The eIF2α kinase HRI triggers the autophagic clearance of cytosolic protein aggregates.

Large cytosolic protein aggregates are removed by two main cellular processes, autophagy and the ubiquitin-proteasome system, and defective clearance of these protein aggregates results in proteotoxicity and cell death. Recently, we found that the eIF2α kinase heme-regulated inhibitory (HRI) induced a cytosolic unfolded protein response to prevent aggregation of innate immune signalosomes, but whether HRI acts as a general sensor of proteotoxicity in the cytosol remains unclear. Here we show that HRI controls autophagy to clear cytosolic protein aggregates when the ubiquitin-proteasome system is inhibited. We further report that silencing the expression of HRI resulted in decreased levels of BAG3 and HSPB8, two proteins involved in chaperone-assisted selective autophagy, suggesting that HRI may control proteostasis in the cytosol at least in part through chaperone-assisted selective autophagy. Moreover, knocking down the expression of HRI resulted in cytotoxic accumulation of overexpressed α-synuclein, a protein known to aggregate in Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In agreement with these data, protein aggregate accumulation and microglia activation were observed in the spinal cord white matter of 7-month-old Hri-/- mice as compared with Hri+/+ littermates. Moreover, aged Hri-/- mice showed accumulation of misfolded α-synuclein in the lateral collateral pathway, a region of the sacral spinal cord horn that receives visceral sensory afferents from the bladder and distal colon, a pathological feature common to α-synucleinopathies in humans. Together, these results suggest that HRI contributes to a general cytosolic unfolded protein response that could be leveraged to bolster the clearance of cytotoxic protein aggregates.

The heme-regulated inhibitor is a cytosolic sensor of protein misfolding that controls innate immune signaling.

Multiple cytosolic innate sensors form large signalosomes after activation, but this assembly needs to be tightly regulated to avoid accumulation of misfolded aggregates. We found that the eIF2α kinase heme-regulated inhibitor (HRI) controls NOD1 signalosome folding and activation through a process requiring eukaryotic initiation factor 2α (eIF2α), the transcription factor ATF4, and the heat shock protein HSPB8. The HRI/eIF2α signaling axis was also essential for signaling downstream of the innate immune mediators NOD2, MAVS, and TRIF but dispensable for pathways dependent on MyD88 or STING. Moreover, filament-forming α-synuclein activated HRI-dependent responses, which suggests that the HRI pathway may restrict toxic oligomer formation. We propose that HRI, eIF2α, and HSPB8 define a novel cytosolic unfolded protein response (cUPR) essential for optimal innate immune signaling by large molecular platforms, functionally homologous to the PERK/eIF2α/HSPA5 axis of the endoplasmic reticulum UPR.

Polymorphisms of a Collagen-Like Adhesin Contributes to Legionella pneumophila Adhesion, Biofilm Formation Capacity and Clinical Prevalence.

Legionellosis is a severe respiratory illness caused by the inhalation of aerosolized water droplets contaminated with the opportunistic pathogen Legionella pneumophila. The ability of L. pneumophila to produce biofilms has been associated with its capacity to colonize and persist in human-made water reservoirs and distribution systems, which are the source of legionellosis outbreaks. Nevertheless, the factors that mediate L. pneumophila biofilm formation are largely unknown. In previous studies we reported that the adhesin Legionella collagen-like protein (Lcl), is required for auto-aggregation, attachment to multiple surfaces and the formation of biofilms. Lcl structure contains three distinguishable regions: An N-terminal region with a predicted signal sequence, a central region containing tandem collagen-like repeats (R-domain) and a C-terminal region (C-domain) with no significant homology to other known proteins. Lcl R-domain encodes tandem repeats of the collagenous tripeptide Gly-Xaa-Yaa (GXY), a motif that is key for the molecular organization of mammalian collagen and mediates the binding of collagenous proteins to different cellular and environmental ligands. Interestingly, Lcl is polymorphic in the number of GXY tandem repeats. In this study, we combined diverse biochemical, genetic, and cellular approaches to determine the role of Lcl domains and GXY repeats polymorphisms on the structural and functional properties of Lcl, as well as on bacterial attachment, aggregation and biofilm formation. Our results indicate that the R-domain is key for assembling Lcl collagenous triple-helices and has a more preponderate role over the C-domain in Lcl adhesin binding properties. We show that Lcl molecules oligomerize to form large supramolecular complexes to which both, R and C-domains are required. Furthermore, we found that the number of GXY tandem repeats encoded in Lcl R-domain correlates positively with the binding capabilities of Lcl and with the attachment and biofilm production capacity of L. pneumophila strains. Accordingly, the number of GXY tandem repeats in Lcl influences the clinical prevalence of L. pneumophila strains. Therefore, the number of Lcl tandem repeats could be considered as a potential predictor for virulence in L. pneumophila isolates.

