ABSTRACT
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.
Subject(s)
Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/metabolism , Lipopolysaccharides/immunology , Lipopolysaccharides/metabolism , Virulence Factors/immunology , Virulence Factors/metabolism , Yersinia pestis/immunology , Yersinia pestis/metabolism , Amino Acid Motifs , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial , Humans , Microbial Sensitivity Tests , Molecular Dynamics Simulation , Mutation , Plague/immunology , Plague/microbiology , Protein Binding , Protein Conformation , Yersinia pestis/drug effectsABSTRACT
To develop a modern plague vaccine, we used hypo-endotoxic Yersinia pestis 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 S and intact genes R-Rz 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 Y. pestis 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.
ABSTRACT
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.
Subject(s)
Multigene Family/genetics , O Antigens/chemistry , O Antigens/genetics , Yersinia/chemistry , Yersinia/genetics , Carbohydrate Sequence , GenomicsABSTRACT
Structures of the O-antigens of Yersinia pseudotuberculosis O2c and O3 were reinvestigated by NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, (1)H,(13)C HSQC, and HMBC experiments. The following revised structure of the O2c tetrasaccharide repeating unit was established, which differs from the structure proposed earlier in the glycosylation pattern of the mannose residue at the branching point: [carbohydrate structure: see text] where Abe stands for 3,6-dideoxy-d-xylo-hexose. The structure of the Y. pseudotuberculosis O3 antigen reported earlier was confirmed.
Subject(s)
O Antigens/chemistry , Yersinia pseudotuberculosis/chemistry , Carbohydrate Sequence , Magnetic Resonance Spectroscopy , Molecular Sequence DataABSTRACT
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: 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.
Subject(s)
Lipopolysaccharides/chemistry , O Antigens/chemistry , Yersinia/chemistry , Multigene Family/geneticsABSTRACT
Yersinia pestis undergoes an obligate flea-rodent-flea enzootic life cycle. The rapidly fatal properties of Y. pestis are responsible for the organism's sustained survival in natural plague foci. Lipopolysaccharide (LPS) plays several roles in Y. pestis pathogenesis, prominent among them being resistance to host immune effectors and induction of a septic-shock state during the terminal phases of infection. LPS is acylated with 4-6 fatty acids, the number varying with growth temperature and affecting the molecule's toxic properties. Y. pestis mutants were constructed with a deletion insertion in the lpxM gene in both virulent and attenuated strains, preventing the organisms from synthesizing the most toxic hexa-acylated lipid A molecule when grown at 25 degrees C. The virulence and/or protective potency of pathogenic and attenuated Y. pestis DeltalpxM mutants were then examined in a mouse model. The DeltalpxM mutation in a virulent strain led to no change in the LD(50) value compared to that of the parental strain, while the DeltalpxM mutation in attenuated strains led to a modest 2.5-16-fold reduction in virulence. LPS preparations containing fully hexa-acylated lipid A were ten times more toxic in actinomycin D-treated mice then preparations lacking this lipid A isoform, although this was not significant (P>0.05). The DeltalpxM mutation in vaccine strain EV caused a significant increase in its protective potency. These studies suggest there is little impact from lipid A modifications on the virulence of Y. pestis strains but there are potential improvements in the protective properties in attenuated vaccine strains.
Subject(s)
Gene Deletion , Plague Vaccine/immunology , Plague/microbiology , Plague/prevention & control , Yersinia pestis/immunology , Yersinia pestis/pathogenicity , Animals , Genes, Bacterial/genetics , Lipid A/genetics , Mice , Virulence/genetics , Yersinia pestis/geneticsABSTRACT
Disruption of lipopolysaccharide (LPS) biosynthesis genes in an epidemiologically significant Yersinia pestis strain showed that the ability to synthesize the full inner core of the LPS is crucial for resistances to the bactericidal action of antimicrobial peptides and to complement-mediated serum killing. Resistance to polymyxin B also requires a high content of the cationic sugar, 4-amino-4-deoxy-L-arabinose, in lipid A.
Subject(s)
Lipopolysaccharides/chemistry , Yersinia pestis/chemistry , Antimicrobial Cationic Peptides/pharmacology , Blood Bactericidal Activity , Carbohydrate Sequence , Drug Resistance, Microbial/genetics , Genes, Bacterial , Humans , In Vitro Techniques , Molecular Sequence Data , Molecular Structure , Mutation , Polymyxin B/pharmacology , Spectrometry, Mass, Electrospray Ionization , Yersinia pestis/drug effects , Yersinia pestis/geneticsABSTRACT
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.
