Immunogenic and diagnostic potential of recombinant apical membrane antigen-1 from Plasmodium malariae.

The apical membrane antigen-1 (AMA-1) is a crucial target for malaria management and prevention strategies. While the immunogenicity of AMA-1 has been extensively studied for Plasmodium falciparum and Plasmodium vivax, there is a notable scarcity of information for Plasmodium malariae. In this study, recombinant PmAMA-1 was expressed in Escherichia coli, and its integrity was confirmed via western blotting and indirect immunofluorescence assays. Immunization of BALB/c mice with rPmAMA-1 emulsified in Freund's adjuvant resulted in significantly elevated specific IgG antibodies, predominantly IgG1. The immune response exhibited Th1, Th2, and Th17 phenotypes, with a notable Th1 bias. Antisera from immunized mice effectively recognized native PmAMA-1 on P. malariae. These results suggest that PmAMA-1 is a promising target for both vaccine development and diagnostic applications for P. malariae infections, offering dual preventive and diagnostic benefits in malaria control.

Genomic deletions in Aureobasidium pullulans by an AMA1 plasmid for gRNA and CRISPR/Cas9 expression.

BACKGROUND: Aureobasidium pullulans is a generalist polyextremotolerant black yeast fungus. It tolerates temperatures below 0 °C or salt concentrations up to 18%, among other stresses. A. pullulans genome sequencing revealed a high potential for producing bioactive metabolites. Only few molecular tools exist to edit the genome of A. pullulans, hence it is important to make full use of its potential. Two CRISPR/Cas9 methods have been proposed for the protoplast-based transformation of A. pullulans. These methods require the integration of a marker gene into the locus of the gene to be deleted, when the deletion of this gene does not yield a selectable phenotype. We present the adaptation of a plasmid-based CRISPR/Cas9 system developed in Aspergillus niger for A. pullulans to create deletion strains. RESULTS: The A. niger CRISPR/Cas9 plasmid led to efficient genomic deletions in A. pullulans. In this study, strains with deletions ranging from 30 to 862 bp were obtained by using an AMA1 plasmid-based genome editing strategy. CONCLUSION: The CRISPR/Cas9 transformation system presented in this study provides new opportunities for strain engineering of A. pullulans. This system allows expression of Cas9 and antibiotic resistance while being easy to adapt. This strategy could open the path to intensive genomic engineering in A. pullulans.

Application of gold immunochromatographic assay strip combined with digital evaluation for early detection of Toxoplasma gondii infection in multiple species.

BACKGROUND: Timely diagnosis of Toxoplasma gondii infection is necessary to prevent and control toxoplasmosis transmission. The gold immunochromatographic assay (GICA) is a means of rapidly detecting pathogen in samples. GICA-based diagnostic methods have been developed to accurately detect pathogens with high sensitivity and specificity, and their application in T. gondii diagnosis is expected to yield good results. METHODS: Colloidal gold test strips were produced using T. gondii C-terminal truncated apical membrane antigen 1 (AMA1C). Colloidal gold-AMA1C and colloidal gold-murine protein conjugate were synthesized under optimal conditions. A nitrocellulose membrane was treated with AMA1C and goat anti-mouse antibody as the test line and control line, respectively. In total, 90 cat serum samples were tested using AMA1C-GICA and a commercial enzyme linked immunosorbent assay (ELISA) kit. The GICA results were digitally displayed using a portable colloidal gold immunochromatographic test strip analyzer (HMREADER). The sensitivity, specificity, and stability of AMA1C-GICA were assessed, and this was then used to examine clinical samples, including 203 human sera, 266 cat sera, and 81 dog sera. RESULTS: AMA1C-GICA had a detection threshold of 1:32 for T. gondii-positive serum. The GICA strips specifically detected T. gondii antibodies and exhibited no reactivity with Plasmodium vivax, Paragonimus kellicotti, Schistosoma japonicum, Clonorchis sinensis, and Schistosoma mansoni. Consequently, 15 (16.7%) positive samples were detected using the AMA1C-GICA and commercial ELISA kits for each of the assays. The receiver-operating characteristic curve showed that GICA had a relative sensitivity of 85.3% and specificity of 92%, with an area under the curve of 98%. After analyzing clinical samples using HMREADER, 1.2%-23.4% of these samples were found to be positive for T. gondii. CONCLUSIONS: This study presents a novel assay that enables timely and efficient detection of serum antibodies against T. gondii, thereby allowing for its early clinical diagnosis. Furthermore, the integration of digital detection using HMREADER can enhance the implementation of GICA.

