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1.
Sci Rep ; 11(1): 17205, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34446740

RESUMO

Novel approaches in synthetic biology focus on the bottom-up modular assembly of natural, modified natural or artificial components into molecular systems with functionalities not found in nature. A possible application for such techniques is the bioremediation of natural water sources contaminated with small organic molecules (e.g., drugs and pesticides). A simple molecular system to actively accumulate and degrade pollutants could be a bionanoreactor composed of a liposome or polymersome scaffold combined with energizing- (e.g., light-driven proton pump), transporting- (e.g., proton-driven transporter) and degrading modules (e.g., enzyme). This work focuses on the engineering of a transport module specific for ß-lactam antibiotics. We previously solved the crystal structure of a bacterial peptide transporter, which allowed us to improve the affinity for certain ß-lactam antibiotics using structure-based mutagenesis combined with a bacterial uptake assay. We were able to identify specific mutations, which enhanced the affinity of the transporter for antibiotics containing certain structural features. Screening of potential compounds allowed for the identification of a ß-lactam antibiotic ligand with relatively high affinity. Transport of antibiotics was evaluated using a solid-supported membrane electrophysiology assay. In summary, we have engineered a proton-driven ß-lactam antibiotic translocation module, contributing to the growing toolset for bionanotechnological applications.


Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Yersinia enterocolitica/metabolismo , beta-Lactamas/metabolismo , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico/genética , Biotecnologia/métodos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Estrutura Molecular , Mutação , Nanotecnologia/métodos , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas/métodos , Prótons , Yersinia enterocolitica/genética , beta-Lactamas/química
2.
PLoS One ; 16(6): e0252800, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34143799

RESUMO

Type three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which secretion substrates pass the inner membrane. While the complex formed by four of the EA proteins has been well characterised structurally, little is known about the structure of the membrane domain of the largest subunit, FlhA in flagella, SctV in injectisomes. Furthermore, the biologically relevant nonameric assembly of FlhA/SctV has been infrequently observed and differences in conformation of the cytoplasmic portion of FlhA/SctV between open and closed states have been suggested to reflect secretion system specific differences. FlhA has been shown to bind to chaperone-substrate complexes in an open state, but in previous assembled ring structures, SctV is in a closed state. Here, we identify FlhA and SctV homologues that can be recombinantly produced in the oligomeric state and study them using cryo-electron microscopy. The structures of the cytoplasmic domains from both FlhA and SctV are in the open state and we observe a conserved interaction between a short stretch of residues at the N-terminus of the cytoplasmic domain, known as FlhAL/SctVL, with a groove on the adjacent protomer's cytoplasmic domain, which stabilises the nonameric ring assembly.


Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Proteínas de Membrana/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Microscopia Crioeletrônica/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia de Fluorescência/métodos , Modelos Moleculares , Conformação Proteica , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/ultraestrutura , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo
3.
Nat Commun ; 12(1): 1625, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712575

RESUMO

Many bacterial pathogens use a type III secretion system (T3SS) to manipulate host cells. Protein secretion by the T3SS injectisome is activated upon contact to any host cell, and it has been unclear how premature secretion is prevented during infection. Here we report that in the gastrointestinal pathogens Yersinia enterocolitica and Shigella flexneri, cytosolic injectisome components are temporarily released from the proximal interface of the injectisome at low external pH, preventing protein secretion in acidic environments, such as the stomach. We show that in Yersinia enterocolitica, low external pH is detected in the periplasm and leads to a partial dissociation of the inner membrane injectisome component SctD, which in turn causes the dissociation of the cytosolic T3SS components. This effect is reversed upon restoration of neutral pH, allowing a fast activation of the T3SS at the native target regions within the host. These findings indicate that the cytosolic components form an adaptive regulatory interface, which regulates T3SS activity in response to environmental conditions.


