Two Clostridiumperfringens Type E Isolates in France.

Clostridiumperfringens type E is a less frequently isolated C.perfringens type and has not previously been reported in France. We have characterized two recent type E isolates, C.perfringens 508.17 from the intestinal content of a calf that died of enterotoxemia, and 515.17 from the stool of a 60-year-old woman, subsequent to food poisoning, which contained the plasmid pCPPB-1 with variant iota toxin and C. perfringens enterotoxin genes.

C3 Transferase Gene Therapy for Continuous RhoA Inhibition.

The identification of RhoA inhibition as a therapeutic target in neurodegenerative diseases and traumatic central nervous system (CNS) injuries has introduced a need to develop tools that effectively modulate intracellular RhoA-dependent signaling. In neurons, the bacterial exoenzyme C3 transferase irreversibly inactivates RhoA GTPase signaling to promote neuritogenesis and axon regeneration following an injury. Thus, we have adopted a gene therapy approach for the targeted inhibition of RhoA activity in the CNS by expressing C3 transferase. Herein we describe the construction of adeno-associated viral vectors for the expression of cell-permeable-C3 transferase and their functional characterization in vitro.

Synthetic Cells Synthesize Therapeutic Proteins inside Tumors.

Synthetic cells, artificial cell-like particles, capable of autonomously synthesizing RNA and proteins based on a DNA template, are emerging platforms for studying cellular functions and for revealing the origins-of-life. Here, it is shown for the first time that artificial lipid-based vesicles, containing the molecular machinery necessary for transcription and translation, can be used to synthesize anticancer proteins inside tumors. The synthetic cells are engineered as stand-alone systems, sourcing nutrients from their biological microenvironment to trigger protein synthesis. When pre-loaded with template DNA, amino acids and energy-supplying molecules, up to 2 × 107 copies of green fluorescent protein are synthesized in each synthetic cell. A variety of proteins, having molecular weights reaching 66 kDa and with diagnostic and therapeutic activities, are synthesized inside the particles. Incubating synthetic cells, encoded to secrete Pseudomonas exotoxin A (PE) with 4T1 breast cancer cells in culture, resulted in killing of most of the malignant cells. In mice bearing 4T1 tumors, histological evaluation of the tumor tissue after a local injection of PE-producing particles indicates robust apoptosis. Synthetic cells are new platforms for synthesizing therapeutic proteins on-demand in diseased tissues.

Identification of GntR as regulator of the glucose metabolism in Pseudomonas aeruginosa.

In contrast to Escherichia coli, glucose metabolism in pseudomonads occurs exclusively through the Entner-Doudoroff (ED) pathway. This pathway, as well as the three routes to generate the initial ED pathway substrate, 6-phosphogluconate, is regulated by the PtxS, HexR and GtrS/GltR systems. With GntR (PA2320) we report here the identification of an additional regulator in Pseudomonas aeruginosa PAO1. GntR repressed its own expression as well as that of the GntP gluconate permease. In contrast to PtxS and GtrS/GltR, GntR did not modulate expression of the toxA gene encoding the exotoxin A virulence factor. GntR was found to bind to promoters PgntR and PgntP and the consensus sequence of its operator was defined as 5'-AC-N-AAG-N-TAGCGCT-3'. Both operator sites overlapped with the RNA polymerase binding site and we show that GntR employs an effector mediated de-repression mechanism. The release of promoter bound GntR is induced by gluconate and 6-phosphogluconate that bind with similar apparent affinities to the GntR/DNA complex. GntR and PtxS are paralogous and may have evolved from a common ancestor. The concerted action of four regulatory systems in the regulation of glucose metabolism in Pseudomonas can be considered as a model to understand complex regulatory circuits in bacteria.

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.

CdtR Regulates TcdA and TcdB Production in Clostridium difficile.

