Examination of ELISA against PCR for assessing treatment efficacy against Cryptosporidium in a clinical trial context.

BACKGROUND: Cryptosporidium is a gastrointestinal pathogen that presents a serious opportunistic infection in immunocompromised individuals including those living with human immunodeficiency syndrome. The CRYPTOFAZ trial, previously published, was conducted in Malawi to evaluate the efficacy of clofazimine in response to an unmet need for drugs to treat cryptosporidiosis in HIV populations. A combination of rapid diagnostic tests, ELISA, qPCR, and conventional sequencing were employed to detect Cryptosporidium in 586 individuals during pre-screening and monitor oocyst shedding and identify enteric co-pathogens in 22 enrolled/randomized participants during the in-patient period and follow-up visits. METHODOLOGY: Oocyst shedding as measured by qPCR was used to determine primary trial outcomes, however pathogen was detected even at trial days 41-55 in individuals randomized to either clofazimine or placebo arms of the study. Therefore, in this work we re-examine the trial outcomes and conclusions in light of data from the other diagnostics, particularly ELISA. ELISA data was normalized between experiments prior to comparison to qPCR. The amount of all identified enteric pathogens was examined to determine if co-pathogens other than Cryptosporidium were major causative agents to a participant's diarrhea. CONCLUSION: ELISA had higher sample-to-sample variability and proved to be equally or less sensitive than qPCR in detecting Cryptosporidium positive samples. Compared to qPCR, ELISA had equal or greater specificity in detecting Cryptosporidium negative samples. Sequencing identified several Cryptosporidium species including viatorum which has never been identified in Malawi and Southern Africa. In addition to Cryptosporidium, enterotoxigenic E. coli was also identified as a pathogen in diarrheagenic amounts in 4 out of 22 participants.

Impact of leachate on quality of ground water around Chunga Landfill, Lusaka, Zambia and possible health risks.

We report the characteristics and possible impact of leachate on quality of groundwater in the Chunga Landfill area of Lusaka, Zambia. Water and leachate samples were collected within and around the landfill for analysis. The pH, biological oxygen demand (BOD), chemical oxygen demand (COD), nitrates, sulphates, chlorides for the leachate and groundwater samples were (6.6 ± 0.1 to 8.7 ± 0.0), (1.7 ± 0.3 to 1,569.6 ± 4.9 mg/L), (4.0 ± 0.0 to 10,378.5 ± 59.2 mg/L), (8.0 ± 0.0 to 37.7 ± 0.4 mg/L), (11.7 ± 0.0 to 273.1 ± 1.7 mg/L), (43.0 ± 1.2 to 974.2 ± 0.8 mg/L) respectively. Heavy metal concentration ranges were cadmium (0.004 ± 0.000 to 1.149 ± 0.021 mg/L, chromium (0.007 ± 0.000 to 2.699 ± 0.039 mg/L), copper (0.013 ± 0.002 to 0.246 ± 0.005 mg/L), lead (0.062 ± 0.005 to 2.591 ± 0.065 mg/L) and zinc (0.008 ± 0.001 to 2.032 ± 0.017 mg/L). The pH of the leachate (8.5 ± 0.0 to 8.7 ± 0.0) meant the landfill was in the methane fermentation phase. An indexing approach was used with the leachate pollution index (LPI) of 30.173, heavy metal pollution index (HPI) of 3,938.92. The heavy metal index (HMI) for copper, lead, chromium, cadmium and zinc were found to be 0.92, 1,124.19, 47.20, 994.17 and 1.48 respectively. Principal component analysis (PCA) showed that anthropogenic activities contributed to pollution with high loading values. Ash from continuous burning of the waste may provide alkalinity which reduces leachate BOD and COD. Results showed that the landfill has outgrown the designed cells capacity as not all leachate was collected by the under-drainage. Results also showed that lack of adequate landfill cover significantly increases rainfall infiltration thereby increasing volumes of leachate produced with a, hence potential for underground water contamination and a human health and environmental problem.

Developing High-Resolution Population and Settlement Data for Impactful Malaria Interventions in Zambia.

