Sandrine Petry

Proteomic analysis and immunogenicity of secreted proteins from Rhodococcus equi ATCC 33701

Rhodococcus equi is one of the most important causes of mortality in foals between 1 and 6 months of age. Although rare, infection also occurs in a variety of other mammals including humans, often following immunosuppression of various causes. Secreted proteins are known to mediate important pathogen-host interactions and consequently are favored candidates for vaccine development as they are the most easily accessible microbial antigens to the immune system. Here, we describe the results of a proteomic analysis based on SDS-PAGE, immunoblot and mass spectrometry, which was carried out aiming the identification of secreted proteins that are differently expressed at 30 degrees C versus 37 degrees C and at mid-exponential versus early-stationary growth phase and antigenic proteins from R. equi ATCC 33701. A total of 48 proteins was identified regardless of growth conditions. The cholesterol oxidase ChoE appears to be the major secretory protein. Moreover, four proteins revealed high homologies with the mycolyl transferases of the Ag85 complex from Mycobacterium tuberculosis. The sequence analysis predicted that 24 proteins are transported by a signal peptide-dependent pathway. Moreover, five antigenic proteins of R. equi were identified by immunoblot, including a novel strongly immunoreactive protein of unknown function. In conclusion, the elucidation of the secretome of R. equi identified several proteins with different biological functions and a new candidate for developing vaccines against R. equi infection in horse.

Genome Sequence of Taylorella equigenitalis MCE9, the Causative Agent of Contagious Equine Metritis

Taylorella equigenitalis is the causative agent of contagious equine metritis (CEM), a sexually transmitted infection of horses. We herein report the genome sequence of T. equigenitalis strain MCE9, isolated in 2005 from the urethral fossa of a 4-year-old stallion in France.

Genomic Characterization of the Taylorella Genus

The Taylorella genus comprises two species: Taylorella equigenitalis, which causes contagious equine metritis, and Taylorella asinigenitalis, a closely-related species mainly found in donkeys. We herein report on the first genome sequence of T. asinigenitalis, analyzing and comparing it with the recently-sequenced T. equigenitalis genome. The T. asinigenitalis genome contains a single circular chromosome of 1,638,559 bp with a 38.3% GC content and 1,534 coding sequences (CDS). While 212 CDSs were T. asinigenitalis-specific, 1,322 had orthologs in T. equigenitalis. Two hundred and thirty-four T. equigenitalis CDSs had no orthologs in T. asinigenitalis. Analysis of the basic nutrition metabolism of both Taylorella species showed that malate, glutamate and alpha-ketoglutarate may be their main carbon and energy sources. For both species, we identified four different secretion systems and several proteins potentially involved in binding and colonization of host cells, suggesting a strong potential for interaction with their host. T. equigenitalis seems better-equipped than T. asinigenitalis in terms of virulence since we identified numerous proteins potentially involved in pathogenicity, including hemagluttinin-related proteins, a type IV secretion system, TonB-dependent lactoferrin and transferrin receptors, and YadA and Hep_Hag domains containing proteins. This is the first molecular characterization of Taylorella genus members, and the first molecular identification of factors potentially involved in T. asinigenitalis and T. equigenitalis pathogenicity and host colonization. This study facilitates a genetic understanding of growth phenotypes, animal host preference and pathogenic capacity, paving the way for future functional investigations into this largely unknown genus.

Phenotypic and 16S ribosomal RNA gene diversity of Taylorella asinigenitalis strains isolated between 1995 and 2008.

The objective of this study was to examine the degree of phenotypic and genotypic diversity between 43 French Taylorella asinigenitalis strains isolated from 22 jacks, two stallions and one mare between 1995 and 2008 by culturing genital swabs obtained during routine diagnosis for contagious equine metritis. This retrospective analysis revealed the existence of T. asinigenitalis species since 1995 and the natural colonization of a mares genital tract in 2001. Despite the presence of 27 different patterns revealed by the combination of API ZYM, antibiogram and 16S rDNA profiles, we show that T. asinigenitalis is a highly homogeneous species. API ZYM diversity only concerns acid phosphatase and naphthol-AS-BI-phosphohydrolase activity. The majority of strains are susceptible to a wide range of antimicrobial agents but most are streptomycin-resistant (95.5%), ampicillin-resistant (88.4%), and four strains are atypical due to a high degree of resistance to at least eight antimicrobial agents. 16S rDNA sequence analysis showed only two clusters and revealed similarity of 99.3-100% between T. asinigenitalis strains. The geographic origin of the 43 isolates correlates to the two 16S rDNA clusters.

Development of a single multi-locus sequence typing scheme for Taylorella equigenitalis and Taylorella asinigenitalis.

We describe here the development of a multilocus sequence typing (MLST) scheme for Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), and Taylorella asinigenitalis, a nonpathogenic bacterium. MLST was performed on a set of 163 strains collected in several countries over 35 years (1977-2012). The MLST data were analyzed using START2, MEGA 5.05 and eBURST, and can be accessed at http://pubmlst.org/taylorella/. Our results revealed a clonal population with 39 sequence types (ST) and no common ST between the two Taylorella species. The eBURST analysis grouped the 27 T. equigenitalis STs into four clonal complexes (CC1-4) and five unlinked STs. The 12 T. asinigenitalis STs were grouped into three clonal complexes (CC5-7) and five unlinked STs, among which CC1 (68.1% of the 113 T. equigenitalis) and CC5 (58.0% of the 50 T. asinigenitalis) were dominants. The CC1, still in circulation in France, contains isolates from the first CEM outbreaks that simultaneously emerged in several countries in the late 1970s. The emergence in different countries (e.g. France, Japan, and United Arab Emirates) of STs without any genetic relationship to CC1 suggests the existence of a natural worldwide reservoir that remains to be identified. T. asinigenitalis appears to behave same way since the American, Swedish and French isolates have unrelated STs. This first Taylorella sp. MLST is a powerful tool for further epidemiological investigations and population biology studies of the Taylorella genus.

