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bioRxiv2018

The Streptococcus pneumoniae Competence-Induced BriC Peptide Promotes Nasopharyngeal Colonization and Impacts Biofilm Development

Streptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that causes otitis media, sinusitis, pneumonia, meningitis and sepsis. The progression to this pathogenic lifestyle is preceded by asymptomatic colonization of the nasopharynx. This colonization is associated with biofilm formation; the competence pathway influences the structure and stability of biofilms. However, the molecules that link the competence pathway to biofilm formation are unknown. Here, we describe a new competence-induced gene, called briC, and demonstrate that its product promotes biofilm development and stimulates colonization in a murine model. We show that expression of briC is induced by the master regulator of competence, ComE. Whereas briC does not substantially influence early biofilm development on abiotic surfaces, it significantly impacts later stages of biofilm development. Specifically, briC expression leads to increases in biofilm biomass and thickness at 72h. Consistent with the role of biofilms in colonization, briC promotes nasopharyngeal colonization in the murine model. The function of BriC appears to be conserved across pneumococci, as comparative genomics reveal that briC is widespread across isolates. Surprisingly, many isolates, including strains from clinically important PMEN1 and PMEN14 lineages, which are widely associated with colonization, encode a long briC promoter. This long form captures an instance of genomic plasticity and functions as a competence-independent expression enhancer that may serve as a precocious point of entry into this otherwise competence-regulated pathway. Moreover, overexpression of briC by the long promoter fully rescues the comE-deletion induced biofilm defect in vitro, and partially in vivo. These findings indicate that BriC may bypass the influence of competence in biofilm development and that such a pathway may be active in a subset of pneumococcal lineages. In conclusion, BriC is a part of the complex molecular network that connects signaling of the competence pathway to biofilm development and colonization.

microbiology↗

bioRxiv2018

Data-mining of Antibiotic Resistance Genes Provides Insight into the Community Structure of Ocean Microbiome

BackgroundAntibiotics have been spread widely in environments, asserting profound effects on environmental microbes as well as antibiotic resistance genes (ARGs) within these microbes. Therefore, investigating the associations between ARGs and bacterial communities become an important issue for environment protection. Ocean microbiomes are potentially large ARG reservoirs, but the marine ARG distribution and its associations with bacterial communities remain unclear.\n\nMethodswe have utilized the big-data mining techniques on ocean microbiome data to analysis the marine ARGs and bacterial distribution on a global scale, and applied comprehensive statistical analysis to unveil the associations between ARG contents, ocean microbial community structures, and environmental factors by reanalyzing 132 metagenomic samples from the Tara Oceans project.\n\nResultsWe identified in total 1,926 unique ARGs and found that: firstly, ARGs are more abundant and diverse in the mesopelagic zone than other water layers. Additionally, ARG-enriched genera are closely connected in co-occurrence network. We also found that ARG-enriched genera are often more abundant than their ARG-less neighbors. Furthermore, we found that samples from the Mediterranean that is surrounded by human activities often contain more ARGs.\n\nConclusionOur research for investigating the marine ARG distribution and revealing the association between ARG and bacterial communities provide a deeper insight into the marine bacterial communities. We found that ARG-enriched genera were often more abundant than their ARG-less neighbors in the same environment, indicating that genera enriched with ARGs might possess an advantage over others in the competition for survival in the oceanic microbial communities.

microbiology↗

bioRxiv2018

SUCCESSIVE BLOODMEALS ENHANCE VIRUS DISSEMINATION WITHIN MOSQUITOES AND INCREASE TRANSMISSION POTENTIAL

SUMMARY PARAGRAPHThe recent Zika virus (ZIKV) and chikungunya virus (CHIKV) epidemics highlight the explosive nature of arthropod-borne (arbo) viruses transmitted by Aedes aegypti mosquitoes1,2. Vector competence and the extrinsic incubation period (EIP) are two key entomological parameters used to assess the public health risk posed by arboviruses3. These are typically measured empirically by offering mosquitoes an infectious bloodmeal and temporally sampling mosquitoes to determine infection and transmission status. This approach has been used for the better part of a century; however, it does not accurately capture the biology and behavior of many mosquito vectors which refeed frequently (every 2-3 days)4. Here we demonstrate that administration of a second non-infectious bloodmeal significantly shortens the EIP of ZIKV-infected Ae. aegypti by enhancing virus escape from the mosquito midgut. Similarly, a second bloodmeal increased the competence of this species for dengue virus and CHIKV. This effect was also observed for ZIKV in Aedes albopictus, suggesting that this species might be a more important vector than once thought and that this phenomenon may be common among other virus-vector pairings. Modeling of these findings reveals that a shortened EIP would result in a significant increase in the basic reproductive number, R0. This increase helps explain how Ae.aegypti can sustain an explosive epidemic like ZIKV despite its relatively poor vector competence in single-feed laboratory trials. Together, these data demonstrate a direct and unrecognized link between mosquito feeding behavior, EIP, and vector competence.