Clostridium difficile is a global health burden and the leading cause of antibiotic-associated diarrhoea worldwide, causing severe gastrointestinal disease and death. Three well characterised toxins are encoded by this bacterium in two genetic loci, specifically, TcdB (toxin B) and TcdA (toxin A) in the Pathogenicity Locus (PaLoc) and binary toxin (CDT) in the genomically distinct CDT locus (CdtLoc). Toxin production is controlled by regulators specific to each locus. The orphan response regulator, CdtR, encoded within the CdtLoc, up-regulates CDT production. Until now there has been no suggestion that CdtR influences TcdA and TcdB production since it is not carried by all PaLoc-containing strains and CdtLoc is not linked genetically to PaLoc. Here we show that, in addition to CDT, CdtR regulates TcdA and TcdB production but that this effect is strain dependent. Of clinical relevance, CdtR increased the production of TcdA, TcdB and CDT in two epidemic ribotype 027 human strains, modulating their virulence in a mouse infection model. Strains traditionally from animal lineages, notably ribotype 078 strains, are increasingly being isolated from humans and their genetic and phenotypic analysis is critical for future studies on this important pathogen. Here we show that CdtR-mediated toxin regulation did not occur in other strain backgrounds, including a ribotype 078 animal strain. The finding that toxin gene regulation is strain dependent highlights the regulatory diversity between C. difficile isolates and the importance of studying virulence regulation in diverse lineages and clinically relevant strains. Our work provides the first evidence that TcdA, TcdB and CDT production is linked by a common regulatory mechanism and that CdtR may act as a global regulator of virulence in epidemic 027 strains.

Regulation of the RhoA/ROCK/AKT/ß-catenin pathway by arginine-specific ADP-ribosytransferases 1 promotes migration and epithelial-mesenchymal transition in colon carcinoma.

Arginine-specific ADP-ribosytransferases 1 (ART1) is able to modify the arginine of specific proteins by mono-ADP-ribosylation. We previously reported that the expression of ART1 in human colon adenocarcinoma tissues was higher than in adjacent tissues. Herein, we primarily revealed that ART1 could regulate the epithelial-mesenchymal transition (EMT) and, therefore, the development of colon carcinoma. In CT26 cells, which overexpressed ART1 by lentiviral transfection, the following were promoted: alterations of spindle-like non-polarization, expression of EMT inducers and mesenchymal markers, migration, invasion and adhesion. However, epithelial marker expression was decreased. Correspondingly, knockdown of ART1 in CT26 cells had the opposite effects. The effect of ART1 on EMT and carcinoma metastasis was also verified in a liver metastasis model of BALB/c mice. To further explore the molecular mechanism of ART1 in EMT, CT26 cells were treated with several specific inhibitors and gene silencing. Our data suggest that ART1 could regulate EMT by regulating the RhoA/ROCK1/AKT/ß-catenin pathway and its downstream factors (snail1, vimentin, N-cadherin and E-cadherin) and that it therefore plays an important role in the progression of colon carcinoma.

Influence of chelation strength and bacterial uptake of gallium salicylidene acylhydrazide on biofilm formation and virulence of Pseudomonas aeruginosa.

Development of antibiotic resistance in bacteria causes major challenges for our society and has prompted a great need for new and alternative treatment methods for infection. One promising approach is to target bacterial virulence using for example salicylidene acylhydrazides (hydrazones). Hydrazones coordinate metal ions such as Fe(III) and Ga(III) through a five-membered and a six-membered chelation ring. One suggested mode of action is via restricting bacterial Fe uptake. Thus, it was hypothesized that the chelating strength of these substances could be used to predict their biological activity on bacterial cells. This was investigated by comparing Ga chelation strength of two hydrazone complexes, as well as bacterial Ga uptake, biofilm formation, and virulence in the form of production and secretion of a toxin (ExoS) by Pseudomonas aeruginosa. Equilibrium constants for deprotonation and Ga(III) binding of the hydrazone N'-(5-chloro-2-hydroxy-3-methylbenzylidene)-2,4-dihydroxybenzhydrazide (ME0329), with anti-virulence effect against P. aeruginosa, were determined and compared to bacterial siderophores and the previously described Ga(III) 2-oxo-2-[N-(2,4,6-trihydroxy-benzylidene)-hydrazino]-acetamide (Ga-ME0163) and Ga-citrate complexes. In comparison with these two complexes, it was shown that the uptake of Ga(III) was higher from the Ga-ME0329 complex. The results further show that the Ga-ME0329 complex reduced ExoS expression and secretion to a higher extent than Ga-citrate, Ga-ME0163 or the non-coordinated hydrazone. However, the effect against biofilm formation by P. aeruginosa, by the ME0329 complex, was similar to Ga-citrate and lower than what has been reported for Ga-ME0163.

