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.     

A variety of novel lipid A structures obtained from Francisella tularensis live vaccine strain

F. tularensis is a Gram-negative coccobacillus that causes tularemia. Its LPS has nominal biological activity. Currently, there is controversy regarding the structure of the lipid A obtained from F. tularensis live vaccine strain (LVS). Therefore, to resolve this controversy, the purification and structural identification of this LPS was crucial. To achieve this, LPS from F. tularensis LVS was acid hydrolyzed to obtain crude lipid A that was methylated and purified by HPLC and the fractions were analyzed by MALDI-TOF MS. The structure of the major lipid A species was composed of a glucosamine disaccharide backbone substituted with four fatty acyl groups and a phosphate (1-position) with a molecular mass of 1505. The major lipid A component contained 18:0[3-O(16:0)] in the distal subunit and two 18:0(3-OH) fatty acyl chains at the 2- or 3-positions of the reducing subunit. Additional variations in the lipid A species include: heterogeneity in fatty acyl groups, a phosphate or a phosphoryl galactosamine at the 1-position, and a hexose at the 4' or 6' position, some of which have not been previously described for F. tularensis LVS. This analysis revealed that lipid A from F. tularensis LVS is far more complex than originally believed.     

Francisella tularensis selectively induces proinflammatory changes in endothelial cells

Naturally acquired infections with Francisella tularensis, the bacterial agent of tularemia, occur infrequently in humans. However, the high infectivity and lethality of the organism in humans raise concerns that it might be exploited as a weapon of bioterrorism. Despite this potential for illicit use, the pathogenesis of tularemia is not well understood. To examine how F. tularensis interacts with cells of its mammalian hosts, we tested the ability of a live vaccine strain (LVS) to induce proinflammatory changes in cultured HUVEC. Living F. tularensis LVS induced HUVEC to express the adhesion molecules VCAM-1 and ICAM-1, but not E-selectin, and to secrete the chemokine CXCL8, but not CCL2. Stimulation of HUVEC by the living bacteria was partially suppressed by polymyxin B, an inhibitor of LPS, but did not require serum, suggesting that F. tularensis LVS does not stimulate endothelium through the serum-dependent pathway that is typically used by LPS from enteric bacteria. In contrast to the living organisms, suspensions of killed F. tularensis LVS acquired the ability to increase endothelial expression of both E-selectin and CCL2. Up-regulation of E-selectin and CCL2 by the killed bacteria was not inhibited by polymyxin B. Exposure of HUVEC to either live or killed F. tularensis LVS for 24 h promoted the transendothelial migration of subsequently added neutrophils. These data indicate that multiple components of F. tularensis LVS induce proinflammatory changes in endothelial cells in an atypical manner that may contribute to the exceptional infectivity and virulence of this pathogen.     

The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages

Intracellular killing of Francisella tularensis by macrophages depends on interferon-gamma (IFN-gamma)-induced activation of the cells. The importance of inducible nitric oxide synthase (iNOS) or NADPH phagocyte oxidase (phox) for the cidal activity was studied. Murine IFN-gamma-activated peritoneal exudate cells (PEC) produced nitric oxide (NO), measured as nitrite plus nitrate, and superoxide. When PEC were infected with the live vaccine strain, LVS, of F. tularensis, the number of viable bacteria was at least 1000-fold lower in the presence than in the absence of IFN-gamma after 48 h of incubation. PEC from iNOS-gene-deficient (iNOS-/-) mice killed F. tularensis LVS less effectively than did PEC from wild-type mice. PEC from phox gene-deficient (p47phox-/-) mice were capable of killing the bacteria, but killing was less efficient, although still significant, in the presence of NG-monomethyl-L-arginine (NMMLA), an inhibitor of iNOS. A decomposition catalyst of ONOO-, FeTPPS, completely reversed the IFN-gamma-induced killing of F. tularensis LVS. Under host cell-free conditions, F. tularensis LVS was exposed to S-nitroso-acetyl-penicillamine (SNAP), which generates NO, or 3-morpholinosydnonimine hydrochloride (SIN-1), which generates NO and superoxide, leading to formation of ONOO-. During 6 h of incubation, SNAP caused no killing of F. tularensis LVS, whereas effective killing occurred in the presence of equimolar concentrations of SIN-1. The results suggest that mechanisms dependent on iNOS and to a minor degree, phox, contribute to the IFN-gamma-induced macrophage killing of F. tularensis LVS. ONOO- is likely to be a major mediator of the killing.     

Phase variations of Francisella tularensis lipopolysaccharide in human infection and immunization

The comparative study of the specificity of antibodies in human sera after tularemia infection and immunization with live tularemia infection was carried out with the use of passive hemagglutination and immunoblotting techniques. The sera of tularemia patients contained two different types of immunoglobulins: strictly specific to the antigenic epitopes of F. tularensis Iipopolysaccharide (LPS) and strictly specific to F. tularensis subsp. novicida LPS. Such phenomenon may be due to phase variations of the antigenic structure of F. tularensis LPS in the body of a slightly susceptible host. The immune sera of vaccinated were found to contain antibodies, strictly specific only to F. tularensis LPS. At the same time in one vaccinee by the presence of pronounced postvaccinal reactions was found sharply defined interaction between serum imunoglobulins and F. tularensis subsp. novicida LPS. As the result, the data on the possibility of the antigenic modification of F. tularensis in tularemia infection in humans were obtained. At the same time antigenic epitopes, characteristic of faintly pathogenic and closely related F. tularensis novicida LPS, appeared in the structure of F. tularensis LPS.     

