The phytopathogen Dickeya dadantii 3937 cpxR locus gene participates in the regulation of virulence and the global c‐di‐GMP network

Bacteria use signal transduction systems to sense and respond to their external environment. The two‐component system CpxA/CpxR senses misfolded envelope protein stress and responds by up‐regulating envelope protein factors and down‐regulating virulence factors in several animal pathogens. Dickeya dadantii is a phytopathogen equipped with a type III secretion system (T3SS) for manipulating the host immune response. We found that deletion of cpxR enhanced the expression of the T3SS marker gene hrpA in a designated T3SS‐inducing minimal medium (MM). In the ∆cpxR mutant, multiple T3SS and c‐di‐GMP regulators were also up‐regulated. Subsequent analysis revealed that deletion of the phosphodiesterase gene egcpB in ∆cpxR abolished the enhanced T3SS expression. This suggested that CpxR suppresses EGcpB levels, causing low T3SS expression in MM. Furthermore, we found that the ∆cpxR mutant displayed low c‐di‐GMP phenotypes in biofilm formation and swimming. Increased production of cellular c‐di‐GMP by in trans expression of the diguanylate cyclase gene gcpA was negated in the ∆cpxR mutant. Here, we propose that CpxA/CpxR regulates T3SS expression by manipulating the c‐di‐GMP network, in turn modifying the multiple physiological activities involved in the response to environmental stresses in D. dadantii.     

Cyclic di‐GMP modulates sessile‐motile phenotypes and virulence in Dickeya oryzae via two PilZ domain receptors

Dickeya oryzae is a bacterial pathogen causing the severe rice stem rot disease in China and other rice‐growing countries. We showed recently that the universal bacterial second messenger c‐di‐GMP plays an important role in modulation of bacterial motility and pathogenicity, but the mechanism of regulation remains unknown. In this study, bioinformatics analysis of the D. oryzae EC1 genome led to the identification of two proteins, YcgR and BcsA, both of which contain a conserved c‐di‐GMP receptor domain, known as the PilZ‐domain. By deleting all the genes encoding c‐di‐GMP‐degrading enzymes in D. oryzae EC1, the resultant mutant 7ΔPDE with high c‐di‐GMP levels became nonmotile, formed hyperbiofilm, and lost the ability to colonize and invade rice seeds. These phenotypes were partially reversed by deletion of ycgR in the mutant 7ΔPDE, whereas deletion of bcsA only reversed the hyperbiofilm phenotype of mutant 7ΔPDE. Significantly, double deletion of ycgR and bcsA in mutant 7ΔPDE rescued its motility, biofilm formation, and virulence to levels of wild‐type EC1. In vitro biochemical experiments and in vivo phenotypic assays further validated that YcgR and BcsA proteins are the receptors for c‐di‐GMP, which together play a critical role in regulating the c‐di‐GMP‐associated functionality. The findings from this study fill a gap in our understanding of how c‐di‐GMP modulates bacterial motility and biofilm formation, and provide useful clues for further elucidation of sophisticated virulence regulatory mechanisms in this important plant pathogen.     

4‐phenylpyridine suppresses UVB‐induced skin inflammation by targeting c‐Src in vitro and in vivo

Acute or repetitive exposure to ultraviolet (UV) cause disruptions to the skin barrier and subsequent inflammatory skin disease. 4‐phenylpyridine (4‐PP) is a constituent of Brassica campestris L. ssp. Pekinensis and its effect on skin inflammation and molecular target remain unclear. The purpose of this study is to confirm the anti‐inflammatory efficacy of 4‐PP on UVB‐induced skin inflammation in human keratinocytes HaCaT and mouse skin and validation of its molecular target. 4‐PP also attenuated UVB‐induced phosphorylation of p38/mitogen‐activated protein kinase kinase (MKK) 3/6, c‐Jun N‐terminal kinase 1/2, MKK 4/7, extracellular‐signal‐regulated kinase 1/2, mitogen‐activated protein kinase 1/2. Additionally, 4‐PP inhibited UVB‐induced phosphorylation of epidermal growth factor receptor (EGFR) Y1068, Y1045 and 854 residues but not the proto‐oncogene tyrosine‐protein kinase c‐Src. Drug affinity responsive target stability assay revealed that 4‐PP directly binds to c‐Src and inhibits pronase c‐proteolysis. Knockdown of c‐Src inhibited UVB‐induced COX‐2 expression and phosphorylation of MAPKs and EGFR in HaCaT cells. Dorsal treatment of 4‐PP prevented UVB (0.5 J/cm²)‐induced skin thickness, phosphorylation of EGFR and COX‐2 expression in mouse skin. Our findings suggest that 4‐PP can be used as anti‐inflammatory agent with an effect of skin inflammation by inhibiting the COX‐2 expression via suppressing the c‐Src/EGFR/MAPKs signalling pathway.     

