Distinct roles of reactive nitrogen and oxygen species to control infection with the facultative intracellular bacterium Francisella tularensis

Reactive nitrogen species (RNS) and reactive oxygen species (ROS) are important mediators of the bactericidal host response. We investigated the contribution of these two mediators to the control of infection with the facultative intracellular bacterium Francisella tularensis. When intradermally infected with the live vaccine strain F. tularensis LVS, mice deficient in production of RNS (iNOS(-/-) mice) or in production of ROS by the phagocyte oxidase (p47(phox-/-) mice) showed compromised resistance to infection. The 50% lethal dose (LD(50)) for iNOS(-/-) mice was <20 CFU, and the LD(50) for p47(phox-/-) mice was 4,400 CFU, compared to an LD(50) of >500,000 CFU for wild-type mice. The iNOS(-/-) mice survived for 26.4 +/- 1.8 days, and the p47(phox-/-) mice survived for 10.1 +/- 1.3 days. During the course of infection, the serum levels of gamma interferon (IFN-gamma) and interleukin-6 were higher in iNOS(-/-) and p47(phox-/-) mice than in wild-type mice. Histological examination of livers of iNOS(-/-) mice revealed severe liver pathology. Splenocytes obtained 5 weeks after primary infection from antibiotic-treated iNOS(-/-) mice showed an in vitro recall response that was similar in magnitude and greater secretion of IFN-gamma compared to cells obtained from wild-type mice. In summary, mice lacking expression of RNS or ROS showed extreme susceptibility to infection with F. tularensis LVS. The roles of RNS and ROS seemed to be distinct since mice deficient in production of ROS showed dissemination of infection and died during the early phase of infection, whereas RNS deficiency led to severe liver pathology and a contracted course of infection.     

Specific antibodies contribute to the host protection against strains of Francisella tularensis subspecies holarctica

T cells are crucial to the control and eradication of the facultative intracellular bacterium Francisella tularensis. A contributory role of humoral antibodies in the host defence remains to be assessed. We used B-cell-deficient mice to study the possible contribution of antibodies to the defence against the live vaccine strain (LVS) or a clinical isolate of F. tularensis, both belonging to the subspecies holarctica (type B). When B-cell-deficient (Igmu(-/-)) mice of the C57BL/10 background were administered immune serum one day before intradermal injection of LVS, they developed lower bacterial numbers in skin, liver, and spleen than did mice receiving normal serum, and survived a challenge inoculum that was lethal for mice given normal serum. Administration of immune serum to C57BL/10 mice afforded protection also against infection with the clinical isolate of F. tularensis subsp. holarctica. Five days after intradermal inoculation of bacteria of the isolate, animals receiving immune serum showed 4log10 lower bacterial counts in liver and spleen than mice administered normal serum. In mice primed by LVS infection, T-cell immunity and host protection were strong and only a marginal contribution of immune serum against a secondary intradermal infection was demonstrated. Together, these findings show that specific antibodies contribute to the host defence of mice against F. tularensis subsp. holarctica.     

Antibodies contribute to effective vaccination against respiratory infection by type A Francisella tularensis strains

