Molecular Surveillance Detects High Prevalence of the Neglected Parasite Mansonella ozzardi in the Colombian Amazon.

BACKGROUND: Mansonellosis is an undermapped insect-transmitted disease caused by filarial nematodes that are estimated to infect hundreds of millions of people. Despite their prevalence, there are many outstanding questions regarding the general biology and health impacts of the responsible parasites. Historical reports suggest that the Colombian Amazon is endemic for mansonellosis and may serve as an ideal location to pursue these questions. METHODS: We deployed molecular and classical approaches to survey Mansonella prevalence among adults belonging to indigenous communities along the Amazon River and its tributaries near Leticia, Colombia. RESULTS: Loop-mediated isothermal amplification (LAMP) assays on whole-blood samples detected a much higher prevalence of Mansonella ozzardi infection (approximately 40%) compared to blood smear microscopy or LAMP performed using plasma, likely reflecting greater sensitivity and the ability to detect low microfilaremias and occult infections. Mansonella infection rates increased with age and were higher among men. Genomic analysis confirmed the presence of M. ozzardi that clusters closely with strains sequenced in neighboring countries. We successfully cryopreserved M. ozzardi microfilariae, advancing the prospects of rearing infective larvae in controlled settings. CONCLUSION: These data suggest an underestimation of true mansonellosis prevalence, and we expect that these methods will help facilitate the study of mansonellosis in endemic and laboratory settings.

Differential susceptibility of Onchocerca volvulus microfilaria to ivermectin in two areas of contrasting history of mass drug administration in Cameroon: relevance of microscopy and molecular techniques for the monitoring of skin microfilarial repopulation within six months of direct observed treatment.

BACKGROUND: Ivermectin is an excellent microfilaricide against Onchocerca volvulus. However, in some regions, long term use of ivermectin has resulted in sub-optimal responses to the treatment. More data to properly document the phenomenon in various contexts of ivermectin mass drug administration (IVM-MDA) is needed. Also, there is a need to accurately monitor a possible repopulation of skin by microfilariae following treatment. Skin snip microscopy is known to have a low sensitivity in individuals with light infections, which can be the case following treatment. This study was designed with two complementary objectives: (i) to assess the susceptibility of O. volvulus microfilariae to ivermectin in two areas undergoing IVM-MDA for different lengths of time, and (ii) to document the repopulation of skin by the O. volvulus microfilariae following treatment, using 3 independent diagnostic techniques. METHOD: Identified microfilaridermic individuals were treated with ivermectin and re-examined after 1, 3, and 6 months using microscopy, actin real-time PCR (actin-qPCR) and O-150 LAMP assays. Susceptibility to ivermectin and trends in detecting reappearance of skin microfilariae were determined using three techniques. Microscopy was used as an imperfect gold standard to determine the performance of actin-qPCR and LAMP. RESULTS: In Bafia with over 20 years of IVM-MDA, 11/51 (21.6%) direct observe treated microfilaridemic participants were still positive for skin microfilariae after 1 month. In Melong, with 10 years of IVM-MDA, 2/29 (6.9%) treated participants were still positive. The microfilarial density reduction per skin biopsy within one month following treatment was significantly lower in participants from Bafia. In both study sites, the molecular techniques detected higher proportions of infected individuals than microscopy at all monitoring time points. LAMP demonstrated the highest levels of sensitivity and real-time PCR was found to have the highest specificity. CONCLUSION: Patterns in skin mirofilariae clearance and repopulation were established. O. volvulus worms from Bafia with higher number of annual MDA displayed a lower clearance and higher repopulation rate after treatment with ivermectin. Molecular assays displayed higher sensitivity in monitoring O. volvulus microfilaridemia within six months following treatment.

Structure of the trehalose-6-phosphate phosphatase from Brugia malayi reveals key design principles for anthelmintic drugs.

Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible.

Interdomain lateral gene transfer of an essential ferrochelatase gene in human parasitic nematodes.