Screening of apical membrane antigen-1 (AMA1), dense granule protein-7 (GRA7) and rhoptry protein-16 (ROP16) antigens for a potential vaccine candidate against Toxoplasma gondii for chickens.

Toxoplasmosis is a zoonotic disease caused by the protozoan parasite, Toxoplasma gondii known to infect almost all animals, including birds and humans globally. This disease has impacted the livestock industry and public health, where infection of domestic animals increases the zoonotic risk of transmission of infection to humans, threatening public health. Hence the need to discover novel and safe vaccines to fight against toxoplasmosis. In the current study, a novel multiepitope vaccine was designed using immunoinformatics techniques targeting T. gondii AMA1, GRA7 and ROP16 antigens, consisting of antigenic, immunogenic, non-allergenic and cytokine inducing T-cell (9 CD8+ and 15 CD4+) epitopes and four (4) B-cell epitopes fused together using AAY, KK and GPGPG linkers. The tertiary model of the proposed vaccine was predicted and validated to confirm the structural quality of the vaccine. The designed vaccine was highly antigenic (antigenicity = 0.6645), immunogenic (score = 2.89998), with molecular weight of 73.35 kDa, instability and aliphatic index of 28.70 and 64.10, respectively; and GRAVY of -0.363. The binding interaction, stability and flexibility were assessed with molecular docking and dynamics simulation, which revealed the proposed vaccine to have good structural interaction (binding affinity = -106.882 kcal/mol) and stability when docked with Toll like receptor-4 (TLR4). The results revealed that the Profilin-adjuvanted vaccine is promising, as it predicted induction of enhanced immune responses through the production of cytokines and antibodies critical in blocking host invasion.

Protective efficacy of recombinant proteins AMA1 and IMP1 in rabbits infected with Eimeria intestinalis.

Eimeria intestinalis is one of the most pathogenic rabbit coccidia species causing severe intestinal damage and increased risk of secondary infection from opportunistic pathogens, which results in huge economic losses to the rabbit industry. Anticoccidial drugs are currently the main method to control coccidiosis; however, increasing resistance and drug residues have fueled research on anticoccidial vaccines. Apical membrane antigen 1 (AMA1) and immune mapped protein 1 (IMP1), as surface proteins, are associated with host invasion and might have the potential as candidate vaccine antigens. In the present study, recombinant IMP1 (rEiIMP1) and AMA1 (rEiAMA1) from E. intestinalis were expressed using Escherichia coli BL21. The immunoreactivity and immunoprotective effects of rEiIMP1 and rEiAMA1 were then analyzed. Fifty rabbits were grouped randomly (n = 10 per group): The unimmunized-unchallenged control group (sterilized phosphate-buffered saline (PBS)), the unimmunized-challenged control group (sterilized PBS), the vector protein-challenged control group (100 µg of pET-32a vector protein per rabbit), the rEiIMP1 immunized group (100 µg of rEiIMP1 per rabbit), and the rEiAMA1 immunized group (100 µg of rEiAMA1 per rabbit). After two immunizations, the rabbits were challenged with homologous oocysts (except for the unimmunized-unchallenged group). Serum specific antibody levels were assessed weekly throughout the experimental period; and the levels of different cytokines in the serum before the challenge were detected. The clinical symptoms, oocysts output, weight gain, feed conversion ratio (FCR), and lesion scores were recorded after experimental infection, and the anticoccidial indexes (ACIs) were calculated. The results showed that both rEiIMP1 and rEiAMA1 had good immunoreactivity. Rabbits immunized with rEiIMP1 and rEiAMA1 displayed 66.74 % and 63.14 % oocyst reduction, respective land 81.79 % and 78.87 % body weight gain, respectively. The rEiIMP1 and rEiAMA1 groups had lower FCRs (3.77:1 and 4.06:1, respectively) and lesion scores (P = 0.00). The rEiIMP1 and rEiAMA1 showed moderate effects, with an ACI of 152.09 and 147.17, respectively. Immunization induced high levels of anti-rEiIMP1 and -rEiAMA1 antibodies. Rabbits immunized with rEiIMP1 and rEiAMA1 displayed significantly increased interleukin (IL)- 2 (P = 0.00), interferon gamma (IFN)- γ (P = 0.00), and IL- 4 (P = 0.00) levels. Therefore, this study provided potential candidate vaccine antigens for E. intestinalis.