Pneumonic tularemia is a life-threatening disease caused by inhalation of the highly infectious intracellular bacterium Francisella tularensis. The most serious form of the disease associated with the type A strains can be prevented in experimental animals through vaccination with the attenuated live vaccine strain (LVS). The protection is largely cell mediated, but the contribution of antibodies remains controversial. We addressed this issue in a series of passive immunization studies in Fischer 344 (F344) rats. Subcutaneous LVS vaccination induced a robust serum antibody response dominated by IgM, IgG2a, and IgG2b antibodies. Prophylactic administration of LVS immune serum or purified immune IgG reduced the severity and duration of disease in naïve rats challenged intratracheally with a lethal dose of the virulent type A strain SCHU S4. The level of resistance increased with the volume of immune serum given, but the maximum survivable SCHU S4 challenge dose was at least 100-fold lower than that shown for LVS-vaccinated rats. Protection correlated with reduced systemic bacterial growth, less severe histopathology in the liver and spleen during the early phase of infection, and bacterial clearance by a T cell-dependent mechanism. Our results suggest that treatment with immune serum limited the sequelae associated with infection, thereby enabling a sterilizing T cell response to develop and resolve the infection. Thus, antibodies induced by LVS vaccination may contribute to the defense of F344 rats against respiratory infection by type A strains of F. tularensis.Pneumonic tularemia is a highly debilitating disease caused by the Gram-negative coccobacillus Francisella tularensis. Strains classified under subspecies tularensis (type A) are the most virulent and pose the biggest challenge from a clinical perspective (28), with a mortality rate estimated to exceed 30% in untreated patients (11). Prophylactic vaccination is the best countermeasure, and there is good historical evidence that pneumonic tularemia can be prevented by vaccination with the attenuated F. tularensis live vaccine strain (LVS) (37). However, LVS is unlikely to be licensed for mass vaccination because the mechanism of attenuation has not been defined. Due to the potential of a major public health threat, there is an urgent need to understand the protective mechanisms associated with an effective immune response so that novel vaccines can be developed.Protective immunity against F. tularensis infection is usually attributed to an effective T cell response. However, F. tularensis has a significant extracellular phase, which makes it accessible to humoral immune responses (18). Indeed, there is ample evidence that B cells and antibodies are necessary for mice to develop their natural resistance to primary and secondary LVS infections. Purified lipopolysaccharide (LPS) from LVS induced a population of B1-a cells within 2 to 3 days of administration that protected mice against intraperitoneal (i.p.) LVS challenge (6, 7, 14). Consistent with these results, μMT mice lacking mature B cells exhibited increased susceptibility to primary intradermal (i.d.) LVS infection and delayed bacterial clearance (15, 40). μMT mice were also more susceptible to secondary i.p. LVS infection, and this defect was corrected by reconstitution with LVS-primed B cells (15). The contribution of antibodies has been addressed repeatedly in passive immunization experiments, which showed that immune serum from humans and mice vaccinated with live or inactivated LVS protected naïve mice against challenges with LVS or other low virulence strains given by a variety of routes (13, 19, 26, 29, 33, 36, 40). The dominant antibody response was directed at LPS, but antibodies against protein antigens have also been found (17, 23, 31, 41, 43). Monoclonal antibodies specific for LPS or the outer membrane protein FopA provided significant protection against LVS challenge when given either prophylactically (38) or therapeutically (30, 38). Together, these results suggest that antibodies contribute toward effective control of attenuated or low-virulence F. tularensis strains.It has been much more difficult to demonstrate antibody-mediated protection against type A strains in mice (1, 20, 21, 38), even though they express many antigens recognized by LVS immune serum (13, 30). This is not surprising given the historical difficulties in generating protective immunity against type A strains in this animal model (5). However, Ray et al. recently showed that oral LVS vaccination protected mice against a pulmonary SCHU S4 challenge in an antibody-dependent manner (35). Klimpel et al. also reported a similar finding using immune serum from mice cured of a lethal intranasal (i.n.) SCHU S4 infection with levofloxacin in a passive immunization model (27). Thus, the protective effects of antibodies appear not to be restricted only to low-virulence strains but may also contribute to the protection against highly virulent type A strains.To further characterize the mechanism of antibody-mediated protection, we utilized the recently characterized Fischer 344 (F344) rat model (45). Since F344 rats developed much stronger resistance to respiratory SCHU S4 challenge after LVS vaccination than previously observed in mice, we speculated that antibodies may provide better protection in this model and allow us to define their protective mechanism more thoroughly. We now show in a passive immunization model that serum antibodies from LVS-vaccinated rats conferred protection against a lethal intratracheal (i.t.) SCHU S4 challenge. Protection correlated with reduced systemic bacterial growth and less severe histopathology during the early phase of infection and bacterial clearance by a T cell-dependent mechanism. Thus, antibodies contribute to but are not sufficient for the effective control of respiratory infections by fully virulent type A strains. Our studies provide valuable insights into the protective mechanisms of antibodies that will guide future development of tularemia vaccine candidates.     

The 17 kDa lipoprotein and encoding gene of Francisella tularensis LVS are conserved in strains of Francisella tularensis.

A T-cell-stimulating 17 kDa protein of the vaccine strain Francisella tularensis LVS has previously been cloned, sequenced and shown to be a lipoprotein. In the present study, it was investigated whether the protein, denoted TUL4, and its gene are present in various strains of the genus Francisella. By Western blot analysis, it was demonstrated that a TUL4-specific monoclonal antibody bound to a protein present in each of the Francisella strains. The immunoreactive proteins had an M(r) of 17 kDa in all F. tularensis strains and in the strain Francisella novicida, whereas the M(r) in strains of Francisella philomiragia was 20 kDa. When genomic preparations were probed with a radioactive DNA fragment of F. tularensis LVS encoding TUL4, hybridization was demonstrated in all strains of Francisella, although the F. philomiragia strains did not hybridize under conditions of high stringency. The hybridizing chromosomal DNA fragment of the F. philomiragia strains was larger than that of the other Francisella strains. No hybridization or Western blot reactivity was seen when various other Gram-negative and Gram-positive bacteria were probed. In summary, the 17 kDa lipoprotein of F. tularensis LVS appears to be Francisella-specific and present in the species F. tularensis and F. novicida, whereas an immunologically related protein is present in F. philomiragia.     

