Comparative Analysis of Streptococcus pneumoniae Type I Restriction-Modification Loci: Variation in hsdS Gene Target Recognition Domains.

Streptococcus pneumoniae (pneumococcus) is a respiratory commensal pathogen that causes a range of infections, particularly in young children and the elderly. Pneumococci undergo spontaneous phase variation in colony opacity phenotype, in which DNA rearrangements within the Type I restriction-modification (R-M) system specificity gene hsdS can potentially generate up to six different hsdS alleles with differential DNA methylation activity, resulting in changes in gene expression. To gain a broader perspective of this system, we performed bioinformatic analyses of Type I R-M loci from 18 published pneumococcal genomes, and one R-M locus sequenced for this study, to compare genetic content, organization, and homology. All 19 loci encoded the genes hsdR, hsdM, hsdS, and at least one hsdS pseudogene, but differed in gene order, gene orientation, and hsdS target recognition domain (TRD) content. We determined the coding sequences of 87 hsdS TRDs and excluded seven from further analysis due to the presence of premature stop codons. Comparative analyses revealed that the TRD 1.1, 1.2, and 2.1 protein sequences had single amino acid substitutions, and TRD 2.2 and 2.3 each had seven differences. The results of this study indicate that variability exists among the gene content and arrangements within Type I R-M loci may provide an additional level of divergence between pneumococcal strains, such that phase variation-mediated control of virulence factors may vary significantly between individual strains. These findings are consistent with presently available transcript profile data.

A Novel Function for the Streptococcus pneumoniae Aminopeptidase N: Inhibition of T Cell Effector Function through Regulation of TCR Signaling.

Streptococcus pneumoniae (Spn) causes a variety of disease states including fatal bacterial pneumonia. Our previous finding that introduction of Spn into an animal with ongoing influenza virus infection resulted in a CD8+ T cell population with reduced effector function gave rise to the possibility of direct regulation by pneumococcal components. Here, we show that treatment of effector T cells with lysate derived from Spn resulted in inhibition of IFNγ and tumor necrosis factor α production as well as of cytolytic granule release. Spn aminopeptidase N (PepN) was identified as the inhibitory bacterial component and surprisingly, this property was independent of the peptidase activity found in this family of proteins. Inhibitory activity was associated with reduced activation of ZAP-70, ERK1/2, c-Jun N-terminal kinase, and p38, demonstrating the ability of PepN to negatively regulate TCR signaling at multiple points in the cascade. These results reveal a novel immune regulatory function for a bacterial aminopeptidase.

Streptococcus pneumoniae TIGR4 Phase-Locked Opacity Variants Differ in Virulence Phenotypes.

Streptococcus pneumoniae (pneumococcus) is a leading human pathogen that can cause serious localized and invasive diseases. Pneumococci can undergo a spontaneous and reversible phase variation that is reflected in colony opacity and which allows the population to adapt to different host environments. Generally, transparent variants are adapted for nasopharyngeal colonization, whereas opaque variants are associated with invasive disease. In recent work, colony phase variation was shown to occur by means of recombination events to generate multiple alleles of the hsdS targeting domain of a DNA methylase complex, which mediates epigenetic changes in gene expression. A panel of isogenic strains were created in the well-studied S. pneumoniae TIGR4 background that are "locked" in the transparent (n = 4) or opaque (n = 2) colony phenotype. The strains had significant differences in colony size which were stable over multiple passages in vitro and in vivo. While there were no significant differences in adherence for the phase-locked mutant strains to immortalized epithelial cells, biofilm formation and viability were reduced for the opaque variants in static assays. Nasopharyngeal colonization was stable for all strains, but the mortality rates differed between them. Transcript profiling by transcriptome sequencing (RNA-seq) analyses revealed that the expression levels of certain virulence factors were increased in a phase-specific manner. As epigenetic regulation of phase variation (often referred to as "phasevarion") is emerging as a common theme for mucosal pathogens, these results serve as a model for future studies of host-pathogen interactions. IMPORTANCE A growing number of bacterial species undergo epigenetic phase variation due to variable expression or specificity of DNA-modifying enzymes. For pneumococci, this phase variation has long been appreciated as being revealed by changes in colony opacity, which are reflected in changes in expression or accessibility of factors on the bacterial surface. Recent work showed that recombination-generated variation in alleles of the HsdS DNA methylase specificity subunit mediated pneumococcal phase variation. We generated phase-locked populations of S. pneumoniae TIGR4 expressing a single nonvariant hsdS allele and observed significant differences in gene expression and virulence. These results highlight the importance of focused pathogenesis studies within specific phase types. Moreover, the generation of single-allele hsdS constructs will greatly facilitate such studies.