Phylogenetic analysis of Babesia gibsoni isolates of south India using apical membrane antigen, 50 kDa surface antigen, and 70 kDa heat shock protein genes.

The prevalence of canine babesiosis due to Babesia gibsoni has increased throughout the world including in southern India. The polymerase chain reaction (PCR) based molecular characterization of B. gibsoni in dogs of Kerala, south India, targeting three specific genes viz., apical membrane antigen (AMA1), 50 kDa surface antigen (P50), and heat shock protein (HSP70) was undertaken in this study. Out of 297 blood samples collected from clinically suspected animals, microscopy detected piroplasms of B. gibsoni in 60 (20.20 per cent), while the PCR targeting the BgP50 gene detected 85 (28.61 per cent). Polymerase chain reaction targeting the BgAMA1 and BgHSP70 detected a lesser number of samples (60 and 65 respectively) as positive. The phylogenetic analysis of BgHSP70 gene sequences did not reveal genetic heterogeneity among the B. gibsoni isolates of South India and from other countries, while the BgP50 gene differentiated the Indian isolates from Japanese isolates. When BgAMA1 was used for phylogenetic analysis, genetic variation was not observed among Indian and Taiwanese isolates, however, differentiated them from the Japanese isolates.

A Simple CRISPR/Cas9 System for Efficiently Targeting Genes of Aspergillus Section Flavi Species, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus terreus, and Aspergillus niger.

For Aspergillus flavus, a pathogen of considerable economic and health concern, successful gene knockout work for more than a decade has relied nearly exclusively on using nonhomologous end-joining pathway (NHEJ)-deficient recipients via forced double-crossover recombination of homologous sequences. In this study, a simple CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease) genome editing system that gave extremely high (>95%) gene-targeting frequencies in A. flavus was developed. It contained a shortened Aspergillus nidulans AMA1 autonomously replicating sequence that maintained good transformation frequencies and Aspergillus oryzae ptrA as the selection marker for pyrithiamine resistance. Expression of the codon-optimized cas9 gene was driven by the A. nidulans gpdA promoter and trpC terminator. Expression of single guide RNA (sgRNA) cassettes was controlled by the A. flavus U6 promoter and terminator. The high transformation and gene-targeting frequencies of this system made generation of A. flavus gene knockouts with or without phenotypic changes effortless. Additionally, multiple-gene knockouts of A. flavus conidial pigment genes (olgA/copT/wA or olgA/yA/wA) were quickly generated by a sequential approach. Cotransforming sgRNA vectors targeting A. flavus kojA, yA, and wA gave 52%, 40%, and 8% of single-, double-, and triple-gene knockouts, respectively. The system was readily applicable to other section Flavi aspergilli (A. parasiticus, A. oryzae, A. sojae, A. nomius, A. bombycis, and A. pseudotamarii) with comparable transformation and gene-targeting efficiencies. Moreover, it gave satisfactory gene-targeting efficiencies (>90%) in A. nidulans (section Nidulantes), A. fumigatus (section Fumigati), A. terreus (section Terrei), and A. niger (section Nigri). It likely will have a broad application in aspergilli. IMPORTANCE CRISPR/Cas9 genome editing systems have been developed for many aspergilli. Reported gene-targeting efficiencies vary greatly and are dependent on delivery methods, repair mechanisms of induced double-stranded breaks, selection markers, and genetic backgrounds of transformation recipient strains. They are also mostly strain specific or species specific. This developed system is highly efficient and allows knocking out multiple genes in A. flavus efficiently either by sequential transformation or by cotransformation of individual sgRNA vectors if desired. It is readily applicable to section Flavi species and aspergilli in other sections ("section" is a taxonomic rank between genus and species). This cross-Aspergillus section system is for wild-type isolates and does not require homologous donor DNAs to be added, NHEJ-deficient strains to be created, or forced recycling of knockout recipients to be performed for multiple-gene targeting. Hence, it simplifies and expedites the gene-targeting process significantly.