The C. elegans CCAAT-Enhancer-Binding Protein Gamma Is Required for Surveillance Immunity.

Pathogens attack host cells by deploying toxins that perturb core host processes. Recent findings from the nematode C. elegans and other metazoans indicate that surveillance or "effector-triggered" pathways monitor functioning of these core processes and mount protective responses when they are perturbed. Despite a growing number of examples of surveillance immunity, the signaling components remain poorly defined. Here, we show that CEBP-2, the C. elegans ortholog of mammalian CCAAT-enhancer-binding protein gamma, is a key player in surveillance immunity. We show that CEBP-2 acts together with the bZIP transcription factor ZIP-2 in the protective response to translational block by P. aeruginosa Exotoxin A as well as perturbations of other processes. CEBP-2 serves to limit pathogen burden, promote survival upon P. aeruginosa infection, and also promote survival upon Exotoxin A exposure. These findings may have broad implications for the mechanisms by which animals sense pathogenic attack and mount protective responses.

Proteome Alterations of Hippocampal Cells Caused by Clostridium botulinum C3 Exoenzyme.

C3bot from Clostridium botulinum is a bacterial mono-ADP-ribosylating enzyme, which transfers an ADP-ribose moiety onto the small GTPases Rho A/B/C. C3bot and the catalytic inactive mutant (C3E174Q) cause axonal and dendritic growth as well as branching in primary hippocampal neurons. In cultured murine hippocampal HT22 cells, protein abundances were analyzed in response to C3bot or C3E174Q treatment using a shotgun proteomics approach. Proteome analyses were performed at four time points over 6 days. More than 4000 protein groups were identified at each time point and quantified in triplicate analyses. On day one, 46 proteins showed an altered abundance, and after 6 days, more than 700 proteins responded to C3bot with an up- or down-regulation. In contrast, C3E174Q had no provable impact on protein abundance. Protein quantification was verified for several proteins by multiple reaction monitoring. Data analysis of altered proteins revealed different cellular processes that were affected by C3bot. They are particularly involved in mitochondrial and lysosomal processes, adhesion, carbohydrate and glucose metabolism, signal transduction, and nuclear proteins of translation and ribosome biogenesis. The results of this study gain novel insights into the function of C3bot in hippocampal cells.

Synergistic effect of arginine-specific ADP-ribosyltransferase 1 and poly(ADP-ribose) polymerase-1 on apoptosis induced by cisplatin in CT26 cells.

Arginine-specific ADP-ribosyltransferase 1 (ART1) and poly(ADP-ribose) polymerase-1 (PARP-1) are both post­translational modification proteins. Inhibition of PARP1 induces apoptosis in cancer cells, and ART1 regulates RhoA which promotes apoptosis in hepatic cancer cells when inhibited. However, the interaction of ART1 and PARP-1 on the effect of apoptosis has not yet been elucidated. In the present study, lentiviral vector-mediated ART1-cDNA was transfected into CT26 cells, and the apoptosis rate was detected by flow cytometric assay and Hoechst 33342 staining. Relevant factors were detected by reverse transcriptase-PCR and western blotting. The results showed that the apoptosis rate in the ART1-cDNA CT26 cells treated with PARP-1 inhibitor 5-aminoisoquinoline (5-AIQ) and cisplatin increased, when compared with the ART1-cDNA CT26 cells treated with cisplatin only or the untreated ART1-cDNA CT26 cells. Further studies have shown that PARP-1 is in the downstream of ART1, and plays a role in ART1-mediated CT26 cell apoptosis through the ROCK1/NF-κB/PARP-1 pathway when induced by cisplatin. We also found that in cisplatin-treated cells, activated caspase 3 cleaved PARP-1 and the decreased level of PARP-1 in turn decreased the expression of nuclear factor (NF)-κB, Cox-2 and increased caspase 3, resulting in the enhanced ability of ART1 to regulate CT26 cell apoptosis. Our research provides initial sight into the synergistic effect of ART1 and PARP-1 on apoptosis induced by cisplatin in murine colon carcinoma CT26 cells.

