Streptococcus equi subsp. zooepidemicus (SEZ) is considered a commensal bacterium able to cause a wide range of diseases in several animal species, especially horses. Recently, virulent strains of SEZ caused high mortality in pigs first in Indonesia, China, and in other Asian regions, and then in the USA. SEZ infection in humans may occur, although severe diseases are rare. Genetic differences and potential virulence genes are suspected to be responsible for the differences in virulence found in SEZ strains isolated from different hosts. Streptokinase is a potent plasminogen activating protein which is produced, among others, by several streptococci belonging to different Lancefield groups. Phylogenetic studies on the streptokinase gene of Streptococcus pyogenes, a group A Streptococcus (GAS) responsible for a wide range of diseases in humans, have revealed two main sequence clusters. Streptokinase variants produced by GAS isolates displayed different mechanisms of plasminogen activation and this process directly affected GAS pathogenesis. Very limited studies on streptokinase produced by SEZ have been carried out so far. The aim of this work was to investigate the variability of the gene coding for the streptokinase in SEZ of equids by in silico and in vitro studies. All sequences of the gene coding for the streptokinase (skc) of SEZ were downloaded, aligned, and used for phylogenetic studies. Skc sequences from other streptococci were used in comparative studies. Primers to amplify the skc gene were designed and tested on different Streptococcus spp. strains. SEZ collected from clinical specimens from horses (n=22) and donkey (n=2) were selected from archival DNA samples for a preliminary study. To investigate the most variable strains as possible, the strains included in the study were epidemiologically unrelated and were selected based on the origin of the specimens and of the results obtained by sequencing of the 16S rRNA and by PCR analysis of the 16S-23S rRNA intergenic spacer region. Strains isolated from the respiratory tract of horses with respiratory diseases (group 1), from the reproductive tract of horses with a history of reproductive disorders (group 2) and from non-respiratory and non-reproductive samples of horses showing various diseases (group 3) were selected to investigate the variability of skc gene among strains isolated from different organs. In addition, archival pool samples from lung, spleen and liver of aborted horse fetuses were tested by PCR to detect SEZ. First, a PCR to amplify a 1112 bp sequence of the 16S rRNA gene variable regions V1-V6 of Streptococcus species was carried out. Positive samples were tested by multiplex PCR for detecting seeH and seeI sequences. SEZ-positive samples were used for amplifying and sequencing the skc gene. All sequences obtained were aligned and used for phylogenetic analysis. Fisher's exact test was used to evaluate the statistical significance of the results. A high genetic variability was observed in the skc protein sequences of SEZ downloaded from GenBank, while skc sequences from Streptococcus equi subsp. equi were more conserved. The skc gene sequences obtained from the samples showed high nucleotide variability. Most sequences were similar to sequences already deposited in GenBank, while other sequences were different from already known sequences. No statistical differences were found among sequences obtained from group 1, group 2 or group 3, suggesting that genetic variability of skc does not affect the SEZ tropism for the respiratory and the reproductive organs. Preliminary sequences obtained from samples of aborted fetuses seem more similar each other’s than samples from other source and clustered in a separated branch, but further analysis and a wider set of samples are required to confirm these findings. The results of this study show that the skc gene of SEZ is highly variable. The study of additional sequences is required for better understanding the impact of this variability on SEZ tropism or virulence. Further studies should investigate the correlation between genetic variability of the skc gene and SEZ activity on plasminogen and virulence in horses.
GENETIC DIVERSITY OF THE GENE ENCODING STREPTOKINASE FROM STREPTOCOCCUS EQUI SUBSP. ZOOEPIDEMICUS INFECTING EQUIDS.
Preziuso
Primo
2023-01-01
Abstract
Streptococcus equi subsp. zooepidemicus (SEZ) is considered a commensal bacterium able to cause a wide range of diseases in several animal species, especially horses. Recently, virulent strains of SEZ caused high mortality in pigs first in Indonesia, China, and in other Asian regions, and then in the USA. SEZ infection in humans may occur, although severe diseases are rare. Genetic differences and potential virulence genes are suspected to be responsible for the differences in virulence found in SEZ strains isolated from different hosts. Streptokinase is a potent plasminogen activating protein which is produced, among others, by several streptococci belonging to different Lancefield groups. Phylogenetic studies on the streptokinase gene of Streptococcus pyogenes, a group A Streptococcus (GAS) responsible for a wide range of diseases in humans, have revealed two main sequence clusters. Streptokinase variants produced by GAS isolates displayed different mechanisms of plasminogen activation and this process directly affected GAS pathogenesis. Very limited studies on streptokinase produced by SEZ have been carried out so far. The aim of this work was to investigate the variability of the gene coding for the streptokinase in SEZ of equids by in silico and in vitro studies. All sequences of the gene coding for the streptokinase (skc) of SEZ were downloaded, aligned, and used for phylogenetic studies. Skc sequences from other streptococci were used in comparative studies. Primers to amplify the skc gene were designed and tested on different Streptococcus spp. strains. SEZ collected from clinical specimens from horses (n=22) and donkey (n=2) were selected from archival DNA samples for a preliminary study. To investigate the most variable strains as possible, the strains included in the study were epidemiologically unrelated and were selected based on the origin of the specimens and of the results obtained by sequencing of the 16S rRNA and by PCR analysis of the 16S-23S rRNA intergenic spacer region. Strains isolated from the respiratory tract of horses with respiratory diseases (group 1), from the reproductive tract of horses with a history of reproductive disorders (group 2) and from non-respiratory and non-reproductive samples of horses showing various diseases (group 3) were selected to investigate the variability of skc gene among strains isolated from different organs. In addition, archival pool samples from lung, spleen and liver of aborted horse fetuses were tested by PCR to detect SEZ. First, a PCR to amplify a 1112 bp sequence of the 16S rRNA gene variable regions V1-V6 of Streptococcus species was carried out. Positive samples were tested by multiplex PCR for detecting seeH and seeI sequences. SEZ-positive samples were used for amplifying and sequencing the skc gene. All sequences obtained were aligned and used for phylogenetic analysis. Fisher's exact test was used to evaluate the statistical significance of the results. A high genetic variability was observed in the skc protein sequences of SEZ downloaded from GenBank, while skc sequences from Streptococcus equi subsp. equi were more conserved. The skc gene sequences obtained from the samples showed high nucleotide variability. Most sequences were similar to sequences already deposited in GenBank, while other sequences were different from already known sequences. No statistical differences were found among sequences obtained from group 1, group 2 or group 3, suggesting that genetic variability of skc does not affect the SEZ tropism for the respiratory and the reproductive organs. Preliminary sequences obtained from samples of aborted fetuses seem more similar each other’s than samples from other source and clustered in a separated branch, but further analysis and a wider set of samples are required to confirm these findings. The results of this study show that the skc gene of SEZ is highly variable. The study of additional sequences is required for better understanding the impact of this variability on SEZ tropism or virulence. Further studies should investigate the correlation between genetic variability of the skc gene and SEZ activity on plasminogen and virulence in horses.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