Foundational high-resolution geospatial data products for population, settlements, infrastructure, and boundaries may greatly enhance the efficient planning of resource allocation during health sector interventions. To ensure the relevance and sustainability of such products, government partners must be involved from the beginning in their creation, improvement, and/or management, so they can be successfully applied to public health campaigns, such as malaria control and prevention. As an example, Zambia had an ambitious strategy of reaching the entire population with malaria vector control campaigns by late 2020 or early 2021, but they lacked the requisite accurate and up-to-date data on infrastructure and population distribution. To address this gap, the Geo-Referenced Infrastructure and Demographic Data for Development (GRID3) program, Akros, and other partners developed maps and planning templates to aid Zambia's National Malaria Elimination Program (NMEP) in operationalizing its strategy.

Knowledge, Attitudes and Practices Towards Human Papillomavirus Vaccination Among Medical Doctors at a Tertiary Hospital: A Cross Sectional Study.

BACKGROUND: Human papillomavirus (HPV) infection is a common sexually transmitted disease, characterised by persistent infection with high-risk strains leading to malignant conditions such as cervical cancer. The HPV vaccine is a well-known primary preventive measure for HPV infections. Previous studies have shown that medical doctors' vaccine recommendation is one of the key strategies in improving HPV vaccine uptake. In 2019, Zambia rolled out the free national HPV vaccination program targeting 14-year-old girls. However, the annual coverage for HPV vaccination is variable, with rates as low as 33% for 2021. MATERIALS AND METHODS: We conducted a cross-sectional study between September and December 2020 at the University Teaching Hospitals in Lusaka, Zambia. We used analysis of variance to assess the mean differences in the overall scores for knowledge, attitude and practices towards the HPV vaccine. In addition, we used structural equation modelling (SEM) to test the traditional education theory as medical doctors' HPV vaccine knowledge, attitude, and practices cover several facets, and SEM can model latent variables. RESULTS: We enrolled 121 medical doctors, of whom 67 (44.6%) were male. The majority, 76 (62.8%), were registrars and 79 (65.3%) had more than ten years of clinical experience. The overall mean knowledge, attitude, and practice score of the HPV vaccine mean (SD) were 70.2 (15.1), 72.1 (18.5) and 77.1 (28.9), respectively. More than half of the medical doctors would advise anyone eligible to take the HPV vaccine 66 (54.6%). There was a positive correlation between attitude and practice towards the HPV vaccine (ß = .03, P = .017). Conversely, there was no evidence of a correlation between overall HPV knowledge and attitude (ß = .01, P = .670) and rank of the medical doctors (ß = -7.87, P = .355). CONCLUSION: Knowledge was high with good attitudes and practices among medical doctors, which are vital in vaccine recommendation and subsequent uptake.

Reducing maternal and neonatal mortality through integrated and sustainability-focused programming in Zambia.

Reducing maternal and neonatal mortality is a critical health priority within Zambia and globally. Although evidence-based clinical interventions can prevent a majority of these deaths, scalable and sustainable delivery of interventions across low-resource settings remains uneven, particularly across rural and marginalized communities. The Zambian Ministry of Health and the Clinton Health Access Initiative implemented an integrated sexual, reproductive, maternal, and newborn health (SRMNH) program in Northern Province aimed at dramatically reducing mortality over four years. Interventions were implemented between 2018 and 2021 across 141 government-owned health facilities covering all 12 districts of Northern Province, the poorest performing province nationwide and home to over 1.4 million people, around six pillars of an integrated health system. Data on institutional delivery and antenatal and postnatal care were collected through the national Health Management Information System (HMIS). A community-based system for capturing birth outcomes was established using existing government tools and community volunteers since HMIS did not include community-based mortality. Baseline and endline population-based mortality rates were compared for program-supported areas. From the earliest period of population-based mortality reporting in 2019 to program end in 2021, there were statistically significant decreases of 41%, 45%, and 43% in maternal, neonatal, and perinatal mortality rates respectively. Between 2017 to 2021, institutional maternal, neonatal, and perinatal mortality rates across entirety of Northern Province reduced by 12%, 40%, and 41%, respectively. Service readiness and coverage for SRMNH services improved dramatically, supporting increased numbers of patients. Significant mortality reductions were achieved over a relatively short period, reinforced through an emphasis on sustainability and strengthening existing government systems. These results were attained through a consciously cost-efficient approach backed by substantially lower levels of external investment relative to prior programs, allowing many of the interventions to be successfully adopted by government within public sector budgets.

Performance and safety of the induced sputum procedure in young children in Malawi: a prospective study.

