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Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX) is a unique exotoxin produced by Mycoplasma pneumoniae (MP) and has been confirmed to possess ADP-ribosyltransferase (ART) and vacuolating activities. CARDS TX binds to receptors on the surfaces of mammalian cells followed by entry into the cells through clathrin-mediated endocytosis, and exerts cytotoxic effects by undergoing retrograde transport and finally cleavage on endosomes and cellular organelles. In addition, CARDS TX can trigger severe inflammatory reactions resulting in airway dysfunction, producing allergic inflammation and asthma-like conditions. As a newly discovered virulence factor of MP, CARDS TX has been extensively studied in recent years. As resistance to macrolide drugs has increased significantly in recent years and there is no vaccine against MP, the development of a vaccine targeting CARDS TX is considered a potential preventive measure. This review focuses on recent studies and insights into this toxin, providing directions for a better understanding of MP pathogenesis and treatment. IMPORTANCE: A serious hazard to worldwide public health in recent years, Mycoplasma pneumoniae (MP) is a prominent bacterium that causes community-acquired pneumonia (CAP) in hospitalized children. Due to their high prevalence and fatality rates, MP infections often cause both respiratory illnesses and extensive extrapulmonary symptoms. It has recently been shown that MP produces a distinct exotoxin known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). Mycoplasma pneumoniae pneumonia (MPP)-like tissue injury is caused by this toxin because it has both ADP-ribosyltransferase and vacuolating properties. A better knowledge of MP etiology and therapy is provided by this review, which focuses on latest research and insights into this toxin., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Ling Li reports financial support was provided by Wuxi Taihu Lake Talent Plan. Yun Guo reports financial support was provided by The Natural Science Foundation of Jiangsu Province. Yun Guo reports financial support was provided by Top Talent Support Program for young and middle-aged people of Wuxi Health Committee. Ling Li reports financial support was provided by Major Program of Wuxi health and Family Planning Commission. Yun Guo reports financial support was provided by Wuxi Science and Technology Development Fund. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study aimed to evaluate the effects of Cinnamaldehyde, Carvacrol, and honey either alone or in combinations on the expression of exoS and ampC genes in multidrug-resistant (MDR) P. aeruginosa isolates. Thirty-five P. aeruginosa isolates were recovered from burn wound infections of patients admitted to the burn ward of Besat hospital of Hamadan, Iran, during 2018. Antibiotic susceptibility testing was performed using the Kirby-Bauer disk diffusion method to identify MDR isolates. The antibacterial effects of Cinnamaldehyde, Carvacrol, and honey either alone or in combinations with each other were compared to Imipenem (as the control group) using the broth dilution method. The expressions of exoS and ampC genes were determined in bacteria treated with sub-minimum inhibitory concentration (MIC) of the ternary combination of Cinnamaldehyde, Carvacrol, and honey by Real-Time-PCR. The data were analyzed using SPSS software applying student t-test, Kruskal-Wallis, and Mann-Whitney U tests. The P-value less than 0.05 was considered as statistically significant. The average MICs of Cinnamaldehyde, Carvacrol, and honey were 0.82-0.01, 0.01-0.6, and 62.5-250 μg/mL, respectively. The average MIC of the mentioned compounds was 430 times lower than that of Imipenem. A synergistic effect was detected between these drugs against 70% of the isolates. At sub-MIC concentration, the triple combination of Cinnamaldehyde, Carvacrol, and honey reduced the expressions of exoS and ampC genes by 6.12 and 2.85 folds, respectively. The combination of Cinnamaldehyde, Carvacrol, and honey showed a higher antibacterial effect than Imipenem. However, it needs confirmation with more isolates., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Three patients with severe Clostridium difficile infection (CDI) caused by an unusual strain of C. difficile, PCR ribotype (RT) 251, were identified in New South Wales, Australia. All cases presented with severe diarrhoea, two had multiple recurrences and one died following a colectomy. C. difficile RT251 strains were isolated by toxigenic culture. Genetic characterisation was performed using techniques including toxin gene profiling, PCR ribotyping, whole genome sequencing (WGS), in-silico multi-locus-sequence-typing (MLST) and core-genome single nucleotide variant (SNV) analyses. Antimicrobial susceptibility was determined using an agar incorporation method. In vitro toxin production was confirmed by Vero cell cytotoxicity assay and pathogenicity was assessed in a murine model of CDI. All RT251 isolates contained toxin A (tcdA), toxin B (tcdB) and binary toxin (cdtA and cdtB) genes. Core-genome analyses revealed the RT251 strains were clonal, with 0-5 SNVs between isolates. WGS and MLST clustered RT251 in the same evolutionary clade (clade 2) as RT027. Despite comparatively lower levels of in vitro toxin production, in the murine model RT251 infection resembled RT027 infection. Mice showed marked weight loss, severe disease within 48 h post-infection and death. All isolates were susceptible to metronidazole and vancomycin. Our observations suggest C. difficile RT251 causes severe disease and emphasise the importance of ongoing surveillance for new and emerging strains of C. difficile with enhanced virulence., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objectives: Clostridioides difficile strains cause severe infection. Previous studies suggested that the virulence of C. difficile is dependent on ribotype; however, this hypothesis is still controversial. We aim to investigate the relationship between ribotypes, restriction endonuclease analysis (REA) types, and toxin gene expression in C. difficile strains., Methods: We utilized 53 clinical C. difficile strains. All strains were assigned a molecular strain type using PCR ribotyping and REA typing and classified into 17 ribotypes and six REA types. The expression of toxin genes (tcdA, tcdB, and cdtB) in C. difficile strains were quantified by real-time PCR using each specific primer set, and expression was normalized to that of the housekeeping gene rpoA., Results: All 53 strains expressed tcdB and four strains expressed cdtB. Five strains did not express tcdA. Most ribotype and REA type strains expressed tcdA and tcdB similar to the BAA-1870 strain. In cdtB-positive strains, the cdtB expression levels were similar to those in the BAA-1870 strain. tcdA and tcdB expression levels were similar in the cdtB-positive and cdtB-negative strains., Conclusion: Toxin gene expression was not associated with the ribotype. Production of binary toxin C. difficile transferase was not related to tcdA and tcdB expression levels., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Clostridium difficile infection causes antibiotics-associated diarrhea and pseudomembranous colitis. Major virulence factors of C. difficile are the Rho-glucosylating toxins TcdA and TcdB. In addition, many, so-called hypervirulent C. difficile strains produce the binary actin-ADP-ribosylating toxin CDT. CDT causes depolymerization of F-actin and rearrangement of the actin cytoskeleton. Thereby, many cellular functions, which depend on actin, are altered. CDT disturbs the dynamic balance between actin and microtubules in target cells. The toxin increases microtubule polymerization and induces the formation of microtubule-based protrusions at the plasma membrane of target cells. Moreover, CDT causes a redistribution of vesicles from the basolateral side to the apical side, where extracellular matrix proteins are released. These processes may increase the adherence of clostridia to target cells. Here, we review the effects of the action of CDT on the actin cytoskeleton and on the microtubule system., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Iota toxin is a binary toxin solely produced by Clostridium perfringens type E strains, and is structurally related to CDT from C. difficile and CST from C. spiroforme. As type E causes hemorrhagic enteritis in cattle, it is usually assumed that associated diseases are mediated by iota toxin, although evidence in this regard has not been provided. In the present report, iota toxin intestinal effects were evaluated in vivo using a mouse model. Histological damage was observed in ileal loops treated with purified iota toxin after 4 h of incubation. Luminal iota toxin induced fluid accumulation in the small intestine in a dose dependent manner, as determined by the enteropooling and the intestinal loop assays. None of these changes were observed in the large intestine. These results suggest that C. perfringens iota toxin alters intestinal permeability, predominantly by inducing necrosis and degenerative changes in the mucosal epithelium of the small intestine, as well as changes in intestinal motility. The obtained results suggest a central role for iota toxin in the pathogenesis of C. perfringens type E hemorrhagic enteritis, and contribute to remark the importance of clostridial binary toxins in digestive diseases., (Published by Elsevier Ltd.)

