Functional characterization and biological significance of Yersinia pestis lipopolysaccharide biosynthesis genes

The Gram negative bacterium Yersinia pestis is the etiological agent of flea transmitted fulminant systemic rodent zoonosis and the reason of the three devastating pandemics of plague Lipopolysaccharide (LPS, endotoxin) is an impor tant factor of pathogenicity of Gram negative bacteria. The full LPS molecule (S form LPS) consists of three well defined domains: i) lipid A composed of sugars, fatty acids, and phosphate; it represents the endotoxic princi ple of the LPS and anchors it in the outer membrane; ii) a core oligosaccharide containing charged groups; and iii) an O specific polysaccharide (O antigen), which carries ISSN 0006 2979, Biochemistry (Moscow), 2011, Vol. 76, No. 7, pp. 808 822. © Pleiades Publishing, Ltd., 2011. Published in Russian in Biokhimiya, 2011, Vol. 76, No. 7, pp. 989 1005 Abstract-In silico analysis of available bacterial genomes revealed the phylogenetic proximity levels of enzymes responsible for biosynthesis of lipopolysaccharide (LPS) of Yersinia pestis, the cause of plague, to homologous proteins of closely relat ed Yersinia spp. and some other bacteria (Serratia proteamaculans, Erwinia carotovora, Burkholderia dolosa, Photorhabdus luminescens and others). Isogenic Y. pestis mutants with single or double mutations in 14 genes of LPS biosynthetic path ways were constructed by site directed mutagenesis on the base of the virulent strain 231 and its attenuated derivative. Using high resolution electrospray ionization mass spectrometry, the full LPS structures were elucidated in each mutant, and the sequence of monosaccharide transfers in the assembly of the LPS core was inferred. Truncation of the core decreased sig nificantly the resistance of bacteria to normal human serum and polymyxin B, the latter probably as a result of a less effi cient incorporation of 4 amino 4 deoxyarabinose into lipid A. Impairing of LPS biosynthesis resulted also in reduction of LPS dependent enzymatic activities of plasminogen activator and elevation of LD 50 and average survival time in mice and guinea pigs infected with experimental plague. Unraveling correlations between biological properties of bacteria and partic ular LPS structures may help a better understanding of pathogenesis of plague and implication of appropriate genes as potential molecular targets for treatment of plague

Competitive wholesale market of the electric power and power: state and new calls

In article features of functioning of the main segments of the wholesale market of the electric power and power (OREM) are considered. The factors exerting impact on competitiveness of the generation companies come to light. Results of functioning of OREM, since the moment of his full liberalization are analyzed, merits and demerits of the operating market mechanisms reveal

Intraspecies and Temperature-Dependent Variations in Susceptibility of Yersinia pestis to the Bactericidal Action of Serum and to Polymyxin B

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

Effect of deletion of the lpxM gene on virulence and vaccine potential of Yersinia pestis in mice