Overexpression of c‐Jun inhibits erastin‐induced ferroptosis in Schwann cells and promotes repair of facial nerve function

Myelin undergoes various changes after nerve injury, and c‐Jun has a close relationship with Schwann cells (SCs). However, it remains unclear whether c‐Jun can be involved in nerve repair by regulating ferroptosis. To explore this, we first set up a facial nerve injury model and detected the changes of ferroptosis‐related proteins and c‐Jun by immunofluorescence and Western blot. Then, we cultured RSC 96 and pSCs, and studied the potential regulatory relationships by a combination of experimental methods such as CCK‐8, ELISA, immunofluorescence, qRT‐PCR, Western blot and viral transfection. Finally, we corroborated the role of c‐Jun through animal experiments. Our experiments revealed that ferroptosis occurs after facial nerve injury. Erastin decreased GPX4, c‐Jun proteins and GSH content, while PTGS2, NRF2, HO‐1 proteins, MDA, Fe²⁺ and ROS contents increased. This effect was inhibited after c‐Jun overexpression but was reversed after the addition of c‐Jun siRNA. Besides, we proved in vivo that c‐Jun could inhibit ferroptosis and promote the recovery of facial nerve function. In conclusion, our study identified the relationship between c‐Jun and ferroptosis during peripheral nerve injury repair, which provides new ideas for studying peripheral nerve injury and repair.     

Hsa_circ_0003945 promotes progression of hepatocellular carcinoma by mediating miR‐34c‐5p/LGR4/β‐catenin axis activity

Accumulating evidence suggests that circular RNAs (circRNAs) play essential roles in regulating cancer progression, but many circRNAs in hepatocellular carcinoma (HCC) remain unknown. Dysregulated circRNAs in HCC were identified through bioinformatics analysis of Gene Expression Omnibus data sets. Quantitative real‐time PCR (qRT‐PCR), Sanger sequencing, RNase R digestion and actinomycin D treatment were conducted to confirm the characterization of circRNAs. CCK‐8, wound‐healing and Transwell assays were performed to assess the functional roles of Hsa_circ_0003945 (Circ_0003945) in HCC cell lines. Subcellular fractionation and fluorescence in situ hybridization (FISH) were performed to locate Circ_0003945 in HCC cells. Dual‐luciferase reporter assay was executed to verify the binding of Circ_0003945 to microRNAs (miRNAs) or the miRNAs to their target genes. In this study, we found that Circ_0003945 was upregulated in HCC tissue, and higher Circ_0003945 expression was positively correlated with tumour size and tumour stage. Furthermore, high plasma levels of circulating Circ_0003945 were confirmed in HCC patients compared with those in non‐HCC groups. The functional experiments revealed that overexpression or knockdown of Circ_0003945 promoted or attenuated tumour growth and migration, respectively. Mechanistically, Circ_0003945 might exert as a miR‐34c‐5p sponge to upregulate the expression of leucine‐rich repeat‐containing G protein‐coupled receptor 4 (LGR4), activating the β‐catenin pathway, and finally facilitating HCC progression. Additionally, a β‐catenin activator could reverse the effect of Circ_0003945 knockdown. In conclusion, Circ_0003945 exerts a tumour‐promoting role in HCC cells by regulating the miR‐34c‐5p/LGR4/β‐catenin axis, which may be a potential target for HCC therapy.     

