Pulmonary Disease Associated With Nonencapsulated Streptococcus pneumoniae.

We discuss 3 patients presenting with pneumonia associated with nonencapsulated Streptococcus pneumoniae (NESp), an emerging pathogen commonly causing upper respiratory infections. Clinical isolates obtained from these patients were characterized to evaluate their respective antibiotic resistance and virulence mechanisms. We demonstrate that NESp resistant to classical drug treatments are isolated during pneumonia.

Pseudomonas aeruginosa Protease IV Exacerbates Pneumococcal Pneumonia and Systemic Disease.

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.IMPORTANCES. 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.

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.

Mucosal Infections and Invasive Potential of Nonencapsulated Streptococcus pneumoniae Are Enhanced by Oligopeptide Binding Proteins AliC and AliD.

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.

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.