A Chimeric Peptide Inhibits Red Blood Cell Invasion by Plasmodium falciparum with Hundredfold Increased Efficacy.

Inhibiting the formation of a tight junction between two malaria parasite proteins, apical membrane antigen 1 and rhoptry neck protein 2, crucial for red blood cell invasion, prevents progression of the disease. In this work, we have used a unique approach to design a chimeric peptide, prepared by fusion of the best features of two peptide inhibitors, that has displayed parasite growth inhibition ex vivo with nanomolar IC50 , which is 100 times better than any of its parent peptides. Furthermore, to gain structural insights, we computationally modelled the hybrid peptide on its receptor.

Vesicular stomatitis virus-based vaccine targeting plasmodium blood-stage antigens elicits immune response and protects against malaria with protein booster strategy.

Merozoite invasion of the erythrocytes in humans is a key step in the pathogenesis of malaria. The proteins involved in the merozoite invasion could be potential targets for the development of malaria vaccines. Novel viral-vector-based malaria vaccine regimens developed are currently under clinical trials. Vesicular stomatitis virus (VSV) is a single-stranded negative-strand RNA virus widely used as a vector for virus or cancer vaccines. Whether the VSV-based malarial vaccine is more effective than conventional vaccines based on proteins involved in parasitic invasion is still unclear. In this study, we have used the reverse genetics system to construct recombinant VSVs (rVSVs) expressing apical membrane protein 1 (AMA1), rhoptry neck protein 2 (RON2), and reticulocyte-binding protein homolog 5 (RH5), which are required for Plasmodium falciparum invasion. Our results showed that VSV-based viral vaccines significantly increased Plasmodium-specific IgG levels and lymphocyte proliferation. Also, VSV-PyAMA1 and VSV-PyRON2sp prime-boost regimens could significantly increase the levels of IL-2 and IFN-γ-producing by CD4+ and CD8+ T cells and suppress invasion in vitro. The rVSV prime-protein boost regimen significantly increase Plasmodium antigen-specific IgG levels in the serum of mice compared to the homologous rVSV prime-boost. Furthermore, the protective efficacy of rVSV prime protein boost immunization in the mice challenged with P. yoelii 17XL was better compared to traditional antigen immunization. Together, our results show that VSV vector is a novel strategy for malarial vaccine development and preventing the parasitic diseases.

Preliminary evaluation of the protective effects of recombinant AMA1 and IMP1 against Eimeria stiedae infection in rabbits.

BACKGROUND: Eimeria stiedae parasitizes the bile duct, causing hepatic coccidiosis in rabbits. Coccidiosis control using anticoccidials led to drug resistance and residues; therefore, vaccines are required as an alternative control strategy. Apical membrane antigen 1 (AMA1) and immune mapped protein 1 (IMP1) are surface-located proteins that might contribute to host cell invasion, having potential as candidate vaccine antigens. METHODS: Herein, we cloned and expressed the E. stiedae EsAMA1 and EsIMP1 genes. The reactogenicity of recombinant AMA1 (rEsAMA1) and IMP1 (rEsIMP1) proteins were investigated using immunoblotting. For the vaccination-infection trial, rabbits were vaccinated with rEsAMA1 and rEsIMP1 (both 100 µg/rabbit) twice at 2-week intervals. After vaccination, various serum cytokines were measured. The protective effects of rEsAMA1 and rEsIMP1 against E. stiedae infection were assessed using several indicators. Sera were collected weekly to detect the specific antibody levels. RESULTS: Both rEsAMA1 and rEsIMP1 showed strong reactogenicity. Rabbits vaccinated with rEsAMA1 and rEsIMP1 displayed significantly increased serum IL-2 (F (4, 25) = 9.53, P = 0.000), IL-4 (F (4, 25) = 7.81, P = 0.000), IL-17 (F (4, 25) = 8.55, P = 0.000), and IFN-γ (F (4, 25) = 6.89, P = 0.001) levels; in the rEsIMP1 group, serum TGF-ß1 level was also elevated (F (4, 25) = 3.01, P = 0.037). After vaccination, the specific antibody levels increased and were maintained at a high level. The vaccination-infection trial showed that compared with the positive control groups, rabbits vaccinated with the recombinant proteins showed significantly reduced oocyst output (F (5, 54) = 187.87, P = 0.000), liver index (F (5, 54) = 37.52, P = 0.000), and feed conversion ratio; body weight gain was significantly improved (F (5, 54) = 28.82, P = 0.000). CONCLUSIONS: rEsAMA1 and rEsIMP1 could induce cellular and humoral immunity, protecting against E. stiedae infection. Thus, rEsAMA1 and rEsIMP1 are potential vaccine candidates against E. stiedae.

