PLoS Pathogens最新文献

Widespread anthelmintic resistance has complicated the management of parasitic nematodes. Resistance to the benzimidazole (BZ) drug class is nearly ubiquitous in many species and is associated with mutations in beta-tubulin genes. However, mutations in beta-tubulin alone do not fully explain all BZ resistance. We performed a genome-wide association study using a genetically diverse panel of Caenorhabditis elegans strains to identify loci that contribute to resistance to the BZ drug thiabendazole (TBZ). We identified a quantitative trait locus (QTL) on chromosome V independent of all beta-tubulin genes and overlapping with two promising candidate genes, the cytochrome P450 gene cyp-35D1 and the nuclear hormone receptor nhr-176. Both genes were previously demonstrated to play a role in TBZ metabolism. NHR-176 binds TBZ and induces the expression of CYP-35D1, which metabolizes TBZ. We generated single gene deletions of cyp-35D1 and nhr-176 and found that both genes play a role in TBZ response. A predicted high-impact lysine-to-glutamate substitution at position 267 (K267E) in CYP-35D1 was identified in a sensitive strain, and reciprocal allele replacement strains in different genetic backgrounds were used to show that the lysine allele conferred increased TBZ resistance. Using competitive fitness assays, we found that neither allele was deleterious, but the lysine allele was selected in the presence of TBZ. Additionally, we found that the lysine allele significantly increased the rate of TBZ metabolism compared to the glutamate allele. Moreover, yeast expression assays showed that the lysine version of CYP-35D1 had twice the enzymatic activity of the glutamate allele. To connect our results to parasitic nematodes, we analyzed four Haemonchus contortus cytochrome P450 orthologs but did not find variation at the 267 position in fenbendazole-resistant populations. Overall, we confirmed that variation in this cytochrome P450 gene is the first locus independent of beta-tubulin to play a role in BZ resistance.

A sustained blood-stage infection of the human malaria parasite P. falciparum relies on the active exit of merozoites from their host erythrocytes. During this process, named egress, the infected red blood cell undergoes sequential morphological events: the rounding-up of the surrounding parasitophorous vacuole, the disruption of the vacuole membrane and finally the rupture of the red blood cell membrane. These events are coordinated by two intracellular second messengers, cGMP and calcium ions (Ca2+), that control the activation of their dedicated kinases, PKG and CDPKs respectively, and thus the secretion of parasitic factors that assist membranes rupture. We had previously identified the serine-threonine phosphatase PP1 as an essential enzyme required for the rupture of the surrounding vacuole. Here, we address its precise positioning and function within the egress signaling pathway by combining chemical genetics and live-microscopy. Fluorescent reporters of the parasitophorous vacuole morphology were expressed in the conditional PfPP1-iKO line which allowed to monitor the kinetics of natural and induced egress, as well as the rescue capacity of known egress inducers. Our results underscore a dual function for PP1 in the egress cascade. First, we provide further evidence that PP1 controls the homeostasis of the second messenger cGMP by modulating the basal activity of guanylyl cyclase alpha and consequently the PKG-dependent downstream Ca2+ signaling. Second, we demonstrate that PP1 also regulates the rounding-up of the parasitophorous vacuole, as this step is almost completely abolished in PfPP1-null schizonts. Strikingly, our data show that rounding-up is the step triggered by egress inducers, and support its reliance on Ca2+, as the calcium ionophore A23187 bypasses the egress defect of PfPP1-null schizonts, restores proper egress kinetics and promotes the initiation of the rounding-up step. Therefore, this study places the phosphatase PP1 upstream of the cGMP-PKG signaling pathway, and sheds new light on the regulation of rounding-up, the first step in P. falciparum blood stage egress cascade.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a diverse family of variant surface antigens, encoded by var genes, that mediates binding of infected erythrocytes to human cells and plays a key role in parasite immune evasion and malaria pathology. The increased availability of parasite genome sequence data has revolutionised the study of PfEMP1 diversity across multiple P. falciparum isolates. However, making functional sense of genomic data relies on the ability to infer binding phenotype from var gene sequence. For P. falciparum rosetting, the binding of infected erythrocytes to uninfected erythrocytes, the analysis of var gene/PfEMP1 sequences encoding the phenotype is limited, with only eight rosette-mediating PfEMP1 variants described to date. These known rosetting PfEMP1 variants fall into two types, characterised by N-terminal domains known as "domain cassette" 11 (DC11) and DC16. Here we test the hypothesis that DC11 and DC16 are the only PfEMP1 types in the P. falciparum genome that mediate rosetting, by examining a set of thirteen recent culture-adapted Kenyan parasite lines. We first analysed the var gene/PfEMP1 repertoires of the Kenyan lines and identified an average of three DC11 or DC16 PfEMP1 variants per genotype. In vitro rosette selection of the parasite lines yielded four with a high rosette frequency, and analysis of their var gene transcription, infected erythrocyte PfEMP1 surface expression, rosette disruption and erythrocyte binding function identified four novel rosette-mediating PfEMP1 variants. Two of these were of the predicted DC11 type (one showing the dual rosetting/IgM-Fc-binding phenotype), whereas two contained DC15 (DBLα1.2-CIDRα1.5b) a PfEMP1 type not previously associated with rosetting. We also showed that a Thai parasite line expressing a DC8-like PfEMP1 binds to erythrocytes to form rosettes. Hence, these data expand current knowledge of rosetting mechanisms and emphasize that the PfEMP1 types mediating rosetting are more diverse than previously recognised.

