Comparative analysis of two complete Corynebacterium ulcerans genomes and detection of candidate virulence factors

Abstract

Background: Corynebacterium ulcerans has been detected as a commensal in domestic and wild animals that may serve as reservoirs for zoonotic infections. During the last decade, the frequency and severity of human infections associated with C. ulcerans appear to be increasing in various countries. As the knowledge of genes contributing to the virulence of this bacterium was very limited, the complete genome sequences of two C. ulcerans strains detected in the metropolitan area of Rio de Janeiro were determined and characterized by comparative genomics: C. ulcerans 809 was initially isolated from an elderly woman with fatal pulmonary infection and C. ulcerans BR-AD22 was recovered from a nasal sample of an asymptomatic dog.

Results: The circular chromosome of C. ulcerans 809 has a total size of 2,502,095 bp and encodes 2,182 predicted proteins, whereas the genome of C. ulcerans BR-AD22 is 104,279 bp larger and comprises 2,338 protein-coding regions. The minor difference in size of the two genomes is mainly caused by additional prophage-like elements in the C. ulcerans BR-AD22 chromosome. Both genomes show a highly similar order of orthologous coding regions; and both strains share a common set of 2,076 genes, demonstrating their very close relationship. A screening for prominent virulence factors revealed the presence of phospholipase D (Pld), neuraminidase H (NanH), endoglycosidase E (EndoE), and subunits of adhesive pili of the SpaDEF type that are encoded in both C. ulcerans genomes. The rbp gene coding for a putative ribosome-binding protein with striking structural similarity to Shiga-like toxins was additionally detected in the genome of the human isolate C. ulcerans 809.

Conclusions: The molecular data deduced from the complete genome sequences provides considerable knowledge of virulence factors in C. ulcerans that is increasingly recognized as an emerging pathogen. This bacterium is apparently equipped with a broad and varying set of virulence factors, including a novel type of a ribosome-binding protein. Whether the respective protein contributes to the severity of human infections (and a fatal outcome) remains to be elucidated by genetic experiments with defined bacterial mutants and host model systems.

Figure 1

The complete genomes of C. ulcerans 809 and C. ulcerans BR-AD22. (A), Circular representation of the chromosomes from C. ulcerans 809 and C. ulcerans BR-AD22. The circles represent the following features: circle 1, DNA base position; circles 2 and 3, predicted coding sequences transcribed clockwise and anticlockwise, respectively; circle 4, G/C skew [(G-C)/(G+C)] plotted using a 10-kb window; circle 5, G+C content plotted using a 10-kb window. Color code in circles 2 and 3: green, predicted protein-coding regions; red, rRNA or tRNA genes. (B), Distribution of actinobacterial architecture imparting sequences on the leading and lagging strands of the two C. ulcerans chromosomes. The deduced position of the putative dif region is indicated in the linear representation of the chromosomes. The position of the origin of replication (oriC) and the nucleotide sequence of the conserved 28-bp sequence of the dif region are indicated.

Figure 2

Comparative analysis of the gene order in C. ulcerans, C. diphtheriae and C. pseudotuberculosis. (A), Synteny between the sequenced chromosomes of C. ulcerans 809 and C. ulcerans BR-AD22. (B), Synteny between the chromosome of C. ulcerans 809 and those from C. diphtheriae NCTC 13129 (blue) and C. pseudotuberculosis FRC41 (red). The graphs represent X-Y plots of dots forming syntenic regions between the selected chromosomes. Each dot represents a predicted protein having an orthologous counterpart in another corynebacterial genome, with co-ordinates corresponding to the position of the respective coding region in each genome. Orthologous proteins were detected by reciprocal best BLASTP matches. The genomic positions of putative prophages detected in C. ulcerans are marked in the synteny plots. Symbols: β, β-corynephage of C. diphtheriae NCTC 13129; asterisk, nitrate reductase gene region of C. diphtheriae NCTC 13129.

Figure 3

Genetic maps of putative prophages detected in the C. ulcerans genomes. The functional annotation of the prophage-like region ΦCULC809I from C. ulcerans 809 and of the prophage-like regions ΦCULC22I, ΦCULC22II, ΦCULC22III, and ΦCULC22IV from C. ulcerans BR-AD22 is shown. The predicted protein functions are indicated by color codes. ΦCULC809I and ΦCULC22I are closely related genetic elements according to the very high overall similarity of their genes and gene products. The nucleotide sequences of putative integration sites of ΦCULC22II, ΦCULC22III, and ΦCULC22IV in the chromosome of C. ulcerans BR-AD22 are shown. tRNA genes flanking the putative prophages are indicated as blue triangles.

Figure 4

Intra- and inter-species comparison of the predicted gene content of the C. ulcerans genomes. (A), Selected examples of genomic regions comprising strain-specific genes in C. ulcerans. Orthologous gene regions are shaded gray. (B), Venn diagram comparing the gene content of C. ulcerans 809, C. diphtheriae NCTC 13129 and C. pseudotuberculosis FRC41. The Venn diagram shows the number of shared and species-specific genes among the three corynebacterial genomes.

Figure 5

Sequence analyses of prominent virulence factors detected in the genome of C. ulcerans 809. (A), Analysis of the corynebacterial protease CP40 (Cpp). An amino acid sequence alignment of the corynebacterial proteases CP40 from C. pseudotuberculosis FRC41 and C. ulcerans 809 with the α domain of endoglycosidase EndoE from E. faecalis is shown. Predicted signal peptides are colored in yellow; predicted protein segments belonging to the α domain of EndoE are shaded gray. The catalytic FGH18 motif of EndoE is indicated by bold letters. The domain organization of EndoE is shown schematically below the sequence alignment. (B), Analysis of the corynebacterial ribosome-binding protein (Rbp). An amino acid sequence alignment of Rbp from C. ulcerans 809 with A chains of the Shiga-like toxins SLT-1 and SLT-2 from E. coli is shown. Conserved amino acids are highlighted in orange, while the conserved catalytic residues are highlighted in blue. The predicted signal peptide of Rbp is labeled yellow; the retranslocation domain of SLT-1 is marked as a green box. The similarity between Rbp and the A chain of SLT-1 is also shown as a 3-D model presented below the sequence alignment. Structural similarities between both proteins are indicated in red.