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1.
Molecules ; 26(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34204994

RESUMO

Chlorophylls and bacteriochlorophylls, together with carotenoids, serve, noncovalently bound to specific apoproteins, as principal light-harvesting and energy-transforming pigments in photosynthetic organisms. In recent years, enormous progress has been achieved in the elucidation of structures and functions of light-harvesting (antenna) complexes, photosynthetic reaction centers and even entire photosystems. It is becoming increasingly clear that light-harvesting complexes not only serve to enlarge the absorption cross sections of the respective reaction centers but are vitally important in short- and long-term adaptation of the photosynthetic apparatus and regulation of the energy-transforming processes in response to external and internal conditions. Thus, the wide variety of structural diversity in photosynthetic antenna "designs" becomes conceivable. It is, however, common for LHCs to form trimeric (or multiples thereof) structures. We propose a simple, tentative explanation of the trimer issue, based on the 2D world created by photosynthetic membrane systems.


Assuntos
Cianobactérias/metabolismo , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Plantas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transferência de Energia , Modelos Moleculares , Fotossíntese , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformação Proteica , Multimerização Proteica
2.
Molecules ; 26(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205065

RESUMO

Bacterial resistance to antibiotics due to increased efficiency of the efflux is a serious problem in clinics of infectious diseases. Knowledge of the factors affecting the activity of efflux pumps would help to find the solution. For this, fast and trustful methods for efflux analysis are needed. Here, we analyzed how the assay conditions affect the accumulation of efflux indicators ethidium (Et+) and tetraphenylphosphonium in Salmonella enterica ser. Typhimurium cells. An inhibitor phenylalanyl-arginyl-ß-naphtylamide was applied to evaluate the input of RND family pumps into the total efflux. In parallel to spectrofluorimetric analysis, we used an electrochemical assessment of Et+ concentration. The results of our experiments indicated that Et+ fluorescence increases immediately after the penetration of this indicator into the cells. However, when cells bind a high amount of Et+, the intensity of the fluorescence reaches the saturation level and stops reacting to the accumulated amount of this indicator. For this reason, electrochemical measurements provide more trustful information about the efficiency of efflux when cells accumulate high amounts of Et+. Measurements of Et+ interaction with the purified DNA demonstrated that the affinity of this lipophilic cation to DNA depends on the medium composition. The capacity of DNA to bind Et+ considerably decreases in the presence of Mg2+, Polymyxin B or when DNA is incubated in high ionic strength media.


Assuntos
DNA/química , Etídio/análise , Salmonella typhimurium/crescimento & desenvolvimento , Espermatozoides/química , Animais , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla , Etídio/química , Masculino , Oniocompostos/química , Compostos Organofosforados/química , Salmão , Salmonella typhimurium/metabolismo , Espectrometria de Fluorescência , Espermatozoides/metabolismo
3.
Nat Commun ; 12(1): 4223, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244518

RESUMO

The bacterial flagellar MS ring is a transmembrane complex acting as the core of the flagellar motor and template for flagellar assembly. The C ring attached to the MS ring is involved in torque generation and rotation switch, and a large symmetry mismatch between these two rings has been a long puzzle, especially with respect to their role in motor function. Here, using cryoEM structural analysis of the flagellar basal body and the MS ring formed by full-length FliF from Salmonella enterica, we show that the native MS ring is formed by 34 FliF subunits with no symmetry variation. Symmetry analysis of the C ring shows a variation with a peak at 34-fold, suggesting flexibility in C ring assembly. Finally, our data also indicate that FliF subunits assume two different conformations, contributing differentially to the inner and middle parts of the M ring and thus resulting in 23- and 11-fold subsymmetries in the inner and middle M ring, respectively. The internal core of the M ring, formed by 23 subunits, forms a hole of the right size to accommodate the protein export gate.


