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. In an effort to characterize the in vivo activity of the LegC7/YlfA SNARE-like effector protein from Legionella in the context of eukaryotic membrane trafficking in yeast, we find that LegC7 interacts with the Emp46p/Emp47p ER-to-Golgi glycoprotein cargo adapter complex, alters ER morphology and induces aberrant ER:endosome interactions, as measured by visualization of ER cargo degradation, reconstitution of split-GFP proteins and enhanced oxidation of the ER lumen. LegC7-dependent toxicity, disruption of ER morphology and ER:endosome fusion events were dependent upon endosomal VPS class C tethering complexes and the endosomal t-SNARE, Pep12p. This work establishes a model in which LegC7 functions to recruit host ER material to the bacterial phagosome during infection by driving ER:endosome contacts, potentially through interaction with host membrane tethering complexes and/or cargo adapters.

Clinical, histopathologic, and immunohistochemical features of 13 cases of canine gallbladder neuroendocrine carcinoma.

In this retrospective descriptive study, we characterized the clinical, histologic, and immunohistochemical features of 13 cases of canine gallbladder neuroendocrine carcinoma (GB-NEC). Immunohistochemical stains for neuroendocrine (neuron-specific enolase [NSE], chromogranin A, synaptophysin) and gastrin markers were evaluated, and clinicopathologic and follow-up data were obtained for all cases. The average age at diagnosis was 8.9 y, and breeds included 6 Boston Terriers, 2 Bichon Frise, 1 Poodle, 1 English Bulldog, 1 French Bulldog, and 2 mixed-breed dogs. Boston Terriers were overrepresented in this cohort, and therefore a breed predilection is possible. Most dogs were presented with emesis and elevated liver enzyme activities: 13 of 13 had elevated alanine aminotransferase and alkaline phosphatase activities; 8 of 13 had elevated aspartate aminotransferase activity; 7 of 13 had elevated gamma-glutamyl transferase activity. Abdominal ultrasound and/or exploratory surgery revealed a gallbladder mass. All neoplasms had similar histologic features and positive immunoreactivity for NSE, chromogranin A, synaptophysin, and gastrin. Vascular invasion was noted in 8 of 13 neoplasms, and metastasis was present in 6 of 13 cases (4 hepatic and 2 pulmonary metastases). The median survival time was 3.7 y in patients who died; 5 of 8 deaths were directly attributed to the GB-NEC, 3 of which had metastatic spread. GB-NECs have the potential to metastasize; however, surgical excision may be curative in a subset of dogs.

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. We demonstrate that the effects of LegC7 are specific to the endosome:vacuole delivery pathways; LegC7 expression does not disrupt other known vacuole-directed pathways. Deletions of the ESCRT-0 complex member, VPS27, provide resistance to the LegC7 toxicity, providing a possible target for LegC7 function in vivo. Furthermore, a single amino acid substitution in LegC7 abrogates both its toxicity and ability to alter endosomal traffic in vivo, thereby identifying a critical functional domain. LegC7 likely inhibits endosomal trafficking during L. pneumophila pathogenesis to prevent entry of the phagosome into the endosomal maturation pathway and eventual fusion with the lysosome.

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. VopQ forms a nonspecific, voltage-gated membrane channel of 18 Å resulting in neutralization of these compartments. We now present data showing that VopQ inhibits yeast vacuole fusion. Furthermore, we identified a unique mutation in VopQ that delineates its two functions, deacidification and inhibition of membrane fusion. The use of VopQ as a membrane fusion inhibitor in this manner now provides convincing evidence that vacuole fusion occurs independently of luminal acidification in vitro.

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). This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis.

Mechanism of protein splicing of the Pyrococcus abyssi lon protease intein.

Protein splicing is a post-translational process by which an intervening polypeptide, the intein, excises itself from the flanking polypeptides, the exteins, coupled to ligation of the exteins. The lon protease of Pyrococcus abyssi (Pab) is interrupted by an intein. When over-expressed as a fusion protein in Escherichia coli, the Pab lon protease intein can promote efficient protein splicing. Mutations that block individual steps of splicing generally do not lead to unproductive side reactions, suggesting that the intein tightly coordinates the splicing process. The intein can splice, although it has Lys in place of the highly conserved penultimate His, and mutants of the intein in the C-terminal region lead to the accumulation of stable branched-ester intermediate.