Recombinant AMA1 Virus-like Particle Antigen for Serodiagnosis of Toxoplasma gondii Infection.

Toxoplasmosis diagnosis predominantly relies on serology testing via enzyme-linked immunosorbent assay (ELISA), but these results are highly variable. Consequently, various antigens are being evaluated to improve the sensitivity and specificity of toxoplasmosis serological diagnosis. Here, we generated Toxoplasma gondii virus-like particles displaying AMA1 of T. gondii and evaluated their diagnostic potential. We found that AMA1 VLPs were highly sensitive and reacted with the sera acquired from mice infected with either T. gondii ME49 or RH strains. The overall IgG and IgM antibody responses elicited by AMA1 VLPs were substantially higher than those induced by the conventionally used T. gondii lysate antigen (TLA). Importantly, AMA1 VLPs were capable of detecting parasitic infection with T. gondii RH and ME49 as early as 1 week post-infection, even when mice were exposed to low infectious doses (5 × 103 and 10 cysts, respectively). AMA1 VLPs also did not cross-react with the immune sera acquired from Plasmodium berghei-infected mice. Compared to TLA, stronger antibody responses were induced by AMA1 VLPs when tested using T. gondii-infected human sera. The sensitivities and specificities of the two antigens were substantially different, with AMA1 VLPs demonstrating over 90% sensitivity and specificity, whereas these values were in the 70% range for the TLA. These results indicated that AMA1 VLPs can detect infections of both T. gondii ME49 and RH at an early stage of infection caused by very low infection doses in mice, and these could be used for serological diagnosis of human toxoplasmosis.

Evasion of Cas9 toxicity to develop an efficient genome editing system and its application to increase ethanol yield in Fusarium venenatum TB01.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) system is a powerful genome editing tool that has been successfully established in some filamentous fungi due to its high flexibility and efficiency. However, the potential toxicity of Cas9 restricts the further popularization and application of this system to some degree. The AMA1 element is a self-replicator derived from Aspergillus nidulans, and its derived vectors can be readily lost without selection. In this study, we eliminated Cas9 toxicity to Fusarium venenatum TB01 based on 100% AMA1-based Cas9 expression vector loss. Meanwhile, two available endogenous Pol III promoters (FvU6374 and Fv5SrRNA) used for sgRNA expression of the CRISPR/Cas9 system were excavated. Compared to FvU6374 (40-50%), Fv5SrRNA exhibited higher single-gene editing efficiency (> 85%), and the efficiency of simultaneous editing of the two genes using Fv5SrRNA was over 75%. Based on this system, a butanediol dehydrogenase encoding gene FvBDH was deleted, and the ethanol yield in variants increased by 52% compared with that of the wild-type. The highly efficient CRISPR/Cas9 system developed here lays the technical foundation for advancing the development of F. venenatum TB01 through metabolic engineering, and the obtained FvBDH gene-edited variants have the potential to simultaneously produce mycoprotein and ethanol by further gene modification and fermentation process optimization in the future.Key points• Cas9 toxicity disappeared and DNA-free gene-edited strains obtained after vector loss• Promoter Fv5SrRNA conferred TB01 higher gene editing efficiency than FvU6374•Deletion of the FvBDH gene resulted in a 52% increase in ethanol yield.

Identification of factors associated with residual malaria transmission using school-based serological surveys in settings pursuing elimination.