Assessing the regulation of Clostridium difficile transferase (CDT), is complicated by the presence of a Pathogenicity locus (PaLoc) which encodes Toxins A and B. Here we developed R20291ΔPaLoc model strains and cell-based assays to quantify CDT-mediated virulence. Their application demonstrated that the transcriptional regulator, CdtR, was required for CDT-mediated cytotoxicity., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Salmonella enterica serovar typhimurium (S. typhimurium) is a facultative intracellular pathogen that can cause gastroenteritis and systemic infection in a wide range of hosts. Salmonella plasmid virulence gene spvB is closely related to bacterial virulence in different cells and animal models, and the encoded protein acts as an intracellular toxin required for ADP-ribosyl transferase activity. However, until now there is no report about the pathogenecity of spvB gene on zebrafish. Due to the outstanding advantages of zebrafish in analyzing bacteria-host interactions, a S. typhimurium infected zebrafish model was set up here to study the effect of spvB on autophagy and intestinal pathogenesis in vivo. We found that spvB gene could decrease the LD50 of S. typhimurium, and the strain carrying spvB promoted bacterial proliferation and aggravated the intestinal damage manifested by the narrowed intestines, fallen microvilli, blurred epithelium cell structure and infiltration of inflammatory cells. Results demonstrated the enhanced virulence induced by spvB in zebrafish. In spvB-mutant strain infected zebrafish, the levels of Lc3 turnover and Beclin1 expression increased, and the double-membraned autophagosome structures were observed, suggesting that spvB can inhibit autophagy activity. In summary, our results indicate that S. typhimurium strain containing spvB displays more virulence, triggering an increase in bacterial survival and intestine injuries by suppressing autophagy for the first time. This model provides novel insights into the role of Salmonella plasmid virulence gene in bacterial pathogenesis, and can help to further elucidate the relationship between bacteria and host immune response., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We studied the contribution of exotoxin A to the virulence of Pseudomonas aeruginosa against the silkworm, Bombyx mori. First, an exotoxin A-deficient mutant strain (PAO1toxA) was created, and its virulence compared with that of the parental PAO1 strain. In a short-term mortality assay, the mutant harboring pBBR1MCS2 did not kill B. mori until 120 h after inoculation and complementation of the corresponding gene in trans restored the strain's virulence. Next, to ascertain whether or not it lost all virulence, PAO1toxA (pBBR1MCS2, pGFP) was used in a long-term mortality assay. B. mori inoculated with the mutant strain did not die until early in the 5th instar (240 h after inoculation). However, 50% of the inoculated B. mori died late in the 5th instar or in the early pupal stage (408 h after inoculation). All had died by the pupal stage (600 h after inoculation). The mutant strain was isolated from dead larvae and cocoons. The bacterial population of PAO1toxA in hemolymph reached 4.77 × 10(7) cfu/ml. These results indicated that exotoxin A acts as a virulence factor in B. mori and that other virulence factor(s) are involved during the late stages of infection., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We recently characterized a T3SS effector, AexU, from a diarrheal isolate SSU of Aeromonas hydrophila, which exhibited ADP-ribosyltransferase (ADPRT) activity. Here we provided evidence that AexU also possessed GTPase-activating protein (GAP) activity, which was mainly responsible for host cell apoptosis and disruption of actin filaments. Earlier, we showed that the DeltaaexU null mutant was attenuated in a mouse model, and we now demonstrated that while the parental A. hydrophila strain could be detected in the lung, liver, and spleen of infected mice, the DeltaaexU mutant was rapidly cleared from these organs resulting in increased survivability of animals. Further, AexU prevented phosphorylation of c-Jun, JNK and IkappaBalpha and inhibited IL-6 and IL-8 secretion from HeLa cells. Our data indicated that AexU operated by inhibiting NF-kappaB and inactivating Rho GTPases. Importantly, however, when the DeltaaexU null mutant was complemented with the mutated aexU gene devoid of ADPRT and GAP activities, a higher mortality rate in mice with concomitant increase in the production of pro-inflammatory cytokines/chemokines was noted. These data indicated that either such a mutated AexU is a potent inducer of them or that AexU possesses yet another unknown activity that is modulated by ADPRT and GAP activities and results in this aberrant cytokine/chemokine production responsible for increased animal death., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Pseudomonas aeruginosa exotoxin A (PEA) is a number of family of bacterial ADP-ribosylating toxins and possesses strong immunogenicity. The detoxified exotoxin A, as a potent vaccine adjuvant and vaccine carrier protein, has been extensively used in human and animal vaccinations. However, the expression level of PEA gene in Escherichia coli is relative low which is likely due to the presence of rare codon and high levels of GC content. In order to enhance PEA gene expression, we optimized PEA gene using E. coli preferred codons and expressed it in E. coli BL21 (DE3) by using pET-20b(+) secretory expression vector. Our results showed that codon optimization significantly reduced GC content and enhanced PEA gene expression (70% increase compared with that of the wild-type). Moreover, the codon-optimized PEA possessed biological activity and had the similar toxic effects on mouse L292 cells compared with the wild-type PEA gene. Codon optimization will not only improve PEA gene expression but also benefit further modification of PEA gene using nucleotide-mediated site-directed mutagenesis. A large number of purified PEA proteins will provide the necessary conditions for further PEA functional research and application., (Copyright 2010 Elsevier Inc. All rights reserved.)

