Noncompetitive expansion of cytotoxic T lymphocytes specific for different antigens during bacterial infection.
(17/47697)
Listeria monocytogenes is an intracellular bacterium that elicits complex cytotoxic T-lymphocyte (CTL) responses in infected mice. The responses of CTL populations that differ in antigen specificity range in magnitude from large, dominant responses to small, subdominant responses. To test the hypothesis that dominant T-cell responses inhibit subdominant responses, we eliminated the two dominant epitopes of L. monocytogenes by anchor residue mutagenesis and measured the T-cell responses to the remaining subdominant epitopes. Surprisingly, the loss of dominant T-cell responses did not enhance subdominant responses. While mice immunized with bacteria lacking dominant epitopes developed L. monocytogenes-specific immunity, their ability to respond to dominant epitopes upon rechallenge with wild-type bacteria was markedly diminished. Recall responses in mice immunized with wild-type or epitope-deficient L. monocytogenes showed that antigen presentation during recall infection is sufficient for activating memory cells yet insufficient for optimal priming of naive T lymphocytes. Our findings suggest that T-cell priming to different epitopes during L. monocytogenes infection is not competitive. Rather, T-cell populations specific for different antigens but the same pathogen expand independently. (+info)
Cryptosporidium parvum sporozoite pellicle antigen recognized by a neutralizing monoclonal antibody is a beta-mannosylated glycolipid.
(18/47697)
The protozoan parasite Cryptosporidium parvum is an important cause of diarrhea in humans, calves, and other mammals worldwide. No approved vaccines or parasite-specific drugs are currently available for the control of cryptosporidiosis. To effectively immunize against C. parvum, identification and characterization of protective antigens are required. We previously identified CPS-500, a conserved, neutralization-sensitive antigen of C. parvum sporozoites and merozoites defined by monoclonal antibody 18.44. In the present study, the biochemical characteristics and subcellular location of CPS-500 were determined. CPS-500 was chloroform extractable and eluted with acetone and methanol in silicic acid chromatography, consistent with being a polar glycolipid. Following chloroform extraction and silicic acid chromatography, CPS-500 was isolated by high-pressure liquid chromatography for glycosyl analysis, which indicated the presence of mannose and inositol. To identify which component of CPS-500 comprised the neutralization-sensitive epitope recognized by 18.44, the ability of the monoclonal antibody to bind CPS-500 treated with proteases, or with alpha- or beta-glycosidases, was determined. Monoclonal antibody 18.44 did not bind antigen treated with beta-D-mannosidase but did bind antigen treated with alpha-D-mannosidase, other alpha- or beta-glycosidases, or a panel of proteases. These data indicated that the target epitope was dependent on terminal beta-D-mannopyranosyl residues. By immunoelectron microscopy, 18.44 binding was localized to the pellicle and an intracytoplasmic tubulovesicular network in sporozoites. Monoclonal antibody 18.44 also bound to antigen deposited and released onto substrate over the course travelled by gliding sporozoites and merozoites. Surface localization, adhesion and release during locomotion, and neutralization sensitivity suggest that CPS-500 may be involved in motility and invasion processes of the infective zoite stages. (+info)
Hyperproduction of alpha-hemolysin in a sigB mutant is associated with elevated SarA expression in Staphylococcus aureus.
(19/47697)
To evaluate the role of SigB in modulating the expression of virulence determinants in Staphylococcus aureus, we constructed a sigB mutant of RN6390, a prototypic S. aureus strain. The mutation in the sigB gene was confirmed by the absence of the SigB protein in the mutant on an immunoblot as well as the failure of the mutant to activate sigmaB-dependent promoters (e.g., the sarC promoter) of S. aureus. Phenotypic analysis indicated that both alpha-hemolysin level and fibrinogen-binding capacity were up-regulated in the mutant strain compared with the parental strain. The increase in fibrinogen-binding capacity correlated with enhanced expression of clumping factor and coagulase on immunoblots. The effect of the sigB mutation on the enhanced expression of the alpha-hemolysin gene (hla) was primarily transcriptional. Upon complementation with a plasmid containing the sigB gene, hla expression returned to near parental levels in the mutant. Detailed immunoblot analysis as well as a competitive enzyme-linked immunosorbent assay of the cell extract of the sigB mutant with anti-SarA monoclonal antibody 1D1 revealed that the expression of SarA was higher in the mutant than in the parental control. Despite an elevated SarA level, the transcription of RNAII and RNAIII of the agr locus remained unaltered in the sigB mutant. Because of a lack of perturbation in agr, we hypothesize that inactivation of sigB leads to increased expression of SarA which, in turn, modulates target genes via an agr-independent but SarA-dependent pathway. (+info)
Role of iron in Nramp1-mediated inhibition of mycobacterial growth.
