Immunogenic dialyzable factor derived from a ribosomal fraction of Salmonella typhimurium III. Analysis of resistance induced by dialyzable factors

Dialyzable factors (DF) were prepared from ribosomal fractions of several organisms including rough mutants of Salmonella typhimurium LT2, salmonella species of different serogroups, other enteric bacteria and gram-positive organisms, and tested for their immunogenicity against S. typhimurium infection in mice. All of them conferred local resistance on mice challenged intramuscularly with S. typhimurium LT2 in the early stage of immunization before the establishment of delayed-type hypersensitivity (DTH) to salmonella antigens. Although DFs of enteric bacteria including rough mutants of S. typhimurium induced DTH to salmonella antigens, only DF of a two-heptose mutant of S. typhimurium LT2 afforded significant mouse protection but others only prolonged the mean time to death. DF of Listeria monocytogenes induced the cross-reacting immunity which afforded the low level of mouse protection as well as an increase in mean time to death without inducing DTH. Passive transfer of anti-O antibody did not enhance the mouse protection provided by each DF. Resistance conferred by DF of S. typhimurium LT2 consisted of two phases: (i) nonspecific macrophage activation resulting in reduction of organisms at the infected site, which became active in the early stage of immunization and (ii) salmonella-specific immunity capable of preventing systemic infection, which became active in the late stage of immunization.     

Mechanisms of acquired resistance in mouse typhoid

A polyvalent radiovaccine of Salmonella was shown to induce protective immunity in mice. The results revealed that the immunized mice progressively eliminated the challenged organisms and no Salmonella could be isolated after a period of 21 days. In contrast, Salmonella grew in the control mice and reached levels of 10(8) to 10(9) cfu/spleen resulting in the death of animals. Sera from both control and the immune mice were found to lack bactericidal activity. It was further observed that the vaccine induced delayed type of hypersensitivity and that antibody production as measured by bacterial agglutination and passive hemagglutination were low in response to the vaccine. However, the phagocytic activity of the reticuloendothelial system was considerably enhanced by the immunization. The results of experiments with immunosuppressed mice suggested the role of thymus derived (T) lymphocytes in the protection.     

Immunogenic dialyzable factor derived from a ribosomal fraction of Salmonella typhimurium. II. Isolation and characterization of the protective moiety in the dialyzable factor

An immunogenic dialyzable factor was obtained by dialysis of the freeze-thawed ribosomal fraction derived from a smooth virulent strain (LT2) of Salmonella typhimurium. Ion exchange chromatography of the dialyzable factor on Dowex 1-X2 (Cl- form) demonstrated the presence of four peaks and the fourth peak eluted with 0.4 M NaCl in 0.005 N HCl was found to be necessary for protection. This effective peak was not obtained by chromatography of nonprotective dialyzable factors such as an RNase digest. Dowex chromatography of the dialyzable factors isolated from rough mutants of strain LT2 revealed that the dialyzable factor of strain SL1004 whose live vaccine is capable of inducing protective immunity contained fairly large amounts of peak IV. DEAE-cellulose for two-dimensional thin layer chromatography was used to identify the composition of the dialyzable factor and peak IV. Eight spots were located under ultraviolet light and seven spots were characterized by their absorption ratios. In peak IV, four nucleotides were located and identified by comparison with a map of the original dialyzable factor. The data show that the effective components of the dialyzable factor are mixed nucleotides and may be unique to ribonucleic acids of strains of S. typhimurium in which live vaccines are capable of affording mouse protection.     

