Toll-like receptor 4 (TLR4) is required for activation of innate immunity upon recognition of lipopolysaccharide (LPS) of Gram-negative bacteria. The ability of TLR4 to respond to a particular LPS ...species is important since insufficient activation may not prevent bacterial growth while excessive immune reaction may lead to immunopathology associated with sepsis. Here, we investigated the biological activity of LPS from Burkholderia mallei that causes glanders, and from the two well-known opportunistic pathogens Acinetobacter baumannii and Pseudomonas aeruginosa (causative agents of nosocomial infections). For each bacterial strain, R-form LPS preparations were purified by hydrophobic chromatography and the chemical structure of lipid A, an LPS structural component, was elucidated by HR-MALDI-TOF mass spectrometry. The biological activity of LPS samples was evaluated by their ability to induce production of proinflammatory cytokines, such as IL-6 and TNF, by bone marrow-derived macrophages. Our results demonstrate direct correlation between the biological activity of LPS from these pathogenic bacteria and the extent of their lipid A acylation.
The outer membrane is a key virulence determinant of gram‐negative bacteria. In Yersinia pestis, the deadly agent that causes plague, the protein Ail and lipopolysaccharide (LPS)6 enhance lethality ...by promoting resistance to human innate immunity and antibiotics, enabling bacteria to proliferate in the human host. Their functions are highly coordinated. Here we describe how they cooperate to promote pathogenesis. Using a multidisciplinary approach, we identify mutually constructive interactions between Ail and LPS that produce an extended conformation of Ail at the membrane surface, cause thickening and rigidification of the LPS membrane, and collectively promote Y. pestis survival in human serum, antibiotic resistance, and cell envelope integrity. The results highlight the importance of the Ail–LPS assembly as an organized whole, rather than its individual components, and provide a handle for targeting Y. pestis pathogenesis.
We identify mutually constructive interactions between the outer membrane protein Ail and lipopolysaccharide from Yersinia pestis that produce an extended conformation of Ail at the membrane surface, cause thickening and rigidification of the outer membrane, and collectively promote Y. pestis survival in human serum, antibiotic resistance and cell envelope integrity. The results highlight the importance of the Ail‐LPS assembly as an organized whole, rather than its individual components, and provide a handle for targeting Y. pestis pathogenesis.
It has been shown previously that several endemic Y. pestis isolates with limited virulence contained the I259 isoform of the outer membrane protease Pla, while the epidemic highly virulent strains ...possessed only the T259 Pla isoform. Our sequence analysis of the pla gene from 118 Y. pestis subsp. microtus strains revealed that the I259 isoform was present exclusively in the endemic strains providing a convictive evidence of more ancestral origin of this isoform. Analysis of the effects of the I259T polymorphism on the intrinsic disorder propensity of Pla revealed that the I259T mutation slightly increases the intrinsic disorder propensity of the C-terminal tail of Pla and makes this protein slightly more prone for disorder-based protein-protein interactions, suggesting that the T259 Pla could be functionally more active than the I259 Pla. This assumption was proven experimentally by assessing the coagulase and fibrinolytic activities of the two Pla isoforms in human plasma, as well as in a direct fluorometric assay with the Pla peptide substrate. The virulence testing of Pla-negative or expressing the I259 and T259 Pla isoforms Y. pestis subsp. microtus and subsp. pestis strains did not reveal any significant difference in LD50 values and dose-dependent survival assays between them by using a subcutaneous route of challenge of mice and guinea pigs or intradermal challenge of mice. However, a significant decrease in time-to-death was observed in animals infected with the epidemic T259 Pla-producing strains as compared to the parent Pla-negative variants. Survival curves of the endemic I259 Pla+ strains fit between them, but significant difference in mean time to death post infection between the Pla-strains and their I259 Pla+ variants could be seen only in the isogenic set of subsp. pestis strains. These findings suggest an essential role for the outer membrane protease Pla evolution in Y. pestis bubonic infection exacerbation that is necessary for intensification of epidemic process from endemic natural focality with sporadic cases in men to rapidly expanding epizootics followed by human epidemic outbreaks, local epidemics or even pandemics.
Mild acid degradation of the lipopolysaccharide of Yersinia kristensenii C-134 afforded a glycerol teichoic acid-like O-polysaccharide, which was studied by sugar analysis, O-deacetylation and ...dephosphorylation along with 1D and 2D NMR spectroscopy. The following structure of the O-polysaccharide was established:
This structure is related to those of other Y. kristensenii O-polysaccharides studied earlier. The O-antigen gene cluster of Y. kristensenii С-134 was analyzed and found to be consistent with the O-polysaccharide structure established.
