An unconventional SNARE complex mediates exocytosis at the plasma membrane and vesicular fusion at the apical annuli in Toxoplasma gondii.

Exocytosis is a key active process in cells by which proteins are released in bulk via the fusion of exocytic vesicles with the plasma membrane. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-mediated vesicle fusion with the plasma membrane is essential in most exocytotic pathways. In mammalian cells, the vesicular fusion step of exocytosis is normally mediated by Syntaxin-1 (Stx1) and SNAP25 family proteins (SNAP25 and SNAP23). However, in Toxoplasma gondii, a model organism of Apicomplexa, the only SNAP25 family protein, with a SNAP29-like molecular structure, is involved in vesicular fusion at the apicoplast. Here, we reveal that an unconventional SNARE complex comprising TgStx1, TgStx20, and TgStx21 mediates vesicular fusion at the plasma membrane. This complex is essential for the exocytosis of surface proteins and vesicular fusion at the apical annuli in T. gondii.

Tumor-Derived PD-L1+ Exosomes with Natural Inflammation Tropism for Psoriasis-Targeted Treatment.

Psoriasis is a chronic inflammatory disease whose etiology is directly related to the dysregulation of cutaneous immune homeostasis. However, how to finely modulate the skin immune microenvironment to restore homeostasis remains an important challenge. Inspired by the natural attribute of tumor exosomes in the immune escape, the tumor-derived exosomes as an active targeting nanoplatform for the effective treatment of inflammatory skin disorder were first reported. As keratinocytes and immune cells express high PD-1 during the onset of psoriasiform skin inflammation, the PD-L1-positive exosomes derived from melanoma cells carrying pristimerin with extremely anti-inflammatory potential were yielded to treat psoriasis. The PD-L1+ exosomes carrying pristimerin were characterized, and the cellular uptake was performed to evaluate the PD-1 target capability. The anti-inflammatory action of PD-L1+ exosomes carrying pristimerin was observed in both in vitro and in vivo models of psoriasis. Our exosomes substantially increased pristimerin uptake with CD4+ T cells and keratinocytes, significantly inhibited the proliferation of Th17 cells, and promoted Treg differentiation in a psoriasis-like model. Obviously, PD-L1+ exosomes carrying pristimerin significantly and safely reversed imiquimod (IMQ)-induced psoriasis in mice, indicated by reducing epidermal thickness, decreasing plaque formation, and suppressed excessive inflammatory response, due to its dual targeting of both CD4+ T cells and keratinocytes gathering around the lesion. The inflammatory cell infiltration and pro-inflammatory cytokine production in psoriasis were suppressed by our engineered exosomes. Besides, PD-L1+ exosomes carrying pristimerin treatment alleviated ferroptosis-related changes in psoriatic skin, thereby dampening excessive inflammation and, in turn, decreasing the abnormal proliferation of keratinocytes in psoriatic lesions. This study demonstrates that our engineered exosomes can not only act as a treat-to-target strategy for psoriasis treatment but also provide insight in clinical application of inflammatory disorders.

The Sec1/Munc18-like proteins TgSec1 and TgVps45 play pivotal roles in assembly of the pellicle and sub-pellicle network in Toxoplasma gondii.

Post-Golgi vesicle trafficking is indispensable for precise movement of proteins to the pellicle, the sub-pellicle network and apical secretory organelles in Apicomplexa. However, only a small number of molecular complexes involved in trafficking, tethering and fusion of vesicles have been identified in Toxoplasma gondii. Consequently, it is unclear how complicated vesicle trafficking is accomplished in this parasite. Sec1/Munc18-like (SM) proteins are essential components of protein complexes involved in vesicle fusion. Here, we found that depletion of the SM protein TgSec1 using an auxin-inducible degron-based conditional knockout strategy led to mislocalization of plasma membrane proteins. By contrast, conditional depletion of the SM protein TgVps45 led to morphological changes, asymmetrical loss of the inner membrane complex and defects in nucleation of sub-pellicular microtubules, polarization and symmetrical assembly of daughter parasites during repeated endodyogeny. TgVps45 interacts with the SNARE protein TgStx16 and TgVAMP4-1. Conditional ablation of TgStx16 causes the similar growth defect like TgVps45 deficiency suggested they work together for the vesicle fusion at TGN. These findings indicate that these two SM proteins are crucial for assembly of pellicle and sub-pellicle network in T. gondii respectively.

