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Funder: Cambridge Trust, Funder: Islamic Development Bank, Funder: AstraZeneca, Lipoteichoic acid synthase (LtaS) is a key enzyme for the cell wall biosynthesis of Gram-positive bacteria. Gram-positive bacteria that lack lipoteichoic acid (LTA) exhibit impaired cell division and growth defects. Thus, LtaS appears to be an attractive antimicrobial target. The pharmacology around LtaS remains largely unexplored with only two small-molecule LtaS inhibitors reported, namely "compound 1771" and the Congo red dye. Structure-based drug discovery efforts against LtaS remain unattempted due to the lack of an inhibitor-bound structure of LtaS. To address this, we combined the use of a molecular docking technique with molecular dynamics (MD) simulations to model a plausible binding mode of compound 1771 to the extracellular catalytic domain of LtaS (eLtaS). The model was validated using alanine mutagenesis studies combined with isothermal titration calorimetry. Additionally, lead optimization driven by our computational model resulted in an improved version of compound 1771, namely, compound 4 which showed greater affinity for binding to eLtaS than compound 1771 in biophysical assays. Compound 4 reduced LTA production in S. aureus dose-dependently, induced aberrant morphology as seen for LTA-deficient bacteria, and significantly reduced bacteria titers in the lung of mice infected with S. aureus. Analysis of our MD simulation trajectories revealed the possible formation of a transient cryptic pocket in eLtaS. Virtual screening (VS) against the cryptic pocket led to the identification of a new class of inhibitors that could potentiate β-lactams against methicillin-resistant S. aureus. Our overall workflow and data should encourage further drug design campaign against LtaS. Finally, our work reinforces the importance of considering protein conformational flexibility to a successful VS endeavor.

Background Pneumococcus kills over one million children annually and over 90 % of these deaths occur in low-income countries especially in Sub-Saharan Africa (SSA) where HIV exacerbates the disease burden. In SSA, serotype 1 pneumococci particularly the endemic ST217 clone, causes majority of the pneumococcal disease burden. To understand the evolution of the virulent ST217 clone, we analysed ST217 whole genomes from isolates sampled from African and Asian countries. Methods We analysed 226 whole genome sequences from the ST217 lineage sampled from 9 African and 4 Asian countries. We constructed a whole genome alignment and used it for phylogenetic and coalescent analyses. We also screened the genomes to determine presence of antibiotic resistance conferring genes. Results Population structure analysis grouped the ST217 isolates into five sequence clusters (SCs), which were highly associated with different geographical regions and showed limited intracontinental and intercontinental spread. The SCs showed lower than expected genomic sequence, which suggested strong purifying selection and small population sizes caused by bottlenecks. Recombination rates varied between the SCs but were lower than in other successful clones such as PMEN1. African isolates showed higher prevalence of antibiotic resistance genes than Asian isolates. Interestingly, certain West African isolates harbored a defective chloramphenicol and tetracycline resistance-conferring element (Tn5253) with a deletion in the loci encoding the chloramphenicol resistance gene (cat pC194), which caused lower chloramphenicol than tetracycline resistance. Furthermore, certain genes that promote colonisation were absent in the isolates, which may contribute to serotype 1’s rarity in carriage and consequently its lower recombination rates. Conclusions The high phylogeographic diversity of the ST217 clone shows that this clone has been in circulation globally for a long time, which allowed its diversification and adaptation in different geographical regions. Such geographic adaptation reflects local variations in selection pressures in different locales. Further studies will be required to fully understand the biological mechanisms which makes the ST217 clone highly invasive but unable to successfully colonise the human nasopharynx for long durations which results in lower recombination rates. Electronic supplementary material The online version of this article (doi:10.1186/s12879-016-1987-z) contains supplementary material, which is available to authorized users.

