Giuseppe Teti

Infections caused by extracellular Gram positive bacteria are still a major health problems. Better understanding of the mechanisms underlying immune responses to these organisms is key to develop pharmacological agents, including vaccines, to control these infections. The objective of this review is to highlight the importance of nucleic acid-sensing, intracellular Toll-like receptors in innate immune recognition and in host defenses against extracellular bacteria. Toll-like receptors 7 and 9 have a major role in inducing host-protective type I interferon responses in conventional dendritic cells in response to streptococci and other extracellular gram positive bacteria. Moreover an as yet unidentified MyD88-dependent receptor is likely responsible for proinflammatory cytokine induction in response to these pathogens.

No vaccine is available for preventing infections by serogroup B Neisseria meningitidis (MenB), which accounts for a major portion of meningococcal cases in developed countries, because of the poor immunogenicity of the capsular polysaccharide (CP) even after protein conjugation. We have previously induced anticapsular antibodies by immunization with a single chain variable fragment (scFv), which mimics a protective CP epitope. This surrogate antigen, however, was ineffective at inducing serum bactericidal activity, an accepted marker of protection in humans. Serum bactericidal activity was consistently achieved by immunizing mice with the scFv-encoding gene. Immunization with vectors without a secretory signal sequence before the scFv resulted in markedly higher bactericidal activity relative to those with such a sequence. The induced antibodies were capsule specific, as shown by complete inhibition of bactericidal activity by purified MenB CP and by resistance to killing of MenA or MenC. Moreover, these antibodies were predominantly of the IgG2a isotype, reflecting a T helper type 1 response. Administration of sera from scFv gene–vaccinated animals protected infant rats against MenB bacteremia. These data illustrate the potential of vaccination with genes encoding capsular mimics in providing protection against MenB and other encapsulated bacteria.

Host defenses against the encapsulated yeast Cryptococcus neoformans involve both humoral and cell-mediated immunity. Mannoproteins (MPs) are a heterogeneous class of immunodominant glycoproteins which have been only incompletely characterized. In this study, we report on the molecular features of two novel MPs that are recognized by serum antibodies during cryptococcosis. After fractionation of extracellular cryptococcal products, MPs reacted more strongly than other components with sera from C. neoformans -infected AIDS patients. Further fractionation and Western blot analysis of MPs evidenced the presence of highly reactive bands with molecular masses of 250, 125, 115, and 84 kDa. The 115- and 84-kDa bands contained significant amounts of N-linked oligosaccharides, as shown by decreased molecular mass after peptide- N -glycosidase F treatment. N-terminal amino acid sequences of the two bands were used to search C. neoformans nucleotide databases. Homologous genomic sequences were used to synthesize DNA probes and isolate cDNA clones containing the full-length genes, which were designated MP84 and MP115. Both genes showed the presence of a serine/threonine-rich region, a potential site for heavy glycosylation. MP84 and MP115 showed homology with, respectively, polysaccharide deacetylases and carboxylesterases from other organisms. Recombinant, deglycosylated proteins expressed in Escherichia coli still reacted with sera from patients, albeit more weakly than natural MPs, indicating that at least some of the reactive epitopes were retained in the recombinant forms. In conclusion, we identified two novel MPs that are important targets of antibody responses during cryptococcosis. These data may be useful to devise alternative immunity-based strategies to control the disease.

Bacteroides fragilis, which is part of the normal intestinal flora, is a frequent cause of serious disease, especially in diabetic and surgical patients. In these conditions,B. fragilislipopolysaccharide (LPS) is likely to play a major pathophysiologic role.B. fragilisLPS is structurally different from classical enterobacterial LPS, whose biological activities are mediated by Toll-like receptor 4 (TLR4) activation. The ability ofB. fragilisLPS to activate TLR4 and TLR2 was investigated here, since evidence on this issue is scarce and controversial. Each of four different protein-freeB. fragilisLPS preparations could induce interleukin-8 responses in cells cotransfected with TLR4/CD14/MD2 but not TLR4/CD14 alone. Two of the preparations also induced cytokine production in cells cotransfected with TLR2/CD14 or in peritoneal macrophages from TLR4 mutant C3H/HeJ mice. Both of these activities, however, were lost after repurification with a modified phenol reextraction procedure. Importantly, all preparations could induce endotoxic shock in TLR2-deficient mice, but not in TLR4 mutant C3H/HeJ mice. Consistent with these findings, anti-TLR4 and anti-CD14, but not anti-TLR2, antibodies could inhibitB. fragilisLPS-induced cytokine production in human monocytes. Collectively, these results indicate thatB. fragilisLPS signals via a TLR4/CD14/MD2-dependent pathway, and it is unable to activate TLR2. Moreover, our data document the occurrence of TLR2-activating contaminants even in highly purifiedB. fragilisLPS preparations. This may explain earlier contradictory findings on the ability ofB. fragilisLPS to activate cells in the absence of functional TLR4. These data may be useful to devise strategies to prevent the pathophysiologic changes observed duringB. fragilissepsis and to better understand structure-activity relationships of LPS.