Expression of immunoproteasome subunits in the brains of Toxoplasma gondii-infected mice.

The immunoproteasomes are specific proteasomes that clear oxidant-damaged proteins under inflammatory conditions in various diseases. Toxoplasma gondii (T. gondii) infects the central nervous system and causeencephalitis. However, the relationship between the immunoproteasomes and brain inflammation during T. gondii infection is not well characterized. In this study, we established an in vivo mouse model of T. gondii PLK strain infection via intraperitoneal injection and evaluated the expression of immunoproteasome subunits in the brains of infected mice. The results demonstrated that first, pathological changes in the brains of infected mice increase in severity over time. Second, following T. gondii infection, activated microglia and astrocytes undergo a series of functional alterations and morphological transformations, including proliferation and migration. Third, T. gondii infection induces expression of inflammatory cytokines, including IFN-γ, IL-1ß, TNF-α, and IL-6. Fourth, the immunoproteasome subunits low-molecular-weight polypeptide 2 (LMP2), LMP7, and LMP10 mRNA and protein levels are significantly upregulated in T. gondii-infected mouse brains, as shown by RT-qPCR and western blot analysis, compared with that in vehicle-treated brains, and their expression is localized in the microglia, astrocytes, and neurons of T. gondii-infected brains, as determined via immunofluorescence staining. Furthermore, the western blot mean gray value for the immunoproteasome subunits and the positive microglia and astrocyte immunohistochemical signals in the brains of T. gondii-infected mice were positively correlated, indicating that the observed relationships were highly significant. Therefore, it was concluded that the induction of the immunoproteasomes is a pathogenic mechanism underlying T. gondii infection-induced inflammation.

GABARAPL2 Is Critical for Growth Restriction of Toxoplasma gondii in HeLa Cells Treated with Gamma Interferon.

Gamma interferon (IFN-γ)-induced innate immune responses play important roles in the inhibition of Toxoplasma gondii infection. It has been reported that IFN-γ stimulates non-acidification-dependent growth restriction of T. gondii in HeLa cells, but the mechanism remains unclear. Here, we found that γ-aminobutyric acid (GABA) receptor-associated protein-like 2 (GABARAPL2) plays a critical role in parasite restriction in IFN-γ-treated HeLa cells. GABARAPL2 is recruited to membrane structures surrounding parasitophorous vacuoles (PV). Autophagy adaptors are required for the proper localization and function of GABARAPL2 in the IFN-γ -induced immune response. These findings provide further understanding of a noncanonical autophagy pathway responsible for IFN-γ-dependent inhibition of T. gondii growth in human HeLa cells and demonstrate the critical role of GABARAPL2 in this response.

Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway.

Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum Therefore, AATs are suggested as drug targets against Plasmodium The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondiiin vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

Localization and enzyme kinetics of aminopeptidase N3 from Toxoplasma gondii.

Aminopeptidase N is an important metalloenzyme from the M1 zinc metallopeptidase family, which is present in numerous apicomplexan parasites, including Plasmodium, Eimeria, and Cryptosporidium. Aminopeptidase N is a potential drug target, and hence, its properties have been widely investigated. In the current study, the cellular localization and enzyme characteristics of Toxoplasma gondii aminopeptidase N3 (TgAPN3) were evaluated in vitro. Cellular localization analysis revealed that TgAPN3 and GRA protein were co-located in the organelle and parasitophorous vacuole of T. gondii. The secretion assay showed that TgAPN3 could be co-secreted from the tachyzoites with GRA protein. A functional recombinant Toxoplasma aminopeptidase N3 (rTgAPN3) was produced in Escherichia coli. The enzyme activity was first determined using a fluorogenic H-Ala-MCA substrate. Some activity of rTgAPN3 was observed between pH 3.0 and 8.0, with a peak at pH 7.0. The activity was significantly enhanced in the presence of Co2+ ions. Substrate specificity of rTgAPN3 was then evaluated. The enzyme showed a preference for substrates containing N-terminal Ala residues, followed by Tyr and Cys. The rTgAPN3 activity was significantly inhibited by bestatin and phebestatin. In general, TgAPN3 was a structurally conserved member of the M1 family, although it also displayed unique biochemical characteristics. These results lay the foundation for a functional study of TgAPN3 and constitute its putative identification as a drug target.

