lipid-a and Plague

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.

Topics: Animals; Carbohydrate Sequence; Humans; Lipid A; Lipopolysaccharides; Molecular Sequence Data; Plague; Yersinia pestis

Significance

Topics: Animals; Antibodies, Bacterial; Antigens, Bacterial; Lethal Dose 50; Lipid A; Mice; Plague; Plague Vaccine; Plasmids; Pore Forming Cytotoxic Proteins; Yersinia pseudotuberculosis

The infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking. Here, we describe our discovery and characterization of a biphenylacetylene-based inhibitor of LpxC, an essential enzyme in the biosynthesis of the lipid A component of the outer membrane of Gram-negative bacteria. The compound LPC-069 has no known adverse effects in mice and is effective

Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Benzamides; Disease Models, Animal; Drug Resistance, Multiple, Bacterial; Enzyme Inhibitors; Female; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Lipid A; Mice; Morpholines; Plague; Yersinia pestis

Yersinia pestis, the causative agent of plague, can be transmitted by fleas by two different mechanisms: by early-phase transmission (EPT), which occurs shortly after flea infection, or by blocked fleas following long-term infection. Efficient flea-borne transmission is predicated upon the ability of Y. pestis to be maintained within the flea. Signature-tagged mutagenesis (STM) was used to identify genes required for Y. pestis maintenance in a genuine plague vector, Xenopsylla cheopis. The STM screen identified seven mutants that displayed markedly reduced fitness in fleas after 4 days, the time during which EPT occurs. Two of the mutants contained insertions in genes encoding glucose 1-phosphate uridylyltransferase (galU) and UDP-4-amino-4-deoxy-l-arabinose-oxoglutarate aminotransferase (arnB), which are involved in the modification of lipid A with 4-amino-4-deoxy-l-arabinose (Ara4N) and resistance to cationic antimicrobial peptides (CAMPs). These Y. pestis mutants were more susceptible to the CAMPs cecropin A and polymyxin B, and produced lipid A lacking Ara4N modifications. Surprisingly, an in-frame deletion of arnB retained modest levels of CAMP resistance and Ara4N modification, indicating the presence of compensatory factors. It was determined that WecE, an aminotransferase involved in biosynthesis of enterobacterial common antigen, plays a novel role in Y. pestis Ara4N modification by partially offsetting the loss of arnB. These results indicated that mechanisms of Ara4N modification of lipid A are more complex than previously thought, and these modifications, as well as several factors yet to be elucidated, play an important role in early survival and transmission of Y. pestis in the flea vector.

Topics: Animals; Bacterial Proteins; Humans; Insect Vectors; Lipid A; Microbial Viability; Plague; Rats; Rats, Sprague-Dawley; Siphonaptera; Yersinia pestis

Fibrin has been demonstrated to function protectively against pathogens in our previous studies, but we observed that a very high level of fibrin played a negative role during infection. We performed this research to address the complication.. After infection, mice were monitored daily and harvested on day 4. The fibrin levels within the tissue samples were quantified by Western-blot. The in situ assay was used to detect plasminogen activators, protein C-ase and prothrombinase activation. PT-PCR was used to test coagulation factors expression.. Mice treated with Coumadin showed that the protection correlates with fibrin levels. By interacting with Toll-like receptor 4, the hexa-acylated lipopolysaccharide, although not the tetra-acylated lipopolysaccharide, activates coagulation and regulates plasminogen activator inhibitor 1, thrombin activatable fibrinolysis inhibitor and thrombomodulin expression through myeloid differentiation factor 88, leading to plasminogen activators, protein C-ase and prothrombinase activation and fibrin formation. Because of the regulation, fibrin formation was controlled to deposit appropriate levels and confer protection.. We demonstrated that the appropriate level of fibrin formation was deployed by hexa-acylated LPS-lipid A through myeloid differentiation factor 88 to confer protection.