Assuntos
Citosol/metabolismo , Transporte Proteico/fisiologia , Sistemas de Secreção Tipo III/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Concentração de Íons de Hidrogênio , Shigella flexneri/metabolismo , Sistemas de Secreção Tipo III/genética , Yersinia enterocolitica/metabolismo
4.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540627

RESUMO

In this study, we found that the loss of OmpR, the response regulator of the two-component EnvZ/OmpR system, increases the cellular level of Fur, the master regulator of iron homeostasis in Y. enterocolitica. Furthermore, we demonstrated that transcription of the fur gene from the YePfur promoter is subject to negative OmpR-dependent regulation. Four putative OmpR-binding sites (OBSs) were indicated by in silico analysis of the fur promoter region, and their removal affected OmpR-dependent fur expression. Moreover, OmpR binds specifically to the predicted OBSs which exhibit a distinct hierarchy of binding affinity. Finally, the data demonstrate that OmpR, by direct binding to the promoters of the fecA, fepA and feoA genes, involved in the iron transport and being under Fur repressor activity, modulates their expression. It seems that the negative effect of OmpR on fecA and fepA transcription is sufficient to counteract the indirect, positive effect of OmpR resulting from decreasing the Fur repressor level. The expression of feoA was positively regulated by OmpR and this mode of action seems to be direct and indirect. Together, the expression of fecA, fepA and feoA in Y. enterocolitica has been proposed to be under a complex mode of regulation involving OmpR and Fur regulators.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Proteínas Repressoras/genética , Transativadores/metabolismo , Yersinia enterocolitica/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Transporte/genética , Simulação por Computador , Homeostase , Proteínas de Ligação ao Ferro/genética , Regiões Promotoras Genéticas , Receptores de Superfície Celular/genética , Yersinia enterocolitica/genética
5.
Molecules ; 25(23)2020 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-33287412

RESUMO

Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria, and its integrity is monitored by various stress response systems. Although the Rcs system is involved in the envelope stress response and regulates genes controlling numerous bacterial cell functions of Yersinia enterocolitica, whether it can sense the truncated LPS in Y. enterocolitica remains unclear. In this study, the deletion of the Y. enterocolitica waaF gene truncated the structure of LPS and produced a deep rough LPS. The truncated LPS increased the cell surface hydrophobicity and outer membrane permeability, generating cell envelope stress. The truncated LPS also directly exposed the smooth outer membrane to the external environment and attenuated the resistance to adverse conditions, such as impaired survival under polymyxin B and sodium dodecyl sulfate (SDS) exposure. Further phenotypic experiment and gene expression analysis indicated that the truncated LPS was correlated with the activation of the Rcs phosphorelay, thereby repressing cell motility and biofilm formation. Our findings highlight the importance of LPS integrity in maintaining membrane function and broaden the understanding of Rcs phosphorelay signaling in response to cell envelope stress, thus opening new avenues to develop effective antimicrobial agents for combating Y. enterocolitica infections.


Assuntos
Cápsulas Bacterianas/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Yersinia enterocolitica/efeitos dos fármacos , Antibacterianos/farmacologia , Cápsulas Bacterianas/metabolismo , Membrana Externa Bacteriana/efeitos dos fármacos , Membrana Externa Bacteriana/metabolismo , Biofilmes/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Polimixina B/farmacologia , Transdução de Sinais/efeitos dos fármacos , Dodecilsulfato de Sódio/farmacologia , Yersiniose/tratamento farmacológico , Yersiniose/microbiologia , Yersinia enterocolitica/metabolismo
6.
Food Microbiol ; 92: 103593, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32950135

RESUMO

Conventional methods for Yersinia enterocolitica detection in food samples are generally considered inadequate. Problems arise from the presence of the so-called "background flora", coupled to the low contamination level of the pathogen. Since, data on the microbial ecology occurring in competitive microflora are still lacking, MALDI TOF MS was used for strains 'identification after enrichment in PSB or ITC broths, and after plating on selective CIN medium at different incubation times. SYBR Green Real time PCR was used for the Y. enterocolitica strains' detection (4/O:3, 1A/O:5) in experimentally contaminated foods, as well as in naturally contaminated samples. A higher number of different bacterial genera (10 on CIN and 18 on PCA) was recorded after enrichment in PSB, whilst enrichment in ITC led to recovery of 6 and 10 genera on CIN and PCA, respectively. Yersiniaceae was the dominant family on the first day of incubation, but on the second day the percentage of isolation considerably decreased. By testing experimentally contaminated samples, substantial difficulties were encountered. The biotype 1A was always detected, whereas strain 4/O:3 proved to be poorly competitive. Based on the data, the enrichment media PSB and ITC, currently proposed for Y. enterocolitica detection, need to be improved to promote a successful pathogen's recovery.


