LPS-deficient Mutant Research Articles

Essential oils of Pinus sylvestris, Citrus limon and Origanum vulgare exhibit high bactericidal and anti-biofilm activities against Neisseria gonorrhoeae and Streptococcus suis

Antimicrobial resistance is a worldwide problem that urges novel alternatives to treat infections. In attempts to find novel molecules, we assess the antimicrobial potential of seven essential oils (EO) of different plants (Pinus sylvestris, Citrus limon, Origanum vulgare, Cymbopogon martini, Cinnamomum cassia, Melaleuca alternifolia and Eucalyptus globulus) against two multidrug-resistant bacteria species, i.e. Neisseria gonorrhoeae and Streptococcus suis. EOs of P. sylvestris and C. limon revealed higher bactericidal activity (MIC ≤ 0.5 mg/mL) and capacity to rapidly disperse biofilms of several N. gonorrhoeae clinical isolates than other EOs. Examination of biofilms exposed to both EO by electron microscopy revealed a reduction of bacterial aggregates, high production of extracellular vesicles, and alteration of cell integrity. This activity was dose-dependent and was enhanced in DNase I-treated biofilms. Antibiotic susceptibility studies confirmed that both EOs affected the outer membrane permeability, and analysis of EO- susceptibility of an LPS-deficient mutant suggested that both EO target the LPS bilayer. Further analysis revealed that α- and β-pinene and d-limonene, components of both EO, contribute to such activity. EO of C. martini, C. cassia, and O. vulgare exhibited promising antimicrobial activity (MIC ≤ 0.5 mg/mL) against S. suis, but only EO of O. vulgare exhibited a high biofilm dispersal activity, which was also confirmed by electron microscopy studies. To conclude, the EO of P. sylvestris, C. limon and O. vulgare studied in this work exhibit bactericidal and anti-biofilm activities against gonococcus and streptococcus, respectively.

Outer Membrane Vesicles (OMVs) of Pseudomonas aeruginosa Provide Passive Resistance but Not Sensitization to LPS-Specific Phages.

Outer membrane vesicles (OMVs) released from gram-negative bacteria are key elements in bacterial physiology, pathogenesis, and defence. In this study, we investigated the role of Pseudomonas aeruginosa OMVs in the anti-phage defence as well as in the potential sensitization to LPS-specific phages. Using transmission electron microscopy, virion infectivity, and neutralization assays, we have shown that both phages efficiently absorb on free vesicles and are unable to infect P. aeruginosa host. Nevertheless, the accompanying decrease in PFU titre (neutralization) was only observed for myovirus KT28 but not podovirus LUZ7. Next, we verified whether OMVs derived from wild-type PAO1 strain can sensitize the LPS-deficient mutant (Δwbpl PAO1) resistant to tested phages. The flow cytometry experiments proved a quite effective and comparable association of OMVs to Δwbpl PAO1 and wild-type PAO1; however, the growth kinetic curves and one-step growth assay revealed no sensitization event of the OMV-associated phage-resistant P. aeruginosa deletant to LPS-specific phages. Our findings for the first time identify naturally formed OMVs as important players in passive resistance (protection) of P. aeruginosa population to phages, but we disproved the hypothesis of transferring phage receptors to make resistant strains susceptible to LPS-dependent phages.

HldE Is Important for Virulence Phenotypes in Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrheal illness in third world countries and it especially affects children and travelers visiting these regions. ETEC causes disease by adhering tightly to the epithelial cells in a concerted effort by adhesins, flagella, and other virulence-factors. When attached ETEC secretes toxins targeting the small intestine host-cells, which ultimately leads to osmotic diarrhea. HldE is a bifunctional protein that catalyzes the nucleotide-activated heptose precursors used in the biosynthesis of lipopolysaccharide (LPS) and in post-translational protein glycosylation. Both mechanisms have been linked to ETEC virulence: Lipopolysaccharide (LPS) is a major component of the bacterial outer membrane and is needed for transport of heat-labile toxins to the host cells, and ETEC glycoproteins have been shown to play an important role for bacterial adhesion to host epithelia. Here, we report that HldE plays an important role for ETEC virulence. Deletion of hldE resulted in markedly reduced binding to the human intestinal cells due to reduced expression of colonization factor CFA/I on the bacterial surface. Deletion of hldE also affected ETEC motility in a flagella-dependent fashion. Expression of both colonization factors and flagella was inhibited at the level of transcription. In addition, the hldE mutant displayed altered growth, increased biofilm formation and clumping in minimal growth medium. Investigation of an orthogonal LPS-deficient mutant combined with mass spectrometric analysis of protein glycosylation indicated that HldE exerts its role on ETEC virulence both through protein glycosylation and correct LPS configuration. These results place HldE as an attractive target for the development of future antimicrobial therapeutics.

