Genetic Evidence for Erythrocyte Receptor Glycophorin B Expression Levels Defining a Dominant Plasmodium falciparum Invasion Pathway into Human Erythrocytes.

Plasmodium falciparum, the parasite that causes the deadliest form of malaria, has evolved multiple proteins known as invasion ligands that bind to specific erythrocyte receptors to facilitate invasion of human erythrocytes. The EBA-175/glycophorin A (GPA) and Rh5/basigin ligand-receptor interactions, referred to as invasion pathways, have been the subject of intense study. In this study, we focused on the less-characterized sialic acid-containing receptors glycophorin B (GPB) and glycophorin C (GPC). Through bioinformatic analysis, we identified extensive variation in glycophorin B (GYPB) transcript levels in individuals from Benin, suggesting selection from malaria pressure. To elucidate the importance of the GPB and GPC receptors relative to the well-described EBA-175/GPA invasion pathway, we used an ex vivo erythrocyte culture system to decrease expression of GPA, GPB, or GPC via lentiviral short hairpin RNA transduction of erythroid progenitor cells, with global surface proteomic profiling. We assessed the efficiency of parasite invasion into knockdown cells using a panel of wild-type P. falciparum laboratory strains and invasion ligand knockout lines, as well as P. falciparum Senegalese clinical isolates and a short-term-culture-adapted strain. For this, we optimized an invasion assay suitable for use with small numbers of erythrocytes. We found that all laboratory strains and the majority of field strains tested were dependent on GPB expression level for invasion. The collective data suggest that the GPA and GPB receptors are of greater importance than the GPC receptor, supporting a hierarchy of erythrocyte receptor usage in P. falciparum

Coordinated regulation of pathogenic phenotypes and host response by the Vibrio cholerae type three secretion system

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2014.Vibrio cholerae strain AM-19226, which causes sporadic cholera-like disease, encodes a horizontally-acquired type three secretion system (T3SS) as its major virulence mechanism. Disease mediated by strain AM-19226 in animal models requires a functional T3SS, which translocates bacterial effectors directly into the host cell cytoplasm. Putative open reading frames within the AM-19226 T3SS genomic island were screened for their ability to inhibit growth when expressed in Saccharomyces cerevisiae, and were further screened in yeast strains deleted for components of MAPK pathways to potentially identify interacting partners, and also tested for T3SS-dependent translocation into HeLa cells. Eleven translocated proteins with varying levels of translocation were identified. Previous studies showed that AM-19226 T3SS transcriptional regulators, VttRA and VttRB, are required for bile-dependent expression of T3SS structural genes in vitro. To better understand the scope of genes that are potentially regulated by VttRA and VttRB, deep RNA sequencing was performed on wild type AM-19226 and derivatives deleted for vttRA and vttRB grown in bile. Comparative transcriptome analysis revealed genes encoded outside the T3SS genomic island, such as those involved in biofilm formation, motility, and type six secretion as potentially regulated by VttRA/B. In addition, T3SS effectors also appeared to be positively regulated by VttRA/B. Infant mouse competition assays showed that strains individually deleted for effectors, Vops H, A, M, I, and W, exhibited a 100-1000 fold defect in colonization whereas others had ≈ 10-fold defect (VopG) or no defect (VopZ). Because colonization can alter host cell signaling pathways and AM-19226-mediated disease has an inflammatory component, we evaluated the chemokine profile of Caco2-BBE cells during AM-19226 infection. A standard ELISA detected increased levels of IL-8 during Caco2-BBE/AM-19226 co-culture that required a functional T3SS and bile. Collective data suggest that colonization by strain AM-19226 is mediated by 7 translocated proteins, 5 of which are encoded within the T3SS structural gene operons, and VttRA and VttRB are global transcriptional regulators that directly or indirectly coordinate T3SS gene expression with diverse phenotypes such as motility and biofilm formation during different stages of infection

Identification of Vibrio cholerae Type III Secretion System Effector Proteins▿ †

AM-19226 is a pathogenic O39 serogroup Vibrio cholerae strain that lacks the typical virulence factors for colonization (toxin-coregulated pilus [TCP]) and toxin production (cholera toxin [CT]) and instead encodes a type III secretion system (T3SS). The mechanism of pathogenesis is unknown, and few effector proteins have been identified. We therefore undertook a survey of the open reading frames (ORFs) within the ∼49.7-kb T3SS genomic island to identify potential effector proteins. We identified 15 ORFs for their ability to inhibit growth when expressed in yeast and then used a β-lactamase (TEM1) fusion reporter system to demonstrate that 11 proteins were bona fide effectors translocated into HeLa cells in vitro in a T3SS-dependent manner. One effector, which we named VopX (A33_1663), is conserved only in V. cholerae and Vibrio parahaemolyticus T3SS-positive strains and has not been previously studied. A vopX deletion reduces the ability of strain AM-19226 to colonize in vivo, and the bile-induced expression of a vopX-lacZ transcriptional fusion in vitro is regulated by the T3SS-encoded transcriptional regulators VttRA and VttRB. An RLM1 yeast deletion strain rescued the growth inhibition induced by VopX expression, suggesting that VopX interacts with components of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway. The collective results show that the V. cholerae T3SS encodes multiple effector proteins, one of which likely has novel activities that contribute to disease via interference with eukaryotic signaling pathways