Assuntos
Produtos da Carne/microbiologia , Yersinia enterocolitica/isolamento & purificação , Animais , Meios de Cultura/química , Meios de Cultura/metabolismo , Contaminação de Alimentos/análise , Produtos da Carne/análise , Suínos , Yersinia enterocolitica/classificação , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo
7.
Sci Rep ; 10(1): 10167, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576927

RESUMO

Yersinia enterocolitica is an enteric bacterium which can cause severe gastroenteritis. Beta-lactams are the most widely used antibiotics against Y. enterocolitica. Y. enterocolitica produces two chromosomal ß-lactamases, BlaA and BlaB. BlaB is an Ambler Class C inducible broad spectrum cephlaosporinase which showed differential enzyme activity in different biotypes of Y. enterocolitica. The expression of blaB is mainly regulated by ampR- the transcriptional regulator and, ampD - which helps in peptidoglycan recycling. The aim of this study was to identify and characterize genetic determinants underlying differential enzyme activity of BlaB in Y. enterocolitica biotypes 1 A, IB, 2 and 4. Thus, ampR, blaB and ampD were PCR-amplified and modeled in silico. The intercistronic region containing promoters of ampR and blaB was also investigated. Our results indicated that blaB was more inducible in biotypes 2 and 4, than in biotypes 1 A and 1B. Superimposition of in silico modeled proteins suggested that variations in amino acid sequences of AmpR, BlaB and AmpD were not responsible for hyper-production of BlaB in biotypes 2 and 4. Analysis of promoter regions of ampR and blaB revealed variations at -30, -37 and -58 positions from blaB transcription start site. Studies on relative expression levels of blaB in different biotypes by qRT-PCR indicated that nucleotide variations at these positions might contribute to a higher enzyme activity of BlaB in biotypes 2 and 4. However, this is a preliminary study and further studies including more strains of each biotype are required to strengthen our findings. Nevertheless, to the best of our knowledge, this is the first study which has investigated the genetic determinants underlying differential inducible production of BlaB in different biotypes of Y. enterocolitica.


Assuntos
Cefalosporinase/genética , Cefalosporinase/metabolismo , Regulação Bacteriana da Expressão Gênica , Expressão Gênica , Yersinia enterocolitica/citologia , Yersinia enterocolitica/enzimologia , Proteínas de Bactérias/fisiologia , Peptidoglicano/metabolismo , Yersinia enterocolitica/metabolismo , Yersinia enterocolitica/patogenicidade
8.
Nat Commun ; 11(1): 2381, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404906

RESUMO

Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. Although the T3SS can efficiently export heterologous cargo proteins, a lack of target cell specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to govern its activity. Using optogenetic interaction switches to control the availability of the dynamic cytosolic T3SS component SctQ, T3SS-dependent effector secretion can be regulated by light. The resulting system, LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the light-regulated translocation of heterologous reporter proteins, and induction of apoptosis in cultured eukaryotic cells. LITESEC-T3SS constitutes a new method to control protein secretion and translocation into eukaryotic host cells with unparalleled spatial and temporal resolution.


Assuntos
Proteínas de Bactérias/metabolismo , Células Eucarióticas/metabolismo , Bactérias Gram-Negativas/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Citosol/metabolismo , Citosol/microbiologia , Células Eucarióticas/microbiologia , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/fisiologia , Humanos , Luz , Microscopia de Fluorescência , Optogenética/métodos , Transporte Proteico/efeitos da radiação , Análise Espacial , Sistemas de Secreção Tipo III/genética , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Yersinia enterocolitica/fisiologia
9.
J Vis Exp ; (151)2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31545311

RESUMO

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.


Assuntos
Citosol/metabolismo , Imagem Individual de Molécula/métodos , Proteínas de Bactérias/metabolismo , Difusão , Fluorescência , Movimento (Física) , Yersinia enterocolitica/metabolismo
10.
Int J Med Microbiol ; 309(5): 331-337, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31176600

RESUMO

Bacteria often express numerous virulence factors. These virulence factors make them successful pathogens, by e.g. mediating attachment to host cells and thereby facilitating persistence or invasion, or by contributing to the evasion of the host immune system to allow proliferation and spread within the host and in the environment. The site of first contact of Gram negative bacteria with the host is the bacterial outer membrane (OM). Consisting of an asymmetrical lipid bilayer with phospholipids forming the inner, and lipopolysaccharides forming the outer leaflet, the OM harbors numerous integral membrane proteins that are almost exclusively ß-barrel proteins. One distinct family of OM ß-barrel proteins strongly linked to bacterial virulence are the autotransporter (AT) proteins. During the last years huge progress has been made to better understand the mechanisms underlying the insertion of AT proteins into the OM and also AT function for interaction with the host. This review shortly summarizes our current knowledge about outer membrane protein (OMP) and more specifically AT biogenesis and function. We focused on the AT proteins that we haved studied in most detail: i.e. the Yersinia adhesin A (YadA) and invasin of Yersinia enterocolitica (Ye) as well as its homolog intimin (Int) expressed by enteropathogenic Escherichia coli. In addition, this review provides a short outlook about how we could possibly use this knowledge to fight infection.


