Toxoplasma gondii actively infects circulating immune cells, including monocytes and DCs, and is thought to use these cells as Trojan horses for parasite dissemination. To investigate the interactions of T. gondii-infected human monocytes with vascular endothelium under conditions of shear stress, we developed a fluidic and time-lapse fluorescence microscopy system. Both uninfected and infected monocytes rolled, decelerated, and firmly adhered on TNF-α-activated endothelium. Interestingly, T. gondii-infected primary human monocytes and THP-1 cells exhibited altered adhesion dynamics compared with uninfected monocytes: infected cells rolled at significantly higher velocities (2.5- to 4.6-fold) and over greater distances (2.6- to 4.8-fold) than uninfected monocytes, before firmly adhering. During monocyte searching, 29-36% of infected monocytes compared with 0-11% of uninfected monocytes migrated >10 μm from the point where they initiated searching, and these "wandering" searches were predominantly in the direction of flow. As infected monocytes appeared delayed in their transition to firm adhesion, we examined the effects of infection on integrin expression and function. T. gondii did not affect the expression of LFA-1, VLA-4, or MAC-1 or the ability of Mn(2+) to activate these integrins. However, T. gondii infection impaired LFA-1 and VLA-4 clustering and pseudopod extension in response to integrin ligands. Surprisingly, a single intracellular parasite was sufficient to mediate these effects. This research has established a system for studying pathogen modulation of human leukocyte adhesion under conditions of physiological shear stress and has revealed a previously unappreciated effect of T. gondii infection on ligand-dependent integrin clustering.

The rodent arenavirus glycoprotein complex encodes a stable signal peptide (SSP) that is an essential structural component of mature virions. The SSP, GP1, and GP2 subunits of the trimeric glycoprotein complex noncovalently interact to stud the surface of virions and initiate arenavirus infectivity. Nascent glycoprotein production undergoes two proteolytic cleavage events: first within the endoplasmic reticulum (ER) to cleave SSP from the remaining precursor GP1/2 (glycoprotein complex [GPC]) glycoprotein and second within the Golgi stacks by the cellular SKI-1/S1P for GP1/2 processing to yield GP1 and GP2 subunits. Cleaved SSP is not degraded but retained as an essential glycoprotein subunit. Here, we defined functions of the 58-amino-acid lymphocytic choriomeningitis virus (LCMV) SSP in regard to glycoprotein complex processing and maturation. Using molecular biology techniques, confocal microscopy, and flow cytometry, we detected SSP at the plasma membrane of transfected cells. Further, we identified a sorting signal (FLLL) near the carboxyl terminus of SSP that is required for glycoprotein maturation and trafficking. In the absence of SSP, the glycoprotein accumulated within the ER and was unable to undergo processing by SKI-1/S1P. Mutation of this highly conserved FLLL motif showed impaired glycoprotein processing and secretory pathway trafficking, as well as defective surface expression and pH-dependent membrane fusion. Immunoprecipitation of SSP confirmed an interaction between the signal peptide and the GP2 subunit; however, mutations within this FLLL motif disrupted the association of the GP1 subunit with the remaining glycoprotein complex.

The glycoprotein of lymphocytic choriomeningitis virus (LCMV) contains nine potential N-linked glycosylation sites. We investigated the function of these N-glycosylations by using alanine-scanning mutagenesis. All the available sites were occupied on GP1 and two of three on GP2. N-linked glycan mutations at positions 87 and 97 on GP1 resulted in reduction of expression and absence of cleavage and were necessary for downstream functions, as confirmed by the loss of GP-mediated fusion activity with T87A, S97A mutants. In contrast, T234A and E379N/A381T mutants impaired GP-mediated cell fusion without altered expression or processing. Infectivity via virus-like particles required glycans and a cleaved glycoprotein. Glycosylation at the first site within GP2, not normally utilized by LCMV, exhibited increased VLP-infectivity. We also confirmed the role of the N-linked glycan at position 173 in the masking of the neutralizing epitope GP-1D. Taken together, our results indicated a strong relationship between fusion and infectivity.

T. whipplei glycosylation is likely to impair antibody-mediated immune recognition in patients. Such an intracellular antigen masking system in bacteria has not previously been described.

The glycoprotein (GP) of arenaviruses is glycosylated at 11 conserved N-glycosylation sites. We constructed recombinant lymphocytic choriomeningitis virus (rLCMV) featuring either additions or deletions of these N-glycans to investigate their role in the viral life cycle. N-glycosylation at two sites, T87 and S97, were found to be necessary to rescue rLCMV. Three of nine successfully rescued mutants, S116A, T234A, and S373A, under selective pressures in either epithelial, neuronal, or macrophage cells reverted to WT sequence. Of the seven stable N-glycan deletion mutants, five of these led to altered viral fitness and cell tropism, assessed as growth in either mouse primary cortical neurons or bone marrow derived macrophages. These results demonstrate that the deletion of N-glycans in LCMV GP may confer an advantage to the virus for infection of neurons but a disadvantage in macrophages.