Escherichia coli strain Nissle 1917 ameliorates experimental colitis via toll-like receptor 2- and toll-like receptor 4-dependent pathways.

Toll-like receptors (TLRs) are key components of the innate immune system that trigger antimicrobial host defense responses. The aim of the present study was to analyze the effects of probiotic Escherichia coli Nissle strain 1917 in experimental colitis induced in TLR-2 and TLR-4 knockout mice. Colitis was induced in wild-type (wt), TLR-2 knockout, and TLR-4 knockout mice via administration of 5% dextran sodium sulfate (DSS). Mice were treated with either 0.9% NaCl or 10(7) E. coli Nissle 1917 twice daily, followed by the determination of disease activity, mucosal damage, and cytokine secretion. wt and TLR-2 knockout mice exposed to DSS developed acute colitis, whereas TLR-4 knockout mice developed significantly less inflammation. In wt mice, but not TLR-2 or TLR-4 knockout mice, E. coli Nissle 1917 ameliorated colitis and decreased proinflammatory cytokine secretion. In TLR-2 knockout mice a selective reduction of gamma interferon secretion was observed after E. coli Nissle 1917 treatment. In TLR-4 knockout mice, cytokine secretion was almost undetectable and not modulated by E. coli Nissle 1917, indicating that TLR-4 knockout mice do not develop colitis similar to the wt mice. Coculture of E. coli Nissle 1917 and human T cells increased TLR-2 and TLR-4 protein expression in T cells and increased NF-kappaB activity via TLR-2 and TLR-4. In conclusion, our data provide evidence that E. coli Nissle 1917 ameliorates experimental induced colitis in mice via TLR-2- and TLR-4-dependent pathways.

Immune responses to intracellular bacteria.

The multifaceted dialogue between intracellular bacteria and the mammalian host continues to be an exciting issue from both the scientific and public-health viewpoint. The recent year has witnessed some particularly impressive progress in knowledge about the two major culprits affecting the health of mankind, Mycobacterium tuberculosis and Salmonella typhi - the causative agents of tuberculosis and typhoid fever.

MHC class Ia-restricted T cells partially account for beta2-microglobulin-dependent resistance to Mycobacterium tuberculosis.

Recent studies have highlighted the heterogeneous nature of the CD8(+) T cell response during human Mycobacterium tuberculosis infection; MHC class Ia, MHC class Ib and CD1 have all been identified as significant restriction elements. Here we have attempted to define the role of MHC class Ia in resistance to M. tuberculosis infection in mice. The course of M. tuberculosis infection in mice deficient in a single MHC class Ia molecule, either H2-K(b) or H2-D(b), was essentially identical to that observed in wild-type mice. In contrast, mice fully deficient in MHC class Ia molecules (H2-K(b) / H2-D(b) double knockout mice) were substantially more susceptible to M. tuberculosis infection. However, the double knockout mice were not as susceptible as beta 2-microglobulin-deficient mice, which have a broader phenotypic deficit. Thus, antigen presentation via MHC class Ia is an important component in resistance to M. tuberculosis, but its absence only partially accounts for the increased susceptibility of beta 2-microglobulin-deficient mice.

Introduction: microbiology and immunology: lessons learned from Salmonella.

Salmonella enterica, a Gram-negative bacterium, causes significant morbidity and mortality worldwide, and is an excellent model to study bacterial pathogenesis and cellular immune responses. With the development of powerful new technologies, there has been a fusion of research on immunology, molecular biology and cellular microbiology of S. enterica infections. This multidisciplinary research will enhance our understanding of the basic mechanisms of bacterial infections and immunity; it also provides new approaches towards therapeutic and control measures.

Bacterial virulence, proinflammatory cytokines and host immunity: how to choose the appropriate Salmonella vaccine strain?

Salmonella infection in its mammalian host can be dissected into two main components. The co-ordinate expression of bacterial virulence genes which are designed to evade, subvert or circumvent the host response on the one hand, and the host defence mechanisms which are designed to restrict bacterial survival and replication on the other hand. The outcome of infection is determined by the one which succeeds in disturbing this equilibrium more efficiently. This delicate balance between Salmonella virulence and host immunity/inflammation has important implications for vaccine development or therapeutic intervention. Novel Salmonella vaccine candidates and live carriers for heterologous antigens are attenuated strains with defined genetic modifications of metabolic or virulence functions. Although genetic defects of different gene loci can lead to similar degrees of attenuation, effects on the course of infection may vary, thereby altering the quality of the elicited immune response. Studies with gene-deficient animals indicate that Salmonella typhimurium strains with mutations in aroA, phoP/phoQ or ssrA/ssrB invoke different immune responses and that a differential repertoire of pro-inflammatory cytokines is required for clearance. Consequently, Salmonella mutants defective in distinct virulence functions offer the potential to specifically modulate the immune response for defined medical applications.

