The outer membranes of Brucella spp. are not barriers to hydrophobic permeants

Tejada, Guillermo Martínez de; Moriyón, Ignacio

doi:10.1128/jb.175.16.5273-5275.1993

Cited by 46 publications

(58 citation statements)

References 18 publications

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“…Levels of novobiocin sensitivity and accumulation of the hydrophobic probes CV and NPN in V. cholerae were significantly higher than in E. coli (Table 1;, Fig. 1 and 2) and were similar to those reported for Brucella, a pathogen that uses a nonenteric route for infection (15). This observation raises fundamental questions about how V. cholerae survives in the intestine.…”

Section: Discussionsupporting

confidence: 51%

Effect of Bile on the Cell Surface Permeability Barrier and Efflux System of Vibrio cholerae

et al. 2004

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Gram-negative bacteria are inherently impermeable to hydrophobic compounds, due to the synergistic activity of the permeability barrier imposed by the outer membrane and energy dependent efflux systems. The gram-negative, enteric pathogen Vibrio cholerae appears to be deficient in both these activities; the outer membrane is not an effective barrier to hydrophobic permeants, presumably due to the presence of exposed phospholipids on the outer leaflet of the outer membrane, and efflux systems are at best only partially active. When V. cholerae was grown in the presence of bile, entry of hydrophobic compounds into the cells was significantly reduced. No difference was detected in the extent of exposed phospholipids on the outer leaflet of the outer membrane between cells grown in the presence or absence of bile. However, in the presence of energy uncouplers, uptake of hydrophobic probes was comparable between cells grown in the presence or absence of bile, indicating that energy-dependent efflux processes may be involved in restricting the entry of hydrophobic permeants into bile grown cells. Indeed, an efflux system(s) is essential for survival of V. cholerae in the presence of bile. Expression of acrAB, encoding an RND family efflux pump, was significantly increased in V. cholerae cells grown in vitro in the presence of bile and also in cells grown in rabbit intestine.Vibrio cholerae, a noninvasive enteric bacterium, is the causative agent of the diarrheal disease cholera. Cholera remains a major cause of human mortality in developing countries, where conditions of poor sanitation, war, famine, and malnourishment contribute to regular episodes of cholera epidemics. For successful infection of its human host, V. cholerae must colonize the small intestine and produce copious amounts of cholera toxin (CT), a potent enterotoxin that causes the massive fluid loss characteristic of the disease. In addition to obvious virulence factors like CT, other toxins, toxin-coregulated pilus, hemolysins, and hemagglutinins (factors essential for survival of the bacteria in vivo and evasion of the host defense system) also contribute to the pathogenecity of V. cholerae (for a review, see references 12 and 24).Enteric pathogens and normal intestinal flora must necessarily survive and colonize the intestine in the presence of bile. Bile salts are surface-active, amphipathic compounds with pronounced detergent-like activity that can cause disaggregation of the lipid bilayer structure of cellular membranes (11). However, gram-negative enteric bacteria are inherently resistant to bile, partly due to the basic, asymmetric structure of their outer membranes (OMs). Although the inner surface of the OM contains phospholipids, a characteristic lipopolysaccharide (LPS) is present on the outer leaflet that significantly retards diffusion of hydrophobic compounds across the OM. The OM thus functions as an effective permeability barrier and gives protection to enteric bacteria from potentially noxious agents present in the intestine, particula...

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Section: Discussionsupporting

confidence: 51%

Effect of Bile on the Cell Surface Permeability Barrier and Efflux System of Vibrio cholerae

et al. 2004

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“…In addition, several OM proteins are accessible on the outer leaflet of the Brucella surface (7,17) and the OM of the smooth (S) Brucella species contains a native hapten polysaccharide possibly intertwined with the O chain of the LPS (1). Some of these features correlate well with the observed permeability of the Brucella OM to hydrophobic compounds (25) and with the resistance of these bacteria to EDTA, Tris, some detergents (32), and oxygen-independent killing mechanisms (3,35). Despite the fact that all of these OM features have been presented as key factors in the intracellular survival of Brucella sp.…”