Translationally controlled tumor protein is required for the fast growth of Toxoplasma gondii and maintenance of its intracellular development.

Translationally controlled tumor protein (TCTP) is a highly conserved, multifunctional protein that has been implicated in a range of cell physiologic processes, especially cell growth and development. A TCTP-like gene has been identified in the Toxoplasma genome [ Toxoplasma gondii TCTP ( TgTCTP)], although its function remains unknown. The sequence analysis of TgTCTP indicated that it is a highly conserved protein in eukaryotes. We found that the expression level of TgTCTP in the virulent RH strain was significantly higher than that in the avirulent PLK strain. Indirect immunofluorescence showed that TgTCTP was expressed in the parasite cytoplasm. The localization of TgTCTP was unchanged during the replication of the parasite. We expressed a functional recombinant TgTCTP (r TgTCTP) protein in Escherichia coli and found that the recombinant protein could form a multimer. We then evaluated the function of TgTCTP using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 knockout (KO) system. Phenotypic analysis of the KO strain (Δ TgTCTP) revealed that TgTCTP is required for the robust growth of the parasites. TgTCTP deficiency also led to early egress of the parasites and subsequent impairment in their invasion and attachment abilities. We subsequently found that the multimer form of TgTCTP might not be necessary for the growth and replication of the parasite. Then the expression profiling of genes in the Δ TgTCTP and complement strains were analyzed. The results revealed that 988 genes were regulated in Δ TgTCTP compared with the complement strain. Overall, although not essential, TgTCTP is required for the fast growth of Tg and maintenance of its intracellular development.-Zheng, J., Chen, Y., Li, Z., Cao, S., Zhang, Z., Jia, H. Translationally controlled tumor protein is required for the fast growth of Toxoplasma gondii and maintenance of its intracellular development.

The Nrf2 pathway is required for intracellular replication of Toxoplasma gondii in activated macrophages.

Reactive oxygen species (ROS) produced by oxidases and nonenzymatic sources are important for host defence against intracellular pathogens. In this study, we knocked out the Nrf2 gene in RAW264.7 cells using the CRISPR/Cas9 system and investigated the antioxidant effects of the Nrf2 pathway in the cells stimulated by IFN-γ and TNF-α. The results indicated that the Nrf2 signalling pathway is necessary for maintaining redox homeostasis in activated RAW264.7 cells. Inactivation of Nrf2 impaired parasite growth. We also found that p62 contributes to Nrf2-mediated pathways involved in T gondii infection. These findings suggest that the Nrf2/Keap1 pathway may be targeted to prevent and treat toxoplasmosis.

The moving junction protein RON4, although not critical, facilitates host cell invasion and stabilizes MJ members.

Toxoplasma gondii is an obligate intracellular parasite of phylum Apicomplexa. To facilitate high-efficiency invasion of host cells, T. gondii secretes various proteins related to the moving junction (MJ) complex from rhoptries and micronemes into the interface between the parasite and host. AMA1/RON2/4/5/8 is an important MJ complex, but its mechanism of assembly remains unclear. In this study, we used the CRISPR-Cas9 system to generate a derivative of T. gondii strain RH with a null mutation in TgRON4, thought to be an essential MJ component. Deficiency of TgRON4 moderately decreased invasion ability relative to that of the wild-type parasite. In addition, expression of the endogenous N-terminal fragment of RON5 decreased in the mutant. Together, the results improve our understanding of the assembly mechanism of the MJ complex of T. gondii and raise the possibility of developing new therapeutic drugs that target this complex.

Functional characterization of X-prolyl aminopeptidase from Toxoplasma gondii.

