HP0018 Has a Potential Role in the Maintenance of the Cell Envelope.

is a bacterial pathogen that colonizes the human stomach, where it can cause a variety of diseases. uses a cluster of sheathed flagella for motility, which is required for host colonization in animal models. The flagellar sheath is continuous with the outer membrane and is found in most species identified to date.

FlgV forms a flagellar motor ring that is required for optimal motility of Helicobacter pylori.

Flagella-driven motility is essential for Helicobacter pylori to colonize the human stomach, where it causes a variety of diseases, including chronic gastritis, peptic ulcer disease, and gastric cancer. H. pylori has evolved a high-torque-generating flagellar motor that possesses several accessories not found in the archetypical Escherichia coli motor.

Control of the flagellation pattern in by FlhF and FlhG.

FlhF and FlhG control the location and number of flagella, respectively, in many polar-flagellated bacteria. The roles of FlhF and FlhG are not well characterized in bacteria that have multiple polar flagella, such as . Deleting in shifted the flagellation pattern where most cells had approximately four flagella to a wider and more even distribution in flagellar number.

Wbm0076, a candidate effector protein of the Wolbachia endosymbiont of Brugia malayi, disrupts eukaryotic actin dynamics.

Brugia malayi, a parasitic roundworm of humans, is colonized by the obligate intracellular bacterium, Wolbachia pipientis. The symbiosis between this nematode and bacterium is essential for nematode reproduction and long-term survival in a human host. Therefore, identifying molecular mechanisms required by Wolbachia to persist in and colonize B.

Legionella pneumophila LegC7 effector protein drives aberrant endoplasmic reticulum:endosome contacts in yeast.

Legionella pneumophila is a facultative intracellular bacterial pathogen, causing the severe form of pneumonia known as Legionnaires' disease. Legionella actively alters host organelle trafficking through the activities of "effector" proteins secreted via a type-IVB secretion system, in order to construct the bacteria-laden Legionella-containing vacuole (LCV) and prevent lysosomal degradation. The LCV is created with membrane derived from host endoplasmic reticulum (ER), secretory vesicles and phagosomes, although the precise molecular mechanisms that drive its synthesis remain poorly understood.

A distinct inhibitory mechanism of the V-ATPase by Vibrio VopQ revealed by cryo-EM.

The Vibrio parahaemolyticus T3SS effector VopQ targets host-cell V-ATPase, resulting in blockage of autophagic flux and neutralization of acidic compartments. Here, we report the cryo-EM structure of VopQ bound to the V subcomplex of the V-ATPase. VopQ inserts into membranes and forms an unconventional pore while binding directly to subunit c of the V-ATPase membrane-embedded subcomplex V.

The Toxoplasma Vacuolar H-ATPase Regulates Intracellular pH and Impacts the Maturation of Essential Secretory Proteins.

Vacuolar-proton ATPases (V-ATPases) are conserved complexes that couple the hydrolysis of ATP to the pumping of protons across membranes. V-ATPases are known to play diverse roles in cellular physiology. We studied the Toxoplasma gondii V-ATPase complex and discovered a dual role of the pump in protecting parasites against ionic stress and in the maturation of secretory proteins in endosomal-like compartments.

5-Diphosphoinositol pentakisphosphate (5-IP) regulates phosphate release from acidocalcisomes and yeast vacuoles.

Acidocalcisomes of and the acidocalcisome-like vacuoles of are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate-sodium symporter (TbPho91 and Pho91p in and yeast, respectively), but the roles of these transporters in growth and orthophosphate (P) transport are unclear. We found here that trypanosomes have a lower growth rate under phosphate starvation and contain larger acidocalcisomes that have increased P content.

Identification of putative effectors of the Type IV secretion system from the Wolbachia endosymbiont of Brugia malayi.

Wolbachia is an unculturable, intracellular bacterium that persists within an extremely broad range of arthropod and parasitic nematode hosts, where it is transmitted maternally to offspring via vertical transmission. In the filarial nematode Brugia malayi, a causative agent of human lymphatic filariasis, Wolbachia is an endosymbiont, and its presence is essential for proper nematode development, survival, and pathogenesis. While the elucidation of Wolbachia:nematode interactions that promote the bacterium's intracellular persistence is of great importance, research has been hampered due to the fact that Wolbachia cannot be cultured in the absence of host cells.

