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1.
Artigo em Inglês | MEDLINE | ID: mdl-30782988

RESUMO

Vancomycin-resistant Enterococcus faecium (VREfm) is a frequent cause of nosocomial outbreaks. In the second half of 2015, a sharp increase in the incidence of VREfm was observed at our university medical center. Next-generation sequencing (NGS) was used to analyze the first isolates of VREfm recovered from patients between 2010 and 2016 (n = 773) in order to decipher epidemiological change, outbreak dynamics, and possible transmission routes. VREfm isolates were analyzed using whole-genome sequencing followed by sequence type extraction and phylogenetic analysis. We examined epidemiological data, room occupancy data, and patient transferals and calculated an intensity score for patient-to-patient contact. Phylogenetic analysis revealed the presence of 38 NGS clusters and 110 single clones. The increase of VREfm was caused mainly by the expansion of two newly introduced NGS clusters, comprising VanB-type strains determined by multilocus sequence typing (MLST) as sequence type 80 (ST80) and ST117. By combining phylogenetic information with epidemiological data, intrahospital transmission could be demonstrated, however to a lesser extent than initially expected based solely on epidemiological data. The outbreak clones were continuously imported from other hospitals, suggesting a change in the epidemiological situation at a regional scale. By tracking intrahospital patient transferals, two major axes could be identified that contributed to the spread of VREfm within the hospital. NGS-based outbreak analysis revealed a dramatic change in the local and regional epidemiology of VREfm, emphasizing the role of health care networks in the spread of VREfm.

2.
J Clin Microbiol ; 56(11)2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30135233

RESUMO

Klebsiella pneumoniae and related species are frequent causes of nosocomial infections and outbreaks. Therefore, quick and reliable strain typing is crucial for the detection of transmission routes in the hospital. The aim of this study was to evaluate Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as rapid methods for typing clinical Klebsiella isolates in comparison to whole-genome sequencing (WGS), which was considered the gold standard for typing and identification. Here, 68 clinical Klebsiella strains were analyzed by WGS, FTIR, and MALDI-TOF MS. FTIR showed high discriminatory power in comparison to the WGS reference, whereas MALDI-TOF MS exhibited a low ability to type the isolates. MALDI-TOF mass spectra were further analyzed for peaks that showed high specificity for different Klebsiella species. Phylogenetic analysis revealed that the Klebsiella isolates comprised three different species: K. pneumoniae, K. variicola, and K. quasipneumoniae Genome analysis showed that MALDI-TOF MS can be used to distinguish K. pneumoniae from K. variicola due to shifts of certain mass peaks. The peaks were tentatively identified as three ribosomal proteins (S15p, L28p, L31p) and one stress response protein (YjbJ), which exhibit amino acid differences between the two species. Overall, FTIR has high discriminatory power to recognize the clonal relationship of isolates, thus representing a valuable tool for rapid outbreak analysis and for the detection of transmission events due to fast turnaround times and low costs per sample. Furthermore, specific amino acid substitutions allow the discrimination of K. pneumoniae and K. variicola by MALDI-TOF MS.

