Effects on childhood infections of promoting safe and hygienic complementary-food handling practices through a community-based programme: A cluster randomised controlled trial in a rural area of The Gambia.

BACKGROUND: The Gambia has high rates of under-5 mortality from diarrhoea and pneumonia, peaking during complementary-feeding age. Community-based interventions may reduce complementary-food contamination and disease rates. METHODS AND FINDINGS: A public health intervention using critical control points and motivational drivers, delivered February-April 2015 in The Gambia, was evaluated in a cluster randomised controlled trial at 6- and 32-month follow-up in September-October 2015 and October-December 2017, respectively. After consent for trial participation and baseline data were collected, 30 villages (clusters) were randomly assigned to intervention or control, stratified by population size and geography. The intervention included a community-wide campaign on days 1, 2, 17, and 25, a reminder visit at 5 months, plus informal community-volunteer home visits. It promoted 5 key complementary-food and 1 key drinking-water safety and hygiene behaviours through performing arts, public meetings, and certifications delivered by a team from local health and village structures to all villagers who attended the activities, to which mothers of 6- to 24-month-old children were specifically invited. Control villages received a 1-day campaign on domestic-garden water use. The background characteristics of mother and clusters (villages) were balanced between the trial arms. Outcomes were measured at 6 and 32 months in a random sample of 21-26 mothers per cluster. There were no intervention or research team visits to villages between 6 and 32 months. The primary outcome was a composite outcome of the number of times key complementary-food behaviours were observed as a proportion of the number of opportunities to perform the behaviours during the observation period at 6 months. Secondary outcomes included the rate of each recommended behaviour; microbiological growth from complementary food and drinking water (6 months only); and reported acute respiratory infections, diarrhoea, and diarrhoea hospitalisation. Analysis was by intention-to-treat analysis adjusted by clustering. (Registration: PACTR201410000859336). We found that 394/571 (69%) of mothers with complementary-feeding children in the intervention villages were actively involved in the campaign. No villages withdrew, and there were no changes in the implementation of the intervention. The intervention improved behaviour adoption significantly. For the primary outcome, the rate was 662/4,351(incidence rate [IR] = 0.15) in control villages versus 2,861/4,378 (IR = 0.65) in intervention villages (adjusted incidence rate ratio [aIRR] = 4.44, 95% CI 3.62-5.44, p < 0.001), and at 32 months the aIRR was 1.17 (95% CI 1.07-1.29, p = 0.001). Secondary health outcomes also improved with the intervention: (1) mother-reported diarrhoea at 6 months, with adjusted relative risk (aRR) = 0.39 (95% CI 0.32-0.48, p < 0.001), and at 32 months, with aRR = 0.68 (95% CI 0.48-0.96, p = 0.027); (2) mother-reported diarrhoea hospitalisation at 6 months, with aRR = 0.35 (95% CI 0.19-0.66, p = 0.001), and at 32 months, with aRR = 0.38 (95% CI 0.18-0.80, p = 0.011); and (3) mother-reported acute respiratory tract infections at 6 months, with aRR = 0.67 (95% CI 0.53-0.86, p = 0.001), though at 32 months improvement was not significant (p = 0.200). No adverse events were reported. The main limitations were that only medium to small rural villages were involved. Obtaining laboratory cultures from food at 32 months was not possible, and no stool microorganisms were investigated. CONCLUSIONS: We found that low-cost and culturally embedded behaviour change interventions were acceptable to communities and led to short- and long-term improvements in complementary-food safety and hygiene practices, and reported diarrhoea and acute respiratory tract infections. TRIAL REGISTRATION: The trial was registered on the 17th October 2014 with the Pan African Clinical Trial Registry in South Africa with number (PACTR201410000859336) and 32-month follow-up as an amendment to the trial.

Impact of pentavalent rotavirus vaccine against severe rotavirus diarrhoea in The Gambia.