Susceptibility of immunodeficient mice to aerosol and systemic infection with virulent strains of Francisella tularensis

Previous studies have shown that IFN-gamma, TNF-alpha and NOS-2, but not B cells, are crucial for host defense against primary systemic infection with the attenuated live vaccine strain (LVS) of Francisella tularensis. In this study, we examined the importance of these and additional immune components in host resistance against infection with virulent strains of F. tularensis initiated by systemic and airborne routes. Wild-type (WT) mice and mice deficient in IFN-gamma, TNFR1R2, NOS-2, or B cells were equally susceptible to low dose ( approximately 10 colony forming units) aerosol or intradermal challenge with virulent type B F. tularensis, and succumbed to the infection between days 6 and 8 post-inoculation. Quantitative bacteriology showed that IFN-gamma-/- and B cell-/- mice consistently harbored up to one log(10) more bacteria in their lungs, spleens and livers than WT mice at day 5 post aerosol exposure. Surprisingly, however, compared to other strains of KO mice and WT control mice, IFN-gamma-/- mice showed only mild liver damage as assessed by histopathology and liver function tests. Additional experiments established that even mice with broad immunodeficiency (SCID, neutropenic, splenectomized or thymectomized mice and mice treated with corticosteroid) were no more susceptible to aerosol-initiated infection with virulent type B or type A F. tularensis than immunosufficient control mice. Combined, our results indicate that, unlike LVS, normal type A and type B F. tularensis strains are so extremely virulent that even immunocompetent mice are virtually defenseless to low dose aerosol and intradermal challenges with them.     

Genome-wide DNA microarray analysis of Francisella tularensis strains demonstrates extensive genetic conservation within the species but identifies regions that are unique to the highly virulent F. tularensis subsp. tularensis

Francisella tularensis is a potent pathogen and a possible bioterrorism agent. Little is known, however, to explain the molecular basis for its virulence and the distinct differences in virulence found between the four recognized subspecies, F. tularensis subsp. tularensis, F. tularensis subsp. mediasiatica, F. tularensis subsp. holarctica, and F. tularensis subsp. novicida. We developed a DNA microarray based on 1,832 clones from a shotgun library used for sequencing of the highly virulent strain F. tularensis subsp. tularensis Schu S4. This allowed a genome-wide analysis of 27 strains representing all four subspecies. Overall, the microarray analysis confirmed a limited genetic variation within the species F. tularensis, and when the strains were compared, at most 3.7% of the probes showed differential hybridization. Cluster analysis of the hybridization data revealed that the causative agents of type A and type B tularemia, i.e., F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, respectively, formed distinct clusters. Despite marked differences in their virulence and geographical origin, a high degree of genomic similarity between strains of F. tularensis subsp. tularensis and F. tularensis subsp. mediasiatica was apparent. Strains from Japan clustered separately, as did strains of F. tularensis subsp. novicida. Eight regions of difference (RD) 0.6 to 11.5 kb in size, altogether comprising 21 open reading frames, were identified that distinguished strains of the moderately virulent subspecies F. tularensis subsp. holarctica and the highly virulent subspecies F. tularensis subsp. tularensis. One of these regions, RD1, allowed for the first time the development of an F. tularensis-specific PCR assay that discriminates each of the four subspecies.     

Reduction of Brucella species and Francisella tularensis cross-reacting agglutinins by dithiothreitol.

Cross-reaction agglutinin titers to Brucella abortus antigen were found in 42 of 128 tularemia serum specimens, and cross-reaction titers to Francisella tularensis antigen were found in 8 of 34 brucellosis serum specimens. The cross-reaction titers were reduced to 10 or less by dithiothreitol, suggesting that the titers are due to immunoglobulin M antibody.     

