ADP-ribosyltransferase Activity Research Articles

An ELISA method to estimate the mono ADP-ribosyltransferase activities: e.g in pertussis toxin and vaccines

ADP-ribosyltransferase activities have been observed in many prokaryotic and eukaryotic species and viruses and are involved in many cellular processes, including cell signalling, DNA repair, gene regulation and apoptosis. In a number of bacterial toxins, mono ADP-ribosyltransferase is the main cause of host cell cytotoxicity. Several approaches have been used to analyse this biological system from measuring its enzyme products to its functions. By using a mono ADP-ribose binding protein we have now developed an ELISA method to estimate native pertussis toxin mono ADP-ribosyltransferase activity and its residual activities in pertussis vaccines as an example. This new approach is easy to perform and adaptable in most laboratories. In theory, this assay system is also very versatile and could measure the enzyme activity in other bacteria such as Cholera, Clostridium, E. coli, Diphtheria, Pertussis, Pseudomonas, Salmonella and Staphylococcus by just switching to their respective peptide substrates. Furthermore, this mono ADP-ribose binding protein could also be used for staining mono ADP-ribosyl products resolved on gels or membranes.

Long noncoding RNA and mRNA profiling in MDA-MB-231 cells following RNAi-mediated knockdown of SIRT7.

Breast cancer is one of the most common malignant cancers among women and a major clinical obstacle. Although studies have reported the abnormal expression of SIRT7 in breast cancer, whether the function of SIRT7 regulates the expression of long noncoding RNAs (lncRNAs) in breast cancer remains unknown. We aimed to determine the differential expressions of mRNAs and lncRNAs associated with SIRT7 and understand the regulatory mechanism of SIRT7 in breast cancer. RNA sequencing was performed to explore the transcriptome in MDA-MB-231 cells after SIRT7 depletion, and a total of 50,634 different transcripts were identified. In comparison with the negative control, siSIRT7 groups showed 240 differentially expressed mRNAs and 26 differentially expressed lncRNAs. Gene ontology analysis revealed that the differentially expressed mRNAs mainly regulated DNA replication, CXCR chemokine receptor binding, and maturation of large subunit rRNA from tricistronic rRNA transcript, nucleoplasm, mitochondrion, and NAD+ ADP-ribosyltransferase activity. Kyoto Encyclopedia of Genes and Genomes analysis showed that the differentially expressed mRNAs were mainly involved in pathways associated with MAPK signaling pathway, tumor necrosis factor signaling pathway, hepatitis B, and cancer. Moreover, the target genes of the differentially expressed lncRNAs mainly regulated the carboxylic acid metabolic processes and were involved in glycolysis pathway. The mRNA-lncRNA coexpression network comprised 186 mRNAs and 23 lncRNAs. Our results provide essential data regarding differentially expressed lncRNAs and mRNAs after the depletion of SIRT7 in breast cancer cells, which may be useful to elucidate the role of SIRT7 in breast cancer development.