INTRODUCTION: Rotavirus vaccines protect against the leading cause of severe childhood diarrhoea, and have been introduced in many low-income African countries. The Gambia introducedRotateq® (RV5) into their national immunization program in 2013. We revieweddata from an active rotavirus sentinel surveillancesitefor early evidence of vaccine impact. METHODS: We compared rotavirus prevalence in diarrhoeal stool in children< 5 years of age admittedat the Edward Francis Small Teaching Hospital sentinel surveillance site before (2013) andafterRV5 introduction (2015-2016) in the Gambia. The rotavirus-percent positive was separately compared for all diarrhoealhospitalizations and for hospitalizations with severe symptoms. Rotavirus prevalence was compared annually for the pre-vaccine year of 2013 with post-vaccine years of 2015 and 2016 using chi-square or Fisher's exact tests and the p-value to establish significant relationship was set at p < 0.05. All analyses were completed in SAS 9.3 (SAS Analytics, North Carolina). RESULTS: Rotavirus prevalence among all diarrhoeahospitalizations decreased from 22% in 2013 to 11% in 2015 (p = 0.04), while remaining unchanged in 2016 (18%, p = 0.56). For hospitalizations that were clinically severe and/or treated with intravenous fluids (mean of 46 per year), the rotavirus prevalence decreased from 33% in 2013 to 8% in 2015 (p = 0.04), and to 15% in 2016 (p = 0.08). The children with age <1 year accounted for 45% the population infected with rotavirus in both pre and post rotavirus vaccination periods. CONCLUSIONS: Rotavirus vaccine introduction in the Gambia could be among factors resulting in decreased diarrhea hospitalizations among children at the Edward Francis Small Teaching Hospital, particularly those with severe disease. These results support the continuation of rotavirus vaccine and additional monitoring of rotavirus hospitalization trends in the country.

Acceptability and feasibility of a screen-and-treat programme for hepatitis B virus infection in The Gambia: the Prevention of Liver Fibrosis and Cancer in Africa (PROLIFICA) study.

BACKGROUND: Despite the introduction of immunisation for hepatitis B virus (HBV) in the 1990s, HBV-related morbidity and mortality remain high in sub-Saharan Africa. Identification and treatment of asymptomatic people with chronic HBV infection should reduce the disease burden. We therefore assessed the feasibility of a screen-and-treat programme for HBV infection in The Gambia, west Africa, and estimated the proportion of HBV-infected people who had significant liver disease in need of treatment. METHODS: Between Dec 7, 2011, and Jan 24, 2014, individuals living in randomly selected communities in western Gambia were offered hepatitis B surface antigen (HBsAg) screening via a point-of-care test. The test was also offered to potential blood donors attending the central hospital in the capital, Banjul. HBsAg-positive individuals were invited for a comprehensive liver assessment and were offered treatment according to international guidelines. We defined linkage to care as visiting the liver clinic at least once. Eligibility for treatment was judged in accordance with the 2012 European Association for the Study of the Liver guidelines. FINDINGS: HBsAg screening was accepted by 5980 (weighted estimate 68·9%, 95% CI 65·0-72·4) of 8170 adults from 27 rural and 27 urban communities and 5559 (81·4%, 80·4-82·3) of 6832 blood donors. HBsAg was detected in 495 (8·8%, 7·9-9·7) individuals in communities and 721 (13·0%, 12·1-13·9) blood donors. Prevalence was higher in men (239 [10·5%, 8·9-12·1] of 2328 men vs 256 [7·6%, 6·5-8·7] of 3652 women; p=0·004) and middle-aged participants. Linkage to care was high in the communities, with 402 (81·3%) of 495 HBsAg-positive individuals attending the clinic. However, only 300 (41·6%) of 721 HBsAg-positive people screened at the blood bank linked into care. Of those who attended the clinic, 18 (4·4%, 2·5-7·7) patients from the communities and 29 (9·7%, 6·8-13·6) from the blood bank were eligible for treatment. Male sex was strongly associated with treatment eligibility (odds ratio 4·35, 1·50-12·58; p=0·007). INTERPRETATION: HBV infection remains highly prevalent in The Gambia. The high coverage of community-based screening, good linkage into care, and the small proportion of HBsAg carriers who need treatment suggest that large-scale screening and treatment programmes are feasible in sub-Saharan Africa. FUNDING: European Commission (FP7).

