Microbiology
Functional characterization of Vip3Aa from Bacillus thuringiensis reveals the contributions of specific domains to its insecticidal activity
Microbially derived, protein-based biopesticides offer a more sustainable pest management alternative to synthetic pesticides. Vegetative insecticidal proteins (Vip3), multidomain proteins secreted by Bacillus thuringiensis, represent a second-generation insecticidal toxin that has been preliminarily used in transgenic crops. However, the molecular mechanism underlying Vip3’s toxicity is poorly understood. Here, we determine the distinct functions and contributions of the domains of the Vip3Aa protein to its toxicity against Spodoptera frugiperda larvae.The main protease of SARS-CoV-2 cleaves histone deacetylases and DCP1A, attenuating the immune defense of the interferon-stimulated genes
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019, constitutes an emerging human pathogen of zoonotic origin. A critical role in protecting the host against invading pathogens is carried out by interferon-stimulated genes (ISGs), the primary effectors of the type I interferon (IFN) response. All coronaviruses studied thus far have to first overcome the inhibitory effects of the IFN/ISG system before establishing efficient viral replication. However, whether SARS-CoV-2 evades IFN antiviral immunity by manipulating ISG activation remains to be elucidated.Residues E53, L55, H59, and G70 of the cellular receptor protein Tva mediate cell binding and entry of the novel subgroup K avian leukosis virus
Subgroup K avian leukosis virus (ALV-K) is a novel subgroup of ALV isolated from Chinese native chickens. As for a retrovirus, the interaction between its envelope protein and cellular receptor is a crucial step in ALV-K infection. Tva, a protein previously determined to be associated with vitamin B12/cobalamin uptake, has been identified as the receptor of ALV-K. However, the molecular mechanism underlying the interaction between Tva and the envelope protein of ALV-K remains unclear. In this study, we identified the C-terminal loop of the LDL-A module of Tva as the minimal functional domain that directly interacts with gp85, the surface component of the ALV-K envelope protein.Lanpepsy is a novel lanthanide-binding protein involved in the lanthanide response of the obligate methylotroph Methylobacillus flagellatus
Lanthanides were recently discovered as metals required in the active site of certain methanol dehydrogenases. Since then, the characterization of the lanthanome, that is, proteins involved in sensing, uptake, and utilization of lanthanides, has become an active field of research. Initial exploration of the response to lanthanides in methylotrophs has revealed that the lanthanome is not conserved and that multiple mechanisms for lanthanide utilization must exist. Here, we investigated the lanthanome in the obligate model methylotroph Methylobacillus flagellatus.The unique N-terminal region of Mycobacterium tuberculosis sigma factor A plays a dominant role in the essential function of this protein
SigA (σA) is an essential protein and the primary sigma factor in Mycobacterium tuberculosis (Mtb). However, due to the absence of genetic tools, our understanding of the role and regulation of σA activity and its molecular attributes that help modulate Mtb survival is scant. Here, we generated a conditional gene replacement of σA in Mtb and showed that its depletion results in a severe survival defect in vitro, ex vivo, and in vivo in a murine infection model. Our RNA-seq analysis suggests that σA either directly or indirectly regulates ∼57% of the Mtb transcriptome, including ∼28% of essential genes.Enolpyruvate transferase MurAAA149E, identified during adaptation of Enterococcus faecium to daptomycin, increases stability of MurAA–MurG interaction
Daptomycin (DAP) is an antibiotic frequently used as a drug of last resort against vancomycin-resistant enterococci. One of the major challenges when using DAP against vancomycin-resistant enterococci is the emergence of resistance, which is mediated by the cell-envelope stress system LiaFSR. Indeed, inhibition of LiaFSR signaling has been suggested as a strategy to “resensitize” enterococci to DAP. In the absence of LiaFSR, alternative pathways mediating DAP resistance have been identified, including adaptive mutations in the enolpyruvate transferase MurAA (MurAAA149E), which catalyzes the first committed step in peptidoglycan biosynthesis; however, how these mutations confer resistance is unclear.MceG stabilizes the Mce1 and Mce4 transporters in Mycobacterium tuberculosis