VapA of Rhodococcus equi binds phosphatidic acid.

Rhodococcus equi is a multihost, facultative intracellular bacterial pathogen that primarily causes pneumonia in foals less than six months in age and immunocompromised people. Previous studies determined that the major virulence determinant of R. equi is the surface bound virulence associated protein A (VapA).

The Legionella pneumophila effector protein, LegC7, alters yeast endosomal trafficking.

The intracellular pathogen, Legionella pneumophila, relies on numerous secreted effector proteins to manipulate host endomembrane trafficking events during pathogenesis, thereby preventing fusion of the bacteria-laden phagosome with host endolysosomal compartments, and thus escaping degradation. Upon expression in the surrogate eukaryotic model Saccharomyces cerevisiae, we find that the L. pneumophila LegC7/YlfA effector protein disrupts the delivery of both biosynthetic and endocytic cargo to the yeast vacuole.

Vibrio effector protein VopQ inhibits fusion of V-ATPase-containing membranes.

Vesicle fusion governs many important biological processes, and imbalances in the regulation of membrane fusion can lead to a variety of diseases such as diabetes and neurological disorders. Here we show that the Vibrio parahaemolyticus effector protein VopQ is a potent inhibitor of membrane fusion based on an in vitro yeast vacuole fusion model. Previously, we demonstrated that VopQ binds to the V(o) domain of the conserved V-type H(+)-ATPase (V-ATPase) found on acidic compartments such as the yeast vacuole.

The pore-forming bacterial effector, VopQ, halts autophagic turnover.

Vibrio parahemolyticus Type III effector VopQ is both necessary and sufficient to induce autophagy within one hour of infection. We demonstrated that VopQ interacts with the Vo domain of the conserved vacuolar H(+)-ATPase. Membrane-associated VopQ subsequently forms pores in the membranes of acidic compartments, resulting in immediate release of protons without concomitant release of lumenal protein contents.

Vibrio effector protein, VopQ, forms a lysosomal gated channel that disrupts host ion homeostasis and autophagic flux.

Defects in normal autophagic pathways are implicated in numerous human diseases--such as neurodegenerative diseases, cancer, and cardiomyopathy--highlighting the importance of autophagy and its proper regulation. Herein we show that Vibrio parahaemolyticus uses the type III effector VopQ (Vibrio outer protein Q) to alter autophagic flux by manipulating the partitioning of small molecules and ions in the lysosome. This effector binds to the conserved Vo domain of the vacuolar-type H(+)-ATPase and causes deacidification of the lysosomes within minutes of entering the host cell.

LegC3, an effector protein from Legionella pneumophila, inhibits homotypic yeast vacuole fusion in vivo and in vitro.

During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes).

HOPS proofreads the trans-SNARE complex for yeast vacuole fusion.

The fusion of yeast vacuoles, like other organelles, requires a Rab-family guanosine triphosphatase (Ypt7p), a Rab effector and Sec1/Munc18 (SM) complex termed HOPS (homotypic fusion and vacuole protein sorting), and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The central 0-layer of the four bundled vacuolar SNAREs requires the wild-type three glutaminyl (Q) and one arginyl (R) residues for optimal fusion. Alterations of this layer dramatically increase the K(m) value for SNAREs to assemble trans-SNARE complexes and to fuse.

Excess vacuolar SNAREs drive lysis and Rab bypass fusion.

Although concentrated soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) drive liposome fusion and lysis, the fusion of intracellular membranes also requires Rab GTPases, Rab effectors, SM proteins, and specific regulatory lipids and is accompanied by little or no lysis. To rationalize these findings, we generated yeast strains that overexpress all four vacuolar SNAREs (4SNARE(++)). Although vacuoles with physiological levels of Rab, Rab effector/SM complex, and SNAREs support rapid fusion without Rab- and SNARE-dependent lysis, vacuoles from 4SNARE(++) strains show extensive lysis and a reduced need for the Rab Ypt7p or regulatory lipids for fusion.

Reversible, cooperative reactions of yeast vacuole docking.

Homotypic yeast vacuole fusion occurs in three stages: (i) priming reactions, which are independent of vacuole clustering, (ii) docking, in which vacuoles cluster and accumulate fusion proteins and fusion regulatory lipids at a ring-shaped microdomain surrounding the apposed membranes of each docked vacuole, where fusion will occur, and (iii) bilayer fusion/compartment mixing. These stages require vacuolar SNAREs, SNARE-chaperones, GTPases, effector complexes, and chemically minor but functionally important lipids. For each, we have developed specific ligands that block fusion and conditions that reverse each block.

