Development of a platform-based approach for the clinical production of HIV gp120 envelope glycoprotein vaccine candidates.

Preclinical development of vaccine candidates is an important link between the discovery and manufacture of vaccines for use in human clinical trials. Here, an exploratory clinical study utilizing multiple gp120 envelope proteins as vaccine antigens was pursued, which required a harmonized platform development approach for timely and efficient manufacture of the combined HIV vaccine product. Development of cell lines, processes, and analytical methods was initiated with a transmitted founder envelope protein (CH505TF), then applied to produce three subsequent gp120 Env (envelope) variants. Cell lines were developed using the commercially available Freedom CHO DG44 kit (Life Technologies). The fed-batch cell culture production process was based on a commercially-available medium with harmonized process parameters across the variants. A platform purification process was developed utilizing a mixed mode chromatography capture step, with ceramic hydroxyapatite and ion exchange polishing steps. A suite of analytical methods was developed to establish and monitor the Quality Target Profile (QTP), release and long-term stability testing of the vaccine products. The platform development strategy was successfully implemented to produce four gp120 envelope protein variants. In some cases, minor changes to the platform were required to optimize for a particular variant; however, baseline conditions for the processes (cell line type, media & feed system, chromatography resins, and analytical approaches) remained constant, leading to successful transfer and manufacture of all four proteins in a cGMP facility. This body of work demonstrates successful pursuit of a platform development approach to manufacture important vaccine candidates and can be used as a model for other vaccine glycoproteins, such as HIV gp140 trimers or other viral glycoproteins with global health implications. Clinical trial identifier. NCT03220724, NCT03856996.

Efficacy and safety of intermittent preventive treatment and intermittent screening and treatment versus single screening and treatment with dihydroartemisinin-piperaquine for the control of malaria in pregnancy in Indonesia: a cluster-randomised, open-label, superiority trial.

BACKGROUND: Plasmodium falciparum and Plasmodium vivax infections are important causes of adverse pregnancy outcomes in the Asia-Pacific region. We hypothesised that monthly intermittent preventive treatment (IPT) or intermittent screening and treatment (IST) with dihydroartemisinin-piperaquine is more effective in reducing malaria in pregnancy than the existing single screening and treatment (SST) strategy, which is used to screen women for malaria infections at the first antenatal visit followed by passive case detection, with management of febrile cases. METHODS: We did an open-label, three-arm, cluster-randomised, superiority trial in Sumba (low malaria transmission site) and Papua (moderate malaria transmission site), Indonesia. Eligible participants were 16-30 weeks pregnant. Clusters (antenatal clinics with at least ten new pregnancies per year matched by location, size, and malaria risk) were randomly assigned (1:1:1) via computer-generated lists to IPT, IST, or SST clusters. In IPT clusters, participants received the fixed-dose combination of dihydroartemisinin-piperaquine (4 and 18 mg/kg per day). In IST clusters, participants were screened with malaria rapid diagnostic tests once a month, whereas, in SST clusters, they were screened at enrolment only. In all groups, participants with fever were tested for malaria. Any participant who tested positive received dihydroartemisinin-piperaquine regardless of symptoms. The primary outcome was malaria infection in the mother at delivery. Laboratory staff were unaware of group allocation. Analyses included all randomly assigned participants contributing outcome data and were adjusted for clustering at the clinic level. This trial is complete and is registered with ISRCTN, number 34010937. FINDINGS: Between May 16, 2013, and April 21, 2016, 78 clusters (57 in Sumba and 21 in Papua) were randomly assigned to SST, IPT, or IST clusters (26 clusters each). Of 3553 women screened for eligibility, 2279 were enrolled (744 in SST clusters, 681 in IPT clusters, and 854 in IST clusters). At enrolment, malaria prevalence was lower in IST (5·7%) than in SST (12·6%) and IPT (10·6%) clusters. At delivery, malaria prevalence was 20·2% (128 of 633) in SST clusters, compared with 11·6% (61 of 528) in IPT clusters (relative risk [RR] 0·59, 95% CI 0·42-0·83, p=0·0022) and 11·8% (84 of 713) in IST clusters (0·56, 0·40-0·77, p=0·0005). Conditions related to the pregnancy, the puerperium, and the perinatal period were the most common serious adverse events for the mothers, and infections and infestations for the infants. There were no differences between groups in serious adverse events in the mothers or in their infants. INTERPRETATION: IST was associated with a lower prevalence of malaria than SST at delivery, but the prevalence of malaria in this group was also lower at enrolment, making interpretation of the effect of IST challenging. Further studies with highly sensitive malaria rapid diagnostic tests should be considered. Monthly IPT with dihydroartemisinin-piperaquine is a promising alternative to SST in areas in the Asia-Pacific region with moderate or high transmission of malaria. FUNDING: Joint Global Health Trials Scheme of the Medical Research Council, Department for International-Development, and the Wellcome Trust.