Assuntos
Proteínas de Bactérias/ultraestrutura , Flagelos/ultraestrutura , Proteínas de Membrana/ultraestrutura , Sistemas de Secreção Tipo III/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Fracionamento Celular , Microscopia Crioeletrônica , Flagelos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/ultraestrutura , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo
4.
Nat Commun ; 12(1): 4254, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253723

RESUMO

Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis.


Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Membrana Celular/metabolismo , Lipoproteínas/biossíntese , Acilação , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Sequência Conservada , Cisteína/metabolismo , Análise Mutacional de DNA , Processamento de Proteína Pós-Traducional , Relação Estrutura-Atividade , Especificidade por Substrato
5.
Nat Commun ; 12(1): 4064, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210966

RESUMO

The intrinsically disordered region (IDR) is a preserved signature of phytobacterial type III effectors (T3Es). The T3E IDR is thought to mediate unfolding during translocation into the host cell and to avoid host defense by sequence diversification. Here, we demonstrate a mechanism of host subversion via the T3E IDR. We report that the Xanthomonas campestris T3E XopR undergoes liquid-liquid phase separation (LLPS) via multivalent IDR-mediated interactions that hijack the Arabidopsis actin cytoskeleton. XopR is gradually translocated into host cells during infection and forms a macromolecular complex with actin-binding proteins at the cell cortex. By tuning the physical-chemical properties of XopR-complex coacervates, XopR progressively manipulates multiple steps of actin assembly, including formin-mediated nucleation, crosslinking of F-actin, and actin depolymerization, which occurs through competition for actin-depolymerizing factor and depends on constituent stoichiometry. Our findings unravel a sophisticated strategy in which bacterial T3E subverts the host actin cytoskeleton via protein complex coacervation.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Bactérias/metabolismo , Xanthomonas/metabolismo , Agrobacterium , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Plantas Geneticamente Modificadas , Análise de Sequência , Tabaco , Xanthomonas/genética , Xanthomonas campestris/metabolismo
6.
Int J Mol Sci ; 22(14)2021 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-34299060

RESUMO

Pseudomonas syringae-secreted HopA1 effectors are important determinants in host range expansion and increased pathogenicity. Their recent acquisitions via horizontal gene transfer in several non-pathogenic Pseudomonas strains worldwide have caused alarming increase in their virulence capabilities. In Arabidopsis thaliana, RESISTANCE TO PSEUDOMONAS SYRINGAE 6 (RPS6) gene confers effector-triggered immunity (ETI) against HopA1pss derived from P. syringae pv. syringae strain 61. Surprisingly, a closely related HopA1pst from the tomato pathovar evades immune detection. These responsive differences in planta between the two HopA1s represents a unique system to study pathogen adaptation skills and host-jumps. However, molecular understanding of HopA1's contribution to overall virulence remain undeciphered. Here, we show that immune-suppressive functions of HopA1pst are more potent than HopA1pss. In the resistance-compromised ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) null-mutant, transcriptomic changes associated with HopA1pss-elicited ETI are still induced and carry resemblance to PAMP-triggered immunity (PTI) signatures. Enrichment of HopA1pss interactome identifies proteins with regulatory roles in post-transcriptional and translational processes. With our demonstration here that both HopA1 suppress reporter-gene translations in vitro imply that the above effector-associations with plant target carry inhibitory consequences. Overall, with our results here we unravel possible virulence role(s) of HopA1 in suppressing PTI and provide newer insights into its detection in resistant plants.


Assuntos
Arabidopsis/imunologia , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Degradação do RNAm Mediada por Códon sem Sentido , Doenças das Plantas/imunologia , Imunidade Vegetal , Pseudomonas syringae/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Bactérias/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Virulência
7.
Int J Mol Sci ; 22(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299141

