Isolation of Atypical Lipopolysaccharides from Purified Cell Walls of Pseudomonas cepacia

Manniello, J.M.; Heymann, H; Adair, Frank W.

doi:10.1099/00221287-112-2-397

Cited by 22 publications

(15 citation statements)

References 21 publications

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“…This idea is supported by the observation that P. cepacia was not permeabilized by EDTA (Table 1). Finally, it is possible that LPS from P. cepacia Mg2 (mM) the phosphate content of P. cepacia LPS was determined in one study (8) to be only one-third of that of P. aeruginosa LPS.…”

Section: Resultsmentioning

confidence: 99%

“…The n value (Hill number) calculated from the Hill plots ( b Assuming LPS had a molecular weight of 9,000. For P. aeruginosa this was obtained by assuming that a measured weight of LPS contained two reactive 2-keto-3-deoxyoctonate molecules per LPS molecule (2,8). Since P. cepacia LPS had a similar pattern of distribution of LPS species on sodium dodecyl sulfate-polyacrylammide gel electrophoretograms as P. aeruginosa LPS (unpublished data), we assumed these LPS species had similar average molecular weights.…”

Section: Resultsmentioning

confidence: 99%

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Involvement of outer membrane of Pseudomonas cepacia in aminoglycoside and polymyxin resistance

Moore¹,

Hancock²

1986

Antimicrob Agents Chemother

131

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Pseudomonas cepacia was found to be resistant to the outer membrane-permeabilizing effects of aminoglycoside antibiotics, polymyxin B, and EDTA. Permeabilization of P. cepacia to the fluorescent probe l-N-phenylnaphthylamine was not achieved at concentrations 100-to 1,000-fold above those required to permeabilize Pseudomonas aeruginosa. Furthermore, in contrast to P. aeruginosa cells, intact cells of P. cepacia did not bind the fluorescent probe dansyl-polymyxin. However, purified lipopolysaccharide (LPS) from P. cepacia bound dansyl-polymyxin with approximately the same affinity as did LPS from P. aeruginosa. Also, binding of dansyl-polymyxin to P. cepacia (and P. aeruginosa) LPS was inhibited by polymyxin B, streptomycin, gentamicin, and Mg2+. These data suggest that P. cepacia does not utilize the self-promoted pathway for aminoglycoside uptake and that the outer membrane is arranged in a way that conceals or protects cation-binding sites on LPS which are capable of binding polycations such as aminoglycosides or polymxyin.Pseudomonas cepacia has become increasingly important as an opportunistic pathogen. Clinical isolates are usually highly resistant to polymyxin as well as many 1-lactam and aminoglycoside antibiotics (3). Consequently, P. cepacia infections are difficult to treat and often life threatening.Penetration of polymyxin B and, at least in some cases, aminoglycosides into the gram-negative bacterium involves an initial interaction with the outer membrane (5,10,12,14). For Pseudomonas aeruginosa, a self-promoted uptake pathway has been proposed to explain the mechanism of aminoglycoside uptake across the outer membrane (4, 5, 10). In this model, an aminoglycoside or other polycation must interact with a divalent-cation-binding site which is involved in the stabilization of outer membranes by cross bridging adjacent lipopolysaccharide (LPS) molecules. The result of this interaction is the displacement of the divalent cation (9) and subsequent disruption and permeabilization of the outer membrane (5-7, 10).The reported inherent resistance of P. cepacia to aminoglycosides (3) and polymyxin (12) led us to examine the interaction of these compounds with this organism. Our findings suggest that a major factor explaining aminoglycoside resistance in P. cepacia is the inability of the antibiotic to disrupt and permeabilize the outer membrane. MATERIALS AND METHODSBacterial strains and growth conditions. P. aeruginosa PAO1 strain H103 was used in this study and has been previously described (11). P. cepacia ATCC 25609, the type strain, was obtained from the American Type Culture Collection (Rockville, Md). P. cepacia K61-3 and PC715J were clinical isolates from cystic fibrosis patients and were obtained from D. Woods, University of Calgary, Calgary, Alberta, Canada. Cells were grown in 1% Proteose Peptone no. 2 (Difco Laboratoies, Detroit, Mich.) medium. For the experiments desribed below, fresh medium (20 ml) was inoculated with an overnight culture to a final dilution of 1:20 * Corresponding author. and gr...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Involvement of outer membrane of Pseudomonas cepacia in aminoglycoside and polymyxin resistance