Scheduled Intermittent Screening with Rapid Diagnostic Tests and Treatment with Dihydroartemisinin-Piperaquine versus Intermittent Preventive Therapy with Sulfadoxine-Pyrimethamine for Malaria in Pregnancy in Malawi: An Open-Label Randomized Controlled Trial.

BACKGROUND: In Africa, most plasmodium infections during pregnancy remain asymptomatic, yet are associated with maternal anemia and low birthweight. WHO recommends intermittent preventive therapy in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP). However, sulfadoxine-pyrimethamine (SP) efficacy is threatened by high-level parasite resistance. We conducted a trial to evaluate the efficacy and safety of scheduled intermittent screening with malaria rapid diagnostic tests (RDTs) and treatment of RDT-positive women with dihydroartemisinin-piperaquine (DP) as an alternative strategy to IPTp-SP. METHODS AND FINDINGS: This was an open-label, two-arm individually randomized superiority trial among HIV-seronegative women at three sites in Malawi with high SP resistance. The intervention consisted of three or four scheduled visits in the second and third trimester, 4 to 6 wk apart. Women in the IPTp-SP arm received SP at each visit. Women in the intermittent screening and treatment in pregnancy with DP (ISTp-DP) arm were screened for malaria at every visit and treated with DP if RDT-positive. The primary outcomes were adverse live birth outcome (composite of small for gestational age, low birthweight [<2,500 g], or preterm birth [<37 wk]) in paucigravidae (first or second pregnancy) and maternal or placental plasmodium infection at delivery in multigravidae (third pregnancy or higher). Analysis was by intention to treat. Between 21 July 2011 and 18 March 2013, 1,873 women were recruited (1,155 paucigravidae and 718 multigravidae). The prevalence of adverse live birth outcome was similar in the ISTp-DP (29.9%) and IPTp-SP (28.8%) arms (risk difference = 1.08% [95% CI -3.25% to 5.41%]; all women: relative risk [RR] = 1.04 [95% CI 0.90-1.20], p = 0.625; paucigravidae: RR = 1.10 [95% CI 0.92-1.31], p = 0.282; multigravidae: RR = 0.92 [95% CI 0.71-1.20], p = 0.543). The prevalence of malaria at delivery was higher in the ISTp-DP arm (48.7% versus 40.8%; risk difference = 7.85%, [95% CI 3.07%-12.63%]; all women: RR = 1.19 [95% CI 1.07-1.33], p = 0.007; paucigravidae: RR = 1.16 [95% CI 1.04-1.31], p = 0.011; multigravidae: RR = 1.29 [95% CI 1.02-1.63], p = 0.037). Fetal loss was more common with ISTp-DP (2.6% versus 1.3%; RR = 2.06 [95% CI 1.01-4.21], p = 0.046) and highest among non-DP-recipients (3.1%) in the ISTp-DP arm. Limitations included the open-label design. CONCLUSIONS: Scheduled screening for malaria parasites with the current generation of RDTs three to four times during pregnancy as part of focused antenatal care was not superior to IPTp-SP in this area with high malaria transmission and high SP resistance and was associated with higher fetal loss and more malaria at delivery. TRIAL REGISTRATION: Pan African Clinical Trials Registry PACTR201103000280319; ISRCTN Registry ISRCTN69800930.