3.
BMC Genomics ; 18(1): 859, 2017 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-29126393

RESUMO

BACKGROUND: Pseudomonas putida is a Gram-negative, non-fermenting bacterium frequently encountered in various environmental niches. P. putida rarely causes disease in humans, though serious infections and outbreaks have been reported from time to time. Some have suggested that P. putida functions as an exchange platform for antibiotic resistance genes (ARG), and thus represents a serious concern in the spread of ARGs to more pathogenic organisms within a hospital. Though poorly understood, the frequency of ARG exchange between P. putida and the more virulent Pseudomonas aeruginosa and its clinical relevance are particularly important for designing efficient infection control strategies, such as deciding whether high-risk patients colonized with a multidrug resistant but typically low pathogenic P. putida strain should be contact isolated or not. RESULTS: In this study, 21,373 screening samples (stool, rectal and throat swab) were examined to determine the presence of P. putida in a high-risk group of haemato-oncology patients during a 28-month period. A total of 89 P. putida group strains were isolated from 85 patients, with 41 of 89 (46.1%) strains harbouring the metallo-beta-lactamase gene bla VIM. These 41 clinical isolates, plus 18 bla VIM positive environmental P. putida isolates, and 17 bla VIM positive P. aeruginosa isolates, were characterized by whole genome sequencing (WGS). We constructed a maximum-likelihood tree to separate the 59 bla VIM positive P. putida group strains into eight distinct phylogenetic clusters. Bla VIM-1 was present in 6 clusters while bla VIM-2 was detected in 4 clusters. Five P. putida group strains contained both, bla VIM-1 and bla VIM-2 genes. In contrast, all P. aeruginosa strains belonged to a single genetic cluster and contained the same ARGs. Apart from bla VIM-2 and sul genes, no other ARGs were shared between P. aeruginosa and P. putida. Furthermore, the bla VIM-2 gene in P. aeruginosa was predicted to be only chromosomally located. CONCLUSION: These data provide evidence that no exchange of comprehensive ARG harbouring mobile genetic elements had occurred between P. aeruginosa and P. putida group strains during the study period, thus eliminating the need to implement enhanced infection control measures for high-risk patients colonized with a bla VIM positiv P. putida group strains in our clinical setting.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Meio Ambiente , Transferência Genética Horizontal , Genômica , Pseudomonas aeruginosa/genética , Pseudomonas putida/genética , Humanos , Filogenia , Pseudomonas putida/efeitos dos fármacos , Pseudomonas putida/fisiologia
4.
J Clin Microbiol ; 55(7): 2116-2126, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28446572

RESUMO

Bloodstream infections (BSI) are an important cause of morbidity and mortality. Increasing rates of antimicrobial-resistant pathogens limit treatment options, prompting an empirical use of broad-range antibiotics. Fast and reliable diagnostic tools are needed to provide adequate therapy in a timely manner and to enable a de-escalation of treatment. The Accelerate Pheno system (Accelerate Diagnostics, USA) is a fully automated test system that performs both identification and antimicrobial susceptibility testing (AST) directly from positive blood cultures within approximately 7 h. In total, 115 episodes of BSI with Gram-negative bacteria were included in our study and compared to conventional culture-based methods. The Accelerate Pheno system correctly identified 88.7% (102 of 115) of all BSI episodes and 97.1% (102 of 105) of isolates that are covered by the system's identification panel. The Accelerate Pheno system generated an AST result for 91.3% (95 of 104) samples in which the Accelerate Pheno system identified a Gram-negative pathogen. The overall category agreement between the Accelerate Pheno system and culture-based AST was 96.4%, the rates for minor discrepancies 1.4%, major discrepancies 2.3%, and very major discrepancies 1.0%. Of note, ceftriaxone, piperacillin-tazobactam, and carbapenem resistance was correctly detected in blood culture specimens with extended-spectrum beta-lactamase-producing Escherichia coli (n = 7) and multidrug-resistant Pseudomonas aeruginosa (n = 3) strains. The utilization of the Accelerate Pheno system reduced the time to result for identification by 27.49 h (P < 0.0001) and for AST by 40.39 h (P < 0.0001) compared to culture-based methods in our laboratory setting. In conclusion, the Accelerate Pheno system provided fast, reliable results while significantly improving turnaround time in blood culture diagnostics of Gram-negative BSI.


Assuntos
Técnicas Bacteriológicas/métodos , Sangue/microbiologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/diagnóstico , Sepse/diagnóstico , Automação Laboratorial/métodos , Bactérias Gram-Negativas/classificação , Humanos , Sensibilidade e Especificidade , Fatores de Tempo
5.
Artigo em Inglês | MEDLINE | ID: mdl-27956426

RESUMO

The metallo-beta-lactamase GIM-1 has been found in various bacterial host species nearly exclusively in western Germany. However, not much is known about the epidemiology of GIM-1-positive Serratia marcescens Here we report on a surprisingly protracted regional dissemination. In-hospital transmission was investigated by using conventional epidemiological tools to identify spatiotemporal links. Strain typing was performed using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). Bayesian phylogeny was used to infer the time axis of the observed occurrence. Thirteen S. marcescens strains from 10 patients from 6 different German hospitals were investigated. Suspected in-hospital transmissions were confirmed by molecular typing at a higher resolution by WGS than by PFGE. A detailed sequence analysis demonstrated the spread of one predominant strain variant but also provided evidence for transfer of the blaGIM-1 gene cassette between different strains. A Bayesian phylogenetic analysis showed that the most recent common ancestor of the identified clonal cluster could be dated back to April 1993 (95% highest posterior density interval, January 1973 to March 2003) and that this strain might have already harbored the blaGIM-1 at that time and, therewith, years before the first detection of this resistance gene in clinical specimens. This study shows a long-standing clonal and plasmid-mediated expansion of GIM-1-producing S. marcescens that might have gone unnoticed in the absence of a standardized and effective molecular screening for carbapenemases. The systematic and early detection of resistance is thus highly advisable, especially for the prevention of potentially long-term dissemination that may progress beyond control.


