Characterization of the lipopolysaccharide and beta-glucan of the fish pathogen Francisella victoria

Lipopolysaccharide (LPS) and beta-glucan from Francisella victoria, a fish pathogen and close relative of highly virulent mammal pathogen Francisella tularensis, have been analyzed using chemical and spectroscopy methods. The polysaccharide part of the LPS was found to contain a nonrepetitive sequence of 20 monosaccharides as well as alanine, 3-aminobutyric acid, and a novel branched amino acid, thus confirming F. victoria as a unique species. The structure identified composes the largest oligosaccharide elucidated by NMR so far, and was possible to solve using high field NMR with cold probe technology combined with the latest pulse sequences, including the first application of H2BC sequence to oligosaccharides. The non-phosphorylated lipid A region of the LPS was identical to that of other Francisellae, although one of the lipid A components has not been found in Francisella novicida. The heptoseless core-lipid A region of the LPS contained a linear pentasaccharide fragment identical to the corresponding part of F. tularensis and F. novicida LPSs, differing in side-chain substituents. The linkage region of the O-chain also closely resembled that of other Francisella. LPS preparation contained two characteristic glucans, previously observed as components of LPS preparations from other strains of Francisella: amylose and the unusual beta-(1-6)-glucan with (glycerol)2phosphate at the reducing end.     

Characterisation of the core part of the lipopolysaccharide O-antigen of Francisella novicida (U112)

Francisella novicida (U112), a close relative of the highly virulent bacterium F. tularensis, is known to produce a lipopolysaccharide that is significantly different in biological properties from the LPS of F. tularensis. Here we present the results of the structural analysis of the F. novicida LPS core part, which is found to be similar to that of F. tularensis, differing only by one additional alpha-Glc residue:where R is an O-chain, linked via a beta-bacillosamine (2,4-diamino-2,4,6-trideoxyglucose) residue. The lipid part of F. novicida LPS contains no phosphate substituent and apparently has a free reducing end, a feature also noted in F. tularensis LPS.     

Immunogenicity and toxicity of lipopolysaccharide from Francisella tularensis LVS

Abstract Lipopolysaccharide (LPS) from the live vaccine strain of Francisella tularensis ( F . tularensis LVS) was isolated and purified. The LPS did not stimulate lymphocytes from previously tularaemia-vaccinated individuals or lymphocytes from nonprimed individuals. However, serum antibodies from tularaemia vaccines reacted with the LPS whereas virtually no reactivity was found with antibodies from individuals not exposed to F. tularensis LVS. Antibodies of immunoglobulin class M displayed the antibody reactivity predominantly. The LPS failed to induce the mononuclear cell-derived cytokine interleukin-1 and only low levels of tumour necrosis factor were detected. Furthermore, no LPS endotoxin properties were found in galactosamine-treated mice or in the Limulus amoebocyte lysate assay. From these results it can be concluded that F. tularensis LVS possesses a lipopolysaccharide-like molecule, which does not exhibit properties of a classical endotoxin.     

Species- and genus-specific antigenic epitopes of Francisella tularensis lipopolysaccharides

Lipopolysaccharide (LPS) antigenic epitopes of natural virulent and isogenic avirulent Francisella tularensis strains and other species of the Francisella genus (F. novicida, F. novicida-like, and F. philomiragia) were studied by dot and immunoblotting. Polyclonal rabbit and human sera to virulent F. tularensis strains and monoclonal antibodies to F. tularensis LPS O-side chain were used for detecting species- and genus-specific LPS epitopes. Typical virulent F. tularensis strains produce two types of S-LPS with different antigenic specificity simultaneously. Antigenic determinants of two LPS types were located in LPS O-polysaccharide but not in the core oligosaccharide. The epitopes of the first LPS type were characterized by species specificity for F. tularensis in contrast to determinants of the second LPS type, which had epitopes common with F. novicida. Cross exhaustion of human and rabbit antitularemic sera by F. tularensis and F. novicida LPS showed that F. novicida LPS molecules contained at least two epitopes--highly specific for F. novicida and common with the second type of F. tularensis LPS. The immune response of rabbits and humans to F. tularensis LPS epitopes was different in principle. Sera from rabbits immunized with vaccine and virulent F. tularensis strains contained antibodies "recognizing" antigenic epitopes of two S-LPS forms of the bacterium: type 1 species-specific (in high titers) and type 2 epitopes common with F. novicida LPS (in low titers). In addition to these, sera from patients with tularemia contain immunoglobulins to species-specific epitopes of F. novicida LPS in high titers. Experiments on avirulent mutants showed that in some cases attenuation of F. tularensis can involve loss of species-specific LPS form, while S-LPS with epitopes common with F. novicida LPS will be retained. The difference in specificity of human and rabbit antitularemic antibodies is due to individual features in the host immune system.     

