Proteasome inhibition triggers tissue-specific immune responses against different pathogens in C. elegans.

Protein quality control pathways play important roles in resistance against pathogen infection. For example, the conserved transcription factor SKN-1/NRF up-regulates proteostasis capacity after blockade of the proteasome and also promotes resistance against bacterial infection in the nematode Caenorhabditis elegans. SKN-1/NRF has 3 isoforms, and the SKN-1A/NRF1 isoform, in particular, regulates proteasomal gene expression upon proteasome dysfunction as part of a conserved bounce-back response. We report here that, in contrast to the previously reported role of SKN-1 in promoting resistance against bacterial infection, loss-of-function mutants in skn-1a and its activating enzymes ddi-1 and png-1 show constitutive expression of immune response programs against natural eukaryotic pathogens of C. elegans. These programs are the oomycete recognition response (ORR), which promotes resistance against oomycetes that infect through the epidermis, and the intracellular pathogen response (IPR), which promotes resistance against intestine-infecting microsporidia. Consequently, skn-1a mutants show increased resistance to both oomycete and microsporidia infections. We also report that almost all ORR/IPR genes induced in common between these programs are regulated by the proteasome and interestingly, specific ORR/IPR genes can be induced in distinct tissues depending on the exact trigger. Furthermore, we show that increasing proteasome function significantly reduces oomycete-mediated induction of multiple ORR markers. Altogether, our findings demonstrate that proteasome regulation keeps innate immune responses in check in a tissue-specific manner against natural eukaryotic pathogens of the C. elegans epidermis and intestine.

A PAX6-regulated receptor tyrosine kinase pairs with a pseudokinase to activate immune defense upon oomycete recognition in Caenorhabditis elegans.

Oomycetes were recently discovered as natural pathogens of Caenorhabditis elegans, and pathogen recognition alone was shown to be sufficient to activate a protective transcriptional program characterized by the expression of multiple chitinase-like (chil) genes. However, the molecular mechanisms underlying oomycete recognition in animals remain fully unknown. We performed here a forward genetic screen to uncover regulators of chil gene induction and found several independent loss-of-function alleles of old-1 and flor-1, which encode receptor tyrosine kinases belonging to the C. elegans-specific KIN-16 family. We report that OLD-1 and FLOR-1 are both necessary for mounting the immune response and act in the epidermis. FLOR-1 is a pseudokinase that acts downstream of the active kinase OLD-1 and regulates OLD-1 levels at the plasma membrane. Interestingly, the old-1 locus is adjacent to the chil genes in the C. elegans genome, thereby revealing a genetic cluster important for oomycete resistance. Furthermore, we demonstrate that old-1 expression at the anterior side of the epidermis is regulated by the VAB-3/PAX6 transcription factor, well known for its role in visual system development in other animals. Taken together, our study reveals both conserved and species-specific factors shaping the activation and spatial characteristics of the immune response to oomycete recognition.

A pals-25 gain-of-function allele triggers systemic resistance against natural pathogens of C. elegans.

Regulation of immunity throughout an organism is critical for host defense. Previous studies in the nematode Caenorhabditis elegans have described an "ON/OFF" immune switch comprised of the antagonistic paralogs PALS-25 and PALS-22, which regulate resistance against intestinal and epidermal pathogens. Here, we identify and characterize a PALS-25 gain-of-function mutant protein with a premature stop (Q293*), which we find is freed from physical repression by its negative regulator, the PALS-22 protein. PALS-25(Q293*) activates two related gene expression programs, the Oomycete Recognition Response (ORR) against natural pathogens of the epidermis, and the Intracellular Pathogen Response (IPR) against natural intracellular pathogens of the intestine. A subset of ORR/IPR genes is upregulated in pals-25(Q293*) mutants, and they are resistant to oomycete infection in the epidermis, and microsporidia and virus infection in the intestine, but without compromising growth. Surprisingly, we find that activation of PALS-25 seems to primarily stimulate the downstream bZIP transcription factor ZIP-1 in the epidermis, with upregulation of gene expression in both the epidermis and in the intestine. Interestingly, we find that PALS-22/25-regulated epidermal-to-intestinal signaling promotes resistance to the N. parisii intestinal pathogen, demonstrating cross-tissue protective immune induction from one epithelial tissue to another in C. elegans.

Infection of C. elegans by Haptoglossa Species Reveals Shared Features in the Host Response to Oomycete Detection.