Assuntos
Infecção Hospitalar/transmissão , Genoma Bacteriano , Filogenia , Infecções por Serratia/transmissão , Serratia marcescens/genética , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Teorema de Bayes , Células Clonais , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Eletroforese em Gel de Campo Pulsado , Expressão Gênica , Genótipo , Alemanha , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Infecções por Serratia/tratamento farmacológico , Infecções por Serratia/epidemiologia , Infecções por Serratia/microbiologia , Serratia marcescens/classificação , Serratia marcescens/efeitos dos fármacos , Serratia marcescens/crescimento & desenvolvimento , beta-Lactamases/metabolismo
6.
Sci Rep ; 6: 39053, 2016 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-27982054

RESUMO

ß-Barrel proteins are found in the outer membrane (OM) of Gram-negative bacteria, chloroplasts and mitochondria. The assembly of these proteins into the corresponding OM is facilitated by a dedicated protein complex that contains a central conserved ß-barrel protein termed BamA in bacteria and Tob55/Sam50 in mitochondria. BamA and Tob55 consist of a membrane-integral C-terminal domain that forms a ß-barrel pore and a soluble N-terminal portion comprised of one (in Tob55) or five (in BamA) polypeptide transport-associated (POTRA) domains. Currently the functional significance of this difference and whether the homology between BamA and Tob55 can allow them to replace each other are unclear. To address these issues we constructed hybrid Tob55/BamA proteins with differently configured N-terminal POTRA domains. We observed that constructs harboring a heterologous C-terminal domain could not functionally replace the bacterial BamA or the mitochondrial Tob55 demonstrating species-specific requirements. Interestingly, the various hybrid proteins in combination with the bacterial chaperones Skp or SurA supported to a variable extent the assembly of bacterial ß-barrel proteins into the mitochondrial OM. Collectively, our findings suggest that the membrane assembly of various ß-barrel proteins depends to a different extent on POTRA domains and periplasmic chaperones.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Evolução Molecular , Mitocôndrias/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência , Especificidade da Espécie
7.
J Biol Chem ; 291(38): 20096-112, 2016 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-27466361

RESUMO

Intimin is an essential adhesin of attaching and effacing organisms such as entropathogenic Escherichia coli It is also the prototype of type Ve secretion or inverse autotransport, where the extracellular C-terminal region or passenger is exported with the help of an N-terminal transmembrane ß-barrel domain. We recently reported a stalled secretion intermediate of intimin, where the passenger is located in the periplasm but the ß-barrel is already inserted into the membrane. Stalling of this mutant is due to the insertion of an epitope tag at the very N terminus of the passenger. Here, we examined how this insertion disrupts autotransport and found that it causes misfolding of the N-terminal immunoglobulin (Ig)-like domain D00. We could also stall the secretion by making an internal deletion in D00, and introducing the epitope tag into the second Ig-like domain, D0, also resulted in reduced passenger secretion. In contrast to many classical autotransporters, where a proximal folding core in the passenger is required for secretion, the D00 domain is dispensable, as the passenger of an intimin mutant lacking D00 entirely is efficiently exported. Furthermore, the D00 domain is slightly less stable than the D0 and D1 domains, unfolding at ∼200 piconewtons (pN) compared with ∼250 pN for D0 and D1 domains as measured by atomic force microscopy. Our results support a model where the secretion of the passenger is driven by sequential folding of the extracellular Ig-like domains, leading to vectorial transport of the passenger domain across the outer membrane in an N to C direction.