Comparative analysis of the immune response of a rabbit to antigens to live and killed Francisella species bacteria]

Serum antibodies were analyzed in rabbits immunized with live and formalin-killed Francisella (F. tularensis, F. novicida, F. novicida-like, and F. philomiragia). Passive hemagglutination test with erythrocytes sensitized by these bacteria' LPS showed much higher titers of species-specific antibodies in all sera to live microorganisms than sera to killed bacteria. The results of immunoblotting with purified LPS and bacterial lysates indicate that sera to live bacteria contained mainly immunoglobulins to species-specific antigenic epitopes of LPS O-polysaccharide chain and few antibodies to the protein component of the cell. By contrast, killed bacterial cells induced weak production of antibodies to S-LPS and a pronounced antibody response to protein antigens. Besides the quantitative differences, live and killed bacteria differed by the qualitative spectrum of immunodominant proteins. Serum to live F. tularensis 15/10 contained antibodies to at least 3 immunodominant antigens of the cell, while serum to killed bacteria contained antibodies to only two of these. Immunoglobulins to protein antigens, absent in homologous sera to live bacteria, were detected in the sera to killed F. novicida and F. novicida-like bacteria. Both sera to F. philomiragia had antibodies reacting with LPS epitopes and immunodominant complex containing protein. In contrast to other Francisella, F. philomiragia was found to synthesize an uncommon LPS representing two major lipooligosaccharides with different molecular weights and antigenic specificity. Therefore, immune response of the host to live and killed Francisella is different: live cells more effectively induce the production of antibodies to S-LPS epitopes, while killed ones to protein antigens.     

MsbA transporter-dependent lipid A 1-dephosphorylation on the periplasmic surface of the inner membrane: topography of francisella novicida LpxE expressed in Escherichia coli

The lipid A anchor of Francisella tularensis lipopolysaccharide (LPS) lacks both phosphate groups present in Escherichia coli lipid A. Membranes of Francisella novicida (an environmental strain related to F. tularensis) contain enzymes that dephosphorylate lipid A and its precursors at the 1- and 4'-positions. We now report the cloning and characterization of a membrane-bound phosphatase of F. novicida that selectively dephosphorylates the 1-position. By transferring an F. novicida genomic DNA library into E. coli and selecting for low level polymyxin resistance, we isolated FnlpxE as the structural gene for the 1-phosphatase, an inner membrane enzyme of 239 amino acid residues. Expression of FnlpxE in a heptose-deficient mutant of E. coli caused massive accumulation of a previously uncharacterized LPS molecule, identified by mass spectrometry as 1-dephospho-Kdo2-lipid A. The predicted periplasmic orientation of the FnLpxE active site suggested that LPS export might be required for 1-dephosphorylation of lipid A. LPS and phospholipid export depend on the activity of MsbA, an essential inner membrane ABC transporter. Expression of FnlpxE in the msbA temperature-sensitive E. coli mutant WD2 resulted in 90% 1-dephosphorylation of lipid A at the permissive temperature (30 degrees C). However, the 1-phosphate group of newly synthesized lipid A was not cleaved at the nonpermissive temperature (44 degrees C). Our findings provide the first direct evidence that lipid A 1-dephosphorylation catalyzed by LpxE occurs on the periplasmic surface of the inner membrane.     

Use of a zwitterionic detergent for the restoration of the antibody binding capacity of immunoblotted Francisella tularensis lipopolysaccharide.

