T cell recognition of listeriolysin O is induced during infection with Listeria monocytogenes

During bacterial multiplication, Listeria monocytogenes (strain EGD) secretes sulfhydryl-dependent cytotoxin, termed listeriolysin O, a virulence factor presumable promoting intracellular growth of this ubiquitous pathogen. The role of this exotoxin in the process of T cell activation was studied in vivo during the course of an experimental infection in the mouse. By using highly purified listeriolysin O, it was found that infection with viable, replicative bacteria induced in vivo the emergence of T cells specifically reacting against this exotoxin, as demonstrated by eliciting the expression of delayed-type hypersensitivity to listeriolysin O in Listeria-immune mice. The kinetics of this inflammatory reaction followed the same pattern as that observed with crude Listeria antigenic preparation classically used for the detection of delayed-type hypersensitivity, with a peak of expression by day 6 and a slow decline over the next 3 wk to a residual level, indicating the presence of memory T cells reacting with the exotoxin. This result, therefore, allowed us to identify for the first time that a pure immunogenic molecule secreted by L. monocytogenes is specifically recognized by sensitized T cells induced during the course of infection by L. monocytogenes. The expression of T cell-mediated immunity to listeriolysin O was generated by very low amounts of replicative bacteria, indicating that the exotoxin released in host tissues during the process of intracellular growth is highly immunogenic. Our data favor the view that the binding of listeriolysin O to the membrane cholesterol might be a critical event potentiating the in vivo expression of delayed sensitivity against this exotoxin. Indeed, the insertion of listeriolysin O into the cell membrane induced resistance to enzymatic proteolysis and membrane-bound listeriolysin O was significantly more effective in inducing delayed inflammatory reaction in Listeria-immune mice.     

Injection of Pseudomonas aeruginosa Exo toxins into host cells can be modulated by host factors at the level of translocon assembly and/or activity

Pseudomonas aeruginosa type III secretion apparatus exports and translocates four exotoxins into the cytoplasm of the host cell. The translocation requires two hydrophobic bacterial proteins, PopB and PopD, that are found associated with host cell membranes following infection. In this work we examined the influence of host cell elements on exotoxin translocation efficiency. We developed a quantitative flow cytometry based assay of translocation that used protein fusions between either ExoS or ExoY and the ?-lactamase reporter enzyme. In parallel, association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. A pro-myelocytic cell line (HL-60) and a pro-monocytic cell line (U937) were found resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to either macrophage- or neutrophil-like cell lines resulted in injection-sensitive phenotype without significantly changing the level of membrane-inserted translocon proteins. As previous in vitro studies have indicated that the lysis of liposomes by PopB and PopD requires both cholesterol and phosphatidyl-serine, we first examined the role of cholesterol in translocation efficiency. Treatment of sensitive HL-60 cells with methyl-?-cyclodextrine, a cholesterol-depleting agent, resulted in a diminished injection of ExoS-Bla. Moreover, the PopB translocator was found in the membrane fraction, obtained from sucrose-gradient purifications, containing the lipid-raft marker flotillin. Examination of components of signalling pathways influencing the toxin injection was further assayed through a pharmacological approach. A systematic detection of translocon proteins within host membranes showed that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore. In conclusion, we provide new insights in regulation of translocation process and suggest possible cross-talks between eukaryotic cell and the pathogen at the level of exotoxin translocation.     

Construction and use of a nontoxigenic strain of Pseudomonas aeruginosa for the production of recombinant exotoxin A.

To express recombinant forms of Pseudomonas aeruginosa exotoxin A in high yield, we have developed a nontoxigenic strain of P. aeruginosa derived from the hypertoxigenic strain PA103. The nontoxigenic strain, designated PA103A, was produced by the excision marker rescue technique to replace the toxA structural gene in PA103 with an insertionally inactivated toxA gene. The PA103A strain (ToxA-) was used subsequently as the host strain for the expression and production of several recombinant versions of exotoxin A, and the results were compared with exotoxin A production in other P. aeruginosa and Escherichia coli strains. Use of the PA103A strain transformed with the high-copy-number pRO1614 plasmid bearing various toxA alleles resulted in final purification yields of exotoxin A averaging 23 mg/liter of culture. By comparison, exotoxin A production in other expression systems and host strains yields approximately 1/4 to 1/10 as much toxin.     

Cloning and expression of a gene segment encoding the enzymatic moiety of Pseudomonas aeruginosa exotoxin A.