In the present study, a recombinant aminopeptidase P (rTgAPP) from Toxoplasma gondii was expressed in Escherichia coli to evaluate its enzyme parameters. The rTgAPP showed strong activity against a synthetic substrate for aminopeptidase P at pH 8·0 with a K m value of 0·255 µ m and a k cat value of 35·6 s-1. The overall catalytic efficiency (k cat/K m) of the rTgAPP was 139·6 × 105 M-1 s-1. The activity of rTgAPP was enhanced by the addition of divalent cations and inhibited by bestatin. Deletion of TgAPP gene in the parasite through a CRISPR/Cas9 system resulted in inhibition of growth indicating the importance of TgAPP. Thus our findings reveal that TgAPP is an active enzyme in T. gondii and provide an insight into the function of TgAPP.

First characterization in China of Encephalitozoon cuniculi in the blue fox (Alopex lagopus).

Encephalitozoon cuniculi is a microsporidian parasite that infects a wide range of vertebrates, including primates. It has recently emerged as an opportunistic parasite of patients infected with the human immunodeficiency virus. The blue fox (Alopex lagopus; also known as the arctic fox) is one of the most susceptible species for encephalitozoonosis. Here, we report an outbreak of encephalitozoonosis at a fox farm in China. The isolated parasites displayed the typical morphology of E. cuniculi as assessed by Masson's trichrome staining. Analysis of the internal transcribed spacer sequence indicated that the isolated parasite is a genotype III strain of E. cuniculi. Furthermore, phylogenetic analysis of the PTP1 gene verifies classification of this new strain (termed LN-1) with other genotype III E. cuniculi strains, though the PTP3 and SWP1 sequences diverge from the reference strain. This is the first report of encephalitozoonosis in farmed blue foxes in China.

Comparison of the performance of SAG2, GRA6, and GRA7 for serological diagnosis of Toxoplasma gondii infection in cats.

Toxoplasmosis is an important zoonotic disease caused by Toxoplasma gondii that can infect almost all warm-blooded animals worldwide, including humans. The high prevalence of T. gondii infection and its ability to cause serious harm to humans and animals, especially immunodeficient individuals, make it a key public health issue. Accurate diagnostic tools with high sensitivity are needed for controlling T. gondii infection. In the current study, we compared the performance of recombinant SAG2, GRA6, and GRA7 in ELISA for the serological diagnosis of T. gondii infection in cats. We further investigated the antigenicity of recombinant dense granule protein 3 (rGRA3), rGRA5, rGRA8, and rSRS29A expressed in a plant-based, cell-free expression system for detecting antibodies in T. gondii-infected cats. In summary, our data suggest that GRA7 is more sensitive than the other two antigens for the serodiagnosis of T. gondii infection in cats, and GRA3 expressed in the cell-free system is also a priming antigen in serological tests for detecting T. gondii infection in cats.

Kanglexin counters vascular smooth muscle cell dedifferentiation and associated arteriosclerosis through inhibiting PDGFR.

BACKGROUND: Dysregulation of vascular smooth muscle cell (VSMC) function leads to a variety of diseases such as atherosclerosis and hyperplasia after injury. However, antiproliferative drug targeting VSMC exhibits poor specificity. Therefore, there is an urgent to develop highly specific antiproliferative drugs to prevention and treatment VSMC dedifferentiation associated arteriosclerosis. Kanglexin (KLX), a new anthraquinone compound designed by our team, has potential to regulate VSMC phenotype according to the physicochemical properties. PURPOSE: This project aims to evaluate the therapeutic role of KLX in VSMC dedifferentiation and atherosclerosis, neointimal formation and illustrates the underlying molecular mechanism. METHODS: In vivo, the ApoE-/- mice were fed with high-fat diet (HFD) for a duration of 13 weeks to establish the atherosclerotic model. And rat carotid artery injury model was performed to establish the neointimal formation model. In vitro, PDGF-BB was used to induce VSMC dedifferentiation. RESULTS: We found that KLX ameliorated the atherosclerotic progression including atherosclerotic lesion formation, lipid deposition and collagen deposition in aorta and aortic sinus in atherosclerotic mouse model. In addition, The administration of KLX effectively ameliorated neointimal formation in the carotid artery following balloon injury in SD rats. The findings derived from molecular docking and surface plasmon resonance (SPR) experiments unequivocally demonstrate that KLX had potential to bind PDGFR-ß. Mechanism research work proved that KLX prevented VSMC proliferation, migration and dedifferentiation via activating the PDGFR-ß-MEK -ERK-ELK-1/KLF4 signaling pathway. CONCLUSION: Collectively, we demonstrated that KLX effectively attenuated the progression of atherosclerosis in ApoE-/- mice and carotid arterial neointimal formation in SD rats by inhibiting VSMC phenotypic conversion via PDGFR-ß-MEK-ERK-ELK-1/KLF4 signaling. KLX exhibits promising potential as a viable therapeutic agent for the treatment of VSMC phenotype conversion associated arteriosclerosis.

