Cyclic β-glucans at the bacteria–host cells interphase: One sugar ring to rule them all

Cyclic β‐1,2‐D‐glucans (CβG) are natural bionanopolymers present in the periplasmic space of many Proteobacteria. These molecules are sugar rings made of 17 to 25 D‐glucose units linked exclusively by β‐1,2‐glycosidic bonds. CβG are important for environmental sensing and osmoadaptation in bacteria, but most importantly, they play key roles in complex host–cell interactions such as symbiosis, pathogenesis, and immunomodulation. In the last years, the identification and characterisation of the enzymes involved in the synthesis of CβG allowed to know in detail the steps necessary for the formation of these sugar rings. Due to its peculiar structure, CβG can complex large hydrophobic molecules, a feature possibly related to its function in the interaction with the host. The capabilities of the CβG to function as molecular boxes and to solubilise hydrophobic compounds are attractive for application in the development of drugs, in food industry, nanotechnology, and chemistry. More importantly, its excellent immunomodulatory properties led to the proposal of CβG as a new class of adjuvants for vaccine development.Fil: Guidolin, Leticia Soledad. 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: Arce Gorvel, Vilma. Centre National de la Recherche Scientifique; FranciaFil: Ciocchini, Andres Eduardo. 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: 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; Argentina. Comisión Nacional de Energía Atómica. Centro Atómico Ezeiza; ArgentinaFil: Gorvel, Jean-Pierre. Centre National de la Recherche Scientifique; Franci

COX-2 inhibition reduces Brucella bacterial burden in draining lymph nodes

International audienceBrucella is a Gram-negative facultative intracellular bacterium responsible for a chronic disease known as brucellosis, the most widespread re-emerging zoonosis worldwide. Establishment of a Th1-mediated immune response characterized by the production of IL-12 and IFN gamma is essential to control the disease. Leukotrienes derived from arachidonic acid (AA) metabolism are known to negatively regulate a protective Th1 immune response against bacterial infections. Here, using genomics approaches we demonstrate that Brucella abortus strongly stimulates the prostaglandin (PG) pathway in dendritic cells (DC). We also show an induction of AA production by infected cells. This correlates with the expression of Ptgs2, a gene encoding the downstream cyclooxygenase-2 (COX-2) enzyme in infected DC. By comparing different infection routes (oral, intradermal, intranasal and conjunctival), we identified the intradermal inoculation route as the more potent in inducing Ptgs2 expression but also in inducing a local inflammatory response in the draining cervical lymph nodes (CLN). NS-398, a specific inhibitor of COX-2 enzymatic activity decreased B. melitensis burden in the CLN after intradermal infection. This effect was accompanied by a decrease of II10 and a concomitant increase of Ifng expression. Altogether, these results suggest that Brucella has evolved to take advantage of the PG pathway in the harsh environment of the CLN in order to persist and subvert immune responses. This work also proposes that novel strategies to control brucellosis may include the use of COX-2 inhibitors

What have we learned from brucellosis in the mouse model?

Brucellosis is a zoonosis caused by Brucella species. Brucellosis research in natural hosts is often precluded by practical, economical and ethical reasons and mice are widely used. However, mice are not natural Brucella hosts and the course of murine brucellosis depends on bacterial strain virulence, dose and inoculation route as well as breed, genetic background, age, sex and physiological statu of mice. Therefore, meaningful experiments require a definition of these variables. Brucella spleen replication profiles are highly reproducible and course in four phases: i), onset or spleen colonization (first 48 h); ii), acute phase, from the third day to the time when bacteria reach maximal numbers; iii), chronic steady phase, where bacterial numbers plateaus; and iv), chronic declining phase, during which brucellae are eliminated. This pattern displays clear physiopathological signs and is sensitive to small virulence variations, making possible to assess attenuation when fully virulent bacteria are used as controls. Similarly, immunity studies using mice with known defects are possible. Mutations affecting INF-γ, TLR9, Myd88, Tγδ and TNF-β favor Brucella replication; whereas IL-1β, IL-18, TLR4, TLR5, TLR2, NOD1, NOD2, GM-CSF, IL/17r, Rip2, TRIF, NK or Nramp1 deficiencies have no noticeable effects. Splenomegaly development is also useful: it correlates with IFN-γ and IL-12 levels and with Brucella strain virulence. The genetic background is also important: Brucella-resistant mice (C57BL) yield lower splenic bacterial replication and less splenomegaly than susceptible breeds. When inoculum is increased, a saturating dose above which bacterial numbers per organ do not augment, is reached. Unlike many gram-negative bacteria, lethal doses are large (≥ 108 bacteria/mouse) and normally higher than the saturating dose. Persistence is a useful virulence/attenuation index and is used in vaccine (Residual Virulence) quality control. Vaccine candidates are also often tested in mice by determining splenic Brucella numbers after challenging with appropriate virulent brucellae doses at precise post-vaccination times. Since most live or killed Brucella vaccines provide some protection in mice, controls immunized with reference vaccines (S19 or Rev1) are critical. Finally, mice have been successfully used to evaluate brucellosis therapies. It is concluded that, when used properly, the mouse is a valuable brucellosis model

