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1.
Infect Immun ; 88(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31712269

RESUMO

Helicobacter pylori colonizes the stomach in about half of the world's population. H. pylori strains containing the cag pathogenicity island (cag PAI) are associated with a higher risk of gastric adenocarcinoma or peptic ulcer disease than cag PAI-negative strains. The cag PAI encodes a type IV secretion system (T4SS) that mediates delivery of the CagA effector protein as well as nonprotein bacterial constituents into gastric epithelial cells. H. pylori-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and interleukin-8 (IL-8) secretion are attributed to T4SS-dependent delivery of lipopolysaccharide metabolites and peptidoglycan into host cells, and Toll-like receptor 9 (TLR9) activation is attributed to delivery of bacterial DNA. In this study, we analyzed the bacterial energetic requirements associated with these cellular alterations. Mutant strains lacking Cagα, Cagß, or CagE (putative ATPases corresponding to VirB11, VirD4, and VirB4 in prototypical T4SSs) were capable of T4SS core complex assembly but defective in CagA translocation into host cells. Thus, the three Cag ATPases are not functionally redundant. Cagα and CagE were required for H. pylori-induced NF-κB activation, IL-8 secretion, and TLR9 activation, but Cagß was dispensable for these responses. We identified putative ATP-binding motifs (Walker-A and Walker-B) in each of the ATPases and generated mutant strains in which these motifs were altered. Each of the Walker box mutant strains exhibited properties identical to those of the corresponding deletion mutant strains. These data suggest that Cag T4SS-dependent delivery of nonprotein bacterial constituents into host cells occurs through mechanisms different from those used for recruitment and delivery of CagA into host cells.


Assuntos
Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Epiteliais/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo , Transporte Biológico , DNA Bacteriano/metabolismo , Humanos , Interleucina-8/metabolismo , Lipopolissacarídeos/metabolismo , NF-kappa B/metabolismo , Peptidoglicano/metabolismo , Receptor Toll-Like 9/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
2.
PLoS Pathog ; 15(9): e1007651, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31513674

RESUMO

Bacterial type IV secretion systems (T4SS) are a highly diversified but evolutionarily related family of macromolecule transporters that can secrete proteins and DNA into the extracellular medium or into target cells. It was recently shown that a subtype of T4SS harboured by the plant pathogen Xanthomonas citri transfers toxins into target cells. Here, we show that a similar T4SS from the multi-drug-resistant opportunistic pathogen Stenotrophomonas maltophilia is proficient in killing competitor bacterial species. T4SS-dependent duelling between S. maltophilia and X. citri was observed by time-lapse fluorescence microscopy. A bioinformatic search of the S. maltophilia K279a genome for proteins containing a C-terminal domain conserved in X. citri T4SS effectors (XVIPCD) identified twelve putative effectors and their cognate immunity proteins. We selected a putative S. maltophilia effector with unknown function (Smlt3024) for further characterization and confirmed that it is indeed secreted in a T4SS-dependent manner. Expression of Smlt3024 in the periplasm of E. coli or its contact-dependent delivery via T4SS into E. coli by X. citri resulted in reduced growth rates, which could be counteracted by expression of its cognate inhibitor Smlt3025 in the target cell. Furthermore, expression of the VirD4 coupling protein of X. citri can restore the function of S. maltophilia ΔvirD4, demonstrating that effectors from one species can be recognized for transfer by T4SSs from another species. Interestingly, Smlt3024 is homologous to the N-terminal domain of large Ca2+-binding RTX proteins and the crystal structure of Smlt3025 revealed a topology similar to the iron-regulated protein FrpD from Neisseria meningitidis which has been shown to interact with the RTX protein FrpC. This work expands our current knowledge about the function of bacteria-killing T4SSs and increases the panel of effectors known to be involved in T4SS-mediated interbacterial competition, which possibly contribute to the establishment of S. maltophilia in clinical and environmental settings.


Assuntos
Proteínas de Bactérias/fisiologia , Stenotrophomonas maltophilia/fisiologia , Stenotrophomonas maltophilia/patogenicidade , Sistemas de Secreção Tipo IV/fisiologia , Sequência de Aminoácidos , Antibiose/genética , Antibiose/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência Conservada , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Genes Bacterianos , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Proteínas Reguladoras do Ferro/química , Proteínas Reguladoras do Ferro/genética , Proteínas Reguladoras do Ferro/fisiologia , Modelos Moleculares , Infecções Oportunistas/microbiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Stenotrophomonas maltophilia/genética , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/genética , Xanthomonas/genética , Xanthomonas/crescimento & desenvolvimento
3.
Vet Res ; 50(1): 43, 2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31164171

