RESUMO
In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular diversification of Cas9 proteins.
Assuntos
Bacteriófagos/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/metabolismo , Streptococcus thermophilus/enzimologia , Proteínas Virais/metabolismo , Regulação Alostérica , Bacteriófagos/genética , Sítios de Ligação , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/ultraestrutura , DNA/genética , DNA/ultraestrutura , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Células K562 , Cinética , Mutação , Ligação Proteica , Conformação Proteica , Streptococcus thermophilus/genética , Relação Estrutura-Atividade , Proteínas Virais/genética , Proteínas Virais/ultraestruturaRESUMO
Until the late 2000s, lactococci substantially contributed to the discovery of various plasmid-borne phage defence systems, rendering these bacteria an excellent antiphage discovery resource. Recently, there has been a resurgence of interest in identifying novel antiphage systems in lactic acid bacteria owing to recent reports of so-called 'defence islands' in diverse bacterial genera. Here, 321 plasmid sequences from 53 lactococcal strains were scrutinized for the presence of antiphage systems. Systematic evaluation of 198 candidates facilitated the discovery of seven not previously described antiphage systems, as well as five systems, of which homologues had been described in other bacteria. All described systems confer resistance against the most prevalent lactococcal phages, and act post phage DNA injection, while all except one behave like abortive infection systems. Structure and domain predictions provided insights into their mechanism of action and allow grouping of several genetically distinct systems. Although rare within our plasmid collection, homologues of the seven novel systems appear to be widespread among bacteria. This study highlights plasmids as a rich repository of as yet undiscovered antiphage systems.
RESUMO
In Gram-positive bacteria, cell wall polysaccharides (CWPS) play critical roles in bacterial cell wall homeostasis and bacterial interactions with their immediate surroundings. In lactococci, CWPS consist of two components: a conserved rhamnan embedded in the peptidoglycan layer and a surface-exposed polysaccharide pellicle (PSP), which are linked together to form a large rhamnose-rich CWPS (Rha-CWPS). PSP, whose structure varies from strain to strain, is a receptor for many bacteriophages infecting lactococci. Here, we examined the first two steps of PSP biosynthesis, using in vitro enzymatic tests with lipid acceptor substrates combined with LC-MS analysis, AlfaFold2 modeling of protein 3D-structure, complementation experiments, and phage assays. We show that the PSP repeat unit is assembled on an undecaprenyl-monophosphate (C55P) lipid intermediate. Synthesis is initiated by the WpsA/WpsB complex with GlcNAc-P-C55 synthase activity and the PSP precursor GlcNAc-P-C55 is then elongated by specific glycosyltransferases that vary among lactococcal strains, resulting in PSPs with diverse structures. Also, we engineered the PSP biosynthesis pathway in lactococci to obtain a chimeric PSP structure, confirming the predicted glycosyltransferase specificities. This enabled us to highlight the importance of a single sugar residue of the PSP repeat unit in phage recognition. In conclusion, our results support a novel pathway for PSP biosynthesis on a lipid-monophosphate intermediate as an extracellular modification of rhamnan, unveiling an assembly machinery for complex Rha-CWPS with structural diversity in lactococci.