Acetate excretion during growth of Salmonella enterica on ethanolamine requires phosphotransacetylase (EutD) activity, and acetate recapture requires acetyl-CoA synthetase (Acs) and phosphotransacetylase (Pta) activities.

This report shows that Salmonella enterica catabolizes ethanolamine to acetyl-CoA (Ac-CoA), which enters the glyoxylate bypass and tricarboxylic acid cycle for the generation of energy and central metabolites. During growth on ethanolamine, S. enterica excreted acetate, whose recapture depended on Ac-CoA synthetase (Acs) and the housekeeping phosphotransacetylase (Pta) enzyme activities.

Residue Leu-641 of Acetyl-CoA synthetase is critical for the acetylation of residue Lys-609 by the Protein acetyltransferase enzyme of Salmonella enterica.

Posttranslational regulation of protein function by acetylation is present throughout nature. Regulation of protein function by Sir2 protein (sirtuin) deacetylases is conserved in all domains of life. In the prokaryote Salmonella enterica, the metabolic enzyme acetyl-coenzyme A synthetase (Acs) is regulated by a Sir2-dependent protein acetylation/deacetylation system (SDPADS).

Ion regulation of homotypic vacuole fusion in Saccharomyces cerevisiae.

Biological membrane fusion employs divalent cations as protein cofactors or as signaling ligands. For example, Mg2+ is a cofactor for the N-ethylmaleimide-sensitive factor (NSF) ATPase, and the Ca2+ signal from neuronal membrane depolarization is required for synaptotagmin activation. Divalent cations also regulate liposome fusion, but the role of such ion interactions with lipid bilayers in Rab- and soluble NSF attachment protein receptor (SNARE)-dependent biological membrane fusion is less clear.

Acetyl-coenzyme A synthetase (AMP forming).

Acetyl-coenzyme A synthetase (AMP forming; Acs) is an enzyme whose activity is central to the metabolism of prokaryotic and eukaryotic cells. The physiological role of this enzyme is to activate acetate to acetyl-coenzyme A (Ac-CoA). The importance of Acs has been recognized for decades, since it provides the cell the two-carbon metabolite used in many anabolic and energy generation processes.

Identification of the protein acetyltransferase (Pat) enzyme that acetylates acetyl-CoA synthetase in Salmonella enterica.

Post-translational modification of proteins is an efficient way cells use to control the activity of structural proteins, gene expression regulatory proteins, and enzymes. In eukaryotes, the Sir2-dependent system of protein acetylation/deacetylation controls a number of processes that affect cell longevity. Sir2 proteins have NAD(+)-dependent protein deacetylase activity and are found in all forms of life.

A link between transcription and intermediary metabolism: a role for Sir2 in the control of acetyl-coenzyme A synthetase.

The silent information regulator protein (Sir2) and its homologs (collectively known as sirtuins) are NAD+-dependent deacetylase enzymes involved in chromosome stability, gene silencing and cell aging in eukaryotes and archaea. The discovery that sirtuin-dependent protein deacetylation is a NAD+-consuming reaction established a link with the energy generation systems of the cell. This link to metabolism was recently extended to the post-translational control of the activity of short-chain fatty acyl-coenzyme A (adenosine monophosphate-forming) synthetases in bacteria and yeast.

Propionyl coenzyme A is a common intermediate in the 1,2-propanediol and propionate catabolic pathways needed for expression of the prpBCDE operon during growth of Salmonella enterica on 1,2-propanediol.

The studies reported here identify propionyl coenzyme A (propionyl-CoA) as the common intermediate in the 1,2-propanediol and propionate catabolic pathways of Salmonella enterica serovar Typhimurium LT2. Growth on 1,2-propanediol as a carbon and energy source led to the formation and excretion of propionate, whose activation to propionyl-CoA relied on the activities of the propionate kinase (PduW)/phosphotransacetylase (Pta) enzyme system and the CobB sirtuin-controlled acetyl-CoA and propionyl-CoA (Acs, PrpE) synthetases. The different affinities of these systems for propionate ensure sufficient synthesis of propionyl-CoA to support wild-type growth of S.