Subject(s)
Bacterial Proteins/genetics , DNA Transposable Elements , Drug Resistance, Multiple, Bacterial , Klebsiella pneumoniae/genetics , Porins/genetics , Anti-Bacterial Agents/pharmacology , Cloning, Molecular , Databases, Genetic , Hospitals , Humans , Imipenem/pharmacology , Integrons , Klebsiella Infections/drug therapy , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests , Sequence Analysis, DNA , beta-Lactamases/geneticsABSTRACT
Data on the structure and temperature-dependent variations of the lipopolysaccharide (LPS) of Yersinia pestis are summarized and compared with data of other enteric bacteria, including other Yersinia spp. A correlation between the LPS structure and properties of the LPS and bacterial cultures as well as the LPS biosynthesis control are briefly discussed.
Subject(s)
Lipopolysaccharides/chemistry , Plague/microbiology , Yersinia pestis/chemistry , Animals , Carbohydrate Sequence , Humans , Lipid A/biosynthesis , Lipid A/chemistry , Lipopolysaccharides/biosynthesis , Molecular Sequence Data , Yersinia pestis/metabolismABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , Isoenzymes/metabolism , Plasminogen Activators/metabolism , Yersinia pestis/enzymology , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Female , Guinea Pigs , Male , Mice , Mice, Inbred BALB C , Plasminogen Activators/chemistry , Sequence Homology, Amino Acid , Species Specificity , Virulence , Yersinia pestis/pathogenicityABSTRACT
Following a report of variations in the lipopolysaccharide (LPS) structure of Yersinia pestis at mammalian (37 degrees C) and flea (25 degrees C) temperatures, a number of changes to the LPS structure were observed when the bacterium was cultivated at a temperature of winter-hibernating rodents (6 degrees C). In addition to one of the known Y. pestis LPS types, LPS of a new type was isolated from Y. pestis KM218 grown at 6 degrees C. The core of the latter differs in: (i) replacement of terminal galactose with terminal d-glycero-d-manno-heptose; (ii) phosphorylation of terminal oct-2-ulosonic acid with phosphoethanolamine; (iii) a lower content of GlcNAc, and; (iv) the absence of glycine; lipid A differs in the lack of any 4-amino-4-deoxyarabinose and presumably partial (di)oxygenation of a fatty acid(s). The data obtained suggest that cold temperature switches on an alternative mechanism of control of the synthesis of Y. pestis LPS.
Subject(s)
Cold Temperature , Lipopolysaccharides/chemistry , Yersinia pestis/metabolism , Carbohydrate Sequence , Lipopolysaccharides/metabolism , Molecular Sequence Data , Molecular Structure , Yersinia pestis/growth & developmentABSTRACT
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 (1)H and (13)C NMR spectroscopy. The following structure of the linear trisaccharide repeating unit of the O-polysaccharide was established: â3)-ß-D-GalpNAc4PEtN-(1â4)-ß-D-GlcpA-(1â3)-ß-D-FucpNAc4N-(1â where GlcA indicates glucuronic acid, FucNAc4N 2-acetamido-4-amino-2,4,6-trideoxygalactose, and PEtN 2-aminoethyl phosphate.
Subject(s)
O Antigens/chemistry , Photorhabdus/metabolism , Carbohydrate Sequence , Magnetic Resonance Spectroscopy , Photorhabdus/immunologyABSTRACT
O-Polysaccharide was isolated from the lipopolysaccharide of an entomopathogenic bacterium Photorhabdus temperata subsp. temperata XlNach(T). Sugar analysis after full acid hydrolysis of the polysaccharide revealed D-glucose, D-mannose, D-galactose, D-GalNAc, and a branched monosaccharide, 3,6-dideoxy-4-C-[(S)-1',2'-dihydroxyethyl]-D-xylo-hexose (Sug), which was isolated as a 1,2'-anhydro furanose derivative. The following structure of the polysaccharide was established by 1D and 2D 1H and 13C NMR spectroscopy:
Subject(s)
Hexoses/chemistry , Photorhabdus/chemistry , Polysaccharides, Bacterial/chemistry , Carbohydrate Sequence , Molecular Sequence DataABSTRACT
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.