The Cooking and Pneumonia Study (CAPS) in Malawi: Implementation of Remote Source Data Verification.

BACKGROUND: Source data verification (SDV) is a data monitoring procedure which compares the original records with the Case Report Form (CRF). Traditionally, on-site SDV relies on monitors making multiples visits to study sites requiring extensive resources. The Cooking And Pneumonia Study (CAPS) is a 24- month village-level cluster randomized controlled trial assessing the effectiveness of an advanced cook-stove intervention in preventing pneumonia in children under five in rural Malawi (www.capstudy.org). CAPS used smartphones to capture digital images of the original records on an electronic CRF (eCRF). In the present study, descriptive statistics are used to report the experience of electronic data capture with remote SDV in a challenging research setting in rural Malawi. METHODS: At three monthly intervals, fieldworkers, who were employed by CAPS, captured pneumonia data from the original records onto the eCRF. Fieldworkers also captured digital images of the original records. Once Internet connectivity was available, the data captured on the eCRF and the digital images of the original records were uploaded to a web-based SDV application. This enabled SDV to be conducted remotely from the UK. We conducted SDV of the pneumonia data (occurrence, severity, and clinical indicators) recorded in the eCRF with the data in the digital images of the original records. RESULT: 664 episodes of pneumonia were recorded after 6 months of follow-up. Of these 664 episodes, 611 (92%) had a finding of pneumonia in the original records. All digital images of the original records were clear and legible. CONCLUSION: Electronic data capture using eCRFs on mobile technology is feasible in rural Malawi. Capturing digital images of the original records in the field allows remote SDV to be conducted efficiently and securely without requiring additional field visits. We recommend these approaches in similar settings, especially those with health endpoints.

Effectiveness of antenatal clinics to deliver intermittent preventive treatment and insecticide treated nets for the control of malaria in pregnancy in Mali: a household survey.

BACKGROUND: WHO recommends intermittent-preventive-treatment (IPTp) with sulphadoxine-pyrimethamine (SP) and insecticide-treated-nets (ITNs) to prevent malaria in pregnancy in sub-Saharan Africa, however uptake remains unacceptably low. We evaluated the effectiveness of antenatal clinics (ANC) to deliver two doses of IPTp and ITNs to pregnant women in Segou district, Mali. METHODS: We used household data to assess the systems effectiveness of ANC to deliver IPTp and ITNs to pregnant women and used logistic regression to identify predictors of ANC attendance, receipt of IPTp and ITN use during pregnancy, and the impact on community effectiveness. RESULTS: Of 81% of recently pregnant women who made at least one ANC visit, 59% of these attended during the eligible gestational age for IPTp. Of these, 82% reported receiving one dose of SP and 91% attended ANC again, of whom 66% received a second dose, resulting in a cumulative effectiveness for 2-dose IPTp of 29%, most of whom used an ITN (90%). Cumulative effectiveness of 2-dose SP by directly observed therapy (DOT) was very low (6%). ITN use was 92%, and ANC was the main source (81%). Reported and ANC-card data showed some doses of SP are given to women in their first trimester. Women were less likely to receive two doses by DOT if they were married (OR 0.10; CI 0.03, 0.40), or lived <5 km from the health facility (OR 0.34; CI 0.14, 0.83). A high household person-LLIN ratio predicted low ITN use in pregnant women (OR 0.16; CI 0.04, 0.55). CONCLUSION: Our findings suggest poor adherence by health workers to provision of IPTp by eligible gestational age and DOT, contributing to low effectiveness of this strategy in this setting. ITN delivery and use among women was substantially higher. Efforts to improve health worker adherence to IPTp guidelines are needed to improve service delivery of IPTp.

Effectiveness of antenatal clinics to deliver intermittent preventive treatment and insecticide treated nets for the control of malaria in pregnancy in Kenya.

