One of the intensively studied immunotoxins is a fu- sion protein composed of an antibody fragment, such as variable fragment (Fv), single-chain Fv (scFv), or antigen binding fragment (Fab), and an engineered Pseudomonasexotoxin A (PE) [3, 9]. After endocytosis via interaction with a ubiquitous cell-surface receptor (CD91), the toxin translocates into the cytoplasm and then ADP ribosy- lates eukaryotic elongation factor-2 (eEF-2) [10, 11]. The modified eEF-2 displays defects in GDP-GTP exchange, GFP hydrolysis, and binding to pre-translocation ribo- some, which results in inhibition of the protein synthesis and finally cellular death . Immunotoxins have been developed by replacing the domain of PE interacting with CD91 with antibody fragments. For example, an anti-CD22 immunotoxin recombinant designated moxe- tumomab pasudotox has recently been approved for the treatment of hairy cell leukemia . To improve the ef- ficacy and the toxicity of PE, portions of the toxin that were not necessary for the translocation and the modifi- cation of eEF-2 were deleted, resulting in PE24, which showed much lower off-target toxicity than the original , and its immunogenicity was reduced by removing T- and B-cell epitopes [15, 16].
Background: Antibodies raised against selected antigens over-expressed at the cell surface of malignant cells have been chemically conjugated to protein toxin domains to obtain immunotoxins (ITs) able to selectively kill cancer cells. Since latest generation immunotoxins are composed of a toxic domain genetically fused to antibody fragment(s) which confer on the IT target selective specificity, we rescued from the hydridoma 4KB128, a recombinant single-chain variable fragment (scFv) targeting CD22, a marker antigen expressed by B-lineage leukaemias and lymphomas. We constructed several ITs using two enzymatic toxins both able to block protein translation, one of bacterial origin (a truncated version of Pseudomonasexotoxin A, PE40) endowed with EF-2 ADP-ribosylation activity, the other being the plant ribosome-inactivating protein saporin, able to specifically depurinate 23/26/28S ribosomal RNA. PE40 was selected because it has been widely used for the construction of recombinant ITs that have already undergone evaluation in clinical trials. Saporin has also been evaluated clinically and has recently been expressed successfully at high levels in a Pichia pastoris expression system. The aim of the present study was to evaluate optimal microbial expression of various IT formats.
ABSTRACT Anthrax toxin proteins from Bacillus anthracis constitute a highly efficient system for delivering cytotoxic enzymes to the cytosol of tumor cells. However, exogenous proteins delivered to the cytosol of cells are subject to ubiquitination on lysines and proteasomal degradation, which limit their potency. We created fusion proteins containing modified ubiquitins with their C-terminal regions fused to the Pseudomonasexotoxin A catalytic domain (PEIII) in order to achieve delivery and release of PEIII to the cytosol. Fusion proteins in which all seven lysines of wild-type ubiquitin were retained while the site cleaved by cyto- solic deubiquitinating enzymes (DUBs) was removed were nontoxic, apparently due to rapid ubiquitination and proteasomal degradation. Fusion proteins in which all lysines of wild-type ubiquitin were substituted by arginine had high potency, exceed- ing that of a simple fusion lacking ubiquitin. This variant was less toxic to nontumor tissues in mice than the fusion protein lack- ing ubiquitin and was very efficient for tumor treatment in mice. The potency of these proteins was highly dependent on the number of lysines retained in the ubiquitin domain and on retention of the C-terminal ubiquitin sequence cleaved by DUBs. It appears that rapid cytosolic release of a cytotoxic enzyme (e.g., PEIII) that is itself resistant to ubiquitination is an effective strat- egy for enhancing the potency of tumor-targeting toxins.
Two immunotoxins were constructed by chemically coupling the monoclonal antibody C242 to Pseudomonasexotoxin A (PE) or a modified form, NlysPE40, that lacks the cell binding domain of PE. Monoclonal antibody C242 recognizes a specific sialylated carbohydrate epitope on a high molecular weight membrane glycoprotein present on cells of human colon, pancreatic, and cervical cancers. C242-PE and C242-NlysPE40 were very cytotoxic for cells expressing this antigen with 50% inhibition of protein synthesis occurring on
The CD4 molecule is a high affinity receptor for the human immunodeficiency virus (HIV) envelope glycoprotein (gp160 or gp120). This glycoprotein is expressed on the surface membrane of cells infected with HIV. It has, therefore, been suggested that a soluble form of CD4 might be used as a targeting agent to deliver toxins selectively to cells infected with HIV. We demonstrate that CD4-Pseudomonasexotoxin A (PE) conjugates inhibit the proliferation of gp160-transfected Chinese hamster ovary cells and block HIV replication in virus-infected H9 cells. However, this inhibition of HIV replication appears to be incomplete since virus replication occurs following removal of the toxin conjugates from these cultures. Moreover, CD4-PE conjugates delay but do not inhibit HIV replication in human peripheral blood lymphocytes. These studies suggest that such conjugates should be assessed only as potential adjunctive therapies in the acquired immunodeficiency syndrome.
