Differential antibody response to the NTPase of the protozoan parasite Toxoplasma gondii

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NO FULL TEXT AVAILABLE. Access is restricted indefinitely. ----- Toxoplasma gondii is an obligate intracellular protozoan parasite that can infect most, if not all, warm blooded vertebrates. The effects of T. gondii infection have been widely studied in the murine host, which is highly susceptible to infection and where isolates have shown a remarkable dichotomy in virulence, allowing strains to be classified as virulent or avirulent based on the number of tachyzoites required to cause severe disease. In humans, T. gondii causes a wide range of clinical manifestations that are dependent on factors that are intrinsic to both the host and the parasite. Most infections are asymptomatic in the immunocompetent host, however, a small number can suffer from symptomatic ocular infection. Severe symptomatic illness is most common in immunocompromised hosts and can cause serious disease leading to death if left untreated. For instance, congenital toxoplasmosis, which can occur if the mother is infected for the first time during pregnancy, may result in spontaneous miscarriage, stillbirths or foetal abnormalities. Patients who are immunocompromised by HIV infection or who are organ donor recipients can experience a recrudescence of latent T. gondii infection often leading to fatal encephalitis. A number of studies have attempted to elucidate if the infecting strain influences the clinical disease outcome. These studies have relied on isolation of the parasite from symptomatic patients followed by either a murine virulence test or strain genotyping. Given that symptomatic patients may only comprise about 1 % of infected individuals, data on the infecting strain type in the larger infected population is needed to give a complete picture of the epidemiology of T. gondii strains in human infection that is not subject to sampling and parasite isolation biases. The aim of this project was to determine whether a serological based assay could be developed for identifying the infecting strain type in human toxoplasmosis. In this project the nucleoside triphosphate hydrolase (NTPase) of T. gondii was evaluated as a potential serological marker of the virulence of the infecting strain. The NTPase of T. gondii demonstrates an unusually high level of ATP hydrolysis, which, among the apicomplexan parasites, has only been observed in T. gondii and the closely related Neospora caninum. This highly active NTPase was chosen for this study because it is highly expressed (constituting up to 8% of the tachyzoite protein) and is an immunodominant antigen in mice and humans. Two isoforms (NTPaseI and NTPaseII), which correlate to parasite virulence and exhibit different activities with respect to hydrolysis of ATP, exist. Previous studies suggested that all strains of T. gondii contained the less active NTPaseII whilst only virulent strains contained the more active NTPaseI isoform. In order to further investigate the correlation between NTPase isoform and biological significance, the full length NTPaseI and NTPaseII isoforms were cloned and expressed as glutathione S-transferase fusion proteins in Escherichia coli. Enzyme linked immunosorbant assays (ELISAs) were then used, with the full length recombinant NTPases as antigens, to examine 188 naturally infected T. gondii positive sera and 83 T. gondii negative sera for antibody reactivity. All positive sera reacted to T. gondii whole tachyzoite lysate antigen, 31 sera reacted to both NTPase isoforms, three sera reacted specifically to NTPaseI and two sera specifically to NTPaseII. Immunoblot analysis of the five sera reacting to either NTPase I or NTPaseII revealed both quantitative and qualitative differences in reactivity to the two isoforms. One of the five serum samples reacted only with NTPaseI and did not show any reactivity to NTPaseII. When this serum sample was used in comparative immunoblot analysis using truncations of the NTPase I isoform, a presumptive differential epitope between the NTPaseI and NTPaseII isoforms was identified within an 81 amino acid region (amino acids 445 - 526) at the C-terminus of the NTPaseI isoform. This differential reactivity was further localised to the 12 residue region of greatest variability between the two isoforms (residues 488 - 499) using synthetic peptides. This is the first report of naturally infected human sera being used to identify a differential epitope. Competitive antigen ELISAs using the synthetic peptides revealed that this serum sample reacted specifically to the NTPaseI epitope, whilst the corresponding region on NTPaseII isoform was a less specific cross-reactive epitope. These results were consistent with the hypothesis that this patient had been infected with a virulent strain of T. gondii. Further evidence of the differential reactivity of these peptides was observed when sera from rats that were immunised with either recombinant NTPaseI or NTPaseII reacted specifically and differentially to the peptides in ELISAs. To determine if it was possible to identify the infective strain type (virulent or avirulent) in T. gondii infections by serological means, the isoform-specific peptides from the NTPaseI and NTPaseII were used in ELISAs with sera from rats and humans where the infecting strain was known. The ELISAs were used to test six groups of rats that were infected with one of three virulent (RH, P strain or Ent) or three avirulent (Me49, C strain or TPR) strains of T. gondii. No differential antibody reactivity was detected by either whole recNTPase ELISA or peptide ELISA in the sera of rats infected by either virulent or avirulent strains of T. gondii. A panel of human sera from patients infected with known laboratory strains of T.gondii or naturally infected patients where the parasite was isolated and its virulence determined in mice was also studied. Differential reactivity to whole recNTPase isoforms was detected in some human sera but this reactivity was not detected by the isoform specific peptide ELISAs. Overall, although the NTPase peptides exhibited differential antibody reactivity, this was not correlated to the virulence status of the infecting strain and they cannot, therefore, be used as serological markers of virulence of the infecting strain.
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