Using the broad-host-range plasmid vector pRO1614, we cloned a segment of the gene from Pseudomonas aeruginosa PA103 encoding the enzymatically active part of the exotoxin A protein. Expression of the cloned gene segment has been achieved both in Escherichia coli and in a nontoxigenic P. aeruginosa host, as assayed by the production of exotoxin A-related antigen and by the ability of the gene product to ADP-ribosylate elongation factor 2. Western blot hybridization analysis revealed a series of polypeptides antigenically related to exotoxin A, the largest of which had a molecular weight of ca. 50,000.     

Host-parasite relationships among group A streptococci. IV. Suppression of antibody response by streptococcal pyrogenic exotoxin

In rabbits, purified streptococcal pyrogenic exotoxin, at 0.002 of the ld(50) dose, suppressed the antibody response to injected sheep erythrocytes. The antibody suppressed was determined by density gradient ultracentrifugal analysis to be of the 19S class. Background serum antibody (50% hemolytic units), as determined photometrically, correlated well with background antibody-forming spleen cells, as determined by the hemolytic-plaque technique. The exotoxin induced neither positive nor negative changes in background antibody levels, but suppressed the early secondary response to injected antigen. A comparison and control experiment showed that purified gram-negative bacterial endotoxin at identical protocol did not induce antibody suppression, but did induce the well-known adjuvant effect. Because streptococcal pyrogenic exotoxin is known to inhibit the phagocytic function of the reticuloendothelial system (RES), these data strongly support the concept that antigen is processed by cells of the RES before it evokes a secondary immune response. The results also demonstrated that streptococcal pyrogenic exotoxin may play a unique role in lowering the acquired defense of the host against infection. If the anamnestic immune response of the host is temporarily suppressed, then the host-parasite balance would be upset in favor of the parasite.     

A periplasmic intermediate in the extracellular secretion pathway of Pseudomonas aeruginosa exotoxin A.

Pseudomonas aeruginosa exotoxin A is synthesized with a secretion signal peptide typical of proteins whose final destination is the periplasm. However, exotoxin A is released from the cell without a detectable periplasmic pool, suggesting that additional determinants in this protein are important for recognition by a specialized machinery of extracellular secretion. The role of the N terminus of the mature exotoxin A in this recognition was investigated. A series of exotoxin A proteins with amino acid substitutions for the glutamic acid pair at the +2 and +3 positions were constructed by mutagenesis of the exotoxin A gene. These N-terminal acidic residues of the mature exotoxin A protein were found to be important not only for efficient processing of the precursor protein but also for extracellular localization of the toxin. The mutated exotoxin A proteins, in which a glutamic acid at the +2 position was replaced by a lysine or a double substitution of lysine and glutamine for the pair of adjacent glutamic acids, accumulated in precursor forms in the mixed cytoplasmic and membrane fractions, which was not seen with the wild-type exotoxin A. The processing of the precursor form of one exotoxin A mutant, in which the glutamic acid at the +2 position was replaced with a glutamine, was not affected. Moreover, a substantial fraction of the mature forms of all three mutants of exotoxin A accumulated in the periplasm, while wild-type exotoxin A could be detected only extracellularly. The periplasmic pools of these variants of exotoxin A could therefore represent the intermediate state during extracellular secretion. The signal for extracellular localization may be located in a small region near the amino terminus of the mature protein or could consist of several regions that are brought together after the polypeptide has folded. Alternatively, the acidic residues may be important for ensuring a conformation essential for exotoxin A to traverse the outer membrane.     

Inhibition of macromolecular synthesis in cultured macrophages by Pseudomonas pseudomallei exotoxin

Pseudomonas pseudomallei exotoxin was found to be a potent inhibitor of protein and DNA synthesis in cultured macrophages. Inhibition of DNA synthesis occurred at toxin concentrations as low as 1-2 micrograms/ml and inhibition of 3H-thymidine uptake was almost complete at concentrations of 8 micrograms/ml or more. A close correlation between cell damage and inhibition by DNA synthesis was observed. For protein synthesis, inhibition was obtained at much lower doses (0.06-2.0 micrograms/ml) of the toxin. At similar toxin concentrations, DNA synthesis was marginally affected. Further, it was shown that protein synthesis inhibition occurred almost immediately after incubation, reaching its maximal inhibitory effect of 70% after 6 hr. DNA synthesis, however, was minimally affected by a similar toxin concentration even after 10 hr of incubation. The inhibition of macromolecular synthesis in macrophages by P. pseudomallei exotoxin may be relevant to its modulatory effect on the host defense mechanism.     

A recombinant form of Pseudomonas exotoxin directed at the epidermal growth factor receptor that is cytotoxic without requiring proteolytic processing.