Lipids are important nutrients for Mycobacterium tuberculosis (Mtb) to support bacterial survival in mammalian tissues and host cells. Fatty acids and cholesterol are imported across the Mtb cell wall via the dedicated Mce1 and Mce4 transporters, respectively. It is thought that the Mce1 and Mce4 transporters are comprised of subunits that confer substrate specificity and proteins that couple lipid transport to ATP hydrolysis, similar to other bacterial ABC transporters. However, unlike canonical bacterial ABC transporters, Mce1 and Mce4 appear to share a single ATPase, MceG.The crystal structure of the EspB-EspK virulence factor-chaperone complex suggests an additional type VII secretion mechanism in Mycobacterium tuberculosis
Pathogenic species from the Mycobacterium genus are responsible for a number of adverse health conditions in humans and animals that threaten health security and the economy worldwide. Mycobacteria have up to five specialized secretion systems (ESX-1 to ESX-5) that transport virulence factors across their complex cell envelope to facilitate manipulation of their environment. In pathogenic species, these virulence factors influence the immune system’s response and are responsible for membrane disruption and contributing to cell death.An evolutionarily conserved N-terminal leucine is essential for MX1 GTPase antiviral activity against different families of RNA viruses
Myxovirus resistance protein 1 (MX1) and MX2 are homologous, dynamin-like large GTPases, induced upon interferon exposure. Human MX1 (HsMX1) is known to inhibit many viruses, including influenza A virus, by likely acting at various steps of their life cycles. Despite decades of studies, the mechanism(s) of action with which MX1 proteins manage to inhibit target viruses is not fully understood. MX1 proteins are mechano-enzymes and share a similar organization to dynamin, with a GTPase domain and a carboxy-terminal stalk domain, connected by a bundle signaling element.Toll-like receptor 4 and lipopolysaccharide from commensal microbes regulate Tembusu virus infection
Unlike most flaviviruses transmitted by arthropods, Tembusu virus (TMUV) is still active during winter and causes outbreaks in some areas, indicating vector-independent spread of the virus. Gastrointestinal transmission might be one of the possible routes of vector-free transmission, which also means that the virus has to interact with more intestinal bacteria. Here, we found evidence that TMUV indeed can transmit through the digestive tract. Interestingly, using an established TMUV disease model by oral gavage combined with an antibiotic treatment, we revealed that a decrease in intestinal bacteria significantly reduced local TMUV proliferation in the intestine, revealing that the bacterial microbiome is important in TMUV infection.Ornate, large, extremophilic (OLE) RNA forms a kink turn necessary for OapC protein recognition and RNA function
Ornate, large, extremophilic (OLE) RNAs represent a class of noncoding RNAs prevalent in Gram-positive, extremophilic/anaerobic bacterial species. OLE RNAs (∼600 nt), whose precise biochemical functions remain mysterious, form an intricate secondary structure interspersed with regions of highly conserved nucleotides. In the alkali-halophilic bacterium Bacillus halodurans, OLE RNA is a component of a ribonucleoprotein (RNP) complex involving at least two proteins named OapA and OapB, but additional components may exist that could point to functional roles for the RNA.Herpes simplex virus 1 protein pUL21 alters ceramide metabolism by activating the interorganelle transport protein CERT
Herpes simplex virus (HSV)-1 dramatically alters the architecture and protein composition of cellular membranes during infection, but its effects upon membrane lipid composition remain unclear. HSV-1 pUL21 is a virus-encoded protein phosphatase adaptor that promotes dephosphorylation of multiple cellular and virus proteins, including the cellular ceramide (Cer) transport protein CERT. CERT mediates nonvesicular Cer transport from the endoplasmic reticulum to the trans-Golgi network, whereupon Cer is converted to sphingomyelin (SM) and other sphingolipids that play important roles in cellular proliferation, signaling, and membrane trafficking.Tick-borne encephalitis virus capsid protein induces translational shutoff as revealed by its structural–biological analysis
