Brucella abortusand Its Closest Phylogenetic Relative,Ochrobactrumspp., Differ in Outer Membrane Permeability and Cationic Peptide Resistance

Abstract

The outer membrane (OM) of the intracellular parasite Brucella abortus is permeable to hydrophobic probes and resistant to destabilization by polycationic peptides and EDTA. The significance of these unusual properties was investigated in a comparative study with the opportunistic pathogens of the genus Ochrobactrum, the closest known Brucella relative. Ochrobactrum spp. OMs were impermeable to hydrophobic probes and sensitive to polymyxin B but resistant to EDTA. These properties were traced to lipopolysaccharide (LPS) because ( Brucella spp. are gram-negative bacteria of the ␣-2 subgroup of the domain Proteobacteria characteristically able to multiply intracellularly in a variety of cells (35). The pathogenicity of Brucella is not related to production of classical toxin, antiphagocytic capsules, proteinaceous adhesins, or plasmid-encoded virulence factors. On the other hand, the low endotoxicity of the lipopolysaccharide (LPS) (49), the resistance to a wide range of bactericidal cationic peptides (13,32), and the ability to prevent lysosome fusion and to locate in autophagosome-like compartments (46, 47) are characteristics of Brucella in all likelihood related to pathogenicity. Reduced endotoxicity and polycation resistance reflect properties of the LPS, and this molecule is also linked to additional peculiarities of the Brucella outer membrane (OM), such as its resistance to EDTA (13, 31, 38) and permeability to hydrophobic compounds (31). Because the OM plays a critical role in interaction of the bacteria with the environment, we have proposed (38,39,49) that the unusual set of properties of the Brucella OM is linked to its intracellular parasitism.A first approach to test this possibility is to obtain mutants altered in OM properties. We have recently produced polycation-sensitive mutants of B. abortus which, in support of our hypothesis, also show increased OM permeability to hydrophobic dyes, cannot penetrate into HeLa cells, and are avirulent in the mouse model (55). These mutants are disrupted in either of the two genes of a sensory/regulatory system (BvrR/BvrS) highly homologous to sensory/regulatory systems essential for virulence and endosymbiosis in Agrobacterium and Rhizobium, two plant-associated bacteria also belonging to the ␣-2 Proteobacteria (55). Moreover, we found that the divergence in BvrS is concentrated in the periplasmic sensory section (55); this shows that in the ␣-2 Proteobacteria, slight modifications of some basic systems reflect the adaptation to seemingly very different hosts and environments. Thus, a complementary approach to assess the relevance of the OM properties in the biology of Brucella is to perform comparative studies with phylogenetically related bacteria differing in pathogenicity. For this purpose, the members of the genus Ochrobactrum are particularly suitable. These bacteria are very close to Brucella by DNA, rRNA, and protein analysis (12,23,59,62,65), but they are primarily soil dwellers known to be pathogenic only in critically ill or immunocompromised patient...

The identification of wadB, a new glycosyltransferase gene, confirms the branched structure and the role in virulence of the lipopolysaccharide core of Brucella abortus

“…The MICs we found were 1.5 µg/mL for Ba-parental, 0.75 µg/mL for BaΔwadC, 0.30 µg/mL for BaΔwadB and 1.5 µg/mL for the pwadB-complemented Ba∆wadB strain. To confirm that the defective core LPS is responsible for the increased sensitivity of the wadB mutant to polycationic peptides, we also probed the binding of purified LPS to the potent Limulus anti-LPS peptide 19.8-4 [12,17]. We found that the LPS of BaΔwadB and BaΔwadC bound more peptide than the LPS of Ba-parental (Figure 2, left panel).…”

“…LPS was then sedimented by ultracentrifugation, extracted four times with chloroform-methanol (2:1 [vol/vol]) to remove free lipids, and freeze-dried [12]. Alternatively, LPS was extracted from whole bacteria by the sodium dodecylsulfate (SDS) -proteinase K protocol [13].…”

Rough vaccines in animal brucellosis: Structural and genetic basis and present status

“…With regard to point (iii), it cannot be assumed in the present state of knowledge that all R mutations (and R mutants) result in attenuation in the natural host or, if so, that they are all equivalent in this aspect. Both the results of comparative studies [130] and the existence of the Brucella R species virulent for their hosts (B. canis in dogs and B. ovis in sheep) show that not only the O-PS but also the LPS core is implicated in Brucella virulence. Thus, different core deficiencies may result in different degrees of attenuation and, therefore, the structure, biosynthesis and genetics of Brucella S-LPS has to be taken into account to design the best possible R vaccine candidates.…”

Lipopolysaccharide as a target for brucellosis vaccine design

“…The reduced affinity of bactericidal peptides for Brucella LPS could be accounted for by the low density of negatively charged groups that is perceived in the determination of the zeta potential 2 of B. abortus LPS aggregates [31], and this same feature could also contribute to a low affinity for MD2. In keeping with this, qualitative chemical analyses indicate that this core contains Kdo as the only or major negatively charged sugar plus glucose and mannose and, noteworthy, glucosamine [29].…”

“…Although the precise structure of B. abortus core is knot known, genetic and biophysical evidence (see Figure 2) shows that it is a structure with a lateral branch hampering access to the inner negative groups of Kdo and lipid A. [32] and [31]). …”