Natural history of chronic HBV infection in West Africa: a longitudinal population-based study from The Gambia.

BACKGROUND: The natural history of chronic HBV infection in sub-Saharan Africa is unknown. Data are required to inform WHO guidelines that are currently based on studies in Europe and Asia. METHODS: Between 1974 and 2008, serosurveys were repeated in two Gambian villages, and an open cohort of treatment-naive chronic HBV carriers was recruited. Participants were followed to estimate the rates of hepatitis B e (HBeAg) and surface antigen (HBsAg) clearance and incidence of hepatocellular carcinoma (HCC). In 2012-2013, a comprehensive liver assessment was conducted to estimate the prevalence of severe liver disease. RESULTS: 405 chronic carriers (95% genotype E), recruited at a median age of 10.8 years, were followed for a median length of 28.4 years. Annually, 7.4% (95% CI 6.3% to 8.8%) cleared HBeAg and 1.0% (0.8% to 1.2%) cleared HBsAg. The incidence of HCC was 55.5/100 000 carrier-years (95% CI 24.9 to 123.5). In the 2012-2013 survey (n=301), 5.5% (95% CI 3.4% to 9.0%) had significant liver fibrosis. HBV genotype A (versus E), chronic aflatoxin B1 exposure and an HBsAg-positive mother, a proxy for mother-to-infant transmission, were risk factors for liver fibrosis. A small proportion (16.0%) of chronic carriers were infected via mother-to-infant transmission; however, this population represented a large proportion (63.0%) of the cases requiring antiviral therapy. CONCLUSIONS: The incidence of HCC among chronic HBV carriers in West Africa was higher than that in Europe but lower than rates in East Asia. High risk of severe liver disease among the few who are infected by their mothers underlines the importance of interrupting perinatal transmission in sub-Saharan Africa.

The gamma-glutamyl transpeptidase to platelet ratio (GPR) predicts significant liver fibrosis and cirrhosis in patients with chronic HBV infection in West Africa.

BACKGROUND: Simple and inexpensive non-invasive fibrosis tests are highly needed but have been poorly studied in sub-Saharan Africa. METHODS: Using liver histology as a gold standard, we developed a novel index using routine laboratory tests to predict significant fibrosis in patients with chronic HBV infection in The Gambia, West Africa. We prospectively assessed the diagnostic accuracy of the novel index, Fibroscan, aspartate transaminase-to-platelet ratio index (APRI), and Fib-4 in Gambian patients with CHB (training set) and also in French and Senegalese CHB cohorts (validation sets). RESULTS: Of 135 consecutive treatment-naïve patients with CHB who had liver biopsy, 39% had significant fibrosis (Metavir fibrosis stage ≥F2) and 15% had cirrhosis (F4). In multivariable analysis, gamma-glutamyl transpeptidase (GGT) and platelet count were independent predictors of significant fibrosis. Consequently, GGT-to-platelet ratio (GPR) was developed. In The Gambia, the area under the receiver operating characteristic curve (AUROC) of the GPR was significantly higher than that of APRI and Fib-4 to predict ≥F2, ≥F3 and F4. In Senegal, the AUROC of GPR was significantly better than Fib-4 and APRI for ≥F2 (0.73, 95% CI 0.59 to 0.86) and better than Fib-4 and Fibroscan for ≥F3 (0.93, 0.87 to 0.99). In France, the AUROC of GPR to diagnose ≥F2 (0.72, 95% CI 0.59 to 0.85) and F4 (0.87, 0.76 to 0.98) was equivalent to that of APRI and Fib-4. CONCLUSIONS: The GPR is a more accurate routine laboratory marker than APRI and Fib-4 to stage liver fibrosis in patients with CHB in West Africa. The GPR represents a simple and inexpensive alternative to liver biopsy and Fibroscan in sub-Saharan Africa.