microbiology↗

bioRxiv2018

Direct Imaging of the circular chromosome of a live bacterium

New assays for quantitative imaging1-6 and sequencing7-11 have yielded great progress towards understanding the organizational principles of chromosomes. Yet, even for the well-studied model bacterium Escherichia coli, many basic questions remain unresolved regarding chromosomal (sub-)structure2,11, its mechanics1,2,12 and dynamics13,14, and the link between structure and function1,15,16. Here we resolve the spatial organization of the circular chromosome of bacteria by directly imaging the chromosome in live E. coli cells with a broadened cell shape. The chromosome was observed to exhibit a torus topology with a 4.2 m toroidal length and 0.4 m bundle thickness. On average, the DNA density along the chromosome shows dense right and left arms that branch from a lower-density origin of replication, and are connected at the terminus of replication by an ultrathin flexible string of DNA. At the single-cell level, the DNA density along the torus is found to be strikingly heterogeneous, with blob-like Mbp-size domains that undergo major dynamic rearrangements, splitting and merging at a minute timescale. We show that prominent domain boundaries at the terminus and origin of replication are induced by MatP proteins, while weaker transient domain boundaries are facilitated by the global transcription regulators HU and Fis. These findings provide an architectural basis for the understanding of the spatial organization of bacterial genomes.

microbiology↗

bioRxiv2018

Insights into circovirus host range from the genomic fossil record.

A diverse range of DNA sequences derived from circoviruses (family Circoviridae) have been identified in samples obtained from humans and domestic animals, often in association with pathological conditions. In the majority of cases, however, little is known about the natural biology of the viruses from which these sequences are derived. Endogenous circoviral elements (CVe) are DNA sequences derived from circoviruses that occur in animal genomes and provide a useful source of information about circovirus-host relationships. In this study we screened genome assemblies of 675 animal species and identified numerous circovirus-related sequences, including the first examples of CVe derived from cycloviruses. We confirmed the presence of these CVe in the germline of the elongate twig ant (Pseudomyrmex gracilis), thereby establishing that cycloviruses infect insects. We examined the evolutionary relationships between CVe and contemporary circoviruses, showing that CVe from ants and mites group relatively closely with cycloviruses in phylogenies. Furthermore, the relatively random interspersal of CVe from insect genomes with cyclovirus sequences recovered from vertebrate samples, suggested that contamination might be an important consideration in studies reporting these viruses. Our study demonstrates how endogenous viral sequences can inform metagenomics-based virus discovery. In addition, it raises doubts about the role of cycloviruses as pathogens of humans and other vertebrates.

microbiology↗

bioRxiv2018

The Viral protein Corona Directs Viral Pathogenesis and Amyloid Aggregation

Artificial nanoparticles accumulate a protein corona layer in biological fluids, which significantly influences their bioactivity. As nanosized obligate intracellular parasites, viruses share many biophysical properties with artificial nanoparticles in extracellular environments and here we show that respiratory syncytial virus (RSV) and herpes simplex virus 1 (HSV-1) accumulate a rich and distinctive protein corona in different biological fluids. Moreover, we show that corona pre-coating differentially affects viral infectivity and immune cell activation. Additionally, we demonstrate that viruses bind amyloidogenic peptides in their corona and catalyze amyloid formation via surface-assisted heterogeneous nucleation. Importantly, we show that HSV-1 catalyzes the aggregation of the amyloid beta peptide (A{beta}42), a major constituent of amyloid plaques in Alzheimers disease, in-vitro and in animal models. Our results highlight the viral protein corona as an acquired structural layer that is critical for viral-host interactions and illustrate a mechanistic convergence between viral and amyloid pathologies.

microbiology↗

bioRxiv2018

Coronavirus S protein-induced fusion is blocked prior to hemifusion by Abl kinase inhibitors

