After initiation of an infective cycle, spread of virus infection can occur in two fundamentally different ways: (i) viral particles can be released into the external environment and diffuse through ...the extracellular space until they interact with a new host cell, and (ii) virions can remain associated with infected cells, promoting the direct passage between infected and uninfected cells that is referred to as direct cell-to-cell transmission. Although evidence of cell-associated transmission has accumulated for many different viruses, the ability of members of the genus Pestivirus to use this mode of transmission has not been reported. In the present study, we used a novel recombinant virus expressing the envelope glycoprotein E2 fused to mCherry fluorescent protein to monitor the spreading of bovine viral diarrhea virus (BVDV) (the type member of the pestiviruses) infection. To demonstrate direct cell-to-cell transmission of BVDV, we developed a cell coculture system that allowed us to prove direct transmission from infected to uninfected cells in the presence of neutralizing antibodies. This mode of transmission requires cell-cell contacts and clathrin-mediated receptor-dependent endocytosis. Notably, it overcomes antibody blocking of the BVDV receptor CD46, indicating that cell-to-cell transmission of the virus involves the engagement of coreceptors on the target cell.
BVDV causes one of the most economically important viral infections for the cattle industry. The virus is able to cross the placenta and infect the fetus, leading to the birth of persistently infected animals, which are reservoirs for the spread of BVDV. The occurrence of persistent infection has hampered the efficacy of vaccination because it requires eliciting levels of protection close to sterilizing immunity to prevent fetal infections. While vaccination prevents disease, BVDV can be detected if animals with neutralizing antibodies are challenged with the virus. Virus cell-to-cell transmission allows the virus to overcome barriers to free virus dissemination, such as antibodies or epithelial barriers. Here we show that BVDV exploits cell-cell contacts to propagate infection in a process that is resistant to antibody neutralization. Our results provide new insights into the mechanisms underlying the pathogenesis of BVDV infection and can aid in the design of effective control strategies.
Host cell egress is a critical step in the life cycle of intracellular pathogens, especially in microbes capable of establishing chronic infections. The Gram-negative bacterium Brucella belongs to ...such a group of pathogens. Even though much has been done to understand how Brucella avoids killing and multiplies in its intracellular niche, the mechanism that this bacterium deploys to egress from the cell to complete its cycle has been poorly studied. In the manuscript, we quantify the kinetics of bacterial egress and show that Brucella exploits multivesicular bodies to exit host cells. For the first time, we visualized the process of egress in real time by live video microscopy and showed that a population of intracellular bacteria exit from host cells in vacuoles containing multivesicular body-like features. We observed the colocalization of Brucella with two multivesicular markers, namely, CD63 and LBPA, both during the final stages of the intracellular life cycle and in egressed bacteria. Moreover, drugs that either promote or inhibit multivesicular bodies either increased or decreased the number of extracellular bacteria, respectively. Our results strongly suggest that Brucella hijacks multivesicular bodies to exit the host cells to initiate new infection events.
How intracellular bacterial pathogens egress from host cells has been poorly studied. This is particularly important because this stage of the infectious cycle can have a strong impact on how the host resolves the infection. Brucella is an intracellular pathogen that infects mammals, including humans, and causes a chronic debilitating illness. The bacterium has evolved a plethora of mechanisms to invade host cells, avoid degradation in the endocytic pathway, and actively multiply within a specialized intracellular compartment. However, how this pathogen exits from infected cells to produce reinfection and complete its life cycle is poorly understood. In the manuscript, we shed some light on the mechanisms that are exploited by Brucella to egress from host cells. We observed for the first time the egress of Brucella from infected cells by time-lapse video microscopy, and we found that the bacterium exits in vesicles containing multivesicular bodies (MVBs) features. Moreover, the drug manipulation of MVBs resulted in the alteration of bacterial egress efficiency. Our results indicate that Brucella hijacks MVBs to exit host cells and that this strongly contributes to the reinfection cycle.
