Computational protocol: Insight into cross-talk between intra-amoebal pathogens

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Protocol publication

[…] To identify genes that were present in both L. drancourtii and P. acanthamoebae, we performed BLASTP comparisons between both genomes and the best reciprocal hits were retrieved if they exhibited a similarity > 30% and a query and hit length coverage > 60%. These conserved genes were searched for in one amoebal genome (Dictyostelium discoideum AX4 [NC_007087-NC_007092], the only fully sequenced amoebal genome available to date) and six bacterial genomes with a decreasing degree of phylogenetic relatedness respectively to L. drancourtii and P. acanthamoebae. Model organisms or medically important bacteria were selected within the same family (Legionella pneumophila Paris [NC_006368], respectively Pr. amoebophila UWE25 [NC_005861]), within the same order (Coxiella burnetii RSA 493 [NC_002971], respectively Chlamydia trachomatis D/UW-3/CX [NC_000117]) and within the same phylum (Escherichia coli K12 substr. MG1655 [NC_000913.2], respectively Rhodopirellula baltica SH1 [NC_005027]). Each gene was considered present if it exhibited > 30% identity and had a length coverage > 60% on both the query and hit. The genes were classified according to the number of hits obtained using P. acanthamoebae or L. drancourtii as the query against each of the six bacterial genomes based on a score of 0 (no hits in any f the screened genomes) to 6 (hits in all of the screened bacterial genomes). [...] A BLASTP search against the nr database was performed for genes with scores between 0 and 3. For each gene, a phylogenetic analysis was conducted with the 20 best BLASTP hits of P. acanthamoebae and L. drancourtii that was restricted to one representative species per genus. Sequence alignment was performed using Muscle []. Phylogenetic relationships among species were inferred with PhyML (PHYlogenetic inferences using Maximum Likelihood) [] using the WAG model. For particularly interesting cases, a phylogeny based on a Bayesian inference was performed using MrBayes [] on alignments curated by Gblocks []. [...] Genes exhibiting a topology different from that expected for core genes were classified according to one of the five following topologies: i) P. acanthamoebae genes clustering with L. drancourtii; ii) P. acanthamoebae genes clustering with Legionellales; iii) Legionellales genes clustering with Chlamydiales; iv) P. acanthamoebae and/or L. drancourtii genes clustering with Rickettsia, Ehrlichia and Wolbachia and (v) genes matching eukaryotic proteins by BLAST homology searches.To characterize further the genes exhibiting an unexpected phylogenetic topology, their GC content was examined relative to the genomic mean with R [] using the seqinR package []. A BLASTN comparison was achieved versus ISfinder [] with an e-value of 1e-5 to search for insertion sequences in the genomes of L. drancourtii and P. acanthamoebae. […]

Pipeline specifications

Software tools BLASTP, MUSCLE, PhyML, MrBayes, Gblocks, seqinr, BLASTN
Databases ISfinder
Applications Phylogenetics, Nucleotide sequence alignment, Genome data visualization
Organisms Homo sapiens, Legionella pneumophila, Parachlamydia acanthamoebae
Diseases Chlamydia Infections