Flexible survival strategies of Pseudomonas aeruginosa in biofilms result in increased fitness compared with Candida albicans.

The majority of microorganisms persist in nature as surface-attached communities often surrounded by an extracellular matrix, called biofilms. Most natural biofilms are not formed by a single species but by multiple species. Microorganisms not only cooperate as in some multispecies biofilms but also compete for available nutrients. The Gram-negative bacterium Pseudomonas aeruginosa and the polymorphic fungus Candida albicans are two opportunistic pathogens that are often found coexisting in a human host. Several models of mixed biofilms have been reported for these organisms showing antagonistic behavior. To investigate the interaction of P. aeruginosa and C. albicans in more detail, we analyzed the secretome of single and mixed biofilms of both organisms using MALDI-TOF MS/MS at several time points. Overall 247 individual proteins were identified, 170 originated from P. aeruginosa and 77 from C. albicans. Only 39 of the 131 in mixed biofilms identified proteins were assigned to the fungus whereby the remaining 92 proteins belonged to P. aeruginosa. In single-species biofilms, both organisms showed a higher diversity of proteins with 73 being assigned to C. albicans and 154 to P. aeruginosa. Most interestingly, P. aeruginosa in the presence of C. albicans secreted 16 proteins in significantly higher amounts or exclusively among other virulence factors such as exotoxin A and iron acquisition systems. In addition, the high affinity iron-binding siderophore pyoverdine was identified in mixed biofilms but not in bacterial biofilms, indicating that P. aeruginosa increases its capability to sequester iron in competition with C. albicans. In contrast, C. albicans metabolism was significantly reduced, including a reduction in detectable iron acquisition proteins. The results obtained in this study show that microorganisms not only compete with the host for essential nutrients but also strongly with the present microflora in order to gain a competitive advantage.

Characterization of an actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila.

The mono-ADP-ribosyltransferase (mART) toxins are contributing factors to a number of human diseases, including cholera, diphtheria, traveler's diarrhea, and whooping cough. VahC is a cytotoxic, actin-targeting mART from Aeromonas hydrophila PPD134/91. This bacterium is implicated primarily in diseases among freshwater fish species but also contributes to gastrointestinal and extraintestinal infections in humans. VahC was shown to ADP-ribosylate Arg-177 of actin, and the kinetic parameters were K(m)(NAD(+)) = 6 µM, K(m)(actin) = 24 µM, and k(cat) = 22 s(-1). VahC activity caused depolymerization of actin filaments, which induced caspase-mediated apoptosis in HeLa Tet-Off cells. Alanine-scanning mutagenesis of predicted catalytic residues showed the predicted loss of in vitro mART activity and cytotoxicity. Bioinformatic and kinetic analysis also identified three residues in the active site loop that were critical for the catalytic mechanism. A 1.9 Å crystal structure supported the proposed roles of these residues and their conserved nature among toxin homologues. Several small molecules were characterized as inhibitors of in vitro VahC mART activity and suramin was the best inhibitor (IC(50) = 20 µM). Inhibitor activity was also characterized against two other actin-targeting mART toxins. Notably, these inhibitors represent the first report of broad spectrum inhibition of actin-targeting mART toxins.