Abstract 820: Potential pro-proliferative role of SIRT6 in melanoma

Abstract Melanoma is one of the deadliest form of skin cancer that can rapidly metastasize to become lethal, if not diagnosed early or left untreated. In 2016, approximately 76,380 new melanoma cases and 10,130 melanoma-related deaths are predicted in the United States. Current preventive and therapeutic strategies have not been sufficiently effective in the management of melanoma. Therefore, novel molecular targets and treatments are required for an effective management of this neoplasm. In our laboratory, we are assessing the role and functional and therapeutic significance of sirtuin family of proteins in melanoma. Sirtuins (SIRTs) have been conserved through evolution from prokaryotic to eukaryotic cells. SIRTs are nicotinamide adenine dinucleotide (NAD+) dependent protein deacetylases and belong to class III of the histone deacetylase (HDAC) family. Seven members of the mammalian SIRT family are known to date, and despite structural similarities, each SIRT has their own biological niche, performing unique functions via regulating critical mechanisms in the cell. The role of SIRTs in cancer is somewhat controversial, as they have exhibited conflicting functions (tumor promoter vs. tumor suppressor) depending on cell and tissue contexts. The sirtuin SIRT6, a predominantly nuclear protein, has been shown to conduct ADP-ribosyl transferase and histone deacetylase activities. SIRT6 plays key roles in DNA repair, inflammation and metabolic diseases such as cancer. Currently, the role of SIRT6 in melanoma is not known. The objective of this study was to determine the role of SIRT6 in melanoma. Using a panel of human melanoma cell lines (A375, Hs 294T, G361, SK-MEL-2, SK-MEL-28, SK-MEL-31, WM115 and WM35) differing in genetic complexity and disease progression stage, and normal human epidermal melanocytes (NHEMs), we determined the endogenous expression levels of SIRT6. We found that compared to NHEMs, SIRT6 is significantly upregulated in melanoma cell lines, at mRNA as well as protein levels, as shown by quantitative Real-Time PCR and western blot analyses. Further, employing a human tissue microarray (TMA) coupled with quantitative Vectra™ analysis, we determined the expression profile of SIRT6 protein in human melanoma and melanocytic nevus tissues. Our data demonstrated that SIRT6 is significantly overexpressed in human melanoma tissues when compared to nevi. Furthermore, lentiviral short hairpin RNA-mediated knockdown of SIRT6 in human melanoma cells was found to result in a marked anti-proliferative response in melanoma cells. Taken together, our data suggest that SIRT6 overexpression could potentially be a contributing factor in melanoma progression. Further detailed studies are underway to understand the functional significance of SIRT6 in melanoma development and progression. Citation Format: Liz Garcia-Peterson, Mary A. Ndiaye, Chandra K. Singh, Wei Huang, Nihal Ahmad. Potential pro-proliferative role of SIRT6 in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 820. doi:10.1158/1538-7445.AM2017-820

Bioengineered silkworms with butterfly cytotoxin-modified silk glands produce sericin cocoons with a utility for a new biomaterial

Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm (Bombyx mori) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly (Pieris rapae) cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in B. mori-derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential.

The mitochondrial SIR2 related protein 2 (SIR2RP2) impacts Leishmania donovani growth and infectivity.

BackgroundLeishmania donovani, a protozoan parasite is the major causative agent of visceral leishmaniasis. Increased toxicity and resistance to the existing repertoire of drugs has been reported. Hence, an urgent need exists for identifying newer drugs and drug targets. Previous reports have shown sirtuins (Silent Information Regulator) from kinetoplastids as promising drug targets. Leishmania species code for three SIR2 (Silent Information Regulator) related proteins. Here, we for the first time report the functional characterization of SIR2 related protein 2 (SIR2RP2) of L. donovani.MethodologyRecombinant L. donovani SIR2RP2 was expressed in E. coli and purified. The enzymatic functions of SIR2RP2 were determined. The subcellular localization of LdSIR2RP2 was done by constructing C-terminal GFP-tagged full-length LdSIR2RP2. Deletion mutants of LdSIR2RP2 were generated in Leishmania by double targeted gene replacement methodology. These null mutants were tested for their proliferation, virulence, cell cycle defects, mitochondrial functioning and sensitivity to known SIR2 inhibitors.ConclusionOur data suggests that LdSIR2RP2 possesses NAD+-dependent ADP-ribosyltransferase activity. However, NAD+-dependent deacetylase and desuccinylase activities were not detected. The protein localises to the mitochondrion of the promastigotes. Gene deletion studies showed that ΔLdSIR2RP2 null mutants had restrictive growth phenotype associated with accumulation of cells in the G2/M phase and compromised mitochondrial functioning. The null mutants had attenuated infectivity. Deletion of LdSIR2RP2 resulted in increased sensitivity of the parasites to the known SIR2 inhibitors. The sirtuin inhibitors inhibited the ADP-ribosyltransferase activity of recombinant LdSIR2RP2. In conclusion, sirtuins could be used as potential new drug targets for visceral leishmaniasis.

SIRT6 inhibits TNF-α-induced inflammation of vascular adventitial fibroblasts through ROS and Akt signaling pathway

