Role of Paracoccidioides brasiliensis cell wall fraction containing beta-glucan in tumor necrosis factor-alpha production by human monocytes: correlation with fungicidal activity.

The polysaccharide fraction of Paracoccidioides brasiliensis mycelial cell wall (F1 fraction), the active component of which is composed of beta-glucan, was investigated in regard to the activation of human monocytes for fungal killing. The cells were primed with interferon-gamma (IFN-gamma) or F1 (100 and 200 microg ml(-1)) or F1 (100 and 200 microg ml(-1)) plus IFN-gamma for 24 h and then evaluated for H2O2 release. In other experiments, the cells were pretreated with the same stimuli, challenged with a virulent strain of P. brasiliensis and evaluated for fungicidal activity and levels of tumor necrosis factor (TNF-alpha) in the supernatants. F1 increased the levels of H2O2 in a similar manner to IFN-gamma. However, a synergistic effect between these two activators was not detected. On the contrary, a significant fungicidal activity was only obtained after priming with IFN-gamma plus F1. This higher activity was associated with high levels of TNF-alpha in the supernatants of the cocultures. Overall, P. brasiliensis F1 fraction induced human monocytes to release relatively high levels of TNF-alpha, which, in combination with IFN-gamma, is responsible for the activation of human monocytes for effective killing of P. brasiliensis.     

Tumour necrosis factor and interferon-gamma are required in host resistance against virulent Rhodococcus equi infection in mice: cytokine production depends on the virulence levels of R. equi

Rhodococcus equi is a facultative intracellular bacterial pathogen that causes pneumonia in foals and immunosuppressed humans. There are at least three virulence levels of R. equi and these pathogenicities are associated, in mice, with the presence of virulence plasmids. This study focused on cytokine secretion, in mice, in the course of a primary infection with sublethal doses of R. equi strains of different virulence levels (virulent, intermediately virulent and avirulent). Tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma), but not interleukin-4 (IL-4) and interleukin-10 (IL-10), were induced endogenously in mice in relation to the multiplication and clearance of virulent and intermediately virulent strains of R. equi. These cytokines were not detected in mice infected with avirulent R. equi. Deaths occurred among mice treated with monoclonal antibodies (mAbs) against either TNF or IFN-gamma prior to sublethal dose infection with virulent and intermediately virulent strains of R. equi, but not with avirulent R. equi. These results suggested that cytokine production depended largely on the virulence levels of R. equi: TNF and IFN-gamma were required early during infection with virulent R. equi to limit replication and clearance of bacteria within the organs, but they were not necessary for limiting infection with avirulent R. equi.     

Synthesis and immunological activity of a branched peptide carrying the T-cell epitope of gp43, the major exocellular antigen of Paracoccidioides brasiliensis

The 43 kDa glycoprotein (gp43) of Paracoccidioides brasiliensis is the major diagnostic antigen of paracoccidioidomycosis (PCM), a prevalent fungal infection in South America. A 15-mer sequence from gp43, denominated P10, induced T-CD4+ T helper 1 cellular immune responses in mice of three different haplotypes and protected against intratracheal challenge by a virulent isolate of P. brasiliensis. In an attempt to improve delivery of P10, a promiscuous antigen also presented by human leucocyte antigen-DR alleles, aiming at immunotherapy, we synthesized a multiple antigen peptide with the protective T-cell epitope expressed in a tetravalent 13-mer analog of P10 (M10). M10 induced specific lymph node cell proliferation in mice preimmunized with peptides in complete Freund's adjuvant (CFA). In addition, M10 immunization without CFA significantly protected intratracheally infected mice. We conclude that M10 is a candidate for an anti-PCM vaccine. In this report we describe: (1) the synthesis of M10; (2) the induction of M10-elicited T-cell response and (3) in vivo protection of mice immunized with M10 and challenged by a virulent strain of P. brasiliensis.     

Effect of pyochelin on the virulence of Pseudomonas aeruginosa.