Assuntos
Parede Celular , Lactococcus , Polissacarídeos Bacterianos , Ramnose , Proteínas de Bactérias/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Glicosiltransferases/metabolismo , Lactococcus/classificação , Lactococcus/citologia , Lactococcus/metabolismo , Lactococcus/virologia , Lipídeos , Peptidoglicano/metabolismo , Polissacarídeos Bacterianos/metabolismo , Conformação Proteica , Ramnose/metabolismo , Especificidade por Substrato , Bacteriófagos/fisiologiaRESUMO
Although more than 12,000 bacteriophages infecting mycobacteria (mycobacteriophages) have been isolated so far, there is a knowledge gap on their structure-function relationships. Here, we have explored the architecture of host-binding machineries from seven representative mycobacteriophages of the Siphoviridae family infecting Mycobacterium smegmatis, Mycobacterium abscessus, and Mycobacterium tuberculosis, using AlphaFold2 (AF2). AF2 enables confident structural analyses of large and flexible biological assemblies resistant to experimental methods, thereby opening new avenues to shed light on phage structure and function. Our results highlight the modularity and structural diversity of siphophage host-binding machineries that recognize host-specific receptors at the onset of viral infection. Interestingly, the studied mycobacteriophages' host-binding machineries present unique features compared with those of phages infecting other Gram-positive actinobacteria. Although they all assemble the classical Dit (distal tail), Tal (tail-associated lysin), and receptor-binding proteins, five of them contain two potential additional adhesion proteins. Moreover, we have identified brush-like domains formed of multiple polyglycine helices which expose hydrophobic residues as potential receptor-binding domains. These polyglycine-rich domains, which have been observed in only five native proteins, may be a hallmark of mycobacteriophages' host-binding machineries, and they may be more common in nature than expected. Altogether, the unique composition of mycobacteriophages' host-binding machineries indicate they might have evolved to bind to the peculiar mycobacterial cell envelope, which is rich in polysaccharides and mycolic acids. This work provides a rational framework to efficiently produce recombinant proteins or protein domains and test their host-binding function and, hence, to shed light on molecular mechanisms used by mycobacteriophages to infect their host. IMPORTANCE Mycobacteria include both saprophytes, such as the model system Mycobacterium smegmatis, and pathogens, such as Mycobacterium tuberculosis and Mycobacterium abscessus, that are poorly responsive to antibiotic treatments and pose a global public health problem. Mycobacteriophages have been collected at a very large scale over the last decade, and they have proven to be valuable tools for mycobacteria genetic manipulation, rapid diagnostics, and infection treatment. Yet, molecular mechanisms used by mycobacteriophages to infect their host remain poorly understood. Therefore, exploring the structural diversity of mycobacteriophages' host-binding machineries is important not only to better understand viral diversity and bacteriophage-host interactions, but also to rationally develop biotechnological tools. With the powerful protein structure prediction software AlphaFold2, which was publicly released a year ago, it is now possible to gain structural and functional insights on such challenging assemblies.
Assuntos
Bacteriófagos , Micobacteriófagos , Mycobacterium tuberculosis , Siphoviridae , Micobacteriófagos/genética , Furilfuramida , Bacteriófagos/genéticaRESUMO
Temperate P335 phage TP901-1 represents one of the best-characterized Gram-positive phages regarding its structure and host interactions. Following its reversible adsorption to the polysaccharidic side-chain of the cell wall polysaccharide of its host Lactococcus cremoris 3107, TP901-1 requires a glucosylated cell envelope moiety to trigger its genome delivery into the host cytoplasm. Here, we demonstrate that three distinct single amino acid substitutions in the Tal protein of TP901-1 baseplate are sufficient to overcome the TP901-1 resistance of three L. cremoris 3107 derivatives, whose resistance is due to impaired DNA release of the phage. All of these Tal alterations are located in the N-terminally located gp27-like domain of the protein, conserved in many tailed phages. AlphaFold2 predictions of the Tal mutant proteins suggest that these mutations favor conformational changes necessary to reposition the Tal fiber and thus facilitate release of the tape measure protein from the tail tube and subsequent DNA ejection in the absence of the trigger otherwise required for phage genome release. IMPORTANCE: Understanding the molecular mechanisms involved in phage-host interactions is essential to develop phage-based applications in the food and probiotic industries, yet also to reduce the risk of phage infections in fermentations. Lactococcus, extensively used in dairy fermentations, has been widely employed to unravel such interactions. Phage infection commences with the recognition of a suitable host followed by the release of its DNA into the bacterial cytoplasm. Details on this latter, irreversible step are still very scarce in lactococci and other Gram-positive bacteria. We demonstrate that a component of the baseplate of the lactococcal phage TP901-1, the tail-associated lysin (Tal), is involved in the DNA delivery into its host, L. cremoris 3107. Specifically, we have found that three amino acid changes in Tal appear to facilitate structural rearrangements in the baseplate necessary for the DNA release process, even in the absence of an otherwise required host trigger.
