Search

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.

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.

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.

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. [ABSTRACT FROM AUTHOR]

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., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)