Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry.

Autophagy is emerging as a crucial defense mechanism against bacteria, but the host intracellular sensors responsible for inducing autophagy in response to bacterial infection remain unknown. Here we demonstrated that the intracellular sensors Nod1 and Nod2 are critical for the autophagic response to invasive bacteria. By a mechanism independent of the adaptor RIP2 and transcription factor NF-kappaB, Nod1 and Nod2 recruited the autophagy protein ATG16L1 to the plasma membrane at the bacterial entry site. In cells homozygous for the Crohn's disease-associated NOD2 frameshift mutation, mutant Nod2 failed to recruit ATG16L1 to the plasma membrane and wrapping of invading bacteria by autophagosomes was impaired. Our results link bacterial sensing by Nod proteins to the induction of autophagy and provide a functional link between Nod2 and ATG16L1, which are encoded by two of the most important genes associated with Crohn's disease.

Protein aggregation as a cellular response to oxidative stress induced by heme and iron.

Hemolytic diseases include a variety of conditions with diverse etiologies in which red blood cells are destroyed and large amounts of hemeproteins are released. Heme has been described as a potent proinflammatory molecule that is able to induce multiple innate immune responses, such as those triggered by TLR4 and the NLRP3 inflammasome, as well as necroptosis in macrophages. The mechanisms by which eukaryotic cells respond to the toxic effects induced by heme to maintain homeostasis are not fully understood, however. Here we describe a previously uncharacterized cellular response induced by heme: the formation of p62/SQTM1 aggregates containing ubiquitinated proteins in structures known as aggresome-like induced structures (ALIS). This action is part of a response driven by the transcription factor NRF2 to the excessive generation of reactive oxygen species induced by heme that results in the expression of genes involved in antioxidant responses, including p62/SQTM1. Furthermore, we show that heme degradation by HO-1 is required for ALIS formation, and that the free iron released on heme degradation is necessary and sufficient to induce ALIS. Moreover, ferritin, a key protein in iron metabolism, prevents excessive ALIS formation. Finally, in vivo, hemolysis promotes an increase in ALIS formation in target tissues. Our data unravel a poorly understood aspect of the cellular responses induced by heme that can be explored to better understand the effects of free heme and free iron during hemolytic diseases such as sickle cell disease, dengue fever, malaria, and sepsis.

Post-transcriptional inhibition of luciferase reporter assays by the Nod-like receptor proteins NLRX1 and NLRC3.

Luciferase reporter assays (LRAs) are widely used to assess the activity of specific signal transduction pathways. Although powerful, rapid and convenient, this technique can also generate artifactual results, as revealed for instance in the case of high throughput screens of inhibitory molecules. Here we demonstrate that the previously reported inhibitory effect of the Nod-like receptor (NLR) protein NLRX1 on NF-κB- and type I interferon-dependent pathways in LRAs was a nonspecific consequence of the overexpression of the NLRX1 leucine-rich repeat (LRR) domain. By comparing luciferase activity and luciferase gene expression using quantitative PCR from the same samples, we showed that NLRX1 inhibited LRAs in a post-transcriptional manner. In agreement, NLRX1 also repressed LRAs if luciferase was expressed under the control of a constitutive promoter, although the degree of inhibition by NLRX1 seemed to correlate with the dynamic inducibility of luciferase reporter constructs. Similarly, we observed that overexpression of another NLR protein, NLRC3, also resulted in artifactual inhibition of LRAs; thus suggesting that the capacity to inhibit LRAs at a post-transcriptional level is not unique to NLRX1. Finally, we demonstrate that host type I interferon response to Sendai virus infection was normal in NLRX1-silenced human HEK293T cells. Our results thus highlight the fact that LRAs are not a reliable technique to assess the inhibitory function of NLRs, and possibly other overexpressed proteins, on signal transduction pathways.

The Unfolded Protein Response and Autophagy on the Crossroads of Coronaviruses Infections.

The ability to sense and adequately respond to variable environmental conditions is central for cellular and organismal homeostasis. Eukaryotic cells are equipped with highly conserved stress-response mechanisms that support cellular function when homeostasis is compromised, promoting survival. Two such mechanisms - the unfolded protein response (UPR) and autophagy - are involved in the cellular response to perturbations in the endoplasmic reticulum, in calcium homeostasis, in cellular energy or redox status. Each of them operates through conserved signaling pathways to promote cellular adaptations that include re-programming transcription of genes and translation of new proteins and degradation of cellular components. In addition to their specific functions, it is becoming increasingly clear that these pathways intersect in many ways in different contexts of cellular stress. Viral infections are a major cause of cellular stress as many cellular functions are coopted to support viral replication. Both UPR and autophagy are induced upon infection with many different viruses with varying outcomes - in some instances controlling infection while in others supporting viral replication and infection. The role of UPR and autophagy in response to coronavirus infection has been a matter of debate in the last decade. It has been suggested that CoV exploit components of autophagy machinery and UPR to generate double-membrane vesicles where it establishes its replicative niche and to control the balance between cell death and survival during infection. Even though the molecular mechanisms are not fully elucidated, it is clear that UPR and autophagy are intimately associated during CoV infections. The current SARS-CoV-2 pandemic has brought renewed interest to this topic as several drugs known to modulate autophagy - including chloroquine, niclosamide, valinomycin, and spermine - were proposed as therapeutic options. Their efficacy is still debatable, highlighting the need to better understand the molecular interactions between CoV, UPR and autophagy.

