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Abstract Background Sialic acid (N-acetylneuraminic acid; NeuNAc) is one of the most important carbohydrates for Streptococcus pneumoniae due of its role as a carbon and energy source, receptor for adhesion and invasion and molecular signal for promotion of biofilm formation, nasopharyngeal carriage and invasion of the lung. Results In this work, NeuNAc and its metabolic derivative N-acetyl mannosamine (ManNAc) were used to analyze regulatory mechanisms of the neuraminidase locus expression. Genomic and metabolic comparison to Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii and Streptococcus sanguinis elucidates the metabolic association of the two amino sugars to different parts of the locus coding for the two main pneumococcal neuraminidases and confirms the substrate specificity of the respective ABC transporters. Quantitative gene expression analysis shows repression of the locus by glucose and induction of all predicted transcriptional units by ManNAc and NeuNAc, each inducing with higher efficiency the operon encoding for the transporter with higher specificity for the respective amino sugar. Cytofluorimetric analysis demonstrated enhanced surface exposure of NanA on pneumococci grown in NeuNAc and ManNAc and an activity assay allowed to quantify approximately twelve times as much neuraminidase activity on induced cells as opposed to glucose grown cells. Conclusions The present data increase the understanding of metabolic regulation of the nanAB locus and indicate that experiments aimed at the elucidation of the relevance of neuraminidases in pneumococcal virulence should possibly not be carried out on bacteria grown in glucose containing media.

Abstract Background Different models for biofilm in Streptococcus pneumoniae have been described in literature. To permit comparison of experimental data, we characterised the impact of the pneumococcal quorum-sensing competence system on biofilm formation in three models. For this scope, we used two microtiter and one continuous culture biofilm system. Results In both microtiter models the competence system influences stability and structure of biofilm in the late attachment phase and synthetic competence stimulating peptide (CSP) restored wild type phenotypes in the comC mutants unable to produce the peptide. Early attachment of single cells to well bottoms was found for both systems to be competence independent, while later phases, including microcolony formation correlated to an intact competence system. The continuous culture biofilm model was not affected by mutations in the competence locus, but deletion of capsule had a significant impact in this model. Conclusions Since biofilm remains a largely uncharacterised multi-parameter phenotype it appears to be advisable to exploit more than one model in order to draw conclusion of possible relevance of specific genotypes on pneumococcal physiology.

Abstract Background Streptococcus pneumoniae is an important human pathogen representing a major cause of morbidity and mortality worldwide. We sequenced the genome of a serotype 11A, ST62 S. pneumoniae invasive isolate (AP200), that was erythromycin-resistant due to the presence of the erm(TR) determinant, and carried out analysis of the genome organization and comparison with other pneumococcal genomes. Results The genome sequence of S. pneumoniae AP200 is 2,130,580 base pair in length. The genome carries 2216 coding sequences (CDS), 56 tRNA, and 12 rRNA genes. Of the CDSs, 72.9% have a predicted biological known function. AP200 contains the pilus islet 2 and, although its phenotype corresponds to serotype 11A, it contains an 11D capsular locus. Chromosomal rearrangements resulting from a large inversion across the replication axis, and horizontal gene transfer events were observed. The chromosomal inversion is likely implicated in the rebalance of the chromosomal architecture affected by the insertions of two large exogenous elements, the erm(TR)-carrying Tn1806 and a functional prophage designated ϕSpn_200. Tn1806 is 52,457 bp in size and comprises 49 ORFs. Comparative analysis of Tn1806 revealed the presence of a similar genetic element or part of it in related species such as Streptococcus pyogenes and also in the anaerobic species Finegoldia magna, Anaerococcus prevotii and Clostridium difficile. The genome of ϕSpn_200 is 35,989 bp in size and is organized in 47 ORFs grouped into five functional modules. Prophages similar to ϕSpn_200 were found in pneumococci and in other streptococcal species, showing a high degree of exchange of functional modules. ϕSpn_200 viral particles have morphologic characteristics typical of the Siphoviridae family and are capable of infecting a pneumococcal recipient strain. Conclusions The sequence of S. pneumoniae AP200 chromosome revealed a dynamic genome, characterized by chromosomal rearrangements and horizontal gene transfers. The overall diversity of AP200 is driven mainly by the presence of the exogenous elements Tn1806 and ϕSpn_200 that show large gene exchanges with other genetic elements of different bacterial species. These genetic elements likely provide AP200 with additional genes, such as those conferring antibiotic-resistance, promoting its adaptation to the environment.

