Rifampicin resistance and mutation of the rpoB gene in Mycobacterium tuberculosis

Abstract The bradyzoite and tachyzoite forms of Toxoplasma gondii , purified from infected animals, were analysed for their activities of phosphofructokinase, pyruvate kinase, lactate dehydrogenase, NAD⁺- and NADH-linked isocitrate dehydrogenases, and succinic dehydrogenase. Both developmental stages contained high activities of phosphofructokinase (specific for pyrophosphate rather than ATP), pyruvate kinase and lactate dehydrogenase, suggesting that energy metabolism in both forms may centre around a high glycolytic flux linked to lactate production. The markedly higher activity of the latter two enzymes in bradyzoites suggests that lactate production is particularly important in this developmental form. NAD⁺-specific isocitrate dehydrogenase was not detectable in either stage of the parasite (and proved useful as a measure of the purity of the bradyzoite preparation), whereas both parasite forms contained low activities of NADP⁺-linked isocitrate dehydrogenase. The results are consistent with the bradyzoites lacking a functional TCA cycle and respiratory chain and are suggestive of a lack of susceptibility of this developmental stage to atovaquone.     

A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.     

Toxoplasma gondii-positive human sera recognise intracellular tachyzoites and bradyzoites with diverse patterns of immunoreactivity

Antibody detection assays have long been the first line test to confirm infection with the zoonotic parasite Toxoplasma gondii. However, challenges exist with serological diagnosis, especially distinguishing between acute, latent and reactivation disease states. The sensitivity and specificity of serological tests might be improved by testing for antibodies against parasite antigens other than those typically found on the parasite surface during the acute stage. To this end, we analysed the reactivity profile of human sera, identified as positive for anti-Toxoplasma gondii IgG in traditional assays, by indirect immunofluorescence reactivity to acute stage intracellular tachyzoites and in vitro-induced latent stage bradyzoites. The majority of anti-Toxoplasma gondii IgG positive sera recognised both intracellularly replicating tachyzoites and in vitro-induced bradyzoites with varying patterns of immune-reactivity. Furthermore, anti-bradyzoite antibodies were not detected in sera that were IgM-positive/IgG-negative. These results demonstrate that anti-Toxoplasma gondii-positive sera may contain antibodies to a variety of antigens in addition to those traditionally used in serological tests, and suggest the need for further investigations into the utility of anti-bradyzoite-specific antibodies to aid in diagnosis of Toxoplasma gondii infection.     

Toxoplasma gondii: DNA vaccination with bradyzoite antigens induces protective immunity in mice against oral infection with parasite cysts

Infection of the host by Toxoplasma gondii leads to an acute systemic dissemination of tachyzoites, followed by a chronic phase, in which bradyzoites, enclosed in cysts, persist in the brain, the heart, and other tissues. Among putative vaccine candidates, the bradyzoite antigens BAG1 and MAG1 look promising since they are preferentially expressed during the chronic stage of the parasite. This work focused on studying the immunogenicity of bradyzoite antigens in a mouse model of chronic toxoplasmosis. A mixture of plasmids directing the cytoplasmic expression of MAG1 and BAG1 in mammalian cells was used to immunize mice. We show here that immunized mice developed, preferentially, specific anti-MAG1 and anti-BAG1 IgG2a subclass antibodies, indicating a shift towards a Th1-like response after DNA immunization. We then demonstrated that DNA immunization followed by challenge infection elicited effective protection in mice, suggesting that bradyzoite antigens should be considered in the design of vaccines against toxoplasmosis.     