BACKGROUND: Malaria in pregnancy can have devastating consequences for mother and baby. Coverage with the WHO prevention strategy for sub-Saharan Africa of intermittent-preventive-treatment (IPTp) with two doses of sulphadoxine-pyrimethamine (SP) and insecticide-treated-nets (ITNs) in pregnancy is low. We analysed household survey data to evaluate the effectiveness of antenatal clinics (ANC) to deliver IPTp and ITNs to pregnant women in Nyando district, Kenya. METHODS: We assessed the systems effectiveness of ANC to deliver IPTp and ITNs to pregnant women and the impact on low birthweight (LBW). Logistic regression was used to identify predictors of receipt of IPTp and ITN use during pregnancy. RESULTS: Among 89% of recently pregnant women who attended ANC at least once between 4-9 months gestation, 59% reported receiving one dose of SP and 90% attended ANC again, of whom 57% received a second dose, resulting in a cumulative effectiveness for IPTp of 27%, most of whom used an ITN (96%). Overall ITN use was 89%, and ANC the main source (76%). Women were less likely to receive IPTp if they had low malaria knowledge (0.26, 95% CI 0.08-0.83), had a child who had died (OR 0.36, 95% CI 0.14-0.95), or if they first attended ANC late (OR 0.20, 95% CI 0.06-0.67). Women who experienced side effects to SP (OR 0.18, CI 0.03-0.90) or had low malaria knowledge (OR 0.78, 95% CI 0.11-5.43) were less likely to receive IPTp by directly observed therapy. Ineffective delivery of IPTp reduced its potential impact by 231 LBW cases averted (95% CI 64-359) per 10,000 pregnant women. CONCLUSION: IPTp presents greater challenges to deliver through ANC than ITNs in this setting. The reduction in public health impact on LBW resulting from ineffective delivery of IPTp is estimated to be substantial. Urgent efforts are required to improve service delivery of this important intervention.

Cysteine-scanning mutagenesis supports the importance of Clostridium perfringens enterotoxin amino acids 80 to 106 for membrane insertion and pore formation.

Clostridium perfringens enterotoxin (CPE) causes the gastrointestinal symptoms of the second most common bacterial food-borne illness. Previous studies suggested that a region named TM1, which has amphipathic characteristics and spans from amino acids 81 to 106 of the native CPE protein, forms a ß-hairpin involved in ß-barrel pore formation. To further explore the potential role of TM1 in pore formation, the single Cys naturally present in CPE at residue 186 was first altered to alanine by mutagenesis; the resultant rCPE variant, named C186A, was shown to retain cytotoxic properties. Cys-scanning mutagenesis was then performed in which individual Cys mutations were introduced into each TM1 residue of the C186A variant. When those Cys variants were characterized, three variants were identified that exhibit reduced cytotoxicity despite possessing binding and oligomerization abilities similar to those of the C186A variant from which they were derived. Pronase challenge experiments suggested that the reduced cytotoxicity of those two Cys variants, i.e., the F91C and F95C variants, which model to the tip of the ß-hairpin, was attributable to a lessened ability of these variants to insert into membranes after oligomerization. In contrast, another Cys variant, i.e., the G103C variant, with impaired cytotoxicity apparently inserted into membranes after oligomerization but could not form a pore with a fully functional channel. Collectively, these results support the TM1 region forming a ß-hairpin as an important step in CPE insertion and pore formation. Furthermore, this work identifies the first amino acid residues specifically involved in those two steps in CPE action.

Structure of the food-poisoning Clostridium perfringens enterotoxin reveals similarity to the aerolysin-like pore-forming toxins.

Clostridium perfringens enterotoxin (CPE) is a major cause of food poisoning and antibiotic-associated diarrhea. Upon its release from C. perfringens spores, CPE binds to its receptor, claudin, at the tight junctions between the epithelial cells of the gut wall and subsequently forms pores in the cell membranes. A number of different complexes between CPE and claudin have been observed, and the process of pore formation has not been fully elucidated. We have determined the three-dimensional structure of the soluble form of CPE in two crystal forms by X-ray crystallography, to a resolution of 2.7 and 4.0 Å, respectively, and found that the N-terminal domain shows structural homology with the aerolysin-like ß-pore-forming family of proteins. We show that CPE forms a trimer in both crystal forms and that this trimer is likely to be biologically relevant but is not the active pore form. We use these data to discuss models of pore formation.