Assuntos
Escherichia coli Enteropatogênica/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Biológicos , Dobramento de Proteína , Adesinas Bacterianas/genética , Escherichia coli Enteropatogênica/genética , Proteínas de Escherichia coli/genética , Domínios Proteicos
8.
PLoS One ; 11(5): e0154828, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27149272

RESUMO

Phenotypic diversity is an important trait of bacterial populations and can enhance fitness of the existing genotype in a given environment. To characterize different subpopulations, several studies have analyzed differential gene expression using fluorescent reporters. These studies visualized either single or multiple genes within single cells using different fluorescent proteins. However, variable maturation and folding kinetics of different fluorophores complicate the study of dynamics of gene expression. Here, we present a proof-of-principle study for an alternative gene expression system in a wbaP mutant of Salmonella Typhimurium (S. Tm) lacking the O-sidechain of the lipopolysaccharide. We employed the hemagglutinin (HA)-tagged inverse autotransporter invasin (invAHA) as a transcriptional reporter for the expression of the type three secretion system 1 (T1) in S. Tm. Using a two-reporter approach with GFP and the InvAHA in single cells, we verify that this reporter system can be used for T1 gene expression analysis, at least in strains lacking the O-antigen (wbaP), which are permissive for detection of the surface-exposed HA-epitope. When we placed the two reporters gfp and invAHA under the control of either one or two different promoters of the T1 regulon, we were able to show correlative expression of both reporters. We conclude that the invAHA reporter system is a suitable tool to analyze T1gene expression in S. Tm and propose its applicability as molecular tool for gene expression studies within single cells.


Assuntos
Proteínas de Transporte/genética , Epitopos/genética , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Mutação , Salmonella typhimurium/genética , Cromossomos Bacterianos , Deleção de Genes , Plasmídeos
9.
Oncotarget ; 7(10): 10990-1001, 2016 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-26934329

RESUMO

The specific and rapid detection of Enterobacteriaceae, the most frequent cause of gram-negative bacterial infections in humans, remains a major challenge. We developed a non-invasive method to rapidly detect systemic Yersinia enterocolitica infections using immunoPET (antibody-targeted positron emission tomography) with [64Cu]NODAGA-labeled Yersinia-specific polyclonal antibodies targeting the outer membrane protein YadA. In contrast to the tracer [18F]FDG, [64Cu]NODAGA-YadA uptake co-localized in a dose dependent manner with bacterial lesions of Yersinia-infected mice, as detected by magnetic resonance (MR) imaging. This was accompanied by elevated uptake of [64Cu]NODAGA-YadA in infected tissues, in ex vivo biodistribution studies, whereas reduced uptake was observed following blocking with unlabeled anti-YadA antibody. We show, for the first time, a bacteria-specific, antibody-based, in vivo imaging method for the diagnosis of a Gram-negative enterobacterial infection as a proof of concept, which may provide new insights into pathogen-host interactions.


Assuntos
Imagem Molecular/métodos , Tomografia por Emissão de Pósitrons/métodos , Yersiniose/diagnóstico por imagem , Acetatos/farmacologia , Adesinas Bacterianas/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Radioisótopos de Cobre , Feminino , Compostos Heterocíclicos com 1 Anel/farmacologia , Imagem por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Compostos Radiofarmacêuticos/farmacologia , Yersinia enterocolitica
10.
Methods Mol Biol ; 1329: 17-31, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26427673

RESUMO

Beta-barrel proteins are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. The evolutionary conservation in the biogenesis of these proteins allows mitochondria to assemble bacterial ß-barrel proteins in their functional form. In this chapter, we describe exemplarily how the capacity of yeast mitochondria to process the trimeric autotransporter YadA can be used to study the role of bacterial periplasmic chaperones in this process.


Assuntos
Adesinas Bacterianas/biossíntese , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Interleucina-8/metabolismo , Estrutura Secundária de Proteína , Transformação Genética
11.
Methods Mol Biol ; 1329: 157-67, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26427683

RESUMO

In addition to the cytoplasmic membrane, Gram-negative bacteria have a second lipid bilayer, the outer membrane, which is the de facto barrier between the cell and the extracellular milieu. Virtually all integral proteins of the outer membrane form ß-barrels, which are inserted into the outer membrane by the BAM complex. Some outer membrane proteins, like the porins and trimeric autotransporter adhesins, are multimeric. In the former case, the porin trimer consists of three individual ß-barrels, whereas in the latter, the single autotransporter ß-barrel domain is formed by three separate polypeptides. This chapter reviews methods to investigate the folding and membrane insertion of multimeric OMPs and further explains the use of a BamA depletion strain to study the effects of the BAM complex on multimeric OMPs in E. coli.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Dobramento de Proteína , Multimerização Proteica , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas de Escherichia coli/isolamento & purificação , Temperatura Alta , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína
12.
Int J Med Microbiol ; 305(2): 276-82, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25596886