BACKGROUND: Targeted research on residual malaria transmission is important to improve strategies in settings pursuing elimination, where transmission reductions prove challenging. This study aimed to detect and characterize spatial heterogeneity and factors associated with Plasmodium falciparum infections and exposure, P. falciparum apical membrane antigen 1 (PfAMA1) antibody (Ab) response, in the Central Highlands of Madagascar (CHL). METHODS: From May to July 2014, a cross-sectional school-based survey was carried out in 182 fokontany (villages) within 7 health districts of the CHL. Rapid diagnostic tests (RDTs) and a bead-based immunoassay including PfAMA1 antigen biomarker were used to estimate malaria prevalence and seroprevalence, respectively. Local Moran's I index was used to detect spatial "hotspots". Remotely sensed environmental data-temperature, vegetation indices, land covers, and elevation-were used in multivariable mixed-effects logistic regression models to characterize factors associated with malaria infection and cumulative exposure. RESULTS: Among 6,293 school-children ages 2-14 years surveyed, RDT prevalence was low at 0.8% (95% CI 0.6-1.1%), while PfAMA1 Ab seroprevalence was 7.0% (95% CI 6.4-7.7%). Hotspots of PfAMA1 Ab seroprevalence were observed in two districts (Ankazobe and Mandoto). Seroprevalence increased for children living > 5 km from a health centre (adjusted odds ratio (OR) = 1.6, 95% CI 1.2-2.2), and for those experiencing a fever episode in the previous 2 weeks (OR 1.7, 95% CI 1.2-2.4), but decreased at higher elevation (for each 100-m increase, OR = 0.7, 95% CI 0.6-0.8). A clear age pattern was observed whereby children 9-10 years old had an OR of 1.8 (95% CI 1.2-2.4), children 11-12 years an OR of 3.7 (95% CI 2.8-5.0), and children 13-14 years an OR of 5.7 (95% CI 4.0-8.0) for seropositivity, compared with younger children (2-8 years). CONCLUSION: The use of serology in this study provided a better understanding of malaria hotspots and associated factors, revealing a pattern of higher transmission linked to geographical barriers in health care access. The integration of antibody-assays into existing surveillance activities could improve exposure assessment, and may help to monitor the effectiveness of malaria control efforts and adapt elimination interventions.

Immunization of Cattle With Recombinant Structural Ectodomains I and II of Babesia bovis Apical Membrane Antigen 1 [BbAMA-1(I/II)] Induces Strong Th1 Immune Response.

Both strong innate and adaptive immune responses are an important component of protection against intraerythrocytic protozoan parasites. Resistance to bovine babesiosis is associated with interferon (IFN)-γ mediated responses. CD4+ T cells and macrophages have been identified as major effector cells mediating the clearance of pathogens. Previously, the apical membrane antigen 1 (AMA-1) was found to significantly induce the immune response inhibiting B. bovis merozoite growth and invasion. However, a detailed characterization of both humoral and cellular immune responses against the structure of B. bovis AMA-1 (BbAMA-1) has not yet been established. Herein, the present study aimed to express the recombinant BbAMA-1 domain I+II protein [rBbAMA-1(I/II)], which is the most predominant immune response region, and to characterize its immune response. As a result, cattle vaccinated with BbAMA-1(I/II) significantly developed high titters of total immunoglobulin (Ig) G antibodies and a high ratio of IgG2/IgG1 when compared to control groups. Interestingly, the BbAMA-1(I/II)-based formulations produced in our study could elicit CD4+ T cells and CD8+ T cells producing IFN-γ and tumor necrosis factor (TNF)-α. Collectively, the results indicate that immunization of cattle with BbAMA-1(I/II) could induce strong Th1 cell responses. In support of this, we observed the up-regulation of Th1 cytokine mRNA transcripts, including IFN-γ, TNF-α, Interleukin (IL)-2 and IL-12, in contrast to down regulation of IL-4, IL-6 and IL-10, which would be indicative of a Th2 cytokine response. Moreover, the up-regulation of inducible nitric oxide synthase (iNOS) was observed. In conclusion, this is the first report on the in-depth immunological characterization of the response to BbAMA-1. According to our results, BbAMA-1 is recognized as a potential candidate vaccine against B. bovis infection. As evidenced by the Th1 cell response, it could potentially provide protective immunity. However, further challenge-exposure with virulent B. bovis strain in immunized cattle would be needed to determine its protective efficacy.

Heterologous Expression of Fungal Biosynthetic Pathways in Aspergillus nidulans Using Episomal Vectors.