A method for the partial restoration of the antibody binding capacity of Francisella tularensis lipopolysaccharide (LPS) following denaturation (dissociation) in boiling sodium dodecyl sulfate (SDS) is described. The method relies on the presence of a zwitterionic detergent in the matrix of an SDS-polyacrylamide gel and in the transfer buffer during an immunoblot. F. tularensis LPS, which had lost its earlier capacity to bind to a particular monoclonal antibody in the normal blot procedure, did bind following the addition of the zwitterionic detergent to the polyacrylamide gel and transfer buffer. A number of detergents were tested but most success in restoring antibody binding was achieved with Zwittergent 3-08. This simple modification to the immunoblot procedure proved helpful in identifying a monoclonal antibody specific to hot phenol-extracted F. tularensis LPS.     

Francisella strains express hemolysins of distinct characteristics

Historically, Francisella strains have been described as nonhemolytic. In this study, we show by use of solid and liquid hemolysis assays that some Francisella strains have hemolytic properties. The Francisella novicida type strain U112 is hemolytic to horse erythrocytes and Francisella philomiragia type strain FSC144 is hemolytic towards both human and horse erythrocytes. The F. novicida strain U112 released a protein (novilysin A) into the culture supernatant which cross-reacted with antiserum against Escherichia coli HlyA whereas there was no similar protein detectable with this cross-reactive property from the supernatant of the F. philomiragia strain.     

Proteomic characterization and functional analysis of outer membrane vesicles of Francisella novicida suggests possible role in virulence and use as a vaccine

We have isolated and characterized outer membrane vesicles (OMVs) from Francisella. Transport of effector molecules through secretion systems is a major mechanism by which Francisella tularensis alters the extracellular proteome and interacts with the host during infection. Outer membrane vesicles produced by Francisella were examined using TEM and AFM and found to be 43-125 nm in size, representing another potential mechanism for altering the extracellular environment. A proteomic analysis (LC-MS/MS) of OMVs from F. novicida and F. philomiragia identified 416 (F. novicida) and 238 (F. philomiragia) different proteins, demonstrating that OMVs are an important contributor to the extracellular proteome. Many of the identified OMV proteins have a demonstrated role in Francisella pathogenesis. Biochemical assays demonstrated that Francisella OMVs possess acid phosphatase and hemolytic activities that may affect host cells during infection, and are cytotoxic toward murine macrophages in cell culture. OMVs have been previously used as a human vaccine against Neisseria meningitidis . We hypothesized that Francisella OMVs could be useful as a novel Francisella vaccine. Vaccinated BALB/C mice challenged with up to 50 LD50 of Francisella showed statistically significant protection when compared to control mice. In the context of these new findings, we discuss the relevance of OMVs in Francisella pathogenesis as well as their potential use as a vaccine.     

Outer membranes of a lipopolysaccharide-protein complex (LPS-17 kDa protein) as chemical tularemia vaccines

Abstract Immunisation with outer membranes of Francisella tularensis induced an efficient protection in guinea pigs against challenge with the virulent strains 503 or 144/713 (type B biovar holarctica ), both clinical isolates, and prevented the development of typical signs of infection in hamadryads (baboons), challenged with the virulent strain Schu (type A, biovar tularensis ) of F. tularensis . Immunisation with a lipopolysaccharide protein complex isolated from the outer membranes afforded protection in CBA mice against challenge with strain 503. Another LPS-protein complex obtained by the simple mixture of LPS preparations from strain 503 and a 17-kDa membrane protein from the avirulent R-variant of the vaccine strain 15 also demonstrated protective properties against experimental tularemia in mice.     

Interaction of S- and R-lipopolysaccharides of Francisella tularensis with lypopolysaccharide-binding protein of human serum

Investigation of ability of Francisella tularensis S- and R-lypopolysaccharide (LPS) preparations as well as the live bacteria with different chemotypes to interact with human lypopolysaccharide-binding protein (LBP) was carried out. It was found that LPS preparations derived from virulent(S-LPS) or isogenic avirulent mutant (R-LPS) strains of F. tularensis had markedly lower affinity to LBP as compared with typical S-LPS of Salmonella abortus and R-LPS of Yersinia pestis. It was shown that R-LPS preparation from avirulent mutant binds LPB more effectively than S-LPS from F. tularensis virulent strain. Differences in S- and R-LPS affinity were also confirmed for LPS represented by the live cells. Thus, bacteria with S-chemotype of LPS (F. tularensis 15/10) bound only 20.3% of LBP, whereas cells with R-LPS (F. tularensis 543 cap(-)) bound 39.9%. Such pattern was observed in experiments with both normal non-immune human serum and sera from people immunized with live tularemia vaccine. The latter indicates that opsonization of LPS by specific antibodies does not change its affinity to LBP. The observed more efficient binding of avirulent strain R-LPS to LBP is likely determines the more intensive host response directed to destruction and rapid elimination of the causative agent. At the same time, weak affinity of the vaccine and virulent strains S-LPS to LBP probably allows the bacterium to avoid activation of host defense mechanisms thus contributing to its long-term persistence in microorganism and development of specific immunity against tularemia.     