(20/47697)
Innate resistance to mycobacterial growth is mediated by a gene, Nramp1. We have previously reported that Nramp1 mRNA from macrophages of Mycobacterium bovis BCG-resistant (Bcgr) mice is more stable than Nramp1 mRNA from macrophages of BCG-susceptible (Bcgs) mice. Based on these observations and on reports that show that the closely related Nramp2 gene is a metal ion transporter, we evaluated the effect of iron on the growth of Mycobacterium avium within macrophages as well as on the stability of Nramp1 mRNA. The addition of iron to macrophages from Bcgs mice resulted in a stimulation of mycobacterial growth. In contrast, iron increased the capacity of macrophages from Bcgr mice to control the growth of M. avium. When we treated recombinant gamma interferon (IFN-gamma)-activated macrophages with iron, we found that iron abrogated the growth inhibitory effect of IFN-gamma-activated macrophages from Bcgs mice but that it did not affect the capacity of macrophages from Bcgr mice to control microbial growth. A more detailed examination of the effect of iron on microbial growth showed that the addition of small quantities of iron to resident macrophages from Bcgr mice stimulated antimicrobial activity within a very narrow dose range. The effect of iron on the growth inhibitory activity of macrophages from Bcgr mice was abrogated by the addition of catalase or mannitol to the culture medium. These results are consistent with an Fe(II)-mediated stimulation of the Fenton/Haber-Weiss reaction and hydroxyl radical-mediated inhibition of mycobacterial growth. (+info)
Effect of transforming growth factor beta on experimental Salmonella typhimurium infection in mice.
(21/47697)
We have investigated the effect of the in vivo administration of recombinant transforming growth factor beta (rTGF-beta) on the pathogenic mechanisms involved in Salmonella typhimurium experimental infection in mice. The protective response elicited by macrophages was induced by rTGF-beta1 by 2 days after experimental infection, as demonstrated by an increased NO production, while the humoral protective effect began with cytokine mRNA expression 2 days after the challenge and continued after 5 days with cytokine release and lymphocyte activation. We demonstrated that all mice who received rTGF-beta1 survived 7 days after infection. The number of bacteria recovered in the spleens and in the livers of rTGF-beta1-treated mice 2 and 5 days after infection was significantly smaller than that found in the same organs after phosphate-buffered saline (PBS) inoculation. Furthermore, 2 and 5 days after infection, splenic macrophages from rTGF-beta1-treated mice showed a greater NO production than did those from PBS-treated mice. The effect of rTGF-beta1 on S. typhimurium infection in mice was correlated with the expression of cell costimulatory CD28 molecules. Five days after S. typhimurium infection, the percentage of CD28(+)-expressing T cells in splenic lymphocytes from rTGF-beta1-treated mice increased with respect to that from control mice. Gamma interferon (IFN-gamma) mRNA was present in a greater amount in spleen cells from rTGF-beta1-treated mice after 2 days, although the intensity of the band decreased 5 days after the challenge. A similar pattern was obtained with the mRNAs for interleukin-1alpha (IL-1alpha), IL-6, TGF-beta, and inducible nitric oxide synthase, which showed greater expression in cells obtained from rTGF-beta1-treated and S. typhimurium-infected mice 2 days after challenge. The treatment with rTGF-beta1 induced an increase in IL-1alpha and IFN-gamma release in the supernatant of splenocyte cultures 5 days after the experimental infection with S. typhimurium. Moreover, we demonstrated that 5 days after infection, the IFN-gamma titer was significantly greater in the sera of rTGF-beta-treated mice than in those of PBS-treated mice. Also, hsp60 showed greater expression 2 days after the challenge in splenocytes from rTGF-beta1-treated mice. The role played by proinflammatory and immunoregulatory cytokines and by CD28 is discussed. (+info)
Probing the function of Bordetella bronchiseptica adenylate cyclase toxin by manipulating host immunity.