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confidence: 62%

“…We have recently employed an additional strategy involving the construction of OM bacterial chimeras by incorporating foreign molecules in the OM of Brucella mutants (26). In the present study, we combined several of the above approaches to determine the role of LPS in the previously described (26,32) resistance of Brucella cells to polymyxin B, lactoferricin B (two bactericidal peptides with different structures and biological activities), and EDTA, as well as in the characteristic permeability of Brucella cells to hydrophobic compounds (25). The results demonstrated that LPS plays a determinant role in all of these properties.…”

mentioning

confidence: 92%

“…The fluorescent probe N-phenyl-1-naphthylamine (NPN) (Sigma) has been used in OM permeability studies because its quantum yield increases when it is transferred from a hydrophilic to a hydrophobic environment (24,40,43). Fluorimetric assays were carried out as described by Martínez de Tejada and Moriyón (25), with some modifications. Exponentially growing native bacterial cells or B. abortus-LPS chimeras were resuspended in 1 mM KCl-10 mM N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid (HEPES; pH 7.2) at an optical density at 600 nm of 0.1 and transferred to fluorimetric cuvettes.…”

Section: Methodsmentioning

confidence: 99%

“…The outer membrane (OM) of Brucella organisms shares many structural features with the OM of their close relatives in the genera Ochrobactrum, Bartonella, Agrobacterium, Rhizobium, and Phyllobacterium, all of which are eukaryotic-cell-associated ␣-2 proteobacteria (27, 31). However, the Brucella OM differs from that of other pathogenic groups (6,(25)(26)(27), including several facultative intracellular bacteria, such as Salmonella or Shigella spp. For instance, both phosphatidylcholine and ornithine lipids constitute a major part of the Brucella OM lipids (6, 42).…”

mentioning

confidence: 99%

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Brucella-Salmonella lipopolysaccharide chimeras are less permeable to hydrophobic probes and more sensitive to cationic peptides and EDTA than are their native Brucella sp. counterparts

et al. 1996

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A rough (R) Brucella abortus 45/20 mutant was more sensitive to the bactericidal activity of polymyxin B and lactoferricin B than was its smooth (S) counterpart but considerably more resistant than Salmonella montevideo. The outer membrane (OM) and isolated lipopolysaccharide (LPS) of S. montevideo showed a higher affinity for these cationic peptides than did the corresponding B. abortus OM and LPS. We took advantage of the moderate sensitivity of R B. abortus to cationic peptides to construct live R B. abortus-S-LPS chimeras to test the activities of polymyxin B, lactoferricin B, and EDTA. Homogeneous and abundant peripheral distribution of the heterologous S-LPS was observed on the surface of the chimeras, and this coating had no effect on the viability or morphology of the cells. When the heterologous LPS corresponded to the less sensitive bacterium S B. abortus S19, the chimeras were more resistant to cationic peptides; in contrast, when the S-LPS was from the more sensitive bacterium S. montevideo, the chimeras were more susceptible to the action of peptides and EDTA. A direct correlation between the amount of heterologous S-LPS on the surface of chimeric Brucella cells and peptide sensitivity was observed. Whereas the damage produced by polymyxin B in S. montevideo and B. abortus-S. montevideo S-LPS chimeras was manifested mainly as OM blebbing and inner membrane rolling, lactoferricin B caused inner membrane detachment, vacuolization, and the formation of internal electron-dense granules in these cells. Native S and R B. abortus strains were permeable to the hydrophobic probe N-phenyl-1-naphthylamine (NPN). In contrast, only reduced amounts of NPN partitioned into the OMs of the S. montevideo and B. abortus-S. montevideo S-LPS chimeras. Following peptide exposure, accelerated NPN uptake similar to that observed for S. montevideo was detected for the B. abortus-S. montevideo LPS chimeras. The partition of NPN into native or EDTA-, polymyxin B-, or lactoferricin B-treated LPS micelles of S. montevideo or B. abortus mimicked the effects observed with intact cells, and this was confirmed by using micelle hybrids of B. abortus and S. montevideo LPSs. The results showed that LPS is the main cause of B. abortus' resistance to bactericidal cationic peptides, the OM-disturbing action of divalent cationic chelants, and OM permeability to hydrophobic substances. It is proposed that these three features are related to the ability of Brucella bacteria to multiply within phagocytes.The genus Brucella comprises gram-negative, facultative intracellular pathogens that produce disease in a large number of mammals, including humans (8). The outer membrane (OM) of Brucella organisms shares many structural features with the OM of their close relatives in the genera Ochrobactrum, Bartonella, Agrobacterium, Rhizobium, and Phyllobacterium, all of which are eukaryotic-cell-associated ␣-2 proteobacteria (27, 31). However, the Brucella OM differs from that of other pathogenic groups (6,(25)(26)(27), including several facultative in...

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