A lysozyme-like protein in Brucella abortus is involved in the early stages of intracellular replication

Secretion of proteins in Gram-negative bacteria is a high-energy consuming process that requires the translocation across two membranes and a periplasmic space composed of a mesh like layer, the peptidoglycan. To achieve this, bacteria have evolved complex secretion systems that cross these barriers and, in many cases, there are specific peptidoglycanases that degrade the peptidoglycan to allow the proper assembly of the secretion machinery. We describe here the identification and characterization of a muramidase in Brucella abortus that participates in the intracellular multiplication in professional and non-professional phagocytes. We demonstrated that this protein has peptidoglycanase activity, that a strain with a clean deletion of the gene displayed a defect in the early stages of the intracellular multiplication curve and that this is dependent on the lytic activity. While neither the attachment nor the invasion of the strain was affected we demonstrated that it had a defect in excluding the lysosomal marker LAMP-1 but not in acquiring the reticulum endoplasmic marker calnexin, indicating that the gene participates in the early stages of the intracellular trafficking but not in the establishment of the replicative niche. Analysis of the assembly status and functionality of the VirB secretion apparatus indicated that the mutant has affected the proper function of this central virulence factor.Fil: del Giudice, Mariela Giselda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ugalde, Juan Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Czibener, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Cell-to-cell transmission is the main mechanism supporting bovine viral diarrhea virus spread in cell culture

After initiation of an infective cycle, spread of virus infection can occur fundamentally in two 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, or (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 for 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. Here 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 pestiviuses) infection. To demonstrate direct cell-to-cell transmission of BVDV we developed a cell co-culture system that allowed us to prove the 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 co-receptors on the target cell.Fil: Merwaiss, Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Czibener, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

A genomic island in Brucella involved in the adhesion to host cells: identification of a new adhesin and a translocation factor

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.Fil: Lopez, Paula Veronica. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Guaimas, Francisco Fernando. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Czibener, Cecilia. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ugalde, Juan Esteban. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin

PhiA, a peptidoglycan hydrolase inhibitor of brucella involved in the virulence process

The peptidoglycan in Gram-negative bacteria is a dynamic structure in constant remodeling. This dynamism, achieved through synthesis and degradation, is essential because the peptidoglycan is necessary to maintain the structure of the cell but has to have enough plasticity to allow the transport and assembly of macromolecular complexes in the periplasm and outer membrane. In addition, this remodeling has to be coordinated with the division process. Among the multiple mechanisms bacteria have to degrade the peptidoglycan are the lytic transglycosidases, enzymes of the lysozyme family that cleave the glycan chains generating gaps in the mesh structure increasing its permeability. Because these enzymes can act as autolysins, their activity has to be tightly regulated, and one of the mechanisms bacteria have evolved is the synthesis of membrane bound or periplasmic inhibitors. In the present study, we identify a periplasmic lytic transglycosidase inhibitor (PhiA) in Brucella abortus and demonstrate that it inhibits the activity of SagA, a lytic transglycosidase we have previously shown is involved in the assembly of the type IV secretion system. A phiA deletion mutant results in a strain with the incapacity to synthesize a complete lipopolysaccharide but with a higher replication rate than the wild-type parental strain, suggesting a link between peptidoglycan remodeling and speed of multiplication.Fil: del Giudice, Mariela Giselda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Romani, Alexis Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ugalde, Juan Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Czibener, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

RomA, A Periplasmic Protein Involved in the Synthesis of the Lipopolysaccharide, Tunes Down the Inflammatory Response Triggered by Brucella

Brucellaceae are stealthy pathogens with the ability to survive and replicate in the host in the context of a strong immune response. This capacity relies on several virulence factors that are able to modulate the immune system and in their structural components that have low proinflammatory activities. Lipopolysaccharide (LPS), the main component of the outer membrane, is a central virulence factor of Brucella, and it has been well established that it induces a low inflammatory response. We describe here the identification and characterization of a novel periplasmic protein (RomA) conserved in alpha-proteobacteria, which is involved in the homeostasis of the outer membrane. A mutant in this gene showed several phenotypes, such as membrane defects, altered LPS composition, reduced adhesion, and increased virulence and inflammation. We show that RomA is involved in the synthesis of LPS, probably coordinating part of the biosynthetic complex in the periplasm. Its absence alters the normal synthesis of this macromolecule and affects the homeostasis of the outer membrane, resulting in a strain with a hyperinflammatory phenotype. Our results suggest that the proper synthesis of LPS is central to maximize virulence and minimize inflammation.Fil: Valguarnera, Pablo Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Spera, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Czibener, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Fulgenzi, Fabiana Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Casabuono, Adriana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Altabe, Silvia Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Pasquevich, Karina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Guaimas, Francisco Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Cassataro, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Couto, Alicia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Ugalde, Juan Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Ca2+ and synaptotagmin VII–dependent delivery of lysosomal membrane to nascent phagosomes

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

Development of COVID-19 monoclonal antibodies and recombinant proteins as reagents for biomedical research and diagnostic test