RESUMO
The type VI secretion system (T6SS) delivers enzymatic effectors into target cells to destroy them. Cells of the same strain protect themselves against effectors with immunity proteins that specifically inhibit effectors. Here, we report the identification and characterization of a Tle3 phospholipase effector and its cognate immunity protein Tli3-an outer membrane lipoprotein from adherent-invasive Escherichia coli (AIEC). Enzymatic assays demonstrate that purified Tle3AIEC has a phospholipase A1, and not A2, activity and that its toxicity is neutralized by the cognate immunity protein Tli3AIEC. Tli3AIEC binds Tle3 in a 1:1 stoichiometric ratio. Tle3AIEC, Tli3AIEC and the Tle3AIEC-Tli3AIEC complex were purified and subjected to crystallization. The Tle3AIEC-Tli3AIEC complex structure could not be solved by SeMet phasing, but only by molecular replacement when using an AlphaFold2 prediction model. Tle3AIEC exhibits an α/ß-hydrolase fold decorated by two protruding segments, including a N-terminus loop. Tli3AIEC displays a new fold of three stacked ß-sheets and a protruding loop that inserts in Tle3AIECcatalytic crevice. We showed, experimentally, that Tle3AIEC interacts with the VgrG AIEC cargo protein and AlphaFold2 prediction of the VgrGAIEC-Tle3AIEC complex reveals a strong interaction between the VgrGAIEC C-terminus adaptor and Tle3AIEC N-terminal loop.
Assuntos
Infecções por Escherichia coli , Sistemas de Secreção Tipo VI , Humanos , Escherichia coli/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Proteínas de Bactérias/metabolismo , Aderência Bacteriana , Proteínas Correpressoras/metabolismoRESUMO
Point-of-care (POC) technologies and testing programs hold great potential to significantly improve diagnosis and disease surveillance. POC tests have the intrinsic advantage of being able to be performed near the patient or treatment facility, owing to their portable character. With rapid results often in minutes, these diagnostic platforms have a high positive impact on disease management. POC tests are, in addition, advantageous in situations of a shortage of skilled personnel and restricted availability of laboratory-based analytics. While POC testing programs are widely considered in addressing health care challenges in low-income health systems, the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections could largely benefit from fast, efficient, accurate, and cost-effective point-of-care testing (POCT) devices for limiting COVID-19 spreading. The unrestrained availability of SARS-CoV-2 POC tests is indeed one of the adequate means of better managing the COVID-19 outbreak. A large number of novel and innovative solutions to address this medical need have emerged over the last months. Here, we critically elaborate the role of the surface ligands in the design of biosensors to cope with the current viral outbreak situation. Their notable effect on electrical and electrochemical sensors' design will be discussed in some given examples. Graphical abstract.