Malaria Prevalence among Young Infants in Different Transmission Settings, Africa.

The prevalence and consequences of malaria among infants are not well characterized and may be underestimated. A better understanding of the risk for malaria in early infancy is critical for drug development and informed decision making. In a cross-sectional survey in Guinea, The Gambia, and Benin, countries with different malaria transmission intensities, the overall prevalence of malaria among infants <6 months of age was 11.8% (Guinea, 21.7%; The Gambia, 3.7%; and Benin, 10.2%). Seroprevalence ranged from 5.7% in The Gambia to 41.6% in Guinea. Mean parasite densities in infants were significantly lower than those in children 1-9 years of age in The Gambia (p<0.0001) and Benin (p = 0.0021). Malaria in infants was significantly associated with fever or recent history of fever (p = 0.007) and anemia (p = 0.001). Targeted preventive interventions, adequate drug formulations, and treatment guidelines are needed to address the sizeable prevalence of malaria among young infants in malaria-endemic countries.

Birth order and risk of hepatocellular carcinoma in chronic carriers of hepatitis B virus: a case-control study in The Gambia.

BACKGROUND & AIMS: Early age at infection with Hepatitis B virus (HBV) increases the risk of chronic infection. Moreover, early HBV infection may further independently increase the risk of hepatocellular carcinoma (HCC) beyond its effect on chronicity. METHODS: The distribution of birth order, a proxy for mode and timing of HBV transmission, was compared in The Gambia between hepatitis B surface antigen (HBsAg)-positive HCC cases recruited from hospitals (n = 72) and two HBsAg-positive control groups without HCC: population-based controls from a community HBV screening (n = 392) and hospital-based controls (n = 63). RESULTS: HCC risk decreased with increasing birth order in the population-based case-control analysis. Using first birth order as the reference, the odds ratios were 0.52 (95% CI: 0.20-1.36), 0.52 (0.17-1.56), 0.57 (0.16-2.05) and 0.14 (0.03-0.64) for second, third, fourth and greater than fourth birth order respectively (P = 0.01). A similar inverse association was observed in the hospital-based case-control comparison (P = 0.04). CONCLUSIONS: Compared to controls, HCC cases had earlier birth order, a proxy for young maternal age and maternal HBV viraemia at birth. This finding suggests that in chronic HBV carriers perinatal mother-to-infant transmission may increase HCC risk more than horizontal transmission. Providing HBV vaccine within 24 h of birth to interrupt perinatal transmission might reduce the incidence of HCC in The Gambia.

Validation of rapid point-of-care (POC) tests for detection of hepatitis B surface antigen in field and laboratory settings in the Gambia, Western Africa.

Hepatitis B virus (HBV) infection is a leading cause of death in sub-Saharan Africa (SSA). Point-of-care tests for hepatitis B surface antigen (HBsAg) could be an ideal tool for a large-scale HBV screening/treatment program in SSA. Using data from the PROLIFICA (Prevention of Liver Fibrosis and Cancer in Africa) program, we conducted a cross-sectional study to assess the diagnostic accuracy of three point-of-care tests (Determine, Vikia, and Espline) for the detection of HBsAg in the field or a laboratory setting in the Gambia. In the field, we used finger-prick whole blood for the Determine and Vikia tests and dried blood spots for the reference standard test (AxSYM HBsAg enzyme-linked immunosorbent assay [ELISA]). In the laboratory we used serum for the Determine, Espline, and reference test (Architect chemiluminescent microparticle immunoassay). Of 773 participants recruited at the community and 227 known chronic HBV carriers (1,000 subjects in total), 293 were positive for HBsAg. The sensitivity and specificity of the Determine test were 88.5% and 100% in the field and 95.3% and 93.3% in the laboratory setting, respectively. The sensitivity and specificity were 90.0% and 99.8% for the Vikia test (in the field) and 93.9% and 94.7% for the Espline test (in the laboratory). There was no evidence that one kit was better than another. Most of the patients with false-negative results (18/19) were classified as inactive chronic carriers. In summary, the three point-of-care tests had acceptable ranges of diagnostic accuracy. These tests may represent accurate, rapid, and inexpensive alternatives to serology testing for the screening of HBV infection at field level in SSA.