Detection of reactive oxygen and nitrogen species by EPR spin trapping

Electron paramagnetic resonance spin trapping has become an indispensable tool for the specific detection of reactive oxygen free radicals in biological systems. In this review we describe some of the advantages as well as some experimental considerations of this technique and how it can be applied to biological systems to measure oxidative stress.     

iNOS-mediated generation of reactive oxygen and nitrogen species by biomaterial-adherent neutrophils

Infection due to implanted cardiovascular biomaterials is a serious complication initiated by bacterial adhesion to the surface of the implant. The release of reactive oxygen species by neutrophils, particularly superoxide anion, is a well-known bactericidal mechanism. Additionally, nitric oxide (NO) has also been identified as an important cytotoxic mediator in acute and chronic inflammatory responses with enhanced NO production by upregulation of inducible nitric oxide synthase (iNOS). The interaction of NO and superoxide anion will result in the formation of peroxynitrite (OONO-), a potent cytotoxic oxidant. In this study, we have shown that biomaterial-induced neutrophil activation does not cause upregulation of iNOS and activation of iNOS-mediated pathways. However, NO and O2- production does occur over time upon adhesion to a biomaterial and is modulated by biomaterial surface chemistry. With no stimulus, the polyethylene oxide-modified polyurethane induced greater neutrophil activation than did the control as indicated by the increased production of NO and O2- over time. Adherent-stimulated neutrophils generally produced lower amounts of NO over time in comparison with unstimulated cells. Furthermore, there is no evidence of peroxynitrite activity in unstimulated neutrophils adherent to the Elasthane 80A. However, upon stimulation with adherent Staphylococcus epidermidis, peroxynitrite formation did occur. Our results suggest that bactericidal mechanisms in neutrophils involving NO generation (NOS pathway) are further compromised than O2- producing pathways (NADPH oxidase) upon exposure to biomaterials, resulting in a diminished microbial killing capacity, which can increase the probability of device-centered infections.     

Molecular and genetic characterization of Francisella tularensis strains of differing taxonomic status and virulence

Typing of Francisella collection strains by means of PCR on the basis of the tul4 gene coding the protein of the external membrane of 17kD and the genome region of differentiation RD1 is performed. The identification of the species and subspecies of 112 strains of Francisella tularensis was revealed. PCR on DNA targets’ loci of type IV pili genes—pilA, pilE2, pilE3, pilE4, pilE5, pilF, pilT, pilD, and pilQ—was carried out for differentiation of F. tularensis strains by virulence. The possibility was shown of distinguishing F. tularensis strains in PCR (primers A–B) on the basis of the uncovering of the gene pilA in virulent strains of three subspecies F. tularensis and F. tularensis subsp. novicida. The pilA gene was not detected in the vaccine strain 15/10 and its variants, as well as in most of avirulent F. tularensis supsp. holarctica strains. However, the fragment gene pilA was found in the attenuated strains F. tularensis subsp. tularensis and mediasiatica. No differences were found in other targets of pili genes between F. tularensis strains, with the exception of the strain F. tularensis subsp. novicida Utahl12, which did not have a fragment of the gene pilE2. The use of PCR to target the locus of the piliA gene allows differentiation of virulent F. tularensis subsp. holarctica strains from the vaccine 15/10, its variants, and avirulent strains.     

Humoral and cell-mediated immunity to the intracellular pathogen Francisella tularensis

Summary: Francisella tularensis can cause fatal respiratory tularemia in humans and animals and is increasingly being isolated in the United States and several European countries. The correlates of protective immunity against this intracellular bacterium are not known, and currently there are no licensed vaccines available for human use. Cell-mediated immunity has long been believed to be critical for protection, and the importance of humoral immunity is also now recognized. Furthermore, synergy between antibodies, T cell-derived cytokines, and phagocytes appears to be critical to achieve sterilizing immunity against F. tularensis. Thus, novel vaccine approaches should be designed to induce robust antibody and cell-mediated immune responses to this pathogen.     

Production and characterization of monoclonal antibodies directed against the lipopolysaccharide of Francisella tularensis.

Two monoclonal antibodies (FT14 and FT2F11) directed against the lipopolysaccharide (LPS) of Francisella tularensis were produced for use in tests to detect the organism in environmental samples and clinical specimens. The specificity of the antibodies was determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Both antibodies detected LPS from F. tularensis by ELISA, but only one antibody, FT14, was serologically active in an immunoblot. Treatment of the LPS with detergents prior to ELISA eliminated its binding to FT2F11 but not FT14. Qualitatively, both antibodies detected 10 different strains of F. tularensis by ELISA, but quantitatively, FT14 gave a detectable reaction with 10(3) organisms, whereas FT2F11 was able to detect only 10(5) organisms. FT14 did not cross-react with LPS from a range of other gram-negative species of bacteria, whereas FT2F11 cross-reacted against Vibrio cholerae LPS. Neither antibody showed cross-reactions when entire gram-negative organisms were used as antigens. In a competition ELISA, the two monoclonal antibodies were shown to compete for different epitopes. FT14 was strongly inhibited by purified O side chain from F. tularensis LPS, but FT2F11 was only weakly inhibited. It was inferred from those results that FT14 is directed against the O side chain and that FT2F11 is directed against the core.     

