Genomic analysis of Campylobacter fetus subspecies: identification of candidate virulence determinants and diagnostic assay targets

Background: Campylobacter fetus subspecies venerealis is the causative agent of bovine genital campylobacteriosis, asymptomatic in bulls the disease is spread to female cattle causing extensive reproductive loss. The microbiological and molecular differentiation of C. fetus subsp. venerealis from C. fetus subsp. fetus is extremely difficult. This study describes the analysis of the available C. fetus subsp. venerealis AZUL-94 strain genome (~75–80%) to identify elements exclusively found in C. fetus subsp. venerealis strains as potential diagnostic targets and the characterisation of subspecies virulence genes. Results: Eighty Kb of genomic sequence (22 contigs) was identified as unique to C. fetus subsp. venerealis AZUL-94 and consisted of type IV secretory pathway components, putative plasmid genes and hypothetical proteins. Of the 9 PCR assays developed to target C. fetus subsp. venerealis type IV secretion system genes, 4 of these were specific for C. fetus subsp. venerealis biovar venerealis and did not detect C. fetus subsp. venerealis biovar intermedius. Two assays were specific for C. fetus subsp. venerealis AZUL-94 strain, with a further single assay specific for the AZUL-94 strain and C. fetus subsp. venerealis biovar intermedius (and not the remaining C. fetus subsp. venerealis biovar venerealis strains tested). C. fetus subsp. fetus and C. fetus subsp. venerealis were found to share most common Campylobacter virulence factors such as SAP, chemotaxis, flagellar biosynthesis, 2-component systems and cytolethal distending toxin subunits (A, B, C). We did not however, identify in C. fetus the full complement of bacterial adherence candidates commonly found in other Campylobacter spp. Conclusion: The comparison of the available C. fetus subsp. venerealis genome sequence with the C. fetus subsp. fetus genome identified 80 kb of unique C. fetus subsp. venerealis AZUL94 sequence, with subsequent PCR confirmation demonstrating inconsistent amplification of these targets in all other C. fetus subsp. venerealis strains and biovars tested. The assays developed here highlight the complexity of targeting strain specific virulence genes for field studies for the molecular identification and epidemiology of C. fetus

Gene discovery through genomic sequencing of Brucella abortus.

Brucella abortus is the etiological agent of brucellosis, a disease that affects bovines and human. We generated DNA random sequences from the genome of B. abortus strain 2308 in order to characterize molecular targets that might be useful for developing immunological or chemotherapeutic strategies against this pathogen. The partial sequencing of 1,899 clones allowed the identification of 1,199 genomic sequence surveys (GSSs) with high homology (BLAST expect value < 10(-5)) to sequences deposited in the GenBank databases. Among them, 925 represent putative novel genes for the Brucella genus. Out of 925 nonredundant GSSs, 470 were classified in 15 categories based on cellular function. Seven hundred GSSs showed no significant database matches and remain available for further studies in order to identify their function. A high number of GSSs with homology to Agrobacterium tumefaciens and Rhizobium meliloti proteins were observed, thus confirming their close phylogenetic relationship. Among them, several GSSs showed high similarity with genes related to nodule nitrogen fixation, synthesis of nod factors, nodulation protein symbiotic plasmid, and nodule bacteroid differentiation. We have also identified several B. abortus homologs of virulence and pathogenesis genes from other pathogens, including a homolog to both the Shda gene from Salmonella enterica serovar Typhimurium and the AidA-1 gene from Escherichia coli. Other GSSs displayed significant homologies to genes encoding components of the type III and type IV secretion machineries, suggesting that Brucella might also have an active type III secretion machinery.Instituto de Biotecnologia y Biologia Molecula

Identification of chromosomal regions of common evolutionary ancestry between <i>T. cruzi</i> genomes.

<p>Colinear coding sequences in the genomes analyzed were used to define the boundaries of the corresponding IGRs. Given a pair of colinear coding sequences A, B from one genome, if an ortholog of A in the second genome (A′) is also colinear with the ortholog of B, then the boundaries of the IGR are defined from the location of the corresponding translational START and STOP codons.</p

Contribution of repetitive elements to the observed length differences between IGRs.

<p>Repetitive elements detected by RepeatMasker (see Methods) were grouped into two broad categories: Transposon-like repetitive elements, and microsatellite-like repeats. The figure shows the contribution of these two classes of repetitive elements to the overall size of IGRs. The plot shows data for IGRs where the size difference between alleles is 50 bp.</p

Distribution of indels, sequence composition and nucleotide diversity in a long genomic region.