SIRT6, with both deacetylase and ADP-ribosyltransferase activities, is predominantly expressed in the nucleus. It has been revealed that SIRT6 regulates various biological functions including metabolism, aging and stress resistance. This study aims to investigate the role of SIRT6 in vascular inflammation and it molecular mechanism. We found that tumor necrosis factor-α (TNF-α) did not alter the localization of SIRT6 in vascular adventitial fibroblasts (VAFs), vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs). The expression of SIRT1, SIRT6 was decreased in TNF-α-treated VAFs. In contrast, TNF-α significantly increased the expression of monocyte chemotactic protein 1 (MCP-1) and interleukin (IL) −6. Knockdown of SIRT1 and SIRT6 by siRNA significantly enhanced TNF-α-induced expression of MCP-1 and IL-6, respectively. Overexpression of SIRT1 and SIRT6 inhibited TNF-α-induced expression of MCP-1 and IL-6 in VAFs. Moreover, we also found SIRT1 positively regulated the expression of SIRT6 in VAFs. In addition, knockdown of SIRT1 and SIRT6 respectively augmented TNF-α-induced generation of reactive oxygen species (ROS) and phosphorylation of protein kinase B (Akt). ROS scavenger N-acetyl-L-cysteine (NAC) and Akt inhibitor MK2206 reduced TNF-α-induced mRNA expression of MCP-1 and IL-6 in VAFs. In vivo studies indicated that the expression of SIRT1, SIRT6 was decreased and the expression of MCP-1, IL-6 and IL-1β was increased in carotid collar-induced vascular inflammation. Taken together, these findings indicate that SIRT1 and SIRT6 inhibit TNF-α-induced inflammation in VAFs by ROS and Akt pathway.

Pseudomonas aeruginosa Effector ExoS Inhibits ROS Production in Human Neutrophils

Neutrophils are the first line of defense against bacterial infections, and the generation of reactive oxygen species is a key part of their arsenal. Pathogens use detoxification systems to avoid the bactericidal effects of reactive oxygen species. Here we demonstrate that the Gram-negative pathogen Pseudomonas aeruginosa is susceptible to reactive oxygen species but actively blocks the reactive oxygen species burst using two type III secreted effector proteins, ExoS and ExoT. ExoS ADP-ribosylates Ras and prevents it from interacting with and activating phosphoinositol-3-kinase (PI3K), which is required to stimulate the phagocytic NADPH-oxidase that generates reactive oxygen species. ExoT also affects PI3K signaling via its ADP-ribosyltransferase activity but does not act directly on Ras. A non-ribosylatable version of Ras restores reactive oxygen species production and results in increased bacterial killing. These findings demonstrate that subversion of the host innate immune response requires ExoS-mediated ADP-ribosylation of Ras in neutrophils.

Catalytic-Independent Functions of PARP-1 Determine Sox2 Pioneer Activity at Intractable Genomic Loci

Pioneer transcription factors (TFs) function as genomic first responders, binding to inaccessible regions of chromatin to promote enhancer formation. The mechanism by which pioneer TFs gain access to chromatin remains an important unanswered question. Here we show that PARP-1, a nucleosome-binding protein, cooperates with intrinsic properties of the pioneer TF Sox2 to facilitate its binding to intractable genomic loci in embryonic stem cells. These actions of PARP-1 occur independently of its poly(ADP-ribosyl) transferase activity. PARP-1-dependent Sox2-binding sites reside in euchromatic regions of the genome with relatively high nucleosome occupancy and low co-occupancy by other transcription factors. PARP-1 stabilizes Sox2 binding to nucleosomes at suboptimal sites through cooperative interactions on DNA. Our results define intrinsic and extrinsic features that determine Sox2 pioneer activity. The conditional pioneer activity observed with Sox2 at a subset of binding sites may be a key feature of other pioneer TFs operating at intractable genomic loci.

Vibrio cholerae Cholix Toxin-Induced HepG2 Cell Death is Enhanced by Tumor Necrosis Factor-Alpha Through ROS and Intracellular Signal-Regulated Kinases.