Lateral gene transfer events between bacteria and animals highlight an avenue for evolutionary genomic loss/gain of function. Herein, we report functional lateral gene transfer in animal parasitic nematodes. Members of the Nematoda are heme auxotrophs, lacking the ability to synthesize heme; however, the human filarial parasite Brugia malayi has acquired a bacterial gene encoding ferrochelatase (BmFeCH), the terminal step in heme biosynthesis. BmFeCH, encoded by a 9-exon gene, is a mitochondrial-targeted, functional ferrochelatase based on enzyme assays, complementation, and inhibitor studies. Homologs have been identified in several filariae and a nonfilarial nematode. RNAi and ex vivo inhibitor experiments indicate that BmFeCH is essential for viability, validating it as a potential target for filariasis control.

Extraction-free LAMP assays for generic detection of Old World Orthopoxviruses and specific detection of Mpox virus.

Mpox is a neglected zoonotic disease endemic in West and Central Africa. The Mpox outbreak with more than 90,000 cases worldwide since 2022 generated great concern about future outbreaks and highlighted the need for a simple and rapid diagnostic test. The Mpox virus, MPV, is a member of the Orthopoxvirus (OPV) genus that also contains other pathogenic viruses including variola virus, vaccinia virus, camelpox virus, and cowpox virus. Phylogenomic analysis of 200 OPV genomes identified 10 distinct phylogroups with the New World OPVs placed on a very long branch distant from the Old World OPVs. Isolates derived from infected humans were found to be distributed across multiple phylogroups interspersed with isolates from animal sources, indicating the zoonotic potential of these viruses. In this study, we developed a simple and sensitive colorimetric LAMP assay for generic detection of Old World OPVs. We also developed an MPV-specific probe that differentiates MPV from other OPVs in the N1R LAMP assay. In addition, we described an extraction-free protocol for use directly with swab eluates in LAMP assays, thereby eliminating the time and resources needed to extract DNA from the sample. Our direct LAMP assays are well-suited for low-resource settings and provide a valuable tool for rapid and scalable diagnosis and surveillance of OPVs and MPV.

Multiple lineages of nematode-Wolbachia symbiosis in supergroup F and convergent loss of bacterioferritin in filarial Wolbachia.

The intracellular endosymbiotic proteobacteria Wolbachia have evolved across the phyla nematoda and arthropoda. In Wolbachia phylogeny, supergroup F is the only clade known so far with members from both arthropod and filarial nematode hosts and therefore can provide unique insights into their evolution and biology. In this study, 4 new supergroup F Wolbachia genomes have been assembled using a metagenomic assembly and binning approach, wMoz and wMpe from the human filarial parasites Mansonella ozzardi and Mansonella perstans, and wOcae and wMoviF from the blue mason bee Osmia caerulescens and the sheep ked Melophagus ovinus respectively. A comprehensive phylogenomic analysis revealed two distinct lineages of filarial Wolbachia in supergroup F, indicating multiple horizontal transfer events between arthropod and nematode hosts. The analysis also reveals that the evolution of Wolbachia-filaria symbioses is accompanied by a convergent pseudogenization and loss of the bacterioferritin gene, a phenomenon found to be shared by all filarial Wolbachia, even those outside supergroup F. These observations indicate that differences in heme metabolism might be a key feature distinguishing filarial and arthropod Wolbachia. The new genomes provide a valuable resource for further studies on symbiosis, evolution, and the discovery of new antibiotics to treat mansonellosis.

Development and Validation of a Colorimetric Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Detection of Eastern Equine Encephalitis Virus in Mosquitoes.