Rapid neutrophil response controls fast-replicating intracellular bacteria but not slow-replicating Mycobacterium tuberculosis.

Being one of the first cells to invade the site of infection, neutrophils play an important role in the control of various bacterial and viral infections. In the present work, the contribution of neutrophils to the control of infection with different intracellular bacteria was investigated. Mice were treated with the neutrophil-depleting monoclonal antibody RB6-8C5, and the time course of infection in treated and untreated mice was compared by using intracellular bacterial species and strains varying in virulence and replication rate. The results indicate that neutrophils are crucial for the control of fast-replicating intracellular bacteria, whereas early neutrophil effector mechanisms are dispensable for the control of the slow-replicating Mycobacterium tuberculosis.

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.

Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens.

In 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year. The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced. We compared the proteomes of two non-virulent vaccine strains of M. bovis BCG (Chicago and Copenhagen) with two virulent strains of M. tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics. The mycobacterial strains were analysed by two-dimensional electrophoresis (2-DE) combining non-equilibrium pH gradient electrophoresis (NEPHGE) with SDS-PAGE. Distinct and characteristic proteins were identified by mass spectrometry and introduced into a dynamic 2-DE database (http://www.mpiib-berlin.mpg.de/2D-PAGE). Silver-stained 2-DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identified. Of these, 54 belong to the culture supernatant. Sixteen and 25 proteins differing in intensity or position between M. tuberculosis H37Rv and Erdman, and H37Rv and M. bovis BCG Chicago, respectively, were identified and categorized into protein classes. It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis.

A dynamic two-dimensional polyacrylamide gel electrophoresis database: the mycobacterial proteome via Internet.

Proteome analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometry, in combination with protein chemical methods, is a powerful approach for the analysis of the protein composition of complex biological samples. Data organization is imperative for efficient handling of the vast amount of information generated. Thus we have constructed a 2-D PAGE database to store and compare protein patterns of cell-associated and culture-supernatant proteins of different mycobacterial strains. In accordance with the guidelines for federated 2-DE databases, we developed a program that generates a dynamic 2-D PAGE database for the World-Wide-Web to organise and publish, via the internet, our results from proteome analysis of different Mycobacterium tuberculosis as well as Mycobacterium bovis BCG strains. The uniform resource locator for the database is http://www.mpiib-berlin.mpg.de/2D-PAGE and can be read with a Java compatible browser. The interactive hypertext markup language documents displayed are generated dynamically in each individual session from a rational data file, a 2-D gel image file and a map file describing the protein spots as polygons. The program consists of common gateway interface scripts written in PERL, minimizing the administrative workload of the database. Furthermore, the database facilitates not only interactive use, but also worldwide active participation of other scientific groups with their own data, requiring only minimal computer hardware and knowledge of information technology.

The Yersinia Yops inhibit invasion of Listeria, Shigella and Edwardsiella but not Salmonella into epithelial cells.

Yersinia virulence is dependent on the expression of plasmid-encoded secreted proteins called Yops. After bacterial adherence to receptors on the mammalian cell membrane, several Yops are transported by a type III secretion pathway into the host cell cytoplasm. Two Yops, YopH and YopE, prevent macrophages from phagocytosing Yersinia by disrupting the host cell cytoskeleton and signal transduction pathways. In contrast to this active inhibition of phagocytosis by Yersinia, other pathogens such as Salmonella, Shigella, Listeria and Edwardsiella actively promote their entry into mammalian cells by binding to specific host surface receptors and exploiting existing cell cytoskeletal and signalling pathways. We have tested whether Yersinia Yops can prevent the uptake of these diverse invasive pathogens. We first infected epithelial cells with Yersinia to permit delivery of Yops and subsequently with an invasive pathogen. We then measured the level of bacterial invasion. Preinfection with Yersinia inhibited invasion of Edwardsiella, Shigella and Listeria, but not Salmonella. Furthermore, we found that either YopE or YopH prevented Listeria invasion, whereas only YopE prevented Edwardsiella and Shigella invasion. We correlated the inhibitory effect of the Yops with the inhibitory action of the cell-signalling inhibitors Wortmannin, LY294002 and NDGA, and concluded that the four invasive pathogenic species enter epithelial cells using at least three distinct host cell pathways. We also speculate that YopE affects the rho pathway.

Functional analysis of ssaJ and the ssaK/U operon, 13 genes encoding components of the type III secretion apparatus of Salmonella Pathogenicity Island 2.