Neisseria meningitidis and Escherichia coli are protected from leukocyte phagocytosis by binding to erythrocyte complement receptor 1 in human blood

The initial interaction of Gram-negative bacteria with erythrocytes and its implications on leukocyte phagocytosis and oxidative burst in human whole blood were examined. Alexa-labeled Escherichia coli, wild-type H44/76 N. meningitidis and the H44/76lpxA lipopolysaccharide (LPS)-deficient mutant were incubated with whole blood using lepirudin as anticoagulant which has no adverse effects on complement. Bacteria free in plasma, bound to erythrocytes or phagocytized by granulocytes and monocytes were quantified using flow cytometry. The effects of the C3 inhibitor compstatin, a C5a receptor antagonist (C5aRa) and a complement receptor 1 (CR1)-blocking antibody (3D9) were examined. Most bacteria (80%) immediately bound to erythrocytes. The binding gradually declined over time, with a parallel increase in phagocytosis. Complement inhibition with compstatin reduced erythrocyte binding and bacterial C3 opsonization. In contrast, the C5aRa efficiently reduced phagocytosis, but did not affect the binding of bacteria to erythrocytes. The anti-CR1 blocking mAb dose-dependently reduced bacterial binding to erythrocytes to nil, with subsequent increased phagocytosis and oxidative burst. LPS had no effect on these processes since similar results were obtained using an LPS-deficient N. meningitidis mutant. In vivo experiments in a pig model of sepsis showed limited binding of bacteria to erythrocytes, consistent with the facts that erythrocyte CR1 receptors are absent in non-primates and that the bacteria were mainly found in the lungs. In conclusion, complement-dependent binding of Gram-negative bacteria to erythrocyte CR1 decreases phagocytosis and oxidative burst by leukocytes in human whole blood.

Secondary Acylation of Klebsiella pneumoniae Lipopolysaccharide Contributes to Sensitivity to Antibacterial Peptides

Klebsiella pneumoniae is an important cause of nosocomial Gram-negative sepsis. Lipopolysaccharide (LPS) is considered to be a major virulence determinant of this encapsulated bacterium and most mutations to the lipid A anchor of LPS are conditionally lethal to the bacterium. We studied the role of LPS acylation in K. pneumoniae disease pathogenesis by using a mutation of lpxM (msbB/waaN), which encodes the enzyme responsible for late secondary acylation of immature lipid A molecules. A K. pneumoniae B5055 (K2:O1) lpxM mutant was found to be attenuated for growth in the lungs in a mouse pneumonia model leading to reduced lethality of the bacterium. B5055DeltalpxM exhibited similar sensitivity to phagocytosis or complement-mediated lysis than B5055, unlike the non-encapsulated mutant B5055nm. In vitro, B5055DeltalpxM showed increased permeability of the outer membrane and an increased susceptibility to certain antibacterial peptides suggesting that in vivo attenuation may be due in part to sensitivity to antibacterial peptides present in the lungs of BALB/c mice. These data support the view that lipopolysaccharide acylation plays a important role in providing Gram-negative bacteria some resistance to structural and innate defenses and especially the antibacterial properties of detergents (e.g. bile) and cationic defensins.

Chemokine Patterns in Meningococcal Disease

Chemokines are important in regulating leukocyte traffic during infection. We analyzed plasma chemokine levels of monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1 alpha , interleukin (IL)-8, and RANTES in patients with meningococcal infection and correlated these to plasma lipopolysaccharide (LPS) levels, which are closely associated with clinical presentation. In patients with fulminant meningococcal septicemia, versus distinct meningitis or mild systemic meningococcal disease, MCP-1 (both P<.0001), MIP-1 alpha (both P<.0001), and IL-8 (P<.0001 and P=.011) were significantly higher and RANTES significantly lower (P=.007 and P=.021). MCP-1 (r=.88), MIP-1 alpha (r=.82), and IL-8 (r=.89) were positively correlated to plasma LPS levels, whereas RANTES was negatively correlated (r=-.49). In an ex vivo whole-blood model, heat-inactivated wild-type Neisseria meningitidis, purified meningococcal LPS, and (to a negligible extent) heat-inactivated LPS-deficient mutant N. meningitidis induced these chemokines. N. meningitidis LPS is the major cause of chemokine release in meningococcal disease.

