Cytosolic Sensor of Bacterial Lipopolysaccharide in Human Macrophages

Inflammasomes are cytosolic supramolecular organizing centers that, in response to pathogen-derived molecules and endogenous danger signals, assemble and activate innate immune responses. Bacterial lipopolysaccharide (LPS) is an inflammasome trigger and a major mediator of inflammation during infection, including during the potentially lethal condition sepsis. Activation of most inflammasomes is triggered by sensing of pathogen products by a specific host cytosolic nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein (NLRP) or other sensor protein that in turn activates a pro-inflammatory caspase. LPS that accesses the cell cytosol (cLPS) induces cell-autonomous activation of a non-canonical inflammasome that contains caspases-4/5 in humans or caspase-11 in mice1-3. Whereas the NLRPs that sense most pathogen triggers have been identified, no NLRP is known to sense cLPS, which together with the observation that caspases-4, -5, and -11 bind LPS in vitro4, has led to the postulate that inflammasome activation by cLPS occurs independent of an NLRP. Here we show that primate-specific NLRP11 senses cLPS and promotes the activation of caspase-4. We found that in response to infection by each of several gram-negative intracellular bacterial pathogens or to LPS transfection, efficient activation of the non-canonical pathway in human-derived macrophages depends on NLRP11. Further, we found that in both immortalized human-derived macrophages and primary human macrophages, the dependence of the non-canonical pathway on NLRP11 is due to detection of cLPS. Moreover, in cell lysates, NLRP11 binds LPS independently of caspase-4 and binds caspase-4 independently of LPS. Our results demonstrate that NLRP11 senses cLPS and promotes LPS-dependent activation of caspase-4. NLRP11 is a previously missing link in the human non-canonical inflammasome activation pathway.

Zeptomole Imaging of Cytosolic MicroRNA Cancer Biomarkers with A Light-Controlled Nanoantenna

Detecting and quantifying intracellular microRNAs (miRNAs) are a critical step in resolving a cancer diagnostic and resolving the ensemble of gene products that orchestrate the living state of cells. However, the nanoprobe for detecting low abundance miRNAs in cell cytosol is restricted by either the “one-to-one” signal-trigger model or difficulty for cytosol delivery. To address these challenges, we designed a light-harvesting nanoantenna-based nanoprobe, which directs excitation energy to a single molecule to sensitively detect cytosolic miRNA. With light irradiation, the light-harvesting nanoantenna effectively disrupted lysosomal structures by generation of reactive oxygen species, substantially achieved cytosol delivery. The nanoantenna containing > 4000 donor dyes can efficiently transfer excitation energy to one or two acceptors with 99% efficiency, leading to unprecedented signal amplification and biosensing sensitivity. The designed nanoantenna can quantify cytosolic miR-210 at zeptomolar level. The fluorescence lifetime of the donor exhibited good relationship with miR-210 concentration in the range of 0.032 to 2.97 amol/ngRNA. The zeptomole sensitivity of nanoantenna provides accurate bioimaging of miR-210 both in multiple cell lines and in vivo assay, which creates a pathway for the creation of miRNA toolbox for quantitative epigenetics and personalized medicine.

Transport mechanisms at the malaria parasite-host cell interface

Obligate intracellular malaria parasites reside within a vacuolar compartment generated during invasion which is the principal interface between pathogen and host. To subvert their host cell and support their metabolism, these parasites coordinate a range of transport activities at this membrane interface that are critically important to parasite survival and virulence, including nutrient import, waste efflux, effector protein export, and uptake of host cell cytosol. Here, we review our current understanding of the transport mechanisms acting at the malaria parasite vacuole during the blood and liver-stages of development with a particular focus on recent advances in our understanding of effector protein translocation into the host cell by the Plasmodium Translocon of EXported proteins (PTEX) and small molecule transport by the PTEX membrane-spanning pore EXP2. Comparison to Toxoplasma gondii and other related apicomplexans is provided to highlight how similar and divergent mechanisms are employed to fulfill analogous transport activities.

Lumacaftor and Matrine: Possible Therapeutic Combination to Counteract the Inflammatory Process in Cystic Fibrosis

Cystic fibrosis is a monogenic, autosomal, recessive disease characterized by an alteration of chloride transport caused by mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene. The loss of Phe residue in position 508 (ΔF508-CFTR) causes an incorrect folding of the protein causing its degradation and electrolyte imbalance. CF patients are extremely predisposed to the development of a chronic inflammatory process of the bronchopulmonary system. When the cells of a tissue are damaged, the immune cells are activated and trigger the production of free radicals, provoking an inflammatory process. In addition to routine therapies, today drugs called correctors are available for mutations such as ΔF508-CFTR as well as for others less frequent ones. These active molecules are supposed to facilitate the maturation of the mutant CFTR protein, allowing it to reach the apical membrane of the epithelial cell. Matrine induces ΔF508-CFTR release from the endoplasmic reticulum to cell cytosol and its localization on the cell membrane. We now have evidence that Matrine and Lumacaftor not only restore the transport of mutant CFTR protein, but probably also counteract the inflammatory process by improving the course of the disease.