Filamentous fungi produce a wide diversity of secondary metabolites, whose biosynthesis is encoded in biosynthetic gene clusters (BGCs). As novel BGCs are often found in fungal species that are genetically intractable or difficult to cultivate, heterologous expression is increasingly being used for compound discovery. In addition, heterologous expression is a useful strategy to elucidate the function of the genes within a BGC and shed light on their enzymatic mechanisms. Here, we describe a method for BGC elucidation using multi-marker AMA1-based pYFAC vectors for episomal expression in the fungal host Aspergillus nidulans. The pYFAC vectors have the advantage of high transformation efficiency and support high compound production. In addition, different pathway intermediates can be easily evaluated by testing different vector combinations. This protocol encompasses different AMA1-based strategies for BGC expression such as cloning of a BGC native sequence, promoter exchange or transcription factor overexpression. We also describe procedures for A. nidulans protoplasting, transformation, and small-scale culture analysis of strains containing AMA1 vectors.

Amplicon Sequencing as a Potential Surveillance Tool for Complexity of Infection and Drug Resistance Markers in Plasmodium falciparum Asymptomatic Infections.

BACKGROUND: Genotyping Plasmodium falciparum subpopulations in malaria infections is an important aspect of malaria molecular epidemiology to understand within-host diversity and the frequency of drug resistance markers. METHODS: We characterized P. falciparum genetic diversity in asymptomatic infections and subsequent first febrile infections using amplicon sequencing (AmpSeq) of ama1 in Coastal Kenya. We also examined temporal changes in haplotype frequencies of mdr1, a drug-resistant marker. RESULTS: We found >60% of the infections were polyclonal (complexity of infection [COI] >1) and there was a reduction in COI over time. Asymptomatic infections had a significantly higher mean COI than febrile infections based on ama1 sequences (2.7 [95% confidence interval {CI}, 2.65-2.77] vs 2.22 [95% CI, 2.17-2.29], respectively). Moreover, an analysis of 30 paired asymptomatic and first febrile infections revealed that many first febrile infections (91%) were due to the presence of new ama1 haplotypes. The mdr1-YY haplotype, associated with chloroquine and amodiaquine resistance, decreased over time, while the NY (wild type) and the NF (modulates response to lumefantrine) haplotypes increased. CONCLUSIONS: This study emphasizes the utility of AmpSeq in characterizing parasite diversity as it can determine relative proportions of clones and detect minority clones. The usefulness of AmpSeq in antimalarial drug resistance surveillance is also highlighted.

Application of recyclable CRISPR/Cas9 tools for targeted genome editing in the postharvest pathogenic fungi Penicillium digitatum and Penicillium expansum.

Penicillium digitatum and Penicillium expansum are plant pathogenic fungi that cause the green and blue mold diseases, respectively, leading to serious postharvest economic losses worldwide. Moreover, P. expansum can produce mycotoxins, which are hazardous compounds to human and animal health. The development of tools that allow multiple and precise genetic manipulation of these species is crucial for the functional characterization of their genes. In this sense, CRISPR/Cas9 represents an excellent opportunity for genome editing due to its efficiency, accuracy and versatility. In this study, we developed protoplast generation and transformation protocols and applied them to implement the CRISPR/Cas9 technology in both species for the first time. For this, we used a self-replicative, recyclable AMA1-based plasmid which allows unlimited number of genomic modifications without the limitation of integrative selection markers. As test case, we successfully targeted the wetA gene, which encodes a regulator of conidiophore development. Finally, CRISPR/Cas9-derived ΔwetA strains were analyzed. Mutants showed reduced axenic growth, differential pathogenicity and altered conidiogenesis and germination. Additionally, P. digitatum and P. expansum ΔwetA mutants showed distinct sensitivity to fungal antifungal proteins (AFPs), which are small, cationic, cysteine-rich proteins that have become interesting antifungals to be applied in agriculture, medicine and in the food industry. With this work, we demonstrate the feasibility of the CRISPR/Cas9 system, expanding the repertoire of genetic engineering tools available for these two important postharvest pathogens and open up the possibility to adapt them to other economically relevant phytopathogenic fungi, for which toolkits for genetic modifications are often limited.

Genetic diversity of Plasmodium falciparum AMA-1 antigen from the Northeast Indian state of Tripura and comparison with global sequences: implications for vaccine development.

