Migratory activation of parasitized dendritic cells by the protozoan Toxoplasma gondii 14-3-3 protein.

The obligate intracellular parasite Toxoplasma gondii exploits cells of the immune system to disseminate. Upon infection, parasitized dendritic cells (DCs) and microglia exhibit a hypermigratory phenotype in vitro that has been associated with enhancing parasite dissemination in vivo in mice. One unresolved question is how parasites commandeer parasitized cells to achieve systemic dissemination by a 'Trojan-horse' mechanism. By chromatography and mass spectrometry analyses, we identified an orthologue of the 14-3-3 protein family, T. gondii 14-3-3 (Tg14-3-3), as mediator of DC hypermotility. We demonstrate that parasite-derived polypeptide fractions enriched for Tg14-3-3 or recombinant Tg14-3-3 are sufficient to induce the hypermotile phenotype when introduced by protein transfection into murine DCs, human DCs or microglia. Further, gene transfer of Tg14-3-3 by lentiviral transduction induced hypermotility in primary human DCs. In parasites expressing Tg14-3-3 in a ligand-regulatable fashion, overexpression of Tg14-3-3 was correlated with induction of hypermotility in parasitized DCs. Localization studies in infected DCs identified Tg14-3-3 within the parasitophorous vacuolar space and a rapid recruitment of host cell 14-3-3 to the parasitophorous vacuole membrane. The present work identifies a determinant role for Tg14-3-3 in the induction of the migratory activation of immune cells by T. gondii. Collectively, the findings reveal Tg14-3-3 as a novel target for an intracellular pathogen that acts by hijacking the host cell's migratory properties to disseminate.

Chlamydia trachomatis Inclusion Disrupts Host Cell Cytokinesis to Enhance Its Growth in Multinuclear Cells.

Chlamydia trachomatis, the leading cause of bacterial sexually transmitted infections, disrupts cytokinesis and causes significant multinucleation in host cells. Here, we demonstrate that multinuclear cells that result from unsuccessful cell division contain significantly higher Golgi content, an important source of lipids for chlamydiae. Using immunofluorescence and fluorescent live cell imaging, we show that C. trachomatis in multinuclear cells indeed intercept Golgi-derived lipid faster than in mononuclear cells. Moreover, multinuclear cells enhance C. trachomatis inclusion growth and infectious particle formation. Together, these results indicate that C. trachomatis robustly position inclusions to the cell equator to disrupt host cell division in order to acquire host Golgi-derived lipids more quickly in multinucleated progeny cells.

Classically activated macrophages use stable microtubules for matrix metalloproteinase-9 (MMP-9) secretion.

As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule (MT) stabilization and secretion of MMPs. Macrophages up-regulate MMP-9 expression and secretion upon immunological challenge and require its activity for migration during the inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution as well as the mechanisms responsible for its trafficking are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contained calreticulin and protein-disulfide isomerase in activated RAW 264.7 macrophages. We demonstrated vesicular organelles of MMP-9 aligned along stable subsets of MTs and showed that selective modulation of MT dynamics contributes to the enhanced trafficking of MMP-9 extracellularly. We found a Rab3D-dependent association of MMP-9 vesicles with the molecular motor kinesin, whose association with the MT network was greatly enhanced after macrophage activation. Finally, we implicated kinesin 5B and 3B isoforms in the effective trafficking of MMP-9 extracellularly.

Controlled sulfation of mixed-linkage glucan by Response Surface Methodology for the development of biologically applicable polysaccharides.

Endogenous and exogenous sulfated polysaccharides exhibit potent biological activities, including inhibiting blood coagulation and protein interactions. Controlled chemical sulfation of alternative polysaccharides holds promise to overcome limited availability and heterogeneity of naturally sulfated polysaccharides. Here, we established reaction parameters for the controlled sulfation of the abundant cereal polysaccharide, mixed-linkage ß(1,3)/ß(1,4)-glucan (MLG), using Box-Behnken Design of Experiments (BBD) and Response Surface Methodology (RSM). The optimization of the degree-of-substitution (DS) was externally validated through the production of sulfated MLGs (S-MLGs) with observed DS and Mw values deviating less than 20% and 30% from the targeted values, respectively. Simultaneous optimization of DS and Mw resulted in the same range of deviation from the targeted value. S-MLGs with DS > 1 demonstrated a modest anticoagulation effect versus heparin, and a greater P-selectin affinity than fucoidan. As such, this work provides a route to medically important polymers from an economical agricultural polysaccharide.