Streptococcus pneumoniae causes a high burden of invasive pneumococcal disease (IPD) globally, especially in children from resource-poor settings. Like many bacteria, the pneumococcus can import DNA from other strains or even species by transformation and homologous recombination, which has allowed the pneumococcus to evade clinical interventions such as antibiotics and pneumococcal conjugate vaccines (PCVs). Pneumococci are enclosed in a complex polysaccharide capsule that determines the serotype; the capsule varies in size and is associated with properties including carriage prevalence and virulence. We determined and quantified the association between capsule and recombination events using genomic data from a diverse collection of serotypes sampled in Malawi. We determined both the amount of variation introduced by recombination relative to mutation (the relative rate) and how many individual recombination events occur per isolate (the frequency). Using univariate analyses, we found an association between both recombination measures and multiple factors associated with the capsule, including duration and prevalence of carriage. Because many capsular factors are correlated, we used multivariate analysis to correct for collinearity. Capsule size and carriage duration remained positively associated with recombination, although with a reduced P value, and this effect may be mediated through some unassayed additional property associated with larger capsules. This work describes an important impact of serotype on recombination that has been previously overlooked. While the details of how this effect is achieved remain to be determined, it may have important consequences for the serotype-specific response to vaccines and other interventions.IMPORTANCE: The capsule determines >90 different pneumococcal serotypes, which vary in capsule size, virulence, duration, and prevalence of carriage. Current serotype-specific vaccines elicit anticapsule antibodies. Pneumococcus can take up exogenous DNA by transformation and insert it into its chromosome by homologous recombination. This mechanism has disseminated drug resistance and generated vaccine escape variants. It is hence crucial to pneumococcal evolutionary response to interventions, but there has been no systematic study quantifying whether serotypes vary in recombination and whether this is associated with serotype-specific properties such as capsule size or carriage duration. Larger capsules could physically inhibit DNA uptake, or given the longer carriage duration for larger capsules, this may promote recombination. We find that recombination varies among capsules and is associated with capsule size, carriage duration, and carriage prevalence and negatively associated with invasiveness. The consequence of this work is that serotypes with different capsules may respond differently to selective pressures like vaccines.

Summary of the genes found in the genetic recombination regions in clade SC2-WA. Genes present in the regions with recombination events in each clade are summarised. (DOCX 124Â kb)

Distribution of the accessory genes and antibiotic resistance conferring elements in the ST217 isolates. a) Maximum likelihood phylogeny of the isolates rooted using isolates from ST615 as an outgroup (not shown). b) Number of shared accessory open reading frames (ORFs) or genes between pairs of isolates in the phylogeny on the left side and at the top. c) Colour strips showing the continent and country of origin of the isolates and their SCs. d) A heatmap showing the number of shared accessory genes between each pair of isolates in the phylogeny (panel [a] and [e]). (PDF 4076Â kb)

Recombination events identified in the South East African clade (SC3-SEA). (a) Maximum likelihood phylogeny showing genetic relationship of the isolates in the SC. Color strips labeled (b) and (c) shows country and continent of origin of the isolates respectively. (d) Annotations in the reference serotype 1 genome. (e) Horizontal tracks from the phylogenetic tips represent each genome. The coloured blocks shows locations of the recombination events in the chromosome. Red blocks show shared recombination events identified in at least two isolate while the blue blocks show strain specific (unique) recombination events. (PDF 1077Â kb)

Summary of the genes found in the genetic recombination regions in clade SC1-SA. Genes present in the regions with recombination events in each clade are summarised. (DOCX 85Â kb)

Recombination events identified in the West African clade (SC2-WA). (a) Maximum likelihood phylogeny showing genetic relationship of the isolates in the SC. Color strips labeled (b) and (c) shows country and continent of origin of the isolates respectively. (d) Annotations in the reference serotype 1 genome. (e) Horizontal tracks from the phylogenetic tips represent each genome. The coloured blocks shows locations of the recombination events in the chromosome. Red blocks show shared recombination events identified in at least two isolate while the blue blocks show strain specific (unique) recombination events. (PDF 1452Â kb)

Estimated evolutionary parameters for all the clades. Each parameter estimate was calculated using MEGA. (DOC 80Â kb)

Summary of the genes found in the genetic recombination regions in clade SC3-SEA. Genes present in the regions with recombination events in each clade are summarised. (DOCX 133Â kb)

Clade specific phylogenies showing country of origin of the ST217 isolates. Maximum likelihood phylogenetic trees for clade (a) SC1-SA, (b) SC2-WA and (c) SC3-SEA. (PDF 292Â kb)

Recombination events identified in the South African clade (SC1-SA). (a) Maximum likelihood phylogeny showing genetic relationship of the isolates in the SC. Color strips labeled (b) and (c) shows country and continent of origin of the isolates respectively. (d) Annotations in the reference serotype 1 genome. (e) Horizontal tracks from the phylogenetic tips represent each genome. The coloured blocks shows locations of the recombination events in the chromosome. Red blocks show shared recombination events identified in at least two isolate while the blue blocks show strain specific (unique) recombination events. (PDF 641Â kb)