Crystallization and preliminary crystallographic analysis of the Clostridium perfringens enterotoxin.

Clostridium perfringens is a Gram-positive anaerobic species of bacterium that is notable for its ability to produce a plethora of toxins, including membrane-active toxins (alpha-toxins), pore-forming toxins (-toxins) and binary toxins (iota-toxins). Here, the crystallization of the full-length wild-type C. perfringens enterotoxin is reported, which is the causative agent of the second most prevalent food-borne illness in the United States and has been implicated in many other gastrointestinal pathologies. Several crystal forms were obtained. However, only two of these optimized crystal forms (I and II) were useable for X-ray diffraction data collection. The form I crystals diffracted to d(min) = 2.7 A and belonged to space group C2, while the form II crystals diffracted to d(min) = 4 A and belonged to space group P2(1)3.

Identification of a claudin-4 residue important for mediating the host cell binding and action of Clostridium perfringens enterotoxin.

The 24-member claudin protein family plays a key role in maintaining the normal structure and function of epithelial tight junctions. Previous studies with fibroblast transfectants and naturally sensitive Caco-2 cells have also implicated certain claudins (e.g., Claudin-4) as receptors for Clostridium perfringens enterotoxin (CPE). The present study first provided evidence that the second extracellular loop (ECL-2) of claudins is specifically important for mediating the host cell binding and cytotoxicity of native CPE. Rat fibroblast transfectants expressing a Claudin-4 chimera, where the natural ECL-2 was replaced by ECL-2 from Claudin-2, exhibited no CPE-induced cytotoxicity. Conversely, CPE bound to, and killed, CPE-treated transfectants expressing a Claudin-2 chimera with a substituted ECL-2 from Claudin-4. Site-directed mutagenesis was then used to alter an ECL-2 residue that invariably aligns as N in claudins known to bind native CPE but as D or S in claudins that cannot bind CPE. Transfectants expressing a Claudin-4(N149D) mutant lost the ability to bind or respond to CPE, while transfectants expressing a Claudin-1 mutant with the corresponding ECL-2 residue changed from D to N acquired CPE binding and sensitivity. Identifying carriage of this N residue in ECL-2 as being important for native CPE binding helps to explain why only certain claudins can serve as CPE receptors. Finally, preincubating CPE with soluble recombinant Claudin-4, or Claudin-4 fragments containing ECL-2 specifically blocked the cytotoxicity on Caco-2 cells. This result opens the possibility of using receptor claudins as therapeutic decoys to ameliorate CPE-mediated intestinal disease.

Probing the pH-dependent prepore to pore transition of Bacillus anthracis protective antigen with differential oxidative protein footprinting.

The protective antigen (PA) component of the anthrax toxin (ATx) plays an essential role in the pathogenesis of the bioterrorism bacterium Bacillus anthracis. After oligomerization on the cell surface and docking of lethal factor and/or edema factor, PA is internalized and undergoes a conformational change when exposed to the low pH of the endosome to form a membrane-penetrating pore. While the structure of the PA prepore has been determined, precise structural information regarding the pore state remains lacking. Oxidative protein footprinting (OPF) can provide dynamic structural information about a protein complex through analysis of amino acid oxidation both before and after a conformational change. In this study, PA at pH 7.5 and 5.5 was exposed to hydroxyl radicals generated by ionizing radiation. Mass spectrometry was then used to both identify and quantitate the extent of oxidation of differentially modified residues. Several residues were found to be more readily oxidized at pH 7.5, most of which clustered toward the bottom plane of the prepore heptamer. Two amino acids had greater oxidation rates at pH 5.5, both found on the outer periphery of the prepore. When the OPF results were mapped to a current computational model of the pore, the accessibilities of some residues were consistent with their modeled positions in the pore (i.e., Y688 and V619/I620), while data for other residues (W346 and M350) appeared to conflict with the model. The results from this study illustrate the utility of OPF in generating empirical structural information for yet undetermined structures and offering opportunities for refinement for models thereof.