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.     

Galleria mellonella as a model host to study infection by the Francisella tularensis live vaccine strain

We used the killing of Galleria mellonella (Lepidoptera: Pyralidae; the greater wax moth) caterpillar by the live vaccine strain (LVS) of Francisella tularensis to develop an invertebrate host system that can be used to study F. tularensis infection and the in vivo effects of antibacterial compounds on F. tularensis LVS. After injection into the insect hemocoel, F. tularensis LVS, killed caterpillars despite the association of LVS with hemocytes. The rate of killing depended on the number of bacteria injected. Antibiotic therapy with ciprofloxacin, levofloxacin or streptomycin administered before or after inoculation prolonged survival and decreased the tissue burden of F. tularensis in the hemocoel. Delayed drug treatment reduced the efficacy of antibacterials and especially streptomycin. The G. mellonella-F. tularensis LVS system may facilitate the in vivo study of F. tularensis, efficacy with antibacterial agents.     

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.     

Myeloid differentiation factor-88 (MyD88) is essential for control of primary in vivo Francisella tularensis LVS infection, but not for control of intra-macrophage bacterial replication

The means by which Francisella tularensis, the causative agent of tularemia, are recognized by mammalian immune systems are poorly understood. Here we wished to explore the contribution of the MyD88/Toll-like receptor signaling pathway in initiating murine responses to F. tularensis Live Vaccine Strain (LVS). MyD88 knockout (KO) mice, but not TLR2-, TLR4- or TLR9-deficient mice, rapidly succumbed following in vivo bacterial infection via the intradermal route even with a very low dose of LVS (5 x 10(1)) that was 100,000-fold less than the LD(50) of normal wild-type (WT) mice. By day 5 after LVS infection, bacterial organ burdens were 5-6 logs higher in MyD88 knockout mice; further, unlike infected WT mice, levels of interferon-gamma in the sera of LVS-infected MyD88 KO were undetectable. An in vitro culture system was used to assess the ability of bone marrow macrophages derived from either KO or WT mice to support bacterial growth, or to control intracellular bacterial replication when co-cultured with immune lymphocytes. In this assay, bacterial replication was similar in macrophages derived from either WT or any of the TLR KO mice. Bacterial growth was controlled in co-cultures containing macrophages from MyD88 KO mice or TLR KO mice as well as in co-cultures containing immune WT splenic lymphocytes and WT macrophages. Further, MyD88-deficient LVS-immune splenocytes controlled intracellular growth comparably to those from normal mice. Thus MyD88 is essential for innate host resistance to LVS infection, but is not required for macrophage control of intracellular bacterial growth.     

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.     

<Emphasis Type="Italic">Francisella tularensis</Emphasis> strain LVS resides in MHC II-positive autophagic vacuoles in Macrophages

The Francisella tularensis strain LVS phagosome disintegrates during the first few hours after bacterial entry and microbes are released to the cytosol. Within 12 h both rapid multiplication of microbes and a steep increase of apoptosis of infected macrophages occur. We searched for signals involved in the death of macrophages and detected molecules associated with the autophagy machinery cathepsin D, PTEN, p53 and LC3, whose levels or modification were influenced by ongoing in vitro tularemic infection. The sequestration of cytoplasmic F. tularensis LVS into autophagosomes was confirmed by co-localization of the LVS strain containing vacuoles with LC3 (an autophagosomal marker). We also demonstrated the presence of MHC II antigens in these autophagosomes, indicating that they might act as a source of endogenous tularemic antigens for presentation to CD4+ T lymphocytes.     

Construction and characterization of a highly efficient Francisella shuttle plasmid

Francisella tularensis is a facultative intracellular pathogen that infects a wide variety of mammals and causes tularemia in humans. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of transmission. To date, genetic manipulation in Francisella spp. has been limited due to the inefficiency of DNA transformation, the relative lack of useful selective markers, and the lack of stably replicating plasmids. Therefore, the goal of this study was to develop an enhanced shuttle plasmid that could be utilized for a variety of genetic procedures in both Francisella and Escherichia coli. A hybrid plasmid, pFNLTP1, was isolated that was transformed by electroporation at frequencies of >1 x 10(7) CFU mug of DNA(-1) in F. tularensis LVS, Francisella novicida U112, and E. coli DH5alpha. Furthermore, this plasmid was stably maintained in F. tularensis LVS after passage in the absence of antibiotic selection in vitro and after 3 days of growth in J774A.1 macrophages. Importantly, F. tularensis LVS derivatives carrying pFNLTP1 were unaltered in their growth characteristics in laboratory medium and macrophages compared to wild-type LVS. We also constructed derivatives of pFNLTP1 containing expanded multiple cloning sites or temperature-sensitive mutations that failed to allow plasmid replication in F. tularensis LVS at the nonpermissive temperature. In addition, the utility of pFNLTP1 as a vehicle for gene expression, as well as complementation, was demonstrated. In summary, we describe construction of a Francisella shuttle plasmid that is transformed at high efficiency, is stably maintained, and does not alter the growth of Francisella in macrophages. This new tool should significantly enhance genetic manipulation and characterization of F. tularensis and other Francisella biotypes.     