BACKGROUND: Collecting sputum specimens is a challenge in infants and young children. We assessed the performance and safety of induced sputum (IS) collection in this population, embedded in a prospective study evaluating respiratory cryptosporidiosis in Malawian children with diarrheal disease. METHODS: We assessed the sputum quality and correlation with detection of Cryptosporidium spp. and evaluated safety and adverse events in 162 children. RESULTS: Among 159 stool specimens tested, 34 (21%, 95% CI 15.0 to 28%) were positive for Cryptosporidium spp. There were 160 IS and 161 nasopharyngeal (NP) specimens collected. IS and NP specimen collection was performed for each patient. The majority of IS specimens (122/147; 83%) were clear in appearance and 132/147 (90%) were of good quality. Among the respiratory specimens tested, 10 (6.3%, 95% CI 2.5 to 10%) IS and 4 (3%, 95% CI 0 to 5%) NP were positive for Cryptosporidium spp. When stool cryptosporidium PCR was the gold standard, IS PCR sensitivity was higher (29%, 95% CI 22 to 37%) compared with NP PCR (12%, 95% CI 7 to 17%) for detection of Cryptosporidium spp. One (0.4%) adverse event occurred, consisting of a drop in oxygen saturations at the 30-min postprocedure evaluation. Consciousness level, median respiratory rate and oxygen saturations were unchanged, before or after IS. CONCLUSIONS: IS provides good quality specimens, is more sensitive than NP specimens for diagnosis of respiratory cryptosporidiosis, and collection can be performed safely in children hospitalized with diarrheal disease.

Respiratory cryptosporidiosis in Malawian children with diarrheal disease.

BACKGROUND: Respiratory cryptosporidiosis has been documented in children with diarrhea. We sought to describe the dynamics of respiratory involvement in children hospitalized with gastrointestinal (GI) diarrheal disease. METHODS: We conducted a prospective, observational longitudinal study of Malawian children 2-24 months hospitalized with diarrhea. Nasopharyngeal (NP) swabs, induced sputum and stool specimens were collected. Participants that were positive by Cryptosporidium PCR in any of the three compartments were followed up with fortnightly visits up to 8 weeks post-enrollment. RESULTS: Of the 162 children recruited, participants had mild-moderate malnutrition (mean HAZ -1.6 (SD 2.1)), 37 (21%) were PCR-positive for Cryptosporidium at enrollment (37 stool, 11 sputum, and 4 NP) and 27 completed the majority of follow-up visits (73%). Cryptosporidium was detected in all compartments over the 4 post-enrollment visits, most commonly in stool (100% at enrollment with mean cycle thresholds (Ct) of 28.8±4.3 to 44% at 8 weeks with Ct 29.9±4.1), followed by sputum (31% at enrollment with mean Ct 31.1±4.4 to 20% at 8 weeks with Ct 35.7±2.6), then NP (11% with mean Ct 33.5±1.0 to 8% with Ct 36.6±0.7). Participants with Cryptosporidium detection in both the respiratory and GI tract over the study period reported respiratory and GI symptoms in 81% and 62% of study visits, respectively, compared to 68% and 27%, respectively, for those with only GI detection, and had longer GI shedding (17.5±6.6 v. 15.9±2.9 days). CONCLUSION: Cryptosporidium was detected in both respiratory and GI tracts throughout the 8 weeks post-enrollment. The development of therapeutics for Cryptosporidium in children should target the respiratory as well as GI tract.

Exhaustive valorization of cashew nut shell waste as a potential bioresource material.