Oomycetes are a group of eukaryotic organisms that includes many important pathogens of animals and plants. Within this group, the Haptoglossa genus is characterised by the presence of specialised gun cells carrying a harpoon-like infection apparatus. While several Haptoglossa pathogens have been morphologically described, there are currently no host systems developed to study the infection process or host responses in the lab. In this study, we report that Haptoglossa species are potent natural pathogens of Caenorhabditis nematodes. Using electron microscopy, we characterise the infection process in C. elegans and demonstrate that the oomycete causes excessive tissue degradation upon entry in the body cavity, whilst leaving the host cuticle intact. We also report that the host transcriptional response to Haptoglossa infection shares similarities with the response against the oomycete Myzocytiopsis humicola, a key example of which is the induction of chitinase-like (chil) genes in the hypodermis. We demonstrate that this shared feature of the host response can be mounted by pathogen detection without any infection, as previously shown for M. humicola. These results highlight similarities in the nematode immune response to natural infection by phylogenetically distinct oomycetes.

Chemosensory Neurons Modulate the Response to Oomycete Recognition in Caenorhabditis elegans.

Understanding how animals detect and respond to pathogen threats is central to dissecting mechanisms of host immunity. The oomycetes represent a diverse eukaryotic group infecting various hosts from nematodes to humans. We have previously shown that Caenorhabditis elegans mounts a defense response consisting of the induction of chitinase-like (chil) genes in the epidermis to combat infection by its natural oomycete pathogen Myzocytiopsis humicola. We provide here evidence that C. elegans can sense the oomycete by detecting an innocuous extract derived from animals infected with M. humicola. The oomycete recognition response (ORR) leads to changes in the cuticle and reduction in pathogen attachment, thereby increasing animal survival. We also show that TAX-2/TAX-4 function in chemosensory neurons is required for the induction of chil-27 in the epidermis in response to extract exposure. Our findings highlight that neuron-to-epidermis communication may shape responses to oomycete recognition in animal hosts.

Giardia lamblia Hsp90 pre-mRNAs undergo self-splicing to generate mature RNA in an in vitro trans-splicing reaction.

We have previously shown that the Heat Shock Protein 90 (Hsp90) gene in G. lamblia is expressed from two ORFs localized 777 kb apart. The pre-mRNAs transcribed from these ORFs are stitched by a trans-splicing mechanism. Here, we provide mechanistic details of this process by reconstituting the reaction using in vitro synthesized pre-mRNA substrates. Using RT-PCR, northern blot and nanostring technology, we demonstrate that the in vitro synthesized pre-mRNAs have the capability to self-splice in the absence of nuclear proteins. Inhibition of the trans-splicing reaction using a ssDNA oligo corresponding to a 26-nucleotide complementary sequence confirmed their role in juxtapositioning the pre-mRNA substrates during the self-splicing reaction. Our study provides the first example of a self catalysed, trans-splicing reaction in eukaryotes.

Proteome and Structural Organization of the Knob Complex on the Surface of the Plasmodium Infected Red Blood Cell.

PURPOSE: The cell membrane of the erythrocytes infected with the malaria parasite Plasmodium falciparum undergoes several changes during the course of parasite life cycle and forms protrusions known as 'knobs' on its surface during the mature trophozoite and schizont stages. The structural organization of knob components especially PfEMP1 on the iRBC surface is the main determinant for the cytoadhesive and rosetting capacity of the iRBC by binding to various host receptors as well as for the variable antigenicity, which is crucial for immunoevasion. Although several studies report individual interactions among knob constituents, a comprehensive identification of the knob proteome is lacking. EXPERIMENTAL DESIGN: The detergent-resistant membrane (DRM) rafts are isolated from the infected erythrocyte membrane and knob (KAHRP) positive fractions are subjected to proteomics analysis. In addition, structures of various knob components are modeled and assembled ab initio based on experimentally established protein interactions. RESULTS: Proteins of various functional classes are found to be present in the knobs including the newly identified knob constituents which include host Hsp70, elongation factor 1A, acyl CoA synthetase, and some hypothetical proteins. Ab initio structural prediction of PfEMP1, KHARP, PfEMP2, PfEMP3, and PHIST shows that these proteins are intrinsically disordered and can have varying number of protein-protein interactions depending on their lowest energy structure. Further in silico mathematical modeling of a single repeat unit of PfEMP1-PHIST is present 63-112 times along the periphery of a single knob. CONCLUSIONS AND CLINICAL RELEVANCE: This study provides structural insight into the organization of the core knob components and uncovers novel proteins as knob components. This structural information can be used for the development of better vaccine design strategies or drug design to destabilize the knob structure, which is a major virulence determinant in P. falciparum malaria.

Characterization of Precursor PfHsp60 in Plasmodium falciparum Cytosol during Its Asexual Development in Human Erythrocytes.