The ectoenzyme ADP-ribosyltransferase 2.2 (ART2.2) can apoptotically delete various T-cell subsets. Depending on the involved apoptotic T-cell subset, enhanced ART2.2 activity could result in immunosuppression or autoimmunity. Diminished activity of the CD38 ectoenzyme that normally represents a counter-regulatory competitor for the NAD substrate represents one mechanism enhancing ART2.2 activity. Hence, it would be desirable to develop an agent that efficiently blocks ART2.2 activity in vivo. While the llama derived recombinant s+16 single domain antibody overcame the difficulty of specifically targeting the ART2.2 catalytic site potential therapeutic use of this reagent is limited due to short in vivo persistence. Thus, we tested if a modified version of s+16 incorporating the murine IgG1 Fc tail (s+16Fc) mediated long-term efficient in vivo suppression of ART2.2. We reasoned an ideal model to test the s+16Fc reagent were NOD mice in which genetic ablation of CD38 results in an ART2.2 mediated reduction in already sub-normal numbers of immunoregulatory natural killer T-(NKT) cells to a level that no longer allows them when activated by the super-agonist alpha-galactosylceramide (alpha-GalCer) to elicit effects inhibiting autoimmune type 1 diabetes (T1D) development. Treatment with s+16Fc efficiently mediated long-term in vivo inhibition of ART2.2 activity in NOD.CD38(null) mice, restoring their iNKT cell numbers to levels that upon alpha-GalCer activation were capable of inhibiting T1D development., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Bacteremia caused by Clostridium difficile is rare. In this report, we describe a case of C. difficile bacteremia caused by an unusual strain of C. difficile. The isolate contained neither toxin A nor B genes, however, binary toxin genes were present (tcdA(-), tcdB(-), cdtA(+), cdtB(+)) and a 7.2-kb element unrelated to the PaLoc was found inserted within the PaLoc integration site. The clinical relevance of the isolate could not be determined.