C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa coordinates its virulence expression and establishment in the host in response to modification of its environment. During the infectious process, bacteria are exposed to and can detect eukaryotic products including hormones. It has been shown that P. aeruginosa is sensitive to natriuretic peptides, a family of eukaryotic hormones, through a cyclic nucleotide-dependent sensor system that modulates its cytotoxicity. We observed that pre-treatment of P. aeruginosa PAO1 with C-type natriuretic peptide (CNP) increases the capacity of the bacteria to kill Caenorhabditis elegans through diffusive toxin production. In contrast, brain natriuretic peptide (BNP) did not affect the capacity of the bacteria to kill C. elegans. The bacterial production of hydrogen cyanide (HCN) was enhanced by both BNP and CNP whereas the production of phenazine pyocyanin was strongly inhibited by CNP. The amount of 2-heptyl-4-quinolone (HHQ), a precursor to 2-heptyl-3-hydroxyl-4-quinolone (Pseudomonas quinolone signal; PQS), decreased after CNP treatment. The quantity of 2-nonyl-4-quinolone (HNQ), another quinolone which is synthesized from HHQ, was also reduced after CNP treatment. Conversely, both BNP and CNP significantly enhanced bacterial production of acylhomoserine lactone (AHL) [e.g. 3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and butanoylhomoserine lactone (C4-HSL)]. These results correlate with an induction of lasI transcription 1 h after bacterial exposure to BNP or CNP. Concurrently, pre-treatment of P. aeruginosa PAO1 with either BNP or CNP enhanced PAO1 exotoxin A production, via a higher toxA mRNA level. At the same time, CNP led to elevated amounts of algC mRNA, indicating that algC is involved in C. elegans killing. Finally, we observed that in PAO1, Vfr protein is essential to the pro-virulent effect of CNP whereas the regulator PtxR supports only a part of the CNP pro-virulent activity. Taken together, these data reinforce the hypothesis that during infection natriuretic peptides, particularly CNP, could enhance the virulence of PAO1. This activity is relayed by Vfr and PtxR activation, and a general diagram of the virulence activation cascade involving AHL, HCN and exotoxin A is proposed.

New antigen in the Dombrock blood group system, DOYA, ablates expression of Do(a) and weakens expression of Hy, Jo(a), and Gy(a) antigens.

BACKGROUND: The Dombrock (Do) blood group system consists of five distinct antigens: Do(a), Do(b), Gy(a), Hy, and Jo(a). Our finding of a patient whose plasma contained a Do-related alloantibody suggested the presence of a sixth antigen. STUDY DESIGN AND METHODS: Standard hemagglutination, flow cytometry, and polymerase chain reaction (PCR)-based methods were used throughout. Protein homology modeling was used to map the amino acid change on the protein structure. RESULTS: The patient's red blood cells (RBCs) typed as Do(a-b-), Hy+(w), Jo(a+(w)), and Gy(a+(w)). The patient's plasma agglutinated RBCs with common Dombrock phenotypes. Reactivity with Hy- and Jo(a-) RBC samples was weak, and Gy(a-) RBC samples were nonreactive. DNA analysis showed the patient to be DO*793A (DO*A/DO*A), DO*323G, and DO*350C, which predicts the Do(a+b-), Hy+, and Jo(a+) phenotype, and revealed a homozygous single-nucleotide change of 547T>G in Exon 2 that is predicted to change tyrosine at Amino Acid Position 183 to aspartic acid. This missense substitution introduced a BtgZI restriction enzyme site. The sequence data were confirmed with a PCR-restriction fragment length polymorphism assay and revealed that the patient's parents and children were heterozygous DO*547T/G. Homology modeling predicted that the 183Tyr substitution by Asp altered the Cys182 environment and influenced the formation and/or stability of the Cys182-Cys231 disulfide bond. CONCLUSION: The patient's DO genes have a single-nucleotide change, which leads to the absence of the high-prevalence antigen DOYA. The absence of this antigen is associated with 183Asp and silencing of Do(a) and weakening of Gy(a), Hy, and Jo(a) antigens.

A novel composite immunotoxin that suppresses rabies virus production by the infected cells.

