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1.  The Role of Host and Microbial Factors in the Pathogenesis of Pneumococcal Bacteraemia Arising from a Single Bacterial Cell Bottleneck 
PLoS Pathogens  2014;10(3):e1004026.
The pathogenesis of bacteraemia after challenge with one million pneumococci of three isogenic variants was investigated. Sequential analyses of blood samples indicated that most episodes of bacteraemia were monoclonal events providing compelling evidence for a single bacterial cell bottleneck at the origin of invasive disease. With respect to host determinants, results identified novel properties of splenic macrophages and a role for neutrophils in early clearance of pneumococci. Concerning microbial factors, whole genome sequencing provided genetic evidence for the clonal origin of the bacteraemia and identified SNPs in distinct sub-units of F0/F1 ATPase in the majority of the ex vivo isolates. When compared to parental organisms of the inoculum, ex-vivo pneumococci with mutant alleles of the F0/F1 ATPase had acquired the capacity to grow at low pH at the cost of the capacity to grow at high pH. Although founded by a single cell, the genotypes of pneumococci in septicaemic mice indicate strong selective pressure for fitness, emphasising the within-host complexity of the pathogenesis of invasive disease.
Author Summary
Decades of research on bacterial sepsis have been devoted to analysing the steps that lead from a local event, either carriage or a localised infection, to systemic disease. Our work analyses in depth the events determining systemic infection by one of the main human pathogens, Streptococcus pneumoniae. Consistent with similar findings on the pathogenesis of bacteraemia due to other commensal pathogens, our results show that after an intravenous inoculum of a million pneumococci, the resulting septicaemia is often founded by a single bacterial cell. Investigation into the nature of this monoclonal infection identified strong within-host selective pressure for metabolic fitness during outgrowth of the bacterial population.
doi:10.1371/journal.ppat.1004026
PMCID: PMC3961388  PMID: 24651834
2.  Contribution of different pneumococcal virulence factors to experimental meningitis in mice 
BMC Infectious Diseases  2013;13:444.
Background
Pneumococcal meningitis (PM) is a life-threatening disease with a high case-fatality rate and elevated risk for serious neurological sequelae. In this study, we investigated the contribution of three major virulence factors of Streptococcus pneumoniae, the capsule, pneumococcal surface protein A (PspA) and C (PspC), to the pathogenesis of experimental PM.
Methods
Mice were challenged by the intracranial route with the serotype 4 TIGR4 strain (wt) and three isogenic mutants devoid of PspA, PspC, and the capsule. Survival, bacterial counts, and brain histology were carried out. To study the interaction between S. pneumoniae mutants and microglia, phagocytosis and survival experiments were performed using the BV2 mouse microglial cell line.
Results
Virulence of the PspC mutant was comparable to that of TIGR4. In contrast, survival of animals challenged with the PspA mutant was significantly increased compared with the wt, and the mutant was also impaired at replicating in the brain and blood of infected mice. Brain histology indicated that all strains, except for the unencapsulated mutant, caused PM. Analysis of inflammation and damage in the brain of mice infected with TIGR4 or its unencapsulated mutant demonstrated that the rough strain was unable to induce inflammation and neuronal injury, even at high challenge doses. Results with BV2 cells showed no differences in phagocytic uptake between wt and mutants. In survival assays, however, the PspA mutant showed significantly reduced survival in microglia compared with the wt.
Conclusions
PspA contributed to PM pathogenesis possibly by interacting with microglia at early infection stages, while PspC had limited importance in the disease. The rough mutant did not cause brain inflammation, neuronal damage or mouse death, strengthening the key role of the capsule in PM.
doi:10.1186/1471-2334-13-444
PMCID: PMC3848944  PMID: 24059458
Experimental pneumococcal meningitis; Microglia; PspA; PspC; Capsule
3.  Regulation of neuraminidase expression in Streptococcus pneumoniae 
BMC Microbiology  2012;12:200.
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.
doi:10.1186/1471-2180-12-200
PMCID: PMC3509027  PMID: 22963456
Sialic acid; Metabolic regulation; Carbon catabolite repression

Results 1-3 (3)