Pseudomonas exotoxin A is composed of three structural domains that mediate cell recognition (I), membrane translocation (II), and ADP-ribosylation (III). Within the cell, the toxin is cleaved within domain II to produce a 37-kDa carboxyl-terminal fragment, containing amino acids 280-613, which is translocated to the cytosol and causes cell death. In this study, we constructed a mutant protein (PE37), composed of amino acids 280-613 of Pseudomonas exotoxin A, which does not require proteolysis to translocate. PE37 was targeted specifically to cells with epidermal growth factor receptors by inserting transforming growth factor-alpha (TGF-alpha) after amino acid 607 near the carboxyl terminus of Pseudomonas exotoxin A. PE37/TGF-alpha was very cytotoxic to cells with epidermal growth factor receptors. It was severalfold more cytotoxic than a derivative of full-length Pseudomonas exotoxin A containing TGF-alpha in the same position, probably because the latter requires intracellular proteolytic processing to exhibit its cytotoxicity, and proteolytic processing is not 100% efficient. Deletion of 2, 4, or 7 amino acids from the amino terminus of PE37/TGF-alpha greatly diminished cytotoxic activity, indicating the need for a proper amino-terminal sequence. In addition, a mutant containing an internal deletion of amino acids 314-380 was minimally active, indicating that other regions of domain II are also required for the cytotoxic activity of Pseudomonas exotoxin A.     

鳗弧菌毒力相关基因的研究进展

鳗弧菌是引起多种海水鱼类出血性败血症的病原菌。其致病机理与各个毒力基因的协同作用密切相关。文中综述了鳗弧菌的主要毒力基因,包括编码外毒素、粘附因子、侵袭因子、细胞表面成分以及铁吸收系统的基因和部分检测方法。     

Prevalence of antibodies to Pseudomonas pseudomallei exotoxin and whole cell antigens in military personnel in Sabah and Sarawak, Malaysia.

Sera from 420 military personnel serving in Sabah and Sarawk, Malaysia, were tested for antibodies to Pseudomonas pseudomallei exotoxin and whole cell antigens by enzyme-linked immunosorbent assay procedure (ELISA). Data showed that 54.4% of serum samples were positive for antibodies to P. pseudomallei exotoxin and 65.7% were positive for antibodies to the whole cell antigens. Samples gave much lower titers for anti-exotoxin antibodies compared to titers against crude whole cell antigens. The incidence of antibody to exotoxin was highest in the age groups ranging from 26 to 32 years, where the positive rates were higher than 40% and 30% for military personnel serving in Sarawak and Sabah, respectively.     

Response of cultured mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae: differential cytotoxicity.

The sensitivities of 21 mammalian cell lines to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae were measured. Each line exhibited 1-4 log differences in sensitivities to the two toxins. No species-specific sensitivities were noted for Pseudomonas exotoxin while diphtheria exotoxin was most potent in cells of monkey origin, followed by human and hamster cells. Rat- and mouse-derived cell lines were very insensitive to diphtheria exotoxin. The rates of cellular intoxication by both toxins exhibited apparent first-order kinetics and were indistinguishable from one another when equipotent doses were used. Our preparation of diphtheria exotoxin appeared to have a slightly higher ADP-ribosylating efficiency than did Pseudomonas toxin. However, neither toxin exhibited cell line-specific differences in ribosylating efficiencies which could have explained the wide range in potencies for intact cells. Our results suggest that there are significant differences in the mechanisms of cellular intoxication by Pseudomonas and diphtheria exotoxins and that these differences probably exist in the attachment or internalization stages of toxin action.     

A dipeptide insertion in domain I of exotoxin A that impairs receptor binding

Deletions within the structural exotoxin A gene of 27 or 119 amino acids in domain I of the mature polypeptide, or of 88 or 105 amino acids in domains I and II, resulted in the synthesis of exotoxin A (ETA) polypeptides that were not secreted from Pseudomonas aeruginosa hosts but were localized in the cell membrane. Insertions of a hexanucleotide sequence, either pCGAGCT or pCGAATT, at TaqI sites within the gene resulted in variant exotoxin A polypeptides which were secreted normally. pCGAGCT causes insertion of either Glu-Leu or Ser-Ser in the amino acid sequence of the toxin, while pCGAATT causes insertion of either Glu-Phe or Asn-Ser dipeptides. Although the cytotoxicity of eight variants was unimpaired, that of four others was reduced, and one variant which had a Glu-Phe insert between residues 60 and 61 (ETA-60EF61) was 500-fold less cytotoxic than wild-type exotoxin A. Purified ETA-60EF61 dissociated much faster from mouse LMTK- cells than wild-type ETA, suggesting that the insertion impaired the ability of ETA-60EF61 to interact with exotoxin A receptors. The location of the insert is within a major concavity on the surface of domain I of the exotoxin A molecule, suggesting that this concavity is important for toxin-receptor interaction.     