Topics: Animals; Blood Coagulation; Fibrin; Lipid A; Mice; Mice, Inbred C57BL; Myeloid Differentiation Factor 88; Plague; Plasminogen Activator Inhibitor 1; Plasminogen Activators; Protein C; Thrombomodulin; Toll-Like Receptor 4

Here we report the development of a new cationic liposome-hyaluronic acid (HA) hybrid nanoparticle (NP) system and present our characterization of these NPs as an intranasal vaccine platform using a model antigen and F1-V, a candidate recombinant antigen for Yersinia pestis, the causative agent of plague. Incubation of cationic liposomes composed of DOTAP and DOPE with anionic HA biopolymer led to efficient ionic complexation and formation of homogenous liposome-polymer hybrid NPs, as evidenced by fluorescence resonance energy transfer, dynamic light scattering, and nanoparticle tracking analyses. Incorporation of cationic liposomes with thiolated HA allowed for facile surface decoration of NPs with thiol-PEG, resulting in the formation of DOTAP/HA core-PEG shell nanostructures. These NPs, termed DOTAP-HA NPs, exhibited improved colloidal stability and prolonged antigen release. In addition, cytotoxicity associated with DOTAP liposomes (LC50~0.2mg/ml) was significantly reduced by at least 20-fold with DOTAP-HA NPs (LC50>4mg/ml), as measured with bone marrow derived dendritic cells (BMDCs). Furthermore, NPs co-loaded with ovalbumin (OVA) and a molecular adjuvant, monophosphoryl lipid A (MPLA) promoted BMDC maturation and upregulation of co-stimulatory markers, including CD40, CD86, and MHC-II, and C57BL/6 mice vaccinated with NPs via intranasal route generated robust OVA-specific CD8(+) T cell and antibody responses. Importantly, intranasal vaccination with NPs co-loaded with F1-V and MPLA induced potent humoral immune responses with 11-, 23-, and 15-fold increases in F1-V-specific total IgG, IgG1, and IgG2c titers in immune sera by day 77, respectively, and induced balanced Th1/Th2 humoral immune responses, whereas mice immunized with the equivalent doses of soluble F1-V vaccine failed to achieve sero-conversion. Overall, these results suggest that liposome-polymer hybrid NPs may serve as a promising vaccine delivery platform for intranasal vaccination against Y. pestis and other infectious pathogens.

Topics: Adaptive Immunity; Administration, Intranasal; Animals; Antigens; Cations; Colloids; Dendritic Cells; Hyaluronic Acid; Immunity, Humoral; Lipid A; Liposomes; Mice; Mice, Inbred C57BL; Nanoparticles; Plague; Vaccination; Vaccines; Yersinia pestis

Pulmonary immunization poses the unique challenge of balancing vaccine efficacy with minimizing inflammation in the respiratory tract. While previous studies have shown that mice immunized intranasally with F1-V-loaded polyanhydride nanoparticles are protected from a lethal challenge with Yersinia pestis, little is known about the initial interaction between the nanoparticles and immune cells following intranasal administration. Here, the deposition within the lung and internalization by phagocytic cells of polyanhydride nanovaccines encapsulating F1-V are compared with that of soluble F1-V alone or F1-V adjuvanted with monophosphoryl lipid A (MPLA). Encapsulation of F1-V into polyanhydride nanoparticles prolonged its presence while F1-V administered with MPLA is undetectable within 48 h. The inflammation induced by the polyanhydride nanovaccine is mild compared with the marked inflammation induced by the MPLA-adjuvanted F1-V. Even though F1-V delivered with saline is detected in the lung 48 h after administration, it is known that this regimen does not elicit a protective immune response. The prolonged F1-V presence in the lung in concert with the mild inflammatory response provided by the nanovaccine provides new insights into the development of protective immune responses with a single intranasal dose.

Topics: Adjuvants, Immunologic; Animals; Female; Immunization; Lipid A; Lung; Mice; Mice, Inbred C57BL; Nanostructures; Plague; Pneumonia; Polyanhydrides; Vaccines; Yersinia pestis