Synaptotagmin (Syt) VII is a ubiquitously expressed member of the Syt family of Ca2+ sensors. It is present on lysosomes in several cell types, where it regulates Ca2+-dependent exocytosis. Because ...Ca2+i and exocytosis have been associated with phagocytosis, we investigated the phagocytic ability of macrophages from Syt VII-/- mice. Syt VII-/- macrophages phagocytose normally at low particle/cell ratios but show a progressive inhibition in particle uptake under high load conditions. Complementation with Syt VII rescues this phenotype, but only when functional Ca2+-binding sites are retained. Reinforcing a role for Syt VII in Ca2+-dependent phagocytosis, particle uptake in Syt VII-/- macrophages is significantly less dependent on Ca2+i. Syt VII is concentrated on peripheral domains of lysosomal compartments, from where it is recruited to nascent phagosomes. Syt VII recruitment is rapidly followed by the delivery of Lamp1 to phagosomes, a process that is inhibited in Syt VII-/- macrophages. Thus, Syt VII regulates the Ca2+-dependent mobilization of lysosomes as a supplemental source of membrane during phagocytosis.
Immune responses directed towards surface polysaccharides conjugated to proteins are effective in preventing colonization and infection of bacterial pathogens. Presently, the production of these ...conjugate vaccines requires intricate synthetic chemistry for obtaining, activating, and attaching the polysaccharides to protein carriers. Glycoproteins generated by engineering bacterial glycosylation machineries have been proposed to be a viable alternative to traditional conjugation methods.
In this work we expressed the C. jejuni oligosaccharyltansferase (OTase) PglB, responsible for N-linked protein glycosylation together with a suitable acceptor protein (AcrA) in Yersinia enterocolitica O9 cells. MS analysis of the acceptor protein demonstrated the transfer of a polymer of N-formylperosamine to AcrA in vivo. Because Y. enterocolitica O9 and Brucella abortus share an identical O polysaccharide structure, we explored the application of the resulting glycoprotein in vaccinology and diagnostics of brucellosis, one of the most common zoonotic diseases with over half a million new cases annually. Injection of the glycoprotein into mice generated an IgG response that recognized the O antigen of Brucella, although this response was not protective against a challenge with a virulent B. abortus strain. The recombinant glycoprotein coated onto magnetic beads was efficient in differentiating between naïve and infected bovine sera.
Bacterial engineered glycoproteins show promising applications for the development on an array of diagnostics and immunoprotective opportunities in the future.
In an attempt to understand the process mediating K+ transport into roots, we examined the contribution of the NH4
+-sensitive and NH4
+-insensitive components of Rb+ transport to the uptake of Rb+ ...in barley (Hordeum vulgare L.) plants grown in different ionic environments. We found that at low external Rb+ concentrations, an NH4
+-sensitive component dominates Rb+ uptake in plants grown in the absence of NH4
+, while Rb+ uptake preferentially occurs through an NH4
+-insensitive pathway in plants grown at high external NH4
+ concentrations. A comparison of the Rb+-uptake properties observed in roots with those found in heterologous studies with yeast cells indicated that the recently cloned HvHAK1 K+ transporter may provide a major route for the NH4
+-sensitive component. HvHAK1 failed to complement the growth of a yeast strain defective in NH4
+ transport, suggesting that it could not act as an NH4
+ transporter. Heterologous studies also showed that the HKT1 $\text{K}^{+}/\text{Na}^{+}$-cotransporter may act as a pathway for high-affinity Rb+ transport sensitive to NH4
+. However, we found no evidence of an enhancement of Rb+ uptake into roots due to Na+ addition. The possible identity of the systems contributing to the NH4
+-insensitive component in barley plants is discussed.
