Monocyte-driven atypical cytokine storm and aberrant neutrophil activation as key mediators of COVID-19 disease severity.

Epidemiological and clinical reports indicate that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we perform cytokine and multiplex immune profiling in COVID-19 patients. We show that hypercytokinemia in COVID-19 differs from the interferon-gamma-driven cytokine storm in macrophage activation syndrome, and is more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling shows that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Antigen presenting machinery expression is also reduced in critical disease. Furthermore, we report that neutrophils contribute to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together our findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.

Lymphocyte-independent pathways underlie the pathogenesis of murine cytomegalovirus-associated secondary haemophagocytic lymphohistiocytosis.

Haemophagocytic lymphohistiocytosis (HLH) constitutes a spectrum of immunological disorders characterized by uncontrolled immune activation and key symptoms such as fever, splenomegaly, pancytopenia, haemophagocytosis, hyperferritinaemia and hepatitis. In genetic or primary HLH, hyperactivated CD8+ T cells are the main drivers of pathology. However, in acquired secondary HLH, the role of lymphocytes remains vague. In the present study the involvement of lymphocytes in the pathogenesis of a cytomegalovirus-induced model of secondary HLH was explored. We have previously reported CD8+ T cells to be redundant in this model, and therefore focused on CD4+ helper and regulatory T cells. CD4+ T cells were activated markedly and skewed towards a proinflammatory T helper type 1 transcription profile in mice displaying a severe and complete HLH phenotype. Counter to expectations, regulatory T cells were not reduced in numbers and were, in fact, more activated. Therapeutic strategies targeting CD25high hyperactivated T cells were ineffective to alleviate disease, indicating that T cell hyperactivation is not a pathogenic factor in cytomegalovirus-induced murine HLH. Moreover, even though T cells were essential in controlling viral proliferation, CD4+ T cells, in addition to CD8+ T cells, were dispensable in the development of the HLH-like syndrome. In fact, no T or B cells were required for induction and propagation of HLH disease, as evidenced by the occurrence of cytomegalovirus-associated HLH in severe combined immunodeficient (SCID) mice. These data suggest that lymphocyte-independent mechanisms can underlie virus-associated secondary HLH, accentuating a clear distinction with primary HLH.

The sheathed flagellum of Brucella melitensis is involved in persistence in a murine model of infection.

Persistence infection is the keystone of the ruminant and human diseases called brucellosis and Malta fever, respectively, and is linked to the intracellular tropism of Brucella spp. While described as non-motile, Brucella spp. have all the genes except the chemotactic system, necessary to assemble a functional flagellum. We undertook to determine whether these genes are expressed and are playing a role in some step of the disease process. We demonstrated that in the early log phase of a growth curve in 2YT nutrient broth, Brucella melitensis expresses genes corresponding to the basal (MS ring) and the distal (hook and filament) parts of the flagellar apparatus. Under these conditions, a polar and sheathed flagellar structure is visible by transmission electron microscopy (TEM). We evaluated the effect of mutations in flagellar genes of B. melitensis encoding various parts of the structure, MS ring, P ring, motor protein, secretion apparatus, hook and filament. None of these mutants gave a discernible phenotype as compared with the wild-type strain in cellular models of infection. In contrast, all these mutants were unable to establish a chronic infection in mice infected via the intraperitoneal route, raising the question of the biological role(s) of this flagellar appendage.

Fun stories about Brucella: the "furtive nasty bug".

Although Brucella is responsible for one of the major worldwide zoonosis, our understanding of its pathogenesis remains in its infancy. In this paper, we summarize some of the research in progress in our laboratory that we think could contribute to a better understanding of the Brucella molecular virulence mechanisms and their regulation.

Studies on the potential of microparticles entrapping pDNA-poly(aminoacids) complexes as vaccine delivery systems.