Synthesis of Escherichia coli LpxL, which transfers a secondary laurate chain to the 2' position of lipid A, in Yersinia pestis produced bisphosphoryl hexa-acylated lipid A at 37°C, leading to significant attenuation of virulence. Our previous observations also indicated that strain χ10015(pCD1Ap) (ΔlpxP32::P(lpxL) lpxL) stimulated a strong inflammatory reaction but sickened mice before recovery and retained virulence via intranasal (i.n.) infection. The development of live, attenuated Y. pestis vaccines may be facilitated by detoxification of its lipopolysaccharide (LPS). Heterologous expression of the lipid A 1-phosphatase, LpxE, from Francisella tularensis in Y. pestis yields predominantly 1-dephosphorylated lipid A, as confirmed by mass spectrometry. Results indicated that expression of LpxE on top of LpxL provided no significant reduction in virulence of Y. pestis in mice when it was administered i.n. but actually reduced the 50% lethal dose (LD(50)) by 3 orders of magnitude when the strain was administered subcutaneously (s.c.). Additionally, LpxE synthesis in wild-type Y. pestis KIM6+(pCD1Ap) led to slight attenuation by s.c. inoculation but no virulence change by i.n. inoculation in mice. In contrast to Salmonella enterica, expression of LpxE does not attenuate the virulence of Y. pestis.

Topics: Acyltransferases; Animals; Disease Models, Animal; Escherichia coli Proteins; Gene Expression; Lethal Dose 50; Lipid A; Mass Spectrometry; Membrane Proteins; Mice; Phosphoric Monoester Hydrolases; Plague; Recombinant Proteins; Survival Analysis; Virulence; Virulence Factors; Yersinia pestis

Although lipopolysaccharide (LPS) stimulation through the Toll-like receptor (TLR)-4/MD-2 receptor complex activates host defense against Gram-negative bacterial pathogens, how species-specific differences in LPS recognition impact host defense remains undefined. Herein, we establish how temperature dependent shifts in the lipid A of Yersinia pestis LPS that differentially impact recognition by mouse versus human TLR4/MD-2 dictate infection susceptibility. When grown at 37°C, Y. pestis LPS is hypo-acylated and less stimulatory to human compared with murine TLR4/MD-2. By contrast, when grown at reduced temperatures, Y. pestis LPS is more acylated, and stimulates cells equally via human and mouse TLR4/MD-2. To investigate how these temperature dependent shifts in LPS impact infection susceptibility, transgenic mice expressing human rather than mouse TLR4/MD-2 were generated. We found the increased susceptibility to Y. pestis for "humanized" TLR4/MD-2 mice directly paralleled blunted inflammatory cytokine production in response to stimulation with purified LPS. By contrast, for other Gram-negative pathogens with highly acylated lipid A including Salmonella enterica or Escherichia coli, infection susceptibility and the response after stimulation with LPS were indistinguishable between mice expressing human or mouse TLR4/MD-2. Thus, Y. pestis exploits temperature-dependent shifts in LPS acylation to selectively evade recognition by human TLR4/MD-2 uncovered with "humanized" TLR4/MD-2 transgenic mice.

Topics: Acylation; Animals; Cell Line; Chromosomes, Artificial, Bacterial; Cytokines; Escherichia coli; Escherichia coli Infections; HEK293 Cells; Humans; Lipid A; Lipopolysaccharides; Lymphocyte Antigen 96; Mice; Mice, Inbred C57BL; Mice, Transgenic; Plague; Salmonella enterica; Salmonella Infections, Animal; Signal Transduction; Temperature; Toll-Like Receptor 4; Yersinia pestis

Yersinia pestis, the causative agent of plague, is a potential weapon of bioterrorism. Y. pestis evades the innate immune system by synthesizing tetra-acylated lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity at 37°C, whereas hexa-acylated lipid A, a potent TLR4 agonist, is made at lower temperatures. Synthesis of Escherichia coli LpxL, which transfers the secondary laurate chain to the 2'-position of lipid A, in Y. pestis results in production of hexa-acylated lipid A at 37°C, leading to significant attenuation of virulence. Previously, we described a Y. pestis vaccine strain in which crp expression is under the control of the arabinose-regulated araC P(BAD) promoter, resulting in a 4-5 log reduction in virulence. To reduce the virulence of the crp promoter mutant further, we introduced E. coli lpxL into the Y. pestis chromosome. The χ10030(pCD1Ap) (ΔlpxP32::P(lpxL)lpxL ΔP(crp21)::TT araC P(BAD)crp) construct likewise produced hexa-acylated lipid A at 37°C and was significantly more attenuated than strains harboring each individual mutation. The LD(50) of the mutant in mice, when administered subcutaneously or intranasally was >10(7)-times and >10(4)-times greater than wild type, respectively. Mice immunized subcutaneously with a single dose of the mutant were completely protected against a subcutaneous challenge of 3.6×10(7) wild-type Y. pestis and significantly protected (80% survival) against a pulmonary challenge of 1.2×10(4) live cells. Intranasal immunization also provided significant protection against challenges by both routes. This mutant is an immunogenic, highly attenuated live Y. pestis construct that merits further development as a vaccine candidate.

