IDENTIFICATION OF THE GENES CONTROLLED BY THE FAS AND MGA TRANSCRIPTIONAL REGULATORS OF STREPTOCOCCUS EQUI USING A TRANSCRIPTOMICS APPROACH

Streptococcus equi subspecies equi (S. equi) is the causative agent of strangles, but despite its economic impact the only available vaccine, Equilis StrepE, is rarely used because it causes adverse reactions [1]. Recently, a technique known as transposon directed insertion-site sequencing (TraDIS) [2] was used to identify which genes in S. equi were required for the formation of adverse reactions following vaccination with three transposon mutant pools of a new live attenuated strangles vaccine, Se4592. Interestingly, rather than identifying genes lost from the population that were essential for survival at the injection site, 43% of surviving mutants contained disrupted fas or mga genes. These data suggest that the loss of function of these genes promotes the formation of adverse reactions. The genes fas and mga encode the putative transcriptional regulators: fibronectin/fibrinogen binding/haemolytic activity/streptokinase regulator and multiple virulence gene regulator of Group A Streptococcus (GAS), respectively. The aim of the present study was to use a transcriptomics approach to identify genes controlled by Fas and Mga, which may influence the ability of live S. equi vaccines to cause adverse reactions. Transposon mutants of fas and mga were identified from individual S. equi colonies recovered from an adverse reaction by PCR using a transposon-specific forward primer and a set of fas or mga reverse primers. The PCR products were sequenced to confirm the insertion sites. RNA was isolated from the two mutants and the wild-type bacteria (Se4592) for transcriptomic analysis. The indexed sequencing reads for each isolate were mapped against the reference genome of Se4592 using Bowtie2, and transcriptomes were reconstructed using Cufflinks. The transcriptomes of the mutant strains were compared to the transcriptome of Se4592 using Cuffdiff [3]. Genes controlled by the regulators were identified by virtue of decreased or increased copies of mRNA sequence in the fas and mga mutant strains relative to Se4592. Nine genes were differentially transcribed in the mga mutant and seven genes in the fas mutant (P< 0.001), compared to the vaccine strain. These results are very interesting because show that a mutation of fas and mga alters the transcription of a series of genes that deserve further investigation. Our data shed light on the processes that underlie the formation of adverse vaccine reactions towards improving the safety of live attenuated strangles vaccines. [1] Waller A. New perspectives for the diagnosis, control, treatment, and prevention of strangles in horses, Veterinary Clinics of North America: Equine Practice, 30:591-607, 2014. [2] Langridge GC et al. Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants, Genome Research, 19:2308-16, 2009 [3] Külahoglu C, Bräutigam A. Quantitative transcriptome analysis using RNA-seq, Methods in Molecular Biology 1158:71-91, 2014.