In this paper, we report extraction of cashew nut shell liquid (CNSL) from cashew nut shell waste (CNSW) and further use of residues for generation of activated carbon for removal of heavy metals and methylene blue (MB). Solvent extraction yielded 24.6 ± 0.4%, 38.2 ± 0.4% and 40.1 ± 0.9% for petroleum ether, hexane and ethanol respectively. Phytochemical screening showed presence of alkaloids, carbohydrates, saponins, phenols, tannins, flavonoids, amino acids, terpenoids, proteins, steroids, glycosides and carboxylic acids. The CNSL had a pH of 3.2, viscosity (104.6 ± 1.8 mPa s), moisture (6.5%), ash (1.6 ± 0.1%), refractive index (1.52 ± 0.001), specific density (0.9561 ± 0.0002 g/cm3), acid value (118.7 ± 9.2 mg KOH/g), free fatty acid value (60.1 ± 4.7%), saponification number (138.1 ± 3.2 mg KOH/g) and iodine value (188.1 ± 2.3 mgI 2/100 g). The average percentage removal of Cu (II), Pb (II), Cd (II) and Zn (II) was 99.4 ± 0.5, 95.4 ± 1.5, 99.5 ± 0.1, 98.4 ± 0.1%, and removal efficiency of MB at 50, 150, 250 and 350 mg/L was 99.63, 97.66, 96.48 and 94.81%, respectively. Equilibrium data were best described by the Freundlich isotherm model. The maximum monolayer adsorption capacity was 12.1 mg/g. The adsorption kinetics conformed to pseudo-second-order model. ∆G° was negative and a ∆H° of + 22.76 kJ/mol indicated that adsorption was endothermic. The ΔS° (+ 0.086 kJ/mol/K) showed that there was spontaneous interaction of the solution and adsorbate. These results show that CNSW is a potential bioresource for CNSL production for use in the paints, varnishes, surface coatings, agrochemicals and ethnomedicine industries. Residual shells can be exploited as fuels or converted to activated carbon for use as low-cost filters in water purification.

Distinct clinical and immunological profiles of patients with evidence of SARS-CoV-2 infection in sub-Saharan Africa.

Although the COVID-19 pandemic has left no country untouched there has been limited research to understand clinical and immunological responses in African populations. Here we characterise patients hospitalised with suspected (PCR-negative/IgG-positive) or confirmed (PCR-positive) COVID-19, and healthy community controls (PCR-negative/IgG-negative). PCR-positive COVID-19 participants were more likely to receive dexamethasone and a beta-lactam antibiotic, and survive to hospital discharge than PCR-negative/IgG-positive and PCR-negative/IgG-negative participants. PCR-negative/IgG-positive participants exhibited a nasal and systemic cytokine signature analogous to PCR-positive COVID-19 participants, predominated by chemokines and neutrophils and distinct from PCR-negative/IgG-negative participants. PCR-negative/IgG-positive participants had increased propensity for Staphylococcus aureus and Streptococcus pneumoniae colonisation. PCR-negative/IgG-positive individuals with high COVID-19 clinical suspicion had inflammatory profiles analogous to PCR-confirmed disease and potentially represent a target population for COVID-19 treatment strategies.

Assessing the biodegradability of common pharmaceutical products (PPs) on the Zambian market.

Biodegradation is the breakdown of complex organic compounds into simpler molecules like carbon dioxide and water by microorganisms like bacteria and fungi. Biodegradation studies of pharmaceuticals are initially done to assess which pharmaceuticals are persistent in the environment. Whole pharmaceuticals or their metabolites are excreted from the human body via urine or fecal matter after administration. These go into the Wastewater Treatment Plants (WWTP) and are later released into the environment with the treated wastewater. Recent studies have reported a number of pharmaceuticals in the ecosystem and the effects of these on non-target species has become an issue of environmental concern. The biodegradation studies of eight pharmaceuticals were carried out in this research. The choice of pharmaceuticals was based on the most commonly prescribed medications at the University of Zambia (UNZA) Clinic in seven therapeutic groups: anti-hypertensives, antibiotic, antimalarial drugs, anti-tuberculosis, antihelminthics, antifungals and antiretroviral drugs. The biodegradability tests were carried out using a modified carbon dioxide evolution method (modified Sturm test). The inoculum was derived from the secondary effluent of the UNZA WWTP plant and Dextrose monohydrate was used as a system control. Using this guideline, the system control, dextrose monohydrate biodegraded 77 ± 0.270% in seven days. All the pharmaceuticals except ciprofloxacin were found to be non-biodegradable: Atenolol degraded 6.8 ± 0.026%, ketoconazole degraded 1.0 ± 0.003%, isoniazid/rifampicin degraded 0.8 ± 0.003%, mebendazole degraded 13.0 ± 0.050%, nevirapine degraded 1.3 ± 0.005%, pen-v degraded 1.0 ± 0.004% and quinine sulfate degraded 1.8 ± 0.008%. Ciprofloxacin showed a negative carbon dioxide evolution and it was noted that bacteria were not viable as the drug proved to be very potent against bacterial strains in the inoculum used.

Distinct Biomarker Profiles Distinguish Malawian Children with Malarial and Non-malarial Sepsis.