We investigated here the potential role of Toll‐like receptors (TLR) and the adaptor protein MyD88 in innate immunity responses to Cryptococcus neoformans, a pathogenic encapsulated yeast. Peritoneal macrophages from MyD88^–/– or TLR2^–/– mice released significantly less TNF‐α, compared with wild‐type controls, after in vitro stimulation with whole yeasts. In contrast, no differences in TNF‐α release were noted between macrophages from C3H/HeJ mice, which have a loss of function mutation in TLR4, relative to C3H/HeN controls. When MyD88‐ or TLR2‐deficient mice were infected with low doses of the H99 serotype A strain, all of the control animals, but none of MyD88^–/– and only 38% of the TLR2^–/– animals survived, in association with higher fungal burden in the mutant mice. Both MyD88^–/– and TLR2^–/– animals showed decreased TNF‐α, IL‐12p40 and/or IFN‐γ expression in various organs during infection. No difference in susceptibility to experimental cryptococcosis was found between C3H/HeJ mice and C3H/HeN controls. In conclusion, our data indicate that TLR2 and MyD88, but not TLR4, critically contribute to anti‐cryptococcal defenses through the induction of increased TNF‐α, IL‐12 and IFN‐γ expression.

A 25-kDa cryptococcal deacetylase (d25) was found here to induce cell proliferation, as well as secretion of interleukin 2 and gamma interferon, but not interleukin 4, in spleen cells from d25-immunized orCryptococcus neoformans-infected mice. The gamma interferon, but not the interleukin 2, response was required for the protective activities of d25 immunization in a murine cryptococcosis model.

Cell-mediated immunity plays a crucial role in host defenses againstCryptococcus(Filobasidiella)neoformans. Therefore, the identification of cryptococcal antigens capable of producing T-cell-mediated responses, such as delayed-type hypersensitivity (DTH) reactions, may be useful in the development of immune-based strategies to control cryptococcosis. In order to characterize DTH-producing antigens, culture supernatants from the unencapsulated Cap-67 strain were separated by anion-exchange chromatography. After further fractionation by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a purified protein with an apparent molecular mass of 25 kDa was found to produce DTH, as evidenced by increased footpad swelling in mice immunized with culture supernatants, relative to unimmunized mice. The 20-amino-acid N-terminal sequence of the 25-kDa protein was used to search data of theC. neoformansGenome Project. Based on the genomic DNA sequence, a DNA probe was used to screen a λ cDNA library prepared from strain B3501. Clones were isolated containing the full-length gene (d25), which showed homology with a number of polysaccharide deacetylases from fungi and bacteria. The recombinant d25 protein expressed inEscherichia coliwas similar to the natural one in DTH-producing activity. Moreover, immunization with either the natural or the recombinant protein prolonged survival and decreased fungal burden in mice challenged with the highly virulentC. neoformansstrain H99. In conclusion, we have described the first cryptococcal gene whose product, a 25-kDa extracellular polysaccharide deacetylase, has been shown to induce protective immunity responses.

Several group B streptococcal products have been previously found to stimulate human monocytes to produce tumor necrosis factor alpha. In order to identify the receptors involved in these responses, monocytes were stimulated with purified group- or type-specific carbohydrates or lipoteichoic acid in the presence of anti-receptor monoclonal antibodies, soluble CD14, or lipopolysaccharide-binding protein. Results indicate that CD14 plays an important role in tumor necrosis factor alpha responses to all of the stimuli tested. Moreover, both CD14 and complement receptor type 3 may be involved in responses to the group-antigen.

This study was undertaken to test the hypothesis that altered IL-10 production plays a role in the increased susceptibility of neonates to listeriosis. Plasma IL-10 levels were measured in neonatal and adult mice at various times after infection with Listeria monocytogenes. Relative to adults, neonatal mice had markedly increased IL-10 levels early in the course of infection with Listeria using a 90% lethal dose. Higher neonatal IL-10 responses were also observed after injecting adults and pups with equal doses of killed organisms. Splenic macrophages from neonates produced higher IL-10 levels than those of adults after in vitro stimulation with killed bacteria, confirming in vivo observations. Moreover, IL-10 blockade had differential effects in neonates and adults infected with live Listeria. In adult mice, anti-IL-10 Abs decreased bacterial burden early in the course of infection, but were no longer effective at 6 days or later after challenge. In the pups, however, the same treatment had beneficial effects both early and late during infection and resulted in increased survival. Collectively, our data suggest that an overproduction of IL-10 by macrophages may at least partially explain the increased susceptibility of neonates to listeriosis, and provide further evidence that cytokine production is different in adults and neonates.