The human nasopharynx is an important ecological niche for Streptococcus pneumoniae, and asymptomatic nasopharyngeal carriage is a common precursor to invasive disease. However, knowledge of the immunological events, which occur during carriage, both on a cellular and humoral level, remains limited. Here, we present a long-term stable model of asymptomatic nasopharyngeal carriage using outbred naïve mice, in which we have investigated the effect of previous nasopharyngeal exposure to pneumococci, in the prevention of subsequent carriage and invasive disease. Carriage of D39 wildtype pneumococci restricted to the nasopharynx could be detected for at least 28 days post-infection, whereas nasopharyngeal carriage of a pneumolysin negative isogenic mutant (PLN-A) was cleared in 7-14 days. Both carriage events induced total and capsule specific IgA mucosal antibodies and increased levels of systemic antibodies (IgG against pneumococcal surface protein A (PspA) and IgM capsular polysaccharide), which increased over time and correlated to reduced nasopharyngeal pneumococcal numbers. Prior nasopharyngeal colonisation with PLN-A significantly reduced the duration of subsequent D39 wildtype carriage, and significantly increased survival following invasive pneumococcal challenge. In this case systemic anti-PspA and anti-capsular antibody IgM concentrations showed a strong correlation with reduced bacterial numbers in the lungs and nasopharynx, respectively and also with increased levels of IL17A and CD4+ T cells in lungs of pre-colonised mice. Prior nasopharyngeal colonisation with PLN-A also resulted in significant cross-serotype protection with mice protected from invasive disease with serotype 3 strain (A66) after pre-colonisation with a serotype 2 strain (D39). Our results suggest that both mucosal and systemic antibody as well as cellular host factors have a role in long-term protection against both colonisation and invasive pneumococcal challenge.

The pneumococcus obtains its energy from the metabolism of host glycosides. Therefore efficient degradation of host glycoproteins is integral to pneumococcal virulence. In search of novel pneumococcal glycosidases, we characterised the S. pneumoniae strain D39 protein encoded by SPD0065 and found that this gene encodes a beta-galactosidase. The SPD0065 recombinant protein released galactose from desialylated fetuin, used here as a model of glycoproteins found in vivo. A pneumococcal mutant with a mutation in SPD0065 showed diminished beta-galactosidase activity, exhibited an extended lag period in mucin-containing defined medium and it cleaved significantly less galactose than the parental strain during growth on mucin. As pneumococcal beta-galactosidase activity had been previously attributed solely to SPD0562 (bgaA), we evaluated the contribution of SPD0065 and SPD0562 to total beta-galactosidase activity. Mutation of either gene significantly reduced enzymic activity but beta-galactosidase activity in the double mutant, although significantly less than in either of the single mutants, was not completely abolished. The expression of SPD0065 in S. pneumoniae grown in mucin-containing medium or tissues harvested from infected animals was significantly up-regulated compared to pneumococci from glucose-containing medium. The SPD0065 mutant strain was found to be attenuated in virulence in a manner specific to the host tissue.

Streptococcus pneumoniae resides in the oxygen-rich environment of the upper respiratory tract, and therefore the ability to survive in the presence of oxygen is an important aspect of its in vivo survival. To investigate how S. pneumoniae adapts to oxygen, we determined the global gene expression profile of the microorganism in aerobiosis and anaerobiosis. It was found that exposure to aerobiosis elevated the expression of 54 genes, while the expression of 15 genes was down-regulated. Notably there were significant changes in putative genome plasticity and hypothetical genes. In addition, increased expression of rgg, a putative transcriptional regulator, was detected. To test Rgg's role in the pneumococcal oxidative stress response, an isogenic mutant was constructed. It was found that the mutant was sensitive to oxygen and paraquat, but not to H2O2. In addition, the absence of Rgg strongly reduced biofilm forming ability of an unencapsulated pneumococcus. Virulence studies showed that the median survival time of mice infected intranasally with the rgg mutant was significantly longer than in the wild-type infected group, and the animals infected with the mutant developed septicaemia later than those infected intranasally with the wild type.