Presently, it is difficult to accurately diagnose sepsis, a common cause of childhood death in sub-Saharan Africa, in malaria-endemic areas, given the clinical and pathophysiological overlap between malarial and non-malarial sepsis. Host biomarkers can distinguish sepsis from uncomplicated fever, but are often abnormal in malaria in the absence of sepsis. To identify biomarkers that predict sepsis in a malaria-endemic setting, we retrospectively analyzed data and sera from a case-control study of febrile Malawian children (aged 6-60 months) with and without malaria who presented to a community health center in Blantyre (January-August 2016). We characterized biomarkers for 29 children with uncomplicated malaria without sepsis, 25 without malaria or sepsis, 17 with malaria and sepsis, and 16 without malaria but with sepsis. Sepsis was defined using systemic inflammatory response criteria; biomarkers (interleukin-6 [IL-6], tumor necrosis factor receptor-1, interleukin-1 ß [IL-1ß], interleukin-10 [IL-10], von Willebrand factor antigen-2, intercellular adhesion molecule-1, and angiopoietin-2 [Ang-2]) were measured with multiplex magnetic bead assays. IL-6, IL-1ß, and IL-10 were elevated, and Ang-2 was decreased in children with malaria compared with non-malarial fever. Children with non-malarial sepsis had greatly increased IL-1ß compared with the other subgroups. IL-1ß best predicted sepsis, with an area under the receiver operating characteristic (AUROC) of 0.71 (95% CI: 0.57-0.85); a combined biomarker-clinical characteristics model improved prediction (AUROC of 0.77, 95% CI: 0.67-0.85). We identified a distinct biomarker profile for non-malarial sepsis and developed a sepsis prediction model. Additional clinical and biological data are necessary to further explore sepsis pathophysiology in malaria-endemic regions.

Antihyperglycemic and Antihyperlipidemic Effects of Aqueous Extracts of Lannea edulis in Alloxan-Induced Diabetic Rats.

Lannea edulis (Sond.) Engl. commonly known as wild grape is used traditionally for the treatment of diabetes. It is only found in Eastern and Southern Africa. Phytochemical screening, antihyperglycemic and antihyperlipidemic effects of aqueous extracts of L. edulis in alloxan induced diabetic rats were carried out. We report herein the findings of this research work. Lannea edulis crude aqueous extracts were obtained by hot infusion and evaporation method. Phytochemical screening was carried out and subsequently toxicity studies of the aqueous extracts were performed to establish the Lethal Dose 50 (LD50) in albino rats. Alloxan monohydrate was used to induce diabetes in the rats. Lannea edulis positive control group doses of 100, 300, and 500 mg/kg were administered to 3 groups for 14 days. The positive control group was administered 5 mg/kg of glibenclamide. The negative and normal control groups were administered distilled water. To determine fasting blood glucose, blood was drawn on days 0, 1, 3, 5, 7, and 14 while it was drawn on days 0 and 14 for the determination of lipids. Phytochemical screening revealed the presence of flavonoids, saponins, tannins, cardiac glycosides, alkaloids and steroids. L. edulis diabetic positive control groups showed significant (P < 0.05) dose dependent reductions in fasting blood glucose levels. When day 0 mean blood glucose levels were compared to day 3 mean blood glucose levels of their respective groups, the 300 mg/kg L. edulis group showed a 23.3% drop and the 500 mg/kg L. edulis group showed a 52.6% drop. The 100 mg/kg L. edulis diabetic positive control group showed a 25.1% drop by day 5, the day on which it showed statistical significance (P < 0.05) compared to the diabetic control. In addition, administration of aqueous extracts of L. edulis to diabetic rats for 14 days significantly decreased (P < 0.05) the levels of serum total cholesterol, triglycerides, Low Density Lipoprotein (LDL) and Very Low Density Lipoprotein (VLDL) whilst increasing the levels of High Density Lipoprotein (HDL), when compared to the diabetic control group. It was concluded that L. edulis showed significant and dose dependent antihyperglycemic and antihyperlipidemic effects thus confirming its traditional use.

Loss of Humoral and Cellular Immunity to Invasive Nontyphoidal Salmonella during Current or Convalescent Plasmodium falciparum Infection in Malawian Children.