Poly(D,L-lactide-co-glycolide) (PLGA) microparticles containing plasmid DNA (pDNA) have potential uses as vaccine delivery systems. Nevertheless, the established double emulsion and solvent evaporation method used to produce them is characterised by a low encapsulation efficiency (about 20%) and nicks the supercoiled DNA. The aim of this work was to develop an encapsulation process to optimise the overall encapsulation efficiency and the supercoiled DNA content, to obtain a carrier suitable for mucosal delivery of DNA vaccines. Our strategy was to reduce the global negative charge of DNA which was unfavourable to its incorporation into the polymer by condensing it with cationic poly(aminoacids) which were previously reported to improve cell transfection. In this study, after characterisation of the compaction of DNA plasmid encoding for a Green Fluorescent Protein, we demonstrated that resulting complexes were successfully encapsulated into PLGA microparticles presenting a mean size around 4.5 microns. The preliminary step of complexation enhances the yield of the process by a factor 4.1 and protects the supercoiled form. In a bacteria transformation assay, we demonstrated that extracted pDNA (naked or complexed) remained in a transcriptionally active form after encapsulation. Bovine macrophages in culture phagocytosed microparticles loaded with uncomplexed/complexed with poly(L-lysine) pDNA. The production of the Green Fluorescent Protein demonstrated that these carriers could deliver intact and functional plasmid DNA probably by escaping from lysosomal degradation.

Crystallographic analysis of family 11 endo-beta-1,4-xylanase Xyl1 from Streptomyces sp. S38.

Family 11 endo-beta-1,4-xylanases degrade xylan, the main constituent of plant hemicelluloses, and have many potential uses in biotechnology. The structure of Xyl1, a family 11 endo-xylanase from Streptomyces sp. S38, has been solved. The protein crystallized from ammonium sulfate in the trigonal space group P321, with unit-cell parameters a = b = 71.49, c = 130.30 A, gamma = 120.0 degrees. The structure was solved at 2.0 A by X-ray crystallography using the molecular-replacement method and refined to a final R factor of 18.5% (R(free) = 26.9%). Xyl1 has the overall fold characteristic of family 11 xylanases, with two highly twisted beta-sheets defining a long cleft containing the two catalytic residues Glu87 and Glu177.

Open-reading-frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans.

The genome sequences of Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana have been predicted to contain 19,000, 13,600 and 25,500 genes, respectively. Before this information can be fully used for evolutionary and functional studies, several issues need to be addressed. First, the gene number estimates obtained in silico and not yet supported by any experimental data need to be verified. For example, it seems biologically paradoxical that C. elegans would have 50% more genes than Drosophilia. Second, intron/exon predictions need to be tested experimentally. Third, complete sets of open reading frames (ORFs), or "ORFeomes," need to be cloned into various expression vectors. To address these issues simultaneously, we have designed and applied to C. elegans the following strategy. Predicted ORFs are amplified by PCR from a highly representative cDNA library using ORF-specific primers, cloned by Gateway recombination cloning and then sequenced to generate ORF sequence tags (OSTs) as a way to verify identity and splicing. In a sample (n=1,222) of the nearly 10,000 genes predicted ab initio (that is, for which no expressed sequence tag (EST) is available so far), at least 70% were verified by OSTs. We also observed that 27% of these experimentally confirmed genes have a structure different from that predicted by GeneFinder. We now have experimental evidence that supports the existence of at least 17,300 genes in C. elegans. Hence we suggest that gene counts based primarily on ESTs may underestimate the number of genes in human and in other organisms.

Specificity of Cdk activation in vivo by the two Caks Mcs6 and Csk1 in fission yeast.