Activation-dependent modulation of Streptococcus pneumoniae-mediated death in human lymphocytes.

Streptococcus pneumoniae (Spn) is a leading cause of community-acquired pneumonia, with infants and the elderly exhibiting significant susceptibility to the development of severe disease. A growing body of evidence supports the ability of Spn to negatively regulate the host response to infection, e.g. the capacity to induce death in numerous cell types. However, our understanding of the ability of Spn to directly impact lymphocytes remains limited. In this study, we tested the hypothesis that lymphocyte type and activation state influences the susceptibility to pneumococcus-mediated death. We show that in the resting state, CD4+ T cells exhibit a modestly increased susceptibility to Spn-induced death compared to CD8+ T cells or NK cells. In the presence of activating stimuli, the situation most reflective of what would occur in vivo during infection, all subsets demonstrated a significant increase in sensitivity to Spn-mediated death. Importantly, the activated subsets diverged dramatically in susceptibility with natural killer cells exhibiting an 8.6-fold greater sensitivity to pneumococcal components compared to the T-cell subsets. These results significantly expand our understanding of the capacity for pneumococcus to negatively regulate lymphocytes.

Pneumococcal Neuraminidase A (NanA) Promotes Biofilm Formation and Synergizes with Influenza A Virus in Nasal Colonization and Middle Ear Infection.

Even in the vaccine era, Streptococcus pneumoniae (the pneumococcus) remains a leading cause of otitis media, a significant public health burden, in large part because of the high prevalence of nasal colonization with the pneumococcus in children. The primary pneumococcal neuraminidase, NanA, which is a sialidase that catalyzes the cleavage of terminal sialic acids from host glycoconjugates, is involved in both of these processes. Coinfection with influenza A virus, which also expresses a neuraminidase, exacerbates nasal colonization and disease by S. pneumoniae, in part via the synergistic contributions of the viral neuraminidase. The specific role of its pneumococcal counterpart, NanA, in this interaction, however, is less well understood. We demonstrate in a mouse model that NanA-deficient pneumococci are impaired in their ability to cause both nasal colonization and middle ear infection. Coinfection with neuraminidase-expressing influenza virus and S. pneumoniae potentiates both colonization and infection but not to wild-type levels, suggesting an intrinsic role of NanA. Using in vitro models, we show that while NanA contributes to both epithelial adherence and biofilm viability, its effect on the latter is actually independent of its sialidase activity. These data indicate that NanA contributes both enzymatically and nonenzymatically to pneumococcal pathogenesis and, as such, suggest that it is not a redundant bystander during coinfection with influenza A virus. Rather, its expression is required for the full synergism between these two pathogens.

Genetic, biochemical, and serological characterization of a new pneumococcal serotype, 6H, and generation of a pneumococcal strain producing three different capsular repeat units.

Streptococcus pneumoniae clinical isolates were recently described that produced capsular polysaccharide with properties of both serotypes 6A and 6B. Their hybrid serological property correlated with mutations affecting the glycosyltransferase WciP, which links rhamnose to ribitol by an α(1-3) linkage for serotypes 6A and 6C and an α(1-4) linkage for serotypes 6B and 6D. The isolates had mutations in the triad residues of WciP that have been correlated with enzyme specificity. The canonical triad residues of WciP are Ala192-Ser195-Arg254 for serotypes 6A and 6C and Ser192-Asn195-Gly254 for serotypes 6B and 6D. To prove that the mutations in the triad residues are responsible for the hybrid serotype, we introduced the previously described Ala192-Cys195-Arg254 triad into a 6A strain and found that the change made WciP bispecific, resulting in 6A and 6B repeat unit expression, although 6B repeat unit production was favored over production of 6A repeat units. Likewise, this triad permitted a 6C strain to express 6C and 6D repeat units. With reported bispecificity in WciN, which adds either glucose or galactose as the second sugar in the serogroup 6 repeat unit, the possibility exists for a strain to simultaneously produce all four serogroup 6 repeat units; however, when genes encoding both bispecific enzymes were introduced into a 6A strain, only 6A, 6B, and 6D repeat units were detected serologically. Nonetheless, this may be the first example of a bacterial polysaccharide with three different repeat units. This strategy of expressing multiple repeat units in a single polymer is a novel approach to broadening vaccine coverage by eliminating the need for multiple polysaccharide sources to cover multiple serogroup members.

Discovery of Streptococcus pneumoniae serotype 6 variants with glycosyltransferases synthesizing two differing repeating units.

