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.

Aluminum hydroxide adjuvant diverts the uptake and trafficking of genetically detoxified pertussis toxin to lysosomes in macrophages.

Aluminum salts have been successfully utilized as adjuvants to enhance the immunogenicity of vaccine antigens since the 1930s. However, the cellular mechanisms behind the immune adjuvanticity effect of these materials in antigen-presenting cells are poorly understood. In this study, we investigated the uptake and trafficking of aluminum oxy-hydroxide (AlOOH), in RAW 264.

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.

A Novel Single Domain Antibody Targeting FliC Flagellin of for Effective Inhibition of Host Cell Invasion.

The enteric pathogen, is a major cause of human gastroenteritis globally and with increasing bacterial resistance to antibiotics, alternative solutions are urgently needed. Single domain antibodies (sdAbs), the smallest antibody fragments that retain antigen binding specificity and affinity, are derived from variable heavy-chain only fragments (VHH) of camelid heavy-chain-only immunoglobulins. SdAbs typically contain a single disulfide bond simplifying recombinant protein production in microbial systems.

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.

Biosensors for the Detection of Interaction between Collagen-Like Protein and Glycosaminoglycans.

The adhesin Legionella collagen-like (Lcl) protein can bind to extracellular matrix components and mediate the binding of to host cells. In this study, electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR)-based biosensors were employed to characterize these interactions between glycosaminoglycans (GAGs) and the adhesin Lcl protein. Fucoidan displayed a high affinity (K 18 nM) for Lcl protein.

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.

PIKfyve inhibition interferes with phagosome and endosome maturation in macrophages.

Macrophages eliminate pathogens and cell debris through phagocytosis, a process by which particulate matter is engulfed and sequestered into a phagosome. Nascent phagosomes are innocuous organelles resembling the plasma membrane. However, through a maturation process, phagosomes are quickly remodeled by fusion with endosomes and lysosomes to form the phagolysosome.

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.

Comparative Genomics Reveal That Host-Innate Immune Responses Influence the Clinical Prevalence of Legionella pneumophila Serogroups.

Legionella pneumophila is the primary etiologic agent of legionellosis, a potentially fatal respiratory illness. Amongst the sixteen described L. pneumophila serogroups, a majority of the clinical infections diagnosed using standard methods are serogroup 1 (Sg1).

Photonic crystals on copolymer film for bacteria detection.

The development of two-dimensional photonic crystals (PCs) on a copolymer film is described in connection with Fresnel reflection spectroscopy and fluorescence microscopy. Label-free detection of Legionella pneumophila was performed using a PC platform with a detection limit of 200 cells/mL. L.