Growth‐dependent heterogeneity in the DNA damage response in Escherichia coli

In natural environments, bacteria are frequently exposed to sub‐lethal levels of DNA damage, which leads to the induction of a stress response (the SOS response in Escherichia coli). Natural environments also vary in nutrient availability, resulting in distinct physiological changes in bacteria, which may have direct implications on their capacity to repair their chromosomes. Here, we evaluated the impact of varying the nutrient availability on the expression of the SOS response induced by chronic sub‐lethal DNA damage in E. coli. We found heterogeneous expression of the SOS regulon at the single‐cell level in all growth conditions. Surprisingly, we observed a larger fraction of high SOS‐induced cells in slow growth as compared with fast growth, despite a higher rate of SOS induction in fast growth. The result can be explained by the dynamic balance between the rate of SOS induction and the division rates of cells exposed to DNA damage. Taken together, our data illustrate how cell division and physiology come together to produce growth‐dependent heterogeneity in the DNA damage response.     

Vitamin B6 prevents excessive inflammation by reducing accumulation of sphingosine‐1‐phosphate in a sphingosine‐1‐phosphate lyase–dependent manner

Vitamin B6 is necessary to maintain normal metabolism and immune response, especially the anti‐inflammatory immune response. However, the exact mechanism by which vitamin B6 plays the anti‐inflammatory role is still unclear. Here, we report a novel mechanism of preventing excessive inflammation by vitamin B6 via reduction in the accumulation of sphingosine‐1‐phosphate (S1P) in a S1P lyase (SPL)‐dependent manner in macrophages. Vitamin B6 supplementation decreased the expression of pro‐inflammatory cytokines by suppressing nuclear factor‐κB and mitogen‐activated protein kinases signalling pathways. Furthermore, vitamin B6–reduced accumulation of S1P by promoting SPL activity. The anti‐inflammatory effects of vitamin B6 were inhibited by S1P supplementation or SPL deficiency. Importantly, vitamin B6 supplementation protected mice from lethal endotoxic shock and attenuated experimental autoimmune encephalomyelitis progression. Collectively, these findings revealed a novel anti‐inflammatory mechanism of vitamin B6 and provided guidance on its clinical use.     

PML‐NB‐dependent type I interferon memory results in a restricted form of HSV latency

Herpes simplex virus (HSV) establishes latent infection in long‐lived neurons. During initial infection, neurons are exposed to multiple inflammatory cytokines but the effects of immune signaling on the nature of HSV latency are unknown. We show that initial infection of primary murine neurons in the presence of type I interferon (IFN) results in a form of latency that is restricted for reactivation. We also find that the subnuclear condensates, promyelocytic leukemia nuclear bodies (PML‐NBs), are absent from primary sympathetic and sensory neurons but form with type I IFN treatment and persist even when IFN signaling resolves. HSV‐1 genomes colocalize with PML‐NBs throughout a latent infection of neurons only when type I IFN is present during initial infection. Depletion of PML prior to or following infection does not impact the establishment latency; however, it does rescue the ability of HSV to reactivate from IFN‐treated neurons. This study demonstrates that viral genomes possess a memory of the IFN response during de novo infection, which results in differential subnuclear positioning and ultimately restricts the ability of genomes to reactivate.     

Purification and characterization of the receptor‐binding domain of SARS‐CoV‐2 spike protein from Escherichia coli

SARS‐CoV‐2 is responsible for a disruptive worldwide viral pandemic, and renders a severe respiratory disease known as COVID‐19. Spike protein of SARS‐CoV‐2 mediates viral entry into host cells by binding ACE2 through the receptor‐binding domain (RBD). RBD is an important target for development of virus inhibitors, neutralizing antibodies, and vaccines. RBD expressed in mammalian cells suffers from low expression yield and high cost. E. coli is a popular host for protein expression, which has the advantage of easy scalability with low cost. However, RBD expressed by E. coli (RBD‐1) lacks the glycosylation, and its antigenic epitopes may not be sufficiently exposed. In the present study, RBD‐1 was expressed by E. coli and purified by a Ni Sepharose Fast Flow column. RBD‐1 was structurally characterized and compared with RBD expressed by the HEK293 cells (RBD‐2). The secondary structure and tertiary structure of RBD‐1 were largely maintained without glycosylation. In particular, the major β‐sheet content of RBD‐1 was almost unaltered. RBD‐1 could strongly bind ACE2 with a dissociation constant (K_D) of 2.98 × 10^–8 M. Thus, RBD‐1 was expected to apply in the vaccine development, screening drugs and virus test kit.     