Activating phosphorylation of cyclin-dependent kinases (Cdks) is mediated by at least two structurally distinct types of Cdk-activating kinases (Caks): the trimeric Cdk7-cyclin H-Mat1 complex in metazoans and the single-subunit Cak1 in budding yeast. Fission yeast has both Cak types: Mcs6 is a Cdk7 ortholog and Csk1 a single-subunit kinase. Both phosphorylate Cdks in vitro and rescue a thermosensitive budding yeast CAK1 strain. However, this apparent redundancy is not observed in fission yeast in vivo. We have identified mutants that exhibit phenotypes attributable to defects in either Mcs6-activating phosphorylation or in Cdc2-activating phosphorylation. Mcs6, human Cdk7 and budding yeast Cak1 were all active as Caks for Cdc2 when expressed in fission yeast. Although Csk1 could activate Mcs6, it was unable to activate Cdc2. Biochemical experiments supported these genetic results: budding yeast Cak1 could bind and phosphorylate Cdc2 from fission yeast lysates, whereas fission yeast Csk1 could not. These results indicate that Mcs6 is the direct activator of Cdc2, and Csk1 only activates Mcs6. This demonstrates in vivo specificity in Cdk activation by Caks.

Differences in regulation of yeast gluconeogenesis revealed by Cat8p-independent activation of PCK1 and FBP1 genes in Kluyveromyces lactis.

The yeast Kluyveromyces lactis is can utilise a wide range of non-fermentable carbon compounds as sole sources of carbon and energy, and differs from Saccharomyces cerevisiae in being able to carry out oxidative and fermentative metabolism simultaneously. In S. cerevisiae, growth on all non-fermentable carbon sources requires Cat8p, a transcriptional activator that controls the expression of gluconeogenic and glyoxylate cycle genes via CSREs (Carbon Source Responsive Elements). The down-regulation of Cat8p by fermentable carbon sources is the primary factor responsible for the tight repression of gluconeogenesis by glucose in S. cerevisiae. To analyse the regulation of gluconeogenesis in K. lactis, we have cloned and characterised the K. lactis homologue of CAT8 (KlCAT8). The gene was isolated by multicopy suppression of a fog2/klsnf1 mutation, indicating a similar epistatic relationship between KlSNF1 and KlCAT8 as in the case of the S. cerevisiae homologues. KlCAT8 encodes a protein of 1445 amino acids that is 40% identical to ScCat8p. The most highly conserved block is the putative Zn(II)2Cys6 DNA-binding domain, but additional conserved regions shared with members of the zinc-cluster family from Aspergillus define a subfamily of Cat8p-related proteins. KlCAT8 complements the growth defect of a Sccat8 mutant on non-fermentable carbon sources. In K. lactis, deletion of KlCAT8 severely impairs growth on ethanol, acetate and lactate, but not on glycerol. Derepression of enzymes of the glyoxylate cycle--malate synthase and particularly isocitrate lyase--was impaired in a Klcat8 mutant, whereas Northern analysis revealed that derepression of KlFBP1 and KlPCK1 does not require KlCat8p. Taken together, our results indicate that in K. lactis gluconeogenesis is not co-regulated with the glyoxylate cycle, and only the latter is controlled by KlCat8p.

Kluyveromyces marxianus exhibits an ancestral Saccharomyces cerevisiae genome organization downstream of ADH2.

In Saccharomyces cerevisiae, the alcohol dehydrogenase genes ADH1 and ADH5 are part of a duplicated block of genome, thought to originate from a genome-wide duplication posterior to the divergence from the Kluyveromyces lineage. We report here the characterization of Kluyveromyces marxianus ADH2 and the five genes found in its immediate downstream region, MRPS9, YOL087C, RPB5, RIB7 and SPP381. The order of these six genes reflects the structure of the ancestral S. cerevisiae genome before the duplication that formed the blocks including ADH1 on chromosome XV and ADH5 on chromosome II, indicating these ADH genes share a direct ancestor. On the one hand, the two genes found immediately downstream of KmADH2 are located, for the first, downstream ADH5 and, for the second, downstream ADH1 in S. cerevisiae. On the other hand, the order of the paralogs included in the blocks of ADH1 and ADH5 in S. cerevisiae suggests that two of them have been inverted within one block after its formation, and that inversion is confirmed by the gene order observed in K. marxianus.

Antiglucocorticoid activity of hepatocyte nuclear factor-6.