Eastern equine encephalitis virus (EEEV) is a highly pathogenic alphavirus that causes periodic outbreaks in the eastern USA. Mosquito abatement programs are faced with various challenges with surveillance and control of EEEV and other mosquito-borne illnesses. Environmental sampling of mosquito populations can be technically complex. Here we report the identification of biomarkers, development and validation of a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of EEEV. Positive samples are easily visualized by a color change from pink to yellow. The assay was validated using EEEV from viral culture, experimentally spiked mosquito pools, and previously tested mosquito pools. The RT-LAMP assay detected viral titers down to approximately 10% of what would be present in a single infectious mosquito, based upon EEEV viral titers determined by previous competency studies. The RT-LAMP assay efficiently detected EEEV in combined aliquots from previously homogenized pools of mosquitoes, allowing up to 250 individual mosquitoes to be tested in a single reaction. No false positive results were obtained from RNA prepared from negative mosquito pools acquired from known and potential EEEV vectors. The colorimetric RT-LAMP assay is highly accurate, technically simple, and does not require sophisticated equipment, making it a cost-effective alternative to real time reverse transcriptase-polymerase chain reaction (RT-PCR) for vector surveillance.

Development and implementation of a simple and rapid extraction-free saliva SARS-CoV-2 RT-LAMP workflow for workplace surveillance.

Effective management of the COVID-19 pandemic requires widespread and frequent testing of the population for SARS-CoV-2 infection. Saliva has emerged as an attractive alternative to nasopharyngeal samples for surveillance testing as it does not require specialized personnel or materials for its collection and can be easily provided by the patient. We have developed a simple, fast, and sensitive saliva-based testing workflow that requires minimal sample treatment and equipment. After sample inactivation, RNA is quickly released and stabilized in an optimized buffer, followed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and detection of positive samples using a colorimetric and/or fluorescent readout. The workflow was optimized using 1,670 negative samples collected from 172 different individuals over the course of 6 months. Each sample was spiked with 50 copies/µL of inactivated SARS-CoV-2 virus to monitor the efficiency of viral detection. Using pre-defined clinical samples, the test was determined to be 100% specific and 97% sensitive, with a limit of detection of 39 copies/mL. The method was successfully implemented in a CLIA laboratory setting for workplace surveillance and reporting. From April 2021-February 2022, more than 30,000 self-collected samples from 755 individuals were tested and 85 employees tested positive mainly during December and January, consistent with high infection rates in Massachusetts and nationwide.

Characterization of the Caenorhabditis elegans UDP-galactopyranose mutase homolog glf-1 reveals an essential role for galactofuranose metabolism in nematode surface coat synthesis.

Galactofuranose (Gal(f)), the furanoic form of d-galactose produced by UDP-galactopyranose mutases (UGMs), is present in surface glycans of some prokaryotes and lower eukaryotes. Absence of the Gal(f) biosynthetic pathway in vertebrates and its importance in several pathogens make UGMs attractive drug targets. Since the existence of Gal(f) in nematodes has not been established, we investigated the role of the Caenorhabditis elegans UGM homolog glf-1 in worm development. glf-1 mutants display significant late embryonic and larval lethality, and other phenotypes indicative of defective surface coat synthesis, the glycan-rich outermost layer of the nematode cuticle. The glf homolog from the protozoan Leishmania major partially complements C. elegans glf-1. glf-1 mutants rescued by L. major glf, which behave as glf-1 hypomorphs, display resistance to infection by Microbacterium nematophilum, a pathogen of rhabditid nematodes thought to bind to surface coat glycans. To confirm the presence of Gal(f) in C. elegans, we analyzed C. elegans nucleotide sugar pools using online electrospray ionization-mass spectrometry (ESI-MS). UDP-Gal(f) was detected in wild-type animals while absent in glf-1 deletion mutants. Our data indicate that Gal(f) likely has a pivotal role in maintenance of surface integrity in nematodes, supporting investigation of UGM as a drug target in parasitic species.

Rickettsia buchneri, symbiont of the deer tick Ixodes scapularis, can colonise the salivary glands of its host.