Development of a rapid reverse genetics system for feline coronavirus based on TAR cloning in yeast.

Introduction: Reverse genetics has become an indispensable tool to gain insight into the pathogenesis of viruses and the development of vaccines. The yeast-based synthetic genomics platform has demonstrated the novel capabilities to genetically reconstruct different viruses. Methods: In this study, a transformation-associated recombination (TAR) system in yeast was used to rapidly rescue different strains of feline infectious peritonitis virus, which causes a deadly disease of cats for which there is no effective vaccine. Results and discussion: Using this system, the viruses could be rescued rapidly and stably without multiple cloning steps. Considering its speed and ease of manipulation in virus genome assembly, the reverse genetics system developed in this study will facilitate the research of the feline coronaviruses pathogenetic mechanism and the vaccine development.

Specific Activation of CB2R Ameliorates Psoriasis-Like Skin Lesions by Inhibiting Inflammation and Oxidative Stress.

Psoriasis is a chronic inflammatory skin disease. Inflammation and oxidative stress play crucial roles in the pathogenesis of psoriasis. Cannabinoid receptor type 2 (CB2R) is an attractive target for treating various inflammatory disorders. However, the precise role and mechanism of CB2R activation in psoriasis remain to be further elucidated. In this study, imiquimod (IMQ)-induced experimental psoriasis mice and tumor necrosis factor-α (TNF-α)-activated keratinocytes (HaCaT) were used to examine the effect of CB2R activation on psoriasis-like lesions and the mechanism in vivo and in vitro. Our results demonstrated that activation of CB2R by the specific agonist GW842166X (GW) significantly ameliorated IMQ-induced psoriasiform skin lesions in mice by reducing epidermal thickness and decreasing plaque thickness. On the one hand, GW alleviated inflammation by decreasing inflammatory cytokines and abating inflammatory cell infiltration. On the other hand, this treatment reduced the level of iNOS and downregulated the expression of CB2R in psoriatic skin tissue. Further studies suggested that the Kelch-like ECH-associated protein 1/nuclear factor erythroid-2-related factor (Keap1/Nrf2) signaling pathway might be involved. Our findings reveal that selective activation of CB2R may serve as a new strategy for the treatment of psoriasis.

Kidney Mesenchymal stem cells alleviate cisplatin-induced kidney injury and apoptosis in rats.

OBJECTIVE: This experiment was designed to demonstrate Mesenchymal stem cells (MSCs) derived from kidney can alleviate cisplatin-induced kidney injury and renal cell apoptosis through paracrine pathway. METHODS: Firstly, MSCs were isolated from kidney of young rats, and their surface-specific markers were identified by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and immunofluorescence staining. Self-renewal ability of Kidney Mesenchymal Stem Cells (KMSCs) was observed by cell counting and 5-Bromo-2'-deoxyuridine (BrdU) fluorescence staining. KMSCs at logarithmic growth stage were traced and injected into rat through tail vein. RESULTS: The results showed that KMSCs homed in the kidney tissues, decreased the secretion of inflammatory factors (CRP, TNFα, IL-1ß, IL-6), and alleviated renal function. Hematoxylin and Eosin (H＆E), Masson and Periodic Acid-silver Methenamine (PASM) staining showed that KMSCs could alleviate pathological damage in rats. Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling (TUNEL) assay showed that KMSCs could reduce the apoptosis of rat kidney cells induced by cisplatin. Finally, Immunohistochemistry (IHC) results showed that cisplatin could induce higher expression of the pro-apoptotic protein Bax and lower expression of anti-apoptotic Bcl-2 in kidney tissues. However, KMSCs could reverse the pro-apoptotic effect of cisplatin on kidney cells and improve the survival rate of rats. CONCLUSIONS: In conclusion, KMSCs were successfully isolated from kidney tissues, and KMSCs have therapeutic effects on rat kidney injury induced by cisplatin.