Abstract Background The IgA1 protease of Streptococcus pneumoniae is a proteolytic enzyme that specifically cleaves the hinge regions of human IgA1, which dominates most mucosal surfaces and is the major IgA isotype in serum. This protease is expressed in all of the known pneumococcal strains and plays a major role in pathogen's resistance to the host immune response. The present work was focused at identifying the immunodominant regions of pneumococcal IgA1 protease recognized by the human antibody response. Results An antigenic sequence corresponding to amino acids 420–457 (epiA) of the iga gene product was identified by screening a pneumococcal phage display library with patients' sera. The epiA peptide is conserved in all pneumococci and in two out of three S. mitis strains, while it is not present in other oral streptococci so far sequenced. This epitope was specifically recognized by antibodies present in sera from 90% of healthy adults, thus representing an important target of the humoral response to S. pneumoniae and S. mitis infection. Moreover, sera from 68% of children less than 4 years old reacted with the epiA peptide, indicating that the human immune response against streptococcal antigens occurs during childhood. Conclusion The broad and specific recognition of the epiA polypeptide by human sera demonstrate that the pneumococcal IgA1 protease contains an immunodominant B-cell epitope. The use of phage display libraries to identify microbe or disease-specific antigens recognized by human sera is a valuable approach to epitope discovery.

Abstract Background In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery and production of recombinant proteins. Usually, bacteria are manipulated using plasmid expression vectors. The major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. We have developed a genetic system based on suicide vectors on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable Gram-positive bacteria. Results The aim of this work was to select a strong chromosomal promoter from Streptococcus gordonii to improve this genetic system making it suitable for expression of single-copy recombinant genes. To achieve this task, a promoterless gene encoding a chloramphenicol acetyltransferase (cat), was randomly integrated into the S. gordonii chromosome and transformants were selected for chloramphenicol resistance. Three out of eighteen chloramphenicol resistant transformants selected exhibited 100% stability of the phenotype and only one of them, GP215, carried the cat gene integrated as a single copy. A DNA fragment of 600 base pairs exhibiting promoter activity was isolated from GP215 and sequenced. The 5' end of its corresponding mRNA was determined by primer extention analysis and the putative -10 and a -35 regions were identified. To study the possibility of using this promoter (PP) for single copy heterologous gene expression, we created transcriptional fusions of PP with genes encoding surface recombinant proteins in a vector capable of integrating into the conjugative transposon Tn916. Surface recombinant proteins whose expression was controlled by the PP promoter were detected in Tn916-containing strains of S. gordonii and Bacillus subtilis after single copy chromosomal integration of the recombinant insertion vectors into the resident Tn916. The surface recombinant protein synthesized under the control of PP was also detected in Enterococcus faecalis after conjugal transfer of a recombinant Tn916 containing the transcriptional fusion. Conclusion We isolated and characterized a S. gordonii chromosomal promoter. We demonstrated that this promoter can be used to direct expression of heterologous genes in different Gram-positive bacteria, when integrated in a single copy into the chromosome.