The Campylobacter jejuni CiaD effector co-opts the host cell protein IQGAP1 to promote cell entry

Campylobacter jejuni is a foodborne pathogen that binds to and invades the epithelial cells lining the human intestinal tract. Maximal invasion of host cells by C. jejuni requires cell binding as well as delivery of the Cia proteins (Campylobacter invasion antigens) to the host cell cytosol via the flagellum. Here, we show that CiaD binds to the host cell protein IQGAP1 (a Ras GTPase-activating-like protein), thus displacing RacGAP1 from the IQGAP1 complex. This, in turn, leads to the unconstrained activity of the small GTPase Rac1, which is known to have roles in actin reorganization and internalization of C. jejuni. Our results represent the identification of a host cell protein targeted by a flagellar secreted effector protein and demonstrate that C. jejuni-stimulated Rac signaling is dependent on IQGAP1.

Widespread bacterial protein flavinylation in functionally distinct extracytosolic redox biochemistries

Disparate redox biochemistries that take place beyond the bounds of the prokaryotic cell cytosol must connect to membrane or cytosolic electron pools. Proteins post-translationally flavinylated by the enzyme ApbE mediate electron transfer in several characterized extracytosolic redox biochemistries but the breadth of functions of this modification remains unknown. Here we present a comprehensive bioinformatic analysis of 31,910 prokaryotic genomes that provides evidence of extracytosolic ApbEs within ~50% of bacteria and the involvement of flavinylation in numerous uncharacterized biochemistries. By mining flavinylation-associated gene clusters, we identify five protein classes responsible for transmembrane electron transfer and two domains of unknown function (DUF2271 and DUF3570) that are flavinylated by ApbE. We observe flavinylation/iron transporter gene colocalization patterns that suggest functions in iron reduction and assimilation. We find associations with characterized and uncharacterized respiratory oxidoreductases that highlight roles of flavinylation in respiratory electron transport chains. Finally, we identify interspecies gene cluster variability consistent with flavinylation/cytochrome functional redundancies and discover a class of multi-flavinylated proteins that may resemble multiheme cytochromes in facilitating longer distance electron transfer. These findings provide mechanistic insight into an important facet of bacterial physiology and establish flavinylation as a functionally diverse mediator of extracytosolic electron transfer.

Atg5-Deficient Mice Infected with Francisella tularensis LVS Demonstrate Increased Survival and Less Severe Pathology in Internal Organs

Francisella tularensis is a highly virulent intracellular pathogen that proliferates within various cell types and can infect a multitude of animal species. Francisella escapes the phagosome rapidly after infection and reaches the host cell cytosol where bacteria undergo extensive replication. Once cytosolic, Francisella becomes a target of an autophagy-mediated process. The mechanisms by which autophagy plays a role in replication of this cytosolic pathogen have not been fully elucidated. In vitro, F. tularensis avoids degradation via autophagy and the autophagy process provides nutrients that support its intracellular replication, but the role of autophagy in vivo is unknown. Here, we investigated the role of autophagy in the pathogenesis of tularemia by using transgenic mice deficient in Atg5 in the myeloid lineage. The infection of Atg5-deficient mice with Francisella tularensis subsp. holarctica live vaccine strain (LVS) resulted in increased survival, significantly reduced bacterial burden in the mouse organs, and less severe histopathological changes in the spleen, liver and lung tissues. The data highlight the contribution of Atg5 in the pathogenesis of tularemia in vivo.

SARS-CoV-2 infects cells following viral entry via clathrin-mediated endocytosis

With more than 51 million cases and 1.3 million deaths, and with the resulting social upheaval, the COVID-19 pandemic presents one of the greatest challenges ever to human society. It is thus vital to fully understand the biology of SARS-CoV-2, the causative agent of COVID-19. SARS-CoV-2 uses its spike glycoprotein to interact with the cell surface as a first step in the infection process. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, we now demonstrate that following engagement with the plasma membrane, SARS-CoV-2 undergoes rapid clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system, and importantly clathrin-heavy chain knockdown, which blocks clathrin-mediated endocytosis, reduces viral infectivity. This discovery reveals important new information about the basic biology of SARS-CoV-2 infectivity.