Annette Wunderlich

Cathepsin Cs Are Key for the Intracellular Survival of the Protozoan Parasite, Toxoplasma gondii

Cysteine proteases play key roles in apicomplexan invasion, organellar biogenesis, and intracellular survival. We have now characterized five genes encoding papain family cathepsins from Toxoplasma gondii, including three cathepsin Cs, one cathepsin B, and one cathepsin L. Unlike endopeptidases cathepsin B and L, T. gondii cathepsin Cs are exopeptidases and remove dipeptides from unblocked N-terminal substrates of proteins or peptides. TgCPC1 was the most highly expressed cathepsin mRNA in tachyzoites (by real-time PCR), but three cathepsins, TgCPC1, TgCPC2, and TgCPB, were undetectable in in vivo bradyzoites. The specific cathepsin C inhibitor, Gly-Phe-dimethylketone, selectively inhibited the TgCPCs activity, reducing parasite intracellular growth and proliferation. The targeted disruption of TgCPC1 does not affect the invasion and growth of tachyzoites as TgCPC2 is then up-regulated and may substitute for TgCPC1. TgCPC1 and TgCPC2 localize to constitutive secretory vesicles of tachyzoites, the dense granules. T. gondii cathepsin Cs are required for peptide degradation in the parasitophorous vacuole as the degradation of the marker protein, Escherichia coli β-lactamase, secreted into the parasitophorous vacuole of transgenic tachyzoites was completely inhibited by the cathepsin C inhibitor. Cathepsin C inhibitors also limited the in vivo infection of T. gondii in the chick embryo model of toxoplasmosis. Thus, cathepsin Cs are critical to T. gondii growth and differentiation, and their unique specificities could be exploited to develop novel chemotherapeutic agents.

Toxopain-1 is critical for infection in a novel chicken embryo model of congenital toxoplasmosis

ABSTRACT We tested the hypothesis that cathepsins and specifically toxopain-1, a cathepsin B, play a critical role in the pathogenesis of toxoplasmosis. We found that inhibiting the expression of toxopain-1-specific mRNA and protein by >60% significantly decreased the capacity of the parasites to multiply and invade in vitro. To relate these in vitro results to the role of toxopain-1 in pathogenesis in vivo, we developed a novel chicken embryo model of congenital toxoplasmosis. Inhibiting either toxopain-1 expression or specific cysteine proteinase activity significantly reduced congenital infection of chicken embryos, as determined by histopathology and by the number of parasites quantified by real-time PCR. Our new model provides key in vivo validation for the hypothesis that toxopain-1 is a potential drug target in Toxoplasma gondii and also provides a new animal model for rapid, inexpensive screening of antiparasitic compounds.

Genetic manipulation of immunoglobulin binding proteins of Haemophilus somnus.

The relationship of the 76kDa immunoglobulin binding, surface antigen (p76) of Haemophilus somnus to the high molecular weight immunoglobulin binding proteins (HMW IgBPs) was investigated. The kanamycin resistance gene from pLS88 was used via homologous recombination with allelic exchange to replace a portion of the gene encoding IgBPs of H. somnus strain 8025. Recombinants were shown by Western immunoblotting to express and secrete truncated antigens of approximately 200kDa and not to produce p76. The truncated HMW IgBP variants retained the ability to bind bovine IgG2 by the Fc portion as demonstrated by Western immunoblotting against IgG2 anti-DNP. This data indicated that the deleted 1.8kb BglII fragment was not required for secretion or immunoglobulin Fc binding by the HMW IgBPs but was required for expression of the downstream p76 gene. Functional studies showed that, in addition to Fc binding of IgG2 to truncated HMW IgBPs, the mutant strain 8025 Kan1 was equally resistant to killing by mouse complement but less virulent than the wild type parent (8025) in a mouse septicemia model of H. somnus infection. However, mutant strain 8025 Kan1 did adhere less well than the wild type to bovine pulmonary artery endothelial cells. It is probable that p76 and the missing peptides of the HMW IgBPs play a role in this aspect of virulence and perhaps other aspects.