RESUMO

Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Genetic analyses suggest that this pathogen has a novel protein secretion system, known as the "type IX secretion system" (T9SS). We previously reported that deletion of the AS87_RS08465 gene significantly reduced the bacterial virulence of the R. anatipestifer strain Yb2, but the mechanism remained unclear. The AS87_RS08465 gene is predicted to encode the gliding motility protein GldM (GldM) protein, a key component of the T9SS complex. In this study, Western blotting analysis demonstrated that R. anatipestifer GldM was localized to the cytomembrane. Further study revealed that the adhesion and invasion capacities of the mutant strain RA2281 (designated Yb2ΔgldM) in Vero cells and the bacterial loads in the blood of infected ducks were significantly reduced. RNA-Seq and PCR analyses showed that six genes were upregulated and five genes were downregulated in the mutant strain Yb2ΔgldM and that these genes were mainly involved in the secretion of proteins. Yb2ΔgldM was also found to be defective in gliding motility and protein secretion. Liquid chromatography-tandem mass spectrometry analysis revealed that nine of the proteins had a conserved T9SS C-terminal domain and were differentially secreted by Yb2ΔgldM compared to Yb2. The complementation strain cYb2ΔgldM recovered the adhesion and invasion capacities in Vero cells and the bacterial loads in the blood of infected ducks as well as the bacterial gliding motility and most protein secretion in the mutant strain Yb2ΔgldM to the levels of the wild-type strain Yb2. Taken together, these results indicate that R. anatipestifer GldM is associated with T9SS and is important in bacterial virulence.


Assuntos
Aderência Bacteriana/genética , Expressão Gênica , Riemerella/genética , Riemerella/patogenicidade , Sistemas de Secreção Tipo IV/genética , Mutação , Peptídeo Hidrolases/biossíntese , Riemerella/enzimologia , Sistemas de Secreção Tipo IV/metabolismo , Virulência/genética , Fatores de Virulência/genética
4.
mBio ; 10(3)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088930

RESUMO

Helicobacter pylori colonizes about half of humans worldwide, and its presence in the gastric mucosa is associated with an increased risk of gastric adenocarcinoma, gastric lymphoma, and peptic ulcer disease. H. pylori strains carrying the cag pathogenicity island (cagPAI) are associated with increased risk of disease progression. The cagPAI encodes the Cag type IV secretion system (CagT4SS), which delivers the CagA oncoprotein and other effector molecules into human gastric epithelial cells. We visualized structures of native and mutant CagT4SS machines on the H. pylori cell envelope by cryoelectron tomography. Individual H. pylori cells contain multiple CagT4SS nanomachines, each composed of a wheel-shaped outer membrane complex (OMC) with 14-fold symmetry and an inner membrane complex (IMC) with 6-fold symmetry. CagX, CagY, and CagM are required for assembly of the OMC, whereas strains lacking Cag3 and CagT produce outer membrane complexes lacking peripheral components. The IMC, which has never been visualized in detail, is configured as six tiers in cross-section view and three concentric rings surrounding a central channel in end-on view. The IMC contains three T4SS ATPases: (i) VirB4-like CagE, arranged as a hexamer of dimers at the channel entrance; (ii) a hexamer of VirB11-like Cagα, docked at the base of the CagE hexamer; and (iii) VirD4-like Cagß and other unspecified Cag subunits, associated with the stacked CagE/Cagα complex and forming the outermost rings. The CagT4SS and recently solved Legionella pneumophila Dot/Icm system comprise new structural prototypes for the T4SS superfamily.IMPORTANCE Bacterial type IV secretion systems (T4SSs) have been phylogenetically grouped into two subfamilies. The T4ASSs, represented by the Agrobacterium tumefaciens VirB/VirD4T4SS, include "minimized" machines assembled from 12 VirB- and VirD4-like subunits and compositionally larger systems such as the Helicobacter pylori CagT4SS T4BSSs encompass systems closely related in subunit composition to the Legionella pneumophila Dot/IcmT4SS Here, we present structures of native and mutant H. pylori Cag machines determined by in situ cryoelectron tomography. We identify distinct outer and inner membrane complexes and, for the first time, visualize structural contributions of all three "signature" ATPases of T4SSs at the cytoplasmic entrance of the translocation channel. Despite their evolutionary divergence, the CagT4SS aligns structurally much more closely to the Dot/IcmT4SS than an available VirB/VirD4 subcomplex. Our findings highlight the diversity of T4SSs and suggest a structural classification scheme in which T4SSs are grouped as minimized VirB/VirD4-like or larger Cag-like and Dot/Icm-like systems.