Glucocorticoids exert their effects on gene transcription through ubiquitous receptors that bind to regulatory sequences present in many genes. These glucocorticoid receptors are present in all cell types, yet glucocorticoid action is controlled in a tissue-specific way. One mechanism for this control relies on tissue-specific transcriptional activators that bind in the vicinity of the glucocorticoid receptor and are required for receptor action. We now describe a gene-specific and tissue-specific inhibitory mechanism through which glucocorticoid action is repressed by a tissue-restricted transcription factor, hepatocyte nuclear factor-6 (HNF-6). HNF-6 inhibits the glucocorticoid-induced stimulation of two genes coding for enzymes of liver glucose metabolism, namely 6-phosphofructo-2-kinase and phosphoenolpyruvate carboxykinase. Binding of HNF-6 to DNA is required for inhibition of glucocorticoid receptor activity. In vitro and in vivo experiments suggest that this inhibition is mediated by a direct HNF-6/glucocorticoid receptor interaction involving the amino-terminal domain of HNF-6 and the DNA-binding domain of the receptor. Thus, in addition to its known property of stimulating transcription of liver-expressed genes, HNF-6 can antagonize glucocorticoid-stimulated gene transcription.

Glucose repression of the Kluyveromyces lactis invertase gene KlINV1 does not require Mig1p.

Kluyveromyces lactis, a budding yeast related to Saccharomyces cerevisiae, can grow on a wider variety of substrates and shows less sensitivity to glucose repression than does Saccharomyces cerevisiae. Many genes that are subject to glucose repression in S. cerevisiae are repressed only weakly or not at all in K. lactis. The molecular basis for this difference is largely unknown. To compare the mechanisms that regulate glucose repression in K. lactis and S. cerevisiae, we decided to clone and analyse an invertase gene from K. lactis. The SUC2 gene, which encodes invertase in S. cerevisiae, is strongly regulated by glucose and serves as a model system for studies on glucose repression. The invertase gene of K. lactis, KlINV1, was isolated by colony hybridization using a conserved region within the inulinase gene of K. marxianus as a probe. Two independent clones obtained were shown to contain the same ORF of 1827 bp. The deduced amino acid sequence is 59% similar to that of the K. marxianus inulinase and shows 49% similarity to ScSuc2p. Gene disruption experiments and low-stringency Southern analysis indicate that KlINV1 is a unique gene in K. lactis. Northern analysis revealed that the transcription of KlINV1 is strongly repressed in the presence of glucose, but, in contrast to the case in S. cerevisiae, repression is independent of KlMig1p.

Purification and characterization of Aspergillus ficuum endoinulinase.

Endoinulinase from Aspergillus ficuum, which catalyzes the hydrolysis of inulin via an endo-cleavage mode, was purified by chromatography from Novozym 230 as a starting commercial enzyme mixture on CM-Sephadex and DEAE-Sepharose, and by preparative electrophoresis under native conditions. The enzyme was estimated to be pure on the basis of its I/S ratio, whose value was infinite in our assay conditions. Two forms separated by using this method. SDS gel electrophoresis showed the two purified forms to respectively exhibit molecular weights of 64,000 +/- 500 and 66,000 +/- 1,000. The results of deglycosylation indicated that the two forms were originally the same protein but with different sugar contents. A molecular weight of 54,800 +/- 1,500 was found by gel filtration of the native enzyme, indicating the native functional protein to be a monomer. The enzyme showed nearly absolute substrate specificity towards inulin and inulooligosaccharides, and acted via an endo-attack to produce mainly inulotriose during the late stage of the reaction. The apparent Km and Vmax values for inulin hydrolysis were 8.1 +/- 1.0 mM and 773 +/- 60 U/mg, respectively. The internal peptides of the enzyme showed sequence homology to the endoinulinase of Penicillium purpurogenum.

Fission yeast Csk1 is a CAK-activating kinase (CAKAK).