Modulation of osteoclastogenesis with macrophage M1- and M2-inducing stimuli.

Macrophages are generated through the differentiation of monocytes in tissues and they have important functions in innate and adaptive immunity. In addition to their roles as phagocytes, macrophages can be further differentiated, in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), into osteoclasts (multinucleated giant cells that are responsible for bone resorption). In this work, we set out to characterize whether various inflammatory stimuli, known to induce macrophage polarization, can alter the type of multinucleated giant cell obtained from RANKL differentiation. Following a four-day differentiation protocol, along with lipopolysaccharide (LPS)/interferon gamma (IFNγ) as one stimulus, and interleukin-4 (IL-4) as the other, three types of multinucleated cells were generated. Using various microscopy techniques (bright field, epifluorescence and scanning electron), functional assays, and western blotting for osteoclast markers, we found that, as expected, RANKL treatment alone resulted in osteoclasts, whereas the addition of LPS/IFNγ to RANKL pre-treated macrophages generated Langhans-type giant cells, while IL-4 led to giant cells resembling foreign body giant cells with osteoclast-like characteristics. Finally, to gain insight into the modulation of osteoclastogenesis, we characterized the formation and morphology of RANKL and LPS/IFNγ-induced multinucleated giant cells.

Chlamydia trachomatis vacuole maturation in infected macrophages.

Chlamydia trachomatis is an obligate intracellular bacterium responsible for one of the most common sexually transmitted diseases. In epithelial cells, C. trachomatis resides in a modified membrane-bound vacuole known as an inclusion, which is isolated from the endocytic pathway. However, the maturation process of C. trachomatis within immune cells, such as macrophages, has not been studied extensively. Here, we demonstrated that RAW macrophages effectively suppressed C. trachomatis growth and prevented Golgi stack disruption, a hallmark defect in epithelial cells after C. trachomatis infection. Next, we systematically examined association between C. trachomatis and various endocytic pathway markers. Spinning disk confocal time-lapse studies revealed significant and rapid association between C. trachomatis with Rab7 and LAMP1, markers of late endosomes and lysosomes. Moreover, pretreatment with an inhibitor of lysosome acidification led to significant increases in C. trachomatis growth in macrophages. At later stages of infection, C. trachomatis associated with the autophagy marker LC3. TEM analysis confirmed that a significant portion of C. trachomatis resided within double-membrane-bound compartments, characteristic of autophagosomes. Together, these results suggest that macrophages can suppress C. trachomatis growth by targeting it rapidly to lysosomes; moreover, autophagy is activated at later stages of infection and targets significant numbers of the invading bacteria, which may enhance subsequent chlamydial antigen presentation.

Chlamydia trachomatis inclusions induce asymmetric cleavage furrow formation and ingression failure in host cells.

Chlamydia trachomatis infection has been suggested to induce host genome duplication and is linked to increased risks of cervical cancer. We describe here the mechanism by which Chlamydia causes a cleavage furrow defect that consistently results in the formation of multinucleated host cells, a phenomenon linked to tumorigenesis. Host signaling proteins essential for cleavage furrow initiation, ingression, and stabilization are displaced from one of the prospective furrowing cortices after Chlamydia infection. This protein displacement leads to the formation of a unique asymmetrical, unilateral cleavage furrow in infected human cells. The asymmetrical distribution of signaling proteins is caused by the physical presence of the Chlamydia inclusion at the cell equator. By using ingested latex beads, we demonstrate that the presence of a large vacuole at the cell equator is sufficient to cause furrow ingression failure and can lead to multinucleation. Interestingly, internalized latex beads of similar size do not localize to the cell equator as efficiently as Chlamydia inclusions; moreover, inhibition of bacterial protein synthesis with antibiotic reduces the frequency at which Chlamydia localizes to the cell equator. Together, these results suggest that Chlamydia effectors are involved in strategic positioning of the inclusion during cell division.