Identification of a Brucella spp. secreted effector specifically interacting with human small GTPase Rab2.
; ; et al
in Cellular microbiology (2011), 13(7), 1044-58
Bacteria of the Brucella genus are facultative intracellular class III pathogens. These bacteria are able to control the intracellular trafficking of their vacuole, presumably by the use of yet unknown ... [more ▼]
Bacteria of the Brucella genus are facultative intracellular class III pathogens. These bacteria are able to control the intracellular trafficking of their vacuole, presumably by the use of yet unknown translocated effectors. To identify such effectors, we used a high-throughput yeast two-hybrid screen to identify interactions between putative human phagosomal proteins and predicted Brucella spp. proteins. We identified a specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA. This interaction was confirmed by GST-pull-down with the GDP-bound form of Rab2. A TEM-beta-lactamase-RicA fusion was translocated from Brucella abortus to RAW264.7 macrophages during infection. This translocation was not detectable in a strain deleted for the virB operon, coding for the type IV secretion system. However, RicA secretion in a bacteriological culture was still observed in a DeltavirB mutant. In HeLa cells, a DeltaricA mutant recruits less GTP-locked myc-Rab2 on its Brucella-containing vacuoles, compared with the wild-type strain. We observed altered kinetics of intracellular trafficking and faster proliferation of the B. abortusDeltaricA mutant in HeLa cells, compared with the wild-type control. Altogether, the data reported here suggest RicA as the first reported effector with a proposed function for B. abortus. [less ▲]Detailed reference viewed: 10 (0 ULg)
Vaccination of calves using the BRSV nucleocapsid protein in a DNA prime-protein boost strategy stimulates cell-mediated immunity and protects the lungs against BRSV replication and pathology.
; Boxus, Mathieu ; et al
in Vaccine (2008), 26(37), 4840-8
Respiratory syncytial virus (RSV) is a major cause of respiratory disease in both cattle and young children. Despite the development of vaccines against bovine (B)RSV, incomplete protection and ... [more ▼]
Respiratory syncytial virus (RSV) is a major cause of respiratory disease in both cattle and young children. Despite the development of vaccines against bovine (B)RSV, incomplete protection and exacerbation of subsequent RSV disease have occurred. In order to circumvent these problems, calves were vaccinated with the nucleocapsid protein, known to be a major target of CD8(+) T cells in cattle. This was performed according to a DNA prime-protein boost strategy. The results showed that DNA vaccination primed a specific T-cell-mediated response, as indicated by both a lymphoproliferative response and IFN-gamma production. These responses were enhanced after protein boost. After challenge, mock-vaccinated calves displayed gross pneumonic lesions and viral replication in the lungs. In contrast, calves vaccinated by successive administrations of plasmid DNA and protein exhibited protection against the development of pneumonic lesions and the viral replication in the BAL fluids and the lungs. The protection correlated to the cell-mediated immunity and not to the antibody response. [less ▲]Detailed reference viewed: 53 (11 ULg)
DNA immunization with plasmids encoding fusion and nucleocapsid proteins of bovine respiratory syncytial virus induces a strong cell-mediated immunity and protects calves against challenge.
Boxus, Mathieu ; ; et al
in Journal of Virology (2007), 81(13), 6879-89
Respiratory syncytial viruses (RSV) are one of the most important respiratory pathogens of humans and cattle, and there is currently no safe and effective vaccine prophylaxis. In this study, we designed ... [more ▼]
Respiratory syncytial viruses (RSV) are one of the most important respiratory pathogens of humans and cattle, and there is currently no safe and effective vaccine prophylaxis. In this study, we designed two codon-optimized plasmids encoding the bovine RSV fusion (F) and nucleocapsid (N) proteins and assessed their immunogenicity in young calves. Two administrations of both plasmids elicited low antibody levels but primed a strong cell-mediated immunity characterized by lymphoproliferative response and gamma interferon production in vitro and in vivo. Interestingly, this strong cellular response drastically reduced viral replication, clinical signs, and pulmonary lesions after a highly virulent challenge. Moreover, calves that were further vaccinated with a killed-virus vaccine developed high levels of neutralizing antibody and were fully protected following challenge. These results indicate that DNA vaccination could be a promising alternative to the classical vaccines against RSV in cattle and could therefore open perspectives for vaccinating young infants. [less ▲]Detailed reference viewed: 9 (1 ULg)
The stringent response mediator Rsh is required for Brucella melitensis and Brucella suis virulence, and for expression of the type IV secretion system virB.