Using strepavidin as a scaffold, we have assembled a composite immunotoxin that consists of recombinant Pseudomonas exotoxin A subunit (PE38) and recombinant 25-D1.16 Fab fragment which recognizes the SIINFEKL (pOV8) peptide from ovalbumin in association with H-2K(b) MHC class I protein. The composite immunotoxin exercises cytotoxicity against H-2K(b+) cells sensitized with pOV8 peptide but not with irrelevant peptide. Specific binding of the immunotoxin to H-2K(b+) cells infected with recombinant rabies virus (RV) expressing pOV8 epitope (RV-pOV8) resulted in the suppression of the production of virus particles by the infected cells. This strategy allows readily produce different immunotoxins with desired specificity by combining various targeting and toxin molecules. The results provide a proof of concept that composite immunotoxins can be utilized as novel immunotherapeutics to stop virus spread in the acute phase of the infection allowing winning time for the development of protective immune response.

Differential regulation of P2X7 receptor activation by extracellular nicotinamide adenine dinucleotide and ecto-ADP-ribosyltransferases in murine macrophages and T cells.

Extracellular NAD induces the ATP-independent activation of the ionotropic P2X(7) purinergic receptor (P2X(7)R) in murine T lymphocytes via a novel covalent pathway involving ADP-ribosylation of arginine residues on the P2X(7)R ectodomain. This modification is catalyzed by ART2.2, a GPI-anchored ADP-ribosyltransferase (ART) that is constitutively expressed in murine T cells. We previously reported that ART2.1, a related ecto-ART, is up-regulated in inflammatory murine macrophages that constitutively express P2X(7)R. Thus, we tested the hypothesis that extracellular NAD acts via ART2.1 to regulate P2X(7)R function in murine macrophages. Coexpression of the cloned murine P2X(7)R with ART2.1 or ART2.2 in HEK293 cells verified that P2X(7)R is an equivalent substrate for ADP-ribosylation by either ART2.1 or ART2.2. However, in contrast with T cells, the stimulation of macrophages or HEK293 cells with NAD alone did not activate the P2X(7)R. Rather, NAD potentiated ATP-dependent P2X(7)R activation as indicated by a left shift in the ATP dose-response relationship. Thus, extracellular NAD regulates the P2X(7)R in both macrophages and T cells but via distinct mechanisms. Although ADP-ribosylation is sufficient to gate a P2X(7)R channel opening in T cells, this P2X(7)R modification in macrophages does not gate the channel but decreases the threshold for gating in response to ATP binding. These findings indicate that extracellular NAD and ATP can act synergistically to regulate P2X(7)R signaling in murine macrophages and also suggest that the cellular context in which P2X(7)R signaling occurs differs between myeloid and lymphoid leukocytes.

Dombrock genotyping in a native Congolese cohort reveals two novel alleles.

BACKGROUND: Since variant alleles in the Dombrock (DO) blood group system are common in Africans, DNA typing of DO alleles in an uninvestigated Congolese Teke ethnic group was performed. STUDY DESIGN AND METHODS: DO exons were polymerase chain reaction amplified, using genomic DNA extracted from blood samples, and sequenced. Membrane expression in K562 cells transduced with DO-cDNAs using lentiviral vectors was studied by flow cytometry. Amino acid changes were mapped on the protein structure, predicted by homology modeling. RESULTS: In 41 samples investigated, there were 56 DOB or DOB-WL (68%), 15 DOA (18%), 6 HY (7%), and 3 JO (4%) alleles. The remaining two alleles were novel, that is, DOB-SH-Gln149Lys carrying a 445C>A transversion and DOB-(WL)-Ile175Asn showing a 524T>A transversion on a DOB or DOB-WL background. Transduced K562 cells revealed that DOB-SHGln149Lys variant was expressed to the same extent as DOB-SH but to a lesser extent than the DOB control. The DOB-Ile175Asn variant shows equivalent expression to DOB but is not recognized by monoclonal antibodies MIMA-53. As deduced from the protein model, these missense changes would lead to structure similar to the wild-type one, with only modified surface features. CONCLUSION: Molecular screening of Teke individuals revealed a high frequency of HY and JO alleles and two novel alleles, one on the DOB (or DOB-WL) and one on the DOB-SH background. Expression studies highlighted the impact of changes on Do protein expression. These findings suggest that allelic diversity is greater than expected and that expression level of DO alleles should be taken into account in transfusion