Discovery and validation of urinary metabotypes for the diagnosis of hepatocellular carcinoma in West Africans.

UNLABELLED: There is no clinically applicable biomarker for surveillance of hepatocellular carcinoma (HCC), because the sensitivity of serum alpha-fetoprotein (AFP) is too low for this purpose. Here, we determined the diagnostic performance of a panel of urinary metabolites of HCC patients from West Africa. Urine samples were collected from Nigerian and Gambian patients recruited on the case-control platform of the Prevention of Liver Fibrosis and Cancer in Africa (PROLIFICA) program. Urinary proton nuclear magnetic resonance ((1) H-NMR) spectroscopy was used to metabolically phenotype 290 subjects: 63 with HCC; 32 with cirrhosis (Cir); 107 with noncirrhotic liver disease (DC); and 88 normal control (NC) healthy volunteers. Urine samples from a further cohort of 463 subjects (141 HCC, 56 Cir, 178 DC, and 88 NC) were analyzed, the results of which validated the initial cohort. The urinary metabotype of patients with HCC was distinct from those with Cir, DC, and NC with areas under the receiver operating characteristic (AUROC) curves of 0.86 (0.78-0.94), 0.93 (0.89-0.97), and 0.89 (0.80-0.98) in the training set and 0.81 (0.73-0.89), 0.96 (0.94-0.99), and 0.90 (0.85-0.96), respectively, in the validation cohort. A urinary metabolite panel, comprising inosine, indole-3-acetate, galactose, and an N-acetylated amino acid (NAA), showed a high sensitivity (86.9% [75.8-94.2]) and specificity (90.3% [74.2-98.0]) in the discrimination of HCC from cirrhosis, a finding that was corroborated in a validation cohort (AUROC: urinary panel = 0.72; AFP = 0.58). Metabolites that were significantly increased in urine of HCC patients, and which correlated with clinical stage of HCC, were NAA, dimethylglycine, 1-methylnicotinamide, methionine, acetylcarnitine, 2-oxoglutarate, choline, and creatine. CONCLUSION: The urinary metabotyping of this West African cohort identified and validated a metabolite panel that diagnostically outperforms serum AFP.

20-years of population-based cancer registration in hepatitis B and liver cancer prevention in the Gambia, West Africa.

BACKGROUND: The Gambia Hepatitis Intervention Study (GHIS) was designed as a randomised control trial of infant hepatitis B vaccination applied to public health policy, with the main goal of preventing primary liver cancer later in adult life in The Gambia. To that effect, the National Cancer Registry of The Gambia (NCR), a population-based cancer registry (PBCR), was established in 1986 to actively collect data on all cancer diagnosis nation-wide. We extracted 20-years (1990-2009) of data to assess for the first time, the evolution of the most common cancers, also describe and demonstrate the role of the PBCR in a hepatitis B and liver cancer prevention programme in this population. METHODS AND FINDINGS: We estimated Age-Standardised Incidence Rates (ASR (W)) of the most common cancers registered during the period by gender. The registration period was divided into four 5-year intervals and incidence rates were estimated for each interval. The most common cancers in males were liver, prostate, lung plus bronchus, non-Hodgkin lymphoma (NHL) and stomach, accounting for 60%, 5%, 4%, 5% and 3%, respectively. Similarly, cancers of the cervix uteri, liver, breast and NHL, were the most common in females, accounting for 33%, 24%, 11% and 4% of the female cancers, respectively. CONCLUSIONS: Cancer incidence has remained relatively stable over time, but as shown elsewhere in sub-Saharan Africa the disease is a threat in The Gambia. The infection related cancers which are mostly preventable (HBV in men and HPV/HIV in women) were the most common. At the moment the data is not enough to detect an effect of hepatitis B vaccination on liver cancer incidence in The Gambia. However, we observed that monitoring case occurrence through PBCR is a key public health pre-requisite for rational planning and implementation of targeted interventions for improving the health of the population.