Clostridium perfringens iota-toxin is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). We investigated the role of the conserved Ca(2+)-binding motif of Ib in the cytotoxicity of iota-toxin. The cytotoxicity of iota-toxin increased with an increase in the concentration of extracellular Ca(2+). A surface plasmon resonance analysis showed that the binding of Ia to the oligomer of Ib is dependent on the concentration of Ca(2+). However, the addition of Ca(2+) had no effect on the binding of (125)I-labeled Ib to the cells. We replaced Asp-8, -10, and -12 in the Ca(2+)-binding motif of Ib with alanine. D8A, D10A, and D12A bound to the cell and formed an oligomer at about half of the wild-type Ib. The cytotoxicity of Ib variants in the presence of Ia was about 500-fold less than that of wild-type Ib. Immunofluorescence study showed that these variants were internalized in the early endosomes like wild-type Ib. However, wild-type Ib-induced internalization of Ia in the cells, but these variants did not. The result indicates that the conserved Ca(2+)-binding motif in the N-terminal region of Ib plays a role in the interaction of Ib with Ia in the presence of Ca(2+).

Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin therapy. In our previous study, a modified PE38KDEL, denoted PE38KDELKQK, was engineered to eliminate VLS. The PE38KDELKQK-based immunotoxin has been proved to retain potent anti-tumor activity but with a remarkable attenuation in VLS. In the present study, we have constructed and expressed a recombinant immunotoxin CD25-PE38KDELKQK containing humanized anti-CD25 single-chain antibody (scFv) genetically fused to PE38KDELKQK in Escherichia coli. After washing with buffer containing 2 M urea, the purity of inclusion body was about 82%. The denatured inclusion bodies were refolded on-column in Tris buffer (pH 8.0) containing 4mM of GSH and 1 mM of GSSG using a gradient of decreasing urea. We found that the presence of GSH/GSSG (4:1) in the on-column refolding buffer was important for efficient refolding. In addition, slow flow rate was another important factor could increase refolding. Under these conditions, the activity of the refolded protein could reach about 90% of that of the native protein. The refolded proteins were purified to homogeneity ( approximately 95% purity) by a combination of His-Ni(2+) metal affinity chromatography and gel filtration chromatography. The in vitro cytotoxicity assay indicated the purified immunotoxin CD25-PE38KDELKQK had specific cytotoxicity to CD25-positive leukemic cells comparable to wild-type CD25-PE38KDEL (wt). In contrast, CD25-PE38KDELKQK was shown to be much weaker in inducing VLS in mice than wt. The protein expression, purification, and refolding system established in this paper is important for further study on immunotoxin CD25-PE38KDELKQK.

We cloned and sequenced an ADP-ribosylating toxin (AexT) from a mesophilic Aeromonas hydrophila strain AH-3 with a type III secretion system (T3SS). This toxin only showed homology, in genes and proteins, with the first half of A. salmonicida AexT. The A. hydrophila AexT showed ADP-ribosyltransferase activity, translocation through the T3SS system, and this A. hydrophila T3SS system is inducible under calcium-depleted conditions. The A. hydrophila aexT mutant showed a slight reduction in their virulence assayed by several methods when compared to the wild-type strain, while an A. hydrophila T3SS mutant is highly reduced in virulence on the same assays. The A. hydrophila AexT is the first described and the smallest T3SS effector toxin found in mesophilic Aeromonas with a functional T3SS.

A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular characterization of an aexT-like toxin gene (designated as aexU) from a diarrheal isolate SSU of A. hydrophila. The aexU gene was 1539bp in length and encoded a protein of 512 amino acid (aa) residues. The NH(2)-terminus of AexU (aa residues 1-231) exhibited a 67% homology with the NH(2)-terminus of AexT from A. salmonicida. Importantly, its COOH-terminus (aa residues 232-512) had no homology with any known functional proteins in the database; however, the full-length AexU retained ADP-ribosyltransferase activity. The expression and subsequent secretion of AexU was T3SS dependent, as inactivation of the ascV gene that codes for an inner-membrane component of the T3SS channel from the wild-type (WT) bacterium, blocked translocation of AexU in HT-29 human colonic epithelial cells. We provided evidence that inactivation of acrV and axsE genes (homologs of lcrV and exsE in Yersinia species and P. aeruginosa, respectively) from A. hydrophila SSU, altered expression and/or secretion of AexU. We deleted an aexU gene from the WT, as well as from the DeltaaopB mutant, of A. hydrophila, generating a single knockout (DeltaaexU) and a double knockout mutant, DeltaaopB/DeltaaexU. Increased phagocytosis was observed in RAW264.7 murine macrophages infected with the DeltaaopB/DeltaaexU mutant, as compared to macrophages when infected with the parental DeltaaopB strain. Further, mice infected with the DeltaaexU mutant had a 60% survival rate, compared to animals infected with the WT or the DeltaaexU-complemented strain that caused 90-100% of the animals to die at a 2-3 LD(50s) dose. Immunization of mice with the recombinant AexU protected them from subsequent lethal challenge dose by the WT bacterium. Finally, we detected specific anti-AexU antibodies in the sera of mice that survived challenge by the WT bacterium, which may indicate that AexU plays an important role in the pathogenesis of Aeromonas infections.

C3 transferase has been extensively used in the investigation of Rho signaling. It acts as a Rho antagonist by covalently attaching an ADP-ribose group to RhoA, B, and C proteins. Cell-permeable C3 fusion proteins have been developed so that lower doses of C3 can be used. We describe a simple and fast fluorescence-based assay to evaluate the enzymatic activity of cell-permeable C3 proteins purified from Escherichia coli. The assay measures glycohydrolase (GH) activity of C3 that cleaves NAD+ into ADP-ribose and nicotinamide. Results from the GH activity correlate with other tests carried out in tissue culture cells such as neurite outgrowth or ADP ribosylation of RhoA. This method provides reliable measurements of the activity of permeable C3 proteins or other C3-related proteins.