Isoginkgetin, a Natural Biflavonoid Proteasome Inhibitor, Sensitizes Cancer Cells to Apoptosis via Disruption of Lysosomal Homeostasis and Impaired Protein Clearance.

Protein degradation pathways are critical for maintaining proper protein dynamics within the cell, and considerable efforts have been made toward the development of therapeutics targeting these catabolic processes. We report here that isoginkgetin, a naturally derived biflavonoid, sensitized cells undergoing nutrient starvation to apoptosis, induced lysosomal stress, and activated the lysosome biogenesis gene TFEB Isoginkgetin treatment led to the accumulation of aggregates of polyubiquitinated proteins that colocalized strongly with the adaptor protein p62, the 20S proteasome, and the endoplasmic reticulum-associated degradation (ERAD) protein UFD1L. Isoginkgetin directly inhibited the chymotrypsin-like, trypsin-like, and caspase-like activities of the 20S proteasome and impaired NF-κB signaling, suggesting that the molecule may display its biological activity in part through proteasome inhibition. Importantly, isoginkgetin was effective at killing multiple myeloma (MM) cell lines in vitro and displayed a higher rate of cell death induction than the clinically approved proteasome inhibitor bortezomib. We propose that isoginkgetin disturbs protein homeostasis, leading to an excess of protein cargo that places a burden on the lysosomes/autophagic machinery, eventually leading to cancer cell death.

The mitochondrial Nod-like receptor NLRX1 modifies apoptosis through SARM1.

NLRX1, the mitochondrial NOD-like receptor (NLR), modulates apoptosis in response to both intrinsic and extrinsic cues. Insights into the mechanism of how NLRX1 influences apoptosis remain to be determined. Here, we demonstrate that NLRX1 associates with SARM1, a protein with a toll/interleukin-1 receptor (TIR)-containing domain also found in adaptor proteins downstream of toll-like receptors, such as MyD88. While a direct role of SARM1 in innate immunity is unclear, the protein plays essential roles in Wallerian degeneration (WD), a type of neuronal catabolism occurring following axonal severing or damage. In non-neuronal cells, we found that endogenous SARM1 was equally distributed in the cytosol and the mitochondrial matrix, where association with NLRX1 occurred. In these cells, the apoptotic role of NLRX1 was fully dependent on SARM1, indicating that SARM1 was downstream of NLRX1 in apoptosis regulation. In primary murine neurons, however, Wallerian degeneration induced by vinblastine or NGF deprivation occurred in SARM1- yet NLRX1-independent manner, suggesting that WD requires the cytosolic pool of SARM1 or that NLRX1 levels in neurons are too low to contribute to WD regulation. Together, these results shed new light into the mechanisms through which NLRX1 controls apoptosis and provides evidence of a new link between NLR and TIR-containing proteins.

NOD1 and NOD2 in inflammation, immunity and disease.

