The folate antagonist methotrexate (MTX) is extensively used in graft-versus-host disease, rheumatoid arthritis, and other chronic inflammatory disorders. In addition to its antiinflammatory activity associated with increased release of adenosine, MTX exerts antiproliferative properties by inhibition of dihydrofolate reductase and other folate-dependent enzymes. However, the mechanisms of immunosuppressive properties associated with low-dose MTX treatments are still elusive. We report here that MTX (0.1-10 microM) induces apoptosis of in vitro activated T cells from human peripheral blood. PBL exposed to MTX for 8 h, then activated in drug-free medium, underwent apoptosis, which was completely abrogated by addition of folinic acid or thymidine. Apoptosis of activated T cells did not require interaction between CD95 (Fas, APO-1) and its ligand, and adenosine release accounted for only a small part of this MTX activity. Apoptosis required progression of activated T cells to the S phase of the cell cycle, as it was prevented by drugs or antibodies that interfere with IL-2 synthesis or signaling pathways. MTX achieved clonal deletion of activated T cells in mixed lymphocyte reactions. Finally, in vitro activation of PBL taken from rheumatoid arthritis patients after MTX injection resulted in apoptosis. Altogether, the data demonstrate that MTX can selectively delete activated peripheral blood T cells by a CD95-independent pathway. This property could be used as a new pharmacological end point to optimize dosage and timing of MTX administration. It may account for the immunosuppressive effects of low-dose MTX treatments.

The continued evolution of bacterial pathogens has major implications for both human and animal disease, but the exchange of genetic material between host-restricted pathogens is rarely considered. Streptococcus equi subspecies equi (S. equi) is a host-restricted pathogen of horses that has evolved from the zoonotic pathogen Streptococcus equi subspecies zooepidemicus (S. zooepidemicus). These pathogens share approximately 80% genome sequence identity with the important human pathogen Streptococcus pyogenes. We sequenced and compared the genomes of S. equi 4047 and S. zooepidemicus H70 and screened S. equi and S. zooepidemicus strains from around the world to uncover evidence of the genetic events that have shaped the evolution of the S. equi genome and led to its emergence as a host-restricted pathogen. Our analysis provides evidence of functional loss due to mutation and deletion, coupled with pathogenic specialization through the acquisition of bacteriophage encoding a phospholipase A₂ toxin, and four superantigens, and an integrative conjugative element carrying a novel iron acquisition system with similarity to the high pathogenicity island of Yersinia pestis. We also highlight that S. equi, S. zooepidemicus, and S. pyogenes share a common phage pool that enhances cross-species pathogen evolution. We conclude that the complex interplay of functional loss, pathogenic specialization, and genetic exchange between S. equi, S. zooepidemicus, and S. pyogenes continues to influence the evolution of these important streptococci. Streptococci colonize a diverse range of animals and tissues, and this association is normally harmless. Occasionally some strains of streptococci have an increased ability to cause disease that is often associated with a reduction in the ability to colonize and the acquisition of new genes, which enable the strain to inhabit a new niche. S. equi is the causative agent of strangles, one of the most frequently diagnosed and feared infectious diseases of horses, which is believed to have evolved from the closely related and usually harmless S. zooepidemicus. We aim to understand the mechanisms by which S. equi causes disease by studying and comparing the genomes of these different strains. Here we identify specific genes that have been lost and gained by S. equi, which may have directed its transition from colonizer to invader. Several of the novel genes acquired by S. equi have also been identified in strains of the closely related bacterium S. pyogenes that are associated with increased morbidity and mortality in humans. Our research highlights the role of genetic exchange in cross-species bacterial evolution and argues that the evolution of human pathogens cannot be considered in isolation.

Strangles, the most frequently diagnosed infectious disease of horses worldwide, is caused byStreptococcus equi. Despite its prevalence, the global diversity and mechanisms underlying the evolution ofS. equias a host-restricted pathogen remain poorly understood. Here, we define the global population structure of this important pathogen and reveal a population replacement in the late 19th or early 20th Century. Our data reveal a dynamic genome that continues to mutate and decay, but also to amplify and acquire genes despite the organism having lost its natural competence and become host-restricted. The lifestyle ofS. equiwithin the horse is defined by short-term acute disease, strangles, followed by long-term infection. Population analysis reveals evidence of convergent evolution in isolates from post-acute disease samples as a result of niche adaptation to persistent infection within a host. Mutations that lead to metabolic streamlining and the loss of virulence determinants are more frequently found in persistent isolates, suggesting that the pathogenic potential ofS. equireduces as a consequence of long-term residency within the horse post-acute disease. An example of this is the deletion of the equibactin siderophore locus that is associated with iron acquisition, which occurs exclusively in persistent isolates, and rendersS. equisignificantly less able to cause acute disease in the natural host. We identify several loci that may similarly be required for the full virulence ofS. equi, directing future research toward the development of new vaccines against this host-restricted pathogen.