Structure of the aminoterminal domain of the birnaviral multifunctional VP3 protein and its unexplored critical role.

To overcome their limited genetic capacity, numerous viruses encode multifunctional proteins. The birnavirus VP3 protein plays key roles during infection, including scaffolding of the viral capsid during morphogenesis, recruitment, and regulation of the viral RNA polymerase, shielding of the double-stranded RNA genome and targeting of host endosomes for genome replication, and immune evasion. The dimeric form of VP3 is critical for these functions.

Microscopy-Based Tracking and Quantification Methods to Study Phagosome Resolution.

Phagosome resolution is a newly defined, terminal stage in the process of phagocytosis. During this phase, phagolysosomes are fragmented into smaller vesicles, which we called phagosome-derived vesicles (PDVs). PDVs gradually accumulate within macrophages, while the phagosomes diminish in size until the organelles are no longer detectable.

Filamentous Bacteria as Targets to Study Phagocytosis.

Filamentous targets are internalized via phagocytic cups that last for several minutes before closing to form a phagosome. This characteristic offers the possibility to study key events in phagocytosis with greater spatial and temporal resolution than is possible to achieve using spherical particles, for which the transition from a phagocytic cup to an enclosed phagosome occurs within a few seconds after particle attachment. In this chapter, we provide methodologies to prepare filamentous bacteria and describe how they can be used as targets to study different aspects of phagocytosis.

Infection of Human Macrophages Retains Golgi Structure but Reduces O-Glycans.

is an accidental pathogen that replicates intracellularly within the -containing vacuole (LCV) in macrophages. Within an hour of infection, secretes effectors to manipulate Rab1 and intercept ER-derived vesicles to the LCV. The downstream consequences of interrupted ER trafficking on the Golgi of macrophages are not clear.

Rab1b-GBF1-ARF1 Secretory Pathway Axis Is Required for Birnavirus Replication.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide.

Aggregation and Size Attributes Analysis of Unadsorbed and Adjuvant-adsorbed Antigens using a Multispectral Imaging Flow Cytometer Platform.

Various vaccine quality attributes should be monitored to ensure consistency, potency, purity, and safety of vaccine products prior to lot release. Vaccine particle size and protein antigen aggregation are two important considerations for particle-adsorbed vaccines. In this study, we evaluated the use of imaging flow cytometry as a potential all-in-one platform to measure adjuvant particle size and to detect protein aggregates through a combination of brightfield microscopy, side scatter detection, and fluorescence microscopy.

Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes.

Phagocytes engulf unwanted particles into phagosomes that then fuse with lysosomes to degrade the enclosed particles. Ultimately, phagosomes must be recycled to help recover membrane resources that were consumed during phagocytosis and phagosome maturation, a process referred to as "phagosome resolution." Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission.

Phosphatidylinositol 3-Phosphate Mediates the Establishment of Infectious Bursal Disease Virus Replication Complexes in Association with Early Endosomes.

nfectious ursal isease irus (IBDV) is the archetypal member of the family and the etiological agent of Gumboro disease, a highly contagious immunosuppressive infection of concern to the global poultry sector for its adverse health effects in chicks. Unlike most ouble-tranded RNA (dsRNA) viruses, which enclose their genomes within specialized cores throughout their viral replication cycle, birnaviruses organize their bisegmented dsRNA genome in iboucleorotein (RNP) structures. Recently, we demonstrated that IBDV exploits endosomal membranes for replication.

Polymorphisms of a Collagen-Like Adhesin Contributes to Adhesion, Biofilm Formation Capacity and Clinical Prevalence.

Legionellosis is a severe respiratory illness caused by the inhalation of aerosolized water droplets contaminated with the opportunistic pathogen . The ability of to produce biofilms has been associated with its capacity to colonize and persist in human-made water reservoirs and distribution systems, which are the source of legionellosis outbreaks. Nevertheless, the factors that mediate biofilm formation are largely unknown.

Pseudobutyrivibrio xylanivorans adhesion to epithelial cells.

The ruminal bacteria Pseudobutyrivibrio xylanivorans strain 2 (P. xylanivorans 2), that mediates the digestion of plant fiber, is considered an attractive candidate for probiotics. Adherence to the epithelium of the digestive tract of the host is one of the major requirements for probiotics.

Metabolic control of cytosolic-facing pools of diacylglycerol in budding yeast.

Diacylglycerol (DAG) is a key signaling lipid and intermediate in lipid metabolism. Our knowledge of DAG distribution and dynamics in cell membranes is limited. Using live-cell fluorescence microscopy we investigated the localization of yeast cytosolic-facing pools of DAG in response to conditions where lipid homeostasis and DAG levels were known to be altered.

Phagocytosis: what's on the menu? .

Phagocytosis is an evolutionarily conserved process. In Protozoa, phagocytosis fulfills a feeding mechanism, while in Metazoa, phagocytosis diversified to play multiple organismal roles, including immune defence, tissue homeostasis, and remodeling. Accordingly, phagocytes display a high level of plasticity in their capacity to recognize, engulf, and process targets that differ in composition and morphology.

Small Rho GTPases and the Effector VipA Mediate the Invasion of Epithelial Cells by Filamentous .

(Lp) exhibits different morphologies with varying degrees of virulence. Despite their detection in environmental sources of outbreaks and in respiratory tract secretions and lung autopsies from patients, the filamentous morphotype of Lp remains poorly studied. We previously demonstrated that filamentous Lp invades lung epithelial cells (LECs) and replicates intracellularly in a containing vacuole.

