| Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia in swine. Infection with M. hyopneumoniae results in a mild, chronic disease and is further complicated by the establishment of other secondary infections. M. hyopneumoniae has finite biosynthetic capabilities, including a deficit in de novo nucleotide synthesis. Recent research has demonstrated that neutrophils can produce extracellular traps (ETs), chromatin nets decorated with granular proteins released into the extracellular environment for the purpose of interacting and sequentially eliminating pathogens. We hypothesized that M. hyopneumoniae was able to utilize a membrane nuclease to obtain nucleotides from extracellular traps to synthesize its own DNA. The human monocytic cell line THP-1 was used as a model since macrophage extracellular traps (METs) are structurally similar to neutrophil extracellular traps (NETs). The thymidine analog ethynyl deoxyuridine (EdU) was used as a DNA label and was detected using a fluorescently labeled azide in a copper-catalyzed reaction coined "Click" chemistry. Prior to beginning the MET studies, the M. hyopneumoniae membrane nuclease activity was validated using a DNA degradation assay. Then, THP-1 cells were incubated with 10 ?M EdU for 24 h and induced with 200 nM phorbal myristate acetate for 36 h for the production of EdU-labeled METs. M. hyopneumoniae was incubated with these METs for 6 h followed by EdU was detected using an Alexa555-labeled azide, and fluorescence confocal microscopy. To test whether the nucleases were required for procurement of the EdU, nuclease co-factors Mg2+ and Ca2+ were chelated using EDTA and EGTA prior to incubating M. hyopneumoniae with the EdU-labeled METs for 6 h and visualizing the bacterial cells for EdU transfer. The results showed that when M. hyopneumoniae is incubated with EdU-labeled METs, METs are degraded and the EdU can be visualized co-localized within M. hyopneumoniae DNA. Additionally, when the nucleases are deprived of Mg2+ and Ca2+, METs remain intact and little to no EdU is visualized in M. hyopneumoniae DNA. The transfer of EdU from METs to M. hyopneumoniae DNA allows us to conclude that M. hyopneumoniae degrades host ETs likely via a membrane nuclease and uses the free nucleotides for its own DNA synthesis. |