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•Structure of the O-polysaccharide of Yersinia kristensenii C-134 was established.•The O-polysaccharide has a glycerol teichoic acid-like structure.•Dephosphorylation of the O-polysaccharide afforded a glycerol-linked hexasaccharide.•The O-antigen gene cluster was found to be consistent with the O-polysaccharide structure.
Hospital Klebsiella pneumoniae strains (n = 196) were collected in 2012-16 from the patients of a Moscow neurosurgical intensive care unit. Klebsiella pneumoniae strains were multidrug-resistant and ...carried beta-lactamase genes blaSHV (97.4% of strains), blaCTX-M (84.7%), blaTEM (56.1%), blaOXA-48-like (49.0%) and blaNDM-1 (one strain), class 1 integrons (43.4% of strains) and porin protein ompK36 gene (100% of strains). The ompK36 porin protein gene disruption by insertion sequence (IS) elements and OmpK36 production loss in two strains were detected in this study. Outer membrane proteins were isolated according to Carlone et al. (Rapid microprocedure for isolating detergent-insoluble outer membrane proteins from Haemophilus species. J Clin Microbiol 1986;24:330-2). The IS10R element belonging to the IS4 family, IS10 group was detected at the position of the 41st nucleotide of the ompK36 gene in K. pneumoniae strain KPB-2304K/15 (the first report for a certain IS element in K. pneumoniae). The IS1R element belonging to the IS1 family was identified at the position of the 86th nucleotide of the ompK36 gene in the K. pneumoniae strain KPB-367K/15 (novel insertion site for IS1 element into ompK36 gene). DNA transfer of the intact ompK36 gene into the strain KPB-367K/15 by vector plasmid restored OmpK36 porin protein production and resulted in a decrease of imipenem minimal inhibitory concentration. Such data confirm the importance of IS elements in ongoing multidrug-resistant evolution in hospital Klebsiella.
Lipopolysaccharide was isolated from bacteria Yersinia intermedia H9-36/83 (O:17) and degraded with mild acid to give an O-specific polysaccharide, which was isolated by GPC on Sephadex G-50 and ...studied by sugar analysis and 1D and 2D NMR spectroscopy. The polysaccharide was found to contain 3-deoxy-3-(R)-3-hydroxybutanoylamino-d-fucose (d-Fuc3NR3Hb) and the following structure of the heptasaccharide repeating unit was established:
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The structure established is consistent with the gene content of the O-antigen gene cluster. The O-polysaccharide structure and gene cluster of Y. intermedia are related to those of Hafnia alvei 1211 and Escherichia coli O:103.
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•Structure of the O-polysaccharide of Y. intermedia H9-36/83 was elucidated.•The polysaccharide was found to contain 3-deoxy-3-(R)-3-hydroxybutanoylamino-d-fucose.•The O-antigen gene cluster of Y. intermedia H9-36/83 is consistent with the O-polysaccharide structure.
Yersinia pestis spread throughout the Americas in the early 20th century, and it occurs predominantly as a single clone within this part of the world. However, within Eurasia and parts of Africa ...there is significant diversity among Y. pestis strains, which can be classified into different biovars (bv.) and/or subspecies (ssp.), with bv. orientalis/ssp. pestis most closely related to the American clone. To determine one aspect of the relatedness of these different Y. pestis isolates, the structure of the lipopolysaccharide (LPS) of four wild-type and one LPS-mutant Eurasian/African strains of Y. pestis was determined, evaluating effects of growth at mammalian (37 °C) or flea (25 °C) temperatures on the structure and composition of the core oligosaccharide and lipid A. In the wild-type clones of ssp. pestis, a single major core glycoform was synthesized at 37 °C whereas multiple core oligosaccharide glycoforms were produced at 25 °C. Structural differences occurred primarily in the terminal monosaccharides. Only tetraacyl lipid A was made at 37 °C, whereas at 25 °C additional pentaacyl and hexaacyl lipid A structures were produced. 4-Amino-4-deoxyarabinose levels in lipid A increased with lower growth temperatures or when bacteria were cultured in the presence of polymyxin B. In Y. pestis ssp. caucasica, the LPS core lacked d-glycero-d-manno-heptose and the content of 4-amino-4-deoxyarabinose showed no dependence on growth temperature, whereas the degree of acylation of the lipid A and the structure of the oligosaccharide core were temperature dependent. A spontaneous deep-rough LPS mutant strain possessed only a disaccharide core and a slightly variant lipid A. The diversity and differences in the structure of the Y. pestis LPS suggest important contributions of these variations to the pathogenesis of this organism, potentially related to innate and acquired immune recognition of Y. pestis and epidemiologic means to detect, classify, control and respond to Y. pestis infections.
