Cell-Pathogen Interactions (Viruses and Bacteria)

175

The Chlamydial Toxin CPAF Promotes Early Exit from Mitosis in Infected Cells.

H. Brown1, A. Knowlton1, S. Grieshaber1; 1Oral Biology, University of Florida, Gainesville, FL Chlamydia trachomatis has been epidemiologically linked to cervical cancer in patients with a prior human papilomavirus (HPV) infection. Chlamydia causes multinucleation of the host cell, a potential pathway for chromosomal instability. Two mechanisms that are known to initiate multinucleation are cell fusion and cytokinesis failure. Our studies ruled out cell fusion and demonstrated that multinucleation is caused by cytokinesis failure. From this data, we aimed to determine the mechanism of cytokinesis failure due to Chlamydia infection. We have previously reported that chlamydial infection of the host cell overrides the Spindle Assembly Checkpoint (SAC) causing mitosis to be shortened. Our findings demonstrate that the Chlamydial Protease- like Activity Factor (CPAF) functionally inactivates cyclin B1 and cyclin dependent kinase 1 (Cdk1) through cleavage, effectively bypassing the SAC. This checkpoint is necessary for an organized cell division. In this study we demonstrate that premature entry into anaphase causes a increased rate of DNA tangles that results in cytokinesis failure of chlamydial infected cells. 176

Brush border microvilli-derived vesicles detoxify bacterial products and regulate epithelial-microbial interactions.

D. Shifrin1, R. McConnell2, R. Nambiar1, J. Higginbotham3, R. Coffey3, M. Tyska1; 1Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN,

2_{Department of Biology, Massachusetts Institute of Technology, Cambridge, MA,} 3_Department

of Medicine, Vanderbilt University Medical Center, Nashville, TN

The continuous monolayer of intestinal epithelial cells (IECs) that lines the gut lumen functions as the site of nutrient absorption and as a physical barrier that prevents the translocation of microbes and associated toxic compounds into the peripheral vasculature. IECs also express host defense proteins such as intestinal alkaline phosphatase (IAP), which detoxify bacterial products and prevent intestinal inflammation. Our laboratory recently showed that IAP is enriched on vesicles that are released from the tips of IEC microvilli and accumulate in the intestinal lumen (McConnell et al, 2007; McConnell et al, 2009). Here, we show that these native ‘lumenal vesicles’ (LVs): (i) contain catalytically active IAP that can dephosphorylate lipopolysaccharide (LPS), (ii) cluster on the surface of native lumenal bacteria, (iii) prevent the adherence of enteropathogenic E. coli (EPEC) to epithelial monolayers, and (iv) limit bacterial growth. We also find that IECs upregulate LV production in response to EPEC and defects in LV production are associated with significant changes in the intestinal microbiota. Together these results suggest that microvillar vesicle shedding represents a novel mechanism for distributing host defense machinery into the intestinal lumen, and that microvillus-derived LVs modulate epithelial-microbial interactions and exert a direct effect on the lumenal microbiome.

177

Mitochondrial dynamics in Listeria monocytogenes infection.

F. Stavru1, F. Bouillaud2, A. Sartori3, D. Ricquier2, P. Cossart4; 1Bacteria Cell Interactions, Institut Pasteur, Paris, France, 2Mitochondria, bioenergetic, metabolism and signaling, Institut Cochin, Paris, France, 3PFMU - Imagopole, Institut Pasteur, Paris, France, 4Bacteria Cell Interactions, Institut Pasteur, Paris, France

Mitochondria are dynamic organelles central to energy production and several other cellular processes, such as calcium buffering and apoptosis. As such, they are targeted by pathogens to subvert cellular function. We studied the effects of infection by the intracellular pathogen Listeria monocytogenes on mitochondrial dynamics and function and could recently show that it profoundly alters mitochondrial dynamics, causing transient fragmentation of the mitochondrial network. Such mitochondrial fragmentation occurs early during infection, is specific to pathogenic L. monocytogenes, and is not observed upon infection with several other intracellular pathogens. The relevance of mitochondrial dynamics for L. monocytogenes infection is highlighted by the finding that siRNA-mediated inhibition of mitochondrial fusion or fission alters infection efficiency. We identified the secreted pore-forming toxin listeriolysin O as the main bacterial factor responsible for disruption of the mitochondrial network and for mitochondrial function modulation. Our work suggests that transient disruption of mitochondrial dynamics and function represents a novel strategy used by pathogenic bacteria to interfere with cellular physiology at the onset of infection, which opens new avenues to study mitochondrial dynamics and function.