Assuntos
Antígenos Virais/análise , Técnicas Biossensoriais/métodos , Teste para COVID-19/métodos , COVID-19/diagnóstico , Testes Imediatos/tendências , SARS-CoV-2/imunologia , Antígenos Virais/imunologia , COVID-19/virologia , Técnicas Eletroquímicas , Humanos , Ligantes , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
Contractile tails are composed of an inner tube wrapped by an outer sheath assembled in an extended, metastable conformation that stores mechanical energy necessary for its contraction. Contraction is used to propel the rigid inner tube towards target cells for DNA or toxin delivery. Although recent studies have revealed the structure of the contractile sheath of the type VI secretion system, the mechanisms by which its polymerization is controlled and coordinated with the assembly of the inner tube remain unknown. Here we show that the starfish-like TssA dodecameric complex interacts with tube and sheath components. Fluorescence microscopy experiments in enteroaggregative Escherichia coli reveal that TssA binds first to the type VI secretion system membrane core complex and then initiates tail polymerization. TssA remains at the tip of the growing structure and incorporates new tube and sheath blocks. On the basis of these results, we propose that TssA primes and coordinates tail tube and sheath biogenesis.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Polimerização , Cristalografia por Raios X , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Moleculares , Estrutura Terciária de Proteína , Sistemas de Secreção Tipo VI/química , Sistemas de Secreção Tipo VI/metabolismo , Sistemas de Secreção Tipo VI/ultraestruturaRESUMO
The biosynthetic machinery for cell wall polysaccharide (CWPS) production in lactococci is encoded by a large gene cluster, designated cwps. This locus displays considerable variation among lactococcal genomes, previously prompting a classification into three distinct genotypes (A-C). In the present study, the cwps loci of 107 lactococcal strains were compared, revealing the presence of a fourth cwps genotype (type D). Lactococcal CWPSs are comprised of two saccharidic structures: a peptidoglycan-embedded rhamnan backbone polymer to which a surface-exposed, poly/oligosaccharidic side-chain is covalently linked. Chemical structures of the side-chain of seven lactococcal strains were elucidated, highlighting their diverse and strain-specific nature. Furthermore, a link between cwps genotype and chemical structure was derived based on the number of glycosyltransferase-encoding genes in the cwps cluster and the presence of conserved genes encoding the presumed priming glycosyltransferase. This facilitates predictions of several structural features of lactococcal CWPSs including (a) whether the CWPS possesses short oligo/polysaccharide side-chains, (b) the number of component monosaccharides in a given CWPS structure, (c) the order of monosaccharide incorporation into the repeating units of the side-chain (for C-type strains), (d) the presence of Galf and phosphodiester bonds in the side-chain, and (e) the presence of glycerol phosphate substituents in the side-chain.
Assuntos
Parede Celular/genética , Lactococcus/genética , Polissacarídeos Bacterianos/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Glicosiltransferases/metabolismo , Lactococcus/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Família Multigênica/genética , Peptidoglicano/metabolismo , Polissacarídeos/metabolismo , Polissacarídeos Bacterianos/metabolismo , Polissacarídeos Bacterianos/fisiologiaRESUMO
Bacteria share their ecological niches with other microbes. The bacterial type VI secretion system is one of the key players in microbial competition, as well as being an important virulence determinant during bacterial infections. It assembles a nano-crossbow-like structure in the cytoplasm of the attacker cell that propels an arrow made of a haemolysin co-regulated protein (Hcp) tube and a valine-glycine repeat protein G (VgrG) spike and punctures the prey's cell wall. The nano-crossbow is stably anchored to the cell envelope of the attacker by a membrane core complex. Here we show that this complex is assembled by the sequential addition of three type VI subunits (Tss)-TssJ, TssM and TssL-and present a structure of the fully assembled complex at 11.6 Å resolution, determined by negative-stain electron microscopy. With overall C5 symmetry, this 1.7-megadalton complex comprises a large base in the cytoplasm. It extends in the periplasm via ten arches to form a double-ring structure containing the carboxy-terminal domain of TssM (TssMct) and TssJ that is anchored in the outer membrane. The crystal structure of the TssMct-TssJ complex coupled to whole-cell accessibility studies suggest that large conformational changes induce transient pore formation in the outer membrane, allowing passage of the attacking Hcp tube/VgrG spike.