Lessons from an LPS-deficient Neisseria meningitidis mutant.

In the pathogen Neisseria meningitidis, a completely LPS-deficient but viable mutant can be obtained by insertional inactivation of the lpxA gene, encoding UDP-GlcNAc acyltransferase required for the first step of lipid A biosynthesis. The expression and assembly of integral outer membrane proteins in the absence of LPS is largely unaffected. However, the expression of iron limitation-inducible, cell surface-exposed lipoproteins is greatly reduced. Major changes were seen in the phospholipid composition, with a shift towards PE and PG species containing mostly shorter chain, saturated fatty acids. The presence of the capsular polysaccharide turned out to be essential for viability without LPS. The immunogenicity of outer membrane proteins in mice was greatly reduced for the LPS-deficient mutant, showing the importance of LPS as an internal adjuvant in such vaccines. Stimulation of MM6 cells and peripheral blood mononuclear cells showed that induction of TNF-alpha by whole meningococci was greatly reduced for the LPS-deficient mutant. However, even without LPS the mutant strain could still induce a significant inflammatory response.

Lessons from an LPS-deficient Neisseria meningitidis mutant

In the pathogen Neisseria meningitidis, a completely LPS-deficient but viable mutant can be obtained by insertional inactivation of the lpxA gene, encoding UDP-GlcNAc acyltransferase required for the first step of lipid A biosynthesis. The expression and assembly of integral outer membrane proteins in the absence of LPS is largely unaffected. However, the expression of iron limitation-inducible, cell surface-exposed lipoproteins is greatly reduced. Major changes were seen in the phospholipid composition, with a shift towards PE and PG species containing mostly shorter chain, saturated fatty acids. The presence of the capsular polysaccharide turned out to be essential for viability without LPS. The immunogenicity of outer membrane proteins in mice was greatly reduced for the LPS-deficient mutant, showing the importance of LPS as an internal adjuvant in such vaccines. Stimulation of MM6 cells and peripheral blood mononuclear cells showed that induction of TNF-α by whole meningococci was greatly reduced for the LPS-deficient mutant. However, even without LPS the mutant strain could still induce a significant inflammatory response.

Contributions ofNeisseria meningitidisLPS and non-LPS to proinflammatory cytokine response

To determine the relative contribution of lipopolysaccharide (LPS) and non-LPS components of Neisseria meningitidis to the pathogenesis of meningococcal sepsis, this study quantitatively compared cytokine induction by isolated LPS, wild-type serogroup B meningococci (strain H44/76), and LPS-deficient mutant meningococci (strain H44/76[pLAK33]). Stimulation of human peripheral-blood mononuclear cells with wild-type and LPS-deficient meningococci showed that non-LPS components of meningococci are responsible for a substantial part of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta production and virtually all interferon (IFN)-gamma production. Based on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LPS in proteinase K-treated lysates of N. meningitidis H44/76, a quantitative comparison was made between the cytokine-inducing capacity of isolated and purified LPS and LPS-containing meningococci. At concentrations of >10(7) bacteria/mL, intact bacteria were more potent cytokine inductors than equivalent amounts of isolated LPS, and cytokine induction by non-LPS components was additive to that by LPS. Experiments with mice showed that non-LPS components of meningococci were able to induce cytokine production and mortality. The principal conclusion is that non-LPS parts of N. meningitidis may play a role in the pathogenesis of meningococcal sepsis by inducing substantial TNF-alpha, IL-1beta, and IFN-gamma production.

Membrane-associated proteins of a lipopolysaccharide-deficient mutant of Neisseria meningitidis activate the inflammatory response through toll-like receptor 2.