C3 exoenzyme from Clostridium limosum, specifically ADP-ribosylates and inactivates Rho GTPases, but not or much less than Rac and Cdc42. To bypass the poor cell accessibility of the exoenzyme, a chimeric fusion toxin was constructed consisting of C3 exoenzyme and the N-terminal adaptor domain of the enzyme component C2I of the actin-ADP-ribosylating Clostridium botulinum C2 toxin. This fusion toxin C2IN-C3 is transported into cells by interaction with the binding and translocation component (C2II) of C2 toxin. Purification and activity of the chimeric toxin is reported.

Type III secretion (TTS) mediated translocation of exoenzymes is a key virulence strategy utilised by the opportunistic pathogen Pseudomonas aeruginosa to deliver exoenzyme effectors into the eukaryotic cell. We have previously shown that type III mediated translocation is a contact dependent process, which requires the secreted translocator proteins PcrV, PopB and PopD. To further analyse this mechanism, HeLa cells were infected with the wild-type strain PAK as well as isogenic pcrV, popB, popD, pcrG and popN mutants. In the presence of eukaryotic cells, expression of exoenzyme S (ExoS) increased. When cells were infected with the wild-type strain PAK no ExoS was detected in the tissue culture medium. This confirms that ExoS translocation by P. aeruginosa occurs by a polarised mechanism. In contrast, high levels of ExoS were recovered in the tissue culture medium when cells were infected with pcrG, pcrV and popN mutants. Additionally, ExoS expression levels were higher for these mutants regardless of inducing conditions. This suggests that PcrG, PcrV and PopN are involved in negative regulation of ExoS expression and secretion, and are required to ensure polarised delivery of effectors into target cells.

The spv genes carried on the Salmonella virulence plasmid are commonly associated with severe systemic infection in experimental animals. The SpvB virulence-associated protein has been shown to ADP-ribosylate actin, and this enzymatic activity is essential for virulence in mice. Here, we present evidence that intracellular expression of SpvB protein induces not only disruption of actin filaments but also apoptotic cell death in eukaryotic cells.

Nontyphoid Salmonella enterica requires the plasmid-encoded spv genes to establish successful systemic infection in experimental animals. The SpvB virulence-associated protein has recently been shown to contain the ADP-ribosyltransferase domain. SpvB ADP-ribosilates actin and depolymerizes actin filaments when expressed in cultured epithelial cells. However, spontaneous secretion or release of SpvB has not been observed under in vitro growth conditions. In the present study we investigated the secretion of SpvB from Salmonella using in vitro and in vivo assay systems. We showed that SpvB is secreted into supernatant from Salmonella strains that contain the cloned spvB gene on a plasmid when they grew in intracellular salts medium (ISM), a minimal medium mimicing the intracellular iron concentrations of eukaryotic cells. A series of mutant SpvB proteins revealed that an N-terminal region of SpvB located at amino acids 1-229 was sufficient to promote secretion into extracellular milieu. Confocal immunofluorescence microscopy also demonstrated efficient localization of the N-terminal domain of SpvB(1-360) tagged with biotinylated peptide within infected host cell cytosol but not truncated SpvB(1-179) fusion protein. In addition, mutations that inactivate genes within Salmonella pathogenicity island 1 or Salmonella pathogenicity island 2 that encode type III secretion systems (TTSS) could secrete the SpvB protein into the culture medium. These results indicate that SpvB protein is transported from the bacteria and into the host cytoplasm independent of TTSS.

Carcinoembryonic antigen (CEA) is expressed at greatly increased levels in nearly all human colorectal carcinomas. Anti-CEA antibodies have been proved to be useful for targeting several cancer types known to express CEA. A recombinant immunotoxin was constructed, in which the cell-binding domain of Pseudomonas exotoxin (PE) was replaced with the single-chain Fv (scFv) of anti-CEA monoclonal antibody for targeting to colorectal carcinomas. This single-chain immunotoxin was expressed in E. coli and purified under denaturing condition of 6M guanidine hydrochloride (GuHCl). It was found that the immunotoxin maintains a binding activity in denaturing condition of 6M GuHCl and the fused PE contributes to the stability of immunotoxin in such condition. Dialysis against PBS buffer after purification under 6M GuHCl keeps the binding activity of immunotoxin.