Assuntos
Sistemas de Secreção Bacterianos , Proteínas de Escherichia coli/química , Escherichia coli/química , Lipopeptídeos/química , Proteínas de Membrana/química , Complexos Multiproteicos/biossíntese , Complexos Multiproteicos/química , Membrana Celular/química , Membrana Celular/metabolismo , Cristalografia por Raios X , Citoplasma/química , Citoplasma/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Lipopeptídeos/biossíntese , Proteínas de Membrana/biossíntese , Microscopia Eletrônica , Modelos Moleculares , Periplasma/química , Periplasma/metabolismo , Porosidade , Estrutura Terciária de Proteína , Subunidades Proteicas/biossíntese , Subunidades Proteicas/químicaRESUMO
Spider mites are one of the major agricultural pests, feeding on a large variety of plants. As a contribution to understanding chemical communication in these arthropods, we have characterized a recently discovered class of odorant-binding proteins (OBPs) in Tetranychus urticae. As in other species of Chelicerata, the four OBPs of T. urticae contain six conserved cysteines paired in a pattern (C1-C6, C2-C3, C4-C5) differing from that of insect counterparts (C1-C3, C2-C5, C4-C6). Proteomic analysis uncovered a second family of OBPs, including twelve members that are likely to be unique to T. urticae. A three-dimensional model of TurtOBP1, built on the recent X-ray structure of Varroa destructor OBP1, shows protein folding different from that of insect OBPs, although with some common features. Ligand-binding experiments indicated some affinity to coniferyl aldehyde, but specific ligands may still need to be found among very large molecules, as suggested by the size of the binding pocket.
Assuntos
Receptores Odorantes/metabolismo , Tetranychidae/metabolismo , Sequência de Aminoácidos , Animais , Ligantes , Modelos Moleculares , Estrutura Molecular , Odorantes , Filogenia , Ligação Proteica , Conformação Proteica , Proteoma , Proteômica/métodos , Receptores Odorantes/química , Receptores Odorantes/genética , Tetranychidae/genéticaRESUMO
Streptococcus thermophilus is a lactic acid bacterium commonly used for the manufacture of yogurt and specialty cheeses. Virulent phages represent a major risk for milk fermentation processes worldwide, as they can inactivate the added starter bacterial cells, leading to low-quality fermented dairy products. To date, four genetically distinct groups of phages infecting S. thermophilus have been described. Here, we describe a fifth group. Phages P738 and D4446 are virulent siphophages that infect a few industrial strains of S. thermophilus The genomes of phages P738 and D4446 were sequenced and found to contain 34,037 and 33,656 bp as well as 48 and 46 open reading frames, respectively. Comparative genomic analyses revealed that the two phages are closely related to each other but display very limited similarities to other S. thermophilus phages. In fact, these two novel S. thermophilus phages share similarities with streptococcal phages of nondairy origin, suggesting that they emerged recently in the dairy environment.IMPORTANCE Despite decades of research and adapted antiphage strategies such as CRISPR-Cas systems, virulent phages are still a persistent risk for the milk fermentation industry worldwide, as they can cause manufacturing failures and alter product quality. Phages P738 and D4446 are novel virulent phages that infect the food-grade Gram-positive bacterial species Streptococcus thermophilus These two related viruses represent a fifth group of S. thermophilus phages, as they are significantly distinct from other known S. thermophilus phages. Both phages share similarities with phages infecting nondairy streptococci, suggesting their recent emergence and probable coexistence in dairy environments. These findings highlight the necessity of phage surveillance programs as the phage population evolves in response to the application of antiphage strategies.
Assuntos
Siphoviridae/classificação , Fagos de Streptococcus/classificação , Streptococcus thermophilus/virologia , Microscopia Eletrônica de Transmissão , Análise de Sequência de DNA , Siphoviridae/genética , Siphoviridae/ultraestrutura , Fagos de Streptococcus/genética , Fagos de Streptococcus/ultraestruturaRESUMO
ANISEED (www.aniseed.cnrs.fr) is the main model organism database for tunicates, the sister-group of vertebrates. This release gives access to annotated genomes, gene expression patterns, and anatomical descriptions for nine ascidian species. It provides increased integration with external molecular and taxonomy databases, better support for epigenomics datasets, in particular RNA-seq, ChIP-seq and SELEX-seq, and features novel interactive interfaces for existing and novel datatypes. In particular, the cross-species navigation and comparison is enhanced through a novel taxonomy section describing each represented species and through the implementation of interactive phylogenetic gene trees for 60% of tunicate genes. The gene expression section displays the results of RNA-seq experiments for the three major model species of solitary ascidians. Gene expression is controlled by the binding of transcription factors to cis-regulatory sequences. A high-resolution description of the DNA-binding specificity for 131 Ciona robusta (formerly C. intestinalis type A) transcription factors by SELEX-seq is provided and used to map candidate binding sites across the Ciona robusta and Phallusia mammillata genomes. Finally, use of a WashU Epigenome browser enhances genome navigation, while a Genomicus server was set up to explore microsynteny relationships within tunicates and with vertebrates, Amphioxus, echinoderms and hemichordates.