Tick-borne encephalitis virus (TBEV) is the most medically relevant tick-transmitted Flavivirus in Eurasia, targeting the host central nervous system and frequently causing severe encephalitis. The primary function of its capsid protein (TBEVC) is to recruit the viral RNA and form a nucleocapsid. Additional functionality of Flavivirus capsid proteins has been documented, but further investigation is needed for TBEVC. Here, we show the first capsid protein 3D structure of a member of the tick-borne flaviviruses group.Inner membrane YfgM–PpiD heterodimer acts as a functional unit that associates with the SecY/E/G translocon and promotes protein translocation
PpiD and YfgM are inner membrane proteins that are both composed of an N-terminal transmembrane segment and a C-terminal periplasmic domain. Escherichia coli YfgM and PpiD form a stable complex that interacts with the SecY/E/G (Sec) translocon, a channel that allows protein translocation across the cytoplasmic membrane. Although PpiD is known to function in protein translocation, the functional significance of PpiD–YfgM complex formation as well as the molecular mechanisms of PpiD–YfgM and PpiD/YfgM–Sec translocon interactions remain unclear.Reconstitution of the SARS-CoV-2 ribonucleosome provides insights into genomic RNA packaging and regulation by phosphorylation
The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 is responsible for compaction of the ∼30-kb RNA genome in the ∼90-nm virion. Previous studies suggest that each virion contains 35 to 40 viral ribonucleoprotein (vRNP) complexes, or ribonucleosomes, arrayed along the genome. There is, however, little mechanistic understanding of the vRNP complex. Here, we show that N protein, when combined in vitro with short fragments of the viral genome, forms 15-nm particles similar to the vRNP structures observed within virions.Isocitrate binds to the itaconic acid–responsive LysR-type transcriptional regulator RipR in Salmonella pathogenesis
Macrophages produce itaconic acid in phagosomes in response to bacterial cell wall component lipopolysaccharide to eliminate invading pathogenic bacteria. Itaconic acid competitively inhibits the first enzyme of the bacterial glyoxylate cycle. To overcome itaconic acid stress, bacteria employ the bacterial LysR-type transcriptional regulator RipR. However, it remains unknown which molecule activates RipR in bacterial pathogenesis. In this study, we determined the crystal structure of the regulatory domain of RipR from the intracellular pathogen Salmonella.Outer surface lipoproteins from the Lyme disease spirochete exploit the molecular switch mechanism of the complement protease C1s
Proteolytic cascades comprise several important physiological systems, including a primary arm of innate immunity called the complement cascade. To safeguard against complement-mediated attack, the etiologic agent of Lyme disease, Borreliella burgdorferi, produces numerous outer surface–localized lipoproteins that contribute to successful complement evasion. Recently, we discovered a pair of B. burgdorferi surface lipoproteins of the OspEF-related protein family—termed ElpB and ElpQ—that inhibit antibody-mediated complement activation.Inositol phosphorylceramide synthase null Leishmania are viable and virulent in animal infections where salvage of host sphingomyelin predominates
Many pathogens synthesize inositol phosphorylceramide (IPC) as the major sphingolipid (SL), differing from the mammalian host where sphingomyelin (SM) or more complex SLs predominate. The divergence between IPC synthase and mammalian SL synthases has prompted interest as a potential drug target. However, in the trypanosomatid protozoan Leishmania, cultured insect stage promastigotes lack de novo SL synthesis (Δspt2-) and SLs survive and remain virulent, as infective amastigotes salvage host SLs and continue to produce IPC.A dedicated C-6 β-hydroxyacyltransferase required for biosynthesis of the glycolipid anchor for Vi antigen capsule in typhoidal Salmonella
Vi antigen is an extracellular polysaccharide produced by Salmonella enterica Typhi, Citrobacter freundii, and some soil bacteria belonging to the Burkholderiales. In Salmonella Typhi, Vi-antigen capsule protects the bacterium against host defenses, and the glycan is used in a current glycoconjugate vaccine to protect against typhoid. Vi antigen is a glycolipid assembled in the cytoplasm and translocated to the cell surface by an export complex driven by an ABC transporter. In Salmonella Typhi, efficient export and cell-surface retention of the capsule layer depend on a reducing terminal acylated-HexNAc moiety.Serine 13 of the human cytomegalovirus viral cyclin-dependent kinase UL97 is required for regulatory protein 14-3-3 binding and UL97 stability