Pseudomonas aeruginosa Exoenzyme S (ExoS) is a bifunctional type-III cytotoxin. The N-terminus (residues 1-232) possesses Rho GTPase-activating (GAP) activity, while the C-terminus (residues 233-453) comprises an ADP-ribosyltransferase domain. Amino acid residues 51-72 of ExoS are involved in membrane binding and aggregation, which has complicated purification schemes. Here, it is reported on the expression, purification, and characterization of two recombinant forms of ExoS that lack this membrane-binding domain, designated rExoS78-453 and rExoSdelta51-72. Purification of these forms was achieved using sequential NTA/Ni(2+)-affinity, gel filtration, and anion-exchange chromatography. Both forms of ExoS possessed Rho GAP activity and ADP-ribosyltransferase activity comparable to wild-type ExoS. Mass spectrometry showed that rExoS78-453 and rExoSdelta51-72 had molecular masses similar to their predicted molecular masses.

The type III secretion system (TTSS) of the opportunistic pathogen Pseudomonas aeruginosa enables the bacterium to deliver exoenzymes directly into the eukaryotic cell. In this study we have investigated the role of key factors involved in this process. We could demonstrate that the translocators PopB, PopD and PcrV are absolutely required for delivery of Exoenzyme S into host cells. By analyzing different Tfp (type IV pili) mutants we could establish a correlation between the frequency of bacteria binding to the host cell and the levels of translocated ExoS, thereby verifying that the process is contact dependent. However, there was no absolute requirement for the Tfp per se, since the pilus could be substituted with a different type of adhesin, the non-fimbrial adhesin pH6 antigen of Yersinia pestis. Taken together, our results demonstrate that binding to establish close contact between the type III secretion organelle and the host cell is essential for translocation, while the additional activities of Tfp are not essential for the delivery of TTSS proteins.

Redirecting retroviral vector transduction simply by insertion of a ligand into the envelope (Env) protein has met with several obstacles. For example, virions targeted to epidermal growth factor receptor (EGFR), after receptor binding, rapidly traffic to the lysosomes, where they are degraded. Exotoxin A of Pseudomonas aeruginosa has the ability to translocate from endosomes to the cytoplasm by means of a translocation domain (TLD). We generated a series of chimeric Env proteins of Moloney murine leukemia virus containing EGFR ligands, where TLD was inserted into different regions. These chimeric proteins were successfully produced, if the translocation domain was not located at the immediate N-terminus of Env. The ability to transduce murine cells via the ecotropic receptor varied but correlated with the amount of Env proteins incorporated into the virions. Chimeric vector particles could bind to EGFR, demonstrating the functional exposure of the peptide ligand. However, transduction of human cells expressing EGFR but not the ecotropic receptor by virions carrying the chimeric protein was not observed.

The Entamoeba histolytica small GTP-binding protein EhRho1 has an unusual amino acid residue at a conserved site found in all known Ras superfamily proteins. EhRho1 has an isoleucine at position 45, which corresponds to position 28 of human Ras and Rac and position 30 of human Rho and Cdc42. All other known small GTPases have an aromatic residue (typically phenylalanine) at this position, and mutation to a leucine renders other Ras proteins constitutively active by reason of diminished affinity for GDP. It was determined that the EhRho1 protein has a half-time of GDP dissociation similar to that of a human Rho protein, HsRhoA, and therefore an isoleucine at this site in EhRho1 is not likely to render EhRho1 constitutively active. It was also found that EhRho1 is not a substrate for the Rho-specific C3 exoenzyme. Thus EhRho1 appears to be an unusual member of the Ras family.

Myofibroblasts generate the contractile force responsible for wound healing and pathological tissue contracture. In this paper the stress-relaxed collagen lattice model was used to study lysophosphatidic acid (LPA)-promoted myofibroblast contraction and the role of the small GTPase Rho and its downstream targets Rho kinase and myosin light chain phosphatase (MLCPPase) in regulating myofibroblast contraction. In addition, the regulation of myofibroblast contraction was compared with that of smooth muscle cells. LPA-promoted myofibroblast contraction was inhibited by the myosin light chain kinase (MLCK) inhibitors KT5926 and ML-7; however, in contrast to that observed in smooth muscle cells, elevation of intracellular calcium alone was not sufficient to promote myofibroblast contraction. These results suggest that Ca(2+)-mediated activation of MLCK, while necessary, is not sufficient to promote myofibroblast contraction. The specific Rho inactivator C3-transferase and the Rho kinase inhibitor Y-27632 inhibited LPA-promoted myofibroblast contraction, suggesting that contraction depends on activation of the Rho/Rho kinase pathway. Calyculin, a type 1 phosphatase inhibitor known to inhibit MLCPPase, could promote myofibroblast contraction in the absence of LPA, as well as restore contraction in the presence of C3-transferase or Y-27632. Together these results support a model whereby Rho/Rho kinase-mediated inhibition of MLCPPase is necessary for LPA-promoted myofibroblast contraction, in contrast to smooth muscle cells in which Ca(2+) activation of MLCK alone is sufficient to promote contraction., (Copyright 2000 Academic Press.)