Assuntos
Bases de Dados Genéticas , Conjuntos de Dados como Assunto , Genoma , Urocordados/genética , Animais , Evolução Biológica , Ciona intestinalis/genética , DNA/metabolismo , Mineração de Dados , Evolução Molecular , Expressão Gênica , Ontologia Genética , Internet , Anotação de Sequência Molecular , Filogenia , Ligação Proteica , Especificidade da Espécie , Fatores de Transcrição/metabolismo , Transcrição Gênica , Vertebrados/genética , NavegadorRESUMO
The giant panda Ailuropoda melanoleuca belongs to the family of Ursidae; however, it is not carnivorous, feeding almost exclusively on bamboo. Being equipped with a typical carnivorous digestive apparatus, the giant panda cannot get enough energy for an active life and spends most of its time digesting food or sleeping. Feeding and mating are both regulated by odors and pheromones; therefore, a better knowledge of olfaction at the molecular level can help in designing strategies for the conservation of this species. In this context, we have identified the odorant-binding protein (OBP) repertoire of the giant panda and mapped the protein expression in nasal mucus and saliva through proteomics. Four OBPs have been identified in nasal mucus, while the other two were not detected in the samples examined. In particular, AimelOBP3 is similar to a subset of OBPs reported as pheromone carriers in the urine of rodents, saliva of the boar, and seminal fluid of the rabbit. We expressed this protein, mapped its binding specificity, and determined its crystal structure. Structural data guided the design and preparation of three protein mutants bearing single-amino acid replacements in the ligand-binding pocket, for which the corresponding binding affinity spectra were measured. We also expressed AimelOBP5, which is markedly different from AimelOBP3 and complementary in its binding spectrum. By comparing our binding data with the structures of bamboo volatiles and those of typical mammalian pheromones, we formulate hypotheses on which may be the most relevant semiochemicals for the giant panda.
Assuntos
Bambusa/química , Ecologia , Feromônios/metabolismo , Receptores Odorantes/química , Receptores Odorantes/metabolismo , Olfato/fisiologia , Ursidae/metabolismo , Ração Animal , Animais , Comportamento Animal , Cristalografia por Raios X , Modelos Moleculares , Simulação de Acoplamento Molecular , Mucosa Nasal/química , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteômica , Coelhos , Receptores Odorantes/genética , Receptores Odorantes/isolamento & purificação , Saliva/química , Alinhamento de Sequência , Análise de Sequência de Proteína , SuínosRESUMO
With increasing numbers of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and associated functionality of phage adhesion devices. Recently, decorations have been reported in the tail and neck passage structures of members of the so-called 936 group of lactococcal siphophages. In the current report, using bioinformatic analysis we identified a conserved carbohydrate binding module (CBM) among many of the virion baseplate Dit components, in addition to the CBM present in the 'classical' receptor binding proteins (RBPs). We observed that, within these so-called 'evolved' Dit proteins, the identified CBMs have structurally conserved folds, yet can be grouped into four distinct classes. We expressed such modules in fusion with GFP, and demonstrated their binding capability to their specific host using fluorescent binding assays with confocal microscopy. We detected evolved Dits in several phages infecting various Gram-positive bacterial species, including mycobacteria. The omnipresence of CBM domains in siphophages indicates their auxiliary role in infection, as they can assist in the specific recognition of and attachment to their host, thus ensuring a highly efficient and specific phage-host adhesion process as a prelude to DNA injection.