NOD1 and NOD2 are related intracellular sensors of bacterial peptidoglycan and belong to the Nod-like receptor (NLR) family of innate immune proteins that play fundamental and pleiotropic roles in host defense against infection and in the control of inflammation. The importance of these proteins is also highlighted by the genetic association between single nucleotide polymorphisms in NOD2 and susceptibility to Crohn's disease, an inflammatory bowel disease. At the cellular level, recent efforts have delineated the signaling pathways triggered following activation of NOD1 and NOD2, and the interplay with various cellular processes, such as autophagy. In vivo studies have revealed the importance of NOD-dependent host defense in models of infection, and a crucial area of investigation focuses on understanding the role of NOD1 and NOD2 at the intestinal mucosa, as this is of prime importance for understanding the etiology of Crohn's disease.

Complement C3 Drives Autophagy-Dependent Restriction of Cyto-invasive Bacteria.

In physiological settings, the complement protein C3 is deposited on all bacteria, including invasive pathogens. However, because experimental host-bacteria systems typically use decomplemented serum to avoid the lytic action of complement, the impact of C3 coating on epithelial cell responses to invasive bacteria remains unexplored. Here, we demonstrate that following invasion, intracellular C3-positive Listeria monocytogenes is targeted by autophagy through a direct C3/ATG16L1 interaction, resulting in autophagy-dependent bacterial growth restriction. In contrast, Shigella flexneri and Salmonella Typhimurium escape autophagy-mediated growth restriction in part through the action of bacterial outer membrane proteases that cleave bound C3. Upon oral infection with Listeria, C3-deficient mice displayed defective clearance at the intestinal mucosa. Together, these results demonstrate an intracellular role of complement in triggering antibacterial autophagy and immunity against intracellular pathogens. Since C3 indiscriminately associates with foreign surfaces, the C3-ATG16L1 interaction may provide a universal mechanism of xenophagy initiation.

Listeria monocytogenes and Shigella flexneri Activate the NLRP1B Inflammasome.

Activation of the innate immune receptor NLRP1B leads to the formation of an inflammasome, which induces autoproteolytic processing of pro-caspase-1, and ultimately to the release of inflammatory cytokines and to the execution of pyroptosis. One of the signals to which NLRP1B responds is metabolic stress that occurs in cells deprived of glucose or treated with metabolic inhibitors. NLRP1B might therefore sense microbial infection, as intracellular pathogens such as Listeria monocytogenes and Shigella flexneri cause metabolic stress as a result of nutrient scavenging and host cell damage. Here we addressed whether these pathogens activate the NLRP1B inflammasome. We found that Listeria infection activated the NLRP1B inflammasome in a reconstituted fibroblast model. Activation of NLRP1B by Listeria was diminished in an NLRP1B mutant shown previously to be defective at detecting energy stress and was dependent on the expression of listeriolysin O (LLO), a protein required for vacuolar escape. Infections of either Listeria or Shigella activated NLRP1B in the RAW264.7 murine macrophage line, which expresses endogenous NLRP1B. We conclude that NLRP1B senses cellular infection by distinct invasive pathogens.

The emerging role of mTOR signalling in antibacterial immunity.

Mammalian target of rapamycin (mTOR) is a central regulator of cellular metabolic homeostasis that is highly conserved in evolution. Recent evidence has revealed the existence of a complex interplay between mTOR signalling and immunity. We review here the emerging role of mTOR signalling in the regulation of Toll-like receptor-dependent innate responses and in the activation of T cells and antigen-presenting cells. We also highlight the importance of amino-acid starvation-driven mTOR inhibition in the control of autophagy and intracellular bacterial clearance.

The Legionella pneumophila collagen-like protein mediates sedimentation, autoaggregation, and pathogen-phagocyte interactions.