The recent isolation of a lipopolysaccharide (LPS)-deficient mutant of Neisseria meningitidis has allowed us to explore the roles of other gram-negative cell wall components in the host response to infection. The experiments in this study were designed to examine the ability of this mutant strain to activate cells. Although it was clearly less potent than the parental strain, we found the LPS-deficient mutant to be a capable inducer of the inflammatory response in monocytic cells, inducing a response similar to that seen with Staphylococcus aureus. Cellular activation by the LPS mutant was related to expression of CD14, a high-affinity receptor for LPS and other microbial products, as well as Toll-like receptor 2, a member of the Toll family of receptors recently implicated in host responses to gram-positive bacteria. In contrast to the parental strain, the synthetic LPS antagonist E5564 did not inhibit the LPS-deficient mutant. We conclude that even in the absence of LPS, the gram-negative cell wall remains a potent inflammatory stimulant, utilizing signaling pathways independent of those involved in LPS signaling.

Differential roles of TLR2 and TLR4 in the host response to Gram-negative bacteria: lessons from a lipopolysaccharide-deficient mutant of Neisseria meningitidis

The inflammatory response to bacterial infections plays an important role in the detection and elimination of invading micro-organisms. Various components of the bacterial cell wall are capable of activating this pro-inflammatory response. In the case of Gram-negative bacteria, lipopolysaccharide (LPS) is the dominant trigger, although other bacterial factors are also capable of activating this systemic inflammatory response. Recently, Toll-like receptors (TLRs) have been implicated in host responses to bacterial pathogens. Specifically, TLR4 mediates LPS responses while TLR2 plays a broader role in the recognition of a variety of bacteria and bacterial antigens. The experiments in this study were designed to examine the role of Gram-negative cell wall components, other than LPS, and their cellular receptors in the host response to infection using an LPS-deficient mutant of Neisseria meningitidis. Although less potent than the parental strain, we found the LPS-deficient mutant to be a capable inducer of the inflammatory response in a variety of cell types. Moreover, cellular activation by this mutant required expression of CD14 and TLR2.

Cytokine induction by non-lipopolysaccharide components of Neisseria meningitidis

Abstract Background Lipopolysaccharide (LPS), or endotoxin, is considered responsible for the pattern of cytokine activation and the subsequent pathophysiological events during Gram-negative septic shock. To determine whether non-LPS components of Neisseria meningitidis are able to induce cytokine activation, the cytokine-inducing capacities of serogroup B wild-type N. meningitidis H44/76, of an LPS-deficient mutant (Plak 33) and of meningococcal LPS in human peripheral blood mononuclear cells were studied. Methods 2-Keto-3-deoxyoctanate spectrophotometry was used to obtain a molar-based quantitative comparison between LPS and bacteria. Results Whole meningococci H44/76 were more potent inducers of cytokine production than isolated LPS; the LPS-deficient meningococci, Plak 33, were also able to induce cytokine production. LPS-deficient meningococci supplemented with an amount of LPS equivalent to that present in the wild-type strain produced the same amount of cytokines as the wild-type meningococcus. Conclusion LPS is not the sole toxic element in meningococcal sepsis, because non-LPS components of N. meningitidis are able to induce cytokine activation substantially. Antiendotoxin therapy alone may not be sufficient to inhibit the pattern of cytokine activation and the subsequent pathophysiological events during meningococcal septic shock.

Immunogenicity of outer membrane proteins in a lipopolysaccharide-deficient mutant of Neisseria meningitidis: influence of adjuvants on the immune response.

The immunogenicity of outer membrane complexes (OMCs) or heat-inactivated bacteria of a lipopolysaccharide (LPS)-deficient mutant derived from meningococcal strain H44/76 was studied. The immune response in BALB/c mice to the major outer membrane proteins was poor compared to the immune response elicited by wild-type immunogens. However, addition of external H44/76 LPS to mutant OMCs entirely restored the immune response. By using an LPS-deficient mutant, it may be possible to substitute a less toxic compound as adjuvant in meningococcal outer membrane vaccines. Therefore, a broad panel of adjuvants were tested for their potential to enhance the immunogenicity of LPS-deficient OMCs. AlPO(4), Rhodobacter sphaeroides LPS, monophosphoryl lipid A and alkali-hydrolyzed meningococcal LPS showed significantly lower adjuvant activity than did H44/76 LPS. Adjuvant activity similar to H44/76 LPS was found for Escherichia coli LPS, meningococcal icsB and rfaC LPS, QuilA, subfractions of QuilA, and MF59. Good adjuvant activity was also found with meningococcal htrB1 LPS, containing penta-acylated lipid A. Antisera elicited with the less active adjuvants showed relatively high immunoglobulin G1 (IgG1) titers, whereas strong adjuvants also induced high IgG2a and IgG2b responses in addition to IgG1. Antisera with the IgG2a and IgG2b isotypes showed high bactericidal activity, indicating that adjuvants promoting the IgG2a and IgG2b response contribute most to the protective mechanism. Thus, this study demonstrates that the immunogenicity of meningococcal LPS-deficient OMCs can be restored by using less toxic adjuvants, which opens up new avenues for development of vaccines against meningococcal disease.