Assuntos
Proteínas de Bactérias/genética , Helicobacter pylori/genética , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/ultraestrutura , Antígenos de Bactérias/genética , Microscopia Crioeletrônica , Ilhas Genômicas , Humanos
5.
Vet Microbiol ; 231: 93-99, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30955831

RESUMO

Riemerella anatipestifer is an important pathogen of waterfowl, causing septicemic and exudative diseases. In our previous study, we demonstrated that the deletion of the AS87_08785 gene significantly reduced the virulence of R. anatipestifer strain Yb2, but the mechanism remained unclear. In this study, R. anatipestifer strains with mutated or complemented AS87_08785 genes were constructed and characterized. A sequence analysis indicated that the AS87_08785 gene encoded a putative GldK protein, which localized to the membrane fraction in a western blotting analysis. The mutant strain Yb2ΔgldK displayed defective gliding motility on agar plates, reduced protease activity, and a reduced capacity for protein secretion. RNA sequencing and quantitative PCR analyses indicated that the transcription of 13 genes was downregulated in mutant Yb2ΔgldK. Animal experiments showed that the bacterial loads in the blood of Yb2ΔgldK-infected ducks were significantly reduced relative to those in wild-type strain Yb2 infected ducks. Most of the defective biological properties of the mutant were restored in complementation strain cYb2ΔgldK. Our results demonstrated that R. anatipestifer gene AS87_08785 encoded a component of the type IX secretion system, GldK, which functioned in bacterial gliding motility, protein secretion, and bacterial virulence.


Assuntos
Infecções por Flavobacteriaceae/veterinária , Doenças das Aves Domésticas/microbiologia , Riemerella/genética , Sistemas de Secreção Tipo IV/genética , Animais , Aderência Bacteriana , Carga Bacteriana , Patos/microbiologia , Expressão Gênica , Mutação , Peptídeo Hidrolases/biossíntese , Reação em Cadeia da Polimerase , Riemerella/enzimologia , Análise de Sequência de RNA , Sistemas de Secreção Tipo IV/metabolismo , Virulência/genética , Fatores de Virulência/genética
6.
PLoS Comput Biol ; 15(3): e1006847, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30908487

RESUMO

Bacterial pathogens have evolved numerous strategies to corrupt, hijack or mimic cellular processes in order to survive and proliferate. Among those strategies, Type IV effectors (T4Es) are proteins secreted by pathogenic bacteria to manipulate host cell processes during infection. They are delivered into eukaryotic cells in an ATP-dependent manner via the type IV secretion system, a specialized multiprotein complex. T4Es contain a wide spectrum of features including eukaryotic-like domains, localization signals or a C-terminal translocation signal. A combination of these features enables prediction of T4Es in a given bacterial genome. In this study, we developed a web-based comprehensive suite of tools with a user-friendly graphical interface. This version 2.0 of S4TE (Searching Algorithm for Type IV Effector Proteins; http://sate.cirad.fr) enables accurate prediction and comparison of T4Es. Search parameters and threshold can be customized by the user to work with any genome sequence, whether publicly available or not. Applications range from characterizing effector features and identifying potential T4Es to analyzing the effectors based on the genome G+C composition and local gene density. S4TE 2.0 allows the comparison of putative T4E repertoires of up to four bacterial strains at the same time. The software identifies T4E orthologs among strains and provides a Venn diagram and lists of genes for each intersection. New interactive features offer the best visualization of the location of candidate T4Es and hyperlinks to NCBI and Pfam databases. S4TE 2.0 is designed to evolve rapidly with the publication of new experimentally validated T4Es, which will reinforce the predictive power of the algorithm. The computational methodology can be used to identify a wide spectrum of candidate bacterial effectors that lack sequence conservation but have similar amino acid characteristics. This approach will provide very valuable information about bacterial host-specificity and virulence factors and help identify host targets for the development of new anti-bacterial molecules.


Assuntos
Algoritmos , Genoma Bacteriano/genética , Genômica/métodos , Proteobactérias/genética , Sistemas de Secreção Tipo IV/genética , DNA Bacteriano/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Interface Usuário-Computador
7.
Curr Microbiol ; 76(4): 510-519, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30805699

RESUMO

Brucellosis is the most common zoonotic disease that caused by intracellular parasitic bacteria Brucella. The survival and replication of Brucella in the host depend on the type IV secretion system (T4SS). The T4SS system of Brucella has many components and secreted proteins. But the mechanism helped Brucella to evade the host defense is still not clear. The objective of the present study was to investigate the effects of VceA on autophagy and apoptosis in Brucella-infected embryonic trophoblast cells. We constructed the VceA mutant strain (2308ΔVceA) and complementary strain (2308ΔVceA-C) of Brucella abortus 2308 (S2308). The human trophoblast cells (HPT-8 cells) and mice were infected by S2308, 2308ΔVceA and 2308ΔVceA-C. The cell autophagy and apoptosis were detected. The Atg5, LC3-II and Bcl-2 mRNA expression were significantly increased in 2308ΔVceA group than the S2308 group, and mRNA expression of P62 and Caspase-3 were significantly decreased than the S2308 group. Western blotting, qPCR and flow cytometry analysis showed that 2308ΔVceA promoted autophagy and inhibited apoptosis. Mouse immunohistochemistry experiments showed that P62 protein was scattered coloring and Cytochrome C protein was scarcely in 2308ΔVceA group at the myometrium. These results indicated that 2308ΔVceA promoted autophagy and inhibited apoptosis in HPT-8 cells during Brucella infection.