Noncytotoxic Clostridium perfringens enterotoxin (CPE) variants localize CPE intestinal binding and demonstrate a relationship between CPE-induced cytotoxicity and enterotoxicity.

Clostridium perfringens enterotoxin (CPE) causes the symptoms of a very common food poisoning. To assess whether CPE-induced cytotoxicity is necessary for enterotoxicity, a rabbit ileal loop model was used to compare the in vivo effects of native CPE or recombinant CPE (rCPE), both of which are cytotoxic, with those of the noncytotoxic rCPE variants rCPE D48A and rCPE(168-319). Both CPE and rCPE elicited significant fluid accumulation in rabbit ileal loops, along with severe mucosal damage that starts at villus tips and then progressively affects the entire villus, including necrosis of epithelium and lamina propria, villus blunting and fusion, and transmural edema and hemorrhage. Similar treatment of ileal loops with either of the noncytotoxic rCPE variants produced no visible histologic damage or fluid transport changes. Immunohistochemistry revealed strong CPE or rCPE(168-319) binding to villus tips, which correlated with the abundant presence of claudin-4, a known CPE receptor, in this villus region. These results support (i) cytotoxicity being necessary for CPE-induced enterotoxicity, (ii) the CPE sensitivity of villus tips being at least partially attributable to the abundant presence of receptors in this villus region, and (iii) claudin-4 being an important intestinal receptor for CPE. Finally, rCPE(168-319) was able to partially inhibit CPE-induced histologic damage, suggesting that noncytotoxic rCPE variants might be useful for protecting against some intestinal effects of CPE.

Structure of the claudin-binding domain of Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin is a common cause of food-borne and antibiotic-associated diarrhea. The toxin's receptors on intestinal epithelial cells include claudin-3 and -4, members of a large family of tight junction proteins. Toxin-induced cytolytic pore formation requires residues in the NH(2)-terminal half, whereas residues near the COOH terminus are required for binding to claudins. The claudin-binding COOH-terminal domain is not toxic and is currently under investigation as a potential drug absorption enhancer. Because claudin-4 is overexpressed on some human cancers, the toxin is also being investigated for targeting chemotherapy. Our aim was to solve the structure of the claudin-binding domain to advance its therapeutic applications. The structure of a 14-kDa fragment containing residues 194 to the native COOH terminus at position 319 was solved by x-ray diffraction to a resolution of 1.75A. The structure is a nine-strand beta sandwich with previously unappreciated similarity to the receptor-binding domains of several other toxins of spore-forming bacteria, including the collagen-binding domain of ColG from Clostridium histolyticum and the large Cry family of toxins (including Cry4Ba) of Bacillus thuringiensis. Correlations with previous studies suggest that the claudin-4 binding site is on a large surface loop between strands beta8 and beta9 or includes these strands. The sequence that was crystallized (residues 194-319) binds to purified human claudin-4 with a 1:1 stoichiometry and affinity in the submicromolar range similar to that observed for binding of native toxin to cells. Our results provide a structural framework to advance therapeutic applications of the toxin and suggest a common ancestor for several receptor-binding domains of bacterial toxins.

Clostridium perfringens enterotoxin as a novel-targeted therapeutic for brain metastasis.