RESUMO

Intimin and invasin are adhesins and central virulence factors of attaching and effacing bacteria, such as enterohaemorrhagic Escherichia coli, and enteropathogenic Yersiniae, respectively. These proteins are prototypes of a large family of adhesins distributed widely in Gram-negative bacteria. It is now evident that this protein family represents a previously unrecognized autotransporter secretion system, termed type Ve secretion. In contrast to classical autotransport, where the transmembrane ß-barrel domain or translocation unit is C-terminal to the extracellular region or passenger domain, type Ve-secreted proteins have an inverted topology with the passenger domain C-terminal to the translocation unit; hence the term inverse autotransporter. This minireview covers the recent advances in elucidating the structure and biogenesis of inverse autotransporters.


Assuntos
Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/metabolismo , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Fatores de Virulência/química , Fatores de Virulência/metabolismo
13.
Int J Med Microbiol ; 305(2): 252-8, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25604505

RESUMO

The trimeric autotransporter adhesin Yersinia adhesin A is the prototype of the type Vc secretion systems. It is expressed by enteropathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis strains, but not by Yersinia pestis. A characteristic trait of YadA is its modular composition and trimeric nature. YadA consists of an N-terminal passenger domain which is exposed on the bacterial cell surface. The translocation of this passenger onto the surface is facilitated by a C-terminal ß-barrel domain which concomitantly anchors YadA into the outer membrane with three YadA monomers contributing to the formation of a single ß-barrel. In Y. enterocolitica, but not Y. pseudotuberculosis, YadA is a decisive virulence factor and its deletion renders the bacteria virtually avirulent in mouse models of infection. This striking importance of YadA in infection may derive from its manifold functions in host cell interaction. Presumably the most important function of YadA is that it mediates adhesion to extracellular matrix components of eukaryotic host cells. Only tight adhesion allows for the injection of "anti-host" effector proteins via a type III secretion system into the host cell cytosol. These effector proteins enable Yersinia to subvert the host immune system in order to replicate and establish infection. YadA is also essential for the survival of Y. enterocolitica upon contact with serum, an important immune-evasion mechanism called serum resistance. To this end, YadA interacts with several components of the host complement system, the first line of immune defense. This review will summarize recent findings about the structure and biogenesis of YadA and its interactions with the host complement system.


Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Sistemas de Secreção Bacterianos , Fatores de Virulência/metabolismo , Yersinia enterocolitica/fisiologia , Yersinia pseudotuberculosis/fisiologia , Animais , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Virulência , Yersinia enterocolitica/crescimento & desenvolvimento , Yersinia enterocolitica/metabolismo , Yersinia pseudotuberculosis/metabolismo
14.
J Biol Chem ; 290(3): 1837-49, 2015 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-25488660

RESUMO

Autotransporter proteins comprise a large family of virulence factors that consist of a ß-barrel translocation unit and an extracellular effector or passenger domain. The ß-barrel anchors the protein to the outer membrane of Gram-negative bacteria and facilitates the transport of the passenger domain onto the cell surface. By inserting an epitope tag into the N terminus of the passenger domain of the inverse autotransporter intimin, we generated a mutant defective in autotransport. Using this stalled mutant, we could show that (i) at the time point of stalling, the ß-barrel appears folded; (ii) the stalled autotransporter is associated with BamA and SurA; (iii) the stalled intimin is decorated with large amounts of SurA; (iv) the stalled autotransporter is not degraded by periplasmic proteases; and (v) inverse autotransporter passenger domains are translocated by a hairpin mechanism. Our results suggest a function for the BAM complex not only in insertion and folding of the ß-barrel but also for passenger translocation.