The role of lipopolysaccharide in toxicity of Francisella genus bacteria

It was demonstrated that the lipopolysaccharides (LPS) preparations, which were isolated from all representatives of Francisella Genus bacteria, i.e. F. tularensis, F. novicida, F. novicida-like and F. philomiragia by using the method of R.P. Darveau, R.E. Hancock (1983), were not toxic for white rats and white mice. A comparative study of toxicity of live F. tularensis bacteria (both wild and LPS-defective strains) made it possible to establish a direct correlation between the toxicity of microbes and LPS chemotype. It was found that only typical strains, which synthesize the wild-type S-LPS, caused the death of white rats and white mice in 24 hours after intraperitoneal contamination (10(9), 10(10) CFU/animal). Live bacteria of F tularensis R-mutants were not able to induce a lethal infection of rats and retained only residual virulence for mice. Other representatives of Francissela genus possessed less pronounced pathogenic properties. Thus, the toxic effect was registered, in case of white rats, only for F. novicida but not for F. novicida-like or F. philomiragia. At the same time, the two last mentioned species displayed a certain degree of virulence at high challenge doses (10(9), 10(10) CFU/animal) in respect to white mice. F. philomiragia, which generated lipoolygosaccharide (LOS) with an unusual structure, was found to be least pathogenic (25-75% of dead mice). The toxicity of bacteria, killed experimentally by different means (heating, UV-light, chloroform, acetone and formalin), was studied to define the role of bacterial proteins in the realisation of F. tularensis toxic potential in vivo. No lethal effect was exerted on experimental animals by killed microbes or purified LPS preparations. Finally, the study results show a priority role of the LPS molecule in the toxic effect of F. tularensis, which is possible in vivo only if structurally valuable molecules of live bacterial cells are available.     

Francisella novicida LPS has greater immunobiological activity in mice than F. tularensis LPS,and contributes to F. novicida murine pathogenesis

To further understand the role of LPS in the pathogenesis of Francisella infection, we characterized murine infection with F. novicida, and compared immunobiological activities of F. novicida LPS and the LPS from F. tularensis live vaccine strain (LVS). F. novicida had a lower intradermal LD(50) in BALB/cByJ mice than F. tularensis LVS, and mice given a lethal F. novicida dose intraperitoneally died faster than those given the same lethal F. tularensis LVS dose. However, the pattern of in vivo dissemination was similar, and in vitro growth of both bacteria in bone marrow-derived macrophages was comparable. F. novicida LPS stimulated very modest in vitro proliferation of mouse splenocytes at high doses, but F. tularensis LVS LPS did not. Murine bone marrow macrophages treated in vitro with F. novicida LPS produced IL12 and TNF-alpha, but did not produce detectable interferon-gamma, IL10, or nitric oxide; in contrast, murine macrophages treated with F. tularensis LVS LPS produced none of these mediators. In contrast to clear differences in stimulation of proliferation and especially cytokines, both types of purified LPS stimulated early protection against lethal challenge of mice with F. tularensis LVS, but not against lethal challenge with F. novicida. Thus, although LPS recognition may not be a major factor in engendering protection, the ability of F. novicida LPS to stimulate the production of proinflammatory cytokines including TNF-alpha likely contributes to the increased virulence for mice of F. novicida compared to F. tularensis LVS.     