Actin cross-linking domain of Aeromonas hydrophila repeat in toxin A (RtxA) induces host cell rounding and apoptosis

The repeat in toxin (Rtx) of an environmental isolate ATCC 7966 of Aeromonas hydrophila consists of six genes (rtxACHBDE) organized in an operon similar to the gene organization found for the Rtx of the Vibrio species. The first gene in this operon (rtxA) encodes an exotoxin in vibrios, while other genes code for proteins needed for proper activation of RtxA and in secretion of this toxin from Vibrio cholerae. However, the RtxA of ATCC 7966, as well as from the clinical isolate SSU of A. hydrophila, was exclusively expressed and produced during co-infection of this pathogen with the host, e.g., HeLa cells, indicating that rtxA gene expression required host cell contact. Within the RtxA, an actin cross-linking domain (ACD) exists and to investigate the functionality of this domain, several truncated versions of ACD were generated to discern its minimal biological active region. Such genetically modified genes encoding ACD, which were truncated on either the NH(2) or the COOH terminal, as well as on both ends, were expressed from a bidirectional promoter of the pBI-enhanced green fluorescent protein (EGFP) vector in a HeLa-Tet-Off cell system. We demonstrated that only the full-length ACD of RtxA from A. hydrophila catalyzed the covalent cross-linking of the host cellular actin, whereas the ACD truncated on the NH(2), COOH or both ends did not exhibit such actin cross-linking characteristics. Further, we showed that the full-length ACD of A. hydrophila RtxA disrupted the actin cytoskeleton of HeLa cells, resulting in their rounding phenotype. Finally, our data provided evidence that the full-length ACD of RtxA induced host cell apoptosis. Our study is the first to report that A. hydrophila possesses a functional RtxA having an ACD that contributes to the host cell apoptosis, and hence could represent a potential virulence factor of this emerging human pathogen.     

Human alpha-defensins neutralize toxins of the mono-ADP-ribosyltransferase family

Various bacterial pathogens secrete toxins, which are not only responsible for fatal pathogenesis of disease, but also facilitate evasion of host defences. One of the best-known bacterial toxin groups is the mono-ADP-ribosyltransferase family. In the present study, we demonstrate that human neutrophil alpha-defensins are potent inhibitors of the bacterial enzymes, particularly against DT (diphtheria toxin) and ETA (Pseudomonas exotoxin A). HNP1 (human neutrophil protein 1) inhibited DT- or ETA-mediated ADP-ribosylation of eEF2 (eukaryotic elongation factor 2) and protected HeLa cells against DT- or ETA-induced cell death. Kinetic analysis revealed that inhibition of DT and ETA by HNP1 was competitive with respect to eEF2 and uncompetitive against NAD+ substrates. Our results reveal that toxin neutralization represents a novel biological function of HNPs in host defence.     

Nonpurulent response to toxic shock syndrome toxin 1-producing Staphylococcus aureus. Relationship to toxin-stimulated production of tumor necrosis factor

Infection of surgical wounds with toxic shock syndrome toxin 1 (TSST-1)-producing Staphylococcus aureus does not usually elicit a purulent response from the host. Because S. aureus is normally a pyogenic pathogen, this phenomenon suggests that strains of staphylococci that produce the exotoxin are able to inhibit the migration of polymorphonuclear neutrophils (PMN) to sites of infection. We have considered that inhibition of leukocyte migration may be an effect of secreted TSST-1 and have studied direct and indirect effects of the exotoxin on migratory functions of PMN in vitro. Preincubation of PMN with TSST-1 produced no inhibition of random motility or FMLP- or C5a-stimulated chemotaxis under agarose. Supernatant fluids from mononuclear leukocytes incubated with TSST-1, however, were potently inhibitory for both PMN random and chemotactic migratory functions. The inhibitor of migration was identified as TNF based upon neutralization by anti-TNF antiserum and its presence in the culture supernatant fluids assayed in terms of cytotoxicity for murine TNF-sensitive L-929 cell line cells. Preincubation of PMN with recombinant human TNF also inhibited subsequent PMN random and chemotactic migratory functions. We propose that TSST-1 inhibits the mobilization of PMN to sites of infection by stimulation of monocyte/macrophage TNF production and suggest that TNF may also contribute to some other effects of toxic shock syndrome.     