Assuntos
Lactococcus lactis/virologia , Siphoviridae/genética , Siphoviridae/metabolismo , Proteínas da Cauda Viral/genética , Vírion/genética , Carboidratos/química , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
The Type VI secretion system (T6SS) is a multiprotein and mosaic apparatus that delivers protein effectors into prokaryotic or eukaryotic cells. Recent data on the enteroaggregative Escherichia coli (EAEC) T6SS have provided evidence that the TssA protein is a key component during T6SS biogenesis. The T6SS comprises a trans-envelope complex that docks the baseplate, a cytoplasmic complex that represents the assembly platform for the tail. The T6SS tail is structurally, evolutionarily and functionally similar to the contractile tails of bacteriophages. We have shown that TssA docks to the membrane complex, recruits the baseplate complex and initiates and coordinates the polymerization of the inner tube with that of the sheath. Here, we review these recent findings, discuss the variations within TssA-like proteins, speculate on the role of EAEC TssA in T6SS biogenesis and propose future research perspectives.
Assuntos
Sistemas de Secreção Tipo VI/metabolismo , Bacteriófagos/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMO
The transport of proteins at the cell surface of Bacteroidetes depends on a secretory apparatus known as type IX secretion system (T9SS). This machine is responsible for the cell surface exposition of various proteins, such as adhesins, required for gliding motility in Flavobacterium, S-layer components in Tannerella forsythia, and tooth tissue-degrading enzymes in the oral pathogen Porphyromonas gingivalis Although a number of subunits of the T9SS have been identified, we lack details on the architecture of this secretion apparatus. Here we provide evidence that five of the genes encoding the core complex of the T9SS are co-transcribed and that the gene products are distributed in the cell envelope. Protein-protein interaction studies then revealed that these proteins oligomerize and interact through a dense network of contacts.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Porphyromonas gingivalis/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/análise , Sistemas de Secreção Bacterianos/genética , Infecções por Bacteroidaceae/microbiologia , Cristalografia por Raios X , Genes Bacterianos , Humanos , Porphyromonas gingivalis/química , Porphyromonas gingivalis/genética , Mapas de Interação de Proteínas , Subunidades Proteicas/análise , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
Bacteriophage replication requires specific host-recognition. Some siphophages harbour a large complex, the baseplate, at the tip of their non-contractile tail. This baseplate holds receptor binding proteins (RBPs) that can recognize the host cell-wall polysaccharide (CWPS) and specifically attach the phage to its host. While most phages possess a dedicated RBP, the phage J-1 that infects Lactobacillus casei seemed to lack one. It has been shown that the phage J-1 distal tail protein (Dit) plays a role in host recognition and that its sequence comprises two inserted modules compared with 'classical' Dits. The first insertion is similar to carbohydrate-binding modules (CBMs), whereas the second insertion remains undocumented. Here, we determined the structure of the second insertion and found it also similar to several CBMs. Expressed insertion CBM2, but not CBM1, binds to L. casei cells and neutralize phage attachment to the bacterial cell wall and the isolated and purified CWPS of L. casei BL23 prevents CBM2 attachment to the host. Electron microscopy single particle reconstruction of the J-1 virion baseplate revealed that CBM2 is projected at the periphery of Dit to optimally bind the CWPS receptor. Taken together, these results identify J-1 evolved Dit as the phage RBP.