Knowledge of the in vivo physiology and metabolism of Streptococcus pneumoniae is limited, even though pneumococci rely on efficient acquisition and metabolism of the host nutrients for growth and survival. Because the nutrient-limited, hypoxic host tissues favour mixed acid fermentation, we studied the role of the pneumococcal pyruvate formate lyase (PFL), a key enzyme in mixed acid fermentation, which is activated post-translationally by pyruvate formate lyase activating enzyme (PFL-AE). Mutations were introduced to two putative pfl, SPD0235 and SPD0420, and two putative pfl-ae, SPD0229 and SPD1774. End-product analysis showed there was no formate, the main end product of the reaction catalysed by PFL, produced by mutants defective in SPD0420 and SPD1774, indicating that SPD0420 encodes for PFL and SPD1774 for putative PFL-AE. Expression of SPD0420 was elevated in galactose-containing medium in anaerobiosis compared to growth in glucose, and the mutation of SPD0420 resulted in up-regulation of fba and pyk, encoding respectively fructose 1,6-bisphosphate aldolase and pyruvate kinase, under the same conditions. In addition, an altered fatty acid composition was detected in SPD0420 and SPD1774 mutants. Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type infected cohort, and bacteraemia developed later than the wild type infected group. Furthermore, the numbers of cfu of SPD0420 mutant were lower in the nasopharynx and the lungs after intranasal infection, and fewer mutant cfu were recovered from the blood compared to the wild type, after intravenous infection. The results demonstrate that there is a direct link between pneumococcal fermentative metabolism and virulence.

Anti-polysaccharide immunity is a key facet of protection against several bacterial pathogens. Problems exist with current polysaccharide vaccines and alternative strategies that deliver a protective response are needed. We have identified immunological peptide mimics of type 6B and 9V pneumococcal capsular polysaccharides that could be used as vaccine antigens. Peptides mimicking antigenic properties of serotype 6B capsular polysaccharide were obtained from a phage-displayed peptide library expressing dodecameric peptides, using a human monoclonal antibody (Db3G9). A murine monoclonal antibody (206, F-5) against the serotype 9V capsular polysaccharide identified three peptide mimotopes from the dodecameric peptide library and one from a random pentadecameric peptide library. In ELISA, binding of 206, F-5 and Db3G9 to phage displaying the selected mimotopes was significantly inhibited by type-specific pneumococcal polysaccharide. Peptides were conjugated to keyhole limpet haemocyanin and were used to immunise mice. Two peptides, MP13 and MP7, induced specific anti-6B and 9V polysaccharide antibodies, respectively. Mice immunised with MP7-keyhole limpet hemocyanin or MP13-keyhole limpet hemocyanin conjugate were significantly and specifically protected against a lethal challenge with pneumococci of the appropriate serotype. This study provides strong in vivo evidence that peptide mimics are alternatives to polysaccharide vaccines.

The correlation between carbohydrate availability, pneumococcal biofilm formation, nasopharyngeal colonization, and invasion of the host has been investigated. Of a series of sugars, only sialic acid (i.e., N-acetylneuraminic acid) enhanced pneumococcal biofilm formation in vitro, at concentrations similar to those of free sialic acid in human saliva. In a murine model of pneumococcal carriage, intranasal inoculation of sialic acid significantly increased pneumococcal counts in the nasopharynx and instigated translocation of pneumococci to the lungs. Competition of both sialic acid-dependent phenotypes was found to be successful when evaluated using the neuraminidase inhibitors DANA (i.e., 2,3-didehydro-2-deoxy-N-acetylneuraminic acid), zanamivir, and oseltamivir. The association between levels of free sialic acid on mucosae, pneumococcal colonization, and development of invasive disease shows how a host-derived molecule can influence a colonizing microbe and also highlights a molecular mechanism that explains the epidemiologic correlation between respiratory infections due to neuraminidase-bearing viruses and bacterial pneumonia. The data provide a new paradigm for the role of a host compound in infectious diseases and point to new treatment strategies.