Ionic binding of 3H-gentamicin and short-term bactericidal activity of gentamicin against Pseudomonas aeruginosa isolates differing in lipopolysaccharide structure

The clinical isolates of Pseudomonas aeruginosa can be roughly classified into long- and short-lipopolysaccharide (LPS) strains and LPS-deficient strains, based on the silver-stained patterns of their LPSs after SDS-PAGE. The ionic binding of 3H-gentamicin, a polycationic antibiotic, to the negatively charged sites on the surface structures of P. aeruginosa strains, often differing in LPS structure, was the highest in the long-LPS strains followed in descending order by the short-LPS strains and LPS-deficient strains. It was presumed that a clinical isolate of P. aeruginosa No. 45 is lacking in the O-polysaccharide chains and some structures of the core-regions consisting of its LPS-structure after SDS-PAGE. On the other hand, the binding of 3H-gentamicin to this strain was quite. high, i.e., similar to that to of the long-LPS strains. To clarify this finding, P. aeruginosa PAC1R and its LPS-deficient mutants were used as reference strains because the chemical structures of their LPSs containing the repeated units of O-polysaccharides and the neutral sugar contents in the core-regions were previously confirmed. The PAC605 strain of the LPS-mutants of the PAC series, was completely lacking in the repeated units of O-polysaccharide and also lacking in some neutral sugar residues of the core-oligosaccharide region. However, this strain was highly bound to 3H-gentamicin, suggesting that the negatively charged sites on the deep core-oligosaccharide region and/or on lipid A participated in the binding of 3H-gentamicin. This manner of binding may be also applied to P. aeruginosa No. 45. When P. aeruginosa PAC1R, PAC605 and No. 45 strains were each exposed to gentamicin (20 micrograms/ml) for 10 minutes, the viable cell counts of PAC1R decreased to about 70% of the initial count, whereas the viable cell counts of PAC605 and No. 45 strains decreased to 3.6 and 11.0% respectively, indicating the vulnerability of both types of the strains to be enhanced by the bactericidal action of gentamicin with short-term incubation.

Gentamicin-induced alteration in drug susceptibility and lipopolysaccharide-composition of Pseudomonas aeruginosa isolates

Emergency of multi-drug resistance in Pseudomonas aeruginosa isolates after exposure to gentamicin was investigated with reference to their LPS-compositions. Three strains each of P. aeruginosa, different in LPS-compositions (long- and short-LPS strains, and LPS-deficient strain) were once or repeatedly incubated overnight at 35 degrees C in Mueller-Hinton broth including different concentrations of gentamicin, a poly-cationic antibiotic. LPS-compositions of the bacteria which survived and were grown after exposure to gentamicin were analyzed and the susceptibility to some of anti-pseudomonal drugs was determined. Of the test 9 strains after single exposure, No. 4 (long-LPS) and No. 41 (short-LPS) formed the LPS-deficient mutants. The resistance to ceftazidime and gentamicin was found in No. 4 strain, but not in No. 41 strain. Drug resistance in different patterns was also found in Nos. 43, 29, 45 and 52 strains without any change in their LPS-compositions. Contrary to these results, the susceptibility to norflxacin increased in Nos. 21 and 45 strains without any change in their LPS-compositions. After repeated exposures to gentamicin, the LPS-compositions of Nos. 4 and 14 (short-LPS) were altered and formed the LPS-deficient and the Intermediate-LPS (between short and long compositions) mutants, respectively. The LPS-alteration in both strains were accompanied by the development of resistance to ceftazidime and gentamicin, respectively. The drug resistance in different patterns were also found in Nos. 43, 156, 29, 41, 21, 45 and 52 strains, without any change in the LPS-compositions, whereas the increase in susceptibility to norfloxacin was simultaneously found in No. 45 strain. The ionic-binding of [3H]-gentamicin to the cell surfaces of two LPS-deficient mutants of P. aeruginosa decreased markedly, in comparison with that of the respective parents, and these results might lead to the drug resistance in the LPS-deficient mutants. However, the gentamicin-induced resistance of some strains of P. aeruginosa (but no change in the LPS-compositions) to the test drugs including gentamicin indicates a possibility that negative charges on the bacterial surface were decreased by the drug exposure. The drug resistance and LPS-compositions in the gentamicin-induced mutants of P. aeruginosa were stable during or after the 15th in vitro serial transfers.