Vertically-transmitted bacterial symbionts are widespread in ticks and have manifold impacts on the epidemiology of tick-borne diseases. For instance, they may provide essential nutrients to ticks, affect vector competence, induce immune responses in vertebrate hosts, or even evolve to become vertebrate pathogens. The deer or blacklegged tick Ixodes scapularis harbours the symbiont Rickettsia buchneri in its ovarian tissues. Here we show by molecular, proteomic and imaging methods that R. buchneri is also capable of colonising the salivary glands of wild I. scapularis. This finding has important implications for the diagnosis of rickettsial infections and for pathogen-symbiont interactions in this notorious vector of Lyme borreliosis.

Computational prediction of essential genes in an unculturable endosymbiotic bacterium, Wolbachia of Brugia malayi.

BACKGROUND: Wolbachia (wBm) is an obligate endosymbiotic bacterium of Brugia malayi, a parasitic filarial nematode of humans and one of the causative agents of lymphatic filariasis. There is a pressing need for new drugs against filarial parasites, such as B. malayi. As wBm is required for B. malayi development and fertility, targeting wBm is a promising approach. However, the lifecycle of neither B. malayi nor wBm can be maintained in vitro. To facilitate selection of potential drug targets we computationally ranked the wBm genome based on confidence that a particular gene is essential for the survival of the bacterium. RESULTS: wBm protein sequences were aligned using BLAST to the Database of Essential Genes (DEG) version 5.2, a collection of 5,260 experimentally identified essential genes in 15 bacterial strains. A confidence score, the Multiple Hit Score (MHS), was developed to predict each wBm gene's essentiality based on the top alignments to essential genes in each bacterial strain. This method was validated using a jackknife methodology to test the ability to recover known essential genes in a control genome. A second estimation of essentiality, the Gene Conservation Score (GCS), was calculated on the basis of phyletic conservation of genes across Wolbachia's parent order Rickettsiales. Clusters of orthologous genes were predicted within the 27 currently available complete genomes. Druggability of wBm proteins was predicted by alignment to a database of protein targets of known compounds. CONCLUSION: Ranking wBm genes by either MHS or GCS predicts and prioritizes potentially essential genes. Comparison of the MHS to GCS produces quadrants representing four types of predictions: those with high confidence of essentiality by both methods (245 genes), those highly conserved across Rickettsiales (299 genes), those similar to distant essential genes (8 genes), and those with low confidence of essentiality (253 genes). These data facilitate selection of wBm genes for entry into drug design pipelines.

The Wolbachia endosymbiont of Brugia malayi has an active phosphoglycerate mutase: a candidate target for anti-filarial therapies.

Phosphoglycerate mutases (PGM) interconvert 2- and 3-phosphoglycerate in the glycolytic and gluconeogenic pathways. A putative cofactor-independent phosphoglycerate mutase gene (iPGM) was identified in the genome sequence of the Wolbachia endosymbiont from the filarial nematode, Brugia malayi (wBm). Since iPGM has no sequence or structural similarity to the cofactor-dependent phosphoglycerate mutase (dPGM) found in mammals, it may represent an attractive Wolbachia drug target. In the present study, wBm-iPGM cloned and expressed in Escherichia coli was mostly insoluble and inactive. However, the protein was successfully produced in the yeast Kluyveromyces lactis and the purified recombinant wBm-iPGM showed typical PGM activity. Our results provide a foundation for further development of wBm-iPGM as a promising new drug target for novel anti-filarial therapies that selectively target the endosymbiont.

Complete Genome Sequence of the Wolbachia wAlbB Endosymbiont of Aedes albopictus.