; ; et al
in Cellular Microbiology (2006), 8(11), 1791-802
Physiological adaptation of intracellular bacteria is critical for timely interaction with eukaryotic host cells. One mechanism of adaptation, the stringent response, is induced by nutrient stress via its ... [more ▼]
Physiological adaptation of intracellular bacteria is critical for timely interaction with eukaryotic host cells. One mechanism of adaptation, the stringent response, is induced by nutrient stress via its effector molecule (p)ppGpp, synthesized by the action of RelA/SpoT homologues. The intracellular pathogen Brucella spp., causative agent of brucellosis, possesses a gene homologous to relA/spoT, named rsh, encoding a (p)ppGpp synthetase as confirmed by heterologous complementation of a relA mutant of Sinorhizobium meliloti. The Rsh deletion mutants in Brucella suis and Brucella melitensis were characterized by altered morphology, and by reduced survival under starvation conditions and in cellular and murine models of infection. Most interestingly, we evidenced that expression of virB, encoding the type IV secretion system, a major virulence factor of Brucella, was Rsh-dependent. All mutant phenotypes, including lack of VirB proteins, were complemented with the rsh gene of Brucella. In addition, RelA of S. meliloti functionally replaced Brucella Rsh, describing the capacity of a gene from a plant symbiont to restore virulence in a mammalian pathogen. We therefore concluded that in the intramacrophagic environment encountered by Brucella, Rsh might participate in the adaptation of the pathogen to low-nutrient environments, and indirectly in the VirB-mediated formation of the final replicative niche. [less ▲]Detailed reference viewed: 16 (2 ULg)
A quorum-sensing regulator controls expression of both the type IV secretion system and the flagellar apparatus of Brucella melitensis.
; ; et al
in Cellular Microbiology (2005), 7(8), 1151-61
Both a type IV secretion system and a flagellum have been described in Brucella melitensis. These two multimolecular surface appendages share several features. Their expression in bacteriological medium ... [more ▼]
Both a type IV secretion system and a flagellum have been described in Brucella melitensis. These two multimolecular surface appendages share several features. Their expression in bacteriological medium is growth curve dependent, both are induced intracellularly and are required for full virulence in a mouse model of infection. Here we report the identification of VjbR, a quorum sensing-related transcriptional regulator. A vjbR mutant has a downregulated expression of both virB operon and flagellar genes either during vegetative growth or during intracellular infection. In a cellular model, the vacuoles containing the vjbR mutant or a virB mutant are decorated with the same markers at similar times post infection. The vjbR mutant is also strongly attenuated in a mouse model of infection. As C(12)-homoserine lactone pheromone is known to be involved in virB repression, we postulated that VjbR is mediating this effect. In agreement with this hypothesis, we observed that, as virB operon, flagellar genes are controlled by the pheromone. All together these data support a model in which VjbR acts as a major regulator of virulence factors in Brucella. [less ▲]Detailed reference viewed: 56 (1 ULg)
The Ton system, an ABC transporter, and a universally conserved GTPase are involved in iron utilization by Brucella melitensis 16M.
DANESE, Isabelle ; ; et al
in Infection and Immunity (2004), 72(10), 5783-90
Brucella spp. are gram-negative intracellular facultative pathogens that are known to produce 2,3-dihydroxybenzoic acid (DHBA), a catechol siderophore that is essential for full virulence in the natural ... [more ▼]
Brucella spp. are gram-negative intracellular facultative pathogens that are known to produce 2,3-dihydroxybenzoic acid (DHBA), a catechol siderophore that is essential for full virulence in the natural host. The mechanism of DHBA entry into Brucella and other gram-negative bacteria is poorly understood. Using mini-Tn5Kmcat mutagenesis, we created a transposon library of Brucella melitensis 16M and isolated 32 mutants with a defect in iron acquisition or assimilation. Three of these transposon mutants are deficient in utilization of DHBA. Analysis of these three mutants indicated that the ExbB, DstC, and DugA proteins are required for optimal assimilation of DHBA and/or citrate. ExbB is part of the Ton complex, and DstC is a permease homologue of an iron(III) ABC transporter; in gram-negative bacteria these two complexes are involved in the uptake of iron through the outer and inner membranes, respectively. DugA is a new partner in iron utilization that exhibits homology with the bacterial conserved GTPase YchF. Based on this homology, DugA could have a putative regulatory function in iron assimilation in Brucella. None of the three mutants was attenuated in cellular models or in the mouse model of infection, which is consistent with the previous suggestion that DHBA utilization is not required in these models. [less ▲]Detailed reference viewed: 15 (2 ULg)
Effect of omp10 or omp19 deletion on Brucella abortus outer membrane properties and virulence in mice.