Assuntos
Apoptose/genética , Proteínas de Bactérias/genética , Brucella abortus/genética , Brucelose/microbiologia , Brucelose/patologia , Trofoblastos/patologia , Sistemas de Secreção Tipo IV/genética , Animais , Autofagia/genética , Proteínas de Bactérias/metabolismo , Brucella abortus/metabolismo , Brucelose/metabolismo , Linhagem Celular , Feminino , Teste de Complementação Genética , Humanos , Imuno-Histoquímica , Camundongos Endogâmicos BALB C , Miométrio/metabolismo , Miométrio/patologia , RNA Mensageiro/metabolismo , Deleção de Sequência , Trofoblastos/metabolismo
8.
Arch Microbiol ; 201(3): 377-388, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30683956

RESUMO

Metagenomic surveys across microbial mat (~ 55 °C) samples of high-altitude (1760 m above sea level) Himalayan geothermal springs have revealed specialized community enriched with niche-specific functions. In this study, we have performed metagenomic sequence-based analyses to get insights into taxonomic composition and functional potential of hyperthermophiles in water (~ 95 °C) and sediment samples (78-98 °C). Community analyses revealed predominance of thermophilic bacterial and archeal genera dwelling in water in contrast to microbial mats (55 °C), namely Methylophilus, Methyloversatilis, Emticicia, Caulobacter, Thermus, Enhydrobacter and Pyrobaculum. Sediment samples having surface temperature (~ 78 °C) were colonized by Pyrobaculum and Chloroflexus while genus Massilia was found to be inhabited in high-temperature sediments (~ 98 °C). Functional analyses of metagenomic sequences revealed genetic enrichment of genes such as type IV secretion system, flagellar assembly and two-component system in contrast to mats. Furthermore, inter-sample comparison of enriched microbial diversity among water, sediment and microbial mats revealed habitat-specific clustering of the samples within same environment highlighting the role of temperature dynamics in modulating community structure across different habitats in same niche. However, function-based analysis demonstrated site-specific clustering among sediment, microbial mat and water samples. Furthermore, a novel thermophilic genotype of the genus Emticicia (designated as strain MM) was reconstructed from metagenome data. This is a correlative study between three major habitats present in geothermal spring environment, i.e., water, sediment and microbial mats revealing greater phylogenetic and functional dispersion emphasizing changing habitat-specific dynamics with temperature.


Assuntos
Archaea/genética , Bactérias/genética , Sedimentos Geológicos/microbiologia , Fontes Termais/microbiologia , Metagenoma/genética , Microbiota/genética , Altitude , Archaea/classificação , Bactérias/classificação , Flagelos/genética , Temperatura Alta , Índia , Metagenômica/métodos , Filogenia , Temperatura Ambiente , Sistemas de Secreção Tipo IV/genética
9.
PLoS One ; 14(1): e0202312, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30682021

RESUMO

Type IV secretion systems exist in a number of bacterial pathogens and are used to secrete effector proteins directly into host cells in order to change their environment making the environment hospitable for the bacteria. In recent years, several machine learning algorithms have been developed to predict effector proteins, potentially facilitating experimental verification. However, inconsistencies exist between their results. Previously we analysed the disparate sets of predictive features used in these algorithms to determine an optimal set of 370 features for effector prediction. This study focuses on the best way to use these optimal features by designing three machine learning classifiers, comparing our results with those of others, and obtaining de novo results. We chose the pathogen Legionella pneumophila strain Philadelphia-1, a cause of Legionnaires' disease, because it has many validated effector proteins and others have developed machine learning prediction tools for it. While all of our models give good results indicating that our optimal features are quite robust, Model 1, which uses all 370 features with a support vector machine, has slightly better accuracy. Moreover, Model 1 predicted 472 effector proteins that are deemed highly probable to be effectors and include 94% of known effectors. Although the results of our three models agree well with those of other researchers, their models only predicted 126 and 311 candidate effectors.


