Mycoplasma and host interaction: in vitro gene expression modulation in Mycoplasma synoviae and infected chicken chondrocytes

The complex interplay between Mycoplasma synoviae and chicken chondrocytes (CCH), which come into direct contact during infectious synovitis, has been examined at the level of gene expression. Our previous studies demonstrated a significant influence of M. synoviae on the level of CCH gene expression. Here, we show for the first time that in vitro co-cultivation of M. synoviae and CCH also induces upregulation of gene expression in this mycoplasma. We observed significantly increased expression of genes important for M. synoviae pathogenicity, including cysteine protease cysP, neuraminidase nanH, haemagglutinin vlhA, and the putative nuclease MS53_0284. Moreover, the pattern of gene expression was dependent on the infection environment. In CCH, significant changes in the expression of genes encoding catabolic enzymes of the cartilage extracellular matrix (cathepsins B, K and L, aggrecanase ADAM10, and matrix metalloproteinase MMP2) were demonstrated. Infection of CCH with M. synoviae also elevated the expression of the gene encoding peptidyl arginine deiminase, type III (PADI3), which is responsible for the post-translational citrullination of proteins

Mycoplasma synoviae induces upregulation of apoptotic genes, secretion of nitric oxide and appearance of an apoptotic phenotype in infected chicken chondrocytes

The role of chondrocytes in the development of infectious arthritis is not well understood. Several examples of mycoplasma-induced arthritis in animals indicate that chondrocytes come into direct contact with bacteria. The objective of this study was to analyze the interaction of an arthrogenic Mycoplasma synoviae strain WVU 1853 with chicken chondrocytes. We found that M. synoviae significantly reduces chondrocyte respiration. This was accompanied by alterations in chondrocyte morphology, namely cell shrinkage and cytoplasm condensation, as well as nuclear condensation and formation of plasma membrane invaginations containing nuclear material, which appeared to cleave off the cell surface. In concordance with these apoptosis-like events in chondrocytes, transcription was increased in several pro-apoptotic genes. Twenty-four hours after infection, strong upregulation was assayed in NOS2, Mapk11, CASP8 and Casp3 genes. Twenty-four and 72 h incubation of chondrocytes with M. synoviae induced upregulation of AIFM1, NFκB1, htrA3 and BCL2. Casp3 and NOS2 remained upregulated, but upregulation ceased for Mapk11 and CASP8 genes. Increased production of nitric oxide was also confirmed in cell supernates. The data suggests that chicken chondrocytes infected with M. synoviae die by apoptosis involving production of nitric oxide, caspase 3 activation and mitochondrial inactivation. The results of this study show for the first time that mycoplasmas could cause chondrocyte apoptosis. This could contribute to tissue destruction and influence the development of arthritic conditions. Hence, the study gives new insights into the role of mycoplasma infection on chondrocyte biology and development of infectious arthritis in chickens and potentially in humans

Response of adult honey bees treated in larval stage with prochloraz to infection with Nosema ceranae

Among numerous factors that contribute to honey bee colony losses and problems in beekeeping, pesticides and Nosema ceranae have been often reported. In contrast to insecticides, whose effects on bees have been widely studied, fungicides did not attract considerable attention. Prochloraz, an imidazole fungicide widely used in agriculture, was detected in honey and pollen stored inside hives and has been already proven to alter immune gene expression of honey bees at different developmental stages. The aim of this study was to simulate the realistic conditions of migratory beekeeping, where colonies, both uninfected and infected with N. ceranae, are frequently transported to the vicinity of crop fields treated with prochloraz. We investigated the combined effect of prochloraz and N. ceranae on honey bees that faced fungicide during the larval stage through food consumption and microsporidium infection afterwards. The most pronounced changes in gene expression were observed in newly emerged Nosema-free bees originating from colonies previously contaminated with prochloraz. As exclusively upregulation was registered, prochloraz alone most likely acts as a challenge that induces activation of immune pathways in newly emerged bees. The combination of both stressors (prochloraz and Nosema infection) exerted the greatest effect on six-day-old honey bees. Among ten genes with significantly altered expression, half were upregulated and half downregulated. N. ceranae as a sole stressor had the weakest effects on immune gene expression modulation with only three genes significantly dysregulated. In conclusion, food contaminated with prochloraz consumed in larval stage could present a threat to the development of immunity and detoxification mechanisms in honey bees

Mycoplasma gallisepticum and Mycoplasma synoviae express a cysteine protease CysP, which can cleave chicken IgG into Fab and Fc