RESUMO

The bacterial flagellum is a complex and dynamic nanomachine that propels bacteria through liquids. It consists of a basal body, a hook, and a long filament. The flagellar filament is composed of thousands of copies of the protein flagellin (FliC) arranged helically and ending with a filament cap composed of an oligomer of the protein FliD. The overall structure of the filament core is preserved across bacterial species, while the outer domains exhibit high variability, and in some cases are even completely absent. Flagellar assembly is a complex and energetically costly process triggered by environmental stimuli and, accordingly, highly regulated on transcriptional, translational and post-translational levels. Apart from its role in locomotion, the filament is critically important in several other aspects of bacterial survival, reproduction and pathogenicity, such as adhesion to surfaces, secretion of virulence factors and formation of biofilms. Additionally, due to its ability to provoke potent immune responses, flagellins have a role as adjuvants in vaccine development. In this review, we summarize the latest knowledge on the structure of flagellins, capping proteins and filaments, as well as their regulation and role during the colonization and infection of the host.


Assuntos
Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Flagelos/fisiologia , Flagelina/metabolismo , Nanoestruturas/química , Citoesqueleto , Modelos Moleculares
8.
Int J Mol Sci ; 22(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299161

RESUMO

Prostaglandin E2 (PGE2) is an important biological mediator involved in the defense against Mycobacterium tuberculosis (Mtb) infection. Currently, there are no reports on the mycobacterial components that regulate PGE2 production. Previously, we have reported that RpfE-treated dendritic cells (DCs) effectively expanded the Th1 and Th17 cell responses simultaneously; however, the mechanism underlying Th1 and Th17 cell differentiation is unclear. Here, we show that PGE2 produced by RpfE-activated DCs via the MAPK and cyclooxygenase 2 signaling pathways induces Th1 and Th17 cell responses mainly via the EP4 receptor. Furthermore, mice administered intranasally with PGE2 displayed RpfE-induced antigen-specific Th1 and Th17 responses with a significant reduction in bacterial load in the lungs. Furthermore, the addition of optimal PGE2 amount to IL-2-IL-6-IL-23p19-IL-1ß was essential for promoting differentiation into Th1/Th17 cells with strong bactericidal activity. These results suggest that RpfE-matured DCs produce PGE2 that induces Th1 and Th17 cell differentiation with potent anti-mycobacterial activity.


Assuntos
Proteínas de Bactérias/metabolismo , Diferenciação Celular , Células Dendríticas/metabolismo , Dinoprostona/metabolismo , Mycobacterium tuberculosis/fisiologia , Células Th1/citologia , Células Th17/citologia , Animais , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Células Th1/imunologia , Células Th17/imunologia , Tuberculose/imunologia , Tuberculose/metabolismo , Tuberculose/microbiologia
9.
Int J Mol Sci ; 22(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299177

RESUMO

Adaptation of bacteria to a changing environment is often accompanied by remodeling of the transcriptome. In the facultative phototroph Rhodobacter sphaeroides the alternative sigma factors RpoE, RpoHI and RpoHII play an important role in a variety of stress responses, including heat, oxidative stress and nutrient limitation. Photooxidative stress caused by the simultaneous presence of chlorophylls, light and oxygen is a special challenge for phototrophic organisms. Like alternative sigma factors, several non-coding sRNAs have important roles in the defense against photooxidative stress. RNAseq-based transcriptome data pointed to an influence of the stationary phase-induced StsR sRNA on levels of mRNAs and sRNAs with a role in the photooxidative stress response. Furthermore, StsR also affects expression of photosynthesis genes and of genes for regulators of photosynthesis genes. In vivo and in vitro interaction studies revealed that StsR, that is under control of the RpoHI and RpoHII sigma factors, targets rpoE mRNA and affects its abundance by altering its stability. RpoE regulates expression of the rpoHII gene and, consequently, expression of stsR. These data provide new insights into a complex regulatory network of protein regulators and sRNAs involved in defense against photooxidative stress and the regulation of photosynthesis genes.