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 6C. The virulence and/or protective potency of pathogenic and attenuated Y. pestis DlpxM mutants were then examined in a mouse model. The DlpxM mutation in a virulent strain led to no change in the LD 50 value compared to that of the parental strain, while the DlpxM 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 DlpxM 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. INTRODUCTION Yersinia pestis survives within an enzootic cycle involving infection of susceptible rodent populations and transmission via flea vectors The triggering event of septic shock caused by Gramnegative bacteria is most likely the release of lipopolysaccharide (LPS). Lipid A, the toxic portion of the LPS molecule, causes the release of numerous host proinflammatory cytokines, and activates the complement cascade and the coagulation cascade. Recent studies suggest that Toll-like receptors, inflammatory cytokines, eicosanoids, free radicals, macrophage migration inhibitory factor, signal protein kinases and transcription factors all play an important part in the pathobiology of Gram-negativemediated septic shock lpxM (alternatively msbB or waaN) Recently it was shown that although LPSs from DlpxM mutants had decreased endotoxic activity, they were still as potent adjuvants of the immune response as hexa-acylated molecules from the parent strains In this study, we investigated the effects of variations in the lipid A structure on the endotoxic activity of the LPS and on the pathogenicity of Y. pestis strains that differ in their initial virulence potential. To perform these investigations, we created DlpxM mutants in wild-type strain 231 as well as in attenuated strains, including the Russian vaccine strain, EV, line NIIEG. We also investigated the effect of a DlpxM mutation on the vaccine properties of strain EV. This mutation resulted in an inability to synthesize the hexa-acyl lipid A (LA hexa ) structure found in the parental strains when organisms are grown at 25 u C. Overall, we found a tenfold increase in the LD 50 of the LPS from the DlpxM mutant, but this was not significant at P . 0.05. Inability of the virulent Y. pestis 231 DlpxM mutant to produce a hexa-acylated LPS did not change its ability to cause a lethal infection, while in the attenuated strains there was a modest reduction in virulence. Of note, in the vaccine strain, there was improved protective efficacy and decreased reactogenicity following immunization with the DlpxM mutant. METHODS Bacterial strains, plasmids and primers. The characteristics of the Y. pestis and E. coli strains used in this study are given in 444 Journal of Medical Microbiology 56 the mutant strains contained genes for LPS biogenesis, and did not exert influence on LPS structure when compared with the 'wild-type' in any of the strains Medium and culture conditions. E. coli strains were routinely grown on Luria-Bertani (LB) agar or in LB broth The pMSB3K plasmid was introduced into Y. pestis strains by conjugation using polymyxin for counter-selection, the Km R Ap R exoconjugants were then counter-selected by growing on plates containing 5 % sucrose, and the Km R Ap S colonies in which allelic exchange had occurred were selected and deletion of the chromosomal lpxM gene confirmed by PCR Isolation of LPS and SDS-PAGE. LPSs were extracted from dried cells with phenol/chloroform/light petroleum ether Mass spectrometry. High-resolution electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) MS was performed in the negative ion mode using an Apex II instrument (Bruker Daltonics) equipped with a 7 T actively shielded magnet and an Apollo electrospray ion source as described previously 21 . Capillary entrance voltage was set to 3.8 kV, and dry gas temperature to 150 uC. The spectra were charge deconvoluted, and mass numbers given refer to the monoisotopic molecular masses. Animals. Outbred Swiss Webster mice weighing approximately 20 g were used in animal experiments that were approved by the ethical committee of the State Research Center for Applied Microbiology and Biotechnology. Animals were kept in cages in groups of four to eight, and allowed to feed and drink ad libitum. Median lethal doses (LD 50 ), 50 % immunizing doses (ImD 50 ) and 95 % confidence intervals (CI) were measured according to the method of Kärber Immunization and challenge. Bacterial cultures of either the EV line NIIEG strain or the EVDlpxM mutant were grown for 48 h at 25 uC and administered subcutaneously in a total volume of 0.2 ml 0.9 % NaCl solution (with tenfold dilutions from 10 9 to 10 5 c.f.u.) as a single injection on day 0 (70 mice, divided into 10 groups of 7). Two groups of seven mice were treated only with 0.9 % NaCl solution. At day 21 post-immunization the mice were challenged subcutaneously with 3. RESULTS Isolation and SDS-PAGE characterization of the LPSs Each Y. pestis strain was grown at 25 u C and the corresponding LPS samples were isolated by phenol/chloroform/ light petroleum extraction and fractionated by SDS-PAGE. Examples of LPS migration for two pairs of isogenic strains are shown in Structural studies of the LPSs LPS parent and LPS DlpxM from Y. pestis KM218 and KM260(11) were degraded under mild acid conditions to cleave the linkage between the core and lipid A. The isolated and purified lipid A and core oligosaccharide, as well as the whole LPS samples, were studied by ESI FT-ICR MS. The mass spectrum of the lipid A sample from Y. pestis KM218 LPS parent No significant changes in the core composition and the content of the cationic sugar, 4-amino-4-deoxy-L-arabinose (Ara4N), were observed in LPS parent and LPS DlpxM from both strains studied (data not shown). Therefore, our data confirmed the finding of Rebeil et al. (2006) that Y. pestis DlpxM mutants are unable to incorporate the dodecanoyl group into lipid A and showed that the other LPS biosynthesis pathways are unaffected by the mutation. Toxicity of LPSs in actinomycin D-sensitized mice Several substances Affect of the deletion of lpxM on the lethality of virulent and attenuated Y. pestis strains in a murine system The Y. pestis parental and DlpxM strains were used to assess the contribution to virulence made by having the genetic ability to synthesize a LA hexa . There was no effect on virulence of the Y. pestis wild-type strain 231 due to deletion of lpxM When attenuated strains (i.e. lacking the pCD plasmid or with further mutations) of Y. pestis are injected into mice, the residual virulence can be evaluated with either the use of very high infective doses or immunocompromised animals treated with immunosuppressant