Lance E. Keller

PspK of Streptococcus pneumoniae Increases Adherence to Epithelial Cells and Enhances Nasopharyngeal Colonization

ABSTRACT 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.

Nonencapsulated Streptococcus pneumoniae: Emergence and Pathogenesis

ABSTRACT 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.

Polyamine transporter potABCD is required for virulence of encapsulated but not nonencapsulated Streptococcus pneumoniae

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.

Nonencapsulated Streptococcus pneumoniae Cause Acute Otitis Media in the Chinchilla That Is Enhanced by Pneumococcal Surface Protein K.

Nonencapsulated Streptococcus pneumoniae (NESp) is an emerging human pathogen, and NESp produced acute otitis media in the chinchilla. Increased epithelial cell adhesion correlated to pathogenesis. The expression of pneumococcal surface protein K (PspK) increased NESp virulence.

Draft Genome Sequences of Five Multilocus Sequence Types of Nonencapsulated Streptococcus pneumoniae.

ABSTRACT Nonencapsulated Streptococcus pneumoniae can colonize the human nasopharynx and cause conjunctivitis and otitis media. Different deletions in the capsular polysaccharide biosynthesis locus and different multilocus sequence types have been described for nonencapsulated strains. Draft genome sequences were generated to provide insight into the genomic diversity of these strains.

Immunization with Pneumococcal Surface Protein K of Nonencapsulated Streptococcus pneumoniae Provides Protection in a Mouse Model of Colonization

ABSTRACT Current vaccinations are effective against encapsulated strains of Streptococcus pneumoniae, but they do not protect against nonencapsulated Streptococcus pneumoniae (NESp), which is increasing in colonization and incidence of pneumococcal disease. Vaccination with pneumococcal proteins has been assessed for its ability to protect against pneumococcal disease, but several of these proteins are not expressed by NESp. Pneumococcal surface protein K (PspK), an NESp virulence factor, has not been assessed for immunogenic potential or host modulatory effects. Mammalian cytokine expression was determined in an in vivo mouse model and in an in vitro cell culture system. Systemic and mucosal mouse immunization studies were performed to determine the immunogenic potential of PspK. Murine serum and saliva were collected to quantitate specific antibody isotype responses and the ability of antibody and various proteins to inhibit epithelial cell adhesion. Host cytokine response was not reduced by PspK. NESp was able to colonize the mouse nasopharynx as effectively as encapsulated pneumococci. Systemic and mucosal immunization provided protection from colonization by PspK-positive (PspK+) NESp. Anti-PspK antibodies were recovered from immunized mice and significantly reduced the ability of NESp to adhere to human epithelial cells. A protein-based pneumococcal vaccine is needed to provide broad protection against encapsulated and nonencapsulated pneumococci in an era of increasing antibiotic resistance and vaccine escape mutants. We demonstrate that PspK may serve as an NESp target for next-generation pneumococcal vaccines. Immunization with PspK protected against pneumococcal colonization, which is requisite for pneumococcal disease.

AdcAII of Streptococcus pneumoniae Affects Pneumococcal Invasiveness.

Across bacterial species, metal binding proteins can serve functions in pathogenesis in addition to regulating metal homeostasis. We have compared and contrasted the activities of zinc (Zn2+)-binding lipoproteins AdcA and AdcAII in the Streptococcus pneumoniae TIGR4 background. Exposure to Zn2+-limiting conditions resulted in delayed growth in a strain lacking AdcAII (ΔAdcAII) when compared to wild type bacteria or a mutant lacking AdcA (ΔAdcA). AdcAII failed to interact with the extracellular matrix protein laminin despite homology to laminin-binding proteins of related streptococci. Deletion of AdcA or AdcAII led to significantly increased invasion of A549 human lung epithelial cells and a trend toward increased invasion in vivo. Loss of AdcAII, but not AdcA, was shown to negatively impact early colonization of the nasopharynx. Our findings suggest that expression of AdcAII affects invasiveness of S. pneumoniae in response to available Zn2+ concentrations.

Nonencapsulated Streptococcus pneumoniae as a cause of chronic adenoiditis

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.

Surface Proteins and Pneumolysin of Encapsulated and Nonencapsulated Streptococcus pneumoniae Mediate Virulence in a Chinchilla Model of Otitis Media

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 S. 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 h with resolution by 48 h 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.

Increased Virulence of an Encapsulated Streptococcus pneumoniae Upon Expression of Pneumococcal Surface Protein K.

Background Current Streptococcus pneumoniae vaccines selectively target capsular polysaccharide of specific serotypes, leading to an increase in nonencapsulated S. pneumoniae (NESp). Cocolonization by encapsulated pneumococci and NESp increases the opportunity for intraspecies genetic exchange. Acquisition of NESp genes by encapsulated pneumococci could alter virulence and help vaccine-targeted serotypes persist in the host. Methods Adhesion and invasion assays were performed using immortalized human pharyngeal or lung epithelial cells. In vivo models assessing murine nasopharyngeal colonization and pneumonia, as well as chinchilla otitis media (OM), were also used. Results Pneumococcal surface protein K (PspK) expression increased encapsulated pneumococcal adhesion and invasion of lung cells and enhanced virulence during pneumonia and OM. Additionally, PspK increased nasopharyngeal colonization, persistence in the lungs, and persistence in the middle ear when expressed in a capsule deletion mutant. Competition experiments demonstrated encapsulated pneumococci expressing PspK also had a selective advantage in both the lungs and nasopharynx. Conclusions PspK increases pneumococcal virulence during pneumonia and OM. PspK also partially compensates for loss of virulence in the absence of capsule. Additionally, PspK provides a selective advantage in a competitive environment. Therefore, acquisition of PspK increases encapsulated virulence in a condition-dependent manner. Together, these studies demonstrate risks associated with pneumococcal intraspecies genetic exchange.