The human cytomegalovirus (HCMV) UL97 protein is a conserved herpesvirus protein kinase (CHPK) and a viral cyclin-dependent kinase (v-CDK). However, mechanisms regulating its activity in the context of infection are unknown. Here, we identified several cellular regulatory 14-3-3 proteins as UL97-interacting partners that promote UL97 stability. Humans are known to encode seven isoforms of 14-3-3 proteins (β, ε, η, γ, σ, θ, and ζ) that bind phosphoserines or phosphothreonines to impact protein structure, stability, activity, and localization.SARS-CoV-2 hijacks macropinocytosis to facilitate its entry and promote viral spike–mediated cell-to-cell fusion
Revealing the mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry and cell-to-cell spread might provide insights for understanding the underlying mechanisms of viral pathogenesis, tropism, and virulence. The signaling pathways involved in SARS-CoV-2 entry and viral spike–mediated cell-to-cell fusion remain elusive. In the current study, we found that macropinocytosis inhibitors significantly suppressed SARS-CoV-2 infection at both the entry and viral spike–mediated cell-to-cell fusion steps.Coprinopsis cinerea dioxygenase is an oxygenase forming 10(S)-hydroperoxide of linoleic acid, essential for mushroom alcohol, 1-octen-3-ol, synthesis
1-Octen-3-ol is a volatile oxylipin found ubiquitously in Basidiomycota and Ascomycota. The biosynthetic pathway forming 1-octen-3-ol from linoleic acid via the linoleic acid 10(S)-hydroperoxide was characterized 40 years ago in mushrooms, yet the enzymes involved are not identified. The dioxygenase 1 and 2 genes (Ccdox1 and Ccdox2) in the mushroom Coprinopsis cinerea contain an N-terminal cyclooxygenase-like heme peroxidase domain and a C-terminal cytochrome P450-related domain. Herein, we show that recombinant CcDOX1 is responsible for dioxygenation of linoleic acid to form the 10(S)-hydroperoxide, the first step in 1-octen-3-ol synthesis, whereas CcDOX2 conceivably forms linoleic acid 8-hydroperoxide.The SET-domain protein CgSet4 negatively regulates antifungal drug resistance via the ergosterol biosynthesis transcriptional regulator CgUpc2a
Invasive fungal infections, which pose a serious threat to human health, are increasingly associated with a high mortality rate and elevated health care costs, owing to rising resistance to current antifungals and emergence of multidrug-resistant fungal species. Candida glabrata is the second to fourth common cause of Candida bloodstream infections. Its high propensity to acquire resistance toward two mainstream drugs, azoles (inhibit ergosterol biosynthesis) and echinocandins (target cell wall), in clinical settings, and its inherent low azole susceptibility render antifungal therapy unsuccessful in many cases.The Natural Product Domain Seeker version 2 (NaPDoS2) webtool relates ketosynthase phylogeny to biosynthetic function
The Natural Product Domain Seeker (NaPDoS) webtool detects and classifies ketosynthase (KS) and condensation domains from genomic, metagenomic, and amplicon sequence data. Unlike other tools, a phylogeny-based classification scheme is used to make broader predictions about the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes in which these domains are found. NaPDoS is particularly useful for the analysis of incomplete biosynthetic genes or gene clusters, as are often observed in poorly assembled genomes and metagenomes, or when loci are not clustered, as in eukaryotic genomes.A conserved loop sequence of the proteasome system depupylase Dop regulates substrate selectivity in Mycobacterium tuberculosis
Mycobacteria use a proteasome system that is similar to a eukaryotic proteasome but do not use ubiquitin to target proteins for degradation. Instead, mycobacteria encode a prokaryotic ubiquitin-like protein (Pup) that posttranslationally modifies proteins to mark them for proteolysis. Pupylation occurs on lysines of targeted proteins and is catalyzed by the ligase PafA. Like ubiquitylation, pupylation can be reversed by the depupylase Dop, which shares high structural similarity with PafA. Unique to Dop near its active site is a disordered loop of approximately 40 amino acids that is highly conserved among diverse dop-containing bacterial genera.