Role of lipopolysaccharide in colonization of the mouse intestine by Salmonella typhimurium studied by in situ hybridization.

An avirulent, streptomycin-resistant Salmonella typhimurium strain, SL5319, and its lipopolysaccharide (LPS)-deficient mutant strain, SL5325, differ in their ability to colonize the large intestines of streptomycin-treated mice. When fed to mice independently, the strains colonize equally well, but when fed together, the LPS-deficient mutant is outcompeted by the wild-type strain during establishment in the gut (J.J. Nevola, B.A.D. Stocker, D.C. Laux, and P.S. Cohen, Infect. Immun. 50:152-159, 1985). In the present study, the spatial distribution in the intestinal mucosal layer of the two strains was visualized by specific hybridization to bacterial rRNA in histological sections of mouse colon and cecum. The first day after infection, 9.8% of the smooth SL5319 cells observed in mucus were found to be associated with the mouse epithelial cells, but three days after infection, the corresponding fraction of adhering bacteria was reduced to 2.1%. The LPS-deficient S. typhimurium strain was confined to the part of the mucosal layer closest to the colonic lumen and was not observed to adhere to the epithelium either at day 1 or 3 after infection. Quantitative determinations of the distance from the S. typhimurium cells to the epithelial wall confirmed that the average distance for the rough S. typhimurium SL5325 was much larger than for its smooth counterpart, S. typhimurium SL5319. Quantification of the hybridization signal from bacteria isolated from the cecal mucus revealed that the two strains had the same ribosome concentration, indicating that they have the same potential for growth in the intestinal environment. On the basis of these observations, we suggest that the better colonization ability of the strain carrying wild-type LPS is due to the better abilities to penetrate the intestinal mucosal layer and to subsequently bind to the epithelial cells in vivo.

Role of LPS length in clearance rate of bacteria from the bloodstream in mice.

Strains of Pseudomonas aeruginosa isolated from patients with cystic fibrosis (CF) never spread systemically. This may be due to serum sensitivity since these strains are very sensitive to complement-mediated bactericidal activity. A serum-resistant mutant, P. aeruginosa TUM3 HSR, was obtained from serum-sensitive strain TUM3 from a CF patient in order to clarify the mechanism of failure of systemic spread. LPS profiles on silver-stained gels and immunological analysis revealed that a long O-polysaccharide side chain was overproduced on the LPS molecules of TUM3 HSR as compared with the LPS of TUM3. The clearance rate from the bloodstream in mice was compared in the two strains. The number of TUM3 bacteria in 1 ml of blood, 10 min after injection into the tail vein, significantly decreased from 1.7 x 10(8) to 3.7 x 10(5) c.f.u. ml-1. In contrast, TUM3 HSR was not eliminated during the same period (decrease from 1.9 x 10(8) to 3.4 x 10(7) c.f.u. ml-1). Interestingly, these isogenic strains were not killed by 40% murine serum, probably reflecting immaturity of the complement-mediated killing system in mice. These results pointed to a correlation between LPS structure and blood clearance rate in mice. This was confirmed by examining blood clearance kinetics using the smooth-LPS strain Salmonella typhimurium LT2 and LPS-deficient mutants derived from it. S. typhimurium LT2 resisted blood clearance while the LPS-deficient mutants were cleared rapidly. None of the S. typhimurium strains were killed by murine serum. The number of P. aeruginosa TUM3 and S. typhimurium LPS-deficient mutants trapped in the liver following injection into the peripheral circulation was greater than that of their counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered Colonizing Ability for the Ceca of Broiler Chicks by Lipopolysaccharide-Deficient Mutants of Salmonella typhimurium