BACKGROUND: Malaria continues to be a major public health problem in the Northeastern part of India despite the implementation of vector control measures and changes in drug policies. To develop successful vaccines against malaria, it is important to assess the diversity of vaccine candidate antigens in field isolates. This study was done to assess the diversity of Plasmodium falciparum AMA-1 vaccine candidate antigen in a malaria-endemic region of Tripura in Northeast India and compare it with previously reported global isolates with a view to assess the feasibility of developing a universal vaccine based on this antigen. METHODS: Patients with fever and malaria-like illness were screened for malaria and P. falciparum positive cases were recruited for the current study. The diversity of PfAMA-1 vaccine candidate antigen was evaluated by nested PCR and RFLP. A selected number of samples were sequenced using the Sanger technique. RESULTS: Among 56 P. falciparum positive isolates, Pfama-1 was successfully amplified in 75% (n = 42) isolates. Allele frequencies of PfAMA-1 antigen were 16.6% (n = 7) for 3D7 allele and 33.3% (n = 14) in both K1 and HB3 alleles. DNA sequencing revealed 13 haplotypes in the Pfama-1 gene including three unique haplotypes not reported earlier. No unique amino-acid substitutions were found. Global analysis with 2761 sequences revealed 435 haplotypes with a very complex network composition and few clusters. Nucleotide diversity for Tripura (0.02582 ± 0.00160) showed concordance with South-East Asian isolates while recombination parameter (Rm = 8) was lower than previous reports from India. Population genetic structure showed moderate differentiation. CONCLUSIONS: Besides documenting all previously reported allelic forms of the vaccine candidate PfAMA-1 antigen of P. falciparum, new haplotypes not reported earlier, were found in Tripura. Neutrality tests indicate that the Pfama-1 population in Tripura is under balancing selection. This is consistent with global patterns. However, the high haplotype diversity observed in the global Pfama-1 network analysis indicates that designing a universal vaccine based on this antigen may be difficult. This information adds to the existing database of genetic diversity of field isolates of P. falciparum and may be helpful in the development of more effective vaccines against the parasite.

A bivalent vaccine against avian necrotic enteritis and coccidiosis.

AIMS: In this study, we attempted to design a recombinant vaccine harbouring domain with a key role in enterocyte attachment and cell invasion in necrotic enteritis (NE) and coccidiosis. METHODS AND RESULTS: In this study, we investigated whether a recombinant protein consisting of necrotic enteritis B-like toxin, C-terminal domain of alpha-toxin, apical membrane antigen 1 (AMA1), and Rhoptry neck protein 2 (RON2) which we call "NeCoVac" hereafter, can improve protection against both diseases compared to vaccination with each antigen in previous studies. Birds intestinal lesion scores and specific antibody levels were measured to determine protection after oral gavage challenges with virulent Clostridium perfringens and LIVACOX® T. Birds immunized with NeCoVac were protected up to 84% against NE and coccidiosis compared to unimmunized and even positive groups (groups treated with LIVACOX® T [coccidiosis live vaccine] and tylosin as routine veterinary interventions) (p < 0.05). CONCLUSIONS: Our findings suggest that vaccination with NeCoVac is highly efficient in protecting birds from NE, coccidiosis and a combination of both diseases. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first one to describe the combinatorial use of AMA1 and RON2 against coccidiosis, and the first report using NeCoVac against NE and coccidiosis together.

A universal ELISA assay for detecting six strains of ovine Babesia species in China.

Ovine babesiosis, caused by genus of Babesia, is a zoonotic disease and mainly transmitted by hard ticks. It has led to enormous economic losses to the sheep industry in China. In the present study, an ELISA assay for simultaneous detection six strains of Babesia spp., including B. motasi Lintan, B. motasi Tianzhu, B. motasi Hebei, B. motasi Ningxian, Babesia sp. Xinjiang and Babesia sp. Dunhuang, was developed using Apical Membrane Antigen 1 (AMA1) as candidate diagnostic antigen. The sensitivity and specificity of the established ELISA were 97.4 % and 98.0 %, respectively. Relatively high level of specific antibodies could be detected from 12th day to 126th day after sheep experimentally infected with Babesia spp.. A small scale of field sera was investigated using the developed ELISA assay, and the average positive rate was 51.98 %. This study provides an easy to operate, cost effective and time saving approach, which is suitable for both field and experimental samples, thus it could be a useful tool in epidemiological investigations and diagnoses of ovine babesiosis.