Enveloped viruses gain entry into host cells by fusing with cellular membranes, a step required for virus replication. Coronaviruses, including the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and infectious bronchitis virus (IBV), fuse at the plasma membrane or use receptor-mediated endocytosis and fuse with endosomes depending on the cell or tissue type. The virus Spike (S) protein mediates fusion with the host cell membrane. We have shown previously that an Abl kinase inhibitor, imatinib, significantly reduces SARS-CoV and MERS-CoV viral titers and prevents endosomal entry by HIV SARS S and MERS S pseudotyped virions. SARS-CoV and MERS-CoV are classified as BSL-3 viruses, which can make experimentation into the cellular mechanisms involved in infection more challenging. Here, we use IBV, a BSL-2 virus, as a model for studying the role of Abl kinase activity during coronavirus infection. We found that imatinib and two specific Abl kinase inhibitors, GNF2 and GNF5, reduce IBV titers by blocking the first round of virus infection. Additionally, all three drugs prevented IBV S-induced syncytia formation prior to the hemifusion step. Our results indicate that membrane fusion (both virus-cell and cell-cell) is blocked in the presence of Abl kinase inhibitors. Studying the effects of Abl kinase inhibitors on IBV will be useful in identifying host cell pathways required for coronavirus infection. This will provide insight into possible therapeutic targets to treat infections by current as well as newly emerging coronaviruses.

microbiology↗

bioRxiv2018

A genomic island of Streptomyces coelicolor harbors the self-contained regulon of an ECF sigma factor

Streptomycetes constitute the largest genus of actinobacteria, living predominantly in soil and decaying vegetation. The bacteria are widely known for their filamentous morphologies and their capacity to synthesize antibiotics and other biologically active molecules. More than a decade ago, we and others identified 22 genomic islands that Streptomyces coelicolor M145 possesses and other Streptomyces strains lack. One of these genomic islands, Genomic Island (GI) 6, encodes an extracytoplasmic function (ECF) sigma factor that we were characterizing in separate work. Here we report that artificial induction of the ECF sigma factor, which is encoded by SCO3450, causes the transcription of approximately one-fourth of GI 6, or ~26 mostly contiguous genes, to increase. More than half of the regulon encodes putative enzymes involved in small molecule metabolism. A putative haloacid dehalogenase is present. Genes encoding two putative anti-sigma factors flank SCO3450, the three genes residing within the regulon. Our data suggest that the ECF sigma factor and its regulon are a self-contained transcriptional unit that can be transferred by horizontal gene transfer. To our knowledge, only one other example has been identified of an ECF sigma factor and its contiguous regulon appearing to be transferrable by horizontal gene transfer [18,19]. Because the regulon appears not to be induced by the 44 growth conditions recently examined by Byung-Kwan Cho and colleagues [20], if it confers fitness to S. coelicolor, the regulon likely does so in as-yet unknown situations. Those situations might range from scavenging to detoxification to even communication within microbial communities.\n\nIMPORTANCEStreptomyces bacteria grow as hyphae that colonize soil and differentiate into spores when nutrients become scarce. In their terrestrial habitats, the bacteria encounter diverse conditions. Presumably so that the bacteria can cope with those conditions, the chromosomes of streptomycetes are highly dynamic, varying greatly in structure not only between species but also between closely related strains of a single species. The bacteria also have large numbers of extracytoplasmic function (ECF) sigma factors, which undoubtedly help the microorganisms respond to the plethora of challenges coming from the environment. This work illustrates these two threads of Streptomyces biology dovetailing: Genetic adaptability through horizontal gene transfer seems to have enabled Streptomyces coelicolor to acquire a self-contained transcriptional unit that consists of an ECF sigma factor and its regulon. The suggested facile movement of the regulon between microbial hosts indicates the value of the metabolism of small molecules possibly mediated by the regulon.

microbiology↗

bioRxiv2018

Single Cell Observations Show Persister Cells Wake Based on Ribosome Content

Since persister cells survive antibiotic treatments through dormancy and resuscitate to reconstitute infections, it is imperative to determine the rate at which these cells revive. Using two sets of Escherichia coli persister cells, those arising naturally at low levels and those generated at high levels by ceasing transcription via rifampicin pretreatment (shown to be bona fide persisters through seven sets of experiments), we used microscopy of single cells to determine that persisters have low levels of antibiotic-corrupting proteins and that their resuscitation is heterogeneous and includes cells that grow immediately. In all, five phenotypes were found for persister cell resuscitation: (i) immediate division, (ii) immediate elongation followed by division, (iii) immediate elongation but no division, (iv) delayed elongation/division, and (v) no growth. In addition, once cell division begins, the growth rate is that of exponential cells. Critically, the greater the ribosome content, the faster the persister cells resuscitate.