Assuntos
Proteínas de Bactérias/genética , Legionella pneumophila/genética , Modelos Genéticos , Máquina de Vetores de Suporte , Sistemas de Secreção Tipo IV/genética , Fatores de Virulência/genética , Humanos , Doença dos Legionários/genética
10.
J Biol Chem ; 294(13): 4806-4814, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30692196

RESUMO

Mycobacteria use type VII secretion systems to secrete proteins across their highly hydrophobic diderm cell envelope. Pathogenic mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium marinum, have up to five of these systems, named ESX-1 to ESX-5. Most of these systems contain a set of five conserved membrane components, of which the four Ecc proteins form the core membrane-embedded secretion complex. The fifth conserved membrane protein, mycosin protease (MycP), is not part of the core complex but is essential for secretion, as it stabilizes this membrane complex. Here we investigated which MycP domains are required for this stabilization by producing hybrid constructs between MycP1 and MycP5 in M. marinum and analyzed their effect on ESX-1 and ESX-5 secretion. We found that both the protease and transmembrane domain are required for the ESX system-specific function of mycosins. In addition, we observed that the transmembrane domain strongly affects MycP protein levels. We also show that the extended loops 1 and 2 in the protease domain are probably primarily involved in MycP stability, whereas loop 3 and the MycP5-specific loop 5 are dispensable. The atypical propeptide, or N-terminal extension, is required only for MycP stability. Finally, we show that the protease domain of MycPP1, encoded by the esx-P1 locus on the pRAW plasmid, is functionally redundant to the protease domain of MycP5 These results provide the first insight into the regions of mycosins involved in interaction with and stabilization of their respective ESX complexes.


Assuntos
Proteínas de Bactérias , Mycobacterium marinum , Mycobacterium tuberculosis , Subtilisinas , Sistemas de Secreção Tipo IV , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mycobacterium marinum/enzimologia , Mycobacterium marinum/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Subtilisinas/química , Subtilisinas/genética , Subtilisinas/metabolismo , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo
11.
Biochem Cell Biol ; 97(3): 215-223, 2019 06.
Artigo em Francês | MEDLINE | ID: mdl-30142282

RESUMO

Agrobacterium tumefaciens is a well studied phytopathogen given its various applications for deciphering host-pathogen interactions, bacterial communication, and capacity to transfer DNA fragments into host cells via a membrane protein system, the type IV secretion system (T4SS). T4SS mechanism is similar to the one responsible for antibiotic resistance gene transmission, and new knowledge gained could be applied to other organisms using such a mechanism. As well, A. tumefaciens is of economic importance in biotechnology due to its capacity to generate genetically modified plants. Agrobacterium tumefaciens harbours a plasmid known as Ti plasmid encoding T4SS function genes used for transferring genetic information and plant colonization. In this review, the authors describe the molecular basis of infection, from detection of host signals, to the description of different regions of Ti plasmid key to infection, ending with substrate transfer through bacterial wall. [Journal translation].


Assuntos
Agrobacterium tumefaciens/metabolismo , Doenças das Plantas , Sistemas de Secreção Tipo IV/metabolismo , Interações Hospedeiro-Parasita , Doenças das Plantas/genética , Sistemas de Secreção Tipo IV/genética
12.
Mol Microbiol ; 111(1): 254-268, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30338585

RESUMO

Clusters of Neisseria meningitidis (Nm) urethritis among primarily heterosexual males in multiple US cities have been attributed to a unique non-encapsulated meningococcal clade (the US Nm urethritis clade, US_NmUC) within the hypervirulent clonal complex 11. Resistance to antimicrobial peptides (AMPs) is a key feature of urogenital pathogenesis of the closely related species, Neisseria gonorrhoeae. The US_NmUC isolates were found to be highly resistant to the model AMP, polymyxin B (PmB, MICs 64-256 µg ml-1 ). The isolates also demonstrated stable subpopulations of heteroresistant colonies that showed near total resistant to PmB (MICs 384-1024 µg ml-1 ) and colistin (MIC 256 µg ml-1 ) as well as enhanced LL-37 resistance. This is the first observation of heteroresistance in N. meningitidis. Consistent with previous findings, overall PmB resistance in US_NmUC isolates was due to active Mtr efflux and LptA-mediated lipid A modification. However, whole genome sequencing, variant analyses and directed mutagenesis revealed that the heteroresistance phenotypes and very high-level AMP resistance were the result of point mutations and IS1655 element movement in the pilMNOPQ operon, encoding the type IV pilin biogenesis apparatus. Cross-resistance to other classes of antibiotics was also observed in the heteroresistant colonies. High-level resistance to AMPs may contribute to the pathogenesis of US_NmUC.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Proteínas de Fímbrias/genética , Mutação , Neisseria meningitidis/efeitos dos fármacos , Polimixina B/farmacologia , Uretrite/microbiologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Cidades/epidemiologia , Colistina/farmacologia , Heterossexualidade , Humanos , Masculino , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Neisseria meningitidis/isolamento & purificação , Óperon , Sistemas de Secreção Tipo IV/genética , Estados Unidos/epidemiologia , Sequenciamento Completo do Genoma
13.
Microb Pathog ; 126: 343-350, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30468852