Assuntos
Proteínas de Bactérias/metabolismo , Estresse Oxidativo , Oxigênio/metabolismo , RNA Bacteriano/genética , Rhodobacter sphaeroides/crescimento & desenvolvimento , Fator sigma/metabolismo , Transcriptoma , Proteínas de Bactérias/genética , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , Fator sigma/genética
10.
Nat Commun ; 12(1): 4394, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285211

RESUMO

Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (Agp1). Whereas Agp1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While Agp1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes suggest the use of light-controllable histidine kinases and phosphatases for optogenetics.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina Quinase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fotorreceptores Microbianos/metabolismo , Transdução de Sinais/efeitos da radiação , Agrobacterium/enzimologia , Proteínas de Bactérias/ultraestrutura , Deinococcus/enzimologia , Histidina Quinase/ultraestrutura , Luz , Simulação de Dinâmica Molecular , Monoéster Fosfórico Hidrolases/ultraestrutura , Fotorreceptores Microbianos/ultraestrutura , Domínios Proteicos
11.
Front Cell Infect Microbiol ; 11: 679982, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235094

RESUMO

Sulfate Transport Anti-Sigma antagonist domains (Pfam01740) are found in all branches of life, from eubacteria to mammals, as a conserved fold encoded by highly divergent amino acid sequences. These domains are present as part of larger SLC26/SulP anion transporters, where the STAS domain is associated with transmembrane anchoring of the larger multidomain protein. Here, we focus on STAS Domain only Proteins (SDoPs) in eubacteria, initially described as part of the Bacillus subtilis Regulation of Sigma B (RSB) regulatory system. Since their description in B. subtilis, SDoPs have been described to be involved in the regulation of sigma factors, through partner-switching mechanisms in various bacteria such as: Mycobacterium. tuberculosis, Listeria. monocytogenes, Vibrio. fischeri, Bordetella bronchiseptica, among others. In addition to playing a canonical role in partner-switching with an anti-sigma factor to affect the availability of a sigma factor, several eubacterial SDoPs show additional regulatory roles compared to the original RSB system of B. subtilis. This is of great interest as these proteins are highly conserved, and often involved in altering gene expression in response to changes in environmental conditions. For many of the bacteria we will examine in this review, the ability to sense environmental changes and alter gene expression accordingly is critical for survival and colonization of susceptible hosts.


Assuntos
Proteínas de Transporte de Ânions , Genes Bacterianos , Animais , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Imidazóis , Estrutura Terciária de Proteína , Fator sigma/genética
12.
Int J Mol Sci ; 22(13)2021 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-34281246

RESUMO

Engineering biological processes has become a standard approach to produce various commercially valuable chemicals, therapeutics, and biomaterials. Among these products, bacterial cellulose represents major advances to biomedical and healthcare applications. In comparison to properties of plant cellulose, bacterial cellulose (BC) shows distinctive characteristics such as a high purity, high water retention, and biocompatibility. However, low product yield and extensive cultivation times have been the main challenges in the large-scale production of BC. For decades, studies focused on optimization of cellulose production through modification of culturing strategies and conditions. With an increasing demand for BC, researchers are now exploring to improve BC production and functionality at different categories: genetic, bioprocess, and product levels as well as model driven approaches targeting each of these categories. This comprehensive review discusses the progress in BC platforms categorizing the most recent advancements under different research focuses and provides systematic understanding of the progress in BC biosynthesis. The aim of this review is to present the potential of 'modern genetic engineering tools' and 'model-driven approaches' on improving the yield of BC, altering the properties, and adding new functionality. We also provide insights for the future perspectives and potential approaches to promote BC use in biomedical applications.


