Identification of genes required for the fitness of Streptococcus equi subsp. equi in whole equine blood and hydrogen peroxide

Abstract

The availability of next-generation sequencing techniques provides an unprecedented opportunity for the assignment of gene function. Streptococcus equi subspecies equi is the causative agent of strangles in horses, one of the most prevalent and important diseases of equids worldwide. However, the live attenuated vaccines that are utilized to control this disease cause adverse reactions in some animals. Here, we employ transposon-directed insertion-site sequencing (TraDIS) to identify genes that are required for the fitness of S. equi in whole equine blood or in the presence of H₂O₂ to model selective pressures exerted by the equine immune response during infection. We report the fitness values of 1503 and 1471 genes, representing 94.5 and 92.5 % of non-essential genes in S. equi, following incubation in whole blood and in the presence of H₂O₂, respectively. Of these genes, 36 and 15 were identified as being important to the fitness of S. equi in whole blood or H₂O₂, respectively, with 14 genes being important in both conditions. Allelic replacement mutants were generated to validate the fitness results. Our data identify genes that are important for S. equi to resist aspects of the immune response in vitro, which can be exploited for the development of safer live attenuated vaccines to prevent strangles.

Keywords: Streptococcus equi; hydrogen peroxide; transposon-directed insertion-site sequencing; whole blood.

Fig. 1.

Fitness scores and COG categories of S. equi subsp. equi genes required for survival in whole equine blood. (a) Fitness scores (log₂FC) per gene of S. equi subsp. equi ISS1 mutants post-incubation in whole equine blood, as determined by TraDIS. Blue dots, genes with significantly reduced fitness (log₂FC < −2 and q<0.05); red dots, genes significantly reduced in fitness of which deletion mutants were made and retested to confirm TraDIS results; green dot, eqbE exhibiting no fitness effect that was used as a control for validation; grey dots, genes exhibiting no significant fitness effect. (b) Functional COG categories of the 36 fitness genes identified in whole equine blood. L, Replication, recombination and repair; K, transcription; C, energy production and conversion; T, signal transduction mechanisms; R, general function prediction only; F, nucleotide transport and metabolism; V, defence mechanisms; N, cell motility; M, cell wall/membrane/envelope biogenesis; G, carbohydrate transport and metabolism; D, cell cycle control, cell division, chromosome partitioning; U, intracellular trafficking, secretion and vesicular transport; O, posttranslational modification, protein turnover, chaperones; J, translation, ribosomal structure and biogenesis; E, amino acid transport and metabolism.

Fig. 2.

Fitness scores and COG categories of S. equi subsp. equi genes required for survival in hydrogen peroxide (H₂O₂). (a) Fitness scores (log₂FC) per gene of S. equi subsp. equi ISS1 mutants post-incubation in H₂O₂, as determined by TraDIS. Blue dots, genes with significantly reduced fitness (log₂FC < −2 and q<0.05); red dots, genes significantly reduced in fitness of which deletion mutants were made and retested to confirm TraDIS results; green dots, genes exhibiting no fitness effect that acted as negative controls for validation; grey dots, genes exhibiting no significant fitness effect. (b) Functional COG categories of the fitness genes identified in H₂O₂. C, Energy production and conversion; L, replication, recombination and repair; R, general function prediction only; E, amino acid transport and metabolism; D, cell cycle control, cell division, chromosome partitioning; O, posttranslational modification, protein turnover, chaperones; K, transcription; J, translation, ribosomal structure and biogenesis.

Fig. 3.

Venn diagram of the 36 genes required for the survival of S. equi subsp. equi in whole equine blood compared to the 15 genes required for survival in hydrogen peroxide. The genes that were deleted by allelic replacement mutagenesis to validate the results are indicated.

Fig. 4.

Growth curves of the parental S. equi subsp. equi strain Se4047 and ΔpyrP, ΔhasA, ΔeqbE, ΔaddA, ΔrecG and ΔmnmE deletion mutation strains grown in THB at 37 °C in a humidified atmosphere in the presence of 5 % CO₂. Error bars indicate the se. The significance of changes in doubling time using a two-sided Student’s t-test are indicated.

Fig. 5.

Validation of the S. equi subsp. equi TraDIS screen in whole equine blood. Strains with deletion mutations of whole equine blood fitness genes, as identified by TraDIS, were incubated in blood for 3 h and their survival measured each hour. (a) ΔhasA, (b) ΔaddA, (c) ΔrecG, (d) ΔpyrP, (e) ΔmnmE and (f) ΔeqbE deletion mutation strains compared to the wild-type parental strain, Se4047. Error bars indicate the se. The significance of changes in doubling time using a two-sided Student’s t-test are indicated.

Fig. 6.

Validation of the S. equi subsp. equi TraDIS screen in THB containing hydrogen peroxide. Strains with deletion mutations in H₂O₂ fitness genes, as identified by TraDIS, were incubated in THB containing H₂O₂ for 3 h and their survival measured each hour. (a) ΔhasA, (b) ΔaddA, (c) ΔrecG, (d) ΔpyrP, (e) ΔmnmE and (f) ΔeqbE deletion mutation strains compared to the wild-type parental strain, Se4047. Error bars indicate the se. The significance of changes in doubling time using a two-sided Student’s t-test are indicated.