Salmonella typhimurium strain 3333/O was used to assess the role of bacterial lipopolysaccharide (LPS) in intestinal colonization of broiler chicks by salmonellae. LPS-defective TnPhoA mutants of this strain were isolated. The sensitivities of the mutants to smooth and rough phages and LPS banding patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a defect in the polysaccharide side chain of the LPS molecule. Colonization was determined by orally administering 10(8) cells each of the wild-type and/or the mutant strains per chick and counting the colony-forming units (CFU) from the ceca 1 to 3 weeks after gavage. CFU of chicks given the LPS-deficient strains either were not detected or were significantly lower than the CFU from chicks given the wild-type strain. The incidence of the wild-type strain in spleens was higher than incidence of the mutant strains. In vitro binding studies with LPS-deficient mutants derived in this study and from S. typhimurium LT2 suggest that LPS side-chain components may shield the bacterial cell from entrapment in the chicken mucus. The LPS layer appears to enhance persistence of Salmonella in the avian intestinal tract.

Characterization of lipopolysaccharide-deficient mutants of Pseudomonas aeruginosa derived from serotypes O3, O5, and O6.

Well-characterized rough mutants are important for the understanding of structures, functions, and biosynthesis of lipopolysaccharide (LPS) in gram-negative organisms. In this study, three series of Pseudomonas aeruginosa LPS-deficient mutants, namely PAC strains derived from serotype O3, AK strains derived from strain PAO1 (serotype O5), and serotype O6-derived mutants were subjected to biochemical analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining as well as immunochemical characterization using LPS-specific monoclonal antibodies. The O-side-chain deficiency among the O6-derived mutants was also examined, and three mutants, A28, R5, and H4, were subsequently chosen for the elucidation of component sugars of the core structure of serotype O6 LPS. LPS of strain A28 has L-rhamnose and proportionally higher amounts of D-glucose, a feature shared by the O5-derived mutant, strain AK1401 (previously demonstrated as a mutant with a core-plus-one O repeat). In contrast strains R5 and H4 were shown to be devoid of L-rhamnose and have low and undetectable amounts of D-glucose, respectively, which indicated their core deficiency. The LPS-deficient or -sufficient characteristics of the P. aeruginosa strains examined correlated will with serum sensitivity data. This report represents a comprehensive analysis of rough mutants derived from O3 and O5 strains that have been used by others in many studies and a first look at the core oligosaccharide region of serotype O6 LPS obtained with the O6-derived mutants generated in this study.

Ultrastructural examination of the lipopolysaccharides of Pseudomonas aeruginosa strains and their isogenic rough mutants by freeze-substitution.

The majority of Pseudomonas aeruginosa strains synthesize two antigenically distinct types of lipopolysaccharide (LPS), namely, a serotype-specific B-band LPS and a common antigen A-band LPS. A-band LPS consists of uncharged poly-D-rhamnan, which does not bind uranyl ions and is difficult to stain for electron microscopy; the highly charged B-band LPS is more easily visualized. We selected two wild-type strains, PAO1 (serotype O5) and IATS O6 (serotype O6), generated isogenic mutants from them, and examined the distribution of LPS on the surface of these organisms by freeze-substitution and electron microscopy. On PAO1 cells, which express both A-band and B-band LPSs, a 31- to 36-nm-wide fringe extending perpendicularly from the outer membrane was observed. A fine fibrous material was also observed on the surface of serotype O6 (A+ B+) cells, although this material did not form a uniform layer. When the LPS-deficient mutants, strains AK1401 (A+ B-), AK 1012 (A- B-), rd7513 (A- B-), and R5 (an IATS O6-derived rough mutant; A- B-), were examined, no extraneous material was apparent above the bilayer. However, an asymmetrical staining pattern was observed on the outer leaflet of the outer membrane of each of these mutants, presumably conforming to the anionic charge distribution of the core region of the rough LPS. In all cases, expression of the LPS types was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. When optical densitometry on electron microscopy negatives was used to analyze the outer membrane staining profiles, subtle differences in the degrees of core deficiency among rough mutants were detectable. This is the first time an electron microscopy technique has preserved the infrastructure produced in the outer membrane by its constituent macromolecules. We conclude that freeze-substitution electron microscopy is effective in the visualization of LPS morphotypes.