Cholix toxin (Cholix) from Vibrio cholerae is a potent virulence factor exhibiting ADP-ribosyltransferase activity on eukaryotic elongation factor 2 (eEF2) of host cells, resulting in the inhibition of protein synthesis. Administration of Cholix or its homologue Pseudomonas exotoxin A (PEA) to mice causes lethal hepatocyte damage. In this study, we demonstrate cytotoxicity of Cholix on human hepatocytes in the presence of tumor necrosis factor α (TNF-α), which has been reported to play a fatal role in PEA administered to mice. Compared with incubating HepG2 cells with Cholix alone, co-treatment with TNF-α and Cholix (TNF-α/Cholix) significantly enhanced the activation of caspases, cytochrome c release from mitochondria into cytoplasm, and poly-ADP-ribose polymerase (PARP) cleavage, while incubation with TNF-α alone or co-treatment with TNF-α/catalytically inactive Cholix did not. In the early stage of cell death, Cholix increased phosphorylation of mitogen-activated protein kinases (e.g., p38, ERK, JNK) and Akt, which was not affected by TNF-α alone. MAPK inhibitors (SP600125, SB20852, and U0126) suppressed PARP cleavage induced by TNF-α/Cholix. Protein kinase inhibitor Go6976 suppressed JNK phosphorylation and PARP cleavage by TNF-α/Cholix. In contrast, PKC activator PMA in the absence of TNF-α promoted Cholix-induced PARP cleavage. Reactive oxygen species (ROS) inhibitor, N-acetyl cysteine (NAC), suppressed TNF-α/Cholix-induced JNK and ERK phosphorylation, resulting in inhibition of PARP cleavage. These data suggest that ROS and JNK pathways are important mediators of TNF-α/Cholix-induced HepG2 cell death.

Development of an in vitro Biochemical Assay System for the Measurement of Residual Toxin Activities in Pertussis Toxin Containing Vaccines

Pertussis (whooping cough) vaccines play an essential role in immunisation programmes worldwide and the histamine sensitisation test (HIST) is currently the official batch-release test for acellular pertussis containing combination vaccines. HIST, being a murine animal test, has large assay variability problems and ethical issues. Therefore, in response to the 3Rs, a biochemical assay system has been developed based on the pertussis toxin (PTx)’s two functional domains: an enzyme active A-protomer and a carbohydrate binding B-oligomer. The clinical relevance of these two biochemical tests in relation to the PTx toxic effects has been demonstrated. In order to assess the practicality of this assay system, an international collaborative study involving 9 countries on method transferability had also been performed and showed that this assay system could be adopted readily in control laboratories and vaccine manufacturing facilities. In this update, we provide further optimised assay conditions for measuring both the residual PTx ADP-ribosyltransferase and carbohydrate binding activities in specific vaccines. In general, appropriate dilution of vaccines would help to increase test sensitivity. Spiking experiments showed carbohydrate binding assay could detect low levels of PTx similar to HIST. Although positive trend with increasing spikes could also be demonstrated in ADP-ribosyltransferase activity assay for some vaccines, the resultant values in a number of spiked vaccines were not statistically significant and this is due to high baseline activity of the unspiked vaccines. Since all the reagents of this biochemical assay system are available worldwide, readily controlled and with good test consistency, both assays could be used for monitoring product consistency. The carbohydrate binding assay could even be considered as a potential alternative to HIST for batch-release of acellular pertussis vaccines. Protocols for this in vitro assay system are supplied as Supplement Documents 1 & 2.

Intracellular trafficking of diphtheria toxin and its mutated form, CRM197, in the endocytic pathway.

OBJECTIVE:Diphtheria toxin (DTx) is a well-characterized bacterial toxin. However, the endocytic pathway of the mutant of DTx, CRM197, which is used as an immunological adjuvant, has not yet been fully explained. The aim of this study was to investigate the intracellular trafficking of CRM197-loaded endosomes.METHODS:Human umbilical vein endothelial cells (HUVECs) were used in a cell culture. The effective incubation time was determined by transmission electron microscopy in toxin-treated cells. Density gradient centrifugation and ADP-ribosylation assay were used to isolate and detect toxin-loaded endosomal fractions. Endosomal fractions from CRM197-treated cells were elicited after 15 minutes of incubation and the presence of fragment A was demonstrated using Western blot. Immunofluorescence microscopy was used to identify endosomes in CRM197-treated endothelial cells.RESULTS:DTx-loaded endosomes were detected as enlarged vesicles in the perinuclear area with 15 minutes of toxin treatment. DTx-loaded endosomal fractions were determined by ADP-ribosyltransferase activity test and Western blot analysis. Enzymatic activity of the toxin-loaded endosomal fraction increased by 20% in actin cytoskeletal-damaged cells treated with cytochalasin D. The steps for the toxin treatment of HUVECs with DTx and obtaining endosomal fractions were repeated for CRM197. In the CRM197-loaded endosomal fraction, actin and Hsp90 were identified in addition to fragment A. Fluorescent images revealed that CRM197-loaded endosomes were co-localized with actin filaments and that Rab11, which signals the return to the plasma membrane, was more prominent than Rab7, the lysosomal pathway indicator.CONCLUSION:These results suggest that CRM197-loaded endosomes participate in the recycling pathway.