A virulent isolate of Pseudomonas aeruginosa PAO1, which had been obtained from eight sequential intraperitoneal infections in mice compromised with iron and methotrexate, expressed greater lethality than the avirulent parent strain when both strains were injected into mice treated with iron. The present study demonstrates that pyochelin, a siderophore produced by P. aeruginosa, also increases the lethality of the virulent bacteria but not of the avirulent bacteria. Analysis of the growth and clearance of both virulent and avirulent strains in mice revealed that pyochelin increased the growth and lethality of virulent bacteria but only increased the survival of the avirulent bacteria. A streptomycin-dependent mutant of strain PAO1 (strd1) was used to demonstrate that pyochelin did not affect the clearance activity of mice. This strongly suggests that the effects of pyochelin in stimulating the persistence of avirulent bacteria and in increasing the lethality of virulent bacteria are due solely to the promotion of bacterial growth. Since the virulent bacteria were equivalent to the avirulent bacteria in utilizing pyochelin during in vitro growth in the presence of transferrin, it appears that the stimulation of growth by pyochelin allows the expression of additional virulence properties by the virulent bacteria.     

Altered expression of surface alpha-1,3-glucan in genetically related strains of Blastomyces dermatitidis that differ in virulence.

Recent studies of the dimorphic fungal pathogens Histoplasma capsulatum and Paracoccidioides brasiliensis have suggested a role in virulence for the cell surface carbohydrate alpha-(1,3)-glucan. To investigate a possible basis for alpha-(1,3)-glucan in the pathogenicity and virulence of the dimorphic fungus Blastomyces dermatitidis, we examined three genetically related strains of B. dermatitidis that differ in their virulence for mice: wild-type virulent strain ATCC 26199; mutant strain ATCC 60915, which is 10,000-fold reduced in virulence; and mutant strain ATCC 60916, which is avirulent. Immunologic quantitation of cell wall alpha-(1,3)-glucan revealed that the mutant yeasts were almost devoid of this sugar moiety, in contrast to the high concentration of alpha-(1,3)-glucan on the cell wall of the wild-type yeasts. These differences are discussed in relation to previous studies of yeast surface expression of the WI-1 antigen and recognition and binding of the related strains by human monocyte-derived macrophages.     

Modulatory effect of prostaglandins on human monocyte activation for killing of high- and low-virulence strains of Paracoccidioides brasiliensis

The effect of indomethacin (Indo), a cyclo-oxygenase inhibitor, on the monocyte-mediated killing of a low- (Pb265) and a high- (Pb18) virulence strain of Paracoccidioides brasiliensis was examined. The Pb18 strain was not killed by either non-activated or interferon-gamma (IFN-gamma) -activated human monocytes but these cells did show fungicidal activity if pretreated with Indo. In contrast with IFN-gamma, tumour necrosis factor-alpha (TNF-alpha) was very effective at stimulating the fungicidal activity of monocytes. While the low-virulence strain, Pb265, could not be killed by monocytes, cells preincubated with IFN-gamma demonstrated fungicidal activity. The killing of this strain was also induced by pretreatment of monocytes with Indo. The results suggest a negative role for prostaglandins, which are synthesized via the cyclo-oxygenase pathway, in the regulation of monocyte-mediated killing of virulent and avirulent strains of P. brasiliensis and that TNF-alpha generation during the fungus-monocyte interaction is more important in the killing of Pb265 than Pb18.     

Natural killer cells mediate protection induced by a Salmonella aroA mutant.

We have previously shown that an avirulent strain of Salmonella typhimurium, SL3235, blocked in aromatic synthesis, confers high levels of resistance to challenge with virulent Salmonella as early as 3 days postvaccination. In the present studies, it was found that immunization with SL3235 resulted in high levels of natural killer (NK) cell activity in the spleens and peritoneal cavities of C3H/HeJ mice, as measured by cytotoxicity against YAC-1 targets. NK cell activity was at its maximum 2 to 4 days after immunization and was ablated by in vivo or in vitro treatment with anti-asialo GM1. In vivo treatment with anti-asialo GM1 during the first week after immunization with SL3235 depleted NK cell activity and markedly increased mortality in mice challenged with a virulent Salmonella strain. These results are compatible with a role for NK cells as one important component in the resistance against virulent Salmonella infection induced by a live, attenuated vaccine.     