Detecting Foci of Malaria Transmission with School Surveys: A Pilot Study in the Gambia.

BACKGROUND: In areas of declining malaria transmission such as in The Gambia, the identification of malaria infected individuals becomes increasingly harder. School surveys may be used to identify foci of malaria transmission in the community. METHODS: The survey was carried out in May-June 2011, before the beginning of the malaria transmission season. Thirty two schools in the Upper River Region of The Gambia were selected with probability proportional to size; in each school approximately 100 children were randomly chosen for inclusion in the study. Each child had a finger prick blood sample collected for the determination of antimalarial antibodies by ELISA, malaria infection by microscopy and PCR, and for haemoglobin measurement. In addition, a simple questionnaire on socio-demographic variables and the use of insecticide-treated bed nets was completed. The cut-off for positivity for antimalarial antibodies was obtained using finite mixture models. The clustered nature of the data was taken into account in the analyses. RESULTS: A total of 3,277 children were included in the survey. The mean age was 10 years (SD = 2.7) [range 4-21], with males and females evenly distributed. The prevalence of malaria infection as determined by PCR was 13.6% (426/3124) [95% CI = 12.2-16.3] with marked variation between schools (range 3-25%, p<0.001), while the seroprevalence was 7.8% (234/2994) [95%CI = 6.4-9.8] for MSP119, 11.6% (364/2997) [95%CI = 9.4-14.5] for MSP2, and 20.0% (593/2973) [95% CI = 16.5-23.2) for AMA1. The prevalence of all the three antimalarial antibodies positive was 2.7% (79/2920). CONCLUSIONS: This survey shows that malaria prevalence and seroprevalence before the transmission season were highly heterogeneous.

Malaria in infants aged less than six months - is it an area of unmet medical need?

Despite the protection provided by several factors, including maternal antibodies, the burden of malaria in young infants may be higher than previously thought. Infants with congenital or neonatal malaria may have a different clinical presentation than older children, and diagnosis may be confused with other neonatal diseases due to an overlap of clinical manifestations. In addition, there is little information on the use of artemisinin-based combination therapy in young infants. There is the need for a more accurate estimate of the parasite prevalence and the incidence of clinical malaria in infants under 6 months old, as well as a better characterization of risk factors, pharmacokinetic profiles, safety and efficacy of currently available anti-malarial treatments, in order to develop evidence-based treatment guidelines for this population.

Continued decline of malaria in The Gambia with implications for elimination.

BACKGROUND: A substantial decline in malaria was reported to have occurred over several years until 2007 in the western part of The Gambia, encouraging consideration of future elimination in this previously highly endemic region. Scale up of interventions has since increased with support from the Global Fund and other donors. METHODOLOGY/PRINCIPAL FINDINGS: We continued to examine laboratory records at four health facilities previously studied and investigated six additional facilities for a 7 year period, adding data from 243,707 slide examinations, to determine trends throughout the country until the end of 2009. We actively detected infections in a community cohort of 800 children living in rural villages throughout the 2008 malaria season, and assayed serological changes in another rural population between 2006 and 2009. Proportions of malaria positive slides declined significantly at all of the 10 health facilities between 2003 (annual mean across all sites, 38.7%) and 2009 (annual mean, 7.9%). Statistical modelling of trends confirmed significant seasonality and decline over time at each facility. Slide positivity was lowest in 2009 at all sites, except two where lowest levels were observed in 2006. Mapping households of cases presenting at the latter sites in 2007-2009 indicated that these were not restricted to a few residual foci. Only 2.8% (22/800) of a rural cohort of children had a malaria episode in the 2008 season, and there was substantial serological decline between 2006 and 2009 in a separate rural area. CONCLUSIONS: Malaria has continued to decline in The Gambia, as indicated by a downward trend in slide positivity at health facilities, and unprecedented low incidence and seroprevalence in community surveys. We recommend intensification of control interventions for several years to further reduce incidence, prior to considering an elimination programme.