Assuntos
Celulose/biossíntese , Celulose/química , Celulose/genética , Bactérias/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Materiais Biocompatíveis/síntese química , Metabolismo dos Carboidratos/fisiologia , Engenharia Genética/métodos , Biologia Sintética/métodos
13.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199128

RESUMO

Flavobacterium johnsoniae forms a thin spreading colony on nutrient-poor agar using gliding motility. As reported in the first paper, WT cells in the colony were sparsely embedded in self-produced extracellular polymeric matrix (EPM), while sprB cells were densely packed in immature biofilm with less matrix. The colony surface is critical for antibiotic resistance and cell survival. We have now developed the Grid Stamp-Peel method whereby the colony surface is attached to a TEM grid for negative-staining microscopy. The images showed that the top of the spreading convex WT colonies was covered by EPM with few interspersed cells. Cells exposed near the colony edge made head-to-tail and/or side-to-side contact and sometimes connected via thin filaments. Nonspreading sprB and gldG and gldK colonies had a more uniform upper surface covered by different EPMs including vesicles and filaments. The EPM of sprB, gldG, and WT colonies contained filaments ~2 nm and ~5 nm in diameter; gldK colonies did not include the latter. Every cell near the edge of WT colonies had one or two dark spots, while cells inside WT colonies and cells in SprB-, GldG-, or GldK-deficient colonies did not. Together, our results suggest that the colony surface structure depends on the capability to expand biofilm.


Assuntos
Adesinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Matriz Extracelular/metabolismo , Flavobacterium/fisiologia , Adesinas Bacterianas/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Sistemas de Secreção Bacterianos/metabolismo , Flavobacterium/efeitos dos fármacos , Flavobacterium/ultraestrutura , Testes de Sensibilidade Microbiana , Mutação , Fenótipo
14.
Molecules ; 26(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199200

RESUMO

Glycan-targeting antibodies and pseudo-antibodies have been extensively studied for their stoichiometry, avidity, and their interactions with the rapidly modifying glycan shield of influenza A. Broadly neutralizing antiviral agents bind in the same order when they neutralize enveloped viruses regardless of the location of epitopes to the host receptor binding site. Herein, we investigated the binding of cyanovirin-N (CV-N) to surface-expressed glycoproteins such as those of human immunodeficiency virus (HIV) gp120, hemagglutinin (HA), and Ebola (GP)1,2 and compared their binding affinities with the binding response to the trimer-folded gp140 using surface plasmon resonance (SPR). Binding-site knockout variants of an engineered dimeric CV-N molecule (CVN2) revealed a binding affinity that correlated with the number of (high-) affinity binding sites. Binding curves were specific for the interaction with N-linked glycans upon binding with two low-affinity carbohydrate binding sites. This biologically active assembly of a domain-swapped CVN2, or monomeric CV-N, bound to HA with a maximum KD of 2.7 nM. All three envelope spike proteins were recognized at a nanomolar KD, whereas binding to HIV neutralizing 2G12 by targeting HA and Ebola GP1,2 was measured in the µM range and specific for the bivalent binding scheme in SPR. In conclusion, invariant structural protein patterns provide a substrate for affinity maturation in the membrane-anchored HA regions, as well as the glycan shield on the membrane-distal HA top part. They can also induce high-affinity binding in antiviral CV-N to HA at two sites, and CVN2 binding is achieved at low-affinity binding sites.


Assuntos
Proteínas de Bactérias/metabolismo , Ebolavirus/metabolismo , HIV-1/metabolismo , Orthomyxoviridae/metabolismo , Polissacarídeos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas de Bactérias/farmacologia , Sítios de Ligação , Ebolavirus/imunologia , Ebolavirus/isolamento & purificação , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/isolamento & purificação , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/metabolismo , Doença pelo Vírus Ebola/virologia , Humanos , Influenza Humana/imunologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Orthomyxoviridae/imunologia , Orthomyxoviridae/isolamento & purificação , Polissacarídeos/imunologia , Ligação Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas do Envelope Viral/imunologia
15.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200430