Crystal structure of the ADP-ribosylating component of BEC, the binary enterotoxin of Clostridium perfringens

Binary enterotoxin of Clostridium perfringens (BEC), consisting of the components BECa and BECb, was recently identified as a novel enterotoxin produced by C. perfringens that causes acute gastroenteritis in humans. Although the detailed mechanism of cell intoxication by BEC remains to be defined, BECa shows both NAD+-glycohydrolase and actin ADP-ribosyltransferase activities in the presence of NAD+. In this study, we determined the first crystal structure of BECa in its apo-state and in complex with NADH. The structure of BECa shows striking resemblance with other binary actin ADP-ribosylating toxins (ADPRTs), especially in terms of its overall protein fold and mechanisms of substrate recognition. We present a detailed picture of interactions between BECa and NADH, including bound water molecules located near the C1′-N glycosidic bond of NADH and the catalytically important ADP-ribosylating turn-turn (ARTT) loop. We observed that the conformational rearrangement of the ARTT loop, possibly triggered by a conformational change involving a conserved tyrosine residue coupled with substrate binding, plays a crucial role in catalysis by properly positioning a catalytic glutamate residue in the E-X-E motif of the ARTT loop in contact with the nucleophile. Our results for BECa provide insight into the common catalytic mechanism of the family of binary actin ADPRTs.

Functional significance of active site residues in the enzymatic component of the Clostridium difficile binary toxin

Clostridium difficile binary toxin (CDT) is an ADP-ribosyltransferase which is linked to enhanced pathogenesis of C. difficile strains. CDT has dual function: domain a (CDTa) catalyses the ADP-ribosylation of actin (enzymatic component), whereas domain b (CDTb) transports CDTa into the cytosol (transport component). Understanding the molecular mechanism of CDT is necessary to assess its role in C. difficile infection. Identifying amino acids that are essential to CDTa function may aid drug inhibitor design to control the severity of C. difficile infections. Here we report mutations of key catalytic residues within CDTa and their effect on CDT cytotoxicity. Rather than an all-or-nothing response, activity of CDTa mutants vary with the type of amino acid substitution; S345A retains cytotoxicity whereas S345Y was sufficient to render CDT non-cytotoxic. Thus CDTa cytotoxicity levels are directly linked to ADP-ribosyltransferase activity.

The NarE protein of Neisseria gonorrhoeae catalyzes ADP-ribosylation of several ADP-ribose acceptors despite an N-terminal deletion.

The ADP-ribosylating enzymes are encoded in many pathogenic bacteria in order to affect essential functions of the host. In this study, we show that Neisseria gonorrhoeae possess a locus that corresponds to the ADP-ribosyltransferase NarE, a previously characterized enzyme in N. meningitidis The 291 bp coding sequence of gonococcal narE shares 100% identity with part of the coding sequence of the meningococcal narE gene due to a frameshift previously described, thus leading to a49-amino-acid deletion at the N-terminus of gonococcal NarE protein. However, we found a promoter region and a GTG start codon, which allowed expression of the protein as demonstrated by RT-PCR and western blot analyses. Using a gonococcal NarE-6xHis fusion protein, we demonstrated that the gonococcal enzyme underwent auto-ADP-ribosylation but to a lower extent than meningococcal NarE. We also observed that gonoccocal NarE exhibited ADP-ribosyltransferase activity using agmatine and cell-free host proteins as ADP-ribose acceptors, but its activity was inhibited by human β-defensins. Taken together, our results showed that NarE of Neisseria gonorrhoeae is a functional enzyme that possesses key features of bacterial ADP-ribosylating enzymes.

(1)H(N), (13)C, and (15)N resonance assignments of the CDTb-interacting domain (CDTaBID) from the Clostridium difficile binary toxin catalytic component (CDTa, residues 1-221).

