T helper type 2-polarized invariant natural killer T cells reduce disease severity in acute intra-abdominal sepsis.

Sepsis is characterized by a severe systemic inflammatory response to infection that is associated with high morbidity and mortality despite optimal care. Invariant natural killer T (iNK T) cells are potent regulatory lymphocytes that can produce pro- and/or anti-inflammatory cytokines, thus shaping the course and nature of immune responses; however, little is known about their role in sepsis. We demonstrate here that patients with sepsis/severe sepsis have significantly elevated proportions of iNK T cells in their peripheral blood (as a percentage of their circulating T cells) compared to non-septic patients. We therefore investigated the role of iNK T cells in a mouse model of intra-abdominal sepsis (IAS). Our data show that iNK T cells are pathogenic in IAS, and that T helper type 2 (Th2) polarization of iNK T cells using the synthetic glycolipid OCH significantly reduces mortality from IAS. This reduction in mortality is associated with the systemic elevation of the anti-inflammatory cytokine interleukin (IL)-13 and reduction of several proinflammatory cytokines within the spleen, notably interleukin (IL)-17. Finally, we show that treatment of sepsis with OCH in mice is accompanied by significantly reduced apoptosis of splenic T and B lymphocytes and macrophages, but not natural killer cells. We propose that modulation of iNK T cell responses towards a Th2 phenotype may be an effective therapeutic strategy in early sepsis.

Attenuation of massive cytokine response to the staphylococcal enterotoxin B superantigen by the innate immunomodulatory protein lactoferrin.

Staphylococcal enterotoxin B (SEB) is a pyrogenic exotoxin and a potent superantigen which causes massive T cell activation and cytokine secretion, leading to profound immunosuppression and morbidity. The inhibition of SEB-induced responses is thus considered a goal in the management of certain types of staphylococcal infections. Lactoferrin (LF) is a multi-functional glycoprotein with both bacteriostatic and bactericidal activities. In addition, LF is known to have potent immunomodulatory properties. Given the anti-microbial and anti-inflammatory properties of this protein, we hypothesized that LF can modulate T cell responses to SEB. Here, we report that bovine LF (bLF) was indeed able to attenuate SEB-induced proliferation, interleukin-2 production and CD25 expression by human leucocyte antigen (HLA)-DR4 transgenic mouse T cells. This inhibition was not due to bLF's iron-binding capacity, and could be mimicked by the bLF-derived peptide lactoferricin. Cytokine secretion by an engineered SEB-responsive human Jurkat T cell line and by peripheral blood mononuclear cells from healthy donors was also inhibited by bLF. These findings reveal a previously unrecognized property of LF in modulation of SEB-triggered immune activation and suggest a therapeutic potential for this naturally occurring protein during toxic shock syndrome.

Digoxigenin-labelled peptide nucleic acid to detect lactobacilli PCR amplicons immobilized on membranes from denaturing gradient gel electrophoresis.

AIMS: To develop a digoxigenin (DIG)-labeled peptide nucleic acid (PNA) probe for the detection of Lactobacillus-related genera amongst eubacterial amplicons obtained from vaginal samples using denaturing gradient gel electrophoresis (DGGE) blots. METHODS AND RESULTS: Part of the 16S rRNA gene sequence was used as a target for the PNA probe. After confirming probe specificity using chromosomal DNA from species and isolates that have been detected in the urogenital tract, it was successfully used to detect lactobacilli amplicons generated using eubacterial-specific 16S rRNA gene-targeted primers from vaginal tract samples immobilized on membranes from DGGE. CONCLUSIONS: The Lactobacillus-specific PNA probe could distinguish between DNA fragments from lactobacilli in a DGGE gel from other bacterial species, including those that migrated to a similar position. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of the DIG-labelled PNA probe on blots of eubacterial PCR products from DGGE gels can be used to specifically detect lactobacilli in complex vaginal samples.

Toxic shock syndrome and bacterial superantigens: an update.