Brain metastasis is the most commonly occurring intracranial tumor whose incidence seems to be increasing. With standard therapy, the average survival time of patients is approximately 8 months, and treatment often leads to neurologic dysfunction in long-term survivors, emphasizing the need for novel therapeutics. Clostridium perfringens enterotoxin (CPE) has recently been shown to rapidly and specifically destroy cancer cells expressing CPE receptors claudin-3 and claudin-4. Unfortunately, the utility of CPE is precluded by systemic toxicity because its receptors are expressed in numerous organs. Here, we provide the first preclinical evidence that CPE may be uniquely suited to the local treatment of brain metastasis. By immunohistochemical analysis, claudin-3 and claudin-4 were expressed frequently in metastases from breast (15 of 18), lung (15 of 20), and colon (12 of 14) carcinoma, and infrequently in metastases from renal cell carcinoma (2 of 16) and melanoma (2 of 16). In contrast, expression of claudin-3 and claudin-4 was absent in adjacent normal brain tissue. Further examination of the central nervous system (CNS) revealed low or undetectable levels of claudin-3 and claudin-4 in all regions tested by Western and immunohistochemical analysis. Treatment of breast cancer cell lines (MCF-7, MDA-MB-468, NT2.5-luc) and normal human astrocytes with CPE in vitro resulted in rapid and dose-dependent cytolysis exclusively in breast cancer cells, correlating with claudin-3 and claudin-4 expression. Moreover, intracranial CPE treatment significantly inhibited tumor growth and increased survival in two murine models of breast cancer brain metastasis, without any apparent local or systemic toxicity. These data suggest that CPE therapy may have efficacy against a wide variety of brain metastases without CNS toxicity.

Compositional and stoichiometric analysis of Clostridium perfringens enterotoxin complexes in Caco-2 cells and claudin 4 fibroblast transfectants.

Clostridium perfringens enterotoxin (CPE) binds to host cell receptors, forming a small complex precursor for two large complexes reportedly having molecular masses of approximately 155 or approximately 200 kDa. Formation of the approximately 155 kDa complex causes a Ca(2+) influx that leads to apoptosis or oncosis. CPE complex composition is currently poorly understood, although occludin was identified in the approximately 200 kDa complex. The current study used heteromer gel shift analysis to show both CPE large complexes contain six CPE molecules. Ferguson plots and size exclusion chromatography re-sized the approximately 155 and approximately 200 kDa complexes as approximately 425-500 kDa and approximately 550-660 kDa respectively. Co-immunoprecipitation and electroelution studies demonstrated both CPE-binding and non-CPE-binding claudins are associated with all three CPE complexes in Caco-2 cells and with small complex and approximately 425-500 kDa complex of claudin 4 transfectants. Fibroblast transfectants expressing claudin 4 or C-terminal truncated claudin 4 were CPE-sensitive and formed the approximately 425 kDa complex, indicating claudin-induced cell signalling is not required for CPE action and that expression of a single receptor claudin suffices for approximately 425-500 kDa CPE complex formation. These results identify CPE as a unique toxin that combines with tight junction proteins to form high-molecular-mass hexameric pores and alter membrane permeability.

Claudin-4 overexpression in epithelial ovarian cancer is associated with hypomethylation and is a potential target for modulation of tight junction barrier function using a C-terminal fragment of Clostridium perfringens enterotoxin.

BACKGROUND: Claudin-4, a tight junction (TJ) protein and receptor for the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), is overexpressed in epithelial ovarian cancer (EOC). Previous research suggests DNA methylation is a mechanism for claudin-4 overexpression in cancer and that C-CPE acts as an absorption-enhancing agent in claudin-4-expressing cells. We sought to correlate claudin-4 overexpression in EOC with clinical outcomes and TJ barrier function, investigate DNA methylation as a mechanism for overexpression, and evaluate the effect of C-CPE on the TJ. METHODS: Claudin-4 expression in EOC was quantified and correlated with clinical outcomes. Claudin-4 methylation status was determined, and claudin-4-negative cell lines were treated with a demethylating agent. Electric cell-substrate impedance sensing was used to calculate junctional (paracellular) resistance (Rb) in EOC cells after claudin-4 silencing and after C-CPE treatment. RESULTS: Claudin-4 overexpression in EOC does not correlate with survival or other clinical endpoints and is associated with hypomethylation. Claudin-4 overexpression correlates with Rb and C-CPE treatment of EOC cells significantly decreased Rb in a dose- and claudin-4-dependent noncytotoxic manner. CONCLUSIONS: C-CPE treatment of EOC cells leads to altered TJ function. Further research is needed to determine the potential clinical applications of C-CPE in EOC drug delivery strategies.