Cell cycle progression is dependent on the sequential activity of cyclin-dependent kinases (CDKs). For full activity, CDKs require an activating phosphorylation of a conserved residue (corresponding to Thr160 in human CDK2) carried out by the CDK-activating kinase (CAK). Two distinct CAK kinases have been described: in budding yeast Saccharomyces cerevisiae, the Cak1/Civ1 kinase is responsible for CAK activity. In several other species including human, Xenopus, Drosophila and fission yeast Schizosaccharomyces pombe, CAK has been identified as a complex homologous to CDK7-cyclin H (Mcs6-Mcs2 in fission yeast). Here we identify the fission yeast Csk1 kinase as an in vivo activating kinase of the Mcs6-Mcs2 CAK defining Csk1 as a CAK-activating kinase (CAKAK).

Four hydrophobic amino acid residues in the C-terminal effector domain of the yeast Mig1p repressor are important for its in vivo activity.

The Mig1 repressor is a zinc finger protein that mediates glucose repression in yeast. Previous work in Saccharomyces cerevisiae has shown that two domains in Miglp are required for repression: the N-terminal zinc finger region and a C-terminal effector domain. Both domains are also conserved in Miglp homologs from the distantly related yeasts Kluyveromyces lactis and K. marxianus, and these Mig1 proteins can fully replace the endogenous Mig1p in S. cerevisiae. We have now made a detailed analysis of the conserved C-terminal effector domain in Mig1p from K. marxianus, using expression in S. cerevisiae to monitor its function. First, a series of small deletions were made within the effector domain. Second, an alanine scan mutagenesis was carried out across the effector domain. Third, double, triple and quadruple mutants were made that affect certain residues within the effector domain. Our results show that four conserved residues within the effector domain, three leucines and one isoleucine, are particularly important for its function in vivo. The analysis further revealed that while the C-terminal effector domain of KmMig1p mediates a seven- to nine-fold repression of the reporter gene, a five- to sixfold residual effect also exists that is independent of the C-terminal effector domain. Similar results were obtained when the corresponding mutations were made in ScMig1p. Moreover, we found that mutations in these residues affect the interaction between Mig1p and the general corepressor subunit Cyc8p (Ssn6p). Modeling of the C-terminal effector domain using a protein of known structure suggests that it may be folded into an alpha-helix.

Cloning and characterization of the KlDIM1 gene from Kluyveromyces lactis encoding the m2(6)A dimethylase of the 18S rRNA.

The KlDIM1 gene encoding the m2(6)A rRNA dimethylase was cloned from a Kluyveromyces lactis genomic library using a PCR amplicon from the Saccharomyces cerevisiae ScDIM1 gene as probe. The KlDIM1 gene encodes a 320-amino acid protein which shows 81% identity to ScDim1p from S. cerevisiae and 25% identity to ksgAp from Escherichia coli. Complementation of the kasugamycin-resistant ksgA-mutant of E. coli lacking dimethylase activity demonstrates that KlDim1p is the functional homologue of the bacterial enzyme. Multiple alignment of dimethylases from prokaryotes and yeasts shows that the two yeast enzymes display distinctive structural motives including a putative nuclear localization signal.

Comparative analysis in three fungi reveals structurally and functionally conserved regions in the Mig1 repressor.

The Mig1 repressor is a key effector in glucose repression in the yeast Saccharomyces cerevisiae. To gain further insights into structure-function relationships, we have now cloned the MIG1 homologue from the yeast Kluyveromyces marxianus. The amino acid sequence deduced from KmMIG1 differs significantly from ScMig1p outside the highly conserved zinc fingers. However, 12 discrete conserved motifs could be identified in a multiple alignment that also included the K. lactis Mig1p sequence. We further found that KmMig1p is fully functional when expressed in S. cerevisiae. First, it represses the SUC2 promoter almost as well as ScMig1p. This repression requires the Cyc8 and Tup1 proteins and is dependent on a C-terminal region comprising several conserved leucine-proline repeats. Second, KmMig1p is regulated by glucose in S. cerevisiae, and a KmMig1-VP16 hybrid activator is inhibited by the ScSnf1p kinase in the absence of glucose. This suggests that KmMig1p has retained the ability to interact with several S. cerevisiae proteins, and reinforces the notion that the conserved motifs are functionally important. Finally, we found that the physiological role of Mig1p also is conserved in K. marxianus, since KmMig1p represses INU1, the counterpart of SUC2 in this organism.