Topics: Acyltransferases; Administration, Intranasal; Animals; Antibodies, Bacterial; Cytokines; Escherichia coli Proteins; Female; Injections, Subcutaneous; Lipid A; Mice; Plague; Plague Vaccine; Plasmids; Promoter Regions, Genetic; Vaccines, Attenuated; Virulence; Yersinia pestis

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 related 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 pathways 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 significantly the resistance of bacteria to normal human serum and polymyxin B, the latter probably as a result of a less efficient 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 particular LPS structures may help a better understanding of pathogenesis of plague and implication of appropriate genes as potential molecular targets for treatment of plague.

Topics: Amino Sugars; Animals; Blood Bactericidal Activity; Drug Resistance, Bacterial; Female; Genes, Bacterial; Guinea Pigs; Humans; Lipid A; Lipopolysaccharides; Male; Mice; Plague; Plasminogen Activators; Polymyxin B; Spectrometry, Mass, Electrospray Ionization; Virulence; Yersinia pestis

This study analysed the effect of priming the innate immune system using synthetic lipid A mimetics in a Yersinia pestis murine pulmonary infection model. Two aminoalkyl glucosaminide 4-phosphate (AGP) Toll-like receptor 4 (TLR4) ligands, delivered intranasally, extended time to death or protected against a lethal Y. pestis CO92 challenge. The level of protection was dependent upon the challenge dose of Y. pestis and the timing of AGP therapy. Protection correlated with cytokine induction and a decreased bacterial burden in lung tissue. AGP protection was TLR4-dependent and was not evidenced in transgenic TLR4-deficient mice. AGP therapy augmented with subtherapeutic doses of gentamicin produced dramatically enhanced survival. Combined, these results indicated that AGPs may be useful in protection of immunologically naive individuals against plague and potentially other infectious agents, and that AGP therapy may be used synergistically with other therapies.

Topics: Animals; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Glucosamine; Humans; Immunity, Innate; Lipid A; Lung; Mice; Mice, Inbred BALB C; Mice, Knockout; Organophosphorus Compounds; Plague; Specific Pathogen-Free Organisms; Time Factors; Toll-Like Receptor 4; Yersinia pestis

An effective intranasal (i.n.) vaccine against pneumonic plague was developed. The formulation employed two synthetic lipid A mimetics as adjuvant combined with Yersinia pestis-derived V- and F1-protective antigens. The two nontoxic lipid A mimetics, classed as amino-alkyl glucosaminide 4-phosphates (AGPs) are potent ligands for the Toll-like receptor (TLR) 4. Using a murine (BALB/c) pneumonic plague model, we showed a single i.n. application of the vaccine provided 63% protection within 21 days against a Y. pestis CO92 100 LD50 challenge. Protection reached 100% by 150 days. Using a homologous i.n. 1 degrees /2 degrees dose regimen, with the boost administered at varying times, 63% protection was achieved within 7 days and 100% protection was achieved by 21 days after the first immunization. Little or no protection was observed in animals that received antigens alone, and no protection was observed when the vaccine was administered to BALB/c TLR4 mutant mice. Vaccine-induced serum IgG titers to F1 and V-antigen were reflected in high titers for IgG1 and IgG2a, the latter reflecting a bias for a cell-mediated (TH1) immune response. This intranasal vaccine showed 90% protection in Sprague-Dawley rats challenged with 1000 LD50. We conclude that lipid A mimetics are highly effective adjuvants for an i.n. plague vaccine.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Antibodies, Bacterial; Disease Models, Animal; Female; Glucosamine; Humans; Lipid A; Male; Mice; Mice, Inbred BALB C; Molecular Mimicry; Plague; Plague Vaccine; Rats; Rats, Sprague-Dawley; Toll-Like Receptor 4; Yersinia pestis