RESUMO

The virus-host interaction requires a complex interplay between the phage strategy of reprogramming the host machinery to produce and release progeny virions, and the host defense against infection. Using RNA sequencing, we investigated the phage-host interaction to resolve the phenomenon of improved lytic development of P1vir phage in a DksA-deficient E. coli host. Expression of the ant1 and kilA P1vir genes in the wild-type host was the highest among all and most probably leads to phage virulence. Interestingly, in a DksA-deficient host, P1vir genes encoding lysozyme and holin are downregulated, while antiholins are upregulated. Gene expression of RepA, a protein necessary for replication initiating at the phage oriR region, is increased in the dksA mutant; this is also true for phage genes responsible for viral morphogenesis and architecture. Still, it seems that P1vir is taking control of the bacterial protein, sugar, and lipid metabolism in both, the wild type and dksA- hosts. Generally, bacterial hosts are reacting by activating their SOS response or upregulating the heat shock proteins. However, only DksA-deficient cells upregulate their sulfur metabolism and downregulate proteolysis upon P1vir infection. We conclude that P1vir development is enhanced in the dksA mutant due to several improvements, including replication and virion assembly, as well as a less efficient lysis.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/patogenicidade , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Interações entre Hospedeiro e Microrganismos/genética , Transcriptoma , Proteínas de Bactérias/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Virulência
16.
Nat Commun ; 12(1): 3748, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145250

RESUMO

C. difficile is a major cause of antibiotic-associated gastrointestinal infections. Two C. difficile exotoxins (TcdA and TcdB) are major virulence factors associated with these infections, and chondroitin sulfate proteoglycan 4 (CSPG4) is a potential receptor for TcdB, but its pathophysiological relevance and the molecular details that govern recognition remain unknown. Here, we determine the cryo-EM structure of a TcdB-CSPG4 complex, revealing a unique binding site spatially composed of multiple discontinuous regions across TcdB. Mutations that selectively disrupt CSPG4 binding reduce TcdB toxicity in mice, while CSPG4-knockout mice show reduced damage to colonic tissues during C. difficile infections. We further show that bezlotoxumab, the only FDA approved anti-TcdB antibody, blocks CSPG4 binding via an allosteric mechanism, but it displays low neutralizing potency on many TcdB variants from epidemic hypervirulent strains due to sequence variations in its epitopes. In contrast, a CSPG4-mimicking decoy neutralizes major TcdB variants, suggesting a strategy to develop broad-spectrum therapeutics against TcdB.


Assuntos
Antígenos/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/patogenicidade , Enterocolite Pseudomembranosa/patologia , Proteoglicanas/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antígenos/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Sítios de Ligação/fisiologia , Anticorpos Amplamente Neutralizantes/farmacologia , Microscopia Crioeletrônica , Enterocolite Pseudomembranosa/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Proteoglicanas/genética
17.
Methods Mol Biol ; 2268: 159-178, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085268

RESUMO

A wealth of assays for screening GPCR activity have been developed. Biosensors that employ Förster Resonance Energy transfer (FRET) are specific and enable dynamic measurements. Moreover, FRET biosensors are ideally suited for the analysis of single living cells. The FRET biosensors described in this manuscript are entirely genetically encoded by plasmids. Here, protocols for employing FRET-based biosensors to detect G protein activity upon GPCR activation are reported. The protocols include details on the isolation of plasmids, transfection, generation of stable cell lines with the FRET biosensors, FRET ratio imaging, and data analysis.


Assuntos
Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias/química , Proteínas de Fluorescência Verde/química , Células HEK293 , Humanos , Proteínas Luminescentes/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transdução de Sinais
18.
Res Vet Sci ; 138: 1-10, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34087563

RESUMO

The lprG-p55 operon of Mycobacterium tuberculosis, M. bovis and M. avium strain D4ER has been identified as a virulence factor involved in the transport of toxic compounds. LprG is a lipoprotein that modulates the host immune response against mycobacteria, whereas P55 is an efflux pump that provides resistance to several drugs. In the present study we search for, and characterize, lprg and p55, putative virulence genes in Mycobacterium avium subsp. paratuberculosis (MAP) to generate a live-attenuated strain of MAP that may be useful in the future as live-attenuated vaccine. For this purpose, we generated and evaluated two mutants of MAP strain K10: one mutant lacking the lprG gene (ΔlprG) and the other lacking both genes lprG and p55 (ΔlprG-p55). None of the mutant strains showed altered susceptibility to first-line and second-line antituberculosis drugs or ethidium bromide, only the double mutant had two-fold increase in clarithromycin susceptibility compared with the wild-type strain. The deletion of lprG and of lprG-p55 reduced the replication of MAP in bovine macrophages; however, only the mutant in lprG-p55 grew faster in liquid media and showed reduced viability in macrophages and in a mouse model. Considering that the deletion of both genes lprG-p55, but not that of lprG alone, showed a reduced replication in vivo, we can speculate that p55 contributes to the survival of MAP in this animal model.


