Growth of Streptococcus pneumoniae on human glycoconjugates is dependent upon the sequential activity of bacterial exoglycosidases

In the human host, Streptococcus pneumoniae encounters a variety of glycoconjugates, including mucin, host defense molecules, and glycans associated with the epithelial surface. S. pneumoniae is known to encode a number of glycosidases that may modify these glycoconjugates in vivo. Three exoglycosidases, a neuraminidase (NanA), β-galactosidase (BgaA), and N-acetylglucosaminidase (StrH), have been previously demonstrated to sequentially deglycosylate N-linked glycans on host defense molecules, which coat the pneumococcal surface in vivo. This cleavage is proposed to alter the clearance function of these molecules, allowing pneumococci to persist in the airway. However, we propose that the exoglycosidase-dependent liberation of monosaccharides from these glycoconjugates in close proximity to the pneumococcal surface provides S. pneumoniae with a convenient source of fermentable carbohydrate in vivo. In this study, we demonstrate that S. pneumoniae is able to utilize complex N-linked human glycoconjugates as a sole source of carbon to sustain growth and that efficient growth is dependent upon the sequential deglycosylation of the glycoconjugate substrate by pneumococcal exoglycosidases. In addition to demonstrating a role for NanA, BgaA, and StrH, we have identified a function for the second pneumococcal neuraminidase, NanB, in the deglycosylation of host glycoconjugates and have demonstrated that NanB activity can partially compensate for the loss or dysfunction of NanA. To date, all known functions of pneumococcal neuraminidase have been attributed to NanA. Thus, this study describes the first proposed role for NanB by which it may contribute to S. pneumoniae colonization and pathogenesis.     

Streptococcus pneumoniae in Saliva of Dutch Primary School Children

While nasopharyngeal sampling is the gold standard for the detection of Streptococcus pneumoniae carriage, historically seen, saliva sampling also seems highly sensitive for pneumococcal detection. We investigated S. pneumoniae carriage in saliva from fifty schoolchildren by conventional and molecular methods. Saliva was first culture-enriched for pneumococci, after which, DNA was extracted from all bacterial growth and tested by quantitative-PCR (qPCR) for pneumococcus-specific genes lytA and piaA. Next, serotype composition of the samples was determined by serotype-specific qPCRs, conventional-PCRs (cPCR) and sequencing of cPCR amplicons. Although only 2 (4%) of 50 samples were positive by conventional diagnostic culture, 44 (88%) were positive for pneumococci by qPCR. In total, we detected the presence of at least 81 pneumococcal strains representing 20 serotypes in samples from 44 carriers with 23 carriers (52%) positive for multiple (up to 6) serotypes. The number of serotypes detected per sample correlated with pneumococcal abundance. This study shows that saliva could be used as a tool for future pneumococcal surveillance studies. Furthermore, high rates of pneumococcal carriage and co-carriage of multiple pneumococcal strains together with a large number of serotypes in circulation suggests a ubiquitous presence of S. pneumoniae in saliva of school-aged children. Our results also suggest that factors promoting pneumococcal carriage within individual hosts may weaken competitive interactions between S. pneumoniae strains.     

Structural and Functional Analysis of Fucose-Processing Enzymes from Streptococcus pneumoniae

Fucose metabolism pathways are present in many bacterial species and typically contain the central fucose-processing enzymes fucose isomerase (FcsI), fuculose kinase (FcsK), and fuculose-1-phosphate aldolase (FcsA). Fucose initially undergoes isomerization by FcsI producing fuculose, which is then phosphorylated by FcsK. FcsA cleaves the fuculose-1-phosphate product into lactaldehyde and dihydroxyacetone phosphate, which can be incorporated into central metabolism allowing the bacterium to use fucose as an energy source. Streptococcus pneumoniae has fucose-processing operons containing homologs of FcsI, FcsK, and FcsA; however, this bacterium appears unable to utilize fucose as an energy source. To investigate this contradiction, we performed biochemical and structural studies of the S. pneumoniae fucose-processing enzymes SpFcsI, SpFcsK, and SpFcsA. These enzymes are demonstrated to act in a sequential manner to ultimately produce dihydroxyacetone phosphate and have structural features entirely consistent with their observed biochemical activities. Analogous to the regulation of the Escherichia coli fucose utilization operon, fuculose-1-phosphate appears to act as an inducing molecule for activation of the S. pneumoniae fucose operon. Despite our evidence that S. pneumoniae appears to have the appropriate regulatory and biochemical machinery for fucose metabolism, we confirmed the inability of the S. pneumoniae TIGR4 strain to grow on fucose or on the H-disaccharide, which is the probable substrate of the transporter for the pathway. On the basis of these observations, we postulate that the S. pneumoniae fucose-processing pathway has a non-metabolic role in the interaction of this bacterium with its human host.     