At the temperature of its flea vector (approximately 20-30 degrees C), the causative agent of plague, Yersinia pestis, expresses a profile of genes distinct from those expressed in a mammalian host (37 degrees C). When dendritic cells (DC) are exposed to Y. pestis grown at 26 degrees C (Y. pestis-26 degrees), they secrete copious amounts of IL-12p40 homodimer (IL-12(p40)(2)). In contrast, when DCs are exposed to Y. pestis grown at 37 degrees C (Y. pestis-37 degrees), they transcribe very little IL-12p40, which is secreted as IL-12p40 monomer (IL-12p40). Y. pestis-26 degrees also induces migration of DCs to the homeostatic chemokine CCL19, whereas Y. pestis-37 degrees does not; migratory DCs are positive for IL-12p40 transcription and secrete mostly IL-12(p40)(2); DCs lacking IL-12p40 do not migrate. Expression of acyltransferase LpxL from Escherichia coli in Y. pestis-37 degrees results in the production of a hexa-acylated lipid A, also seen in Y. pestis-26 degrees, rather than tetra-acylated lipid A normally seen in Y. pestis-37 degrees. The LpxL-expressing Y. pestis-37 degrees promotes DC IL-12(p40)(2) production and induction of DC migration. In addition, absence of TLR4 ablates production of IL-12(p40)(2) in DC exposed to Y. pestis-26 degrees. The data demonstrate the molecular pathway by which Y. pestis evades induction of early DC activation as measured by migration and IL-12(p40)(2) production.

Topics: Acetylation; Acyltransferases; Animals; Cell Movement; Cells, Cultured; Chemokine CCL19; Dendritic Cells; Dimerization; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Hot Temperature; Humans; Insect Vectors; Interleukin-12 Subunit p40; Lipid A; Mice; Mice, Knockout; Plague; Siphonaptera; Toll-Like Receptor 4; Yersinia pestis

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.

Topics: Animals; Gene Deletion; Genes, Bacterial; Lipid A; Mice; Plague; Plague Vaccine; Virulence; Yersinia pestis

The lpxM mutant of the live vaccine Yersinia pestis EV NIIEG strain synthesising a less toxic penta-acylated lipopolysaccharide was found to be avirulent in mice and guinea pigs, notably showing no measurable virulence in Balb/c mice which do retain some susceptibility to the parental strain itself. Twenty-one days after a single injection of the lpxM-mutant, 85-100% protection was achieved in outbred mice and guinea pigs, whereas a 43% protection rate was achieved in Balb/c mice given single low doses (10(3) to 2.5 x 10(4) CFU) of this vaccine. A subcutaneous challenge with 2000 median lethal doses (equal to 20,000 CFU) of fully virulent Y. pestis 231 strain, is a 6-10-fold higher dose than that which the EV NIIEG itself can protect against.

Topics: Animals; Female; Gene Deletion; Guinea Pigs; Lipid A; Mice; Mice, Inbred BALB C; Plague; Plague Vaccine; Vaccines, Attenuated; Virulence; Yersinia pestis

At mammalian body temperature, the plague bacillus Yersinia pestis synthesizes lipopolysaccharide (LPS)-lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity. To address the effect of weak TLR4 stimulation on virulence, we modified Y. pestis to produce a potent TLR4-stimulating LPS. Modified Y. pestis was completely avirulent after subcutaneous infection even at high challenge doses. Resistance to disease required TLR4, the adaptor protein MyD88 and coreceptor MD-2 and was considerably enhanced by CD14 and the adaptor Mal. Both innate and adaptive responses were required for sterilizing immunity against the modified strain, and convalescent mice were protected from both subcutaneous and respiratory challenge with wild-type Y. pestis. Despite the presence of other established immune evasion mechanisms, the modified Y. pestis was unable to cause systemic disease, demonstrating that the ability to evade the LPS-induced inflammatory response is critical for Y. pestis virulence. Evading TLR4 activation by lipid A alteration may contribute to the virulence of various Gram-negative bacteria.

Topics: Acyltransferases; Animals; Cells, Cultured; Escherichia coli Proteins; Humans; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Mice; Mice, Inbred Strains; Plague; Plague Vaccine; Toll-Like Receptor 4; Vaccination; Virulence; Virulence Factors; Yersinia pestis