; ; et al
in Infection and Immunity (2002), 70(10), 5540-6
The distinctive properties of Brucella outer membrane have been considered to be critical for Brucella sp. virulence. Among the outer membrane molecules possibly related to these properties, Omp10 and ... [more ▼]
The distinctive properties of Brucella outer membrane have been considered to be critical for Brucella sp. virulence. Among the outer membrane molecules possibly related to these properties, Omp10 and Omp19 are immunoreactive outer membrane lipoproteins. Moreover, these proteins of Brucella could constitute a new family of outer membrane proteins specifically encountered in the family RHIZOBIACEAE: We evaluated the impact of omp10 or omp19 deletion on Brucella abortus outer membrane properties and virulence in mice. The omp10 mutant was dramatically attenuated for survival in mice and was defective for growth in minimal medium but was not impaired in intracellular growth in vitro, nor does it display clear modification of the outer membrane properties. Significantly fewer brucellae were recovered from the spleens of mice infected with the omp19 mutant than from those of mice infected with the parent strain at 4 and 8 weeks postinfection. The omp19 mutant exhibited an increase in sensitivity to the polycation polymyxin B and to sodium deoxycholate. These results indicate that inactivation of the omp19 gene alters the outer membrane properties of B. abortus. [less ▲]Detailed reference viewed: 9 (0 ULg)
How to substantiate eradication of bovine brucellosis when aspecific serological reactions occur in the course of brucellosis testing.
; Saegerman, Claude ; et al
in Veterinary Microbiology (2002), 90(1-4), 461-77
Collaborative work was financed by the EU to develop and assess new diagnostic tools that can differentiate between bovine brucellosis and bovine infections due to Yersinia enterocolitica O:9 either in ... [more ▼]
Collaborative work was financed by the EU to develop and assess new diagnostic tools that can differentiate between bovine brucellosis and bovine infections due to Yersinia enterocolitica O:9 either in conjunction with, or as an alternative to, the classical serological, bacteriological or allergic skin tests. Sixteen heifers were experimentally infected with Brucella abortus biovar 1 (five heifers), Brucella suis biovar 2 (two heifers), Y. enterocolitica O:9 (six heifers) and Y. enterocolitica O:3 (three heifers). Four heifers, naturally infected with Y. enterocolitica O:9 that presented aspecific brucellosis serological reactions were also included in the experiment. A self-limited infection was induced in cattle by B. suis biovar 2. All the brucellosis serological tests used, i.e. the slow agglutination test (SAW), the Rose Bengal test (RB), the complement fixation test (CFT), indirect and competitive ELISA's, lacked specificity when used to analyze sera from Y. enterocolitica O:9 infected animals. A Yersinia outer membrane proteins (YOPs)-ELISA was also used and although the test is able to detect a Yersinia group infection, it provided no evidence of whether or not there is a possible brucellosis infection when dual infections are present. The brucellergen IFN-gamma test showed a lack of specificity also. The only test that was proven to be specific is the brucellergen skin test. All brucellosis serological tests, except the indirect ELISA, were limited in their ability to detect B. abortus persistently infected animals. Based on these experimental studies, a strategy was implemented as part of the year 2001 Belgian Brucellosis Eradication Program to substantiate the eradication of bovine brucellosis. Epidemiological inquiries have identified risk factors associated with aspecific serological reactions, possible transmission and infection of cattle by B. suis biovar 2 from infected wild boars; and both legal and administrative measures taken by the veterinary services. No cases of bovine brucellosis have been confirmed in Belgium since March 2000. [less ▲]Detailed reference viewed: 56 (18 ULg)
Effects of Bovine Herpesvirus Type 1 Infection in Calves with Maternal Antibodies on Immune Response and Virus Latency
; ; et al
in Journal of Clinical Microbiology (2000), 38(5), 1885-94
The presence of maternally derived antibodies can interfere with the development of an active antibody response to antigen. Infection of seven passively immunized young calves with a virulent strain of ... [more ▼]
The presence of maternally derived antibodies can interfere with the development of an active antibody response to antigen. Infection of seven passively immunized young calves with a virulent strain of bovine herpesvirus type 1 (BHV-1) was performed to determine whether they could become seronegative after the disappearance of maternal antibodies while latently infected with BHV-1. Four uninfected calves were controls. All calves were monitored serologically for 13 to 18 months. In addition, the development of a cell-mediated immune response was assessed by an in vitro antigen-specific gamma interferon (IFN-gamma) production assay. All calves had positive IFN-gamma responses as early as 7 days until at least 10 weeks after infection. However, no antibody rise was observed after infection in the three calves with the highest titers of maternal antibodies. One of the three became seronegative by virus neutralization test at 7 months of age like the control animals. This calf presented negative IFN-gamma results at the same time and was classified seronegative by enzyme-linked immunosorbent assay at around 10 months of age. This calf was latently infected, as proven by virus reexcretion after dexamethasone treatment at the end of the experiment. In conclusion, this study demonstrated that BHV-1-seronegative latent carriers can be obtained experimentally. In addition, the IFN-gamma assay was able to discriminate calves possessing only passively acquired antibodies from those latently infected by BHV-1, but it could not detect seronegative latent carriers. The failure to easily detect such animals presents an epidemiological threat for the control of BHV-1 infection. [less ▲]Detailed reference viewed: 10 (0 ULg)