Assuntos
Adesinas Bacterianas/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/metabolismo , Adesinas Bacterianas/química , Transporte Biológico , Membrana Celular/metabolismo , Clonagem Molecular , Reagentes para Ligações Cruzadas/química , Epitopos/química , Proteínas de Escherichia coli/química , Células HeLa , Humanos , Microscopia de Fluorescência , Chaperonas Moleculares/química , Mutagênese Sítio-Dirigida , Mutação , Peptídeo Hidrolases/química , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Propriedades de Superfície
15.
Mol Microbiol ; 95(1): 80-100, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25353290

RESUMO

Intimin and Invasin are prototypical inverse (Type Ve) autotransporters and important virulence factors of enteropathogenic Escherichia coli and Yersinia spp. respectively. In addition to a C-terminal extracellular domain and a ß-barrel transmembrane domain, both proteins also contain a short N-terminal periplasmic domain that, in Intimin, includes a lysin motif (LysM), which is thought to mediate binding to peptidoglycan. We show that the periplasmic domain of Intimin does bind to peptidoglycan both in vitro and in vivo, but only under acidic conditions. We were able to determine a dissociation constant of 0.8 µM for this interaction, whereas the Invasin periplasmic domain, which lacks a LysM, bound only weakly in vitro and failed to bind peptidoglycan in vivo. We present the solution structure of the Intimin LysM, which has an additional α-helix conserved within inverse autotransporter LysMs but lacking in others. In contrast to previous reports, we demonstrate that the periplasmic domain of Intimin mediates dimerisation. We further show that dimerisation and peptidoglycan binding are general features of LysM-containing inverse autotransporters. Peptidoglycan binding by the periplasmic domain in the infection process may aid in resisting mechanical and chemical stress during transit through the gastrointestinal tract.


Assuntos
Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Escherichia coli Enteropatogênica/metabolismo , Peptidoglicano/metabolismo , Yersinia/metabolismo , Adesinas Bacterianas/genética , Sítios de Ligação , Biologia Computacional/métodos , Dimerização , Escherichia coli Enteropatogênica/química , Escherichia coli Enteropatogênica/genética , Concentração de Íons de Hidrogênio , Modelos Moleculares , Multimerização Proteica , Estrutura Secundária de Proteína , Fatores de Virulência/química , Fatores de Virulência/metabolismo , Yersinia/química , Yersinia/genética
16.
J Biol Chem ; 289(43): 29457-70, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25190806

RESUMO

Yersinia adhesin A (YadA) belongs to a class of bacterial adhesins that form trimeric structures. Their mature form contains a passenger domain and a C-terminal ß-domain that anchors the protein in the outer membrane (OM). Little is known about how precursors of such proteins cross the periplasm and assemble into the OM. In the present study we took advantage of the evolutionary conservation in the biogenesis of ß-barrel proteins between bacteria and mitochondria. We previously observed that upon expression in yeast cells, bacterial ß-barrel proteins including the transmembrane domain of YadA assemble into the mitochondrial OM. In the current study we found that when expressed in yeast cells both the monomeric and trimeric forms of full-length YadA were detected in mitochondria but only the trimeric species was fully integrated into the OM. The oligomeric form was exposed on the surface of the organelle in its native conformation and maintained its capacity to adhere to host cells. The co-expression of YadA with a mitochondria-targeted form of the bacterial periplasmic chaperone Skp, but not with SurA or SecB, resulted in enhanced levels of both forms of YadA. Taken together, these results indicate that the proper assembly of trimeric autotransporter can occur also in a system lacking the lipoproteins of the BAM machinery and is specifically enhanced by the chaperone Skp.


Assuntos
Adesinas Bacterianas/metabolismo , Sequência Conservada , Evolução Molecular , Mitocôndrias/metabolismo , Multimerização Proteica , Adesinas Bacterianas/química , Células HeLa , Humanos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Proteólise , Saccharomyces cerevisiae/metabolismo
17.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 6): 1779-89, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24914988

RESUMO

Outer membrane protein (OMP) biogenesis is an essential process for maintaining the bacterial cell envelope and involves the ß-barrel assembly machinery (BAM) for OMP recognition, folding and assembly. In Escherichia coli this function is orchestrated by five proteins: the integral outer membrane protein BamA of the Omp85 superfamily and four associated lipoproteins. To unravel the mechanism underlying OMP folding and insertion, the structure of the E. coli BamA ß-barrel and P5 domain was determined at 3 Šresolution. These data add information beyond that provided in the recently published crystal structures of BamA from Haemophilus ducreyi and Neisseria gonorrhoeae and are a valuable basis for the interpretation of pertinent functional studies. In an `open' conformation, E. coli BamA displays a significant degree of flexibility between P5 and the barrel domain, which is indicative of a multi-state function in substrate transfer. E. coli BamA is characterized by a discontinuous ß-barrel with impaired ß1-ß16 strand interactions denoted by only two connecting hydrogen bonds and a disordered C-terminus. The 16-stranded barrel surrounds a large cavity which implies a function in OMP substrate binding and partial folding. These findings strongly support a mechanism of OMP biogenesis in which substrates are partially folded inside the barrel cavity and are subsequently released laterally into the lipid bilayer.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos
18.
PLoS One ; 7(10): e47069, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23056583