Mitochondrial heat shock protein 60 (Hsp60) is a nuclear encoded gene product that gets post-translationally translocated into the mitochondria. Using multiple approaches such as immunofluorescence experiments, isoelectric point analysis with two-dimensional gel electrophoresis, and mass spectrometric identification of the signal peptide, we show that Hsp60 from Plasmodium falciparum (PfHsp60) accumulates in the parasite cytoplasm during the ring, trophozoite, and schizont stages of parasite development before being imported into the parasite mitochondria. Using co-immunoprecipitation experiments with antibodies specific to cytoplasmic PfHsp90, PfHsp70-1, and PfHsp60, we show association of precursor PfHsp60 with cytoplasmic chaperone machinery. Metabolic labeling involving pulse and chase indicates translocation of the precursor pool into the parasite mitochondrion during chase. Analysis of results obtained with Geldanamycin treatment confirmed precursor PfHsp60 to be one of the clients for PfHsp90. Cytosolic chaperones bind precursor PfHsp60 prior to its import into the mitochondrion of the parasite. Our data suggests an inefficient co-ordination in the synthesis and translocation of mitochondrial PfHsp60 during asexual growth of malaria parasite in human erythrocytes.

Endoplasmic reticulum stress triggers gametocytogenesis in the malaria parasite.

The malaria parasite experiences a significant amount of redox stress during its growth in human erythrocytes and heavily relies on secretory functions for pathogenesis. Most certainly, the parasite is equipped with machinery to tackle perturbations in the secretory pathway, like the unfolded protein response pathway in higher eukaryotes. Our bioinformatics analysis revealed the complete absence of genes involved in the canonical unfolded protein response pathway in Plasmodium falciparum. Accordingly, the parasite was unable to up-regulate endoplasmic reticulum (ER) chaperones or ER-associated degradation in response to DTT-mediated ER stress. Global profiling of gene expression upon DTT treatment revealed a network of AP2 transcription factors and their targets being activated. The overall outcome was up-regulation of genes involved in protein export and the sexual stage of the parasite life cycle culminating in gametocytogenesis. Our results suggest that the malaria parasite uses ER stress as a cue to switch to the transmissible sexual stages.

Identification of an exported heat shock protein 70 in Plasmodium falciparum.

Host cell remodelling is a hallmark of malaria pathogenesis. It involves protein folding, unfolding and trafficking events and thus participation of chaperones such as Hsp70s and Hsp40s is well speculated. Until recently, only Hsp40s were thought to be the sole representative of the parasite chaperones in the exportome. However, based on the re-annotated Plasmodium falciparum genome sequence, a putative candidate for exported Hsp70 has been reported, which otherwise was known to be a pseudogene. We raised a specific antiserum against a C-terminal peptide uniquely present in PfHsp70-x. Immunoblotting and immunofluorescence-based approaches in combination with sub-cellular fractionation by saponin and streptolysin-O have been taken to determine the expression and localization of PfHsp70-x in infected erythrocyte. The re-annotated sequence of PfHsp70-x reveals it to be a functional protein with an endoplasmic reticulum signal peptide. It gets maximally expressed at the schizont stage of intra-erythrocytic life cycle. Majority of the protein localizes to the parasitophorous vacuole and some of it gets exported to the erythrocyte compartment where it associates with Maurer's clefts. The identification of an exported parasite Hsp70 chaperone presents us with the fact that the parasite has evolved customized chaperones which might be playing crucial roles in aspects of trafficking and host cell remodelling.

An exported heat shock protein 40 associates with pathogenesis-related knobs in Plasmodium falciparum infected erythrocytes.

Cell surface structures termed knobs are one of the most important pathogenesis related protein complexes deployed by the malaria parasite Plasmodium falciparum at the surface of the infected erythrocyte. Despite their relevance to the disease, their structure, mechanisms of traffic and their process of assembly remain poorly understood. In this study, we have explored the possible role of a parasite-encoded Hsp40 class of chaperone, namely PFB0090c/PF3D7_0201800 (KAHsp40) in protein trafficking in the infected erythrocyte. We found the gene coding for PF3D7_0201800 to be located in a chromosomal cluster together with knob components KAHRP and PfEMP3. Like the knob components, KAHsp40 too showed the presence of PEXEL motif required for transport to the erythrocyte compartment. Indeed, sub-cellular fractionation and immunofluorescence analysis (IFA) showed KAHsp40 to be exported in the erythrocyte cytoplasm in a stage dependent manner localizing as punctuate spots in the erythrocyte periphery, distinctly from Maurer's cleft, in structures which could be the reminiscent of knobs. Double IFA analysis revealed co-localization of PF3D7_0201800 with the markers of knobs (KAHRP, PfEMP1 and PfEMP3) and components of the PEXEL translocon (Hsp101, PTEX150). KAHsp40 was also found to be in a complex with KAHRP, PfEMP3 and Hsp101 as confirmed by co-immunoprecipitation assay. Our results suggest potential involvement of a parasite encoded Hsp40 in chaperoning knob assembly in the erythrocyte compartment.