Plasmodium vivax is the predominant malaria parasite in Latin America. Its colonization history in the region is rich and complex, and is still highly debated, especially about its origin(s). Our study employed cutting-edge population genomic techniques to analyze whole genome variation from 620 P. vivax isolates, including 107 newly sequenced samples from West Africa, Middle East, and Latin America. This sampling represents nearly all potential source populations worldwide currently available. Analyses of the genetic structure, diversity, ancestry, coalescent-based inferences, including demographic scenario testing using Approximate Bayesian Computation, have revealed a more complex evolutionary history than previously envisioned. Indeed, our analyses suggest that the current American P. vivax populations predominantly stemmed from a now-extinct European lineage, with the potential contribution also from unsampled populations, most likely of West African origin. We also found evidence that P. vivax arrived in Latin America in multiple waves, initially during early European contact and later through post-colonial human migration waves in the late 19th-century. This study provides a fresh perspective on P. vivax's intricate evolutionary journey and brings insights into the possible contribution of West African P. vivax populations to the colonization history of Latin America.

Placental malaria is characterized by the massive accumulation and sequestration of infected erythrocytes in the placental intervillous blood spaces, causing severe birth outcomes. The variant surface antigen VAR2CSA is associated with Plasmodium falciparum sequestration in the placenta via its capacity to adhere to chondroitin sulfate A. We have previously shown that the extracellular region of VAR2CSA is phosphorylated on several residues and that the phosphorylation enhances the adhesive properties of CSA-binding infected erythrocytes. Here, we aimed to identify the kinases mediating this phosphorylation. We report that human and Plasmodium falciparum Casein Kinase 2α are involved in the phosphorylation of the extracellular region of VAR2CSA. We notably show that both CK2α can phosphorylate the extracellular region of recombinant and immunoprecipitated VAR2CSA. Mass spectrometry analysis of recombinant VAR2CSA phosphorylated by recombinant Human and P. falciparum CK2α combined with site-directed mutagenesis led to the identification of residue S1068 in VAR2CSA, which is phosphorylated by both enzymes and is associated with CSA binding. Furthermore, using CRISPR/Cas9 we generated a parasite line in which phosphoresidue S1068 was changed to alanine. This mutation strongly impairs infected erythrocytes adhesion by abolishing VAR2CSA translocation to the surface of infected erythrocytes. We also report that two specific CK2 inhibitors reduce infected erythrocytes adhesion to CSA and decrease the phosphorylation of the recombinant extracellular region of VAR2CSA using either infected erythrocytes lysates as a source of kinases or recombinant Human and P. falciparum casein kinase 2. Taken together, these results undoubtedly demonstrate that host and P. falciparum CK2α phosphorylate the extracellular region of VAR2CSA and that this post-translational modification is important for VAR2CSA trafficking and for infected erythrocytes adhesion to CSA.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that has re-emerged to cause large outbreaks of human infections worldwide. There are currently no approved antivirals for treatment of CHIKV infection. Recently, we reported that the ribonucleoside analog 4'-fluorouridine (4'-FlU) is a highly potent inhibitor of CHIKV replication, and targets the viral nsP4 RNA dependent RNA polymerase. In mouse models, oral therapy with 4'-FlU diminished viral tissue burdens and virus-induced disease signs. To provide critical evidence for the potential of 4'-FlU as a CHIKV antiviral, here we selected for CHIKV variants with decreased 4'-FlU sensitivity, identifying two pairs of mutations in nsP2 and nsP4. The nsP4 mutations Q192L and C483Y were predominantly responsible for reduced sensitivity. These variants were still inhibited by higher concentrations of 4'-FlU, and the mutations did not change nsP4 fidelity or provide a virus fitness advantage in vitro or in vivo. Pathogenesis studies in mice showed that the nsP4-C483Y variant caused similar disease and viral tissue burden as WT CHIKV, while the nsP4-Q192L variant was strongly attenuated. Together these results support the potential of 4'-FlU to be an important antiviral against CHIKV.