Effector‐dependent activation and oligomerization of plant NRC class helper NLRs by sensor NLR immune receptors Rpi‐amr3 and Rpi‐amr1

Plant pathogens compromise crop yields. Plants have evolved robust innate immunity that depends in part on intracellular Nucleotide‐binding, Leucine rich‐Repeat (NLR) immune receptors that activate defense responses upon detection of pathogen‐derived effectors. Most “sensor” NLRs that detect effectors require the activity of “helper” NLRs, but how helper NLRs support sensor NLR function is poorly understood. Many Solanaceae NLRs require NRC (NLR‐Required for Cell death) class of helper NLRs. We show here that Rpi‐amr3, a sensor NLR from Solanum americanum, detects AVRamr3 from the potato late blight pathogen, Phytophthora infestans, and activates oligomerization of helper NLRs NRC2 and NRC4 into high‐molecular‐weight resistosomes. In contrast, recognition of P. infestans effector AVRamr1 by another sensor NLR Rpi‐amr1 induces formation of only the NRC2 resistosome. The activated NRC2 oligomer becomes enriched in membrane fractions. ATP‐binding motifs of both Rpi‐amr3 and NRC2 are required for NRC2 resistosome formation, but not for the interaction of Rpi‐amr3 with its cognate effector. NRC2 resistosome can be activated by Rpi‐amr3 upon detection of AVRamr3 homologs from other Phytophthora species. Mechanistic understanding of NRC resistosome formation will underpin engineering crops with durable disease resistance.     

Structural basis for the stereospecific inhibition of the dual proline/hydroxyproline catabolic enzyme ALDH4A1 by trans‐4‐hydroxy‐L‐proline

Aldehyde dehydrogenase 4A1 (ALDH4A1) catalyzes the final steps of both proline and hydroxyproline catabolism. It is a dual substrate enzyme that catalyzes the NAD⁺‐dependent oxidations of L‐glutamate‐γ‐semialdehyde to L‐glutamate (proline metabolism), and 4‐hydroxy‐L‐glutamate‐γ‐semialdehyde to 4‐erythro‐hydroxy‐L‐glutamate (hydroxyproline metabolism). Here we investigated the inhibition of mouse ALDH4A1 by the six stereoisomers of proline and 4‐hydroxyproline using steady‐state kinetics and X‐ray crystallography. Trans‐4‐hydroxy‐L‐proline is the strongest of the inhibitors studied, characterized by a competitive inhibition constant of 0.7 mM, followed by L‐proline (1.9 mM). The other compounds are very weak inhibitors (approximately 10 mM or greater). Insight into the selectivity for L‐stereoisomers was obtained by solving crystal structures of ALDH4A1 complexed with trans‐4‐hydroxy‐L‐proline and trans‐4‐hydroxy‐D‐proline. The structures suggest that the 10‐fold greater preference for the L‐stereoisomer is due to a serine residue that hydrogen bonds to the amine group of trans‐4‐hydroxy‐L‐proline. In contrast, the amine group of the D‐stereoisomer lacks a direct interaction with the enzyme due to a different orientation of the pyrrolidine ring. These results suggest that hydroxyproline catabolism is subject to substrate inhibition by trans‐4‐hydroxy‐L‐proline, analogous to the known inhibition of proline catabolism by L‐proline. Also, drugs targeting the first enzyme of hydroxyproline catabolism, by elevating the level of trans‐4‐hydroxy‐L‐proline, may inadvertently impair proline catabolism by the inhibition of ALDH4A1.     

Cryo‐EM structures of perforin‐2 in isolation and assembled on a membrane suggest a mechanism for pore formation

Perforin‐2 (PFN2, MPEG1) is a key pore‐forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane‐bound pre‐pore complex that converts to a pore‐forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo‐electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre‐pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre‐assembled complete pre‐pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre‐pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 β‐hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre‐pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion.     

A test of frequency‐dependent selection in the evolution of a generalist phenotype

A solitary population of consumers frequently evolves to the middle of a resource gradient and an intermediate mean phenotype compared to a sympatric pair of competing species that diverge to either side via character displacement. The forces governing the distribution of phenotypes in these allopatric populations, however, are little investigated. Theory predicts that the intermediate mean phenotype of the generalist should be maintained by negative frequency‐dependent selection, whereby alternate extreme phenotypes are favored because they experience reduced competition for resources when rare. However, the theory makes assumptions that are not always met, and alternative explanations for an intermediate phenotype are possible. We provide a test of this prediction in a mesocosm experiment using threespine stickleback that are ecologically and phenotypically intermediate between the more specialized stickleback species that occur in pairs. We manipulated the frequency distribution of phenotypes in two treatments and then measured effects on a focal intermediate population. We found a slight frequency‐dependent effect on survival in the predicted direction but not on individual growth rates. This result suggests that frequency‐dependent selection might be a relatively weak force across the range of phenotypes within an intermediate population and we suggest several general reasons why this might be so. We propose that allopatric populations might often be maintained at an intermediate phenotype instead by stabilizing or fluctuating directional selection.     