<p>The plot shows values of thymidine composition, sequence divergence (SNPs) and indels, in a sliding window of 500 bp that was moved in 10 bp intervals. The region corresponds to TcChr2-S (43230–54007), TcChr2-P (46794–57559), and sylviocontig_9 (11357–22135). Annotated coding sequences in these region are TcCLB.507601.40–90 (TcChr2-P), TcCLB.510351.70–120 (TcChr2-S), TCSYLVIO_009877–9880 (GenBank accession ADWP02023504), and TCSYLVIO_009884 (GenBank accession ADWP02023505).</p

Analysis of sequence composition of core CDS and IGR regions.

<p>A) Comparison of base composition of 1,719 selected IGR regions and their associated flanking coding sequences. B) Comparison of ocurrence of homopolymer tracts in CDS vs IGR regions.</p

PCR amplification of selected IGR regions in different strains of <i>T. cruzi</i>.

<p>Selected genomic regions were amplified to validate length and sequence polymorphisms, and resolved in a 2% TBE-agarose gel. Lanes in the gels correspond to: molecular size markers (lanes 1, 23), DNA from <i>T. cruzi</i> strains (lanes 2–21), and negative control (lane 22). Strains used (and the corresponding lanes) are: 92122102R (2); Dog Theis (3); CanIII (4); TU18 (5); Mas1 cl1 (6); IVV cl4 (7); Y9 IIB (8); X109-02 (9); M5631 cl5 (10); M6241 cl6 (11); LL051 (12); Mn cl2 (13); Sc43 cl9 (14); TEH53 (15); Tula cl2 (16); CL Brener (17); P63 cl1 (18); Sylvio X10/1 (19); Palv2 (20); Dm28c (21). Numbers in the leftmost column (1–6) correspond to the six selected cases mentioned in the text. All samples were analyzed in the same electrophoresis run, however for clarity purposes, groups of lanes were digitally re-ordered.</p

Size correlation of orthologous IGR regions.

<p>The plots show the pairwise comparisons of IGR regions between the three analyzed haplotypes/genomes: A) TcI vs TcII-like, B) TcII-like vs TcIII-like, C) TcI vs TcIII-like. The colored dotted line in each plot marks the mean value of each distribution, while the black dotted lines mark the 5th and 95th percentiles, respectively. Plot axes correspond to length (size) of the IGR region, in base pairs, for each haplotype/genome.</p

Plot of Indels vs SNPs in paired orthologous IGRs.

<p>The plot shows how each IGR region maps in space according to its nucleotide diversity and indel ratios. Each dot represents a pair of IGR regions. Colors indicate the different comparisons across haplotypes/genomes. Dotted lines represent the mean and indels/site values. In black dotted lines we also show the 5th and 95th percentile for each distribution.</p

A genomic scale map of genetic diversity in <it>Trypanosoma cruzi</it>

<p>Abstract</p> <p>Background</p> <p><it>Trypanosoma cruzi</it>, the causal agent of Chagas Disease, affects more than 16 million people in Latin America. The clinical outcome of the disease results from a complex interplay between environmental factors and the genetic background of both the human host and the parasite. However, knowledge of the genetic diversity of the parasite, is currently limited to a number of highly studied <it>loci</it>. The availability of a number of genomes from different evolutionary lineages of <it>T. cruzi</it> provides an unprecedented opportunity to look at the genetic diversity of the parasite at a genomic scale.</p> <p>Results</p> <p>Using a bioinformatic strategy, we have clustered <it>T. cruzi</it> sequence data available in the public domain and obtained multiple sequence alignments in which one or two alleles from the reference CL-Brener were included. These data covers 4 major evolutionary lineages (DTUs): TcI, TcII, TcIII, and the hybrid TcVI. Using these set of alignments we have identified 288,957 high quality single nucleotide polymorphisms and 1,480 indels. In a reduced re-sequencing study we were able to validate ~ 97% of high-quality SNPs identified in 47 loci. Analysis of how these changes affect encoded protein products showed a 0.77 ratio of synonymous to non-synonymous changes in the <it>T. cruzi</it> genome. We observed 113 changes that introduce or remove a stop codon, some causing significant functional changes, and a number of tri-allelic and tetra-allelic SNPs that could be exploited in strain typing assays. Based on an analysis of the observed nucleotide diversity we show that the <it>T. cruzi</it> genome contains a core set of genes that are under apparent purifying selection. Interestingly, orthologs of known druggable targets show statistically significant lower nucleotide diversity values.</p> <p>Conclusions</p> <p>This study provides the first look at the genetic diversity of <it>T. cruzi</it> at a genomic scale. The analysis covers an estimated ~ 60% of the genetic diversity present in the population, providing an essential resource for future studies on the development of new drugs and diagnostics, for Chagas Disease. These data is available through the TcSNP database (<url>http://snps.tcruzi.org</url>).</p