We have investigated the structure and transcriptional organization of 13 genes of Salmonella Pathogenicity Island 2 (SPI2) that encode components of the second type III secretion apparatus of Salmonella typhimurium. ssaK, L, M, V, N, O, P, Q, R, S, T, U constitute one operon of 10 kb. ssaJ lles upstream of ssaK and is the terminal gene of another operon. The deduced products of ssaJ, ssaK, ssaV, ssaN, ssaO, ssaQ, ssaR, ssaS, ssaT, and ssaU show greatest similarity to the Yersinia spp. genes yscJ, yscL, lcrD, yscN, yscO, yscQ, yscR, yscS, yscT, and yscU, respectively. The products of the ssaL, ssaM and ssaP genes do not have significant similarity to products of other type III secretion systems, and might be important for the specific function of the SPI2 type III secretion system. Bacterial strains carrying different ssa mutations display minor alterations in terms of serum sensitivity when compared with the wild-type strain, but none are defective in replication within macrophage-like RAW 264.7 cells. However, some of the ssa mutant strains invade HEp2 cells less efficiently and are less cytotoxic to RAW 264.7 macrophages than the wild-type strain. We show that the invasion defect is correlated with a lack of SipC in culture supernatants of these mutant strains. SipC is a product of the SPI1 type III secretion system of S. typhimurium, and is important for epithelial cell invasion. Therefore, mutations in SPI2 can affect the SPI1 secretion system, which raises the possibility of an interaction between the two type III secretion systems.

Salmonella typhimurium invasion induces apoptosis in infected macrophages.

Invasive Salmonella typhimurium induces dramatic cytoskeletal changes on the membrane surface of mammalian epithelial cells and RAW264.7 macrophages as part of its entry mechanism. Noninvasive S. typhimurium strains are unable to induce this membrane ruffling. Invasive S. typhimurium strains invade RAW264.7 macrophages in 2 h with 7- to 10-fold higher levels than noninvasive strains. Invasive S. typhimurium and Salmonella typhi, independent of their ability to replicate intracellularly, are cytotoxic to RAW264.7 macrophages and, to a greater degree, to murine bone marrow-derived macrophages. Here, we show that the macrophage cytotoxicity mediated by invasive Salmonella is apoptosis, as shown by nuclear morphology, cytoplasmic vacuolization, and host cell DNA fragmentation. S. typhimurium that enter cells causing ruffles but are mutant for subsequent intracellular replication also initiate host cell apoptosis. Mutant S. typhimurium that are incapable of inducing host cell membrane ruffling fail to induce apoptosis. The activation state of the macrophage plays a significant role in the response of macrophages to Salmonella invasion, perhaps indicating that the signal or receptor for initiating programmed cell death is upregulated in activated macrophages. The ability of Salmonella to promote apoptosis may be important for the initiation of infection, bacterial survival, and escape of the host immune response.

Host restriction phenotypes of Salmonella typhi and Salmonella gallinarum.

Salmonella typhi and Salmonella gallinarum phenotypes correlated with mouse host restriction have been identified by using in vitro and in vivo systems. S. typhi is capable of entering the murine intestinal epithelium via M cells, as is Salmonella typhimurium, which causes systemic infection in the mouse. But, unlike S. typhimurium, S. typhi does not destroy the epithelium and is cleared from the Peyer's patches soon after M-cell entry. S. gallinarum appears to be incapable of entering the murine Peyer's patch epithelium. Our in vitro evidence suggests that S. gallinarum is taken up in murine phagocytic cells by a mechanism different from that of S. typhimurium. S. typhimurium is taken up at a higher frequency and is maintained at higher viable counts throughout a 24-h time course in a murine macrophage-like cell line than are S. gallinarum and S. typhi.

Elucidation of linear epitopes of pertussis toxin using overlapping synthetic decapeptides: identification of a human B-cell determinant in the S1 subunit indicative of acute infections.

To identify relevant linear epitopes within the immunodominant ADP-ribosyl transferase (S1 subunit) of pertussis toxin (PT), its complete amino acid sequence was synthesized as consecutive, overlapping decapeptides on solid phase and probed for seroreactivity with pertussis specific human antisera in 'peptide scans'. Comparison of the resulting antigenic profiles revealed two distinct types of human antisera, though amino acids 140-200 could not be assessed as the corresponding peptides reacted non-specifically with the detection system. Human anti-pertussis sera predominantly recognized linear immunodominant epitopes located in three separated segments spanning amino acids 3-16, 21-30, and 211-222. Antisera originating from infants with acute B. pertussis infections (type I) identified determinants in all three segments, while type-II antisera from convalescent patients only recognized epitopes in the N-terminal regions. The binding of pertussis specific antisera--both type I and type II--to the holotoxin was inhibited by preincubation of antibodies with synthetic peptides corresponding to two linear determinants located at the N-terminus of S1:R 3-16 and R 21-30. However, competitive binding of antibodies to PT and to synthetic peptides equivalent to the third epitope (R 211-222) was only observed with type I antisera. Thus, the linear immunogenic determinant identified at the C-terminus of the A-protomer represents a human epitope which is apparently specific for antisera from pertussis patients with acute infections. The possible application of this determinant in serologic diagnosis will be a valuable tool to detect and distinguish acute Bordetella pertussis infections.