Toxic shock syndrome (TSS) is an acute onset illness characterized by fever, rash formation, and hypotension that can lead to multiple organ failure and lethal shock, as well as desquamation in patients that recover. The disease is caused by bacterial superantigens (SAGs) secreted from Staphylococcus aureus and group A streptococci. SAGs bypass normal antigen presentation by binding to class II major histocompatibility complex molecules on antigen-presenting cells and to specific variable regions on the beta-chain of the T-cell antigen receptor. Through this interaction, SAGs activate T cells at orders of magnitude above antigen-specific activation, resulting in massive cytokine release that is believed to be responsible for the most severe features of TSS. This review focuses on clinical and epidemiological aspects of TSS, as well as important developments in the genetics, biochemistry, immunology, and structural biology of SAGs. From the evolutionary relationships between these important toxins, we propose that there are five distinct groups of SAGs.

Antibodies to a surface-exposed, N-terminal domain of aggregation substance are not protective in the rabbit model of Enterococcus faecalis infective endocarditis.

The aggregation substance (AS) surface protein from Enterococcus faecalis has been implicated as an important virulence factor for the development of infective endocarditis. To evaluate the role of antibodies specific for Asc10 (the AS protein from the conjugative plasmid pCF10) in protective immunity to infective endocarditis, an N-terminal region of Asc10 lacking the signal peptide and predicted to be surface exposed (amino acids 44 to 331; AS(44-331)) was cloned with a C-terminal histidine tag translational fusion and expressed from Escherichia coli. N-terminal amino acid sequencing of the purified protein revealed the correct sequence, and rabbit polyclonal antisera raised against AS(44-331) reacted specifically to Asc10 expressed from E. faecalis OG1SSp, but not to other proteins as judged by Western blot analysis. Using these antisera, flow cytometry analysis demonstrated that antibodies to AS(44-331) bound to a surface-exposed region of Asc10. Furthermore, antibodies specific for AS(44-331) were opsonic for E. faecalis expressing Asc10 in vitro but not for cells that did not express Asc10. New Zealand White rabbits immunized with AS(44-331) were challenged intravenously with E. faecalis cells constitutively expressing Asc10 in the rabbit model of experimental endocarditis. Highly immune animals did not show significant differences in clearance of organisms from the blood or spleen or in formation of vegetations on the aortic valve, in comparison with nonimmune animals. Although in vivo expression of Asc10 was demonstrated by immunohistochemistry, these experiments provide evidence that immunity to Asc10 does not play a role in protection from experimental infective endocarditis due to E. faecalis and may have important implications for the development of immunological approaches to combat enterococcal endocarditis.

Functional characterization of streptococcal pyrogenic exotoxin J, a novel superantigen.

Streptococcal toxic shock syndrome (STSS) is a highly lethal, acute-onset illness that is a subset of invasive streptococcal disease. The majority of clinical STSS cases have been associated with the pyrogenic toxin superantigens (PTSAgs) streptococcal pyrogenic exotoxin A or C (SPE A or C), although cases have been reported that are not associated with either of these exotoxins. Recent genome sequencing projects have revealed a number of open reading frames that potentially encode proteins with similarity to SPEs A and C and to other PTSAgs. Here, we describe the cloning, expression, purification, and functional characterization of a novel exotoxin termed streptococcal pyrogenic exotoxin J (SPE J). Purified recombinant SPE J (rSPE J) expressed from Escherichia coli stimulated the expansion of both rabbit splenocytes and human peripheral blood lymphocytes, preferentially expanded human T cells displaying Vbeta2, -3, -12, -14, and -17 on their T-cell receptors, and was active at concentrations as low as 5 x 10(-6) microg/ml. Furthermore, rSPE J induced fevers in rabbits and was lethal in two models of STSS. Biochemically, SPE J had a predicted molecular weight of 24,444 and an isoelectric point of 7.7 and lacked the ability to form the cystine loop structure characteristic of many PTSAgs. SPE J shared 19.6, 47.1, 38.8, 18.1, 19.6, and 24.4% identity with SPEs A, C, G, and H, streptococcal superantigen, and streptococcal mitogenic exotoxin Z-2, respectively, and was immunologically cross-reactive with SPE C. The characterization of a seventh functional streptococcal PTSAg raises important questions relating to the evolution of the streptococcal superantigens.

Identification of a novel two-component regulatory system that acts in global regulation of virulence factors of Staphylococcus aureus.