Protein quality control and regulated proteolysis in the genome‐reduced organism Mycoplasma pneumoniae

Protein degradation is a crucial cellular process in all‐living systems. Here, using Mycoplasma pneumoniae as a model organism, we defined the minimal protein degradation machinery required to maintain proteome homeostasis. Then, we conditionally depleted the two essential ATP‐dependent proteases. Whereas depletion of Lon results in increased protein aggregation and decreased heat tolerance, FtsH depletion induces cell membrane damage, suggesting a role in quality control of membrane proteins. An integrative comparative study combining shotgun proteomics and RNA‐seq revealed 62 and 34 candidate substrates, respectively. Cellular localization of substrates and epistasis studies supports separate functions for Lon and FtsH. Protein half‐life measurements also suggest a role for Lon‐modulated protein decay. Lon plays a key role in protein quality control, degrading misfolded proteins and those not assembled into functional complexes. We propose that regulating complex assembly and degradation of isolated proteins is a mechanism that coordinates important cellular processes like cell division. Finally, by considering the entire set of proteases and chaperones, we provide a fully integrated view of how a minimal cell regulates protein folding and degradation.     

RNA sensing via the RIG‐I‐like receptor LGP2 is essential for the induction of a type I IFN response in ADAR1 deficiency

RNA editing by the adenosine deaminase ADAR1 prevents innate immune responses to endogenous RNAs. In ADAR1‐deficient cells, unedited self RNAs form base‐paired structures that resemble viral RNAs and inadvertently activate the cytosolic RIG‐I‐like receptor (RLR) MDA5, leading to an antiviral type I interferon (IFN) response. Mutations in ADAR1 cause Aicardi‐Goutières Syndrome (AGS), an autoinflammatory syndrome characterized by chronic type I IFN production. Conversely, ADAR1 loss and the consequent type I IFN production restricts tumor growth and potentiates the activity of some chemotherapeutics. Here, we show that another RIG‐I‐like receptor, LGP2, also has an essential role in the induction of a type I IFN response in ADAR1‐deficient human cells. This requires the canonical function of LGP2 as an RNA sensor and facilitator of MDA5‐dependent signaling. Furthermore, we show that the sensitivity of tumor cells to ADAR1 loss requires LGP2 expression. Finally, type I IFN induction in tumor cells depleted of ADAR1 and treated with some chemotherapeutics fully depends on LGP2 expression. These findings highlight a central role for LGP2 in self RNA sensing with important clinical implications.     

High diversity in Delta variant across countries revealed by genome‐wide analysis of SARS‐CoV‐2 beyond the Spike protein

The highly contagious Delta variant of SARS‐CoV‐2 has become a prevalent strain globally and poses a public health challenge around the world. While there has been extensive focus on understanding the amino acid mutations in the Delta variant’s Spike protein, the mutational landscape of the rest of the SARS‐CoV‐2 proteome (25 proteins) remains poorly understood. To this end, we performed a systematic analysis of mutations in all the SARS‐CoV‐2 proteins from nearly 2 million SARS‐CoV‐2 genomes from 176 countries/territories. Six highly prevalent missense mutations in the viral life cycle‐associated Membrane (I82T), Nucleocapsid (R203M, D377Y), NS3 (S26L), and NS7a (V82A, T120I) proteins are almost exclusive to the Delta variant compared to other variants of concern (mean prevalence across genomes: Delta = 99.74%, Alpha = 0.06%, Beta = 0.09%, and Gamma = 0.22%). Furthermore, we find that the Delta variant harbors a more diverse repertoire of mutations across countries compared to the previously dominant Alpha variant. Overall, our study underscores the high diversity of the Delta variant between countries and identifies a list of amino acid mutations in the Delta variant’s proteome for probing the mechanistic basis of pathogenic features such as high viral loads, high transmissibility, and reduced susceptibility against neutralization by vaccines.