Infectious Bursal Disease Virus Hijacks Endosomal Membranes as the Scaffolding Structure for Viral Replication.

Birnaviruses are unconventional members of the group of double-stranded RNA (dsRNA) viruses that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the virally encoded RNA-dependent RNA polymerase (RdRp). This and other structural features suggest that birnaviruses may follow a completely different replication program from that followed by members of the family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses.

pH of endophagosomes controls association of their membranes with Vps34 and PtdIns(3)P levels.

Phagocytosis of filamentous bacteria occurs through tubular phagocytic cups (tPCs) and takes many minutes to engulf these filaments into phagosomes. Contravening the canonical phagocytic pathway, tPCs mature by fusing with endosomes. Using this model, we observed the sequential recruitment of early and late endolysosomal markers to the elongating tPCs.

Filamentous Bacteria as Targets to Study Phagocytosis.

Filamentous targets are internalized via phagocytic cups that last for several minutes before closing to form a phagosome. This characteristic offers the possibility to study key events in phagocytosis with greater spatial and temporal resolution than is possible to achieve using spherical particles, for which the transition from a phagocytic cup to an enclosed phagosome occurs within a few seconds after particle attachment. In this chapter, we provide methodologies to prepare filamentous bacteria and describe how they can be used as targets to study different aspects of phagocytosis.

Legionella pneumophila: homeward bound away from the phagosome.

The intracellular pathogen Legionella pneumophila (Lp) survives and replicates inside a specialized vacuolar compartment that evades canonical phagosomal maturation. Through the action of a large number of effectors translocated into the host cytosol via the Dot/Icm type IV secretion system, Lp subverts host cell pathways to convert its nascent phagosome into an ER-derived compartment, the Legionella containing vacuole (LCV), which serves as bacterial replication niche.

Filamentous morphology of bacteria delays the timing of phagosome morphogenesis in macrophages.

Although filamentous morphology in bacteria has been associated with resistance to phagocytosis, our understanding of the cellular mechanisms behind this process is limited. To investigate this, we followed the phagocytosis of both viable and dead Legionella pneumophila filaments. The engulfment of these targets occurred gradually and along the longitudinal axis of the filament, therefore defining a long-lasting phagocytic cup stage that determined the outcome of phagocytosis.

The Legionella pneumophila collagen-like protein mediates sedimentation, autoaggregation, and pathogen-phagocyte interactions.

Although only partially understood, multicellular behavior is relatively common in bacterial pathogens. Bacterial aggregates can resist various host defenses and colonize their environment more efficiently than planktonic cells. For the waterborne pathogen Legionella pneumophila, little is known about the roles of autoaggregation or the parameters which allow cell-cell interactions to occur.

Chlamydia trachomatis vacuole maturation in infected macrophages.

Chlamydia trachomatis is an obligate intracellular bacterium responsible for one of the most common sexually transmitted diseases. In epithelial cells, C. trachomatis resides in a modified membrane-bound vacuole known as an inclusion, which is isolated from the endocytic pathway.

Mechanism of invasion of lung epithelial cells by filamentous Legionella pneumophila.

Legionella, the aetiological agent responsible for Legionellosis, is an opportunistic pathogen that infects humans upon the inhalation of contaminated aerosolized water droplets. Legionella is pleomorphic and its different morphotypes exhibit varying degrees of virulence. While the filamentous forms of Legionella pneumophila (Lp) have been reported in patient samples since the first description of legionellosis, their role in disease has not been studied.

Vacuolating cytotoxin and variants in Atg16L1 that disrupt autophagy promote Helicobacter pylori infection in humans.

Background & Aims: The Helicobacter pylori toxin vacuolating cytotoxin (VacA) promotes gastric colonization, and its presence (VacA(+)) is associated with more-severe disease. The exact mechanisms by which VacA contributes to infection are unclear. We previously found that limited exposure to VacA induces autophagy of gastric cells, which eliminates the toxin; we investigated whether autophagy serves as a defense mechanism against H pylori infection.

Effect of Helicobacter pylori's vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells.

Host cell responses to Helicobacter pylori infection are complex and incompletely understood. Here, we report that autophagy is induced within human-derived gastric epithelial cells (AGS) in response to H. pylori infection.

SopB promotes phosphatidylinositol 3-phosphate formation on Salmonella vacuoles by recruiting Rab5 and Vps34.

Salmonella colonizes a vacuolar niche in host cells during infection. Maturation of the Salmonella-containing vacuole (SCV) involves the formation of phosphatidylinositol 3-phosphate (PI(3)P) on its outer leaflet. SopB, a bacterial virulence factor with phosphoinositide phosphatase activity, was proposed to generate PI(3)P by dephosphorylating PI(3,4)P2, PI(3,5)P2, and PI(3,4,5)P3.

Autophagy: healthy eating and self-digestion for gastroenterologists.

Autophagy is an evolutionary conserved mechanism in eukaryotic cells that is known to process redundant or defective cellular proteins and organelles. The recent renewal of interest in autophagy research has led to a significant expansion in our understanding of the importance of autophagy in cellular health and disease. This invited review summarizes key elements of autophagy research, emphasizes those of particular interest to gastroenterologists, and offers insights into present and future research directions.