Major poultry pathogens M. gallisepticum and M. synoviae share a gene encoding a putative cysteine protease CysP similar to papain cysteine (CaA subfamily). Comparison of the cysP gene sequences of 18 M. synoviae and 10 M. gallisepticum strains sequenced in this study showed polymorphisms, including deletions. Seven M. synoviae strains, including the type strain WVU 1853, had a 39 bp deletion in the 3\u27 end of the cysP gene. In the same cysP region, all M. gallisepticum strains showed a deletion of 66bp. Immunoblot analysis with specific antibpodies demonstrated that M. synoviae strains expressed CysP, which was approximately 65 kDa. both M. synoviae and M. gallisepticum were able to digest chicken IgG (cIgG). Incubation of cIgG (-170 kDa) with M. synoviae or M. gallisepticum cells (-15 h at 37oC) resultes in a papain-like cleavage pattern of cIgG and fragments corresponding to the antigenbinding fragment of IgG (Fab, -45kDa) and the crystallizable region fragment (Fc) of the IgG heavy chain (dimer of -60 kDa). Iodoacetamide (50 mM) prevented cleavage of cIgG by both Mycoplasma species. Following site-directed mutagenesis (eight TGA codons were changed to TGG) the cysP gene of M. synoviae ULB 925 was expressed as a His-taggede protein in a cell-free system. Purified recombinant CysP (rCysp-67kDa, pI-8) cleaved cIgG into Fab and Fc fragments. This indicates that CysP is responsible for the cIgG cleavage caused by M. synoviae and probbaly, by M. gallisepticum. This is the first evidence to our knowledge that mycopasmas have enzymes that can cleave the host IgG and indicates a novel strategy used by M. gallisepticum and M. synoviae for prolomged survival despite the antibody response of their host

Demonstration of neuraminidase activity in Mycoplasma neurolyticum and of neuraminidase proteins in three canine Mycoplasma species

Neuraminidases are virulence factors in many pathogenic microarganisms. They are present also in some Mycoplasma species that cause disease in birds, dogs and alligators. Thirty-seven Mycoplasma species have been examined previously for neuraminidase (sialidase) activity, whereas many of the species causing disease in man, ruminants, pigs, rodent and other animals have not. In this study neuraminidase enzymatic activity (NEAC) was examined in 45 previously untested Mycoplasma species, including those causing diseases in man, farm animals and laboratory animals. The only species in which NEAC was found was Mycoplasma neurolyticum, specifically, its type strain (Type A[up]T) which is capable of inducing neurologic signs in inoculated young mice and rats. The NEAC of washed cells was relatively weak, but it differed even more than 10-fold among cells of cultures derived from individual colonies of M. neurolyticum. A weak NEAC was also detected in the supernatant of the M. neurolyticum broth culture. Canine Mycoplasma spp. with high sialidase activity reported previously, Mycoplasma canis, Mycoplasma cynos and Mycoplasma molare had 100-fold more NEAC than M. neurolyticum, but apparent differences in NEAC levels existed among strains of M. canis and of M. cynos. Zymograms using neuraminidase-specific chromogenic substrate were used to showproteins havingNEAC. In M. canis (a field isolate Larissa and the type strain PG14[up]), M. cynos (isolate 896) and M. molare (type strain H542[up]) proteins with NEAC had molecular mases of - 130 kDA, 105 kDa and 110 kDa, respectively. Identification of these neraminidases could provide the basis for their molecular characterization

Immune related gene expression in worker honey bee (<i>Apis mellifera carnica</i>) pupae exposed to neonicotinoid thiamethoxam and <i>Varroa</i> mites (<i>Varroa destructor</i>)

<div><p><i>Varroa destructor</i> is one of the most common parasites of honey bee colonies and is considered as a possible co-factor for honey bee decline. At the same time, the use of pesticides in intensive agriculture is still the most effective method of pest control. There is limited information about the effects of pesticide exposure on parasitized honey bees. Larval ingestion of certain pesticides could have effects on honey bee immune defense mechanisms, development and metabolic pathways. Europe and America face the disturbing phenomenon of the disappearance of honey bee colonies, termed Colony Collapse Disorder (CCD). One reason discussed is the possible suppression of honey bee immune system as a consequence of prolonged exposure to chemicals. In this study, the effects of the neonicotinoid thiamethoxam on honey bee, <i>Apis mellifera carnica</i>, pupae infested with <i>Varroa destructor</i> mites were analyzed at the molecular level. <i>Varroa</i>-infested and non-infested honey bee colonies received protein cakes with or without thiamethoxam. Nurse bees used these cakes as a feed for developing larvae. Samples of white-eyed and brown-eyed pupae were collected. Expression of 17 immune-related genes was analyzed by real-time PCR. Relative gene expression in samples exposed only to <i>Varroa</i> or to thiamethoxam or simultaneously to both <i>Varroa</i> and thiamethoxam was compared. The impact from the consumption of thiamethoxam during the larval stage on honey bee immune related gene expression in <i>Varroa</i>-infested white-eyed pupae was reflected as down-regulation of <i>spaetzle</i>, AMPs <i>abaecin</i> and <i>defensin-1</i> and up-regulation of <i>lysozyme-2</i>. In brown-eyed pupae up-regulation of <i>PPOact</i>, <i>spaetzle</i>, <i>hopscotch</i> and <i>basket</i> genes was detected. Moreover, we observed a major difference in immune response to <i>Varroa</i> infestation between white-eyed pupae and brown-eyed pupae. The majority of tested immune-related genes were upregulated only in brown-eyed pupae, while in white-eyed pupae they were downregulated.</p></div

The effect of thiamethoxam on immune related gene expression in <i>Varroa</i> infested bees.

<p>The expression level for each gene is presented as difference in average expression level in <i>Varroa</i> infested group and infested-thiamethoxam treated group. The higher expression levels are indicated with red color, lower expression levels are indicated with blue color. Immune pathway diagram for white-eyed pupae (A) and brown-eyed pupae (B) are presented. The range of relative expression ratio is indicated in the legend (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187079#pone.0187079.s003" target="_blank">S1 Fig</a>). Bluer color in panel indicates higher influence of thiamethoxam on <i>Varroa</i> infested pupae.</p