RESUMO

Type IV secretion system (T4SS) is a specialized nanomachine that is utilized for the pathogenicity of gram-negative bacteria. However, the role of T4SS genes in virulence of rice bacterial brown stripe pathogen Acidovorax oryzae (Ao) strain RS-2 is not clear, which contains T4SS gene cluster based on genome-wide analysis. Here we compared the virulence-related phenotypes between the wild-type strain RS-2 and nine T4SS mutants, which were constructed in this study. Results indicated that mutation of pilT, pilM, pilQ, or pilZ3 genes not only significantly reduced bacterial virulence, but also caused a reduction of 20.4-62.0% in biofilm formation and 37.7-47.7% reduction in motility, but had no effect on exopolysaccharide (EPS) production or extracellular enzymatic activities when compared to the wild type. The four T4SS genes had a differential effect on bacterial growth after 24 h post-incubation. The complemented strains of the four T4SS mutants restored similar virulence symptom as the wild type. In addition, no change was observed in bacterial virulence by mutation of the other five T4SS genes. Totally, these results demonstrated that T4SS played vital roles in bacterial virulence, motility and biofilm formation in plant pathogen Ao strain RS-2.


Assuntos
Comamonadaceae/genética , Comamonadaceae/patogenicidade , Genes Bacterianos/genética , Oryza/microbiologia , Doenças das Plantas/microbiologia , Sistemas de Secreção Tipo IV/genética , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Comamonadaceae/efeitos dos fármacos , Comamonadaceae/crescimento & desenvolvimento , DNA Bacteriano/genética , Tolerância a Medicamentos , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Peróxido de Hidrogênio/farmacologia , Família Multigênica , Mutação , Sistemas de Secreção Tipo IV/metabolismo , Virulência/genética
14.
Helicobacter ; 24(1): e12547, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30440093

RESUMO

BACKGROUND: Despite recent advances in studies on the gastric microbiome, the role of the non-Helicobacter pylori gastric microbiome in gastric carcinogenesis remains unclear. We evaluated the characteristics of the gastric microbiome and metagenomic functions in patients with IM. METHODS: Participants were classified into six groups according to disease status (chronic superficial gastritis [CSG], intestinal metaplasia [IM], and cancer) and H. pylori- infection status (H. pylori-positive and H. pylori-negative). The gastric microbiome was analyzed in mucosal tissues at the gastric antrum by 16S rRNA gene sequencing. Moreover, we assessed the metagenome including the type IV secretion system (T4SS) gene, as T4SS proteins are essential for transferring CagA from H. pylori- into the human gastric epithelium. RESULTS: Among the 138 included patients, 48, 9, 23, 14, 12, and 32 were classified into the H. pylori-negative CSG, H. pylori-negative IM, H. pylori-negative cancer, H. pylori-positive CSG, H. pylori-positive IM, and H. pylori-positive cancer groups, respectively. Cyanobacteria were predominant in the H. pylori-negative CSG group compared to in the H. pylori-negative IM and H. pylori-negative cancer groups (H. pylori-negative CSG vs H. pylori-negative IM vs H. pylori-negative cancer: 14.0% vs 4.2% vs 0.04%, P < 0.001). In contrast, Rhizobiales were commonly observed in the H. pylori-negative IM group (H. pylori-negative CSG vs H. pylori-negative IM vs H. pylori-negative cancer: 1.9% vs 15.4% vs 2.8%, P < 0.001). The relative abundance of Rhizobiales increased as H. pylori-infected stomachs progressed from gastritis to IM. In the H. pylori-negative IM group, genes encoding T4SS were prevalent among the metagenome. Additionally, after H. pylori- eradication therapy, the gastric microbiome was similar to the microbiome observed after spontaneous clearance of H. pylori-. CONCLUSIONS: The relative abundance of Rhizobiales was higher in patients with H. pylori-negative IM than in those with H. pylori-negative CSG or cancer. Additionally, T4SS genes were highly observed in the metagenome of patients with IM. Highly abundant T4SS proteins in these patients may promote gastric carcinogenesis.


Assuntos
Microbioma Gastrointestinal/genética , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Intestinos/microbiologia , Intestinos/patologia , Metaplasia/microbiologia , Adulto , Idoso , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Progressão da Doença , Feminino , Gastrite/microbiologia , Gastrite/patologia , Gastrite Atrófica/tratamento farmacológico , Gastrite Atrófica/microbiologia , Gastrite Atrófica/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Humanos , Masculino , Metagenômica , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Sistemas de Secreção Tipo IV/genética , Adulto Jovem
15.
Mol Microbiol ; 111(3): 732-749, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30561149