Adhesion to host cells is the first step in the virulence cycle of any pathogen. In Gram‐negative bacteria, adhesion is mediated, among other virulence factors such as the lipopolysaccharides, by ...specific outer‐membrane proteins generally termed adhesins that belong to a wide variety of families and have different evolutionary origins. In Brucella, a widespread zoonotic pathogen of animal and human health concern, adhesion is central as it may determine the intracellular fate of the bacterium, an essential stage in its pathogenesis. In the present paper, we further characterised a genomic locus that we have previously reported encodes an adhesin (BigA) with a bacterial immunoglobulin‐like domain (BIg‐like). We found that this region encodes a second adhesin, which we have named BigB; and PalA, a periplasmic protein necessary for the proper display in the outer membrane of BigA and BigB. Deletion of bigB or palA diminishes the adhesion of the bacterium and overexpression of BigB dramatically increases it. Incubation of cells with the recombinant BIg‐like domain of BigB induced important cytoskeletal rearrangements and affected the focal adhesion sites indicating that the adhesin targets cell–cell or cell–matrix proteins. We additionally show that PalA has a periplasmic localisation and is completely necessary for the proper display of BigA and BigB, probably avoiding their aggregation and facilitating their transport to the outer membrane. Our results indicate that this genomic island is entirely devoted to the adhesion of Brucella to host cells.
Adhesion to host cells is a central process in the initial steps of all infectious diseases. This activity is mediated by a wide range of surface exposed molecules, generally termed adhesins, that recognize host cell receptors and trigger internalization. Brucella, a widespread zoonotic pathogen, harbors a unique pathogenicity island that codes for at least two adhesins (BigA and BigB) that target the bacterium to the cell‐cell junction membrane or the focal adhesion sites. This locus also encodes a protein (PalA) necessary for the periplasmic stabilization and proper assembly of these adhesins in the outer membrane.
Brucellosis remains one of the most worldwide distributed zoonosis inflicting serious economical and human health problems in many areas of the world. The disease is caused by different species of ...the genus Brucella that have different tropisms towards different mammals being the most relevant for human health Brucella abortus, Brucella melitensis and Brucella suis that infect cows, goats/sheep, and swine respectively. For B. melitensis, considered the species with more zoonotic potential and highly aggressive for animals, only one vaccine is available to date in the market: Rev 1. This attenuated strain has the disadvantage that is has a very high residual virulence for animals and humans and, for this reason, it is applied by ocular instillation which is technically challenging in many productive settings. For this reason, the search for new vaccines for caprine and ovine brucellosis is an active topic of research. We describe here the construction of a novel highly attenuated vaccine strain (Bm Delta-pgm) that confers excellent levels of protection against B. melitensis in the mouse model of infection. This strain is a clean deletion of the phosphoglucomutase (pgm) gene that codes for a protein that catalyzes the conversion of glucose-6-P to glucose-1-P, which is used as a precursor for the biosynthesis of many polysaccharides, including the O-antigen of the lipopolysaccharide and cyclic beta glucans. Our results indicate that vaccination with Bm Delta-pgm induces a robust memory cellular immune response but no antibody production against the O-antigen. Cross protection experiments show that this new vaccine protects against B. abortus and B. suis raising the possibility that Bm Delta-pgm could be used as a universal vaccine for the most important Brucella species.
Syt VII is a Ca²⁺ sensor that regulates lysosome exocytosis and plasma membrane repair. Because it lacks motifs that mediate lysosomal targeting, it is unclear how Syt VII traffics to these ...organelles. In this paper, we show that mutations or inhibitors that abolish palmitoylation disrupt Syt VII targeting to lysosomes, causing its retention in the Golgi complex. In macrophages, Syt VII is translocated simultaneously with the lysosomal tetraspanin CD63 from tubular lysosomes to nascent phagosomes in a Ca²⁺-dependent process that facilitates particle uptake. Mutations in Syt VII palmitoylation sites block trafficking of Syt VII, but not CD63, to lysosomes and phagosomes, whereas tyrosine replacement in the lysosomal targeting motif of CD63 causes both proteins to accumulate on the plasma membrane. Complexes of CD63 and Syt VII are detected only when Syt VII palmitoylation sites are intact. These findings identify palmitoylation-dependent association with the tetraspanin CD63 as the mechanism by which Syt VII is targeted to lysosomes.
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