Evaluation of Media for Differentiating Nonfermenting Gram-negative Bacteria of Medical Significance

The response of the rabbit to viable or killed whole-cell Pasteurella tularensis vaccines was studied. The most practical preparation for the production of anti-P. tularensis antibodies was viable organisms of the live vaccine strain (LVS). The intravenous route of administration proved superior to either the subcutaneous or intradermal routes, and incorporation of LVS into Freund's adjuvants did not result in increased levels of antibody. Short-term hyperimmunization, three injections at weekly intervals, constituted the most efficient method for increasing levels of the antibodies.     

Inhibition of Francisella tularensis LVS infection of macrophages results in a reduced inflammatory response: evaluation of a therapeutic strategy for intracellular bacteria

Francisella tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. A flow cytometric assay was developed to measure bacterial uptake, using a fluorescein isothiocyanate-labelled anti-F. tularensis lipopolysaccharide antibody in conjunction with antibodies to cell surface markers, in order to determine the specific cell phenotypes that were positive for the bacteria. Several phagocytic inhibitors were evaluated in macrophage cell lines and a lung homogenate assay to determine whether the uptake of F. tularensis strain LVS could be altered. Our data show that cytochalasin B, LY294002, wortmannin, nocodazole, MG132 and XVA143 inhibitors reduced LVS uptake by >50% in these assays without having significant cytotoxic effects. Furthermore, a reduction in the inflammatory cytokines monocyte chemoattractant protein-1, interleukin-6 and tumour necrosis factor-α was found in the supernatant of lung tissue infected with LVS when the inhibitory compounds were present. Similarly, there was an alteration in bacterial uptake and a reduction in the inflammatory cytokine response following the administration of wortmannin to LVS-infected mice. Although wortmannin treatment alone did not correlate with the enhanced survival of LVS-infected mice, these inhibitors may have utility in combination therapeutic approaches or against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.     

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.     

Larval exposure to Francisella tularensis LVS affects fitness of the mosquito Culex quinquefasciatus

Francisella tularensis is an environmental bacterium capable of infecting a wide spectrum of species from mammals and birds to reptiles. It has been demonstrated that F. tularensis can invade and survive within protozoa, but an association with aquatic insects has not been thoroughly investigated. We examined the interaction of F. tularensis LVS biofilms and Culex quinquefasciatus larvae to determine the effects on larvae and adults. Our results demonstrate that F. tularensis LVS can form and persist as biofilms in natural water and that the mosquito larvae of C. quinquefasciatus readily feed on biofilm and planktonic forms of F. tularensis LVS. Larvae raised in both bacteria-only cultures suffered significant delays in pupation. Adults resulting from larvae continuously exposed to the bacteria had significantly reduced wing lengths in males and fecundity of both sexes. The bacteria may be exerting these effects through localization and persistence within the midgut and Malpighian tubule cells of the larvae. The study of oral acquisition of pathogens by insect larvae can significantly contribute to the study of environmental persistence of pathogens. We show that oral uptake of F. tularensis LVS by C. quinquefasciatus larvae results in not only larval effects but also has effects on adult mosquitoes. These effects are important in understanding both the ecology of tularemia as well as bacterial interactions with aquatic invertebrates.     

Uptake of serum-opsonized Francisella tularensis by macrophages can be mediated by class A scavenger receptors

The bacterium Francisella tularensis is highly infective, and this is one of the chief attributes that has led to its development as a bioweapon. Establishment of infection requires efficient uptake of F. tularensis by host macrophages, which provide a safe in vivo environment for F. tularensis replication. Little is known, however, about the cellular entry mechanisms employed by this organism. This report shows that efficient uptake of F. tularensis live vaccine strain (LVS) by macrophages is dependent on a heat-sensitive serum component and is mediated in part by types I and II class A scavenger receptors (SRA), demonstrating for the first time that SRA can act as a receptor for opsonized pathogens. Specifically, uptake of serum-opsonized LVS was partially blocked by general scavenger receptor inhibitors [fucoidan and poly(I)] and was largely inhibited by a specific function-blocking antibody against SRA. A role for SRA in LVS binding was confirmed by showing that ectopic expression of SRA in human embryonic kidney cells conferred the capacity for robust serum-dependent LVS binding. Finally, SRA-/- macrophages ingested significantly fewer LVS than did macrophages from wild-type mice. These findings support a novel role for SRA in innate immunity and suggest a potential therapeutic approach for modulating F. tularensis infection, namely, blocking SRA as a means of hindering F. tularensis access to its intracellular niche.