鳗弧菌毒力相关基因的研究进展

鳗弧菌是引起多种海水鱼类出血性败血症的病原菌。其致病机理与各个毒力基因的协同作用密切相关。文中综述了鳗弧菌的主要毒力基因,包括编码外毒素、粘附因子、侵袭因子、细胞表面成分以及铁吸收系统的基因和部分检测方法。     

GPR107, a G-protein-coupled Receptor Essential for Intoxication by Pseudomonas aeruginosa Exotoxin A,Localizes to the Golgi and Is Cleaved by Furin

A number of toxins, including exotoxin A (PE) of Pseudomonas aeruginosa, kill cells by inhibiting protein synthesis. PE kills by ADP-ribosylation of the translation elongation factor 2, but many of the host factors required for entry, membrane translocation, and intracellular transport remain to be elucidated. A genome-wide genetic screen in human KBM7 cells was performed to uncover host factors used by PE, several of which were confirmed by CRISPR/Cas9-gene editing in a different cell type. Several proteins not previously implicated in the PE intoxication pathway were identified, including GPR107, an orphan G-protein-coupled receptor. GPR107 localizes to the trans-Golgi network and is essential for retrograde transport. It is cleaved by the endoprotease furin, and a disulfide bond connects the two cleaved fragments. Compromising this association affects the function of GPR107. The N-terminal region of GPR107 is critical for its biological function. GPR107 might be one of the long-sought receptors that associates with G-proteins to regulate intracellular vesicular transport.     

Superantigen-induced proliferation of human CD4+CD25- T cells is followed by a switch to a functional regulatory phenotype

Bacterial superantigens are potent T cell activators. In humans they cause toxic shock and scarlet fever, and they are implicated in Kawasaki's disease, autoimmunity, atopy, and sepsis. Their function remains unknown, but it may be to impair host immune responses increasing bacterial carriage and transmission. Regulatory (CD25(+)FOXP3(+)) T cells (Tregs) play a role in controlling inflammatory responses to infection. Approximately 2% of circulating T cells are naturally occurring Tregs (nTregs). Conventional Ag stimulation of naive FOXP3(-) T cells induces Ag-specific Tregs. Polyclonal T cell activation has been shown to produce non-Ag-specific Tregs. Because superantigens are unique among microbial virulence factors in their ability to trigger polyclonal T cell activation, we wanted to determine whether superantigen stimulation of T cells could induce non-Ag-specific Tregs. We assessed the effect of superantigen stimulation of human T cells on activation, regulatory markers, and cytokine production by flow cytometry and T cell suppression assays. Stimulation of PBMCs with staphylococcal exotoxin A and streptococcal pyrogenic exotoxins A and K/L resulted in dose-dependent FOXP3 expression. Characterization of this response for streptococcal pyrogenic exotoxin K/L confirmed its Vβ specificity, that CD25(+)FOXP3(+) cells arose from CD25(-) T cells and required APCs. These cells had increased CTLA-4 and CD127 expression, typical of the recently described activated converted Treg-like cells, and exhibited functional suppressor activity comparable to nTregs. Superantigen-stimulated CD25(+)FOXP3(+) T cells expressed IL-10 at lower superantigen concentrations than was required to trigger IFN-γ production. This study provides a mechanism for bacterial evasion of the immune response through the superantigen induction of Tregs.     

The superantigenic activity of streptococcal pyrogenic exotoxin B is independent of the protease activity

The nature of the mitogenic activity of pyrogenic streptococcal exotoxin B, also known as streptococcal cysteine protease, has been debated in the literature. Streptococcal exotoxin B has been shown to cleave interleukin-1beta precursor and create biologically active interleukin-1beta, a major cytokine mediating inflammation and shock. This activity could mimic the mitogenicity and cytokine release induced by superantigens in lymphocyte stimulating experiments. In this study, the protease activity of streptococcal exotoxin B was irreversibly inhibited by covalent binding of a tripeptide and the superantigenic properties of streptococcal exotoxin B were found not to be influenced by this inactivation. Native as well as protease-inactivated streptococcal exotoxin B was shown to stimulate T-cell proliferation without a need of metabolically active antigen presenting cells. Furthermore, streptococcal exotoxin B-induced T-cell proliferation was shown to require HLA-DQ since addition of HLA-DQ monoclonal antibodies totally inhibited the mitogenic activity of streptococcal exotoxin B, indicating that streptococcal exotoxin B, as other superantigens, makes direct contact with the T-cell receptor via HLA class II. The aim of this study was to characterize the relationship between the proteolytic and superantigenic properties of streptococcal exotoxin B.