RESUMO

Type 4a pili (T4aP) are long, thin and dynamic fibres displayed on the surface of diverse bacteria promoting adherence, motility and transport functions. Genomes of many Enterobacteriaceae contain conserved gene clusters encoding putative T4aP assembly systems. However, their expression has been observed only in few strains including Enterohaemorrhagic Escherichia coli (EHEC) and their inducers remain unknown. Here we used EHEC genomic DNA as a template to amplify and assemble an artificial operon composed of four gene clusters encoding 13 pilus assembly proteins. Controlled expressions of this operon in nonpathogenic E. coli strains led to efficient assembly of T4aP composed of the major pilin PpdD, as shown by shearing assays and immunofluorescence microscopy. When compared with PpdD pili assembled in a heterologous Klebsiella T2SS type 2 secretion system (T2SS) by using cryo-electron microscopy (cryoEM), these pili showed indistinguishable helical parameters, emphasizing that major pilins are the principal determinants of the fibre structure. Bacterial two-hybrid analysis identified several interactions of PpdD with T4aP assembly proteins, and with components of the T2SS that allow for heterologous fibre assembly. These studies lay ground for further characterization of the T4aP structure, function and biogenesis in enterobacteria.


Assuntos
Escherichia coli Êntero-Hemorrágica/metabolismo , Fímbrias Bacterianas/metabolismo , Sistemas de Secreção Tipo IV/metabolismo , Microscopia Crioeletrônica , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/ultraestrutura , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/ultraestrutura , Klebsiella/genética , Klebsiella/metabolismo , Microscopia de Fluorescência , Ligação Proteica , Mapeamento de Interação de Proteínas , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/ultraestrutura
16.
Artigo em Inglês | MEDLINE | ID: mdl-30460204

RESUMO

Enteric redmouth disease caused by the pathogen Yersinia ruckeri is a significant problem for fish farming around the world. Despite its importance, only a few virulence factors of Y. ruckeri have been identified and studied in detail. Here, we report and analyze the complete DNA sequence of pYR4, a plasmid from a highly pathogenic Norwegian Y. ruckeri isolate, sequenced using PacBio SMRT technology. Like the well-known pYV plasmid of human pathogenic Yersiniae, pYR4 is a member of the IncFII family. Thirty-one percent of the pYR4 sequence is unique compared to other Y. ruckeri plasmids. The unique regions contain, among others genes, a large number of mobile genetic elements and two partitioning systems. The G+C content of pYR4 is higher than that of the Y. ruckeri NVH_3758 genome, indicating its relatively recent horizontal acquisition. pYR4, as well as the related plasmid pYR3, comprises operons that encode for type IV pili and for a conjugation system (tra). In contrast to other Yersinia plasmids, pYR4 cannot be cured at elevated temperatures. Our study highlights the power of PacBio sequencing technology for identifying mis-assembled segments of genomic sequences. Comparative analysis of pYR4 and other Y. ruckeri plasmids and genomes, which were sequenced by second and the third generation sequencing technologies, showed errors in second generation sequencing assemblies. Specifically, in the Y. ruckeri 150 and Y. ruckeri ATCC29473 genome assemblies, we mapped the entire pYR3 plasmid sequence. Placing plasmid sequences on the chromosome can result in erroneous biological conclusions. Thus, PacBio sequencing or similar long-read methods should always be preferred for de novo genome sequencing. As the tra operons of pYR3, although misplaced on the chromosome during the genome assembly process, were demonstrated to have an effect on virulence, and type IV pili are virulence factors in many bacteria, we suggest that pYR4 directly contributes to Y. ruckeri virulence.


Assuntos
Fímbrias Bacterianas/genética , Doenças dos Peixes/microbiologia , Plasmídeos/análise , Sistemas de Secreção Tipo IV/genética , Yersiniose/veterinária , Yersinia ruckeri/genética , Yersinia ruckeri/isolamento & purificação , Animais , Composição de Bases , Anotação de Sequência Molecular , Noruega , Plasmídeos/classificação , Salmão , Análise de Sequência de DNA , Yersiniose/microbiologia
17.
Int J Mol Sci ; 19(11)2018 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-30428531

RESUMO

Protein ubiquitylation plays a central role in eukaryotic cell physiology. It is involved in several regulatory processes, ranging from protein folding or degradation, subcellular localization of proteins, vesicular trafficking and endocytosis to DNA repair, cell cycle, innate immunity, autophagy, and apoptosis. As such, it is reasonable that pathogens have developed a way to exploit such a crucial system to enhance their virulence against the host. Hence, bacteria have evolved a wide range of effectors capable of mimicking the main players of the eukaryotic ubiquitin system, in particular ubiquitin ligases, by interfering with host physiology. Here, we give an overview of this topic and, in particular, we detail and discuss the mechanisms developed by pathogenic bacteria to hijack the host ubiquitination system for their own benefit.