Streptococcus pneumoniae Invades Erythrocytes and Utilizes Them to Evade Human Innate Immunity

Streptococcus pneumoniae, a Gram-positive bacterium, is a major cause of invasive infection-related diseases such as pneumonia and sepsis. In blood, erythrocytes are considered to be an important factor for bacterial growth, as they contain abundant nutrients. However, the relationship between S. pneumoniae and erythrocytes remains unclear. We analyzed interactions between S. pneumoniae and erythrocytes, and found that iron ion present in human erythrocytes supported the growth of Staphylococcus aureus, another major Gram-positive sepsis pathogen, while it partially inhibited pneumococcal growth by generating free radicals. S. pneumoniae cells incubated with human erythrocytes or blood were subjected to scanning electron and confocal fluorescence microscopic analyses, which showed that the bacterial cells adhered to and invaded human erythrocytes. In addition, S. pneumoniae cells were found associated with human erythrocytes in cultures of blood from patients with an invasive pneumococcal infection. Erythrocyte invasion assays indicated that LPXTG motif-containing pneumococcal proteins, erythrocyte lipid rafts, and erythrocyte actin remodeling are all involved in the invasion mechanism. In a neutrophil killing assay, the viability of S. pneumoniae co-incubated with erythrocytes was higher than that without erythrocytes. Also, H₂O₂ killing of S. pneumoniae was nearly completely ineffective in the presence of erythrocytes. These results indicate that even when S. pneumoniae organisms are partially killed by iron ion-induced free radicals, they can still invade erythrocytes. Furthermore, in the presence of erythrocytes, S. pneumoniae can more effectively evade antibiotics, neutrophil phagocytosis, and H₂O₂ killing.     

Differential Recognition and Hydrolysis of Host Carbohydrate Antigens by Streptococcus pneumoniae Family 98 Glycoside Hydrolases

The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-β-galactosidase activity on the Lewis^Y antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-β-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.Streptococcus pneumoniae asymptomatically colonizes the nasopharynx of 10–40% of people, but given the appropriate opportunity, it can become an extremely aggressive pathogen (1–3). This bacterium causes millions of deaths annually (1), is acquiring antibiotic resistance (4), and shows a disturbing and lethal synergy with the Influenza virus (5). The ability of S. pneumoniae to cause invasive disease is increasingly being linked with the capacity of this bacterium to attack and process the glycans present in host tissues (see Ref. 6 for a review). Indeed, large scale screening of pneumococcal virulence factors has revealed a large complement of genes devoted to complex carbohydrate metabolism that contribute to pneumococcal virulence (7–9). Recent elegant studies have focused on showing how a group of three exo-glycosidases sequentially trim complex human N-glycans (10, 11). These enzymes, however, only make up a fraction of the 39 glycosidases predicted to be in the pneumococcal genome (TIGR4 strain); at least 18 of these 39 are required for full virulence of the bacterium (7). Despite the growing appreciation for the role of carbohydrate metabolism in pneumococcal virulence and the possibility of targeting such metabolic pathways with small molecule therapeutic compounds, the bulk of the carbohydrate-active proteins of S. pneumoniae remain unexamined. As such, we presently have a relatively superficial but growing appreciation for the array of host glycans that S. pneumoniae can degrade.Several S. pneumoniae genes whose protein products are dedicated to the harvesting and processing of the sugar fucose are beginning to emerge as an important set of pneumococcal virulence factors (12). Comparative genomic studies of several S. pneumoniae genomes has suggested genetic variability at this locus; however, some components of the operon were observed to be present in all of the studied isolates (13). Through our recent identification and characterization of a novel solute-binding protein present in an alternate pneumococcal fucose utilization operon, we have made the observation that there are two different fucose utilization operons distributed among pneumococcal strains (14). Although the organization and composition of the two operons is different, both pathways are predicted to be initiated by the action of a family 98 glycoside hydrolase that is probably secreted (for a discussion of the sequence classification system of glycoside hydrolases, see Ref. 15). This GH98 is the same as that identified as a virulence factor in the TIGR4 strain (7). Remarkably, the GH98 enzymes from the two different pathways display different modular architectures, and their shared catalytic modules only have modest amino acid sequence identity. Given the placement of these enzymes in a fucose utilization operon, we hypothesized that they have activity on fucose-containing glycans; however, their divergent sequences and different modular arrangements led us to postulate that they would have different glycan substrate specificities.Here we describe the specificity and catalytic mechanism for these two different types of S. pneumoniae GH98 enzymes, one from the TIGR4 strain (Sp4GH98) and the other from the SP3-BS71 strain (Sp3GH98). Both enzymes act as endo-β-1,4-galactosidases on the galactosyl-β-1,4-N-acetylglucosamine linkage found in type 2 carbohydrate blood group antigens, although Sp4GH98 displays specificity for the Lewis^Y antigen, whereas Sp3GH98 is highly selective for the same linkage in the blood group A/B-antigens. The biochemical analysis of these enzymes in combination with the determination of their structures in complex with products and substrates provides molecular level insight to their catalytic mechanism and how they discriminate between their respective substrates. We discuss these results in the context of the recent association of the pneumococcal fucose utilization operon with the virulence of S. pneumoniae (7, 12) and the possible strain-specific dependence of pneumococcal virulence on the carbohydrate antigens presented by different hosts.     