We have previously demonstrated that the presence of oxygen is necessary for the production of toxic shock syndrome toxin 1 (TSST-1) by Staphylococcus aureus in vitro. To investigate the mechanism by which oxygen might regulate toxin production, we identified homologs in S. aureus of the Bacillus subtilis resDE genes. The two-component regulatory system encoded by resDE, ResD-ResE, has been implicated in the global regulation of aerobic and anaerobic respiratory metabolism in B. subtilis. We have designated the S. aureus homologs srrAB (staphylococcal respiratory response). The effects of srrAB expression on expression of RNAIII (the effector molecule of the agr locus) and on production of TSST-1 (an exotoxin) and protein A (a surface-associated virulence factor) were investigated. Expression of RNAIII was inversely related to expression of srrAB. Disruption of srrB resulted in increased levels of RNAIII, while expression of srrAB in trans on a multicopy plasmid resulted in repression of RNAIII transcription, particularly in microaerobic conditions. Disruption of srrB resulted in decreased production of TSST-1 under microaerobic conditions and, to a lesser extent, under aerobic conditions as well. Overexpression of srrAB resulted in nearly complete repression of TSST-1 production in both microaerobic and aerobic conditions. Protein A production by the srrB mutant was upregulated in microaerobic conditions and decreased in aerobic conditions. Protein A production was restored to nearly wild-type levels by complementation of srrAB into the null mutant. These results indicate that the putative two-component system encoded by srrAB, SrrA-SrrB, acts in the global regulation of staphylococcal virulence factors, and may repress virulence factors under low-oxygen conditions. Furthermore, srrAB may provide a mechanistic link between respiratory metabolism, environmental signals, and regulation of virulence factors in S. aureus.

Crystal structure of a superantigen bound to the high-affinity, zinc-dependent site on MHC class II.

MHC class II molecules possess two binding sites for bacterial superantigens (SAGs): a low-affinity site on the alpha chain and a high-affinity, zinc-dependent site on the beta chain. Only the former has been defined crystallographically. We report the structure of streptococcal pyrogenic exotoxin C (SPE-C) complexed with HLA-DR2a (DRA*0101, DRB5*0101) bearing a self-peptide from myelin basic protein (MBP). SPE-C binds the beta chain through a zinc bridge that links the SAG and class II molecules. Surprisingly, SPE-C also makes extensive contacts with the MBP peptide, such that peptide accounts for one third of the surface area of the MHC molecule buried in the complex, similar to TCR-peptide/MHC complexes. Thus, SPE-C may optimize T cell responses by mimicking the peptide dependence of conventional antigen presentation and recognition.

Development of streptococcal pyrogenic exotoxin C vaccine toxoids that are protective in the rabbit model of toxic shock syndrome.

Streptococcal pyrogenic exotoxin C (SPE C) is a superantigen produced by many strains of Streptococcus pyogenes that (along with streptococcal pyrogenic exotoxin A) is highly associated with streptococcal toxic shock syndrome (STSS) and other invasive streptococcal diseases. Based on the three-dimensional structure of SPE C, solvent-exposed residues predicted to be important for binding to the TCR or the MHC class II molecule, or important for dimerization, were generated. Based on decreased mitogenic activity of various single-site mutants, the double-site mutant Y15A/N38D and the triple-site mutant Y15A/H35A/N38D were constructed and analyzed for superantigenicity, toxicity (lethality), immunogenicity, and the ability to protect against wild-type SPE C-induced STSS. The Y15A/N38D and Y15A/H35A/N38D mutants were nonmitogenic for rabbit splenocytes and human PBMCs and nonlethal in two rabbit models of STSS, yet both mutants were highly immunogenic. Animals vaccinated with the Y15A/N38D or Y15A/H35A/N38D toxoids were protected from challenge with wild-type SPE C. Collectively, these data indicate that the Y15A/N38D and Y15A/H35A/N38D mutants may be useful as toxoid vaccine candidates.

Colicin V can be produced by lactic acid bacteria.

Colicin V is a small, proteinaceous bacterial toxin, produced by many strains of Escherichia coli and other members of the Enterobacteriaceae, that fits the definition of class II bacteriocins of Gram-positive bacteria. Export of colicin V is dependent on specific ABC (ATP-binding cassette) secretion proteins which recognize a double-glycine-type leader peptide on the immature colicin V bacteriocin. Replacement of the colicin V leader peptide by a signal peptide from the signal sequence-dependent bacteriocin divergicin A allowed expression of colicin V in lactic acid bacteria. This system may serve as a model for the heterologous expression of other small bacteriocins active against Gram-negative bacteria and other antibacterial peptides from lactic acid bacteria.