Once considered a relatively harmless bacterium, Clostridium difficile has become a major concern for healthcare facilities, now the most commonly reported hospital-acquired pathogen. C. difficile infection (CDI) is usually contracted when the normal gut microbiome is compromised by antibiotic therapy, allowing the opportunistic pathogen to grow and produce its toxins. The severity of infection ranges from watery diarrhea and abdominal cramping to pseudomembranous colitis, sepsis, or death. The past decade has seen a marked increase in the frequency and severity of CDI among industrialized nations owing directly to the emergence of a highly virulent C. difficile strain, NAP1. Along with the large Clostridial toxins expressed by non-epidemic strains, C. difficile NAP1 produces a binary toxin, C. difficile transferase (CDT). As the name suggests, CDT is a two-component toxin comprised of an ADP-ribosyltransferase (ART) component (CDTa) and a cell-binding/translocation component (CDTb) that function to destabilize the host cytoskeleton by covalent modification of actin monomers. Central to the mechanism of binary toxin-induced pathogenicity is the formation of CDTa/CDTb complexes at the cell surface. From the perspective of CDTa, this interaction is mediated by the N-terminal domain (residues 1-215) and is spatially and functionally independent of ART activity, which is located in the C-terminal domain (residues 216-420). Here we report the (1)H(N), (13)C, and (15)N backbone resonance assignments of a 221 amino acid,~26kDaN-terminal CDTb-interacting domain (CDTaBID) construct by heteronuclear NMR spectroscopy. These NMR assignments represent the first component coordination domain for a family of Clostridium or Bacillus species harboring ART activity. Our assignments lay the foundation for detailed solution state characterization of structure-function relationships, toxin complex formation, and NMR-based drug discovery efforts.

A mutated cholera toxin without the ADP-ribosyltransferase activity induces cytokine production and inhibits apoptosis of splenocytes in mice possibly via toll-like receptor-4 signaling.

Native cholera toxin (CT) and its mutated form (CT-2*) without ADP-ribosyltransferase activity differ in their immunomodulatory effects on host cells, and the mechanisms of these differences are poorly understood. In this study, we demonstrated that CT-2* induced higher levels of cytokine production and down-regulated ex-vivo apoptosis of splenocytes from C57BL/6 mice. After exposure of the splenocytes ex-vivo to CT or CT-2* (2μg/ml) for 48h, CT-2* stimulated expression of the toll-like receptor (TLR-4) gene was much higher and the cells produced increased levels of interleukin (IL)-12, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, compared to splenocytes of mice exposed to native CT. We confirmed these findings by observing that CT-2*, induced much lower levels of IL-12, IFN-γ, and TNF-α in a TLR-4 knockout macrophage cell line derived from C57BL/6 mice. In addition, while CT is known to stimulate apoptosis in splenocytes, we observed that CT-2* significantly down-regulated apoptosis (4.2%), compared to splenocytes exposed to CT (18.7%) or PBS (negative control, 8.5%). On the contrary, we noted both native CT and CT-2* to exhibit similar levels of apoptosis in TLR-4(-/-) cell line. Overall, the evidence supports the conclusion that CT-2* modulated cytokine production and apoptosis in splenocytes of mice possibly through the TLR-4 signaling pathway.

Dynamic Duo—The Salmonella Cytolethal Distending Toxin Combines ADP-Ribosyltransferase and Nuclease Activities in a Novel Form of the Cytolethal Distending Toxin

The cytolethal distending toxin (CDT) is a well characterized bacterial genotoxin encoded by several Gram-negative bacteria, including Salmonella enterica (S. enterica). The CDT produced by Salmonella (S-CDT) differs from the CDT produced by other bacteria, as it utilizes subunits with homology to the pertussis and subtilase toxins, in place of the traditional CdtA and CdtC subunits. Previously, S-CDT was thought to be a unique virulence factor of S. enterica subspecies enterica serotype Typhi, lending to its classification as the “typhoid toxin.” Recently, this important virulence factor has been identified and characterized in multiple nontyphoidal Salmonella (NTS) serotypes as well. The significance of S-CDT in salmonellosis with regards to the: (i) distribution of S-CDT encoding genes among NTS serotypes, (ii) contributions to pathogenicity, (iii) regulation of S-CDT expression, and (iv) the public health implication of S-CDT as it relates to disease severity, are reviewed here.

