Gene flow in a Yersinia pestis vector, Oropsylla hirsuta, during a plague epizootic.

BACKGROUND & OBJECTIVES: Appreciating how Yersinia pestis, the etiological agent of plague, spreads among black - tailed prairie dog (Cynomys ludovicianus) colonies (BTPD), is vital to wildlife conservation programs in North American grasslands. A little - studied aspect of the system is the role of Y. pestis vectors, i.e. fleas, play in the spreading of plague in natural settings. We investigated the genetic structure and variability of a common prairie dog flea (Oropsylla hirsuta) in BTPD colonies in order to examine dispersal patterns. Given that this research took place during a widespread plague epizootic, there was the added advantage of gaining information on the dynamics of sylvatic plague. METHODS & RESULTS: Oropsylla hirsuta were collected from BTPD burrows in nine colonies from May 2005 to July 2005, and eight polymorphic microsatellite markers were used to generate genotypic data from them. Gene flow estimates revealed low genetic differentiation among fleas sampled from different colonies. NestedPCR plague assays confirmed the presence of Y. pestis with the average Y. pestis prevalence across all nine colonies at 12%. No significant correlations were found between the genetic variability and gene flow of O. hirsuta and Y. pestis prevalence on a per -colony basis. CONCLUSION: Oropsylla hirsuta dispersal among BTPD colonies was high, potentially explaining the rapid spread of Y. pestis in our study area in 2005 and 2006.

Mutation of two Mycoplasma arthritidis surface lipoproteins with divergent functions in cytadherence.

Mycoplasma arthritidis is a natural pathogen of rats, causing an acute polyarthritis. Previous studies identified two membrane-bound lipoproteins, Maa1 and Maa2, thought to be associated with cytadherence of M. arthritidis strain 158p10p9. We have since confirmed that Maa1 is a major adhesin, although the role of Maa2 has proven more elusive. Both proteins were capable of eliciting protective immunity in rats against challenge with the virulent strain 158p10p9, suggesting that they may be important in pathogenesis. The purpose of this study was to better understand the roles of Maa1 and Maa2 in cytadherence in vitro. Insertion mutants were created for both genes by transposon mutagenesis. In vitro adherence of the Maa1 mutant KOMaa1 to rat L2 lung cells was reduced to the level previously reported for a spontaneous low-adherence mutant of 158p10p9 in which Maa1 is truncated and nonfunctional. Surprisingly, adherence of the Maa2 mutant KOMaa2 was approximately fivefold greater than that of the wild type. Complementation of KOMaa1 and KOMaa2 with wild-type alleles of maa1 and maa2, respectively, returned adherence to wild-type levels. This work confirms our earlier observation that Maa1 is a major adhesin for M. arthritidis strain 158p10p9. Maa2, on the other hand, may play a suppressive or modulatory role, possibly serving to release organisms from microcolonies at certain stages of infection.

Inflammatory lipoproteins purified from a toxigenic and arthritogenic strain of Mycoplasma arthritidis are dependent on Toll-like receptor 2 and CD14.

Mycoplasma arthritidis is a naturally occurring murine pathogen, and the disease model has been used extensively to understand inflammatory mechanisms. Recently, Triton X-114 extracts of a virulent strain of M. arthritidis were found to be more potent in activating macrophages than were those from an avirulent strain, suggesting a role in disease. Here, octyl glucoside extraction of cells was used to identify four distinct bioactive moieties, with molecular masses of approximately 41, 37, 34, and 17 kDa. Their bioactivities were resistant to proteinase K but were destroyed by alkaline hydrolysis and oxidation. As for MALP-2, all were dependent upon Toll-like receptor 2, but unlike MALP-2, they were also dependent upon CD14. The M. arthritidis lipoproteins exhibited infrared absorbances at 2,900 cm(-1) and 1,662 cm(-1), similar to those seen in Pam(3)-Cys-Ser-(Lys)(4). Edman degradation failed to reveal N-terminal sequences, suggesting that they were blocked and therefore might be triacylated. However, mass spectrometry of fragments revealed that the 41-kDa moiety, which binds to serum apolipoprotein A-1, had similarity with the recently described MlpD lipoprotein of M. arthritidis.

Isolation and partial purification of macrophage- and dendritic cell-activating components from Mycoplasma arthritidis: association with organism virulence and involvement with Toll-like receptor 2.

Mycoplasma arthritidis induces toxicity, arthritis, and dermal necrosis in mice. Virulence factors include a superantigen and membrane adhesins and possibly also a bacteriophage component. Here we compare the biological properties of Triton X-114 extracts derived from avirulent and virulent M. arthritidis strains. Macrophage cell lines and resident peritoneal macrophages were used to assess inflammatory potential as indicated by production of tumor necrosis factor alpha, interleukin-6, and/or nitric oxide. The activity resided exclusively within the hydrophobic detergent phase, was unaffected by heat treatment at 100 degrees C for 30 min, and was resistant to proteinase K digestion, suggesting involvement of a lipopeptide. Contamination of extracts with endotoxin or superantigen was excluded. Extracts of the more virulent strain had higher activity than did those of the avirulent strain. Using CHO cells expressing Toll-like receptor 2 (TLR2) or TLR4, both with transfected CD14, we showed that extracts activated these cells via TLR2 but not by TLR4. Also, macrophages from C57BL/6 TLR2(-/-) mice failed to respond to the extracts, whereas those from TLR2(+/+) cells did respond. The preparations from the virulent strain of M. arthritidis were also more potent in activating dendritic cells, as evidenced by up-regulation of major histocompatibility complex class II, CD40, B7-1, and B7-2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent elution of gel slices revealed the presence of three active moieties which corresponded to molecular masses of approximately 24, 28, and 40 kDa. Three active components were also found by reverse-phase chromatography. We suggest that macrophage activation by M. arthritidis could play a significant role in the inflammatory response induced in the host by this organism.

Restoration of cytoadherence to an adherence-deficient mutant of Mycoplasma arthritidis by genetic complementation.

Mycoplasma arthritidis causes a severe septic arthritis in rats under natural and experimental conditions. An earlier study implicated a membrane lipoprotein designated MAA1 in cytadherence of M. arthritidis. In addition, a spontaneous adherence-deficient mutant was shown to contain a nonsense mutation in the gene encoding MAA1, resulting in production of a truncated product, MAA1Delta. In the present study, a wild-type maa1 gene carried on transposon Tn4001T was introduced into the low-adherence mutant by polyethylene glycol-mediated transformation. The presence of the tranposon and the wild-type maa1 gene in the chromosome of transformants was confirmed by PCR and Southern hybridization. The latter procedure also confirmed that each transformant contained a single copy of the transposon. Western immunoblotting showed that transformants produced both wild-type MAA1 and MAA1Delta, indicating that the introduced wild-type maa1 gene was functional. This phenotype was stably maintained after multiple subcultures even in the absence of antibiotic selection. Finally, transformants were shown to adhere to rat L-2 lung cells in culture at wild-type levels, providing confirmation for an important role for MAA1 in adherence.