The 18S rRNA dimethylase Dim1p is required for pre-ribosomal RNA processing in yeast.

The m6(2)A1779m6(2)A1780 dimethylation at the 3' end of the small subunit rRNA has been conserved in evolution from bacteria to eukaryotes. The yeast 18S rRNA dimethylase gene DIM1 was cloned previously by complementation in Escherichia coli and shown to be essential for viability in yeast. A conditional GAL10::dim1 strain was constructed to allow the depletion of Dim1p from the cell. During depletion, dimethylation of the pre-rRNA is progressively inhibited and pre-rRNA processing at cleavage sites A1 and A2 is concomitantly lost. In consequence, the mature 18S rRNA and its 20S precursor drastically underaccumulate. This has the effect of preventing the synthesis of nonmethylated rRNA. To test whether the processing defect is a consequence of the absence of the dimethylated nucleotides or of the Dim1p dimethylase itself, a cis-acting mutation was created in which both dimethylated adenosines are replaced by guanosine residues. Methylation cannot occur on this mutant pre-rRNA, but no clear pre-rRNA processing defect is seen. Moreover, methylation of the wild-type pre-rRNA predominantly occurs after cleavage at sites A1 and A2. This shows that formation of the m6(2)A1779m6(2)A1780 dimethylation is not required for pre-rRNA processing. We propose that the binding of Dim1p to the pre-ribosomal particle is monitored to ensure that only dimethylated pre-rRNA molecules are processed to 18S rRNA.

The MIG1 repressor from Kluyveromyces lactis: cloning, sequencing and functional analysis in Saccharomyces cerevisiae.

Sequence comparisons between Saccharomyces cerevisiae ScMig1 and Aspergillus nidulans CREA proteins allowed us to design two sets of degenerate primers from the conserved zinc finger loops. PCR amplification on Kluyveromyces marxianus and K. lactis genomic DNA yielded single products with sequences closely related to each other and to the corresponding regions of ScMig1 and CREA. The KIMIG1 gene of K. lactis was cloned from a genomic library using the K. marxianus PCR fragment as probe. KIMIG1 encodes a 474-amino acid protein 55% similar to ScMig1. Besides their highly conserved zinc fingers, the two proteins display short conserved motifs of possible significance in glucose repression. Heterologous complementation of a mig1 mutant of S. cerevisiae by the K. lactis gene demonstrates that the function of the Mig1 protein is conserved in these two distantly related yeasts.

Identification of residues potentially involved in the interactions between subunits in yeast alcohol dehydrogenases.

The lack of crystal structure for tetrameric yeast alcohol dehydrogenases (ADHs) has precluded, until now, the identification of the residues involved in subunit contacts. In order to address this question, we have characterized the thermal stability and dissociation propensity of native ADH I and ADH II isozymes as well as of several chimeric (ADH I-ADH II) enzymes. Three groups of substitutions affecting the thermostability have been identified among the 24 substitutions observed between isozymes I and II. The first group contains a Cys277-->Ser substitution, located at the interface between subunits in a three-dimensional model of ADH I, based on the crystallographic structure of the dimeric horse liver ADH. In the second group, the Asp236-->Asn substitution is located in the same interaction zone on the model. The stabilizing effect of this substitution can result from the removal of a charge repulsion between subunits. It is shown that the effect of these two groups of substitutions correlates with changes in dissociation propensities. The third group contains the Met168-->Arg substitution that increases the thermal stability, probably by the formation of an additional salt bridge between subunits through the putative interface. These data suggest that at least part of the subunit contacts observed in horse liver ADH are located at homologous positions in yeast ADHs.