BACKGROUND: The Sahel region of West Africa has the highest bacterial meningitis attack and case fatality rate in the world. The effect of climatic factors on patterns of invasive respiratory bacterial disease is not well documented.OBJECTIVE: We aimed to assess the link between climatic factors and occurrence of invasive respiratory bacterial disease in a Sahel region of Niger.METHODS: We conducted daily disease surveillance and climatic monitoring over an 8-year period between January 1, 2003, and December 31, 2010, in Niamey, Niger, to determine risk factors for bacterial meningitis and invasive bacterial disease. We investigated the mechanistic effects of these factors on Streptococcus pneumoniae infection in mice.RESULTS: High temperatures and low visibility (resulting from high concentrations of airborne dust) were identified as significant risk factors for bacterial meningitis. Dust inhalation or exposure to high temperatures promoted progression of stable asymptomatic pneumococcal nasopharyngeal carriage to pneumonia and invasive disease. Dust exposure significantly reduced phagocyte-mediated bacterial killing, and exposure to high temperatures increased release of the key pneumococcal toxin pneumolysin through increased bacterial autolysis.CONCLUSION: Our findings show that climatic factors can have a substantial influence on infectious disease patterns, altering density of pneumococcal nasopharyngeal carriage, reducing phagocytic killing, and resulting in increased inflammation and tissue damage and consequent invasiveness. Climatic surveillance should be used to forecast invasive bacterial disease epidemics, and simple control measures to reduce particulate inhalation might reduce the incidence of invasive bacterial disease in regions of the world exposed to high temperatures and increased airborne dust.

Characteristics and assembly statistics for the ST217 study isolates. (DOCX 166Â kb)

Characteristics and assembly statistics for the ST217 study isolates. (DOCX 166Â kb)

Distribution of genes associated with virulence and colonisation in the ST217 in serotype 1 isolates. Presence of the genes was screened in all the isolates using BLAST as described in the methods section. (PDF 1702Â kb)

Deletion of the immunoglobulin A (iga) protease gene in the ST217 isolates. Genomic comparison of one of the serotype 1 isolates (ERS194295) that lacked the iga gene against the TIGR4 reference S. pneumoniae genome with an intact iga gene in its chromosome to determine the location and structure of the genomic deletion in the ST217 isolates. Sequence comparison was performed by BLASTN and visualised with Artemis Comparison Tool (ACT). (PDF 528Â kb)

BACKGROUND: Pneumococcus kills over one million children annually and over 90 % of these deaths occur in low-income countries especially in Sub-Saharan Africa (SSA) where HIV exacerbates the disease burden. In SSA, serotype 1 pneumococci particularly the endemic ST217 clone, causes majority of the pneumococcal disease burden. To understand the evolution of the virulent ST217 clone, we analysed ST217 whole genomes from isolates sampled from African and Asian countries. METHODS: We analysed 226 whole genome sequences from the ST217 lineage sampled from 9 African and 4 Asian countries. We constructed a whole genome alignment and used it for phylogenetic and coalescent analyses. We also screened the genomes to determine presence of antibiotic resistance conferring genes. RESULTS: Population structure analysis grouped the ST217 isolates into five sequence clusters (SCs), which were highly associated with different geographical regions and showed limited intracontinental and intercontinental spread. The SCs showed lower than expected genomic sequence, which suggested strong purifying selection and small population sizes caused by bottlenecks. Recombination rates varied between the SCs but were lower than in other successful clones such as PMEN1. African isolates showed higher prevalence of antibiotic resistance genes than Asian isolates. Interestingly, certain West African isolates harbored a defective chloramphenicol and tetracycline resistance-conferring element (Tn5253) with a deletion in the loci encoding the chloramphenicol resistance gene (cat pC194), which caused lower chloramphenicol than tetracycline resistance. Furthermore, certain genes that promote colonisation were absent in the isolates, which may contribute to serotype 1's rarity in carriage and consequently its lower recombination rates. CONCLUSIONS: The high phylogeographic diversity of the ST217 clone shows that this clone has been in circulation globally for a long time, which allowed its diversification and adaptation in different geographical regions. Such geographic adaptation reflects local variations in selection pressures in different locales. Further studies will be required to fully understand the biological mechanisms which makes the ST217 clone highly invasive but unable to successfully colonise the human nasopharynx for long durations which results in lower recombination rates.

Intragenic number of observed and expected numbers of SNPs in the ST217 isolates. All the genes in the ST217 isolates, sizes, observed (OSNP) and expected (ESNP) number of SNPs, ratio OSNP to ESNP and products of each gene are summarised. (DOCX 315Â kb)