microbiology↗

bioRxiv2018

Mixing is required for uniform reconstitution of filter-dried protein antigens in a single-injection vaccine formulation

Ambient temperature filter dried vaccine formulations have been proposed to simultaneously achieve thermostability and offer a ready-to-use immunisation device that combines reconstitution and injection. Vaccine concentration should be uniform at the point of injection, but the uniformity following direct reconstitution of filter-dried vaccines has not been reported. We present here a study of vaccine mixing and release following dissolution of filter-dried model protein and toxoid antigens within a single syringe, filter and needle unit. Release was better for filters made from glass than cellulose. Without additional mixing, uniformity was poor and only 41% of input protein was released from protein filter-dried onto glass fibre. In contrast, adding a simple glass bead and mixing by inversion, 100% release antigen solution was achieved, with uniform concentration at exit from the needle throughout a simulated injection. Adsorption onto alum adjuvant had no detectable effect on vaccine dissolution and mixing. The uniformity and yield of low doses of diphtheria and tetanus toxoid was also improved by mixing, albeit with a lower yield of 60-68%. We conclude that uniformity and mixing should be studied to ensure safety and efficacy of directly reconstituted filter-dried vaccine formulations.

microbiology↗

bioRxiv2018

Impact of DNA polymerase choice on assessment of bacterial communities by a Legionella genus-specific next-generation sequencing approach

The library preparation step is a major source of bias in NGS-based studies. Several PCR-related factors might negatively influence the application of NGS tools in environmental studies and diagnostics. Among the most understudied factors are DNA polymerases. In our study, we evaluated the effect of DNA polymerase type on the characterisation of bacterial communities, more precisely Legionella, using a genus-specific NGS approach. The assay with proof-reading high fidelity KAPA HiFi showed better amplification yield than the one with widely used non-proofreading HotStarTaq. Legionella community richness metrics were significantly overestimated with HotStarTaq. However, the choice of DNA polymerase did not significantly change the community profiling and composition. These results substantiate the use of proof-reading high fidelity DNA polymerases in NGS assays and highlight the need of considering the impact of different DNA polymerases in comparative studies and future guidelines for NGS-based diagnostic tools.

microbiology↗

bioRxiv2018

Microbial biogeography of 1,000 geothermal springs in New Zealand

Geothermal springs are model ecosystems to systematically investigate microbial biogeography as they i) represent discrete, homogenous habitats; ii) are abundantly distributed across multiple geographical scales; iii) span broad geochemical gradients; and iv) have simple community structures with reduced metazoan interactions. Taking advantage of these traits, we undertook the largest known consolidated study of geothermal ecosystems (http://1000springs.org.nz) to determine factors that influence biogeographical patterns. Rigorously standardised methodologies were used to measure microbial communities, 46 physicochemical parameters, and metadata from 1,019 hotspring samples across New Zealand. pH was found to be the primary influence on diversity in springs < 70 {degrees}C with community similarity decreasing with geographic distance. Surprisingly, community composition was dominated by two genera (Venenivibrio and Acidithiobacillus) in both average relative abundance (11.2 and 11.1 %) and prevalence (74.2 and 62.9 % respectively) across physicochemical spectrums of 13.9 - 100.6 {degrees}C and pH < 1 - 9.7. This study provides an unprecedented insight into the ecological conditions that drive community assembly in geothermal springs, and can be used as a foundation to improve the characterisation of global microbial biogeographical processes.

microbiology↗

bioRxiv2018

Toxoplasma gondii infection triggers chronic cachexia and sustained commensal dysbiosis in mice