Abstract Background Streptococcus pneumoniae is the leading cause of bacterial meningitis. Pneumococcal meningitis is associated with the highest mortality among bacterial meningitis and it may also lead to neurological sequelae despite the use of antibiotic therapy. Experimental animal models of pneumococcal meningitis are important to study the pathogenesis of meningitis, the host immune response induced after infection, and the efficacy of novel drugs and vaccines. Results In the present work, we describe in detail a simple, reproducible and efficient method to induce pneumococcal meningitis in outbred mice by using the intracranial subarachnoidal route of infection. Bacteria were injected into the subarachnoid space through a soft point located 3.5 mm rostral from the bregma. The model was tested with several doses of pneumococci of three capsular serotypes (2, 3 and 4), and mice survival was recorded. Lethal doses killing 50 % of animals infected with type 2, 3 and 4 S. pneumoniae were 3.2 × 10, 2.9 × 10 and 1.9 × 102 colony forming units, respectively. Characterisation of the disease caused by the type 4 strain showed that in moribund mice systemic dissemination of pneumococci to blood and spleen occurred. Histological analysis of the brain of animals infected with type 4 S. pneumoniae proved the induction of meningitis closely resembling the disease in humans. Conclusions The proposed method for inducing pneumococcal meningitis in outbred mice is easy-to-perform, fast, cost-effective, and reproducible, irrespective of the serotype of pneumococci used.

Abstract Background Streptococcus pneumoniae possesses large zinc metalloproteinases on its surface. To analyse the importance in virulence of three of these metalloproteinases, intranasal challenge of MF1 outbred mice was carried out using a range of infecting doses of wild type and knock-out pneumococcal mutant strains, in order to compare mice survival. Results Observation of survival percentages over time and detection of LD50s of knock out mutants in the proteinase genes in comparison to the type 4 TIGR4 wild type strain revealed two major aspects: i) Iga and ZmpB, present in all strains of S. pneumoniae, strongly contribute to virulence in mice; (ii) ZmpC, only present in about 25% of pneumococcal strains, has a lower influence on virulence in mice. Conclusions These data suggest Iga, ZmpB and ZmpC as candidate surface proteins responsible for pneumococcal infection and potentially involved in distinct stages of pneumococcal disease.

The ubiquitous protein LuxS with S-ribosylhomocysteinase activity is involved in S-adenosyl methionine detoxification, C-1 unit recycling and the production of autoinducers that allow the cell to sense and respond to cell density. Independent reports describe the impact of LuxS deficiency on Streptococcus pneumoniae virulence in the mouse. In vitro, LuxS deficiency confers discrete phenotypes. A combined approach using genetic dissection and mixed-culture experiments allowed the involvement of LuxS in the developmental physiology of S. pneumoniae to be investigated. Functional LuxS was found to be related on the one hand to down-regulation of competence, and on the other hand to attenuation of autolysis in cultures entering stationary phase. The competence phenotype of luxS mutant bacteria was complemented by media conditioned by competence-defective ComABO bacteria, but not by BSA. The autolytic phenotype was complemented by BSA, but not by conditioned supernatants. It is suggested that the impact of LuxS on competence, but not on autolysis, involves cell-cell communication. The phenotype of luxS mutant strains reveals a hierarchy in the competence regulatory networks of S. pneumoniae.

Pneumococci display large zinc metalloproteinases on the surface, including the IgA protease, which cleaves human IgA1 in the hinge region, the ZmpC proteinase, which cleaves human matrix metalloproteinase 9 (MMP-9), and two other proteinases, ZmpB and ZmpD, whose substrates have not yet been identified. Surface metalloproteinases are antigenic and have been linked to virulence. The genes encoding these proteinases reside in three distinct loci: two loci specific for zmpB and zmpC, and a third, the iga locus, containing iga and zmpD. Data obtained by this and other groups have shown that pneumococcal metalloproteinase genes are transcribed and yield mature and enzymatically active proteins. Since the presence of the four proteinase genes is variable in the pneumococcal strains whose genomes have been sequenced, the presence of these genes in a collection of 218 pneumococcal isolates, mostly from invasive disease, was investigated. The data showed that zmpB and iga were present in all the isolates examined, while zmpC and zmpD were present in a variable proportion of the isolates (in 18 and 49 %, respectively). Interestingly, isolates carrying both zmpC and zmpD were found to belong mainly to two serotypes (sts), 8 and 11A. By molecular typing, st 8 and st 11A isolates appeared to belong to the same clonal cluster. The presence of these two additional metalloproteinases could contribute to the fitness of particular pneumococcal clones.