Nrc of Streptococcus pneumoniae suppresses capsule expression and enhances anti-phagocytosis

Abstract

Streptococcus pneumoniae is a major pathogen of community-acquired pneumonia and one of its major virulence factors is pneumolysin, which functions as a cholesterol-dependent cytolytic pore-forming toxin. In this study, we identified the ply-like gene spd0729 in a BLAST search. Unexpectedly, hemolytic and cytotoxic assays showed no significant differences between a Δspd0729 mutant strain and the wild-type strain, whereas the mutant strain exhibited weaker anti-phagocytic activity in human peripheral blood. In addition, real-time RT-PCR analysis revealed that four capsular biosynthesis genes in the mutant strain had expressions 7- to 432-fold greater than those of the wild type, while an enzyme-linked immunoassay showed a mean 3-fold greater amount of total capsular polysaccharide in the mutant strain. These results suggest that Spd0729 is not a cytolysin, though it plays crucial roles in anti-phagocytosis and regulation of capsule expression. Thus, we named Spd0729 as a negative regulator of capsular polysaccharide synthesis (Nrc).

Introduction

Streptococcus pneumoniae is a major cause of human diseases, such as pneumonia, meningitis, otitis, and sepsis. Asymptomatic carriage of pneumococci in biofilm in the throat or the nasopharynx is widespread, with rates especially high in children [1], and millions die every year as a result of pneumonia, bacteremia, and meningitis caused by S. pneumoniae[2], [3]. The antigenic and biochemical properties of the polysaccharide capsule have been used to characterize S. pneumoniae strains into at least 90 distinct serotypes, though S. pneumoniae related diseases are most commonly due to 20 of those strains.

S. pneumoniae has a number of virulence factors that contribute to its ability to cause diseases, with the polysaccharide capsule, which is composed of repeating polysaccharide units that help confer resistance to complement-mediated opsonophagocytosis, playing a major role [4], [5]. However, expression of the capsule reduces bacterial attachment to respiratory epithelial cells, which may hamper colonization [4], [6]. Using electron microscopy, Hammerschmidt et al. found that bacteria in intimate contact with epithelial cells have a thinner capsule layer, i.e., they may down-regulate capsule expression in order to enhance adherence [6]. It has been suggested that, at least in serotype 3, polysaccharide chain length can be modulated by sugar concentration in the environment [7]. Furthermore, the polysaccharide capsule has been reported to play a key role in survival in vivo during systemic infection in animal models [4].

The polysaccharide capsule of most S. pneumoniae serotypes is encoded by a gene cluster located between dexB and aliA. Capsule operons of different serotypes show a similar structure with some conservation, particularly within the first four genes, downstream of which are serotype-specific genes [4], [8]. The first gene, cpsA, is the most conserved and may have a role in regulation of capsule expression [9], which is thought to interfere with biofilm formation, while biofilm development may occur with unencapsulated phenotypic variants [10], [11]. Although the ability of pneumococci to regulate capsule expression likely plays an important role in the transition from carriage to invasive disease, the molecular mechanisms involved in the regulation of capsule expression have not been fully elucidated.

Another major virulence factor is the 53-kDa pore-forming toxin pneumolysin (Ply). Ply acts as a cholesterol-dependent cytolysin by binding to cholesterol in the host cell plasma membrane, after which it becomes oligomerized and inserted into the eukaryotic membrane, forming a pore 350–450 Å in diameter [5]. In the present study, we found the ply-like gene spd0729 utilizing the BLAST search algorithms and showed that it encodes a polypeptide with a 44% identity to the cholesterol-binding domain of Ply. To detect the function of spd0729, a spd0729 mutant of S. pneumoniae was generated by allelic exchange. Interestingly, we found that inactivation of spd0729 in S. pneumoniae did not influence hemolytic activity, while it significantly induced capsular synthesis.