Although only partially understood, multicellular behavior is relatively common in bacterial pathogens. Bacterial aggregates can resist various host defenses and colonize their environment more efficiently than planktonic cells. For the waterborne pathogen Legionella pneumophila, little is known about the roles of autoaggregation or the parameters which allow cell-cell interactions to occur. Here, we determined the endogenous and exogenous factors sufficient to allow autoaggregation to take place in L. pneumophila. We show that isolates from Legionella species which do not produce the Legionella collagen-like protein (Lcl) are deficient in autoaggregation. Targeted deletion of the Lcl-encoding gene (lpg2644) and the addition of Lcl ligands impair the autoaggregation of L. pneumophila. In addition, Lcl-induced autoaggregation requires divalent cations. Escherichia coli producing surface-exposed Lcl is able to autoaggregate and shows increased biofilm production. We also demonstrate that L. pneumophila infection of Acanthamoeba castellanii and Hartmanella vermiformis is potentiated under conditions which promote Lcl dependent autoaggregation. Overall, this study shows that L. pneumophila is capable of autoaggregating in a process that is mediated by Lcl in a divalent-cation-dependent manner. It also reveals that Lcl potentiates the ability of L. pneumophila to come in contact, attach, and infect amoebae.

Biofilms: the stronghold of Legionella pneumophila.

Legionellosis is mostly caused by Legionella pneumophila and is defined as a severe respiratory illness with a case fatality rate ranging from 5% to 80%. L. pneumophila is ubiquitous in natural and anthropogenic water systems. L. pneumophila is transmitted by inhalation of contaminated aerosols produced by a variety of devices. While L. pneumophila replicates within environmental protozoa, colonization and persistence in its natural environment are also mediated by biofilm formation and colonization within multispecies microbial communities. There is now evidence that some legionellosis outbreaks are correlated with the presence of biofilms. Thus, preventing biofilm formation appears as one of the strategies to reduce water system contamination. However, we lack information about the chemical and biophysical conditions, as well as the molecular mechanisms that allow the production of biofilms by L. pneumophila. Here, we discuss the molecular basis of biofilm formation by L. pneumophila and the roles of other microbial species in L. pneumophila biofilm colonization. In addition, we discuss the protective roles of biofilms against current L. pneumophila sanitation strategies along with the initial data available on the regulation of L. pneumophila biofilm formation.

Comparative Genomics Reveal That Host-Innate Immune Responses Influence the Clinical Prevalence of Legionella pneumophila Serogroups.

Legionella pneumophila is the primary etiologic agent of legionellosis, a potentially fatal respiratory illness. Amongst the sixteen described L. pneumophila serogroups, a majority of the clinical infections diagnosed using standard methods are serogroup 1 (Sg1). This high clinical prevalence of Sg1 is hypothesized to be linked to environmental specific advantages and/or to increased virulence of strains belonging to Sg1. The genetic determinants for this prevalence remain unknown primarily due to the limited genomic information available for non-Sg1 clinical strains. Through a systematic attempt to culture Legionella from patient respiratory samples, we have previously reported that 34% of all culture confirmed legionellosis cases in Ontario (n = 351) are caused by non-Sg1 Legionella. Phylogenetic analysis combining multiple-locus variable number tandem repeat analysis and sequence based typing profiles of all non-Sg1 identified that L. pneumophila clinical strains (n = 73) belonging to the two most prevalent molecular types were Sg6. We conducted whole genome sequencing of two strains representative of these sequence types and one distant neighbour. Comparative genomics of the three L. pneumophila Sg6 genomes reported here with published L. pneumophila serogroup 1 genomes identified genetic differences in the O-antigen biosynthetic cluster. Comparative optical mapping analysis between Sg6 and Sg1 further corroborated this finding. We confirmed an altered O-antigen profile of Sg6, and tested its possible effects on growth and replication in in vitro biological models and experimental murine infections. Our data indicates that while clinical Sg1 might not be better suited than Sg6 in colonizing environmental niches, increased bloodstream dissemination through resistance to the alternative pathway of complement mediated killing in the human host may explain its higher prevalence.