Moore¹,

Hancock²

1986

Antimicrob Agents Chemother

131

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“…The part of the inner core region adjacent to the lipid A moiety in usual LPS is constructed of one to three molecules of 3-deoxy-D-mannooctulosonic acid (KDO) and is well conserved among various bacterial species. In the case of LPS extracted from B. cepacia, KDO was not detectable by the conventional method (17,33) but was detectable after hydrolyzing the LPS with strong acid (32). This suggests that the KDO portion of the inner core region in LPS of this organism is replaced by some molecules which provide acid resistance.…”

mentioning

confidence: 94%

Lipopolysaccharide ofBurkholderia cepaciaand Its Unique Character To Stimulate Murine Macrophages with Relative Lack of Interleukin-1β-Inducing Ability

et al. 2001

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Lipopolysaccharide (LPS) of Burkholderia cepacia was purified by the conventional phenol-water extraction method (preparation BcLPS-1), followed by enzymatic treatments with DNase, RNase, trypsin, and proteinase K (preparation BcLPS-2), and finally by deoxycholate-phenol-water extraction (preparation BcLPS-3). Cells of LPS-hyporesponsive C3H/HeJ mice were activated by both the BcLPS-1 and the BcLPS-2 preparations but barely activated by BcLPS-3. When LPS-responsive C3H/HeN mice were used as targets, endotoxic activities such as lethal toxicity to galactosamine-sensitized mice, mitogenicity to spleen cells, and activation of macrophages to induce tumor necrosis factor alpha and interleukin-6 (IL-6) were strongly exhibited even by highly purified BcLPS-3 at levels comparable to those of the highly active enterobacterial LPS of Salmonella enterica serovar Abortus-equi (SaeLPS), used as the control. The ability of BcLPS-3 to activate murine macrophages for induction of IL-1␤ was, however, much weaker than that of SaeLPS. Both accumulation of pro-IL-1␤ protein and expression of IL-1␤ mRNA in macrophages by stimulation with BcLPS-3 were much weaker than by stimulation with SaeLPS. These results indicate that LPS of B. cepacia has the potential to play a role as a pathogenic factor with strong activity comparable to that of usual enterobacterial LPS, but unlike the latter, this LPS has a relative lack of ability in the activation of murine macrophages to induce IL-1␤. The lack of IL-1␤-inducing ability appears to be caused by incomplete signal transduction somewhere in the upstream step(s) of IL-1␤ gene transcription.Burkholderia cepacia is an aerobic glucose-nonfermenting gram-negative bacillus that possesses flagella and formerly belonged to the genus Pseudomonas (37). Bacteria of this species are intrinsically resistant to many antibiotics and active as opportunistic pathogens in hospitalized patients and other compromised hosts (8,26). Especially among patients with cystic fibrosis, infection by this organism makes possible the development of serious pneumonia and sepsis with a high mortality rate, i.e., "cepacia syndrome" (9). Lipase, protease, extracellular toxic complex, and lipopolysaccharide (LPS) released from the bacteria have been suggested as candidates for the virulence factors which cause pathological changes, such as accumulation of polymorphonuclear leukocytes and proteinaceous exudate in bronchial and alveolar lumina, injury and necrosis of bronchial epithelium, and disorganization of alveolar structure. The specific crucial trigger of the pneumonia and sepsis in B. cepacia infection, however, remains to be clarified (16,20,31,34,39).Among these four candidates, we were interested in LPS, a major component of the outer membrane in gram-negative bacteria, since it is assumed to play a central role in the induction of various pathophysiological responses, such as fever, disseminated intravascular coagulation, and shock in hosts suffering from gram-negative bacterial infections (22,27). LPSs of members o...

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“…Compared with LPS from P. aeruginosa that from strains of B. cepacia has less phosphorus and more heptose. Glucose and rhamnose were the major saccharide components of LPS from the organisms tested [42]. An extracellular material isolated from a clinical B. cepacia consisted of a surface carbohydrate antigen, LPS and protein, the toxicity of which appeared to be associated with the LPS portion of the complex [46].…”

Section: Extraceilular Virulence Factorsmentioning

confidence: 98%

“…Initial chemical analysis of B. cepacia LPS indicated the absence of detectable 3-deoxy-Dmanno-2-octulosonic acid (KDO) in LPS from B. cepacia [42,43]. However, Straus et al [44] reported the isolation of KDO from the culture supernate of 2 out of 10 strains of B. cepacia and in a further study KDO was demonstrated in 6 clinical isolates of B. cepacia and all 6 LPS preparations were equally toxic for mice when injected intraperitoneally [45].…”

Section: Extraceilular Virulence Factorsmentioning

confidence: 99%

Virulence factors of Burkholderia cepacia

Nelson¹

1994

FEMS Immunology and Medical Microbiology

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Isolation of Atypical Lipopolysaccharides from Purified Cell Walls of Pseudomonas cepacia

Cited by 22 publications

References 21 publications

Involvement of outer membrane of Pseudomonas cepacia in aminoglycoside and polymyxin resistance

Involvement of outer membrane of Pseudomonas cepacia in aminoglycoside and polymyxin resistance

Lipopolysaccharide ofBurkholderia cepaciaand Its Unique Character To Stimulate Murine Macrophages with Relative Lack of Interleukin-1β-Inducing Ability

Virulence factors of Burkholderia cepacia

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