Since SARS-COV-2 virus spread worldwide and COVID-19 turned rapidly into a pandemic illness, the necessity for vaccines and diagnostic tests became crucial. The viral surface is decorated with Spike, the major antigenic determinant and main target for vaccine development. Within Spike, the receptor binding domain (RBD), constitutes the main target of highly neutralizing antibodies found in COVID-19 convalescent plasma. Besides vaccination, another important aspect of Spike (and RBD) is their use as immunogen for the development of poli- and monoclonal antibodies (mAbs) for therapeutic and diagnostic purposes. Here we report the development and preliminary biochemical characterization of a set of monoclonal antibodies against the Spike RBD domain along with the recombinant expression of two mayor COVID-19 protein reagents: the viral Spike RBD domain and the extracellular domain of the human receptor ACE2. RBD and the extracellular domain of ACE2 (aa 1-740) were obtained through transient gene transfection (TGE) in two different mammalian cell culture systems: HEK293T adherent monolayers and Expi293 suspension cultures. Due to its low cost and ease scale-up, all transfections were carried with polyethyleneimine (PEI). Expressed proteins were purified from culture supernatants by immobilized metal affinity chromatography. Anti-RBD mAbs were developed from two different immunization schemes: one aimed to elicit antibodies with viral neutralizing potential, and the other with the ability to recognize denatured RBD for routinary lab immunoassays. To achieve this, the first group of mice was immunized with RBD in aluminium salts (RBD/Al) and the other with RBD emulsified in Freunds adyuvant (RBD/FA). Polyclonal and monoclonal antibody reactivities against native or denatured RBD forms were then assessed by ELISA. Complete RBD denaturation was followed by intrinsic fluorescence spectral changes upon different physicochemical stress treatments. As expected, RBD/Al immunized mice developed an antibody response shifted to native RBD while those immunized with RBD/FA showed a high response against both forms of the protein. In accordance with the observed polyclonal response, RBD/FA derived mAbs recognize both, native and denatured RBD. On the contrary, hybridomas generated from the RBD/Al protocol mostly recognize RBD in its native state. Further ELISA binding assays revealed that all RBD/FA derived mAbs can form a trimeric complex with ACE2 and RBD, denoting they would not have viral neutralizing activity. ELISA competition assays with the RBD/ACE2 complex aimed to determine the neutralization potential of the RBD/Al derived mAbs are under way. Overall, the anti-Spike RBD mAbs and the recombinant RBD and ACE2 proteins presented here constitute valuable tools for diverse COVID-19 academic research projects and local immunity surveillance testing.Fil: Acuña Intrieri, M. E. Universidad Nacional de San Martin. Centro de Rediseño E Ingenieria de Proteinas.; ArgentinaFil: Deriane, M.A. Universidad Nacional de San Martin. Centro de Rediseño E Ingenieria de Proteinas.; ArgentinaFil: Miller, C.. Universidad Nacional de San Martin. Centro de Rediseño E Ingenieria de Proteinas.; ArgentinaFil: Czibener, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Correa, E.. No especifíca;Fil: Cragnaz, L.. No especifíca;Fil: Guerra, L.. No especifíca;Fil: Rodriguez, S.. No especifíca;Fil: Goldbaum, F.A.. Universidad Nacional de San Martin. Centro de Rediseño E Ingenieria de Proteinas.; ArgentinaFil: Seigelchifer, M.. No especifíca;Fil: Comerci, Diego José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Montagna, Georgina Nuri. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Cerutti, Maria Laura. Universidad Nacional de San Martin. Centro de Rediseño E Ingenieria de Proteinas.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaLVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research y XVI Annual Meeting of the Argentinean Society for General MicrobiologyVirtualArgentinaSociedad Argentina de Investigación Bioquímica y Biología MolecularAsociación Civil de Microbiología Genera

Palmitoylation-dependent association with CD63 targets the Ca2+ sensor synaptotagmin VII to lysosomes

Posttranslational lipid modifications promote association of Syt VII with the tetraspanin CD63, determining its exit from the Golgi and targeting to lysosomes

In vivo functional analysis of the Dicistroviridae intergenic region internal ribosome entry sites

Some viral and cellular messages use an alternative mechanism to initiate protein synthesis that involves internal recruitment of the ribosome to an internal ribosome entry site (IRES). The Dicistroviridae intergenic regions (IGR) have been studied as model IRESs to understand the mechanism of IRES-mediated translation. In this study, the in vivo activity of IGR IRESs were compared. Our analysis demonstrates that Class I and II IGR IRESs have comparable translation efficiency in yeast and that Class II is significantly more active in mammalian cells. Furthermore, while Class II IGR IRES activity was enhanced in yeast grown at a higher temperature, temperature did not affect IGR IRES activity in mammalian cells. This suggests that Class II IRESs may not function optimally with yeast ribosomes. Examination of chimeric IGR IRESs, established that the IRES strength and temperature sensitivity are mediated by the ribosome binding domain. In addition, the sequence of the first translated codon is also an important determinant of IRES activity. Our findings provide us with a comprehensive overview of IGR IRES activities and allow us to begin to understand the differences between Classes I and II IGR IRESs