The NSP-interacting kinase (NIK) receptor-mediated defense pathway has been identified recently as a virulence target of the geminivirus nuclear shuttle protein (NSP). However, the NIK1–NSP interaction does not fit into the elicitor–receptor model of resistance, and hence the molecular mechanism that links this antiviral response to receptor activation remains obscure. Here, we identified a ribosomal protein, rpL10A, as a specific partner and substrate of NIK1 that functions as an immediate downstream effector of NIK1-mediated response. Phosphorylation of cytosolic rpL10A by NIK1 redirects the protein to the nucleus where it may act to modulate viral infection. While ectopic expression of normal NIK1 or a hyperactive NIK1 mutant promotes the accumulation of phosphorylated rpL10A within the nuclei, an inactive NIK1 mutant fails to redirect the protein to the nuclei of co-transfected cells. Likewise, a mutant rpL10A defective for NIK1 phosphorylation is not redirected to the nucleus. Furthermore, loss of rpL10A function enhances susceptibility to geminivirus infection, resembling the phenotype of nik1 null alleles. We also provide evidence that geminivirus infection directly interferes with NIK1-mediated nuclear relocalization of rpL10A as a counterdefensive measure. However, the NIK1-mediated defense signaling neither activates RNA silencing nor promotes a hypersensitive response but inhibits plant growth and development. Although the virulence function of the particular geminivirus NSP studied here overcomes this layer of defense in Arabidopsis, the NIK1-mediated signaling response may be involved in restricting the host range of other viruses.

Aspergillus fumigatus causes invasive aspergillosis, the most common life-threatening fungal disease of immuno-compromised humans. The treatment of disseminated infections with antifungal drugs, including echinocandin cell wall biosynthesis inhibitors, is increasingly challenging due to the rise of drug-resistant pathogens. The fungal calcium responsive calcineurin-CrzA pathway influences cell morphology, cell wall composition, virulence, and echinocandin resistance. A screen of 395 A. fumigatus transcription factor mutants identified nine transcription factors important to calcium stress tolerance, including CrzA and ZipD. Here, comparative transcriptomics revealed CrzA and ZipD regulated the expression of shared and unique gene networks, suggesting they participate in both converged and distinct stress response mechanisms. CrzA and ZipD additively promoted calcium stress tolerance. However, ZipD also regulated cell wall organization, osmotic stress tolerance and echinocandin resistance. The absence of ZipD in A. fumigatus caused a significant virulence reduction in immunodeficient and immunocompetent mice. The ΔzipD mutant displayed altered cell wall organization and composition, while being more susceptible to macrophage killing and eliciting an increased pro-inflammatory cytokine response. A higher number of neutrophils, macrophages and activated macrophages were found in ΔzipD infected mice lungs. Collectively, this shows that ZipD-mediated regulation of the fungal cell wall PLOS Genetics | https://doi.contributes to the evasion of pro-inflammatory responses and tolerance of echinocandin antifungals, and in turn promoting virulence and complicating treatment options. Author summaryA. fumigatus is the main ethiological agent of one of the most important life-threatening fungal diseases in immuno-compromised humans, invasive aspergillosis. Treatment commonly involves echinocandin antifungal drugs that inhibit cell wall β-1,3-glucan polysaccharide biosynthesis. Calcium is an important messenger for many signaling pathways regulating the fungal response to stress and antifungal drugs. The calcium responsive calcineurin phosphatase influences the localization and activity of the CrzA transcription factor (TF), regulating the activation of several stress responses and cell wall modifications. For many years, CrzA has been recognized as the sole calcium/calcineurin-dependent TF. Here, we identify nine TFs involved in the calcium/calcineurin metabolism, including a novel A. fumigatus calcium/calcineurin dependent TF named ZipD. Transcriptional profiling of the response of A. fumigatus wild-type, plus the ΔcrzA and ΔzipD mutant, strains shows CrzA and ZipD to have shared and unique functions. ZipD was found to be important for not only calcium metabolism, but also for the cell wall organization, osmotic stress and echinocandin tolerance. During host infection, ZipD plays an important role in modulating fungal cell walls, promoting evasion of the host pro-inflammatory immune responses and virulence. Our work emphasize...

BackgroundThe present study analyzed whether or not the in vitro cultivation for long periods of time of pre-isolated Leishmania amazonensis from lesions of chronically infected BALB/c mice was able to interfere in the parasites' infectivity using in vivo and in vitro experiments. In addition, the proteins that presented a significant decrease or increase in their protein expression content were identified applying a proteomic approach.Methodology/Principal FindingsParasites were cultured in vitro for 150 days. Aliquots were collected on the day 0 of culture (R0), as well as after ten (R10; 50 days of culture), twenty (R20; 100 days of culture), and thirty (R30; 150 days of culture) passages, and were used to analyze the parasites' in vitro and in vivo infectivity, as well as to perform the proteomic approach. Approximately 837, 967, 935, and 872 spots were found in 2-DE gels prepared from R0, R10, R20, and R30 samples, respectively. A total of 37 spots presented a significant decrease in their intensity of expression, whereas a significant increase in protein content during cultivation could be observed for 19 proteins (both cases >2.0 folds). Some of these identified proteins can be described, such as diagnosis and/or vaccine candidates, while others are involved in the infectivity of Leishmania. It is interesting to note that six proteins, considered hypothetical in Leishmania, showed a significant decrease in their expression and were also identified.Conclusions/SignificanceThe present study contributes to the understanding that the cultivation of parasites over long periods of time may well be related to the possible loss of infectivity of L. amazonensis. The identified proteins that presented a significant decrease in their expression during cultivation, including the hypothetical, may also be related to this loss of parasites' infectivity, and applied in future studies, including vaccine candidates and/or immunotherapeutic targets against leishmaniasis.

