Propeptides regulate protein function and trafficking in many eukaryotic systems and have emerged as important features of regulated secretory proteins in parasites of the phylum Apicomplexa. Regulated protein secretion from micronemes and host cell invasion are inextricably linked and essential processes for the apicomplexan parasite Toxoplasma gondii. TgM2AP is a propeptide-containing microneme protein found in a heterohexameric complex with the microneme protein TgMIC2, a protein that has a demonstrated fundamental role in gliding motility and invasion. TgM2AP function is also central to these processes, because disruption of TgM2AP (m2apKO) results in secretory retention of TgMIC2, leading to reduced TgMIC2 secretion from the micronemes and impaired invasion. Because the TgM2AP propeptide is predicted to be processed in an intracellular site near where TgMIC2 is retained in m2apKO parasites, we hypothesized that the propeptide and its proteolytic removal influence trafficking and secretion of the complex. We found that proTgM2AP traffics through endosomal compartments and that deletion of the propeptide leads to defective trafficking of the complex within or near this site, resulting in aberrant processing and decreased secretion of TgMIC2, impaired invasion, and reduced virulence in vivo, mirroring the phenotypes observed in m2apKO parasites. In contrast, mutation of several cleavage site residues resulted in normal localization, but it affected the stability and secretion of the complex from the micronemes. Therefore, the propeptide and its cleavage site influence distinct aspects of TgMIC2-M2AP function, with both impacting the outcome of infection. INTRODUCTIONEukaryotic secretory proteins use an assortment of luminal or cytoplasmic forward targeting signals to navigate the secretory system for eventual delivery to the extracellular environment. Among the least well understood of the luminal signals are cleavable elements known as propeptides, which are typically positioned either internally or, more commonly, at the N terminus of a protein. Propeptides have been shown to serve in several capacities. They can facilitate the folding of their cognate protein, regulate its activity or oligomeric assembly, or direct it to a particular intracellular compartment within the endolysosomal or secretory system. For proproteins destined for the regulated secretory pathway, proteolytic processing (also termed proteolytic maturation) typically accompanies the condensation of immature secretory granule contents (Orci et al., 1987;Kuiper and Martens, 2000). Yet, the precise role of proteolytic maturation in the biogenesis and discharge of regulated secretory organelles remains contentious (Arvan and Halban, 2004).Toxoplasma gondii, the etiological agent of toxoplasmosis, is a genetically tractable protozoan parasite that causes widespread infection in humans resulting in significant economic losses worldwide (Jones et al., 2001). As a member of the phylum Apicomplexa, T. gondii features a distinct apical complex consis...

The initial stage of invasion by apicomplexan parasites involves the exocytosis of the micronemes-containing molecules that contribute to host cell attachment and penetration. MIC4 was previously described as a protein secreted by Toxoplasma gondii tachyzoites upon stimulation of micronemes exocytosis. We have microsequenced the mature protein, purified after discharge from micronemes and cloned the corresponding gene. The deduced amino acid sequence of MIC4 predicts a 61-kDa protein that contains 6 conserved apple domains. Apple domains are composed of six spacely conserved cysteine residues which form disulfide bridges and are also present in micronemal proteins from two closely related apicomplexan parasites, Sarcocystis muris and Eimeria species, and several mammalian serum proteins, including kallikrein. Here we show that MIC4 localizes in the micronemes of all the invasive forms of T. gondii, tachyzoites, bradyzoites, sporozoites, and merozoites. The protein is proteolytically processed both at the N and the C terminus only upon release from the organelle. MIC4 binds efficiently to host cells, and the adhesive motif maps in the most C-terminal apple domain.

HIV-1 establishes latency primarily by infecting activated CD4+ T cells that later return to quiescence as memory cells. Latency allows HIV-1 to evade immune responses and to persist during antiretroviral therapy, which represents an important problem in clinical practice. The lack of a valid cellular model to study HIV-1 latency has hindered advances in the understanding of its biology. In this study, we attempted to model HIV-1 latency using human primary CD4+ T cells infected in vitro with HIV-1 after activation with Ag-loaded dendritic cells and then brought back to quiescence through a resting phase in the presence of IL-7. During the resting phase, expression of cellular activation markers disappeared and cell proliferation and viral replication ceased, but resumed following restimulation of rested cells with Ag or mAbs directed to CD3/CD28. In addition, higher cell death rates were observed in HIV-1-infected than uninfected cultures during secondary but not primary stimulation. Thus, this system may allow us to study the biology of HIV-1 latency, as well as the mechanisms of CD4+ T cell death following HIV-1 reactivation.

Enteroaggregative Escherichia coli (EAEC) is responsible for inflammatory diarrhea in diverse populations, but its mechanisms of pathogenesis have not been fully elucidated. We have used a previously characterized polarized intestinal T84 cell model to investigate the effects of infection with EAEC strain 042 on tight junction integrity. We find that infection with strain 042 induces a decrease in transepithelial electrical resistance (TER) compared to uninfected controls and to cells infected with commensal E. coli strain HS. When the infection was limited after 3 h by washing and application of gentamicin, we observed that the TER of EAEC-infected monolayers continued to decline, and they remained low even as long as 48 h after the infection. Cells infected with the afimbrial mutant strain 042aafA exhibited TER measurements similar to those seen in uninfected monolayers, implicating the aggregative adherence fimbriae II (AAF/II) as necessary for barrier dysfunction. Infection with wild-type strain 042 induced aberrant localization of the tight junction proteins claudin-1 and, to a lesser degree, occludin. EAEC-infected T84 cells exhibited irregular shapes, and some cells became elongated and/or enlarged; these effects were not observed after infection with commensal E. coli strain HS or 042aafA. The effects on tight junctions were also observed with AAF/I-producing strain JM221, and an afimbrial mutant was similarly deficient in inducing barrier dysfunction. Our results show that EAEC induces epithelial barrier dysfunction in vitro and implicates the AAF adhesins in this phenotype.