RESUMO

Invasin and intimin are major virulence factors of enteropathogenic Yersiniae and Escherichia coli, mediating invasion into and intimate adherence to host cells, respectively. Several studies have hinted that extracellular portion of these homologous proteins might be exported via an autotransport mechanism, but rigorous experimental proof has been lacking. Here, we present a topology model for invasin and intimin, consistent with the hypothesis that the N-terminal ß-barrel domain acts as a translocation pore to secrete the C-terminal passenger domain. We confirmed this topology model by inserting epitope tags into the loops of the ß-barrel. We further show that obstructing the pore of ß-barrel hinders the export of the passenger domain. As for classical autotransport, the biogenesis of invasin and intimin is dependent on the Bam complex and the periplasmic chaperone SurA, whereas the chaperone/protease DegP is involved in quality control. However, compared to classical autotransporters (Type Va secretion), the domain structure of intimin and invasin is inverted. We conclude that proteins of the intimin and invasin family constitute a novel group of autotransported proteins, and propose that this class of autotransporters be termed Type Ve secretion.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Western Blotting , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Dicroísmo Circular , Biologia Computacional , Escherichia coli Enteropatogênica/metabolismo , Ensaio de Imunoadsorção Enzimática , Proteínas de Escherichia coli/genética , Citometria de Fluxo , Células HeLa , Humanos , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Fatores de Virulência/genética
19.
Mol Biol Cell ; 22(10): 1638-47, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21460184

RESUMO

ß-barrel proteins are found in the outer membranes of eukaryotic organelles of endosymbiotic origin as well as in the outer membrane of Gram-negative bacteria. Precursors of mitochondrial ß-barrel proteins are synthesized in the cytosol and have to be targeted to the organelle. Currently, the signal that assures their specific targeting to mitochondria is poorly defined. To characterize the structural features needed for specific mitochondrial targeting and to test whether a full ß-barrel structure is required, we expressed in yeast cells the ß-barrel domain of the trimeric autotransporter Yersinia adhesin A (YadA). Trimeric autotransporters are found only in prokaryotes, where they are anchored to the outer membrane by a single 12-stranded ß-barrel structure to which each monomer is contributing four ß-strands. Importantly, we found that YadA is solely localized to the mitochondrial outer membrane, where it exists in a native trimeric conformation. These findings demonstrate that, rather than a linear sequence or a complete ß-barrel structure, four ß-strands are sufficient for the mitochondria to recognize and assemble a ß-barrel protein. Remarkably, the evolutionary origin of mitochondria from bacteria enables them to import and assemble even proteins belonging to a class that is absent in eukaryotes.


Assuntos
Adesinas Bacterianas/biossíntese , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Fragmentos de Peptídeos/biossíntese , Proteínas Recombinantes/biossíntese , Adesinas Bacterianas/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Fragmentos de Peptídeos/metabolismo , Dobramento de Proteína , Multimerização Proteica , Sinais Direcionadores de Proteínas , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae
20.
Plasmid ; 65(1): 20-4, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20875451

RESUMO

Our laboratory strain Yersinia enterocolitica strain WA-314 biogroup 1B serotype O:8 displayed a different adhesion behavior to host cells compared to other Yersinia strains. To investigate whether this is based on differences in the gene content of the large pYV virulence plasmid which contains the major Yersinia adhesin YadA, we set out to sequence pYV(WA-314). pYV(WA-314) is very similar to pYV127/90, with a notable difference in the length of the Type III secretion system component YscP, which determines the needle length of the system. We found that we could improve the annotation of proteins previously described as "hypothetical" in pYV127/90 and other pYV plasmids, and show that pYV plasmids contain several and seemingly redundant plasmid partitioning and stabilization systems, explaining why these plasmids are not easily lost in laboratory cultures of Yersinia strains.


Assuntos
Plasmídeos/genética , Yersinia enterocolitica/genética , Yersinia enterocolitica/patogenicidade , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Replicação do DNA/genética , Anotação de Sequência Molecular , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , Sorotipagem , Virulência/genética , Yersinia enterocolitica/classificação
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