Immunogenic dialyzable factor derived from a ribosomal fraction of Salmonella typhimurium. I. Preparation of the protective dialyzable factor from the ribosomal fraction by the freeze-thaw procedure

The preparation, properties, and immunogenicity of the dialyzable factor from a ribosomal fraction of Salmonella typhimurium are described. The ribosomal fraction was purified to eliminate O-antigenic components, by affinity chromatography (Sepharose-anti-O antibody conjugates used as immunoadsorbent). The dialyzable factor was obtained in the concentrated dialysate of the purified ribosomal fraction which was alternately frozen in dry-ice acetone and thawed in an 80 C water bath, for a total of five or six cycles. When this preparation was tested for its ability to protect mice against challenge with 1,000 LD50 of the homologous bacteria, it afforded 100% protection at a dose equivalent to 5.0 micrograms of RNA. The protection conferred by this factor was mainly cell mediated but immune serum enhanced this immunity despite the fact that no antibodies were detected in it. The protective activity of this factor was sensitive to RNase digestion but resistant to proteolytic enzymes. Ion exchange chromatography of this factor with DEAE-Sephadex A-25 (in 7 M Urea-0.02 M Tris-HCl buffer, pH 7.5) resulted in a single A260 peak which was found to be immunogenic. Chemical analysis of this peak after it was concentrated and desalted revealed that this immunogenic fraction was composed mainly of mixed nucleotides. The data indicate that protective immunity conferred by a ribosomal vaccine is associated with RNA but may not require the intact RNA molecule.     

Role of CD4+ T cells and T-independent mechanisms in acquired resistance to Salmonella typhimurium infection

Two aspects of acquired resistance to Salmonella typhimurium infection in BALB/c mice, i.e., the ability to clear the primary inoculum from the spleen and resistance to a secondary challenge, were studied with the use of mAb against T cell subsets. The ability to clear a temperature-sensitive mutant of S. typhimurium from the spleen (assessed at day 21) was abrogated by in vivo treatment with anti-CD4 mAb. Accelerated bacterial clearance could be adoptively transferred into naive mice. In vitro depletion experiments also showed the role of CD4+ T cells in this phenomenon. Depletion of CD8+ T cells had only a marginal effect. Resistance to reinfection in the late phase of the primary infection (day 50) was markedly depressed by in vivo treatment with anti-CD4 mAb, whereas this was not the case during the early phase (day 14). Furthermore, during the early phase of infection athymic nude mice showed increased nonspecific resistance to reinfection. Taken together these results suggest that T-independent mechanisms play a major role in acquired resistance during the early phase of infection.     

Cutting edge: role of B lymphocytes in protective immunity against Salmonella typhimurium infection

Infection of mice with Salmonella typhimurium gives rise to a disease similar to human typhoid fever caused by S. typhi. Since S. typhimurium is a facultative intracellular bacterium, the requirement of B cells in the immune response against S. typhimurium is a longstanding matter of debate. By infecting mice on a susceptible background and deficient in B cells (Igmu-/- mice) with different strains of S. typhimurium, we could for the first time formally clarify the role of B cells in the response against S. typhimurium. Compared with Igmu+/+ mice, LD50 values in Igmu-/- mice were reduced during primary, and particularly secondary, oral infection with virulent S. typhimurium. After systemic infection, Igmu-/- mice cleared attenuated aroA- S. typhimurium, but vaccine-induced protection against systemic infection with virulent S. typhimurium involved both B cell-dependent and -independent effector mechanisms. Thus, B cell-mediated immunity plays a distinct role in control of S. typhimurium in susceptible mice.     

The role of gamma interferon in acquired host resistance against Staphylococcus aureus infection in mice

We investigated the expression of an acquired host resistance against Staphylococcus aureus infection in mice. When C57BL/6 mice were immunized with viable S. aureus and challenged with S. aureus eight weeks later, the elimination of S. aureus from the spleen and liver was enhanced in the immunized mice compared with the nonimmunized mice. When gamma interferon (IFN-gamma(-/-)) mice were immunized and challenged, the bacterial numbers in the organs of immunized mice were comparable to those in the nonimmunized mice, suggesting that IFN-gamma plays a critical role in an acquired host resistance against S. aureus infection. IFN-gamma(-/-) mice produced the lower level of anti-S. aureus immunoglobulin M (IgM) and IgG2a antibodies compared with C57BL/6 mice. To elucidate the role of IFN-gamma produced during a challenge with S. aureus, a single injection of anti-IFN-gamma monoclonal antibody to mice was carried out 1 h before challenge. An acquired resistance against S. aureus infection was inhibited by injecting with anti-IFN-gamma monoclonal antibody. However, anti-IFN-gamma monoclonal antibody treatment failed to modulate anti-S. aureus IgM, IgG1 or IgG2a responses in these animals. These results demonstrated that IFN-gamma is required for an acquired resistance against S. aureus infection in mice. However, IFN-gamma induced during the challenge failed to affect the secondary antibody responses.     