Effect of different detoxification procedures on the residual pertussis toxin activities in vaccines

Pertussis toxin (PTx) is a major virulence factor produced by Bordetella pertussis and its detoxified form is one of the major protective antigens in vaccines against whooping cough. Ideally, PTx in the vaccine should be completely detoxified while still preserving immunogenicity. However, this may not always be the case. Due to multilevel reaction mechanisms of chemical detoxification that act on different molecular sites and with different production processes, it is difficult to define a molecular characteristic of a pertussis toxoid. PTx has two functional distinctive domains: the ADP-ribosyltransferase enzymatic subunit S1 (A-protomer) and the host cell binding carbohydrate-binding subunits S2–5 (B-oligomer); and in this study, we investigated the effect of different detoxification processes on these two functional activities of the residual PTx in toxoids and vaccines currently marketed worldwide using a recently developed in vitro biochemical assay system. The patho-physiological activities in these samples were also estimated using the in vivo official histamine sensitisation tests. Different types of vaccines, detoxified by formaldehyde, glutaraldehyde or by both, have different residual functional and individual baseline activities. Of the vaccines tested, PT toxoid detoxified by formaldehyde had the lowest residual PTx ADP-ribosyltransferase activity. The carbohydrate binding results detected by anti-PTx polyclonal (pAb) and anti-PTx subunits monoclonal antibodies (mAb) showed specific binding profiles for toxoids and vaccines produced from different detoxification methods. In addition, we also demonstrated that using pAb or mAb S2/3 as detection antibodies would give a better differential difference between these vaccine lots than using mAbs S1 or S4. In summary, we showed for the first time that by measuring the activities of the two functional domains of PTx, we could characterise pertussis toxoids prepared from different chemical detoxification methods and this study also highlights the potential use of this in vitro biochemical assay system for in-process control.

Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria.

Entomopathogenic bacteria produce insecticidal proteins that accumulate in inclusion bodies or parasporal crystals (such as the Cry and Cyt proteins) as well as insecticidal proteins that are secreted into the culture medium. Among the latter are the Vip proteins, which are divided into four families according to their amino acid identity. The Vip1 and Vip2 proteins act as binary toxins and are toxic to some members of the Coleoptera and Hemiptera. The Vip1 component is thought to bind to receptors in the membrane of the insect midgut, and the Vip2 component enters the cell, where it displays its ADP-ribosyltransferase activity against actin, preventing microfilament formation. Vip3 has no sequence similarity to Vip1 or Vip2 and is toxic to a wide variety of members of the Lepidoptera. Its mode of action has been shown to resemble that of the Cry proteins in terms of proteolytic activation, binding to the midgut epithelial membrane, and pore formation, although Vip3A proteins do not share binding sites with Cry proteins. The latter property makes them good candidates to be combined with Cry proteins in transgenic plants (Bacillus thuringiensis-treated crops [Bt crops]) to prevent or delay insect resistance and to broaden the insecticidal spectrum. There are commercially grown varieties of Bt cotton and Bt maize that express the Vip3Aa protein in combination with Cry proteins. For the most recently reported Vip4 family, no target insects have been found yet.

Rin1 restores host phagocytic activity during invasion by Pseudomonas aeruginosa.

Pseudomonas aeruginosa uses a type III secretion system to deliver toxic effector proteins directly into host cells and alter host protein functions. Exoenzyme S (ExoS), a type III effector protein, ADP-ribosylates Rab5 GTPase and impairs early phagocytic events in macrophage cells. In this study, we tested the hypothesis that Rin1, a Ras effector protein and Rab5 guanine nucleotide exchange factor, generates an intrinsic Rab5 activity cycle during phagocytosis of live P. aeruginosa; thus, allowing proper phagocytic killing. We found that Rab5 activity was attenuated at a very early time point (2.5 min) of the phagocytic process of live but not of heat-inactivated P. aeruginosa. However, upon overexpressing Rin1 in macrophages, the Rab5 activity sustained for a prolonged time (∼20 min) counteracting the negative effects during phagocytosis of live P. aeruginosa. Ras, also a substrate of the ADP-ribosyltransferase activity of ExoS, remained active during the early events of phagocytosis of live as well as heat-inactivated P. aeruginosa. Further examinations revealed that the Rin1 : Vps9 domain (the Rab5 nucleotide catalytic domain) and the Rin1 : RA domain (the Ras association domain of Rin1) are both required for optimal Rin1 function. Finally, the time-based analysis of the ADP-ribosylation status of Rab5 and Ras obtained from this study was consistent in the context of the regulation of (i) Rab5 activity by Rin1 : Vps9 domain and (ii) Ras interaction with Rin1 via the Rin1 : RA domain. These observations highlight a novel crosstalk between Rin1-Rab5 and Rin1-Ras complexes that offsets the anti-phagocytic effects of ExoS in macrophages.