Different virulence levels of the species of Sporothrix in a murine model

A comparative study on the experimental pathogenicity of five species of Sporothrix of clinical interest, Sporothrix albicans , Sporothrix brasiliensis , Sporothrix globosa , Sporothrix mexicana , and Sporothrix schenckii sensu stricto, was performed using an immunocompetent murine model. Two strains of each species and two levels of inoculum for each strain (2 × 10⁷ and 2 × 10⁴ conidia/animal) were tested by intravenous inoculation of mice (ten per group). Mortality was caused by the low inoculum of one strain of S. brasiliensis only, and the high inocula of S. brasiliensis and S. schenckii strains. Other inocula and other species tested did not kill any of the experimental animals. Tissue burden studies showed fungal spread to kidneys, lungs, spleen, brain, and testicles. S. brasiliensis was recovered extensively from all of the studied organs, and S. schenckii and S. globosa were recovered in lower amounts. Histopathological studies revealed differences in the lesions, which ranged from local inflammation with a low number of fungal cells at the injection site in mice infected with S. globosa , to massive infiltration of fungal cells in organs of those infected with S. brasiliensis . Our findings showed that S. brasiliensis and S. schenckii were the most virulent species, and suggest that lesional mechanisms could be species-specific.     

Interactions of virulent and avirulent Legionella pneumophila with human monocytes

Four pairs of virulent/avirulent strains of Legionella pneumophila were examined for their adherence/uptake and activation of human monocytes. Oxidative metabolic responses of monocytes were quantitated by measuring intracellular hydrogen peroxide generation using flow cytometry and by assessment of superoxide dismutase-inhibitable superoxide anion generation. All L. pneumophila strains induced less of a response than did Escherichia coli. Within each pair of isolates, virulent strains of L. pneumophila stimulated the oxidative response of monocytes less than avirulent variants. To determine effects of complement fixation by each strain on their adherence to monocytes, a phagocytic index (PI) was determined under various conditions. In autologous donor serum (AS), all L. pneumophila strains had a PI in the range of 2.1-3.1 bacteria per monocyte, with E. coli having a PI of 9.1. No significant differences were observed between virulent L. pneumophila strains and their avirulent variants. In the presence of heat-inactivated AS, all PI fell to 0.13-0.20 for the L. pneumophila strains, and to 2.16 for E. coli. Using heat-inactivated AS reconstituted with exogenous human complement as a source of opsonization, levels of PI were indistinguishable from their respective levels in AS. This suggests that complement fixation plays an important role in the adherence of virulent and avirulent L. pneumophila to human monocytes.     

Characterization and immunogenicity of Salmonella typhimurium SL1344 and UK-1 delta crp and delta cdt deletion mutants.

S. typhimurium SL1344 and UK-1 mutants with deletions of the crp (cyclic AMP receptor protein) and cdt (colonization of deep tissues) genes have been constructed and characterized, and their levels of virulence and immunogenicity have been determined for BALB/c mice. All Crp- Cdt- and Crp+ Cdt- mutants were avirulent, as mice survived oral doses of 10(9) cells without illness. All the mutants colonized the gut-associated lymphoid tissue efficiently, but capacities to colonize deeper tissues, such as those of the spleen and liver, and blood were significantly reduced for the Crp- Cdt- and Crp+ Cdt- mutants compared with the Crp- Cdt+ mutant and the wild-type parent strain. The Crp- Cdt- and Crp+ Cdt- SL1344 strains induced complete protection, as all mice immunized with the mutants survived challenge with approximately 10(4) times the 50% lethal dose of the wild-type SL1344 strain. The Crp- UK-1 strain was least attenuated yet induced the highest level of protective immunity against challenge with the wild-type UK-1 strain. The Crp+ Cdt- and Crp- Cdt- strains, although totally attenuated, differed in immunogenicity to challenge with the highly virulent UK-1 parent, with the apparently hyperattenuated Crp- Cdt- strain inducing a lower level of protective immunity than the Crp+ Cdt- strain. Nevertheless, these UK-1 Crp- Cdt- and Crp+ Cdt- strains induced complete protective immunity to challenge with the less-virulent SL1344 wild-type strain. Taken collectively, the results indicate that the attenuation of a highly virulent S. typhimurium strain can yield a vaccine that induces excellent protective immunity to challenge with less-virulent S. typhimurium strains.     