Toxoplasma gondii is a protozoan parasite with a predation-mediated transmission cycle between rodents and felines. Intermediate hosts acquire Toxoplasma by eating parasite cysts which invade the small intestine, disseminate systemically and finally establish host life-long chronic infection in brain and muscles. Here we show that Toxoplasma infection can trigger a severe form of sustained cachexia: a disease of progressive weight loss that is a causal predictor of mortality in cancer, chronic disease and many infections. Toxoplasma cachexia is characterized by acute anorexia, systemic inflammation and loss of 20% body mass. Although mice recover from symptoms of peak sickness they fail to regain muscle mass or visceral adipose depots. We asked whether the damage to the intestinal microenvironment observed at acute time points was sustained in chronic infection and could thereby play a role the sustaining cachexia. We found that parasites replicate in the same region of the distal jejunum/proximal ileum throughout acute infection, inducing the development of secondary lymphoid structures and severe, regional inflammation. Small intestine pathology was resolved by 5 weeks post-infection. However, changes in the commensal populations, notably an outgrowth of Clostridia spp., were sustained in chronic infection. Importantly, uninfected animals co-housed with infected mice display similar changes in commensal microflora but never display symptoms of cachexia, indicating that altered commensals are not sufficient to explain the cachexia phenotype alone. These studies indicate that Toxoplasma infection is a novel and robust model to study the immune-metabolic interactions that contribute chronic cachexia development, pathology and potential reversal.

microbiology↗

bioRxiv2018

Comparative genomics and phylogeny unveil lineage diversification of Citrobacter rodentium polyvalent bacteriophage

Enteropathogenic Escherichia coli (EPEC) is a major etiology for diarrheal diseases among children. Antibiotics, when used appropriately, are effective; however, their overuse and misuse has led to the rise of antibiotic resistance worldwide. Thus, there are renewed efforts into the development of phage therapy. Due to the drawbacks of EPEC in vivo models, a surrogate is the mouse-restricted gut pathgoen Citrobacter rodentium. In this study, two new phages CrRp3 and CrRp10, which infect C. rodentium, were isolated and characterized. CrRp3 was found to be a new species within the genus Vectrevirus and CrRp10 is a new strain within the genus Tequatrovirus. Neither phage carries known genes associated with bacterial virulence, antibiotic resistance, or lysogeny. CrRp3 and CrRp10 appear to have independently evolved from E. coli phages. CrRp3 appears to be the more potent being 24x more likely to find a host cell and has a shorter lytic cycle, while CrRp10 at MOI 0.001 was able to maintain bacterial density below the limit of detection after 18 h. We found that hypoxia (5% O2 and 5% CO2) inhibited CrRp3 ability to reverse exponential bacterial growth. It is unclear whether the subtle characteristic differences between CrRp3 and CrRp10 will influence treatment efficacy in future phage therapy in vivo investigations.

microbiology↗

bioRxiv2018

Viromic analysis of wastewater input to a river catchment reveals a diverse assemblage of RNA viruses

Detection of viruses in the environment is heavily dependent on PCR-based approaches that require reference sequences for primer design. While this strategy can accurately detect known viruses, it will not find novel genotypes, nor emerging and invasive viral species. In this study, we investigated the use of viromics, i.e. high-throughput sequencing of the biosphere viral fraction, to detect human/animal pathogenic RNA viruses in the Conwy river catchment area in Wales, UK. Using a combination of filtering and nuclease treatment, we extracted the viral fraction from wastewater, estuarine river water and sediment, followed by RNASeq analysis on the Illumina HiSeq platform for the discovery of RNA virus genomes. We found a higher richness of RNA viruses in wastewater samples than in river water and sediment, and assembled a complete norovirus GI.2 genome from wastewater effluent, which was not contemporaneously detected by conventional qRT-PCR. To our knowledge, this is the first environmentally-derived norovirus genome sequence to be available from a public database. The simultaneous presence of diverse rotavirus signatures in wastewater indicated the potential for zoonotic infections in the area and suggested run-off from pig farms as a possible origin of these viruses. Our results show that viromics can be an important tool in the discovery of pathogenic viruses in the environment and can be used to inform and optimize reference-based detection methods provided appropriate and rigorous controls are included.\n\nImportanceEnteric viruses cause gastro-intestinal illness and are commonly transmitted through the faecal-oral route. When wastewater is released into river systems, these viruses can contaminate the environment. Our results show that we can use viromics to find the range of potentially pathogenic viruses that are present in the environment and identify prevalent genotypes. The ultimate goal is to trace the fate of these pathogenic viruses from origin to the point where they are a threat to human health, informing reference-based detection methods and water quality management.

microbiology↗

bioRxiv2018

Baculovirus AC102 is a nucleocapsid protein that is crucial for nuclear actin polymerization and nucleocapsid morphogensis