Wolbachia, an alpha-proteobacterium closely related to Rickettsia, is a maternally transmitted, intracellular symbiont of arthropods and nematodes. Aedes albopictus mosquitoes are naturally infected with Wolbachia strains wAlbA and wAlbB. Cell line Aa23 established from Ae. albopictus embryos retains only wAlbB and is a key model to study host-endosymbiont interactions. We have assembled the complete circular genome of wAlbB from the Aa23 cell line using long-read PacBio sequencing at 500× median coverage. The assembled circular chromosome is 1.48 megabases in size, an increase of more than 300 kb over the published draft wAlbB genome. The annotation of the genome identified 1,205 protein coding genes, 34 tRNA, 3 rRNA, 1 tmRNA, and 3 other ncRNA loci. The long reads enabled sequencing over complex repeat regions which are difficult to resolve with short-read sequencing. Thirteen percent of the genome comprised insertion sequence elements distributed throughout the genome, some of which cause pseudogenization. Prophage WO genes encoding some essential components of phage particle assembly are missing, while the remainder are found in five prophage regions/WO-like islands or scattered around the genome. Orthology analysis identified a core proteome of 535 orthogroups across all completed Wolbachia genomes. The majority of proteins could be annotated using Pfam and eggNOG analyses, including ankyrins and components of the Type IV secretion system. KEGG analysis revealed the absence of five genes in wAlbB which are present in other Wolbachia. The availability of a complete circular chromosome from wAlbB will enable further biochemical, molecular, and genetic analyses on this strain and related Wolbachia.

The Wolbachia endosymbiont of Brugia malayi has an active pyruvate phosphate dikinase.

Genome analysis of the glycolytic/gluconeogenic pathway in the Wolbachia endosymbiont from the filarial parasite Brugia malayi (wBm) has revealed that wBm lacks pyruvate kinase (PK) and may instead utilize the enzyme pyruvate phosphate dikinase (PPDK; ATP:pyruvate, orthophosphate phosphotransferase, EC 2.7.9.1). PPDK catalyses the reversible conversion of AMP, PPi and phosphoenolpyruvate (PEP) into ATP, Pi and pyruvate. The glycolytic pathway of most organisms, including mammals, contains exclusively PK for the production of pyruvate from PEP. Therefore, the absence of PPDK in mammals makes the enzyme an attractive Wolbachia drug target. In the present study, we have cloned and expressed an active wBm-PPDK, thereby providing insight into the energy metabolism of the endosymbiont. Our results support the development of wBm-PPDK as a promising new drug target in an anti-symbiotic approach to controlling filarial infection.

Changes in canine serum N-glycosylation as a result of infection with the heartworm parasite Dirofilaria immitis.

Filariases are diseases caused by infection with filarial nematodes and transmitted by insect vectors. The filarial roundworm Dirofilaria immitis causes heartworm disease in dogs and other carnivores. D. immitis is closely related to Onchocerca volvulus, Wuchereria bancrofti and Brugia malayi, which cause onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis) in humans and are neglected tropical diseases. Serum N-glycosylation is very sensitive to both pathological infections and changes in mammalian biology due to normal aging or lifestyle choices. Here, we report significant changes in the serum N-glycosylation profiles of dogs infected with D. immitis. Our data derive from analysis of serum from dogs with established patent infections and from a longitudinal infection study. Overall, galactosylation and core fucosylation increase, while sialylation decreases in infected dog sera. We also identify individual glycan structures that change significantly in their relative abundance during infection. Notably, the abundance of the most dominant N-glycan in canine serum (biantennary, disialylated A2G2S2) decreases by over 10 percentage points during the first 6 months of infection in each dog analyzed. This is the first longitudinal study linking changes in mammalian serum N-glycome to progression of a parasitic infection.

Identification of putative effectors of the Type IV secretion system from the Wolbachia endosymbiont of Brugia malayi.