Invasive nontyphoidal Salmonella (iNTS) infections are commonly associated with Plasmodium falciparum infections, but the immunologic basis for this linkage is poorly understood. We hypothesized that P. falciparum infection compromises the humoral and cellular immunity of the host to NTS, which increases the susceptibility of the host to iNTS infection. We prospectively recruited children aged between 6 and 60 months at a Community Health Centre in Blantyre, Malawi, and allocated them to the following groups; febrile with uncomplicated malaria, febrile malaria negative, and nonfebrile malaria negative. Levels of Salmonella enterica serovar Typhimurium-specific serum bactericidal activity (SBA) and whole-blood bactericidal activity (WBBA), complement C3 deposition, and neutrophil respiratory burst activity (NRBA) were measured. Levels of SBA with respect to S Typhimurium were reduced in febrile P. falciparum-infected children (median, -0.20 log10 [interquartile range {IQR}, -1.85, 0.32]) compared to nonfebrile malaria-negative children (median, -1.42 log10 [IQR, -2.0, -0.47], P = 0.052). In relation to SBA, C3 deposition on S Typhimurium was significantly reduced in febrile P. falciparum-infected children (median, 7.5% [IQR, 4.1, 15.0]) compared to nonfebrile malaria-negative children (median, 29% [IQR, 11.8, 48.0], P = 0.048). WBBA with respect to S Typhimurium was significantly reduced in febrile P. falciparum-infected children (median, 0.25 log10 [IQR, -0.73, 1.13], P = 0.0001) compared to nonfebrile malaria-negative children (median, -1.0 log10 [IQR, -1.68, -0.16]). In relation to WBBA, S Typhimurium-specific NRBA was reduced in febrile P. falciparum-infected children (median, 8.8% [IQR, 3.7, 20], P = 0.0001) compared to nonfebrile malaria-negative children (median, 40.5% [IQR, 33, 65.8]). P. falciparum infection impairs humoral and cellular immunity to S Typhimurium in children during malaria episodes, which may explain the increased risk of iNTS observed in children from settings of malaria endemicity. The mechanisms underlying humoral immunity impairment are incompletely understood and should be explored further.

Structural elucidation of an asparagine-linked oligosaccharide from the hyperthermophilic archaeon, Archaeoglobus fulgidus.

The genome of the hyperthermophilic archaeon, Archaeoglobus fulgidus, contains three paralogous AglB genes that encode oligosaccharyltransferase (OST) proteins. The OST enzymes catalyze the transfer of an oligosaccharide chain from lipid-linked oligosaccharides (LLO) to asparagine residues in proteins. The detergent-solubilized membrane fractions prepared from cultured A. fulgidus cells contain both OST and LLO. The addition of a peptide containing the glycosylation sequon produced oligosaccharide chains attached to a structurally defined peptide. To facilitate the NMR analysis, the cells were grown in rich medium supplemented with (13)C-glucose, to label the LLOs metabolically. The MS analysis of the glycopeptide revealed that the glucose and galactose residues were nearly fully (13)C-labeled, but the mannose residues were fractionally labeled with about 20% efficiency. An immunodetection experiment revealed that the longest AglB paralog (AfAglB-L) was expressed in the membrane fractions under our cell culture conditions, while the other two shorter AglB paralogs (AfAglB-S1 and AfAglB-S2) were not. Thus, the oligosaccharide chain analyzed in this study was the product of AfAglB-L. The N-glycan consists of eight hexose residues, as follows: The α1,3-linked glucose is an optional residue branching from the distal mannose residue. The MS analysis of the minor HPLC peak of the in vitro oligosaccharyl transfer products also revealed an optional sulfate modification on the glucose residue directly linked to the Asn residue. The present data will be useful for structural and functional studies of the N-glycosylation system of A. fulgidus.

Crystal structures of an archaeal oligosaccharyltransferase provide insights into the catalytic cycle of N-linked protein glycosylation.

Oligosaccharyltransferase transfers an oligosaccharide chain to the asparagine residues in proteins. The archaeal and eubacterial oligosaccharyltransferases are single subunit membrane enzymes, referred to as "AglB" (archaeal glycosylation B) and "PglB" (protein glycosylation B), respectively. Only one crystal structure of a full-length PglB has been solved. Here we report the crystal structures of the full-length AglB from a hyperthermophilic archaeon, Archaeoglobus fulgidus. The AglB and PglB proteins share the common overall topology of the 13 transmembrane helices, and a characteristic long plastic loop in the transmembrane region. This is the structural basis for the formation of the catalytic center, consisting of conserved acidic residues coordinating a divalent metal ion. In one crystal form, a sulfate ion was bound next to the metal ion. This structure appears to represent a dolichol-phosphate binding state, and suggests the release mechanism for the glycosylated product. The structure in the other crystal form corresponds to the resting state conformation with the well-ordered plastic loop in the transmembrane region. The overall structural similarity between the distantly related AglB and PglB proteins strongly indicates the conserved catalytic mechanism in the eukaryotic counterpart, the STT3 (stauroporine and temperature sensitivity 3) protein. The detailed structural comparison provided the dynamic view of the N-glycosylation reaction, involving the conversion between the structured and unstructured states of the plastic loop in the transmembrane region and the formation and collapse of the Ser/Thr-binding pocket in the C-terminal globular domain.