Cellular adhesions to other cells and to the extracellular matrix play crucial roles in the malignant progression of cancer. In this study, we investigated the role of protein kinase C (PKC) in the regulation of cell-substratum adhesion by the breast adenocarcinoma cell line MCF-7. A PKC activator, 12-O-tetradecanoylphorbol-l, 3-acetate (TPA), stimulated cell adhesion to laminin and collagen I in a dose-dependent manner over a 1- to 4-h interval. This enhanced adhesion was mediated by alpha2beta1 integrin, since both anti-alpha2 and anti-beta1 blocking antibodies each completely abrogated the TPA-induced adhesion. FACS analysis determined that TPA treatment does not change the cell surface expression of alpha2beta1 integrin over a 4-h time interval. However, alpha2beta1 levels were increased after 24 h of TPA treatment. Thus, the enhanced avidity of alpha2beta1-dependent cellular adhesion preceded the induction of alpha2beta1 cell surface expression. Northern blot analysis revealed that mRNA levels of both alpha2 and beta1 subunits were increased after exposure to TPA for 4 h, indicating that the induction of alpha2beta1 mRNA preceded that of its cell surface expression. This further suggested that the TPA-induced avidity of alpha2beta1 was independent of increased expression of alpha2beta1. Pretreatment of cells with the PKC inhibitor calphostin C partially antagonized the TPA-induced increase in expression of alpha2beta1 integrin expression and of alpha2beta1-mediated cellular adhesion. To identify a possible mechanism by which TPA could be acting to promote the rapid induction of alpha2beta1 adhesion, we treated the cells with the Rho-GTPase inhibitor Clostridium botulinumexotoxin C3. C3 inhibited TPA-induced adhesion to laminin and collagen I in a dose-dependant manner, suggesting a likely role for Rho in TPA-induced adhesion. Together, these results suggest that PKC can modulate the alpha2beta1-dependent adhesion of MCF-7 cells by two distinct mechanisms: altering the gene expression of integrins alpha2 and beta1 and altering the avidity of the alpha2beta1 integrin by a Rho-dependant mechanism., (Copyright 1999 Academic Press.)

In a previous study, we found that a specific inhibitor of cellular arginine-specific mono(ADP-ribosyl) transferase, meta-iodobenzylguanidine (MIBG), reversibly inhibited both proliferation and differentiation of cultured embryonic chick primary muscle myoblasts. In addition, we observed that arginine-specific ADP-ribosyltransferase activity increased with muscle-cell differentiation in cultures. Therefore, muscle-cell cultures, especially the 96-h myotube cultures that contain the highest levels of ADP-ribosyltransferase, were used as a working system to determine the cellular protein substrates for arginine-specific ADP-ribosyltransferase. When membrane fractions extracted from 96-h chick myotubes were incubated with [32P]NAD at 30 degrees C for 30 min, only a few proteins were labeled. The labeling of two proteins of 36 and 56 kDa was inhibited by the presence of an arginine-specific mono(ADP-ribosyl) transferase inhibitor, MIBG, and by novobiocin. To prove that these proteins are indeed the targets for arginine-specific mono(ADP-ribosyl)ation, active recombinant muscle ADP-ribosyltransferase was incubated with membrane proteins under the same conditions. ADP-ribosylation of these two membrane proteins, as seen in the endogenous reactions, was also catalyzed by the added muscle transferase and was also inhibited by MIBG and novobiocin. By using antibody specific for desmin for immunoprecipitation and immunoblot analysis, we found that a 56-kDa protein associated with the membrane of myotubes is desmin. Our results showed that incorporation of isotope into this protein band from [32P]NAD is due to ADP-ribosylation of desmin.