothiocyanite compared to its wild-type strain. Overall, results shown here indicate that numerous pneumococcal strains are susceptible to LPO-generated hypothiocyanite.

Streptococcus pneumoniae is commonly found in the human nasopharynx and is the causative agent of multiple diseases. Since invasive pneumococcal infections are associated with encapsulated pneumococci, the capsular polysaccharide is the target of licensed pneumococcal vaccines. However, there is an increasing distribution of non-vaccine serotypes, as well as nonencapsulated S. pneumoniae (NESp). Both encapsulated and nonencapsulated pneumococci possess the polyamine oligo-transport operon (potABCD). Previous research has shown inactivation of the pot operon in encapsulated pneumococci alters protein expression and leads to a significant reduction in pneumococcal murine colonization, but the role of the pot operon in NESp is unknown. Here, we demonstrate deletion of potD from the NESp NCC1 strain MNZ67 does impact expression of the key proteins pneumolysin and PspK, but it does not inhibit murine colonization. Additionally, we show the absence of potD significantly increases biofilm production, both in vitro and in vivo. In a chinchilla model of otitis media (OM), the absence of potD does not significantly affect MNZ67 virulence, but it does significantly reduce the pathogenesis of the virulent encapsulated strain TIGR4 (serotype 4). Deletion of potD also significantly reduced persistence of TIGR4 in the lungs but increased persistence of PIP01 in the lungs. We conclude the pot operon is important for the regulation of protein expression and biofilm formation in both encapsulated and NCC1 nonencapsulated Streptococcus pneumoniae. However, in contrast to encapsulated pneumococcal strains, polyamine acquisition via the pot operon is not required for MNZ67 murine colonization, persistence in the lungs, or full virulence in a model of OM. Therefore, NESp virulence regulation needs to be further established to identify potential NESp therapeutic targets.