Identification of Neospora caninum proteins regulated during the differentiation process from tachyzoite to bradyzoite stage by DIGE

Identification of differentially expressed proteins during Neospora caninum tachyzoite–bradyzoite conversion processes may lead to a better knowledge of the pathogenic mechanisms developed by this important parasite of cattle. In the present work, a differential expression proteomic study of tachyzoite and bradyzoite stages was accomplished for the first time by applying DIGE technology coupled with MS analysis. Up to 72 differentially expressed spots were visualized (1.5‐fold in relative abundance, p<0.05, t‐test). A total of 53 spots were more abundant in bradyzoites and 19 spots in tachyzoites. MS analysis identified 26 proteins; 20 of them overexpressed in the bradyzoite stage and 6 in the tachyzoite stage. Among the novel proteins, enolase and glyceraldehyde‐3‐phosphate dehydrogenase (involved in glycolysis), HSP70 and HSP90 (related to stress response) as well as the dense granule protein GRA9, which showed higher abundance in the bradyzoite stage, might be highlighted. On the other hand, isocitrate dehydrogenase 2, involved in the Krebs cycle, was found to be more abundant in tachyzoites extract. Biological functions from most novel proteins were correlated with previously reported processes during the differentiation process in Toxoplasma gondii. Thus, DIGE technology arises as a suitable tool to study mechanisms involved in the N. caninum tachyzoite to bradyzoite conversion.     

Toxoplasma gondii lacks the enzymes required for de novo arginine biosynthesis and arginine starvation triggers cyst formation

Two separate carbamoyl phosphate synthetase activities are required for the de novo synthesis of pyrimidines and arginine in most eukaryotes. Toxoplasma gondii is novel in possessing a single carbamoyl phosphate synthetase II gene that corresponds to a glutamine-dependent form required for pyrimidine biosynthesis. We therefore examined arginine acquisition in T. gondii to determine whether the single carbamoyl phosphate synthetase II activity could provide both pyrimidine and arginine biosynthesis. We found that arginine deprivation efficiently blocks the replication of intracellular T. gondii, yet has little effect on long-term parasite viability. Addition of citrulline, but not ornithine, rescues the growth defect observed in the absence of exogenous arginine. This rescue with citrulline is ablated when parasites are cultured in a human citrullinemia fibroblast cell line that is deficient in argininosuccinate synthetase activity. These results reveal the absence of genes and activities of the arginine biosynthetic pathway and demonstrate that T. gondii is an arginine auxotroph. Arginine starvation was also found to efficiently trigger differentiation of replicative tachyzoites into bradyzoites contained within stable cyst-like structures. These same parasites expressing bradyzoite antigens can be efficiently switched back to rapidly proliferating tachyzoites several weeks after arginine starvation. We hypothesise that the absence of gene activities that are essential for the biosynthesis of arginine from carbamoyl phosphate confers a selective advantage by increasing bradyzoite switching during the host response to T. gondii infection. These findings are consistent with a model of host-parasite evolution that allowed host control of bradyzoite induction by trading off virulence for increased transmission.     

Use of molecular and ultrastructural markers to evaluate stage conversion of Toxoplasma gondii in both the intermediate and definitive host