Streptococcus pneumoniae is a human commensal and pathogen able to cause a variety of diseases that annually result in over a million deaths worldwide. The S. pneumoniae(Spain23F) sequence type 81 lineage was amongst the first recognised pandemic clones and was responsible for almost 40% of penicillin resistant pneumococcal infections in the USA in the late 1990s. Analysis of the chromosome sequence of a representative strain, and comparison with other available genomes, indicates roles for integrative and conjugative elements (ICE) in the evolution of pneumococci and, more particularly, the emergence of the multi-drug resistant Spain 23F ST81 lineage. A number of recently acquired loci within the chromosome appear to encode proteins involved in the production of, or immunity to, antimicrobial compounds, which may contribute to the proficiency of this strain at nasopharyngeal colonisation. However, further sequencing of other pandemic clones will be required to establish whether there are any general attributes shared by these strains that are responsible for their international success.

The Streptococcus pneumoniae genomes encode up to three sialidases (or neuraminidases), NanA, NanB and NanC, which are believed to be involved in removing sialic acid from host cell surface glycans, thereby promoting colonization of the upper respiratory tract. Here, we present the crystal structure of NanB to 1.7 A resolution derived from a crystal grown in the presence of the buffer Ches (2-N-cyclohexylaminoethanesulfonic acid). Serendipitously, Ches was found bound to NanB at the enzyme active site, and was found to inhibit NanB with a K(i) of approximately 0.5 mM. In addition, we present the structure to 2.4 A resolution of NanB in complex with the transition-state analogue Neu5Ac2en (2-deoxy-2,3-dehydro-N-acetyl neuraminic acid), which inhibits NanB with a K(i) of approximately 0.3 mM. The sulphonic acid group of Ches and carboxylic acid group of Neu5Ac2en interact with the arginine triad of the active site. The cyclohexyl group of Ches binds in the hydrophobic pocket of NanB occupied by the acetamidomethyl group of Neu5Ac2en. The topology around the NanB active site suggests that the enzyme would have a preference for alpha2,3-linked sialoglycoconjugates, which is confirmed by a kinetic analysis of substrate binding. NMR studies also confirm this preference and show that, like the leech sialidase, NanB acts as an intramolecular trans-sialidase releasing Neu2,7-anhydro5Ac. All three pneumoccocal sialidases possess a carbohydrate-binding domain that is predicted to bind sialic acid. These studies provide support for a possible differential role for NanB compared to NanA in pneumococcal virulence.

Streptococcus pneumoniae genomes encode three sialidases, NanA, NanB and NanC, which are key virulence factors that remove sialic acids from various glycoconjugates. The enzymes have potential as drug targets and also as vaccine candidates. The 115 kDa NanA is the largest of the three sialidases and is anchored to the bacterial membrane. Although recombinantly expressed full-length NanA was soluble, it failed to crystallize; therefore, a 56.5 kDa domain that retained full enzyme activity was subcloned. The purified enzyme was crystallized in 0.1 M MES pH 6.5, 30%(w/v) PEG 4000 using the sitting-drop vapour-diffusion method. Data were collected at 100 K to 2.5 A resolution from a crystal grown in the presence of the inhibitor 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 49.2, b = 95.6, c = 226.6 A. The structure was solved by molecular replacement and refined to final R and R(free) factors of 0.246 and 0.298, respectively.

In response to a large local school-based outbreak of tuberculosis, we have been evaluating the utility of microarray bacterial genomic analysis in outbreak management. After initial comparison of the isolate from the index case with Mycobacterium tuberculosis H37Rv, it was possible to design robust PCRs directed towards strain-specific deletions. Rapid PCR analysis of isolates proved valuable in determining whether or not other isolates were compatible with the outbreak strain and further microarray studies revealed genetic markers that could be used to discriminate between locally circulating strains.We suggest that this approach forms the basis for developing rapid local genotyping schemes applicable to M. tuberculosis and that application to other pathogens warrants consideration.