Critical role of CD28 in protective immunity against Salmonella typhimurium

Efficient T cell activation requires both TCR signals and costimulatory signals. CD28 is one of the molecules that provide costimulatory signals for T cells. We used mice deficient in CD28 expression (CD28-/- mice) to analyze the role of CD28 in the immune response against the intracellular bacterium Salmonella typhimurium, the causative agent of murine typhoid fever. CD28-/- mice were highly susceptible to infection with wild-type S. typhimurium and even failed to control infection with attenuated aroA- S. typhimurium. More detailed analysis revealed that CD28-/- animals did not mount a T-dependent Ab response and were highly impaired in the production of IFN-gamma. Thus, CD28 cosignaling is crucial for immunity against S. typhimurium. To our knowledge, this is the first report describing an essential role for CD28 in protective immunity against an intracellular microbial pathogen.     

Salmonella typhimurium acrB-like gene: identification and role in resistance to biliary salts and detergents and in murine infection

Abstract Salmonella serotype typhimurium transpositional mutants altered in resistance to biliary salts and detergents were isolated previously. We have characterized further the LX1054 mutant strain, the most sensitive of them. The chromosomal DNA segment flanking transposon insertion was cloned and sequenced. The highest level of identity was found for the acrB (formerly acrE ) gene of Escherichia coli , a gene encoding a drug efflux pump of the Acr family. LX1054 exhibited a reduced capacity to colonize the intestinal tract. After passages in mice, the mutant strain lost the sensitive phenotype. In vitro, a resumption of growth appeared after 17 h of culture in medium with cholate or other tested biological or chemical detergents. Then, the acquired resistant phenotype seemed stable. The data suggested a role of S. typhimurium acrB -like gene in resistance to biliary salts and detergents and in mice intestinal colonization. However, the local and transient sensitivity observed in vivo, and the in vitro adaptations suggest that several detergent-resistance mechanisms operate in S. typhimurium .     

The role of interleukin 1 and interleukin 2 in human T colony formation

We investigated the roles of interleukin 1 (IL1) and interleukin 2 (IL2) on T colony formation by PHA-stimulated peripheral blood lymphocytes (PBL). Purified T cells stimulated by PHA could not generate T colonies as did PBL. Media conditioned by PHA-stimulated PBL (PHA-LCM) contained IL2 and a T colony-promoting activity (TCPA) which induced T colony formation in PHA-stimulated purified T cells. IL2 and TCPA are coeluted in the same peak of 18,000 molecular weight after gel filtration chromatography. Moreover, TCPA present in the PHA-LCM could be absorbed on IL2-sensitive cells which possessed specific receptors for IL2. These results suggest that TCPA and IL2 are related entities. Monocytes or IL1 (a monokine released by activated monocytes) also induced T colony formation in purified T cells. Phorbol myristate acetate (PMA) could replace monocytes in the induction of T colony. Monocytes, IL1, or PMA are known to be crucial requirements for IL2 production by PHA-stimulated T cells. This combined with the fact that IL2 participates in T colony formation suggests that monocytes induce T colony formation through IL2 production.     

