Maria-Teresa Alvarez-Martinez

Major Outer Membrane Protein Omp25 of Brucella suis Is Involved in Inhibition of Tumor Necrosis Factor Alpha Production during Infection of Human Macrophages

ABSTRACT Brucella spp. can establish themselves and cause disease in humans and animals. The mechanisms by whichBrucella spp. evade the antibacterial defenses of their host, however, remain largely unknown. We have previously reported that live brucellae failed to induce tumor necrosis factor alpha (TNF-α) production upon human macrophage infection. This inhibition is associated with a nonidentified protein that is released into culture medium. Outer membrane proteins (OMPs) of gram-negative bacteria have been shown to modulate macrophage functions, including cytokine production. Thus, we have analyzed the effects of two major OMPs (Omp25 and Omp31) of Brucella suis 1330 (wild-type [WT] B. suis) on TNF-α production. For this purpose, omp25and omp31 null mutants of B. suis(Δomp25 B. suis and Δomp31 B. suis, respectively) were constructed and analyzed for the ability to activate human macrophages to secrete TNF-α. We showed that, in contrast to WTB. suis or Δomp31 B. suis, Δomp25 B. suis induced TNF-α production when phagocytosed by human macrophages. The complementation of Δomp25 B. suis with WT omp25 (Δomp25-omp25 B. suis mutant) significantly reversed this effect: Δomp25-omp25 B. suis-infected macrophages secreted significantly less TNF-α than did macrophages infected with the Δomp25 B. suismutant. Furthermore, pretreatment of WT B. suis with an anti-Omp25 monoclonal antibody directed against an epitope exposed at the surface of the bacteria resulted in substancial TNF-α production during macrophage infection. These observations demonstrated that Omp25 of B. suis is involved in the negative regulation of TNF-α production upon infection of human macrophages.

Production of the Type IV Secretion System Differs among Brucella Species as Revealed with VirB5- and VirB8-Specific Antisera

ABSTRACT Expression of the virB operon, encoding the type IV secretion system required for Brucella suis virulence, occurred in the acidic phagocytic vacuoles of macrophages and could be induced in minimal medium at acidic pH values. To analyze the production of VirB proteins, polyclonal antisera against B. suis VirB5 and VirB8 were generated. Western blot analysis revealed that VirB5 and VirB8 were detected after 3 h in acidic minimal medium and that the amounts increased after prolonged incubation. Unlike what occurs in the related organism Agrobacterium tumefaciens, the periplasmic sugar binding protein ChvE did not contribute to VirB protein production, and B. suis from which chvE was deleted was fully virulent in a mouse model. Comparative analyses of various Brucella species revealed that in all of them VirB protein production increased under acidic conditions. However, in rich medium at neutral pH, Brucella canis and B. suis, as well as the Brucella abortus- and Brucella melitensis-derived vaccine strains S19, RB51, and Rev.1, produced no VirB proteins or only small amounts of VirB proteins, whereas the parental B. abortus and B. melitensis strains constitutively produced VirB5 and VirB8. Thus, the vaccine strains were still able to induce virB expression under acidic conditions, but the VirB protein production was markedly different from that in the wild-type strains at pH 7. Taken together, the data indicate that VirB protein production and probably expression of the virB operon are not uniformly regulated in different Brucella species. Since VirB proteins were shown to modulate Brucella phagocytosis and intracellular trafficking, the differential regulation of the production of these proteins reported here may provide a clue to explain their role(s) during the infection process.

Intracellular Survival of Brucella spp. in Human Monocytes Involves Conventional Uptake but Special Phagosomes

ABSTRACT Brucella spp. are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs. We have investigated howB. suis and B. melitensis enter human monocytes and in which compartment they survive. Peripheral blood monocytes readily internalized nonopsonized brucellae and killed most of them within 12 to 18 h. The presence ofBrucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca2+- and Mg2+-free medium reduced the uptake. Engulfment of all Brucella organisms (regardless of bacterial viability or virulence) initially resulted in phagosomes with tightly apposed walls (TP). Most TP were fully fusiogenic and matured to spacious phagolysosomes containing degraded bacteria, whereas some TP (more in monocyte-derived macrophages, HeLa cells, and CHO cells than in monocytes) remained tightly apposed to intact bacteria. Immediate treatment of infected host cells with the lysosomotropic base ammonium chloride caused a swelling of all phagosomes and a rise in the intraphagosomal pH, abolishing the intracellular survival of Brucella. These results indicate that (i) human monocytes readily internalizeBrucella in a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of someBrucella phagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) brucellae are killed in maturing but not in arrested phagosomes, and (iv) survival of internalized Brucella depends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall.