Toxoplasma gondii has a complex life cycle involving definite (cat) and intermediate (all warm blooded animals) hosts. This gives rise to four infectious forms each of which has a distinctive biological role. Two (tachyzoite and merozoite) are involved in propagation within a host and two (bradyzoite and sporozoite) are involved in transmission to new hosts. The various forms can be identified by their structure, host parasite relationship and distinctive developmental processes. In the present in vivo study, the various stages have been evaluated by electron microscopy and immunocytochemistry using a panel of molecular markers relating to surface and cytoplasmic molecules, metabolic iso-enzymes and secreted proteins that can differentiate between tachyzoite, bradyzoite and coccidian development. Tachyzoites were characterised as being positive for surface antigen 1, enolase isoenzyme 2, lactic dehydrogenase isoenzyme 1 and negative for bradyzoite antigen 1. In contrast, bradyzoites were negative for SAG1 but positive for BAG1, ENO1 and LDH2. When stage conversion was followed in brain lesion at 10 and 15 days post-infection, tachyzoites were predominant but a number of single intermediate organisms displaying tachyzoite and certain bradyzoite markers were observed. At later time points, small groups of organisms displaying only bradyzoite markers were also present. A number (9) of dense granule proteins (GRA1-8, NTPase) have also been identified in both tachyzoites and bradyzoites but there were differences in their location during parasite development. All the dense granule proteins extensively label the parasitophorous vacuole during tachyzoite development. In contrast the tissue cyst wall displays variable staining for the dense granule proteins, which also expresses an additional unique cyst wall protein. The molecular differences could be identified at the single cell stage consistent with conversion occurring at the time of entry into a new cell. These molecular differences were reflected in the structural differences in the parasitophorous vacuoles observed by electron microscopy. Stage conversion to enteric (coccidian) development was limited to the enterocytes of the cat small intestine. Although no specific markers were available, this form of development can be identified by the absence of specific tachyzoite (SAG1) and bradyzoite (BAG1) markers although the isoenzymes ENO2 and LHD1 were expressed. There was also a significant difference in the expression of the dense granule proteins. The coccidian stages and merozoites only expressed two (GRA7 and NTPase) of the nine dense granule proteins and this was reflected in significant differences in the structure of the parasitophorous vacuole. The coccidian stages also undergo conversion from asexual to sexual development. The mechanism controlling this process is unknown but does not involve any change in the host cell type or parasitophorous vacuole and may be pre-programmed, since the number of asexual cycles was self-limiting. In conclusion, it was possible using a combination of molecular markers to identify tachyzoite, bradyzoite and coccidian development in tissue sections.     

Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures

Neospora caninum, like Toxoplasma gondii, undergoes stage conversion in chronically infected animals, and forms tissue cysts which contain the slowly proliferating bradyzoite stage. These tissue cysts are delineated by a cyst wall, protect the parasite from physiological and immunological reactions on part of the host, and bradyzoites remain viable within an infected host for many years. However, unlike T. gondii, N. caninum bradyzoites have been difficult to obtain using in vitro culture techniques, and current protocols, based on those developed for T. gondii, have been shown to be not very efficient in promoting tachyzoite-to-bradyzoite stage conversion. We report here an alternative in vitro culture method to obtain stage conversion of N. caninum from the proliferative to the cystic stage by using the Nc-Liverpool isolate, murine epidermal keratinocytes as host cells, and continuous treatment of infected cultures with 70 microM sodium nitroprusside for up to 8 days. This treatment significantly reduced parasite proliferation as assessed by Neospora-specific quantitative real-time PCR. The expression of bradyzoite markers was analysed by immunofluorescence following 4 and 8 days of in vitro culture using antibodies directed against bradyzoite antigen 1, the mAbCC2, and the lectin Dolichos biflorus agglutinin. Expression of the tachyzoite-specific immunodominant antigen NcSAG1 and the tachyzoite antigen NcMIC1 was also assessed. Transmission electron microscopy revealed that the majority of parasitophorous vacuoles were in the process of forming a distinct cyst wall through accumulation of granular material at the periphery of the vacuole, and parasites exhibited the typical features of bradyzoites. These findings demonstrate the usefulness of this culture technique as a promising way to study tachyzoite-to-bradyzoite stage conversion in N. caninum in vitro.     

Microplate assay for screening Toxoplasma gondii bradyzoite differentiation with DUAL luciferase assay

Toxoplasma gondii can differentiate into tachyzoites or bradyzoites. To accelerate the investigation of bradyzoite differentiation mechanisms, we constructed a reporter parasite, PLK/DLUC_1C9, for a high-throughput assay. PLK/DLUC_1C9 expressed firefly luciferase under the bradyzoite-specific BAG1 promoter. Firefly luciferase activity was detected with a minimum of 10² parasites induced by pH 8.1. To normalize bradyzoite differentiation, PLK/DLUC_1C9 expressed Renilla luciferase under the parasite’s α-tubulin promoter. Renilla luciferase activity was detected with at least 10² parasites. By using PLK/DLUC_1C9 with this 96-well format screening system, we found that the protein kinase inhibitor analogs, bumped kinase inhibitors 1NM-PP1, 3MB-PP1, and 3BrB-PP1, had bradyzoite-inducing effects.