Cholera toxin [9,10], Shiga and the very closely related Shiga-like toxins (STx family) , Pseudomonasexotoxin A (PEx)  and the plant toxin ricin  seem unable to disrupt cellular membranes directly. After binding their respective receptors at the cell surface, all travel from the cell surface to the endoplasmic reticulum (ER) [14-17], presumably to take advantage of a pre-existing cytosolic entry mechanism. The toxic portions of all these ER-traf- ficking toxins have unusually low lysine contents so they should be poor substrates for ubiquitination and subse- quent proteasomal degradation in the cytosol. Recogni- tion of this led to the proposal that these toxic subunits somehow subvert the ERAD (ER-associated protein degra- dation) pathway , which is the process by which ter-
ABSTRACT The Pseudomonas quinolone signal (PQS) is an important quorum- sensing molecule in Pseudomonas aeruginosa that also mediates its own packaging and transport by stimulating outer membrane vesicle (OMV) formation. Because OMVs have been implicated in many virulence-associated behaviors, it is critical that we under- stand how they are formed. Our group proposed the bilayer-couple model for OMV bio- genesis, where PQS intercalates into the outer membrane, causing expansion of the outer leaﬂet and consequently inducing curvature. In accordance with the model, we hypothesized that PQS must be transported from the cytoplasm to the outer membrane before it can initiate OMV formation. We initially examined two laboratory strains of P. aeruginosa and found signiﬁcant strain-dependent differences. PQS export correlated strongly with OMV production, even though equivalent amounts of total PQS were pro- duced by both strains. Interestingly, we discovered that poor OMV producers seques- tered the majority of PQS in the inner membrane, which appeared to be the result of early saturation of the export pathway. Further analysis showed that strain-speciﬁc PQS export and OMV biogenesis patterns were stable once established but could be signiﬁ- cantly altered by changing the growth medium. Finally, we demonstrated that the asso- ciations described for laboratory strains also held for three clinical strains. These results suggest that factors controlling the export of PQS dictate OMV biogenesis. This work provides new insight into PQS-controlled virulence in P. aeruginosa and provides impor- tant tools to further study signal export and OMV biogenesis.
infections. Their ability to adapt to different conditions and presence of pool of virulence factors may render their infections delay in healing. During a period of six months 114 wound swabs were collected and inoculated on Pseudomonas chromogenic agar and then Pseudomonas aeruginosa isolated confirmed by PCR using specific primer for 16S rDNA gene of Pseudomonas aeruginosa. Molecular investigation of some virulence factor like ExoA, OprL, OprI, LasI and LasB were performed using a sets of specific primer pairs. The results revealed that only 26 (22.8%) isolates were Pseudomonas aeruginosa and the coexistence of more than one virulence factors within the same isolates was also recorder. OprI and LasB were most common followed by LasI, ExoA and OprL. Occurrence of virulence factor genes were 12(46.15%) for exoA, oprL was 11(42.3%), oprI was 22(84.61%), lasI was 14(53.84%) and lasB was 18(69.23%). Results of this study can lead us to conclude that P. aeruginosa have an arrays of virulence traits via which can adapt to different conditions and so cause a wide-ranging of hard to cured infections and the delay in healing and worseness degree may be attributed to owning multivirulence factors.
that inhabits many environments, including plants, soil, and water surfaces. Pseudomonas is considered to be the most potential group of plant growth promoting rhizobacteria. (Gardner et al., 1984; Moeinzadeh et al., 2010). Fluorescent Pseudomonads are effective biocontrol agents against plant diseases (Howell and Stipanovic 1980; Kloepper et al. 1980; Scher and Baker 1982; Weller and Cook 1983).
The response of canine duodenal loops to challenge by cholera exotoxin differs from responses of jejunal and ileal loops in a) absence of a detectable “lag period” between administration of exotoxin and initiation of net fluid output; b) a longer period of fluid production following exotoxin administration; and c) a significantly greater net fluid output per unit length of gut. The mean bicarbonate concentration of the fluid produced by
Sequence analysis of the 16S-23S rDNA ITS. A multiple nucleotide sequence alignment of the 16S-23S rDNA ITSs of seven Pseudomonas spp. is presented in Fig. 1A, and the se- quence of the X. maltophilia ITS is shown in Fig. 1B. The ITS homologies between species and among sequevars are summa- rized in Table 3 and clearly indicate that the spacers can be divided into two basic homology groups, one consisting of P. aeruginosa and P. mendocina and the other comprising P. ce- pacia, P. gladioli, P. mallei, P. pickettii, and P. pseudomallei, although P. pickettii is not as closely related to these species as they are to each other. The single, 536-bp 16S-23S ITS of X. maltophilia showed , 50% sequence homology with any of the spacers from the other species and stands alone.