Cell wall composition of two strains of Blastomyces dermatitidis exhibiting differences in virulence for mice

Cell walls isolated from two strains of Blastomyces dermatitidis were examined. Whereas strain Ga-1 was practically avirulent for mice, strain KL-1 produced death by 21 days in 50% of the mice inoculated. Analyses of the trypsin-treated cell walls of the two strains revealed a higher chitin and protein content in strain KL-1, whereas a higher polysaccharide content was observed in the cell walls of strain Ga-1. Extraction of the walls with 1 n NaOH revealed a threefold difference in the amount of alkali-soluble cell wall material present. The alkali-soluble material could be further fractionated into a water-soluble and a water-insoluble fraction. Previous reports have indicated that the water-insoluble fraction of B. dermatitidis consists of an alpha-linked glucan; however, we report that in addition a phospholipid moiety is covalently bound to the polysaccharide. Furthermore, on the basis of organic phosphorus content, considerably more phospholipid is associated with the alpha-linked glucan of the more virulent KL-1 strain. These results suggest that this cell wall constituent might be one of the factors related to the virulence of this fungus.     

Studies on early association of Salmonella typhimurium with intestinal mucosa in vivo and in vitro: relationship to virulence

The abilities of six strains of Salmonella typhimurium to associate with rabbit ileal mucosa have been measured in vitro. Two were "virulent" strains (TML and W118 which are invasive and inducers of fluid secretion in rabbit ileal loops); four were "avirulent" (LT7, M206 and SL1027 which are invasive but induce negligible fluid secretion, and Thax-1 which is neither invasive nor an inducer of fluid secretion). A special organ-culture apparatus was designed to expose only the luminal surface of the mucosa to organisms. Viable counts of washed homogenised tissue taken 30 min after challenge showed that virulent strains TML and W118 and avirulent strains LT7 and M206 could not be distinguished from each other. Avirulent strain SL1027 associated less well than the other four strains, and Thax-1 associated less well than SL1027; both these strains were non-motile whereas the other four were motile. Thus, early association with gut mucosa did not discriminate all avirulent strains from the virulent strains. Qualitative examination of tissues by scanning electronmicroscopy did not detect strains LT7 and M206 on the mucosal surface whereas strains TML and W118 were readily seen, suggesting that the nature of association of virulent and avirulent strains was different. Qualitative examination by transmission electronmicroscopy of tissues challenged in vivo for 120 min showed virulent strains TML and W118 invading epithelial cells; similar events were reproduced after 120-min challenge in vitro. In contrast, invasion by avirulent strains was observed only very rarely.     

Protective Effect of Vaccines on Mycoplasma pulmonis-Induced Respiratory Disease of Mice

Mice inoculated intranasally with either a virulent or an avirulent strain of live Mycoplasma pulmonis were resistant to respiratory disease induced by a subsequent intranasal challenge with virulent organisms. Similarly, mice inoculated intravenously with the virulent strain were resistant to intranasal challenge with the same strain. In contrast, mice inoculated intravenously with avirulent M. pulmonis were not resistant to intranasal challenge with the virulent mycoplasma strain. Studies on mice inoculated intravenously with the two strains of M. pulmonis indicated that persistance of mycoplasmas in the respiratory tract may be important in inducing resistance to intranasal challenge with M. pulmonis. These observations, together with the lack of correlation between the level of serum antibodies and resistance to M. pulmonis-induced respiratory disease, suggested that local immune mechanisms were important in resistance. It is proposed that an effective vaccination schedule to protect mice against M. pulmonis-induced respiratory disease may be one that stimulates both systemic and local immune defenses. This suggestion is supported by the observation that systemic followed by local administration of inactivated M. pulmonis was more effective in inducing resistance in mice to intranasal challenge with live organisms than was systemic administration alone. In addition, mice inoculated solely by the intranasal route with inactivated mycoplasmas were resistant to M. pulmonis-induced respiratory disease. These studies indicate the importance of local defense mechanisms in the induction of resistance to M. pulmonis-induced respiratory disease in mice.     