Effects of profilin–annexin I association on some properties of both profilin and annexin I: modification of the inhibitory activity of profilin on actin polymerization and inhibition of the self-association of annexin I and its interactions with liposomes

We have previously shown that annexin I, a member of a family of calcium-dependent phospholipid and membrane binding proteins, interacts with profilin with high specificity and affinity. This finding further suggests that annexin I is involved through profilin in the regulation of membrane-cytoskeleton organization. We have investigated the consequences of a complex formed by these two proteins on the functions of both profilin and annexin I. Annexin I is able to modify the inhibitory effect of profilin on actin polymerization. This action is partial and the mechanism involved appears to be complex. On the other hand, the association between annexin I and profilin is sufficiently strong to inhibit the self-association of annexin I. The binding capacity of annexin I to liposomes containing phosphatidylserine, which mimics annexin I binding to membranes, is also decreased by profilin. This binding is nevertheless restored when phosphatidylinositol 4,5-biphosphate (PtdInsP2) is included in the liposomes. Finally, the capacity of annexin I to aggregate liposomes is also modified. It is worthwhile mentioning that the liposomes-binding and liposomes-aggregating activities of annexin I are independently regulated. The cell localization and functions of annexin I and profilin suggest that interaction between these two proteins may be directly implicated in the regulation of membrane-cytoskeleton. The phospholipid composition of membranes may be one of the modulating factors.

Absence of Evidence for the Participation of the Macrophage Cellular Prion Protein in Infection with Brucella suis

ABSTRACT Brucella spp. are stealthy bacteria that enter host cells without major perturbation. The molecular mechanism involved is still poorly understood, although numerous studies have been published on this subject. Recently, it was reported that Brucella abortus utilizes cellular prion protein (PrPC) to enter the cells and to reach its replicative niche. The molecular mechanisms involved were not clearly defined, prompting us to analyze this process using blocking antibodies against PrPC. However, the behavior of Brucella during cellular infection under these conditions was not modified. In a next step, the behavior of Brucella in macrophages lacking the prion gene and the infection of mice knocked out for the prion gene were studied. We observed no difference from results obtained with the wild-type control. Although some contacts between PrPC and Brucella were observed on the surface of the cells by using confocal microscopy, we could not show that Brucella specifically bound recombinant PrPC. Therefore, we concluded from our results that prion protein (PrPC) was not involved in Brucella infection.

Dynamics of polymerization shed light on the mechanisms that lead to multiple amyloid structures of the prion protein

It is generally accepted that spongiform encephalopathies result from the aggregation into amyloid of a ubiquitous protein, the so-called prion protein. As a consequence, the dynamics of amyloid formation should explain the characteristics of the prion diseases: infectivity as well as sporadic and genetic occurrence, long incubation time, species barriers and strain specificities. The success of this amyloid hypothesis is due to the good qualitative agreement of this hypothesis with the observations. However, a number of difficulties appeared when comparing quantitatively the in vitro experimental results with the theoretical models, suggesting that some differences should hide important discrepancies. We used well defined quantitative models to analyze the experimental results obtained by in vitro polymerization of the recombinant hamster prion protein. Although the dynamics of polymerization resembles a simple nucleus-dependent fibrillogenesis, neither the initial concentration dependence nor off-pathway hypothesis fit with experimental results. Furthermore, seeded polymerization starts after a long time delay suggesting the existence of a specific mechanism that takes place before nucleus formation. On the other hand, polymerization dynamics reveals a highly stochastic mechanism, the origin of which appears to be caused by nucleation heterogeneity. Moreover, the specific structures generated during nucleation are maintained during successive seeding although a clear improvement of the dynamics parameters (polymerization rate and lag time) is observed. We propose that an additional on-pathway reaction takes place before nucleation and it is responsible for the heterogeneity of structures produced during prion protein polymerization in vitro. These amyloid structures behave like prion strains. A model is proposed to explain the genesis of heterogeneity among prion amyloid.

Release of periplasmic proteins of Brucella suis upon acidic shock involves the outer membrane protein Omp25

ABSTRACT The survival and replication of Brucella in macrophages is initially triggered by a low intraphagosomal pH. In order to identify proteins released by Brucella during this early acidification step, we analyzed Brucella suis conditioned medium at various pH levels. No significant proteins were released at pH 4.0 in minimal medium or citrate buffer, whereas in acetate buffer, B. suis released a substantial amount of soluble proteins. Comparison of 13 N-terminal amino acid sequences determined by Edman degradation with their corresponding genomic sequences revealed that all of these proteins possessed a signal peptide indicative of their periplasmic location. Ten proteins are putative substrate binding proteins, including a homologue of the nopaline binding protein of Agrobacterium tumefaciens. The absence of this homologue in Brucella melitensis was due to the deletion of a 7.7-kb DNA fragment in its genome. We also characterized for the first time a hypothetical 9.8-kDa basic protein composed of five amino acid repeats. In B. suis, this protein contained 9 repeats, while 12 were present in the B. melitensis orthologue. B. suis in acetate buffer depended on neither the virB type IV secretory system nor the omp31 gene product. However, the integrity of the omp25 gene was required for release at acidic pH, while the absence of omp25b or omp25c displayed smaller effects. Together, these results suggest that Omp25 is involved in the membrane permeability of Brucella in acidic medium.