Bivalent recombinant vaccine for botulinum neurotoxin types A and B based on a polypeptide comprising their effector and translocation domains that is protective against the predominant A and B subtypes.

The botulinum neurotoxins (BoNTs) are a large family of extremely potent, neuroparalytic, dichain proteins which act at the peripheral nervous system. The wide genetic diversity observed with this neurotoxin family poses a significant challenge for the development of an effective botulinum vaccine. The present study describes a vaccine development platform based on protein fragments representing the N-terminal two-thirds of each toxin molecule. These fragments, designated LH(N), comprise the light chain and translocation domains of each neurotoxin and are devoid of any neuron-binding activity. Using codon-optimized genes, LH(N) fragments derived from BoNT serotypes A and B were expressed in Escherichia coli in high yield with >1 g of purified, soluble fragment recoverable from 4.5 liter-scale fermentations. The protective efficacy of LH(N)/A was significantly enhanced by treatment with formaldehyde, which induced intramolecular cross-linking but virtually no aggregation of the fragment. A single immunization of the modified fragment protected mice from challenge with a 10(3) 50% lethal dose (LD(50)) of BoNT/A(1) with an 50% effective dose (ED(50)) of 50 ng of the vaccine. In similar experiments, the LH(N)/A vaccine was shown to protect mice against challenge with BoNT/A subtypes A(1), A(2), and A(3), which is the first demonstration of single-dose protection by a vaccine against the principal toxin subtypes of BoNT/A. The LH(N)/B vaccine was also highly efficacious, giving an ED(50) of approximately 140 ng to a challenge of 10(3) LD(50) of BoNT/B(1). In addition, LH(N)/B provided single-dose protection in mice against BoNT/B(4) (nonproteolytic toxin subtype).

Substrate-induced conformational changes in Bacillus subtilis glutamate racemase and their implications for drug discovery.

D-glutamate is an essential building block of the peptidoglycan layer in bacterial cell walls and can be synthesized from L-glutamate by glutamate racemase (RacE). The structure of a complex of B. subtilis RacE with D-glutamate reveals that the glutamate is buried in a deep pocket, whose formation at the interface of the enzyme's two domains involves a large-scale conformational rearrangement. These domains are related by pseudo-2-fold symmetry, which superimposes the two catalytic cysteine residues, which are located at equivalent positions on either side of the alpha carbon of the substrate. The structural similarity of these two domains suggests that the racemase activity of RacE arose as a result of gene duplication. The structure of the complex is dramatically different from that proposed previously and provides new insights into the RacE mechanism and an explanation for the potency of a family of RacE inhibitors, which have been developed as novel antibiotics.

Expression, purification and preliminary X-ray analysis of crystals of Bacillus subtilis glutamate racemase.

Glutamate racemase (MurI, RacE; E.C.5.1.1.3) catalyses the cofactor-independent conversion of L-glutamate to D-glutamate, an essential step in the synthesis of components of the bacterial cell wall. The gene for RacE from Bacillus subtilis has been cloned and the protein expressed in Escherichia coli, purified and crystallized in the presence of L-glutamate using the hanging-drop method of vapour diffusion with diammonium tartrate as the precipitant. The crystals belong to the monoclinic space group C2, with approximate unit-cell parameters a = 133.6, b = 60.1, c = 126.2 A, beta = 117.6 degrees . Consideration of the possible values of V(M) suggests that the asymmetric unit contains either two (V(M) = 3.75 A(3) Da(-1)) or three (V(M) = 2.5 A(3) Da(-1)) subunits. The crystals diffract X-rays to at least 2.1 A resolution on a synchrotron-radiation source and are suitable for structural studies. Determination of the structure may provide insight into the molecular basis of substrate recognition and catalysis by this enzyme.