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.

Intracellular bacteriolysis triggers a massive apoptotic cell death in Shigella-infected epithelial cells.

Infected epithelial cells, which act as a first barrier against pathogens, seldom undergo apoptosis. Rather, infected epithelial cells undergo a slow cell death that displays hallmarks of necrosis. Here, we demonstrate that rapid intracellular lysis of Shigella flexneri, provoked by either the use of a diaminopimelic acid auxotroph mutant or treatment of infected cells with antibiotics of the beta-lactam family, resulted in a massive and rapid induction of apoptotic cell death. This intracellular bacteriolysis-mediated apoptotic death (IBAD) was characterized by the specific involvement of the mitochondrial-dependent cytochrome c/Apaf-1 axis that resulted in the activation of caspases-3, -6 and -9. Importantly, Bcl-2 family members and the NF-kappaB pathway seemed to be critical modulators of IBAD. Finally, we identified that IBAD was also triggered by Salmonella enterica serovar Typhimurium but not by the Gram-positive bacteria, Listeria monocytogenes. Together, our results demonstrate that, contrary to previous findings, epithelial cells are intrinsically able to mount an efficient apoptotic cell death response following infection. Indeed, apoptosis in normal circumstances is masked by powerful anti-apoptotic mechanisms, which are overcome in IBAD. Our results also uncover an unexpected consequence of the treatment of infected cells with certain classes of antibiotics.

Integrated Stress Responses to Bacterial Pathogenesis Patterns.

Activation of an appropriate innate immune response to bacterial infection is critical to limit microbial spread and generate cytokines and chemokines to instruct appropriate adaptive immune responses. Recognition of bacteria or bacterial products by pattern recognition molecules is crucial to initiate this response. However, it is increasingly clear that the context in which this recognition occurs can dictate the quality of the response and determine the outcome of an infection. The cross talk established between host and pathogen results in profound alterations on cellular homeostasis triggering specific cellular stress responses. In particular, the highly conserved integrated stress response (ISR) has been shown to shape the host response to bacterial pathogens by sensing cellular insults resulting from infection and modulating transcription of key genes, translation of new proteins and cell autonomous antimicrobial mechanisms such as autophagy. Here, we review the growing body of evidence demonstrating a role for the ISR as an integral part of the innate immune response to bacterial pathogens.

Heme and iron induce protein aggregation.

Heme is an essential molecule expressed in many tissues where it plays key roles as the prosthetic group of several proteins involved in vital physiological and metabolic processes such as gas and electron transport. Structurally, heme is a tetrapyrrole ring containing an atom of iron (Fe) in its center. When released into the extracellular milieu, heme exerts several deleterious effects, which make it an important player in infectious and noninfectious hemolytic diseases where large amounts of free heme are observed such as malaria, dengue fever, ß-thalassemia, sickle cell disease and ischemia-reperfusion. Our recent work has uncovered an unappreciated cellular response triggered by heme or Fe, one of its degradation products, on macrophages, which is the formation of protein aggregates known as aggresome-like induced structres (ALIS). This response was shown to be fully dependent on ROS production and the activation of the transcription factor NFE2L2/NRF2. In addition, we have demonstrated that heme degradation by HMOX1/HO-1 (heme oxygenase 1) is required and that Fe is essential for the formation of ALIS, as heme analogs lacking the central atom of Fe are not able to induce these structures. ALIS formation is also observed in vivo, in a model of phenylhydrazine (PHZ)-induced hemolysis, indicating that it is an integral part of the host response to excessive free heme and that it may play a role in cellular homeostasis.

Autophagy and viral diseases transmitted by Aedes aegypti and Aedes albopictus.