Streptococcus pneumoniae is a persistent, opportunistic commensal of the human nasopharynx and is the leading cause of community-acquired pneumonia. It expresses an anti-phagocytic capsular polysaccharide (PS). Genetic variation of the capsular PS synthesis (cps) locus is the molecular basis for structural and antigenic heterogeneity of capsule types (serotypes). Serogroup 6 has four known members (6A-6D) with distinct serologic properties, homologous cps loci, and structurally similar PSs. cps of serotypes 6A/6B have wciNα, encoding α-1,3-galactosyltransferase, whereas serotypes 6C/6D have wciNß encoding α-1,3-glucosyltransferase. Two atypical serogroup 6 isolates (named 6X11 and 6X12) have been discovered recently in Germany. Flow cytometric studies using monoclonal antibodies show that 6X11 has serologic properties of 6B/6D, whereas 6X12 has 6A/6C. NMR studies of their capsular PSs revealed that 6X11 and 6X12 have two different repeating units with a distribution of ~40:60 6B:6D and 75:25 6A:6C PS, respectively. Sequencing of the wciNα gene in 6X12 and 6X11 revealed single and double nucleotide substitutions, respectively, resulting in the amino acid changes A150T and D38N. Substitution of alanine with threonine at position 150 in a 6A strain was associated with hybrid serologic and chemical profiles like 6X12. The hybrid serotypes represented by 6X12 and 6X11 strains are now named serotypes 6F and 6G. Single amino acid changes in cps genes encoding glycosyltransferases can alter substrate specificities, permit biosynthesis of heterogeneous capsule repeating units, and result in new hybrid capsule types that may differ in their interaction with the immune system of the host.

Streptococcus pneumoniae serotype 11D has a bispecific glycosyltransferase and expresses two different capsular polysaccharide repeating units.

Streptococcus pneumoniae (pneumococcus) expresses a capsular polysaccharide (CPS) that protects against host immunity and is synthesized by enzymes in the capsular polysaccharide synthesis (cps) locus. Serogroup 11 has six members (11A to -E) and the CPS structure of all members has been solved, except for serotype 11D. The cps loci of 11A and 11D differ by one codon (N112S) in wcrL, which putatively encodes a glycosyltransferase that adds the fourth sugar of the CPS repeating unit (RU). Gas chromatography and nuclear magnetic resonance analysis revealed that 11A and 11D PSs contain identical CPS RUs that contain αGlc as the fourth sugar. However, ∼25% of 11D CPS RUs contain instead αGlcNAc as the fourth sugar, suggesting that 11D wcrL encodes a bispecific glycosyltransferase. To test the hypothesis that codon 112 of WcrL determines enzyme specificity, and therefore the fourth sugar in the RU, we generated three isogenic pneumococcal strains with 11A cps loci containing wcrL encoding Ser-112 (MBO128) or Ala-112 (MBO130). MBO128 was serologically and biochemically identical to serotype 11D. MBO130 has a unique serologic profile; has as much αGlcNAc as 11F, 11B, and 11C CPS do; and may represent a new serotype. These findings demonstrate how pneumococci alter their CPS structure and their immunologic properties with a minimal genetic change.

Streptococcus pneumoniae serotype 9A isolates contain diverse mutations to wcjE that result in variable expression of serotype 9V-specific epitope.

BACKGROUND: Streptococcus pneumoniae is a significant pathogen capable of expressing protective and antigenically diverse capsules. To better understand the molecular basis of capsular antigenic diversity, we investigated the hypothetical serological role of wcjE, which encodes a capsule O-acetyltransferase, in the vaccine-targeted serotype 9V and related serotype 9A. METHODS: We inactivated wcjE by recombination in a serotype 9V strain and determined wcjE sequences of 11 serotype 9A clinical isolates. We determined the antigenic phenotypes of these pneumococcal strains with serogroup 9-specific antibodies and flow cytometry. RESULTS: Inactivation of wcjE in a serotype 9V strain resulted in expression of the 9A phenotype. Each serotype 9A clinical isolate contained a distinct mutation to wcjE. Flow cytometry showed that some 9A isolates (herein named 9Aα) expressed trace amounts of 9V-specific epitopes whereas others (named 9Aß) did not express any. Recombination with 9Aα wcjE alleles into a 9Aß strain conferred partial expression of 9V-specific epitopes. CONCLUSIONS: Each serotype 9A strain independently arose from a serotype 9V strain. Furthermore, clinical isolates identified as 9A can contain mutations to wcjE that are either partially functional or completely nonfunctional, demonstrating a previously unidentified antigenic heterogeneity of serotype 9A isolates.