Assuntos
Proteínas de Bactérias/genética , Deleção de Genes , Proteínas de Membrana Transportadoras/genética , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/patogenicidade , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/metabolismo , Bovinos , Feminino , Macrófagos/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Óperon , Virulência/genética , Fatores de Virulência/metabolismo
19.
Res Vet Sci ; 138: 109-115, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34126449

RESUMO

Streptococcus agalactiae (GBS) is an important pathogen that has increasingly received attention for its role in invasive infections and its broad host range. Research on the regulation of gene expression could illuminate GBS pathogenesis. We previously identified a novel transcriptional regulator XtgS, which is a negative regulator of GBS pathogenicity. Here, we demonstrate that XtgS overexpression significantly attenuated GBS virulence in zebrafish infection tests, and XtgS indirectly downregulated the transcription of two iron transport systems based on the results of transcriptomic analysis, electrophoretic mobility shift assays (EMSAs) and lacZ fusion assays. Subsequent studies verified that the inactivation of iron transport system 1 resulted in GBS excessive iron accumulation and attenuated virulence. Thus, we infer that the downregulation of iron transport system 1 caused by XtgS overexpression probably attenuates bacterial virulence, which partially clarifies the mechanism by which XtgS alleviates the pathogenesis. These findings provide new insights into the relationship between exogenous transcriptional regulation and bacterial virulence.


Assuntos
Proteínas de Bactérias/genética , Doenças dos Peixes/microbiologia , Ferro/metabolismo , Infecções Estreptocócicas/veterinária , Streptococcus agalactiae/genética , Streptococcus agalactiae/patogenicidade , Fatores de Transcrição/genética , Peixe-Zebra , Animais , Proteínas de Bactérias/metabolismo , Infecções Estreptocócicas/microbiologia , Fatores de Transcrição/metabolismo , Virulência/genética
20.
Int J Biol Macromol ; 183: 1861-1870, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34089758

RESUMO

Tyrosinase (Ty) and catechol oxidase (CO) are members of type-3 copper enzymes. While Ty catalyzes both phenolase and catecholase reactions, CO catalyzes only the latter reaction. In the present study, Ty was found to catalyze the catecholase reaction, but hardly the phenolase reaction in the presence of the metallochaperon called "caddie protein (Cad)". The ability of the substrates to dissociate the motif shielding the active-site pocket seems to contribute critically to the substrate specificity of Ty. In addition, a mutation at the N191 residue, which forms a hydrogen bond with a water molecule near the active center, decreased the inherent ratio of phenolase versus catecholase activity. Unlike the wild-type complex, reaction intermediates were not observed when the catalytic reaction toward the Y98 residue of Cad was progressed in the crystalline state. The increased basicity of the water molecule may be necessary to inhibit the proton transfer from the conjugate acid to a hydroxide ion bridging the two copper ions. The deprotonation of the substrate hydroxyl by the bridging hydroxide seems to be significant for the efficient catalytic cycle of the phenolase reaction.


Assuntos
Catecol Oxidase/química , Catecol Oxidase/metabolismo , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Catecol Oxidase/genética , Cristalografia por Raios X , Ligação de Hidrogênio , Metalochaperonas/metabolismo , Modelos Moleculares , Monofenol Mono-Oxigenase/genética , Mutação , Ligação Proteica , Conformação Proteica , Streptomyces/genética , Especificidade por Substrato , Água/química
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