Genetic characterization and heterologous expression of brochocin-C, an antibotulinal, two-peptide bacteriocin produced by Brochothrix campestris ATCC 43754.

Brochocin-C, produced by Brochothrix campestris ATCC 43754, is active against many strains of the closely related meat spoilage organism Brochothrix thermosphacta and a wide range of other gram-positive bacteria, including spores of Clostridium botulinum. Purification of the active compound and genetic characterization of brochocin-C revealed that it is a chromosomally encoded, two-peptide nonlantibiotic bacteriocin. Both peptides of brochocin-C are ribosomally synthesized as prepeptides that are typical of class II bacteriocins. They are cleaved following Gly-Gly cleavage sites to yield the mature peptides, BrcA and BrcB, containing 59 and 43 amino acids, respectively. Fusion of the nucleotides encoding the signal peptide of the bacteriocin divergicin A in front of the structural genes for either BrcA or BrcB allowed independent expression of each component by the general protein secretion pathway. This revealed the two-component nature of brochocin-C and the necessity for both peptides for activity. A 53-amino-acid peptide encoded downstream of brcB functions as the immunity protein (BrcI) for brochocin-C. In addition, the cloned chromosomal fragment revealed open reading frames downstream of brcI, designated brcT and brcD, that encode proteins with homology to ATP-binding cassette translocator and accessory proteins, respectively, involved in the secretion of Gly-Gly-type bacteriocins.

Expression of the antimicrobial peptide carnobacteriocin B2 by a signal peptide-dependent general secretory pathway.

Carnobacteriocin B2 is a well-characterized class II bacteriocin produced by a 61-kb plasmid from Carnobacterium piscicola LV17. Export of this bacteriocin is dependent on specific ABC (ATP-binding cassette) secretion proteins. Divergicin A is a strongly hydrophobic narrow-spectrum bacteriocin produced by a 3.4-kb plasmid from Carnobacterium divergens LV13. Predivergicin A contains a signal peptide and utilizes the general secretary pathway for export (R. W. Worobo, M. J. van Belkum, M. Sailer, K. L. Roy, J. C. Vederas, and M. E. Stiles, J. Bacteriol. 177:3143-3149, 1995). Fusion of the carnobacteriocin B2 structural gene (devoid of its natural leader peptide) behind the signal peptide of divergicin A in the expression vector pMG36e permitted production and export of active carnobacteriocin B2 in the absence of the specific secretion genes. N-terminal sequencing of purified carnobacteriocin B2 established that correct processing of the prebacteriocin occurred beyond the Ala-Ser-Ala cleavage site of the signal peptide. Carnobacteriocin B2 was produced by the wild-type strain of C. divergens, LV13, and in C. piscicola LV17C, the nonbacteriocinogenic, plasmidless variant of the original carnobacteriocin B2 producer strain. The corresponding immunity gene was included immediately downstream of the structural gene. Both of the host strains are sensitive to the bacteriocin, and both acquired immunity when they were transformed with the construct. C. divergens LV13 containing the divergicin A signal peptide-carnobacteriocin B2 fusion construct produces both divergicin A and carnobacteriocin B2 and demonstrates the first example of multiple-bacteriocin expression via the general secretory pathway. The small amount of genetic material required for independent bacteriocin expression has implications for the development of a food-grade multiple-bacteriocin expression vector for use in lactic acid bacteria.

Survival and recovery of Aeromonas hydrophila in water: development of methodology for testing bottled water in Canada.

Proposed changes to the Regulations for bottled water in the Food and Drugs Act of Canada include criteria for Aeromonas hydrophila (0 colony-forming units/100 mL water). The development of the methodology used to support these proposed Regulations and the survival of A. hydrophila in inoculated water are described. The methodology used in the isolation of A. hydrophila includes the use of hydrophobic grid membrane filters (HGMF), a resuscitation step on tryptic soy agar, and selective plating on membrane-Aeromonas-trehalose agar and Aeromonas medium. Aeromonas hydrophila proliferated and survived in inoculated water for up to 60 days or longer depending on the other contaminating bacteria. The presence of Pseudomonas aeruginosa enhanced the survival of A. hydrophila and enabled this bacteria to survive for more than 60 days.