Crystallographic and NMR evidence for flexibility in oligosaccharyltransferases and its catalytic significance.

Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of an oligosaccharide to an asparagine residue in glycoproteins. It possesses a binding pocket that recognizes Ser and Thr residues at the +2 position in the N-glycosylation consensus, Asn-X-Ser/Thr. We determined the crystal structures of the C-terminal globular domains of the catalytic subunits of two archaeal OSTs. A comparison with previously determined structures identified a segment with remarkable conformational plasticity, induced by crystal contact effects. We characterized its dynamic properties in solution by (15)N NMR relaxation analyses. Intriguingly, the mobile region contains the +2 Ser/Thr-binding pocket. In agreement, the flexibility restriction forced by an engineered disulfide crosslink abolished the enzymatic activity, and its cleavage fully restored activity. These results suggest the necessity of multiple conformational states in the reaction. The dynamic nature of the Ser/Thr pocket could facilitate the efficient scanning of N-glycosylation sequons along nascent polypeptide chains.

Crystal structure of the C-terminal globular domain of oligosaccharyltransferase from Archaeoglobus fulgidus at 1.75 Å resolution.

Protein N-glycosylation occurs in the three domains of life. Oligosaccharyltransferase (OST) transfers glycan to asparagine in the N-glycosylation sequon. The catalytic subunit of OST is called STT3 in eukaryotes, AglB in archaea, and PglB in eubacteria. The genome of a hyperthermophilic archaeon, Archaeoglobus fulgidus, encodes three AglB paralogs. Two of them are the shortest AglBs across all domains of life. We determined the crystal structure of the C-terminal globular domain of the smallest AglB to identify the minimal structural unit. The Archaeoglobus AglB lacked a ß-barrel-like structure, which had been found in other AglB and PglB structures. In agreement, the deletion in a larger Pyrococcus AglB confirmed its dispensability for the activity. By contrast, the Archaeoglobus AglB contains a kinked helix bearing a conserved motif, called DK/MI motif. The lysine and isoleucine residues in the motif participate in the Ser/Thr recognition in the sequon. The Archaeoglobus AglB structure revealed that the kinked helix contained an unexpected insertion. A revised sequence alignment based on this finding identified a variant type of the DK motif with the insertion. A mutagenesis study of the Archaeoglobus AglB confirmed the contribution of this particular type of the DK motif to the activity. When taken together with our previous results, this study defined the classification of OST: one group consisting of eukaryotes and most archaea possesses the DK-type Ser/Thr pocket, and the other group consisting of eubacteria and the remaining archaea possesses the MI-type Ser/Thr pocket. This classification provides a useful framework for OST studies.

Comparative structural biology of eubacterial and archaeal oligosaccharyltransferases.

Oligosaccharyltransferase (OST) catalyzes the transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. In the bacterium Campylobacter jejuni, a single-subunit membrane protein, PglB, catalyzes N-glycosylation. We report the 2.8 A resolution crystal structure of the C-terminal globular domain of PglB and its comparison with the previously determined structure from the archaeon Pyrococcus AglB. The two distantly related oligosaccharyltransferases share unexpected structural similarity beyond that expected from the sequence comparison. The common architecture of the putative catalytic sites revealed a new catalytic motif in PglB. Site-directed mutagenesis analyses confirmed the contribution of this motif to the catalytic function. Bacterial PglB and archaeal AglB constitute a protein family of the catalytic subunit of OST along with STT3 from eukaryotes. A structure-aided multiple sequence alignment of the STT3/PglB/AglB protein family revealed three types of OST catalytic centers. This novel classification will provide a useful framework for understanding the enzymatic properties of the OST enzymes from Eukarya, Archaea, and Bacteria.