Posttranscriptional repression of the cel gene of the ColE7 operon by the RNA-binding protein CsrA of Escherichia coli

Carbon storage regulator (CsrA) is a eubacterial RNA-binding protein that acts as a global regulator of many functionally diverse chromosomal genes. Here, we reveal that CsrA represses expression from an extrachromosomal element of Escherichia coli, the lysis gene (cel) of the ColE7 operon (cea-cei-cel). This operon and colicin expression are activated upon SOS response. Disruption of csrA caused ∼5-fold increase of the lysis protein. Gel mobility shift assays established that both the single-stranded loop of the T1 stem–loop distal to cei, and the putative CsrA binding site overlapping the Shine–Dalgarno sequence (SD) of the cel gene are important for CsrA binding. Substitution mutations at SD relieved CsrA-dependent repression of the cel gene in vivo. Steady-state levels and half-life of the cel mRNA were not affected by CsrA, implying that regulation is mediated at the translational level. Levels of CsrB and CsrC sRNAs, which bind to and antagonize CsrA, were drastically reduced upon induction of the SOS response, while the CsrA protein itself remained unaffected. Thus, CsrA is a trans-acting modulator that downregulates the expression of lysis protein, which may confer a survival advantage on colicinogenic E. coli under environment stress conditions.     

Superiority of Trans-Oral over Trans-Nasal Sampling in Detecting Streptococcus pneumoniae Colonization in Adults

The human nasopharynx is the main reservoir for Streptococcus pneumoniae. We applied conventional and molecular methods to determine the prevalence of S. pneumoniae nasopharyngeal colonization in adults. Paired trans-orally and trans-nasally obtained nasopharyngeal samples from 268 parents of 24-month-old children were assessed for pneumococcal presence. Parents were classified as colonized when live pneumococci were recovered from either sample cultured on medium selective for S. pneumoniae. Of the 52 (19%) colonized parents 49 (18%) were culture-positive in trans-nasal and 10 (4%) in trans-oral samples. Bacterial growth was harvested from these cultures, DNA isolated and tested by quantitative-PCR (qPCR) targeting lytA and piaA genes specific for S. pneumoniae. A sample was considered positive if signals for both genes were detected. Altogether 105 (39%) individuals were classified as positive for pneumococcus by qPCR including 50 (19%) in trans-nasal and 94 (35%) in trans-oral settings. Although significantly more trans-nasal compared to trans-oral samples were culture-positive for S. pneumoniae at the primary diagnostic step (p<0.001) the opposite was observed in qPCR results (p<0.001). To confirm the presence of live pneumococcus in samples positive by qPCR but negative at the initial diagnostic step, we serially-diluted cell harvests, re-cultured and carefully examined for S. pneumoniae presence. Live pneumococci were recovered from an additional 43 parents including 42 positive in trans-oral and 4 in trans-nasal samples increasing the number of individuals culture- and qPCR-positive to 93 (35%) and positive by either of two methods to 107 (40%). There were significantly more trans-oral than trans-nasal samples positive for pneumococcus by both culture and qPCR (n = 71; 27%; vs. n = 50; 19%; p<0.05). Our data suggest that pneumococcal colonization is more common in adults than previously estimated and point towards the superiority of a trans-oral over a trans-nasal approach when testing adults for colonization with S. pneumoniae.     

The Polysaccharide Capsule of Streptococcus pneumonia Partially Impedes MyD88-Mediated Immunity during Pneumonia in Mice

Toll-like receptors (TLR) and the downstream adaptor protein MyD88 are considered crucial for protective immunity during bacterial infections. Streptococcus (S.) pneumoniae is a human respiratory pathogen and a large majority of clinical pneumococcal isolates expresses an external polysaccharide capsule. We here sought to determine the role of pneumococcal capsule in MyD88-mediated antibacterial defense during S. pneumonia pneumonia. Wild type (WT) and Myd88^-/- mice were inoculated intranasally with serotype 2 S. pneumoniae D39 or with an isogenic capsule locus deletion mutant (D39∆cps), and analysed for bacterial outgrowth and inflammatory responses in the lung. As compared to WT mice, Myd88^-/- mice infected with D39 demonstrated a modestly impaired bacterial clearance accompanied by decreased inflammatory responses in the lung. Strikingly, while WT mice rapidly cleared D39∆cps, Myd88^-/- mice showed 10⁵-fold higher bacterial burdens in their lungs and dissemination to blood 24 hours after infection. These data suggest that the pneumococcal capsule impairs recognition of TLR ligands expressed by S. pneumoniae and thereby partially impedes MyD88-mediated antibacterial defense.