Separate transfer of mouse protection and delayed-type hypersensitivity with Salmonella typhimurium transfer factor

Delayed-type hypersensitivity (DTH) induced with Salmonella typhimurium transfer factor (TF) contributed to an increase in mean survival days of mice challenged with homologous organisms and afforded only a low level of host protection as determined by survival rate, compared with that obtained by active immunization. TF of other enteric bacteria could transfer DTH which is cross-reactive to salmonella antigen but did not afford host protection. Although TF of Listeria monocytogenes did not transfer the cross-reactive DTH, it could confer the significant increase in mean survival days against the lethal challenge with S. typhimurium. Listerial ribosomal vaccine conferred the high level of mouse protection without inducing DTH to salmonella antigen. The resistance generated upon active immunization with listerial ribosomal vaccine could be enhanced by the injection of S. typhimurium TF to the same level as that obtained after immunization with homologous ribosomal vaccine. Among salmonella TF, there could be no cross-reactive immunity between S. typhimurium and S. choleraesuis, although the cross-reactive DTH was observed. The DTH transfer ability of TF was sensitive to Pronase which could not affect the ability to transfer host immunity, but RNase could abolish the ability to transfer host immunity without impairing DTH transfer activity. These results suggest that in mouse typhoid infection, DTH is not associated with host protection as determined by survival rate.     

Global analysis of the eukaryotic pathways and networks regulated by Salmonella typhimurium in mouse intestinal infection in vivo

Background

Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C.

Results

We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein.

Conclusion

We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.     

The role of cAMP in flagellation of Salmonella typhimurium.

A mutational alteration either in adenylate cyclase (cya-) or in cyclic-3'5'-AMP (cAMP) receptor protein (crp-) rendered Salmonella typhimurium incapable of producing flagella. The amount of mRNA specific for flagellin in these mutants was almost negligible when assayed in an in vitro protein synthesizing system. A secondary mutation cfs, partially suppressing the cya- mutation, was identified among the revertants of cya-. A mutation in the same cistron as cfs resulted in a non-flagellate phenotype either by itself or in combination with cfs. The cistron, which was given the gene symbol flaT, was located between flaE and flaL. It was suggested that cAMP receptor protein together with cAMP modulates the gene flaT, which in turn acts as a positive effector on the synthesis of active mRNA specific for flagellin.     

Immunosuppression in viral oncogenesis. III. Effects of virus infection on interleukin 1 and interleukin 2 generation and responsiveness

Malignant rabbit fibroma virus (MV) is an oncogenic immunosuppressive leporipoxvirus. We studied the effects of MV infection and MV-associated tumor-induced suppressor factor (TISF) on the production of and responsiveness to interleukins 1 and 2. Adherent cells from MV tumor-bearing rabbits elaborate adequate amounts of IL 1 in response to E. coli endotoxin. Neither live virus nor TISF alters the production or the responsiveness to IL 1. However, when we examined spleen cells from rabbits 7 days after MV inoculation, we noted that their ability to produce and respond to IL 2 is deficient. Despite their relatively poor capacity to produce IL 2, these spleen cells express receptor for IL 2 in normal amounts, as measured by the monoclonal antibody 7D4. TISF derived from T lymphocytes from MV tumor-bearing rabbits is by itself capable of inhibiting partially normal secretion of IL 2 and also the response of the cloned murine T cell line HT-2 to added IL 2. Full expression of the immunosuppressive capacity of spleen cells from MV tumor-bearing rabbits requires cell-cell contact, however, and cannot be replaced by either live virus or spleen cell supernatants. Such spleen cells inhibit normal mitogen responsiveness, a defect not remedied by adding exogenous IL 2. Immunologic dysfunction induced by MV infection is transient, and by 11 days after virus inoculation, actively mediated recovery from immunosuppression is observed. We found that spleen cells from rabbits studied 11 days postinoculation secreted IL 2 normally. Thus, immunologic dysfunction secondary to infection with malignant rabbit fibroma virus reflects deficiencies in both elaboration of and response to IL 2, and return of immune function later in the course of the infection is associated with return of the ability of lymphocytes to secrete IL 2.