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.

While significant protection from pneumococcal disease has been achieved by the use of polysaccharide and polysaccharide-protein conjugate vaccines, capsule-independent protection has been limited by serotype replacement along with disease caused by nonencapsulated Streptococcus pneumoniae (NESp). NESp strains compose approximately 3% to 19% of asymptomatic carriage isolates and harbor multiple antibiotic resistance genes. Surface proteins unique to NESp enhance colonization and virulence despite the lack of a capsule even though the capsule has been thought to be required for pneumococcal pathogenesis. Genes for pneumococcal surface proteins replace the capsular polysaccharide (cps) locus in some NESp isolates, and these proteins aid in pneumococcal colonization and otitis media (OM). NESp strains have been isolated from patients with invasive and noninvasive pneumococcal disease, but noninvasive diseases, specifically, conjunctivitis (85%) and OM (8%), are of higher prevalence. Conjunctival strains are commonly of the so-called classical NESp lineages defined by multilocus sequence types (STs) ST344 and ST448, while sporadic NESp lineages such as ST1106 are more commonly isolated from patients with other diseases. Interestingly, sporadic lineages have significantly higher rates of recombination than classical lineages. Higher rates of recombination can lead to increased acquisition of antibiotic resistance and virulence factors, increasing the risk of disease and hindering treatment. NESp strains are a significant proportion of the pneumococcal population, can cause disease, and may be increasing in prevalence in the population due to effects on the pneumococcal niche caused by pneumococcal vaccines. Current vaccines are ineffective against NESp, and further research is necessary to develop vaccines effective against both encapsulated and nonencapsulated pneumococci.

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and can cause invasive disease aided by the pneumococcal capsule. Group II nontypeable S. pneumoniae (NTSp) lacks a polysaccharide capsule, and a subgroup of NTSp carriage isolates has been found to have a novel gene, pneumococcal surface protein K ( pspK ), which replaces the capsule locus. A recent rise in the number of NTSp isolates colonizing the human nasopharynx has been observed, but the colonization factors of NTSp have not been well studied. PspK has been shown to play a role in mouse colonization. We therefore examined PspK-mediated immune evasion along with adherence to host cells and colonization. PspK bound human secretory immunoglobulin A (sIgA) but not the complement regulator factor H and did not decrease C3b deposition on the pneumococcal surface. PspK increased binding of pneumococci to epithelial cells and enhanced pneumococcal colonization independently of the genetic background. Understanding how NTSp colonizes and survives within the nasopharynx is important due to the increase in NTSp carriage. Our data suggest that PspK may aid in the persistence of NTSp within the nasopharynx but is not involved in invasion.

Streptococcus pneumoniae is one of the leading causes of community acquired pneumonia and acute otitis media. Certain aspects of S. pneumoniae’s virulence are dependent upon expression and release of the protein toxin pneumolysin (PLY) and upon the activity of the peroxide-producing enzyme, pyruvate oxidase (SpxB). We investigated the possible synergy of these two proteins and identified that release of PLY is enhanced by expression of SpxB prior to stationary phase growth. Mutants lacking the spxB gene were defective in PLY release and complementation of spxB restored PLY release. This was demonstrated by cytotoxic effects of sterile filtered supernatants upon epithelial cells and red blood cells. Additionally, peroxide production appeared to contribute to the mechanism of PLY release since a significant correlation was found between peroxide production and PLY release among a panel of clinical isolates. Exogenous addition of H2O2 failed to induce PLY release and catalase supplementation prevented PLY release in some strains, indicating peroxide may exert its effect intracellularly or in a strain-dependent manner. SpxB expression did not trigger bacterial cell death or LytA-dependent autolysis, but did predispose cells to deoxycholate lysis. Here we demonstrate a novel link between spxB expression and PLY release. These findings link liberation of PLY toxin to oxygen availability and pneumococcal metabolism.

Streptococcus pneumoniae and other streptococci produce a greenish halo on blood agar plates referred to as alpha-hemolysis. This phenotype is utilized by clinical microbiology laboratories to report culture findings of alpha-hemolytic streptococci, including S. pneumoniae, and other bacteria. The alpha-hemolysis halo on blood agar plates has been related to the hemolytic activity of pneumococcal pneumolysin (Ply) or, to a lesser extent, to lysis of erythrocytes by S. pneumoniae-produced hydrogen peroxide. We investigated the molecular basis of the alpha-hemolysis halo produced by S. pneumoniae. Wild-type strains TIGR4, D39, R6, and EF3030 and isogenic derivative Δply mutants produced similar alpha-hemolytic halos on blood agar plates, while cultures of hydrogen peroxide knockout ΔspxB ΔlctO mutants lacked this characteristic halo. Moreover, in the presence of catalase, the alpha-hemolysis halo was absent in cultures of the wild-type (wt) and Δply mutant strains. Spectroscopic studies demonstrated that culture supernatants of TIGR4 released hemoglobin-bound heme (heme-hemoglobin) from erythrocytes and oxidized oxy-hemoglobin to met-hemoglobin within 30 min of incubation. As expected, given Ply hemolytic activity and that hydrogen peroxide contributes to the release of Ply, TIGR4Δply and ΔspxB ΔlctO isogenic mutants had significantly decreased release of heme-hemoglobin from erythrocytes. However, TIGR4Δply that produces hydrogen peroxide oxidized oxy-hemoglobin to met-hemoglobin, whereas TIGR4ΔspxB ΔlctO failed to produce oxidation of oxy-hemoglobin. Studies conducted with all other wt strains and isogenic mutants resulted in similar findings. We demonstrated that the so-called alpha-hemolysis halo is caused by the oxidation of oxy-hemoglobin (Fe⁺²) to a non-oxygen-binding met-hemoglobin (Fe⁺³) by S. pneumoniae-produced hydrogen peroxide. IMPORTANCE There is a misconception that alpha-hemolysis observed on blood agar plate cultures of Streptococcus pneumoniae and other alpha-hemolytic streptococci is produced by a hemolysin or, alternatively, by lysis of erythrocytes caused by hydrogen peroxide. We noticed in the course of our investigations that wild-type S. pneumoniae strains and hemolysin (e.g., pneumolysin) knockout mutants produced the alpha-hemolytic halo on blood agar plates. In contrast, hydrogen peroxide-defective mutants prepared in four different strains lacked the characteristic alpha-hemolysis halo. We also demonstrated that wild-type strains and pneumolysin mutants oxidized oxy-hemoglobin to met-hemoglobin. Hydrogen peroxide knockout mutants, however, failed to oxidize oxy-hemoglobin. Therefore, the greenish halo formed on cultures of S. pneumoniae and other so-called alpha-hemolytic streptococci is caused by the oxidation of oxy-hemoglobin produced by hydrogen peroxide. Oxidation of oxy-hemoglobin to the nonbinding oxygen form, met-hemoglobin, might occur in the lungs during pneumococcal pneumonia.