Efficient production of active and mutated ADP-ribosyltransferase (S1) of pertussis toxin using affinity expression cassette polymerase chain reaction.

We describe an efficient, general approach for cloning, expression and purification of heterologous proteins in Escherichia coli host strains. The affinity expression cassette polymerase chain reaction (AEC-PCR) allows the insertion of virtually any coding sequence in suitable expression vectors. For ease of purification of the (over)produced protein the gene expression cassettes are engineered by specifically designed oligonucleotide primers in the polymerase chain reaction (PCR) to contain either 3' or 5' additional nucleotides coding for a short amino acid sequence constituting an 'affinity block' fused to either end of the protein. This allows a one-step purification by affinity chromatography. In combination with PCR-mediated site-specific mutagenesis this approach is a highly efficient way for the expression and isolation of proteins and for the analysis and dissection of functional domains. The application of AEC-PCR is demonstrated by the cloning, production and purification of the native, active and the mutagenized, inactive ADP-ribosyltransferase (S1 subunit) of pertussis toxin. In this example, a string of six histidines has been engineered to either the N-terminal or the C-terminal end of the protein to serve as 'affinity block' for the isolation of the recombinant protein by immobilized metal ion affinity chromatography (IMAC). Thus, the S1 subunit can now be produced in sufficient quantities to facilitate further studies on its immunological and molecular properties.

Characterization of murine monoclonal antibodies that recognize defined epitopes of pertussis toxin and neutralize its toxic effect on Chinese hamster ovary cells.

Three murine monoclonal antibodies (MAb), E19, E205, and E251, raised against pertussis toxin reacted in Western blots (immunoblots) with the S1, S4, and S2-S3 subunits, respectively, and neutralized the Chinese hamster ovary cell-clustering activity of pertussis toxin. MAb E251 recognized a linear synthetic peptide corresponding to amino acids 107 to 120 of the S2 subunit, suggesting a role for this region in receptor binding.

Identification of linear B-cell determinants of pertussis toxin associated with the receptor recognition site of the S3 subunit.

Receptor recognition of pertussis toxin is mediated by the B oligomer consisting of subunits S2, S3, 2xS4, and S5. One possible way to interfere with toxin action would be the inhibition of recognition and binding of the cellular receptor(s) by preformed toxin-directed antipeptide antibodies. A prerequisite for this approach is the localization of linear antigenic determinants followed by the identification of inhibitory epitopes. Anti-S2 peptide antibodies have been shown to inhibit binding of the holotoxin to in vitro model receptor systems. For the elucidation of linear antigenic and immunogenic determinants harbored in the S3 subunit, synthetic peptides corresponding to selected linear amino acid sequences of S3 have been prepared and used to raise peptide-specific antibodies in rabbits. All peptides elicited a strong homologous response. Four synthetic peptides reacting with anti-pertussis toxin antibodies (R36-51, R87-95, R134-150, and R147-160) have been identified. Seven synthetic peptides (R1-12, R12-23, R14-29m, R36-51, R95-107, R134-150, and R164-178) induced antibodies recognizing pertussis toxin. Thus, these segments correspond to linear antigenic determinants. Analogous to the S2 subunit, the N terminus of S3 proved to be immunorecessive in the native toxin. The highly homologous S2 subunit was only bound strongly in Western blotting (immunoblotting) by antiserum directed at peptide R164-178, which is identical in the S2 and S3 subunits. A weak recognition of S2 in Western blotting was observed with anti-R95-107 antiserum. The ability of affinity-purified anti-S3 peptide antibodies to interfere with pertussis toxin binding was investigated by hemagglutination of goose erythrocytes as a model receptor system for S3-mediated receptor recognition. Antipeptide antibodies directed at R1-12, R12-23, R14-29m, and R36-51 inhibited hemagglutination of goose erythrocytes. This indicates that the corresponding antigenic regions in the S3 subunit are associated with the formation of the receptor binding domain. Inhibition of B-oligomer-mediated pertussis toxin binding to cellular receptors by preformed antipeptide antibodies of sufficient affinity should not only block the detrimental effects of the S1 subunits, but also interfere with the mitogenic effects attributed to the B oligomer.