Assuntos
Sistemas de Secreção Tipo III/metabolismo , Sistemas de Secreção Tipo IV/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Humanos , Modelos Biológicos , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo IV/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética
18.
Nat Microbiol ; 3(12): 1429-1440, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30349081

RESUMO

Type IV secretion (T4S) systems form the most common and versatile class of secretion systems in bacteria, capable of injecting both proteins and DNAs into host cells. T4S systems are typically composed of 12 components that form 2 major assemblies: the inner membrane complex embedded in the inner membrane and the core complex embedded in both the inner and outer membranes. Here we present the 3.3 Å-resolution cryo-electron microscopy model of the T4S system core complex from Xanthomonas citri, a phytopathogen that utilizes this system to kill bacterial competitors. An extensive mutational investigation was performed to probe the vast network of protein-protein interactions in this 1.13-MDa assembly. This structure expands our knowledge of the molecular details of T4S system organization, assembly and evolution.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/química , Microscopia Crioeletrônica/métodos , Complexos Multiproteicos/química , Sistemas de Secreção Tipo IV/química , Xanthomonas/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Complexos Multiproteicos/genética , Mutação , Ligação Proteica , Conformação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Sistemas de Secreção Tipo IV/genética , Xanthomonas/genética
19.
PLoS One ; 13(9): e0204736, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30261054

RESUMO

Wolbachia is an unculturable, intracellular bacterium that persists within an extremely broad range of arthropod and parasitic nematode hosts, where it is transmitted maternally to offspring via vertical transmission. In the filarial nematode Brugia malayi, a causative agent of human lymphatic filariasis, Wolbachia is an endosymbiont, and its presence is essential for proper nematode development, survival, and pathogenesis. While the elucidation of Wolbachia:nematode interactions that promote the bacterium's intracellular persistence is of great importance, research has been hampered due to the fact that Wolbachia cannot be cultured in the absence of host cells. The Wolbachia endosymbiont of B. malayi (wBm) has an active Type IV secretion system (T4SS). Here, we have screened 47 putative T4SS effector proteins of wBm for their ability to modulate growth or the cell biology of a typical eukaryotic cell, Saccharomyces cerevisiae. Five candidates strongly inhibited yeast growth upon expression, and 6 additional proteins showed toxicity in the presence of zinc and caffeine. Studies on the uptake of an endocytic vacuole-specific fluorescent marker, FM4-64, identified 4 proteins (wBm0076 wBm00114, wBm0447 and wBm0152) involved in vacuole membrane dynamics. The WAS(p)-family protein, wBm0076, was found to colocalize with yeast cortical actin patches and disrupted actin cytoskeleton dynamics upon expression. Deletion of the Arp2/3-activating protein, Abp1p, provided resistance to wBm0076 expression, suggesting a role for wBm0076 in regulating eukaryotic actin dynamics and cortical actin patch formation. Furthermore, wBm0152 was found to strongly disrupt endosome:vacuole cargo trafficking in yeast. This study provides molecular insight into the potential role of the T4SS in the Wolbachia endosymbiont:nematode relationship.


Assuntos
Proteínas de Bactérias , Brugia Malayi/microbiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Simbiose , Sistemas de Secreção Tipo IV , Wolbachia , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo , Wolbachia/genética , Wolbachia/metabolismo
20.
Mol Microbiol ; 110(5): 761-776, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30230643

RESUMO

The human gastric pathogen Helicobacter pylori occurs in two basic variants, either exhibiting a functional cagPAI-encoded type-4-secretion-system (T4SS) or not. Only a few cagPAI-positive strains have been successfully adapted for long-term infection of mice, including the pre-mouse Sydney strain 1 (PMSS1). Here we confirm that PMSS1 induces gastric inflammation and neutrophil infiltration in mice, progressing to intestinal metaplasia. Complete genome analysis of PMSS1 revealed 1,423 coding sequences, encompassing the cagPAI gene cluster and, unusually, the location of the cytotoxin-associated gene A (cagA) approximately 15 kb downstream of the island. PMSS1 harbours three genetically exchangeable loci that are occupied by the hopQ coding sequences. HopQ represents a critical co-factor required for the translocation of CagA into the host cell and activation of NF-κB via the T4SS. Long-term colonisation of mice led to an impairment of cagPAI functionality. One of the bacterial clones re-isolated at four months post-infection revealed a mutation in the cagPAI gene cagW, resulting in a frame shift mutation, which prevented CagA translocation, possibly due to an impairment of T4SS function. Rescue of the mutant cagW re-established CagA translocation. Our data reveal intriguing insights into the adaptive abilities of PMSS1, suggesting functional modulation of the H. pylori cagPAI virulence attribute.


Assuntos
Proteínas de Bactérias/genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Sistemas de Secreção Tipo IV/genética , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Família Multigênica , Virulência , Sequenciamento Completo do Genoma/métodos
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