Wolbachia is an unculturable, intracellular bacterium that persists within an extremely broad range of arthropod and parasitic nematode hosts, where it is transmitted maternally to offspring via vertical transmission. In the filarial nematode Brugia malayi, a causative agent of human lymphatic filariasis, Wolbachia is an endosymbiont, and its presence is essential for proper nematode development, survival, and pathogenesis. While the elucidation of Wolbachia:nematode interactions that promote the bacterium's intracellular persistence is of great importance, research has been hampered due to the fact that Wolbachia cannot be cultured in the absence of host cells. The Wolbachia endosymbiont of B. malayi (wBm) has an active Type IV secretion system (T4SS). Here, we have screened 47 putative T4SS effector proteins of wBm for their ability to modulate growth or the cell biology of a typical eukaryotic cell, Saccharomyces cerevisiae. Five candidates strongly inhibited yeast growth upon expression, and 6 additional proteins showed toxicity in the presence of zinc and caffeine. Studies on the uptake of an endocytic vacuole-specific fluorescent marker, FM4-64, identified 4 proteins (wBm0076 wBm00114, wBm0447 and wBm0152) involved in vacuole membrane dynamics. The WAS(p)-family protein, wBm0076, was found to colocalize with yeast cortical actin patches and disrupted actin cytoskeleton dynamics upon expression. Deletion of the Arp2/3-activating protein, Abp1p, provided resistance to wBm0076 expression, suggesting a role for wBm0076 in regulating eukaryotic actin dynamics and cortical actin patch formation. Furthermore, wBm0152 was found to strongly disrupt endosome:vacuole cargo trafficking in yeast. This study provides molecular insight into the potential role of the T4SS in the Wolbachia endosymbiont:nematode relationship.

Molecular and biochemical characterization of nematode cofactor independent phosphoglycerate mutases.

Phosphoglycerate mutase (PGM, EC 5.4.2.1) catalyzes the isomerization of 3-phosphoglycerate and 2-phosphoglycerate in glycolysis and gluconeogenesis. Two distinct types of PGM exist in nature, one that requires 2,3-bisphosphoglycerate as a cofactor (dPGM) and another that does not (iPGM). The two enzymes are structurally distinct and possess different mechanisms of action. In any particular organism, one form may exist or both. Nematodes possess the iPGM form whereas mammals have dPGM. In the present study, we have cloned and expressed iPGM from Onchocerca volvulus and described the catalytic properties of O. volvulus, Brugia malayi and Caenorhabditis elegans iPGM enzymes. Temperature and pH optima were determined for each enzyme. Like other iPGM enzymes, the activities of the nematode iPGM enzymes were dependent on the presence of divalent ions. Inactivation by EDTA could be restored most effectively by magnesium and manganese ions. Kinetic parameters and specific activities of the various recombinant enzymes were determined. The high similarity in catalytic properties among the enzymes indicates that a single enzyme inhibitor would likely be effective against all nematode enzymes. Inhibition of iPGM activity in vivo may lead to lethality as indicated by RNAi studies in C. elegans. Our results support the development of iPGM as a promising drug target in parasitic nematodes.

Macrocycle peptides delineate locked-open inhibition mechanism for microorganism phosphoglycerate mutases.

Glycolytic interconversion of phosphoglycerate isomers is catalysed in numerous pathogenic microorganisms by a cofactor-independent mutase (iPGM) structurally distinct from the mammalian cofactor-dependent (dPGM) isozyme. The iPGM active site dynamically assembles through substrate-triggered movement of phosphatase and transferase domains creating a solvent inaccessible cavity. Here we identify alternate ligand binding regions using nematode iPGM to select and enrich lariat-like ligands from an mRNA-display macrocyclic peptide library containing >1012 members. Functional analysis of the ligands, named ipglycermides, demonstrates sub-nanomolar inhibition of iPGM with complete selectivity over dPGM. The crystal structure of an iPGM macrocyclic peptide complex illuminated an allosteric, locked-open inhibition mechanism placing the cyclic peptide at the bi-domain interface. This binding mode aligns the pendant lariat cysteine thiolate for coordination with the iPGM transition metal ion cluster. The extended charged, hydrophilic binding surface interaction rationalizes the persistent challenges these enzymes have presented to small-molecule screening efforts highlighting the important roles of macrocyclic peptides in expanding chemical diversity for ligand discovery.