ABSTRACT
The intracellular pathogenic bacterium Salmonella enterica serovar typhimurium (Salmonella) relies on acidification of the Salmonella-containing vacuole (SCV) for survival inside host cells. The transport and fusion of membrane-bound compartments in a cell is regulated by small GTPases, including Rac and members of the Rab GTPase family, and their effector proteins. However, the role of these components in survival of intracellular pathogens is not completely understood. Here, we identify Nischarin as a novel dual effector that can interact with members of Rac and Rab GTPase (Rab4, Rab14 and Rab9) families at different endosomal compartments. Nischarin interacts with GTP-bound Rab14 and PI(3)P to direct the maturation of early endosomes to Rab9/CD63-containing late endosomes. Nischarin is recruited to the SCV in a Rab14-dependent manner and enhances acidification of the SCV. Depletion of Nischarin or the Nischarin binding partners--Rac1, Rab14 and Rab9 GTPases--reduced the intracellular growth of Salmonella. Thus, interaction of Nischarin with GTPases may regulate maturation and subsequent acidification of vacuoles produced after phagocytosis of pathogens.
Subject(s)
Endosomes/microbiology , Imidazoline Receptors/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Salmonella typhimurium/growth & development , Vacuoles/microbiology , rab GTP-Binding Proteins/metabolism , rac GTP-Binding Proteins/metabolism , Biological Transport , Blotting, Western , Endosomes/metabolism , Guanosine Triphosphate/metabolism , HeLa Cells , Humans , Imidazoline Receptors/genetics , Immunoprecipitation , Intracellular Signaling Peptides and Proteins/genetics , Lysosomes/metabolism , Lysosomes/microbiology , Phosphatidylinositol Phosphates/metabolism , Protein Transport , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Salmonella Infections/microbiology , Two-Hybrid System Techniques , Vacuoles/metabolism , rab GTP-Binding Proteins/genetics , rac GTP-Binding Proteins/geneticsABSTRACT
Biosynthesis of flavin adenine dinucleotides in most prokaryotes is catalyzed by a family of bifunctional flavin adenine dinucleotide (FAD) synthetases. These enzymes carry out the dual functions of phosphorylation of flavin mononucleotide (FMN) and its subsequent adenylylation to generate FAD. Using various sequence analysis methods, a new domain has been identified in the N-terminal region that is well conserved in all the bacterial FAD synthetases. We also identify remote similarity of this domain to the nucleotidyl transferases and, hence, this domain is suggested to be invloved in the adenylylation reaction of FAD synthetases.