ABSTRACT
The rough-type lipopolysaccharide (LPS) of the plague pathogen, Yersinia pestis, was studied after mild-acid and strong-alkaline degradations by chemical analyses, NMR spectroscopy and electrospray-ionization mass spectrometry, and the following structure of the core region was determined:where L-alpha-D-Hep stands for L-glycero-alpha-D-manno-heptose, Sug1 for either 3-deoxy-alpha-D-manno-oct-2-ulosonic acid (alpha-Kdo) or D-glycero-alpha-D-talo-oct-2-ulosonic acid (alpha-Ko), and Sug2 for either beta-D-galactose or D-glycero-alpha-D-manno-heptose. A minority of the LPS molecules lacks GlcNAc.
Subject(s)
Lipopolysaccharides/chemistry , Yersinia pestis/chemistry , Carbohydrate Sequence , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Molecular Structure , Oligosaccharides/chemistry , Plague/microbiology , Spectrometry, Mass, Electrospray IonizationABSTRACT
An O-polysaccharide was isolated by mild acid degradation at pH 4.5 of the long-chain lipopolysaccharide of Yersinia pseudotuberculosis PB1 (serotype O:1a) and studied using 2D NMR spectroscopy. It was found to contain two uncommon monosaccharides: paratose (3,6-dideoxy-d-ribo-hexose, Par) in the furanose form and 6-deoxy-d-manno-heptose (d-6dmanHep). The following structure of a branched tetrasaccharide repeat (O-unit) with a disaccharide side chain was established: This structure is at variance with the O-polysaccharide structure of Y. pseudotuberculosis O:1a reported earlier (Komandrova, N. A.; Gorshkova, R. P.; Isakov, V. V.; Ovodov, Y. S. Bioorg. Khim.1984, 10, 232-237). A comparative study by high-resolution ESI MS of the short-chain lipopolysaccharides from strain PB1 and a wbyM mutant thereof confirmed the function of wbyM as the paratosyltransferase gene.
Subject(s)
O Antigens/chemistry , O Antigens/metabolism , Transferases/metabolism , Yersinia pseudotuberculosis/enzymology , Carbohydrate Sequence , Molecular Sequence Data , Transferases/genetics , Yersinia pseudotuberculosis/geneticsABSTRACT
The O-polysaccharide from the lipopolysaccharide of a symbiotic bacterium Photorhabdus luminescens subsp. laumondii TT01 from an insect-pathogenic nematode was studied by sugar analysis and (1)H and (13)C NMR spectroscopy and found to contain D-glycero-D-manno-heptose (DDHep) and 3,6-dideoxy-3-formamido-D-glucose (D-Qui3NFo). The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established:
Subject(s)
Glucosamine/analogs & derivatives , Heptoses/chemistry , Photorhabdus/chemistry , Polysaccharides/chemistry , Carbohydrate Sequence , Glucosamine/chemistry , Magnetic Resonance Spectroscopy , Molecular Sequence DataABSTRACT
The O-polysaccharides were isolated from the lipopolysaccharides of emerging human pathogens Photorhabdus asymbiotica subsp. asymbiotica US-86 and US-87 and subsp. australis AU36, AU46, and AU92. Studies by sugar analysis and (1)H and (13)C NMR spectroscopy before and after O-deacetylation showed that the O-polysaccharide structures are essentially identical within, and only slightly different between, the subspecies. The following structures of the repeating units of the O-polysaccharides were established: â3)-ß-d-Quip4NGlyFo-(1â4)-α-d-GalpNAcAN3Ac-(1â4)-α-d-GalpNAcA3R-(1â3)-α-d-QuipNAc-(1â where GalNAcA stands for 2-acetamido-2-deoxygalacturonic acid, GalNAcAN for amide of GalNAcA, QuiNAc for 2-acetamido-2,6-dideoxyglucose, and Qui4NGlyFo for 4,6-dideoxy-4-(N-formylglycyl)aminoglucose; R=Ac in subsp. asymbiotica or H in subsp. australis. The structures established resemble those of a number of taxonomically remote bacteria including Francisella tularensis (Vinogradov, E. V.; Shashkov, A. S.; Knirel, Y. A.; Kochetkov, N. K.; Tochtamysheva, N. V.; Averin, S. P.; Goncharova, O. V.; Khlebnikov, V. S. Carbohydr. Res.1991, 214, 289-297), which differs in (i) the presence of a formyl group on Qui4N rather than the N-formylglycyl group, (ii) the mode of the linkage between the repeating units (ß1â2 vs α1â3), (iii) amidation of both GalNAcA residues rather than one residue, and iv) the lack of O-acetylation.