Identification of a prepore large-complex stage in the mechanism of action of Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin (CPE) is the etiological agent of the third most common food-borne illness in the United States. The enteropathogenic effects of CPE result from formation of large CPE-containing complexes in eukaryotic cell membranes. Formation of these approximately 155- and approximately 200-kDa complexes coincides with plasma membrane permeability changes in eukaryotic cells, causing a Ca2+ influx that drives cell death pathways. CPE contains a stretch of amino acids (residues 81 to 106) that alternates markedly in side chain polarity (a pattern shared by the transmembrane domains of the beta-barrel pore-forming toxin family). The goal of this study, therefore, was to investigate whether this CPE region is involved in pore formation. Complete deletion of the CPE region from 81 to 106 produced a CPE variant that was noncytotoxic for Caco-2 cells and was unable to form CPE pores. However, this variant maintained the ability to form the approximately 155-kDa large complex. This large complex appears to be a prepore present on the plasma membrane surface since it showed greater susceptibility to proteases, increased complex instability, and a higher degree of dissociation from membranes compared to the large complex formed by recombinant CPE. When a D48A mutation was engineered into this prepore-forming CPE variant, the resultant variant was unable to form any prepore approximately 155-kDa large complex. Collectively these findings reveal a new step in CPE action, whereby receptor binding is followed by formation of a prepore large complex, which then inserts into membranes to form a pore.

Influence of pilin glycosylation on Pseudomonas aeruginosa 1244 pilus function.

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial pneumonia. Among its virulence factors, the type IV pili of P. aeruginosa strain 1244 contain a covalently linked, three-sugar glycan of previously unknown significance. The work described in this paper was carried out to determine the influence of the P. aeruginosa 1244 pilin glycan on pilus function, as well as a possible role in pathogenesis. To accomplish this, a deletion was introduced into the pilO gene of this organism. The isogenic knockout strain produced, 1244G7, was unable to glycosylate pilin but could produce pili normal in appearance and quantity. In addition, this strain had somewhat reduced twitching motility, was sensitive to pilus-specific bacteriophages, and could form a normal biofilm. Analysis of whole cells and isolated pili from wild-type P. aeruginosa strain 1244 by transmission electron microscopy with a glycan-specific immunogold label showed that this saccharide was distributed evenly over the fiber surface. The presence of the pilin glycan reduced the hydrophobicity of purified pili as well as whole cells. With regard to pathogenicity, P. aeruginosa strains producing glycosylated pili were commonly found among clinical isolates and particularly among those strains isolated from sputum. Competition index analysis using a mouse respiratory model comparing strains 1244 and 1244G7 indicated that the presence of the pilin glycan allowed for significantly greater survival in the lung environment. These results collectively suggest that the pilin glycan is a significant virulence factor and may aid in the establishment of infection.

Fine mapping of the N-terminal cytotoxicity region of Clostridium perfringens enterotoxin by site-directed mutagenesis.

Clostridium perfringens enterotoxin (CPE) has a unique mechanism of action that results in the formation of large, sodium dodecyl sulfate-resistant complexes involving tight junction proteins; those complexes then induce plasma membrane permeability alterations in host intestinal epithelial cells, leading to cell death and epithelial desquamation. Previous deletion and point mutational studies mapped CPE receptor binding activity to the toxin's extreme C terminus. Those earlier analyses also determined that an N-terminal CPE region between residues D45 and G53 is required for large complex formation and cytotoxicity. To more finely map this N-terminal cytotoxicity region, site-directed mutagenesis was performed with recombinant CPE (rCPE). Alanine-scanning mutagenesis produced one rCPE variant, D48A, that failed to form large complexes or induce cytotoxicity, despite having normal ability to bind and form the small complex. Two saturation variants, D48E and D48N, also had a phenotype resembling that of the D48A variant, indicating that both size and charge are important at CPE residue 48. Another alanine substitution rCPE variant, I51A, was highly attenuated for large complex formation and cytotoxicity, but rCPE saturation variants I51L and I51V displayed a normal large complex formation and cytotoxicity phenotype. Collectively, these mutagenesis results identify a core CPE sequence extending from residues G47 to I51 that directly participates in large complex formation and cytotoxicity.