Mitochondria, recognized as the "powerhouse" of cells, play a vital role in generating cellular energy through dynamic processes such as fission and fusion. Viruses have evolved mechanisms to hijack mitochondrial function for their survival and proliferation. Here, we report that infection with the swine arterivirus porcine reproductive and respiratory syndrome virus (PRRSV), manipulates mitochondria calcium ions (Ca2+) to induce mitochondrial fission and mitophagy, thereby reprogramming cellular energy metabolism to facilitate its own replication. Mechanistically, PRRSV-induced mitochondrial fission is caused by elevated levels of mitochondria Ca2+, derived from the endoplasmic reticulum (ER) through inositol 1,4,5-triphosphate receptor (IP3R)-voltage-dependent anion channel 1 (VDAC1)-mitochondrial calcium uniporter (MCU) channels. This process is associated with increased mitochondria-associated membranes (MAMs), mediated by the upregulated expression of sigma non-opioid intracellular receptor 1 (SIGMAR1). Elevated mitochondria Ca2+ further activates the Ca2+/CaM-dependent protein kinase kinase β (CaMKKβ)-AMP-activated protein kinase (AMPK)-dynamin-related protein 1 (DRP1) signaling pathway, which interacts with mitochondrial fission protein 1 (FIS1) and mitochondrial dynamics proteins of 49 kDa (MiD49) to promote mitochondrial fission. PRRSV infection, alongside mitochondrial fission, triggers mitophagy via the PTEN-induced putative kinase 1 (PINK1)-Parkin RBR E3 ubiquitin (Parkin) pathway, promoting cellular glycolysis and excessive lactate production to facilitate its own replication. This study reveals the mechanism by which mitochondrial Ca2+ regulates mitochondrial function during PRRSV infection, providing new insights into the interplay between the virus and host cell metabolism.

The latent viral reservoir remains the major barrier to HIV cure, placing the burden of strict adherence to antiretroviral therapy (ART) on people living with HIV to prevent recrudescence of viremia. For infants with perinatally acquired HIV, adherence is anticipated to be a lifelong need. In this study, we tested the hypothesis that administration of ART and viral Envelope-specific rhesus-derived IgG1 monoclonal antibodies (RhmAbs) with or without the IL-15 superagonist N-803 early in infection would limit viral reservoir establishment in SIV-infected infant rhesus macaques. Following initiation of ART at 1-2 weeks after oral SIVmac251 infection, we observed biphasic decay of viremia, with first phase decay significantly faster in the ART + SIV RhmAbs-treated group compared to controls that received only ART. In contrast, the addition of N-803 to ART + SIV RhmAbs significantly slowed both the first and second phase viral decay compared to the ART only group. Treatment with a single dose of N-803 resulted in increased frequency of Ki67 expressing NK, CD8+, and CD4+ T cells. Levels of intact SIV proviruses in CD4+ T cells from blood, lymph nodes, and rectum at week 48 of ART did not differ across groups. Similarly, the time to viral rebound following ART interruption was not impacted by the experimental treatments. These results support the concept that the rebound-competent viral reservoir is formed within days after infection and that targeting only productively infected cells for clearance near the time of ART initiation, even during acute infection, may be insufficient to limit reservoir establishment.

Paramyxoviruses are significant human and animal pathogens that include mumps virus (MuV), Newcastle disease virus (NDV) and the murine parainfluenza virus Sendai (SeV). Despite their importance, few host factors implicated in paramyxovirus infection are known. Using a recombinant SeV expressing destabilized eGFP (rSeVCdseGFP) in a loss-of-function CRISPR screen, we identified the CMP-sialic acid transporter (CST) gene SLC35A1 and the UDP-galactose transporter (UGT) gene SLC35A2 as essential for paramyxovirus infection. As expected, SLC35A1 knockout (KO) cells showed drastic reduction in infections with SeV, NDV and MuV due to the lack of cell surface sialic acids receptors. However, SLC35A2 KO cells revealed unknown critical roles for this factor in virus-cell and cell-to-cell fusion events for the different paramyxoviruses. While UGT was essential for virus-cell fusion during SeV entry to the cell, it was not required for NDV or MuV entry. Importantly, UGT promoted the formation of syncytia during MuV infection, suggesting a role in cell-to-cell virus spread. Our findings demonstrate that paramyxoviruses can bind to or enter A549 cells in the absence of canonical galactose-bound sialic-acid decorations and show that UGT facilitates paramyxovirus fusion processes involved in entry and spread.

Identifying cellular and molecular mechanisms maintaining HIV-1 latency in the viral reservoir is crucial for devising effective cure strategies. Here we developed an innovative flow cytometry-fluorescent in situ hybridization (flow-FISH) approach for direct ex vivo reservoir detection without the need for reactivation using a combination of probes detecting abortive and elongated HIV-1 transcripts. Our flow-FISH assay distinguished between HIV-1-infected CD4+ T cells expressing abortive or elongated HIV-1 transcripts in PBMC from untreated and ART-treated PWH from the Amsterdam Cohort Studies. This flow-FISH method was employed to isolate CD4+ T cells expressing abortive or elongated HIV-1 transcripts from five ART-naïve PWH for transcriptomic analysis by 3' RNA sequencing. Supervised cluster analysis identified several differentially expressed mitochondrial genes in infected CD4+ T cells with abortive HIV-1 transcripts compared to cells containing elongated HIV-1 transcripts. Notably, enhancing mitochondrial function induced HIV-1 transcription in PBMC from PWH. Our data strongly suggests that cellular metabolism is involved in maintaining HIV-1 latency and show that improving mitochondrial functions induces HIV-1 transcriptional activity in PWH. These findings underline the relevance of metabolic regulation in HIV-1 infection, and support the development of strategies modulating immunometabolism to target viral latency.