Aspergillus fumigatus is an opportunistic human pathogen causing allergic reactions or systemic infections such as invasive pulmonary aspergillosis, especially in immunocompromised patients. The fungal cell wall is the main component responsible for recognition by the immune system, due to the specific composition of polysaccharide carbohydrates exposed on the surface of the fungal cell wall called pathogen-associated molecular patterns (PAMPs). Key enzymes in the fungal cell wall biosynthesis are a good target for fungal drug development. This report elucidates the cooperation between the HOG and PKA pathways in the mobilization of carbohydrates for fungal cell wall biosynthesis. We suggest that the reduced mobilization of simple sugars causes defects in the structure of the fungal cell wall. In summary, we propose that SakA is important for PKA activity, therefore regulating the availability and mobilization of monosaccharides for fungal cell wall biosynthesis during cell wall damage and the osmotic stress response.

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Trypanosoma cruzi , the etiological agent of Chagas’ disease, affects 8 million people predominantly living in socioeconomic underdeveloped areas. T . cruzi trypomastigotes (Ty), the classical infective stage, interact with the extracellular matrix (ECM), an obligatory step before invasion of almost all mammalian cells in different tissues. Here we have characterized the proteome and phosphoproteome of T . cruzi trypomastigotes upon interaction with ECM (MTy) and the data are available via ProteomeXchange with identifier PXD010970. Proteins involved with metabolic processes (such as the glycolytic pathway), kinases, flagellum and microtubule related proteins, transport-associated proteins and RNA/DNA binding elements are highly represented in the pool of proteins modified by phosphorylation. Further, important metabolic switches triggered by this interaction with ECM were indicated by decreases in the phosphorylation of hexokinase, phosphofructokinase, fructose-2,6-bisphosphatase, phosphoglucomutase, phosphoglycerate kinase in MTy. Concomitantly, a decrease in the pyruvate and lactate and an increase of glucose and succinate contents were detected by GC-MS. These observations led us to focus on the changes in the glycolytic pathway upon binding of the parasite to the ECM. Inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy were observed and this correlated with the phosphorylation levels of the respective enzymes. Putative kinases involved in protein phosphorylation altered upon parasite incubation with ECM were suggested by in silico analysis. Taken together, our results show that in addition to cytoskeletal changes and protease activation, a reprogramming of the trypomastigote metabolism is triggered by the interaction of the parasite with the ECM prior to cell invasion and differentiation into amastigotes, the multiplicative intracellular stage of T . cruzi in the vertebrate host.

Aspergillus fumigatus , a saprophytic filamentous fungus, is a serious opportunistic pathogen of mammals and it is the primary causal agent of invasive aspergillosis (IA). Mitogen activated protein Kinases (MAPKs) are important components involved in diverse cellular processes in eukaryotes. A. fumigatus MpkC and SakA, the homologs of the Saccharomyces cerevisiae Hog1 are important to adaptations to oxidative and osmotic stresses, heat shock, cell wall damage, macrophage recognition, and full virulence. We performed protein pull-down experiments aiming to identify interaction partners of SakA and MpkC by mass spectrometry analysis. In presence of osmotic stress with sorbitol, 118, and 213 proteins were detected as possible protein interactors of SakA and MpkC, respectively. Under cell wall stress caused by congo red, 420 and 299 proteins were detected interacting with SakA and MpkC, respectively. Interestingly, a group of 78 and 256 proteins were common to both interactome analysis. Co-immunoprecipitation (Co-IP) experiments showed that SakA::GFP is physically associated with MpkC:3xHA upon osmotic and cell wall stresses. We also validated the association between SakA:GFP and the cell wall integrity MAPK MpkA:3xHA and the phosphatase PtcB:3xHA, under cell wall stress. We further characterized A. fumigatus PakA, the homolog of the S. cerevisiae sexual developmental serine/threonine kinase Ste20, as a component of the SakA/MpkC MAPK pathway. The ΔpakA strain is more sensitive to cell wall damaging agents as congo red, calcofluor white, and caspofungin. Together, our data supporting the hypothesis that SakA and MpkC are part of an osmotic and general signal pathways involved in regulation of the response to the cell wall damage, oxidative stress, drug resistance, and establishment of infection. This manuscript describes an important biological resource to understand SakA and MpkC protein interactions. Further investigation of the biological roles played by these protein interactors will provide more opportunities to understand and combat IA.

Trypanosoma cruzi strains show distinctive characteristics as genetic polymorphism and infectivity. Large repertoires of molecules, such as the Gp85 glycoproteins, members of the Gp85/Trans-sialidase superfamily, as well as multiple signaling pathways, are associated with invasion of mammalian cells by the parasite. Due to the large number of expressed members, encoded by more than 700 genes, the research focused on this superfamily conserved sequences is discussed. Binding sites to laminin have been identified at the N-terminus of the Gp85 molecules. Interestingly, the T. cruzi protein phosphorylation profile is changed upon parasite binding to laminin (or fibronectin), particularly the cytoskeletal proteins such as those from the paraflagellar rod and the tubulins, which are both markedly dephosphorylated. Detailed analysis of the signaling cascades triggered upon T. cruzi binding to extracellular matrix (ECM) proteins revealed the involvement of the MAPK/ERK pathway in this event. At the C-terminus, the conserved FLY sequence is a cytokeratin-binding domain and is involved in augmented host cell invasion in vitro and high levels of parasitemia in vivo. FLY, which is associated to tissue tropism and preferentially binds to the heart vasculature may somehow be correlated with the severe cardiac form, an important clinical manifestation of chronic Chagas' disease.