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of alphabaculoviruses, is an enveloped DNA virus that infects lepidopteran insects and is commonly known as a vector for protein expression and cell transduction. AcMNPV belongs to a diverse group of viral and bacterial pathogens that target the host cell actin cytoskeleton during infection. AcMNPV is unusual, however, in that it absolutely requires actin translocation into the nucleus early in infection, and actin polymerization within the nucleus late in infection coincident with viral replication. Of the six viral factors that are sufficient, when coexpressed, to induce the nuclear localization of actin, only AC102 is essential for viral replication and the nuclear accumulation of actin. We therefore sought to better understand the role of AC102 in actin mobilization in the nucleus early and late in infection. Although AC102 was thought to function early in infection, we found that AC102 is predominantly expressed as a late protein. In addition, we observed that AC102 is required for F-actin assembly in the nucleus during late infection, as well as for proper formation of viral replication structures and nucleocapsid morphogenesis. Finally, we found that AC102 is a nucleocapsid protein and a newly recognized member of a complex consisting of the viral proteins EC27, C42, and the actin polymerization protein P78/83. Taken together, our findings suggest that AC102 is necessary for nucleocapsid morphogenesis and actin assembly during late infection through its role as a component of the P78/83-C42-EC27-AC102 protein complex.\n\nIMPORTANCEThe baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an important biotechnological tool for protein expression and cell transduction, and related nucleopolyhedroviruses are also employed as environmentally benign insecticides. One impact of our work is to better understand the fundamental mechanisms through which AcMNPV exploits the cellular machinery of the host for replication, which may aid in the development of improved baculovirus-based research and industrial tools. Moreover, AcMNPVs ability to mobilize the host actin cytoskeleton within the cells nucleus during infection makes it a powerful cell biological tool. It is becoming increasingly clear that actin plays important roles in the cells nucleus, yet the regulation and function of nuclear actin is poorly understood. Our work to better understand how AcMNPV relocalizes and polymerizes actin within the nucleus may reveal fundamental mechanisms that govern nuclear actin regulation and function, even in the absence of viral infection.

microbiology↗

bioRxiv2018

Metatranscriptome analysis of the vaginal microbiota reveals potential mechanisms for recurrence and protection against metronidazole in bacterial vaginosis

Bacterial vaginosis (BV) is a prevalent multifactorial disease of women in their reproductive years characterized by a shift from the Lactobacillus spp. dominated microbial community towards a taxonomically diverse anaerobic community. For unknown reasons, some women do not respond to therapy. In our recent clinical study, out of 37 women diagnosed with BV, 31 were successfully treated with metronidazole, while 6 still had BV after treatment. To discover possible reasons for the lack of response in those patients, we performed a metatranscriptome analysis of their vaginal microbiota, comparing them to patients who responded. Seven out of 8 Cas genes of Gardnerella vaginalis were highly up-regulated in non-responding patients. Cas genes, in addition to protecting against phages, might be involved in DNA repair thus mitigating the bactericidal effect of DNA damaging agents like metronidazole. In the second part of our study, we analyzed the vaginal metatranscriptomes of four patients over three months and showed high in vivo expression of genes for pore-forming toxins in L. iners and of genes encoding enzymes for the production of hydrogen peroxide and D-lactate in L. crispatus.

microbiology↗

bioRxiv2018

A high-throughput in vitro translation screen towards discovery of novel antimalarial protein translation inhibitors

Drugs that target protein synthesis are well-validated for use as antimicrobials, yet specific high throughput (HTP) methods to screen for those targeting malaria are lacking. Here, we have developed a cell free in vitro translation (IVT) assay for the human malaria parasite, Plasmodium falciparum, which reconstitutes the native parasite protein translation machinery. Combining clarified IVT lysate with a click beetle luciferase reporter gene fused to untranslated regions of Pf histidine-rich proteins (hrp)-2 and 3, the HTP IVT assay accurately reports protein translation in a 384-well plate format using a standard spectrofluorometer. We validate the assay as effective in detecting compounds targeting the ribosome, ribosome co-factors (elongation factor 2) and cytosolic tRNA synthetases as well as its ability to find translation inhibitors in a blind screen using a high-density assay format amenable for high throughput. This demonstrates an ability to reconstitute the breadth of the parasite eukaryotic protein translation machinery in vitro and use it as a powerful platform for antimalarial drug discovery.

microbiology↗