178

Dynamics of ESCRT protein recruitment during retroviral assembly.

N. Jouvenet1, P. Bieniasz1,2, S. M. Simon3; 1Aaron Diamond Aids Research, New York, NY, 2Lab of Retrovirology, Rockefeller University, New York, NY, 3Rockefeller University, New York, NY The assembly of retroviruses is the dependent upon the interaction of various components encoded both by the virus and the host cell. The separation of retroviral particles from host membranes is promoted by the cellular ESCRT (Endosomal Sorting Complex Required for Transport) complexes and associated proteins. The ESCRT complexes and associated proteins mediate other membrane scission reactions, such as multi-vesicular body formation and the terminal stages of cytokinesis. These proteins are believed to be sequentially recruited to the site of membrane scission, and then complexes are disassembled by the ATPase Vps4A. However these events have never been observed in living cells and their dynamics are unknown. Characterizing the relative time course of these molecules is complicated in cells where each virion is in a different state of assembly. To characterize the dynamics of these molecules, we studied the assembly of individual virions. By quantifying the recruitment of several ESCRT and associated proteins during the assembly of two retroviruses, EIAV and HIV- 1, we show that Alix progressively accumulated at viral assembly sites, coincident with the accumulation of the major viral structural protein, Gag, and was not recycled after assembly. In contrast, ESCRT-III and Vps4A were only transiently recruited when the accumulation of Gag was complete. These data suggest that the rapid and transient recruitment of proteins that act late in the ESCRT pathway and carry out membrane fission is triggered by prior and progressive accumulation of proteins that bridge viral proteins and the late-acting ESCRT proteins.

179

Salmonella enterica and epithelial cells: life in a vacuole or the cytosol?

L. Knodler1, O. Steele-Mortimer1; 1Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, NIH/NIAID, Hamilton, MT

Salmonella enterica is an intracellular bacterial pathogen that resides and proliferates within a membrane-bound vacuole in phagocytic and non-phagocytic cells of its mammalian hosts. Although essential to disease, how Salmonella escapes from its intracellular niche and spreads to secondary cells within the same host, or to a new host, is not known. We recently described that a subpopulation of Salmonella are released from the Salmonella-containing vacuole and hyper-replicate in the cytosol of epithelial cells. These bacteria are transcriptionally distinct from intravacuolar Salmonella. They are induced for the invasion-associated type III secretion system and possess flagella; hence, they are primed for invasion. Epithelial cells laden with these cytosolic bacteria undergo inflammatory programmed cell death and are extruded out of the monolayer, releasing invasion-primed and -competent Salmonella into the lumen, allowing for completion of the infectious cycle. To distinguish cytosolic from vacuolar Salmonella, we have developed a drug-based assay that we are using to define bacterial factors that contribute to vacuole lysis and cytosolic replication in epithelial cells.

180

Viral interior design: Rewiring the host to generate organelle platforms for replication. N-Y. Hsu1, N. Altan-Bonnet1; 1Department of Biological Sciences, Rutgers Univ, Newark, NJ RNA viruses manipulate multiple host components of the secretory pathway to generate organelles that are specialized for replication and are distinct in protein and lipid composition from the host cell. We show that specific picornaviral proteins promote recruitment of host phosphatidylinositol-4-kinases to membranes, while suppressing recruitment of host membrane coat proteins. This results in the biogenesis of uncoated membrane platforms that are highly enriched in phosphatidylinositol-4-phosphate (PI4P) lipids and contain a specific subset of host endocytic and secretory proteins. We find that the PI4P-rich lipid microenvironment is essential for viral RNA replication in both picorna- and flavivirus families; and that this unique lipid microenvironment may regulate the recruitment and assembly of viral replication machinery including viral RNA polymerases which we show have a specific novel binding site for PI4P lipids. Our findings reveal how RNA viruses can selectively exploit specific elements of the host to form specialized organelles, and identify host phosphatidylinositol-4-kinases and PI4P lipids as key panviral regulators of viral RNA replication and hence potential targets for the