Utilization of macrophage extracellular trap nucleotides by Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia in swine, an important disease worldwide. It has finite biosynthetic capabilities, including a deficit in de novo nucleotide synthesis. The source(s) for nucleotides in vivo are unknown, but mycoplasmas are known to carry membrane-bound nucleases thought to participate in the acquisition of nucleotides from host genomic DNA. Recent research has demonstrated that neutrophils can produce extracellular traps (NETs), chromatin NETs decorated with granular proteins to interact with and eliminate pathogens. We hypothesized that M. hyopneumoniae could utilize its membrane nuclease to obtain nucleotides from extracellular traps to construct its own DNA. Using the human monocytic cell line THP-1, we induced macrophage extracellular traps (METs), which are structurally similar to NETs. The thymidine analogue ethynyl deoxyuridine (EdU) was incorporated into THP-1 DNA and METs were induced. When incubated with M. hyopneumoniae, METs were degraded and the modified nucleotide label could be co-localized within M. hyopneumoniae DNA. When the nucleases were inhibited, MET degradation and nucleotide transfer were also inhibited. Controls confirmed that the EdU originated directly from the METs and not from free nucleotides arising from intracellular pools released during extrusion of the chromosomal DNA. M. hyopneumoniae incorporated labelled nucleotides more efficiently when 'fed' on METs than from free nucleotides in the medium, suggesting a tight linkage between nuclease degradation of DNA and nucleotide transport. These results strongly suggest that M. hyopneumoniae could degrade extracellular traps formed in vivo during infection and incorporate those host nucleotides into its own DNA.

Encapsulation into amphiphilic polyanhydride microparticles stabilizes Yersinia pestis antigens.

The design of biodegradable polymeric delivery systems based on polyanhydrides that would provide for improved structural integrity of Yersinia pestis antigens was the main goal of this study. Accordingly, the full-length Y. pestis fusion protein (F1-V) or a recombinant Y. pestis fusion protein (F1(B2T1)-V10) was encapsulated and released from microparticles based on 1,6-bis(p-carboxyphenoxy)hexane (CPH) and sebacic acid (SA) copolymers and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and CPH copolymers fabricated by cryogenic atomization. An enzyme-linked immunosorbent assay was used to measure changes in the antigenicity of the released proteins. The recombinant F1(B2T1)-V10 was unstable upon release from the hydrophobic CPH:SA microparticles, but maintained its structure and antigenicity in the amphiphilic CPTEG:CPH system. The full-length F1-V was stably released by both CPH:SA and CPTEG:CPH microparticles. In order to determine the effect of the anhydride monomers on the protein structure, changes in the primary, secondary, and tertiary structure, as well as the antigenicity of both Y. pestis antigens, were measured after incubation in the presence of saturated solutions of SA, CPH, and CPTEG anhydride monomers. The results indicated that the amphiphilic environment provided by the CPTEG monomer was important to preserve the structure and antigenicity of both proteins. These studies offer an approach by which a thorough understanding of the mechanisms governing antigenic instability can be elucidated in order to optimize the in vivo performance of biodegradable delivery devices as protein carriers and/or vaccine adjuvants.