(22/47697)
We have examined the role of adenylate cyclase-hemolysin (CyaA) by constructing an in-frame deletion in the Bordetella bronchiseptica cyaA structural gene and comparing wild-type and cyaA deletion strains in natural host infection models. Both the wild-type strain RB50 and its adenylate cyclase toxin deletion (DeltacyaA) derivative efficiently establish persistent infections in rabbits, rats, and mice following low-dose inoculation. In contrast, an inoculation protocol that seeds the lower respiratory tract revealed significant differences in bacterial numbers and in polymorphonuclear neutrophil recruitment in the lungs from days 5 to 12 postinoculation. We next explored the effects of disarming specific aspects of the immune system on the relative phenotypes of wild-type and DeltacyaA bacteria. SCID, SCID-beige, or RAG-1(-/-) mice succumbed to lethal systemic infection following high- or low-dose intranasal inoculation with the wild-type strain but not the DeltacyaA mutant. Mice rendered neutropenic by treatment with cyclophosphamide or by knockout mutation in the granulocyte colony-stimulating factor locus were highly susceptible to lethal infection by either wild-type or DeltacyaA strains. These results reveal the significant role played by neutrophils early in B. bronchiseptica infection and by acquired immunity at later time points and suggest that phagocytic cells are a primary in vivo target of the Bordetella adenylate cyclase toxin. (+info)
Surface expression of a protective recombinant pertussis toxin S1 subunit fragment in Streptococcus gordonii.
(23/47697)
In this study, the expression of the Bordetella pertussis S1 subunit was tested in Streptococcus gordonii, a commensal oral bacterium which has the potential to be a live oral vaccine vehicle. The DNA fragment encoding the N-terminal 179 amino acids of the S1 subunit was ligated into the middle part of spaP, the surface protein antigen P1 gene originating from Streptococcus mutans. The resulting construct, carried on the Escherichia coli-Streptococcus shuttle vector pDL276, was introduced into S. gordonii DL-1 by natural transformation. One of the transformants (RJMIII) produced a 187-kDa protein (the predicted size of the SpaP-S1 fusion protein) which was recognized by both the anti-pertussis toxin (anti-PT) and anti-SpaP antibodies, suggesting that an in-frame fusion had been made. Results from immunogold-electron microscopic studies and cellular fractionation studies showed that the fusion protein was surface localized and was mainly associated with the cell wall of RJMIII, indicating that SpaP was able to direct the fusion protein to the cell surface. A rabbit antiserum raised against heat-killed S. gordonii RJMIII recognized the native S1 subunit of PT in Western blotting and showed a weak neutralization titer to PT by the Chinese hamster ovary cell-clustering assay. BALB/c mice immunized with the heat-killed S. gordonii RJMIII were protected from the toxic effect of PT in the leukocytosis-promoting and histamine sensitization assays. In conclusion, a fragment of the S1 subunit of PT was successfully surface expressed in S. gordonii; the recombinant S1 fragment was found to be immunogenic and could induce protection against the toxic effect of PT in mice. (+info)
Correlation of temperature and toxicity in murine studies of staphylococcal enterotoxins and toxic shock syndrome toxin 1.
(24/47697)
This study describes a quick (<12 h) assay for detecting temperature decreases in BALB/c and C57BL/6 mice injected intraperitoneally (i.p. ) with staphylococcal enterotoxin A (SEA), SEB, or SEC3 or toxic shock syndrome toxin 1 and a potentiating dose of lipopolysaccharide (LPS). Toxin-specific antisera effectively neutralized the temperature fluctuations in this model. Orally administered SEA or SEB (50 microg/animal), with or without LPS, did not have an effect on temperature or lethality. Versus wild-type mice, transgenic knockout mice lacking the p55 receptor for tumor necrosis factor (TNF) or gamma interferon were protected against an i.p. challenge of SEA plus LPS. The p75 receptor for TNF and intercellular adhesion molecule 1 have a negligible role in this toxic shock model. (+info)