A nanoformulation for immunosuppression reversal and broad-spectrum self-amplifying antitumor ferroptosis-immunotherapy.

The efficacy of immunotherapy combined with other therapeutic modalities in the management of cancer has been extensively studied. However, no effective strategy to improve the antitumor effects of immunotherapy at the tumor site has been developed. In this study, we describe a nanoformulation (CP) that integrates ferroptosis-inducing cannabinoid nanoparticles with immunostimulatory Poly(I:C) to enhance antitumor immune responses by activating ferroptosis-immunotherapy pathways. The results indicated that CP nanoformulation effectively induced ferroptosis, cellular immunogenic death, and anti-tumor immune responses which initiate T cell responses leading to the inhibition of established tumors. In addition, CP nanoformulations reversed the tumor immunosuppressive microenvironment and promoted tumor ferroptosis. These results indicated that the self-amplifying nanoformulation may be an effective strategy for broad-spectrum cancer immunotherapy.

Apicoplast biogenesis mediated by ATG8 requires the ATG12-ATG5-ATG16L and SNAP29 complexes in Toxoplasma gondii.

In apicomplexan parasites, the macroautophagy/autophagy machinery is repurposed to maintain the plastid-like organelle apicoplast. Previously, we showed that in Toxoplasma and Plasmodium, ATG12 interacts with ATG5 in a non-covalent manner, in contrast to the covalent interaction in most organisms. However, it remained unknown whether apicomplexan parasites have functional orthologs of ATG16L1, a protein that is essential for the function of the covalent ATG12-ATG5 complex in vivo in other organisms. Furthermore, the mechanism used by the autophagy machinery to maintain the apicoplast is unclear. We report that the ATG12-ATG5-ATG16L complex exists in Toxoplasma gondii (Tg). This complex is localized on isolated structures at the periphery of the apicoplast dependent on TgATG16L. Inducible depletion of TgATG12, TgATG5, or TgATG16L caused loss of the apicoplast and affected parasite growth. We found that a putative soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein, synaptosomal-associated protein 29 (TgSNAP29, Qbc SNARE), is required to maintain the apicoplast in T. gondii. TgSNAP29 depletion disrupted TgATG8 localization at the apicoplast. Additionally, we identified a putative ubiquitin-interacting motif-docking site (UDS) of TgATG8. Mutation of the UDS site abolished TgATG8 localization on the apicoplast but not lipidation. These findings suggest that the TgATG12-TgATG5-TgATG16L complex is required for biogenesis of the apicoplast, in which TgATG8 is translocated to the apicoplast via vesicles in a SNARE -dependent manner in T. gondii.Abbreviations: AID: auxin-inducible degron; CCD: coiled-coil domain; HFF: human foreskin fibroblast; IAA: indole-3-acetic acid; LAP: LC3-associated phagocytosis; NAA: 1-naphthaleneacetic acid; PtdIns3P: phosphatidylinositol-3-phosphate; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; UDS: ubiquitin-interacting motif-docking site; UIM: ubiquitin-interacting motif.

A double antibody sandwich enzyme-linked immunosorbent assay for detection of secreted antigen 1 of Babesia microti using hamster model.

A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) targeting secreted antigen 1 of Babesia microti (BmSA1) was developed for detection of B. microti infection. The optimized DAS-ELISA was sensitive enough to detect circulating BmSA1 by day 2 post-infection, in sequential sera of a hamster infected with B. microti. This detection was 4 days earlier than antibody detection by indirect ELISA. The kinetics of circulating BmSA1 coincided with the profile of parasitemia. The specificity of this assay was evaluated using sera from animals experimentally infected with different species of Babesia. The DAS-ELISA had a higher sensitivity than the microscopic examination of Giemsa-stained blood smears for detection of the infection in hamsters. Taken together, these results indicated that BmSA1 could be a potential marker for surveillance of human babesiosis.