The Peyer's patch mononuclear phagocyte system at steady state and during infection

The gut represents a potential entry site for a wide range of pathogens including protozoa, bacteria, viruses, or fungi. Consequently, it is protected by one of the largest and most diversified population of immune cells of the body. Its surveillance requires the constant sampling of its encounters by dedicated sentinels composed of follicles and their associated epithelium located in specialized area. In the small intestine, Peyer's patches (PPs) are the most important of these mucosal immune response inductive sites. Through several mechanisms including transcytosis by specialized epithelial cells called M-cells, access to the gut lumen is facilitated in PPs. Although antigen sampling is critical to the initiation of the mucosal immune response, pathogens have evolved strategies to take advantage of this permissive gateway to enter the host and disseminate. It is, therefore, critical to decipher the mechanisms that underlie both host defense and pathogen subversive strategies in order to develop new mucosal-based therapeutic approaches. Whereas penetration of pathogens through M cells has been well described, their fate once they have reached the subepithelial dome (SED) remains less well understood. Nevertheless, it is clear that the mononuclear phagocyte system (MPS) plays a critical role in handling these pathogens. MPS members, including both dendritic cells and macrophages, are indeed strongly enriched in the SED, interact with M cells, and are necessary for antigen presentation to immune effector cells. This review focuses on recent advances, which have allowed distinguishing the different PP mononuclear phagocyte subsets. It gives an overview of their diversity, specificity, location, and functions. Interaction of PP phagocytes with the microbiota and the follicle- associated epithelium as well as PP infection studies are described in the light of these new criteria of PP phagocyte identification. Finally, known alterations affecting the different phagocyte subsets during PP stimulation or infection are discussed

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections. Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring. In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells. In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated

Escherichia coli α-Hemolysin Counteracts the Anti-Virulence Innate Immune Response Triggered by the Rho GTPase Activating Toxin CNF1 during Bacteremia

International audienceThe detection of the activities of pathogen-encoded virulence factors by the innate immune system has emerged as a new paradigm of pathogen recognition. Much remains to be determined with regard to the molecular and cellular components contributing to this defense mechanism in mammals and importance during infection. Here, we reveal the central role of the IL-1 beta signaling axis and Gr1+ cells in controlling the Escherichia coli burden in the blood in response to the sensing of the Rho GTPase-activating toxin CNF1. Consistently, this innate immune response is abrogated in caspase-1/11-impaired mice or following the treatment of infected mice with an IL-1 beta antagonist. In vitro experiments further revealed the synergistic effects of CNF1 and LPS in promoting the maturation/secretion of IL-1 beta and establishing the roles of Rac, ASC and caspase-1 in this pathway. Furthermore, we found that the Phi-hemolysin toxin inhibits IL-1 beta secretion without affecting the recruitment of Gr1+ cells. Here, we report the first example of anti-virulence-triggered immunity counteracted by a pore-forming toxin during bacteremia

Identification of lptA, lpxE, and lpxO, Three Genes Involved in the Remodeling of Brucella Cell Envelope.