Genome characterisation of the genus Francisella reveals insight into similar evolutionary paths in pathogens of mammals and fish

ABSTRACT: BACKGROUND: Prior to this study, relatively few strains of Francisella had been genome-sequenced. Previously published Francisella genome sequences were largely restricted to the zoonotic agent F. tularensis. Only limited data were available for other members of the Francisella genus, including F. philomiragia, an opportunistic pathogen of humans, F. noatunensis, a serious pathogen of farmed fish, and other less well described endosymbiotic species. RESULTS: We determined the phylogenetic relationships of all known Francisella species, including some for which the phylogenetic positions were previously uncertain. The genus Francisella could be divided into two main genetic clades: one included F. tularensis, F. novicida, F. hispaniensis and Wolbachia persica, and another included F. philomiragia and F. noatunensis. Some Francisella species were found to have significant recombination frequencies. However, the fish pathogen F. noatunensis subsp. noatunensis was an exception due to it exhibiting a highly clonal population structure similar to the human pathogen F. tularensis. CONCLUSIONS: The genus Francisella can be divided into two main genetic clades occupying both terrestrial and marine habitats. However, our analyses suggest that the ancestral Francisella species originated in a marine habitat. The observed genome to genome variation in gene content and IS elements of different species supports the view that similar evolutionary paths of host adaptation developed independently in F. tularensis (infecting mammals) and F. noatunensis subsp. noatunensis (infecting fish).     

The use of solid-phase liposomal immunoassay for determining a bacterial antigen of lipopolysaccharide nature]

The solid-phase liposomal immunoassay procedure for the determination of Francisella tularensis lipopolysaccharide (LPS) has been developed. This assay has been made with the use of monolayer liposomes, on the average, 360 nm in diameter with their phospholipid bilayer modified with F. tularensis LPS and their internal space filled with calcein used as fluorescent marker. The assay is based on the principle of the competitive immunosorption of liposomes and antigenic LPS on the surface of polystyrene plates sensitized with specific monoclonal antibodies. The dynamic range of the method is 50-2,500 mg/ml, the variation index being 2.3-11.5%. Optimization of this method and its comparison with the enzyme immunoassay system for the given antigen-antibody pair has been made.     

TLR4-dependent activation of inflammatory cytokine response in macrophages by Francisella elongation factor Tu

The bacterial determinants of pulmonary Francisella induced inflammatory responses and their interaction with host components are not clearly defined. In this study, proteomic and immunoblot analyses showed presence of a cytoplasmic protein elongation factor Tu (EF-Tu) in the membrane fractions of virulent Francisella novicida, LVS and SchuS4, but not in an attenuated F. novicida mutant. EF-Tu was immunodominant in mice vaccinated and protected from virulent F. novicida. Moreover, recombinant EF-Tu induced macrophages to produce inflammatory cytokines in a TLR4 dependent manner. This study shows immune stimulatory properties of a cytoplasmic protein EF-Tu expressed on the membrane of virulent Francisella strains.     

Active suppression of the pulmonary immune response by Francisella tularensis Schu4

Francisella tularensis is an obligate, intracellular bacterium that causes acute, lethal disease following inhalation. As an intracellular pathogen F. tularensis must invade cells, replicate, and disseminate while evading host immune responses. The mechanisms by which virulent type A strains of Francisella tularensis accomplish this evasion are not understood. Francisella tularensis has been shown to target multiple cell types in the lung following aerosol infection, including dendritic cells (DC) and macrophages. We demonstrate here that one mechanism used by a virulent type A strain of F. tularensis (Schu4) to evade early detection is by the induction of overwhelming immunosuppression at the site of infection, the lung. Following infection and replication in multiple pulmonary cell types, Schu4 failed to induce the production of proinflammatory cytokines or increase the expression of MHCII or CD86 on the surface of resident DC within the first few days of disease. However, Schu4 did induce early and transient production of TGF-beta, a potent immunosuppressive cytokine. The absence of DC activation following infection could not be attributed to the apoptosis of pulmonary cells, because there were minimal differences in either annexin or cleaved caspase-3 staining in infected mice compared with that in uninfected controls. Rather, we demonstrate that Schu4 actively suppressed in vivo responses to secondary stimuli (LPS), e.g., failure to recruit granulocytes/monocytes and stimulate resident DC. Thus, unlike attenuated strains of F. tularensis, Schu4 induced broad immunosuppression within the first few days after aerosol infection. This difference may explain the increased virulence of type A strains compared with their more attenuated counterparts.     