To date the pyocins produced by Pseudomonas aeruginosa are among the best-known bacteriocins in Gram-negative bacteria. Four different types of bacteriocins termed: R- F- S- and M type pyocins have been identified , and differ in their morphology and mode of killing. The smaller: S-type pyocin is a colicin-like protein, soluble and protease- and heat- sensitive. R- and F-type are high-molecular-mass pyocins, resemble bacteriophage tails . They contain no head structures and no DNA, and are used as defence systems . Whereas the R- type pyocin resembles a contractile but non- flexible tail structure of bacteriophage , and
Our results indicated that the inhibition of 1,4 β Exoglucanases, 1,4 β Endoglucanases, and Cellobiases was recorded in Th+Pf ( Trichoderma harzianum + Pseudomonas fluorescens ) treated culture. Our results substantiate earlier reports that in management of plant diseases the use of biocontrol agents in combinations were more effective than individual agents (Thilagavathi et al ., 2007; Ganeshmoorthi et al ., 2008). The application of T.harzianum , captan and neem seed extract two days after pathogen inoculation significantly reduced damping off disease caused by S. rolfsii in greenhouse grown tomato plants (Okereke and Wokocha 2006). Thangavelu and Gopi (2015) found that the combination of bacterial antagonists could provide sustainable management of Fusarium wilt of banana under field conditions. Our results also revealed that inhibition of 1,4 Exoglucanases 1,4 β Endoglucanases and Cellobiases was recorded in Tv+ Th ( Trichoderma viride + Trichoderma harzianum ) treated culture.
to grow in 3, 5 and 7% (W/V) NaCl. The phenotypic, biochemical and nutritional characteristics of the isolates are listed in Table 1. The isolates showed homogeneity in their physiological, biochemical and nutritional characteristics but some of the isolates exhibited variation in the production of levan. Based on morphological, physiological and biochemical characteristics, the isolates were identified as Pseudomonas savastanoi (28). The strains were phenotypically similar to the reference strains of Pseudomonas savastanoi pv. savastanoi (IVIA-1657.8 and IVIA- 1628.3) from olive, P. s. pv. savastanoi, and P. s. pv. nerii from olive and oleander respectively (11 and 12).
Methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is a leading cause of complicated skin and skin struc- ture infections (cSSSI) in the United States (4, 18, 23). In many regions of the United States, the genetically distinct commu- nity-associated MRSA (CA-MRSA) clone USA300 is now the predominant cause of cSSSI (13, 18, 25). Most CA-MRSA isolates isolated from cSSSI carry pvl, the gene encoding Pan- ton-Valentine leukocidin (PVL) (6, 19, 26). PVL is a pore- forming, bicomponent exotoxin known to induce cell death by necrosis or apoptosis (8). Infections caused by S. aureus strains carrying pvl are commonly thought to be associated with worse clinical outcome (6, 16). For example, the presence of the PVL toxin has been shown to cause necrotizing pneumonia in ani- mal models (14) and is associated with necrotizing S. aureus
Recently, the role of plant growth promoting rhizobacterial (PGPR) on plant growth promotion and stress is gaining importance. In present study, plant Pseudomonas fluorescence strain P2 and P16, , Bacillus cereus 5507(1B), Bacillus cereus re tested for their role in enhancing plant growth and induction of stress related Oryza sativa L.). Two PGPRs, Pseudomonas R81 were used as consortia. Most of the PGPR inoculated plants showed enhanced growth parameter as compared to uninoculated plants under drought stress. Quantitative analyses of stress related enzymes indicated that most of the plants inoculated with mutase (SOD), catalase (CAT) and peroxidase (POD) consortia of Pseudomonas jessenii R62 R81 treated plants showed better improvements in most of the growth well as stress related enzymatic activities. The greater induction of stress related enzymes in plants may be the mechanism through which these PGPRs help plants to tolerate the consequences of drought stress and maintenance of plant homeostasis under severe drought.
cancer cells may help generate targeted anti- breast cancer agents. Recently, various fusion proteins have been produced that are designed to target human breast cancers. For example, Hereg- ulin-Pseudomonasexotoxin, which the ligand Heregulin binds to ErbB-2, ErbB-3 and ErbB-4 receptors, is connected to a truncated form of Pseudomonasexotoxin (PE). This cytotoxin is highly cytotoxic in vitro and in vivo to breast can- cer cells that overexpress ErbB-4 or ErbB-2 plus ErbB-3 receptors (2). A recombinant epidermal growth factor (EGF) Genistein conjugate, in which EGF was conjugated to soybean-derived protein tyrosine kinase inhibitor, was targeted to an EGF-receptor and was found to be cytotoxic to the EGF-receptor positive breast cancer cells (3). A recombinant, humanized moncloclonal anti- Her2 antibody (Herceptin) was able to signifi- cantly inhibit growth of breast cancer in an ani- mal model and in the clinic (4). This antibody synergized with paclitaxel when mediating anti- tumor activity against breast tumor xenograft models. Herceptin was recently licensed by the U.S. Food and Drug Administration (FDA) for the treatment of breast cancer. These studies demonstrate that these classes of biotherapeutics can provide an additional mode of breast cancer therapy, although their clinical benefits have yet to be completely explored. It is possible that ad- ditional breast tumor-associated receptors or antigens will be identified that may provide new targets for breast cancer therapy.