Biosynthetic capacity for type-specific antigen synthesis determines the virulence of serotype III strains of group B streptococci

The level of type-specific antigen (that covalently associated with the cell wall peptidoglycan and that released extracellularly) synthesized by virulent and avirulent strains of type III group B streptococci was quantitated and compared. Additionally, the effect of the physiological age of the cells and the influence of the exogenous phosphate ion concentration on the level of antigen synthesis by these organisms were also examined. Approximately 4% of the total antigen synthesized by the organism is noncovalently bound to the cell surface, and the difference in level of the noncovalently associated type-specific antigen between virulent and avirulent strains was negligible. In contrast, when the cell-associated covalently bound type antigens were evaluated, virulent strains were demonstrated to have two- to threefold higher levels than those of avirulent strains during the exponential and stationary phases of growth under various growth conditions. Furthermore, virulent strains that had high levels of cell-associated type antigen also secreted more extracellular type antigen than did avirulent strains. Thus, the data were consistent with the hypothesis that an overall production of type-specific antigen correlated with virulence in mice. However, the cell-associated type-specific antigen probably represented a better indicator for virulence potential since the addition of purified extracellular type-specific antigen to a mutant strain that lacks cell surface type antigen did not alter the 50% lethality value of the organism. To account for variation in the level of type-specific antigen produced by these strains, the kinetics of both the group- and type-specific antigens synthesis was investigated at the cell membrane level by utilizing an intact protoplast system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protection and immune responses induced by attenuated Salmonella typhimurium UK-1 strains

We previously reported that Salmonella typhimurium SR-11 mutants with deletion mutations in the genes encoding adenylate cyclase (cya) and the cAMP receptor protein (crp) are avirulent and protective in mice. Salmonella typhimurium UK-1 is highly virulent for chicks (oral LD50 of 3x10(3) CFU) and mice (oral LD50 of 8.5x10(3) CFU) and is capable of lethal infections in pigs, calves and horses. We postulated that attenuated derivatives of this lethal strain would probably induce a higher level of protective immunity than achieved with attenuated derivatives of less virulent S. typhimurium strains such as SR11. To test this hypothesis, we have constructed S. typhimurium UK-1 Deltacya-12Deltacrp-11 mutant strain chi3985 and its virulence plasmid cured derivative chi4095 to investigate their avirulence and immunogenicity in mice. We found that the mutants are avirulent and able to induce protective immune responses in BALB/c mice. These mutant strains retained wild-type ability to colonize the gut associated lymphoid tissue but reach and persist in spleen and liver at a significantly lower level than the wild-type parent strain. Mice survived oral infection with >1x10(9) CFU of chi3985 (the equivalent to 10(5) 50% lethal doses of wild-type S. typhimurium UK-1) and were fully protected against challenge with 10(5)times the LD50 of the wild-type parent. Immunized mice developed a high level of serum IgG titre to Salmonella LPS and delayed-type hypersensitivity (DTH) response to S. typhimurium outer membrane proteins. Compared to the virulence plasmid-containing strain chi3985, the virulence plasmid cured DeltacyaDeltacrp mutant strain chi4095 was more attenuated and less protective, as some mice immunized with chi4095 died when challenged with the wild-type UK-1 strain. This work demonstrates that S. typhimurium UK-1 Deltacrp Deltacya mutant strain may be a potential live vaccine to induce protective immunity against Salmonella infection or to deliver foreign antigens to the immune system.     