Genetic diversity within the genus Francisella as revealed by comparative analyses of the genomes of two North American isolates from environmental sources

ABSTRACT: BACKGROUND: Francisella tularensis is an intracellular pathogen that causes tularemia in humans and the public health importance of this bacterium has been well documented in recent history. Francisella philomiragia, a distant relative of F. tularensis, is thought to constitute an environmental lineage along with Francisella novicida. Nevertheless, both F. philomiragia and F. novicida have been associated with human disease, primarily in immune-compromised individuals. To understand the genetic relationships and evolutionary contexts among different lineages within the genus Francisella, the genome of Francisella spp. strain TX07-7308 was sequenced and compared to the genomes of F. philomiragia strains ATCC 25017 and 25015, F. novicida strain U112, and F. tularensis strain Schu S4. RESULTS: The size of strain ATCC 25017 chromosome was 2,045,775 bp and contained 1,983 protein-coding genes. The size of strain TX07-7308 chromosome was 2,035,931 bp and contained 1,980 protein-coding genes. Pairwise BLAST comparisons indicated that strains TX07-7308 and ATCC 25017 contained 1700 protein coding genes in common. NUCmer analyses revealed that the chromosomes of strains TX07-7308 and ATCC 25017 were mostly collinear except for a few gaps, translocations, and/or inversions. Using the genome sequence data and comparative analyses with other members of the genus Francisella (e.g., F. novicida strain U112 and F. tularensis strain Schu S4), several strain-specific genes were identified. Strains TX07-7308 and ATCC 25017 contained an operon with six open reading frames encoding proteins related to enzymes involved in thiamine biosynthesis that was absent in F. novicida strain U112 and F. tularensis strain Schu S4. Strain ATCC 25017 contained an operon putatively involved in lactose metabolism that was absent in strain TX07-7308, F. novicida strain U112, and F. tularensis strain Schu S4. In contrast, strain TX07-7308 contained an operon putatively involved in glucuronate metabolism that was absent in the genomes of strain ATCC 25017, F. novicida strain U112, and F. tularensis strain Schu S4. The polymorphic nature of polysaccharide biosynthesis/modification gene clusters among different Francisella strains was also evident from genome analyses. CONCLUSIONS: From genome comparisons, it appeared that genes encoding novel functions have contributed to the metabolic enrichment of the environmental lineages within the genus Francisella. The inability to acquire new genes coupled with the loss of ancestral traits and the consequent reductive evolution may be a cause for, as well as an effect of, niche selection of F. tularensis. Sequencing and comparison of the genomes of more isolates are required to obtain further insights into the ecology and evolution of different species within the genus Francisella.     

Mutations of Francisella novicida that Alter the Mechanism of Its Phagocytosis by Murine Macrophages

Infection with the bacterial pathogen Francisella tularensis tularensis (F. tularensis) causes tularemia, a serious and debilitating disease. Francisella tularensis novicida strain U112 (abbreviated F. novicida), which is closely related to F. tularensis, is pathogenic for mice but not for man, making it an ideal model system for tularemia. Intracellular pathogens like Francisella inhibit the innate immune response, thereby avoiding immune recognition and death of the infected cell. Because activation of inflammatory pathways may lead to cell death, we reasoned that we could identify bacterial genes involved in inhibiting inflammation by isolating mutants that killed infected cells faster than the wild-type parent. We screened a comprehensive transposon library of F. novicida for mutant strains that increased the rate of cell death following infection in J774 macrophage-like cells, as compared to wild-type F. novicida. Mutations in 28 genes were identified as being hypercytotoxic to both J774 and primary macrophages of which 12 were less virulent in a mouse infection model. Surprisingly, we found that F. novicida with mutations in four genes (lpcC, manB, manC and kdtA) were taken up by and killed macrophages at a much higher rate than the parent strain, even upon treatment with cytochalasin D (cytD), a classic inhibitor of macrophage phagocytosis. At least 10-fold more mutant bacteria were internalized by macrophages as compared to the parent strain if the bacteria were first fixed with formaldehyde, suggesting a surface structure is required for the high phagocytosis rate. However, bacteria were required to be viable for macrophage toxicity. The four mutant strains do not make a complete LPS but instead have an exposed lipid A. Interestingly, other mutations that result in an exposed LPS core were not taken up at increased frequency nor did they kill host cells more than the parent. These results suggest an alternative, more efficient macrophage uptake mechanism for Francisella that requires exposure of a specific bacterial surface structure(s) but results in increased cell death following internalization of live bacteria.