The two-component system PrlS/PrlR of Brucella melitensis is required for persistence in mice and appears to respond to ionic strength.

Bacterial adaptation to environmental conditions is essential to ensure maximal fitness in the face of several stresses. In this context, two-component systems (TCSs) represent a predominant signal transduction mechanism, allowing an appropriate response to be mounted when a stimulus is sensed. As facultative intracellular pathogens, Brucella spp. face various environmental conditions, and an adequate response is required for a successful infection process. Recently, bioinformatic analysis of Brucella genomes predicted a set of 15 bona fide TCS pairs, among which some have been previously investigated. In this report, we characterized a new TCS locus called prlS/R, for probable proline sensor-regulator. It encodes a hybrid histidine kinase (PrlS) with an unusual Na(+)/solute symporter N-terminal domain and a transcriptional regulator (belonging to the LuxR family) (PrlR). In vitro, Brucella spp. with a functional PrlR/S system form bacterial aggregates, which seems to be an adaptive response to a hypersaline environment, while a prlS/R mutant does not. We identified ionic strength as a possible signal sensed by this TCS. Finally, this work correlates the absence of a functional PrlR/S system with the lack of hypersaline-induced aggregation in particular marine Brucella spp.

Brucella pathogenesis, genes identified from random large-scale screens.

Pathogenicity islands, specialized secretion systems, virulence plasmids, fimbriae, pili, adhesins, and toxins are all classical bacterial virulence factors. However, many of these factors, though widespread among bacterial pathogens, are not necessarily found among bacteria that colonize eukaryotic cells in a pathogenic/symbiotic relationship. Bacteria that form these relationships have developed other strategies to infect and grow in their hosts. This is particularly true for Brucella and other members of the class Proteobacteria. Thus far the identification of virulence factors for Brucella has been largely dependent on large-scale screens and testing in model systems. The genomes of the facultative intracellular pathogens Brucella melitensis and Brucella suis were sequenced recently. This has identified several more potential virulence factors for Brucella that were not found in large screens. Here, we present an overall view of Brucella virulence by compiling virulence data from the study of 184 attenuated mutants.

Brucellosis vaccines: assessment of Brucella melitensis lipopolysaccharide rough mutants defective in core and O-polysaccharide synthesis and export.

BACKGROUND: The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. METHODOLOGY/PRINCIPAL FINDINGS: To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.

The stringent response mediator Rsh is required for Brucella melitensis and Brucella suis virulence, and for expression of the type IV secretion system virB.

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.

A quorum-sensing regulator controls expression of both the type IV secretion system and the flagellar apparatus of Brucella melitensis.

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.

Transferrin-binding protein B of Neisseria meningitidis: sequence-based identification of the transferrin-Binding site confirmed by site-directed mutagenesis.

A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf). These two domains are conserved in an alignment of different TbpB sequences from N. meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains. Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding. Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements. The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains.

Effect of omp10 or omp19 deletion on Brucella abortus outer membrane properties and virulence in mice.

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.

The Ton system, an ABC transporter, and a universally conserved GTPase are involved in iron utilization by Brucella melitensis 16M.

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.