Despite a long battle that was started by Oswaldo Cruz more than a century ago, in 1903, Brazil still struggles to fight Aedes aegypti and Aedes albopictus, the mosquito vectors of dengue virus (DENV), Chikungynya virus (CHIKV) and Zika virus (ZIKV). Dengue fever has been a serious public health problem in Brazil for decades, with recurrent epidemic outbreaks occurring during summers. In 2015, until November, 1,534,932 possible cases were reported to the Ministry of Healthv. More recently, the less studied CHIKV and ZIKV have gained attention because of a dramatic increase in their incidence (around 400% for CHIKV) and the association of ZIKV infection with a 11-fold increase in the number of cases of microcephaly from 2014 to 2015 in northeast Brazil (1761 cases until December 2015). The symptoms of these three infections are very similar, which complicates the diagnosis. These include fever, headache, nausea, fatigue, and joint pain. In some cases, DENV infection develops into dengue hemorrhagic fever, a life threatening condition characterized by bleeding and decreases in platelet numbers in the blood. As for CHIKV, the most important complication is joint pain, which can last for months.

(1)H, (15)N and (13)C resonance assignments of the RRM1 domain of the key post-transcriptional regulator HuR.

Human antigen R (HuR) is a ubiquitous protein that recognizes adenylate and uridylate-rich elements in mRNA, thereby interfering with the fate of protein translation. This protein plays a central role in the outcome of the inflammatory response as it may stabilize or silence mRNAs of key components of the immune system. HuR is able to interact with other RNA-binding proteins, reflecting a complex network that dictates mRNAs post-transcriptional control. HuR is composed of three functional domains, known as RNA-recognition motifs (RRM1, RRM2 and RRM3). It is known that RRM1 is the most important domain for mRNA-binding affinity. In this study, we completed the NMR chemical shift assignment of the RRM1 domain of HuR, as a first step to further establishing the structure, dynamics and function relationship for this protein.

Innate immune recognition of microbes through Nod1 and Nod2: implications for disease.

Nod1 and Nod2 are cytosolic proteins involved in intracellular recognition of microbes and their products. Recently, it was shown that these proteins recognize different moieties of bacterial peptidoglycan (PGN) mediating non-specific pathogen resistance and possibly generating signals for the adaptive immune response. Moreover, mutations in the gene encoding Nod2 are associated with increased susceptibility to chronic inflammatory disorders.

Antimicrobial resistance in Gram-negative bacilli isolated from infant formulas.

A total of 90 samples of infant formula (IF) were collected from the lactary of a teaching hospital, during a 4-month period from July to August 1999. The sanitary conditions of the formulas were analyzed, and a physiological characterization of Gram-negative bacillus isolates and antimicrobial susceptibility testing were performed. Colony counts were considered to be unacceptable for the majority of the IF samples and the contamination rates were related to inadequate handling. Coliforms (35 degrees C and 45 degrees C growth) were detected in most of the IF tested. Klebsiella pneumoniae, Citrobacter freundii, Cedacea davisae, Klebsiella planticola and Enterobacter cloacae were the isolates most commonly identified. Antimicrobial susceptibility testing showed significant resistance rates, particularly to amoxicillin/clavulanic acid, cefoxitin, cephalotin or ampicillin. One extended-spectrum beta-lactamase-producing K. pneumoniae strain was also recovered.

Antimicrobial-resistance and enterotoxin-encoding genes among staphylococci isolated from expressed human breast milk.

Resistance traits and the presence of enterotoxin-encoding genes were investigated in staphylococcus isolates obtained from expressed human breast milk. A total of 54 staphylococcal isolates identified as Staphylococcus epidermidis (53.6 %), Staphylococcus warneri (20.4 %), Staphylococcus haemolyticus (13 %) and Staphylococcus aureus (13 %) were investigated. By using a disc-diffusion method, higher rates of resistance, including intermediate resistance, were observed for penicillin (87 %) and erythromycin (59.3 %). All strains were susceptible to clindamycin and vancomycin. Minimal inhibitory concentration (MIC) was determined by a macrodilution method for four clinically relevant antimicrobial drugs. High rates of resistance or intermediate resistance were observed for erythromycin, gentamicin and oxacillin. Additionally, three isolates showed reduced susceptibility to vancomycin (MIC, 8 microg ml(-1)). Genetic determinants of resistance were detected by using PCR and the results showed good correlation with the macrodilution tests. Moreover, in four staphylococcus isolates, the presence of enterotoxin-encoding genes (seg, seh and sea) was identified. The results demonstrated that expressed human breast milk can be a reservoir of multiresistant staphylococci that may also harbour important virulent determinants.

The Interplay between NLRs and Autophagy in Immunity and Inflammation.

Since they were first described as cytosolic sensors of microbial molecules a decade ago, the Nod-like receptors (NLRs) have been shown to have many different and important roles in various aspects of immune and inflammatory responses, ranging from antimicrobial mechanisms to control of adaptive responses. In this review, we focus on the interplay between NLRs and autophagy, an evolutionarily conserved mechanism that is crucial for homeostasis and has recently been shown to be involved in the protective response against infections. Furthermore, the association between mutations of NLRs as well as proteins that form the autophagic machinery and inflammatory diseases such as Crohn's disease highlight the importance of these proteins and their interactions in the regulation of inflammation.