Involvement of nucleoprotein, phosphoprotein, and matrix protein genes of rabies virus in virulence for adult mice

Rabies virus Ni-CE strain causes nonlethal infection in adult mice after intracerebral inoculation, whereas the parental Nishigahara strain kills mice. In this study, to identify viral gene(s) related to the difference in pathogenicity between Ni-CE and Nishigahara strains, we generated chimeric viruses with respective genes of the virulent Nishigahara strain in the background of the avirulent Ni-CE genome. Since chimeric viruses, which had the N, P, or M genes of the Nishigahara strain, respectively, killed adult mice after intracerebral inoculation, it became evident that the N, P, and M genes are related to the difference in pathogenicity between Ni-CE and Nishigahara strains. Previously, we showed that the G gene is a major contributor to the difference in pathogenicity between another avirulent strain, RC-HL, and the parental Nishigahara strain. These results imply that the attenuation mechanism of the Ni-CE strain is different from that of the RC-HL strain, thus suggesting that rabies virus can be attenuated by diverse mechanisms. This is the first report of changes in viral genes other than the G gene of rabies virus causing the reversion of pathogenicity of an avirulent strain.     

Loss of virulence in a protease-deficient mutant of Aeromonas salmonicida.

The importance of extracellular protease production by Aeromonas salmonicida, the bacterial pathogen of fish furunculosis, was investigated with four virulent strains (which were autoagglutinative, hemagglutinative, resistant to fish serum, adhesive to fish tissue culture, protease positive, hemolysin positive, and leukocytolysin positive) and three avirulent strains (which were nonagglutinative, nonhemagglutinative, sensitive to serum, nonadhesive, protease positive, hemolysin positive, and leukocytolysin positive). A protease-deficient mutant (NTG-1) was induced by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine from virulent strain A-7301, showing protease production, which was common to the two strain groups. Strain NTG-1 showed loss of virulence in determinations of 50% lethal doses, although it remained autoagglutinative, hemagglutinative, serum resistant, adhesive, hemolysin positive, and leukocytolysin positive. A protease fraction separated from extracellular products of strain A-7301 by DEAE-cellulose column chromatography had the capacity to produce skin lesions (furuncles) and high mortality in sockeye salmon. A comparable protein fraction from extracellular products of NTG-1 resulted in no protease activity and no pathological effects on the fish. The avirulent strains were eliminated from rainbow trout in a short time, whereas the virulent strains (including A-7301) were highly infective and proliferated in hosts. NTG-1 preserved its infectivity, but fish showed no signs of disease and no mortality. These findings indicate that extracellular protease is a major virulence factor and that protease production in the host is closely implicated in the pathogenesis of fish furunculosis.     

Influence of mouse genotype and bacterial virulence in the generation of interferon-gamma-producing cells during the early phase of Salmonella typhimurium infection.

Interferon-gamma (IFN-gamma) is known to play a major role in resistance to Salmonella typhimurium infection. In this study, the IFN-gamma production in spleens of mice infected with S. typhimurium was analysed at the single cell level using an ELISPOT method. The in vivo IFN-gamma production during the early phase of infection with virulent and avirulent S. typhimurium strains was examined in four mouse strains. Data show that infection with a virulent strain of S. typhimurium caused a much greater enhancement in the frequency of IFN-gamma-producing cells in innately resistant (ltyr) mice (CBA and DBA/2) than in susceptible (ltys) mice (C57BL/6 and BALB/c). In contrast, infection with an avirulent strain of S. typhimurium induced a clear increase in the number of IFN-gamma-producing cells in susceptible mice which was even greater than in resistant ones. These results indicate that both the host genetic background and bacterial virulence play a critical role in the regulation of IFN-gamma production during the early phase of S. typhimurium infection.     

Morphological evidence for penetration of anti-O antibody through the capsule of Klebsiella pneumoniae.

The role of the capsule in the reaction with anti-O or anti-K serum was examined morphologically by the techniques of freeze-substitution and immunoelectron microscopy in two strains of Klebsiella pneumoniae differing in virulence for mice. Strain Chedid (O1:K2), an encapsulated virulent strain, has a thicker capsule (150 nm) than the encapsulated avirulent strain 277 (O1:K2) (60 nm). Two morphologically recognizable domains in the capsule created by the arrangement of the capsular filaments were clearly seen in strain Chedid but were less evident in strain 277. Anti-O antibody could penetrate through the capsular layer of both strains. The capsule has no function as a barrier for the penetration of the antibody. Anti-K antibody reacted only on the surface of the capsule and induced swelling of the capsule.