Rhodococcus equi's extreme resistance to hydrogen peroxide is mainly conferred by one of its four catalase genes.

Rhodococcus equi is one of the most widespread causes of disease in foals aged from 1 to 6 months. R. equi possesses antioxidant defense mechanisms to protect it from reactive oxygen metabolites such as hydrogen peroxide (H2O2) generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxify hydrogen peroxide. Recently, an analysis of the R. equi 103 genome sequence revealed the presence of four potential catalase genes. We first constructed ΔkatA-, ΔkatB-, ΔkatC-and ΔkatD -deficient mutants to study the ability of R. equi to survive exposure to H2O2 in vitro and within mouse peritoneal macrophages. Results showed that ΔkatA and, to a lesser extent ΔkatC, were affected by 80 mM H2O2. Moreover, katA deletion seems to significantly affect the ability of R. equi to survive within murine macrophages. We finally investigated the expression of the four catalases in response to H2O2 assays with a real time PCR technique. Results showed that katA is overexpressed 367.9 times (±122.6) in response to exposure to 50 mM of H2O2 added in the stationary phase, and 3.11 times (±0.59) when treatment was administered in the exponential phase. In untreated bacteria, katB, katC and katD were overexpressed from 4.3 to 17.5 times in the stationary compared to the exponential phase. Taken together, our results show that KatA is the major catalase involved in the extreme H2O2 resistance capability of R. equi.

Analysis of plasmid diversity in 96 Rhodococcus equi strains isolated in Normandy (France) and sequencing of the 87-kb type I virulence plasmid.

To characterize the potential epidemiological relationship between the origin of Rhodococcus equi strains and the type of their virulence plasmids, we performed a comparative analysis of virulence plasmid types encountered in 96 R. equi strains isolated from (1) autopsied horses, (2) organic samples (horse faeces, manure and straw) and (3) environmental samples. Our results revealed no clear epidemiological link between virulence plasmid type and the origin of R. equi strains isolated from horse-related environments. To understand this result, we determined the nucleotide sequence of the second most frequently isolated virulence plasmid type: a 87-kb type I (pVAPA116) plasmid and compared it with the previously sequenced (and most commonly encountered) 85-kb type I (pVAPA1037) plasmid. Our results show that the divergence between these two plasmids is mainly due to the presence of three allelic exchange loci, resulting in the deletion of two genes and the insertion of three genes in pVAPA116 compared with pVAPA1037. In conclusion, it appears that the divergence between the two sequenced rhodococcal virulence plasmids is not associated with the vap pathogenicity island and may result from an evolutionary process driven by a mobility-related invertase/resolvase invA-like gene.

First Draft Genome Sequence of the Dourine Causative Agent: Trypanosoma Equiperdum Strain OVI

Trypanosoma equiperdum is the causative agent of dourine, a sexually-transmitted infection of horses. This parasite belongs to the subgenus Trypanozoon that also includes the agent of sleeping sickness (Trypanosoma brucei) and surra (Trypanosoma evansi). We herein report the genome sequence of a T. equiperdum strain OVI, isolated from a horse in South-Africa in 1976. This is the first genome sequence of the T. equiperdum species, and its availability will provide important insights for future studies on genetic classification of the subgenus Trypanozoon.

Development of a multilocus sequence typing scheme for Rhodococcus equi

Rhodococcus equi causes pulmonary and extrapulmonary infections in animals and humans, with endemic situations and significant young foal mortality in stud farms worldwide. Despite its economic impact in the horse-breeding industry, the broad geographic and host distribution, global diversity and population structure of R. equi remain poorly characterised. In this context, we developed a multilocus sequence typing (MLST) scheme using 89 clinical and environmental R. equi of various origins and eight Rhodococcus sp. Data can be accessed at http://pubmlst.org/rhodococcus/. A clonal R. equi population was observed with 16 out of 37 sequence types (STs) grouped into six clonal complexes (CC) based on single-locus variants. One of the six CCs (CC3) is not host-specific, suggesting potential exchanges between different R. equi reservoirs. Most of the virulent equine R. equi CCs/unlinked STs were plasmid-type-specific. Despite this, marked genetic variability with the circulation of multiple R. equi genotypes was generally observed even within the same animal. Focusing on outbreaks, data indicated (i) the potential contagious transmission of R. equi during the 2012-Mayotte equine outbreak because of the poor genotype diversity of clinical strains; (ii) a potential porcine outbreak among the 30 Belgian farms investigated in 2013. This first Rhodococcus equi MLST is a powerful tool for further epidemiological investigations and population biology studies of R. equi isolates.

Immune response to Rhodococcus equi ATCC 33701-secreted proteins in mice and identification of immunogenic recombinant proteins by dot-blotting.

Rhodococcus equi remains a significant pathogen, causing severe pneumonia in foals. The development of vaccines and serologic diagnosis could be greatly facilitated by studying the humoral immune response to this equine pathogen. In this study, a crude extract of R. equi ATCC 33701-secreted proteins combined with the Montanide® ISA70 adjuvant was found to be highly immunogenic in mice with the highest titer of 99,000 on day 42 after the first subcutaneous immunization. This immune response was dependent on the quantity of proteins injected and the presence of adjuvant. By dot-blotting, eight recombinant secreted proteins were identified to react strongly with sera from immunized mice. Of these eight proteins, four were detected as immunogenic only when administered in conjunction with adjuvant. This screening strategy led to the identification of promising new candidates for vaccine development.