The Brucella suis homologue of the Agrobacterium tumefaciens chromosomal virulence operon chvE is essential for sugar utilization but not for survival in macrophages.

Brucella strains possess an operon encoding type IV secretion machinery very similar to that coded by the Agrobacterium tumefaciens virB operon. Here we describe cloning of the Brucella suis homologue of the chvE-gguA-gguB operon of A. tumefaciens and characterize the sugar binding protein ChvE (78% identity), which in A. tumefaciens is involved in virulence gene expression. B. suis chvE is upstream of the putative sugar transporter-encoding genes gguA and gguB, also present in A. tumefaciens, but not adjacent to that of a LysR-type transcription regulator. Although results of Southern hybridization experiments suggested that the gene is present in all Brucella strains, the ChvE protein was detected only in B. suis and Brucella canis with A. tumefaciens ChvE-specific antisera, suggesting that chvE genes are differently expressed in different Brucella species. Analysis of cell growth of B. suis and of its chvE or gguA mutants in different media revealed that ChvE exhibited a sugar specificity similar to that of its A. tumefaciens homologue and that both ChvE and GguA were necessary for utilization of these sugars. Murine or human macrophage infections with B. suis chvE and gguA mutants resulted in multiplication similar to that of the wild-type strain, suggesting that virB expression was unaffected. These data indicate that the ChvE and GguA homologous proteins of B. suis are essential for the utilization of certain sugars but are not necessary for survival and replication inside macrophages.

Low doses of bioherbicide favour prion aggregation and propagation in vivo

Public concerns over the use of synthetic pesticides are growing since many studies have shown their impact on human health. A new environmental movement in occidental countries promoting an organic agriculture favours the rebirth of botanical pesticides. These products confer an effective alternative to chemical pesticides such as glyphosate. Among the biopesticides, the α-terthienyls found in the roots of Tagetes species, are powerful broad-spectrum pesticides. We found that an α-terthienyl analogue with herbicidal properties, called A6, triggers resistant SDS oligomers of the pathogenic prion protein PrPSc (rSDS-PrPSc) in cells. Our main question is to determine if we can induce those rSDS-PrPSc oligomers in vitro and in vivo, and their impact on prion aggregation and propagation. Using wild-type mice challenged with prions, we showed that A6 accelerates or slows down prion disease depending on the concentration used. At 5 mg/kg, A6 is worsening the pathology with a faster accumulation of PrPSc, reminiscent to soluble toxic rSDS-PrPSc oligomers. In contrast, at 10 and 20 mg/kg of A6, prion disease occurred later, with less PrPSc deposits and with rSDS-PrPSc oligomers in the brain reminiscent to non-toxic aggregates. Our results are bringing new openings regarding the impact of biopesticides in prion and prion-like diseases.

Tropismes et barrières d’espèces des ATNC (maladies à prions) : quels dangers pour l’homme?

Resume Les encephalopathies spongiformes subaigues transmissibles (ESST), encore appelees maladies a prions, sont des maladies qui touchent exclusivement les mammiferes. Elles evoluent de facons sporadiques ou epidemiques a l’interieur d’une espece ou d’une sous-famille, suggerant l’existence d’une barriere limitant sa propagation. La transmission vers d’autre famille est rare, mais l’episode de l’encephalopathie spongiforme bovine (ESB), encore appelee maladie de la vache folle, a montre que certaines souches de l’agent infectieux prion pouvaient passer la barriere d’espece. L’ESB a infecte l’homme et plusieurs familles d’animaux domestiques (felins) ou en captivites (primates, ongules, felins). Les etudes experimentales ont montre que le passage d’une espece a l’autre est determine par au moins deux parametres : 1/ la similarite des sequences de la proteine prion et 2/ des specificites liees a souche incriminee. L’hypothese de la « proteine seule », qui explique l’infection par la formation de structures amyloides a partir de la proteine prion, rend compte aussi bien des observations de l’infection naturelle que des etudes de la transmission experimentales. Elle suggere que la specificite est encryptee dans la structure de l’amyloide. La question est donc posee de l’apparition de nouvelles pathologies issues de la proteine prion, mais plus largement aussi de l’apparition de maladies infectieuses resultant de la formation d’amyloides par d’autres proteines. Ces risques hypothetiques sont discutes.