The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by innate immunity because of a reduction of the pathogen-associated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and other envelope molecules. We investigated the Brucella homologs of lptA, lpxE, and lpxO, three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA, which encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides), while B. abortus carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation, a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except B. microti and is intact in O. anthropi. Free-lipid analysis revealed that lpxO corresponded to olsC, the gene coding for the ornithine lipid (OL) acyl hydroxylase active in O. anthropi and B. microti, while B. abortus carrying the olsC of O. anthropi and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE, or olsC were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the presence of the intact homolog genes in O. anthropi and B. microti but not in other brucellae suggests that LptA, LpxE, or OL β-hydroxylase do not significantly alter the PAMP properties of Brucella LPS and free-lipids and are therefore not positively selected during the adaptation to intracellular life

Omp25-dependent engagement of SLAMF1 by Brucella abortus in dendritic cells limits acute inflammation and favours bacterial persistence in vivo

The strategies by which intracellular pathogenic bacteria manipulate innate immunity to establish chronicity are poorly understood. Here, we show that Brucella abortus outer membrane protein Omp25 specifically binds the immune cell receptor SLAMF1 in vitro. The Omp25-dependent engagement of SLAMF1 by B. abortus limits NF-κB translocation in dendritic cells (DCs) with no impact on Brucella intracellular trafficking and replication. This in turn decreases pro-inflammatory cytokine secretion and impairs DC activation. The Omp25-SLAMF1 axis also dampens the immune response without affecting bacterial replication in vivo during the acute phase of Brucella infection in a mouse model. In contrast, at the chronic stage of infection, the Omp25/SLAMF1 engagement is essential for Brucella persistence. Interaction of a specific bacterial protein with an immune cell receptor expressed on the DC surface at the acute stage of infection is thus a powerful mechanism to support microbe settling in its replicative niche and progression to chronicity.Fil: Degos, Clara. Inserm; Francia. Centre National de la Recherche Scientifique; FranciaFil: Hysenaj, Lisiena. Inserm; Francia. Centre National de la Recherche Scientifique; FranciaFil: Gonzalez Espinoza, Gabriela. Inserm; Francia. Centre National de la Recherche Scientifique; FranciaFil: Arce Gorvel, Vilma. Inserm; Francia. Centre National de la Recherche Scientifique; FranciaFil: Gagnaire, Aurélie. Inserm; Francia. Centre National de la Recherche Scientifique; FranciaFil: Papadopoulos, Alexia. Inserm; Francia. Centre National de la Recherche Scientifique; FranciaFil: Pasquevich, Karina Alejandra. 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: Méresse, Stéphane. Inserm; Francia. Centre National de la Recherche Scientifique; FranciaFil: Cassataro, Juliana. 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: Mémet, Sylvie. Inserm; Francia. Centre National de la Recherche Scientifique; FranciaFil: Gorvel, Jean Pierre. Inserm; Francia. Centre National de la Recherche Scientifique; Franci

Transcriptome Analysis of the Brucella abortus BvrR/BvrS Two-Component Regulatory System

International audienceBackgroundThe two-component BvrR/BvrS system is essential for Brucella abortus virulence. It was shown previously that its dysfunction alters the expression of some major outer membrane proteins and the pattern of lipid A acylation. To determine the genes regulated by BvrR/BvrS, we performed a whole-genome microarray analysis using B. abortus RNA obtained from wild type and bvrR mutant cells grown in the same conditions.Methodology/Principal FindingsA total of 127 differentially expressed genes were found: 83 were over expressed and 44 were less expressed in the bvrR mutant. Two operons, the phosphotransferase system and the maltose transport system, were down-regulated. Several genes involved in cell envelope or outer membrane biogenesis were differentially expressed: genes for outer membrane proteins (omp25a, omp25d), lipoproteins, LPS and fatty acid biosynthesis, stress response proteins, chaperones, flagellar genes, and twelve genes encoding ABC transport systems. Ten genes related with carbon metabolism (pckA and fumB among others) were up-regulated in the bvrR mutant, and denitrification genes (nirK, norC and nosZ) were also regulated. Notably, seven transcriptional regulators were affected, including VjbR, ExoR and OmpR that were less expressed in the bvrR mutant. Finally, the expression of eleven genes which have been previously related with Brucella virulence was also altered.Conclusions/SignificanceAll these data corroborate the impact of BvrR/BvrS on cell envelope modulation, confirm that this system controls the carbon and nitrogen metabolism, and suggest a cross-talk among some regulators to adjust the Brucella physiology to the shift expected to occur during the transit from the extracellular to the intracellular niche