Assuntos
Proteínas da Cauda Viral/metabolismo , Proteínas da Cauda Viral/ultraestrutura , Bacteriófagos/metabolismo , Carboidratos , Especificidade de Hospedeiro , Ácido Láctico , Lactobacillus , Lacticaseibacillus casei/metabolismo , Lactococcus lactis/metabolismo , Microscopia Eletrônica , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Proteínas da Cauda Viral/genética , VírionRESUMO
Phages of Streptococcus thermophilus present a major threat to the production of many fermented dairy products. To date, only a few studies have assessed the biodiversity of S. thermophilus phages in dairy fermentations. In order to develop strategies to limit phage predation in this important industrial environment, it is imperative that such studies are undertaken and that phage-host interactions of this species are better defined. The present study investigated the biodiversity and evolution of phages within an Irish dairy fermentation facility over an 11-year period. This resulted in the isolation of 17 genetically distinct phages, all of which belong to the so-called cos group. The evolution of phages within the factory appears to be influenced by phages from other dairy plants introduced into the factory for whey protein powder production. Modular exchange, primarily within the regions encoding lysogeny and replication functions, was the major observation among the phages isolated between 2006 and 2016. Furthermore, the genotype of the first isolate in 2006 was observed continuously across the following decade, highlighting the ability of these phages to prevail in the factory setting for extended periods of time. The proteins responsible for host recognition were analyzed, and carbohydrate-binding domains (CBDs) were identified in the distal tail (Dit), the baseplate proteins, and the Tail-associated lysin (Tal) variable regions (VR1 and VR2) of many isolates. This supports the notion that S. thermophilus phages recognize a carbohydrate receptor on the cell surface of their host.IMPORTANCE Dairy fermentations are consistently threatened by the presence of bacterial viruses (bacteriophages or phages), which may lead to a reduction in acidification rates or even complete loss of the fermentate. These phages may persist in factories for long periods of time. The objective of the current study was to monitor the progression of phages infecting the dairy bacterium Streptococcus thermophilus over a period of 11 years in an Irish dairy plant so as to understand how these phages evolve. A focused analysis of the genomic region that encodes host recognition functions highlighted that the associated proteins harbor a variety of carbohydrate-binding domains, which corroborates the notion that phages of S. thermophilus recognize carbohydrate receptors at the initial stages of the phage cycle.
Assuntos
Produtos Fermentados do Leite/microbiologia , Fagos de Streptococcus/genética , Streptococcus thermophilus/virologia , Evolução Biológica , Indústria de Laticínios , Fermentação , Genótipo , Especificidade de Hospedeiro , Irlanda , Lisogenia , Filogenia , Fagos de Streptococcus/classificação , Fagos de Streptococcus/isolamento & purificação , Fagos de Streptococcus/fisiologia , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Glycerophosphodiester phosphodiesterases (GDPDs; EC 3.1.4.46) typically hydrolyze glycerophosphodiesters to sn-glycerol 3-phosphate (Gro3P) and their corresponding alcohol during patho/physiological processes in bacteria and eukaryotes. GDPD(-like) domains were identified in the structural particle of bacterial viruses (bacteriophages) specifically infecting Gram-positive bacteria. The GDPD of phage 17 (Ld17; GDPDLd17), representative of the group b Lactobacillus delbrueckii subsp. bulgaricus (Ldb)-infecting bacteriophages, was shown to hydrolyze, besides the simple glycerophosphodiester, two complex surface-associated carbohydrates of the Ldb17 cell envelope: the Gro3P decoration of the major surface polysaccharide d-galactan and the oligo(glycerol phosphate) backbone of the partially glycosylated cell wall teichoic acid, a minor Ldb17 cell envelope component. Degradation of cell wall teichoic acid occurs according to an exolytic mechanism, and Gro3P substitution is presumed to be inhibitory for GDPDLd17 activity. The presence of the GDPDLd17 homotrimer in the viral baseplate structure involved in phage-host interaction together with the dependence of native GDPD activity, adsorption, and efficiency of plating of Ca(2+) ions supports a role for GDPDLd17 activity during phage adsorption and/or phage genome injection. In contrast to GDPDLd17, we could not identify any enzymatic activity for the GDPD-like domain in the neck passage structure of phage 340, a 936-type Lactococcus lactis subsp. lactis bacteriophage.