To develop a modern plague vaccine, we used hypo-endotoxic
bacterial ghosts (BGs) with combinations of genes encoding the bacteriophage ɸX174 lysis-mediating protein E and/or holin-endolysin systems ...from λ or L-413C phages. Expression of the protein E gene resulted in the BGs that retained the shape of the original bacterium. Co-expression of this gene with genes coding for holin-endolysin system of the phage L-413C caused formation of structures resembling collapsed sacs. Such structures, which have lost their rigidity, were also formed as a result of the expression of only the L-413C holin-endolysin genes. A similar holin-endolysin system from phage λ containing mutated holin gene
and intact genes
coding for the endolysins caused generation of mixtures of BGs that had (i) practically preserved and (ii) completely lost their original rigidity. The addition of protein E to the work of this system shifted the equilibrium in the mixture towards the collapsed sacs. The collapse of the structure of BGs can be explained by endolysis of peptidoglycan sacculi. Immunizations of laboratory animals with the variants of BGs followed by infection with a wild-type
strain showed that bacterial envelopes protected only cavies. BGs with maximally hydrolyzed peptidoglycan had a greater protectivity compared to BGs with a preserved peptidoglycan skeleton.
Lipopolysaccharide (LPS) structure impacts the bactericidal action of cationic peptides, such as polymyxin B (PMB), and sensitivity to killing by normal human serum (NHS). Cultivation of different ...subspecies strains of Yersinia pestis isolated from unrelated geographic origins at various temperatures (mammals, 37°C; fleas, 25°C; or winter hibernation, 6°C) affects LPS composition and structure. We tested the susceptibilities of various strains of Y. pestis grown at these different temperatures to PMB and serum bactericidal killing. Both properties varied significantly in response to temperature changes. In Y. pestis subsp. pestis (the main subspecies causing human plague), high levels of resistance to PMB and NHS were detected at 25°C. However, at the same temperature, Y. pestis subsp. caucasica was highly sensitive to PMB. At both of the extreme temperatures, all strains were highly susceptible to PMB. At 25°C and 37°C, Y. pestis subsp. caucasica strain 1146 was highly susceptible to the bactericidal activity of 80% NHS. All Y. pestis strains studied were able to grow in heat-inactivated human serum or in 80% normal mouse serum. At 6°C, all strains were highly sensitive to NHS. Variations in the PMB resistance of different bacterial cultures related to both the content of cationic components (4-amino-4-deoxyarabinose in lipid A and glycine in the core) and a proper combination of terminal monosaccharides in the LPS. The NHS resistance correlated with an elevated content of N-acetylglucosamine in the LPS. Structural variation in the LPS of Y. pestis correlates with the organism's ability to resist innate immunity in both fleas and mammals.
A phosphorylated O-polysaccharide was isolated from the lipopolysaccharide of an entomopathogenic bacterium Photorhabdus temperata subsp. cinerea 3240 and studied by sugar analysis, ...dephosphorylation, and 1H and 13C NMR spectroscopy. The following structure of the linear trisaccharide repeating unit of the O-polysaccharide was established:
→3)-β-d-GalpNAc4PEtN-(1→4)-β-d-GlcpA-(1→3)-β-d-FucpNAc4N-(1→
where GlcA indicates glucuronic acid, FucNAc4N 2-acetamido-4-amino-2,4,6-trideoxygalactose, and PEtN 2-aminoethyl phosphate.
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•An O-polysaccharide was isolated from Photorhabdus temperata subsp. ≿inerea 3240.•Structure of the polysaccharide was established by NMR and chemical approaches.•The polysaccharide contains d-FucNAc4N and d-GalNAc 4-(2-aminoethyl phosphate).•The polysaccharide possesses multiple acidic and basic functions in each repeat.•The structure established is not related to the structures found in other Photorhabdus species.
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