Macrophage migration inhibitory factor in protozoan infections.

Macrophage migration inhibitory factor (MIF) is a cytokine that plays a central role in immune and inflammatory responses. In the present paper, we discussed the participation of MIF in the immune response to protozoan parasite infections. As a general trend, MIF participates in the control of parasite burden at the expense of promoting tissue damage due to increased inflammation.

Amino acid starvation induced by invasive bacterial pathogens triggers an innate host defense program.

Autophagy, which targets cellular constituents for degradation, is normally inhibited in metabolically replete cells by the metabolic checkpoint kinase mTOR. Although autophagic degradation of invasive bacteria has emerged as a critical host defense mechanism, the signals that induce autophagy upon bacterial infection remain unclear. We find that infection of epithelial cells with Shigella and Salmonella triggers acute intracellular amino acid (AA) starvation due to host membrane damage. Pathogen-induced AA starvation caused downregulation of mTOR activity, resulting in the induction of autophagy. In Salmonella-infected cells, membrane integrity and cytosolic AA levels rapidly normalized, favoring mTOR reactivation at the surface of the Salmonella-containing vacuole and bacterial escape from autophagy. In addition, bacteria-induced AA starvation activated the GCN2 kinase, eukaryotic initiation factor 2α, and the transcription factor ATF3-dependent integrated stress response and transcriptional reprogramming. Thus, AA starvation induced by bacterial pathogens is sensed by the host to trigger protective innate immune and stress responses.

Fungal surface and innate immune recognition of filamentous fungi.

The innate immune system performs specific detection of molecules from infectious agents through pattern recognition receptors. This recognition triggers inflammatory responses and activation of microbicidal mechanisms by leukocytes. Infections caused by filamentous fungi have increased in incidence and represent an important cause of mortality and morbidity especially in individuals with immunosuppression. This review will discuss the innate immune recognition of filamentous fungi molecules and its importance to infection control and disease.

Nod proteins link bacterial sensing and autophagy.

Autophagy is one of the main cellular degradation systems in eukaryotes, responsible for the elimination of long-lived proteins and damaged organelles. Besides its well-documented role as a housekeeping mechanism, autophagy has recently caught the attention of groups working in the fields of microbiology and immunology, especially those working in innate immunity. In particular, the highly specific segregation and degradation of intracellular bacteria by the autophagic machinery was a matter of great interest. However, it was still unclear how the autophagy machinery could target intracellular bacteria with such specificity. We have recently analyzed the role of the intracellular peptidoglycan (PG) receptors Nod1 and Nod2 as a link between intracellular bacterial sensing and the induction of autophagy. Our results demonstrated that Nod2 recruits the critical autophagy protein ATG16L1 to the plasma membrane during bacterial invasion and that cells expressing mutations in these proteins--two of the most important associated with Crohn disease--autophagy is defective upon infection or stimulation with the bacterial peptidoglycan fragment MDP. Thus, our findings put together two genes previously reported as independent risk factors for the development of Crohn disease and open a venue in the study of new therapies to cure the disease.

Oncolytic targeting of renal cell carcinoma via encephalomyocarditis virus.

Apoptosis is a fundamental host defence mechanism against invading microbes. Inactivation of NF-kappaB attenuates encephalomyocarditis virus (EMCV) virulence by triggering rapid apoptosis of infected cells, thereby pre-emptively limiting viral replication. Recent evidence has shown that hypoxia-inducible factor (HIF) increases NF-kappaB-mediated anti-apoptotic response in clear-cell renal cell carcinoma (CCRCC) that commonly exhibit hyperactivation of HIF due to the loss of its principal negative regulator, von Hippel-Lindau (VHL) tumour suppressor protein. Here, we show that EMCV challenge induces a strong NF-kappaB-dependent gene expression profile concomitant with a lack of interferon-mediated anti-viral response in VHL-null CCRCC, and that multiple established CCRCC cell lines, as well as early-passage primary CCRCC cultured cells, are acutely susceptible to EMCV replication and virulence. Functional restoration of VHL or molecular suppression of HIF or NF-kappaB dramatically reverses CCRCC cellular susceptibility to EMCV-induced killing. Notably, intratumoural EMCV treatment of CCRCC in a murine xenograft model rapidly regresses tumour growth. These findings provide compelling pre-clinical evidence for the usage of EMCV in the treatment of CCRCC and potentially other tumours with elevated HIF/NF-kappaB-survival signature.