Evaluation of the role of reactive oxygen species in male infertility

Reactive oxygen species (ROS) production from spermatozoa hasbeen measured by chemiluminescence in the two fractions of aPercoll gradient column (47 and 90%). Chemiluminescent signalswere recorded in each fraction after the addition of luminoland horse-radish peroxidase (basal state), and after stimulationwith formyl-methionyl-leucyl-phenylalanine and phorbol ester(PMA). Oligozoospermic samples show a higher rate of ROS productionthan the normozoospermic samples in both fractions of Percoll.Also, ROS were generated at a higher rate by asthenozoospermicsamples in the 90% Percoll fraction than by normal samples afterstimulation with PMA. Our data confirm the fact that white bloodcells play a major role in the production of ROS, even afterpurification on a Percoll gradient. Immunological cases werealso found to be associated with an increased production ofROS, which may be caused by the same underlying pathologicalcondition responsible for the production of the antibodies.Repeated centrifugation of the samples triggers a burst of ROSin excess of that produced after Percoll preparation. In addition,superoxide dismutase activity was found to be significantlyincreased in cases with an elevated production of ROS. It isconcluded that measuring the ROS generation by semen may yielduseful information on the functional capacity of spermatozoa,which may be used to improve the success of male infertilitymanagement.     

Diabetic endothelial dysfunction: role of reactive oxygen and nitrogen species production and poly(ADP-ribose) polymerase activation

Peroxynitrite and hydroxyl radicals are potent initiators of DNA single-strand breakage, which is an obligatory stimulus for the activation of the nuclear enzyme poly(ADP ribose) polymerase (PARP). In response to high glucose incubation medium in vitro, or diabetes and hyperglycemia in vivo, reactive nitrogen and oxygen species generation occurs. These reactive species trigger DNA single-strand breakage, which induces rapid activation of PARP. PARP in turn depletes the intracellular concentration of its substrate, NAD+, slowing the rate of glycolysis, electron transport, and ATP formation. This process results in acute endothelial dysfunction in diabetic blood vessels. Accordingly, inhibitors of PARP protect against endothelial injury under these conditions. In addition to the direct cytotoxic pathway regulated by DNA injury and PARP activation, PARP also appears to modulate the course of inflammation by regulating the activation of nuclear factor kappaB, and the expression of a number of genes, including the gene for intercellular adhesion molecule 1 and the inducible nitric oxide synthase. The research into the role of PARP in diabetic vascular injury is now supported by novel tools, such as new classes of potent inhibitors of PARP and genetically engineered animals lacking the gene for PARP. Pharmacological inhibition of PARP emerges as a potential approach for the experimental therapy of diabetic vascular dysfunction.     

Intranasal interleukin-12 treatment for protection against respiratory infection with the Francisella tularensis live vaccine strain

Francisella tularensis is a gram-negative intracellular bacterium that can induce lethal respiratory infection in humans and rodents. However, little is known about the role of innate or adaptive immunity in protection from respiratory tularemia. In the present study, the role of interleukin-12 (IL-12) in inducing protective immunity in the lungs against intranasal infection of mice with the live vaccine strain (LVS) of F. tularensis was investigated. It was found that gamma interferon (IFN-gamma) and IL-12 were strictly required for protection, since mice deficient in IFN-gamma, IL-12 p35, or IL-12 p40 all succumbed to LVS doses that were sublethal for wild-type mice. Furthermore, exogenous IL-12 treatment 24 h before intranasal infection with a lethal dose of LVS (10,000 CFU) significantly decreased bacterial loads in the lungs, livers, and spleens of wild-type BALB/c and C57BL/6 mice and allowed the animals to survive infection; such protection was not observed in IFN-gamma-deficient mice. The resistance induced by IL-12 to LVS infection was still observed in NK cell-deficient beige mice but not in CD8-/- mice. These results demonstrate that exogenous IL-12 delivered intranasally can prevent respiratory tularemia through a mechanism that is at least partially dependent upon the expression of IFN-gamma and CD8 T cells.