Assuntos
Adesinas Bacterianas/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Sistemas de Secreção Tipo V/metabolismo , Escherichia coli Enteropatogênica/metabolismo , Virulência , Fatores de Virulência/metabolismo , Yersinia enterocolitica/metabolismo
11.
J Struct Biol ; 207(3): 279-286, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31200020

RESUMO

Yersinia enterocolitica is a pectinolytic zoonotic foodborne pathogen, the genome of which contains pectin-binding proteins and several different classes of pectinases, including polysaccharide lyases (PLs) and an exopolygalacturonase. These proteins operate within a coordinated pathway to completely saccharify homogalacturonan (HG). Polysaccharide lyase family 2 (PL2) is divided into two major subfamilies that are broadly-associated with contrasting 'endolytic' (PL2A) or 'exolytic' (PL2B) activities on HG. In the Y. enterocolitica genome, the PL2A gene is adjacent to an independent carbohydrate binding module from family 32 (YeCBM32), which possesses a N-terminal secretion tag and is known to specifically bind HG. Independent CBMs are rare in nature and, most commonly, are fused to enzymes in order to potentiate catalysis. The unconventional gene architecture of YePL2A and YeCBM32, therefore, may represent an ancestral relic of a fission event that decoupled PL2A from its cognate CBM. To provide further insight into the evolution of this pectinolytic locus and the molecular basis of HG depolymerisation within Y. enterocolitica, we have resurrected a YePL2A-YeCBM32 chimera and demonstrated that the extant PL2A digests HG more efficiently. In addition, we have engineered a tryptophan from the active site of the exolytic YePL2B into YePL2A (YePL2A-K291W) and demonstrated, using X-ray crystallography of substrate complexes, that it is a structural determinant of exo-activity within the PL2 family. In this manner, surrogate structural platforms may assist in the study of phylogenetic relationships informed by extant and resurrected sequences, and can be used to overcome challenging structural problems within carbohydrate active enzyme families.


Assuntos
Glicosídeo Hidrolases/metabolismo , Pectinas/metabolismo , Polissacarídeo-Liases/metabolismo , Yersinia enterocolitica/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Modelos Moleculares , Pectinas/química , Filogenia , Polissacarídeo-Liases/química , Polissacarídeo-Liases/genética , Conformação Proteica , Triptofano/química , Triptofano/genética , Triptofano/metabolismo , Yersinia enterocolitica/enzimologia , Yersinia enterocolitica/genética
12.
Cell Microbiol ; 21(9): e13046, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31099152

RESUMO

The virulence strategy of pathogenic Yersinia spp. involves cell-invasive as well as phagocytosis-preventing tactics to enable efficient colonisation of the host organism. Enteropathogenic yersiniae display an invasive phenotype in early infection stages, which facilitates penetration of the intestinal mucosa. Here we show that invasion of epithelial cells by Yersinia enterocolitica is followed by intracellular survival and multiplication of a subset of ingested bacteria. The replicating bacteria were enclosed in vacuoles with autophagy-related characteristics, showing phagophore formation, xenophagy, and recruitment of cytoplasmic autophagosomes to the bacteria-containing compartments. The subsequent fusion of these vacuoles with lysosomes and concomitant vesicle acidification were actively blocked by Yersinia. This resulted in increased intracellular proliferation and detectable egress of yersiniae from infected cells. Notably, deficiency of the core autophagy machinery component FIP200 impaired the development of autophagic features at Yersinia-containing vacuoles as well as intracellular replication and release of bacteria to the extracellular environment. These results suggest that Y. enterocolitica may take advantage of the macroautophagy pathway in epithelial cells to create an autophagosomal niche that supports intracellular bacterial survival, replication, and, eventually, spread of the bacteria from infected cells.


Assuntos
Autofagossomos/microbiologia , Células Epiteliais/microbiologia , Yersinia enterocolitica/patogenicidade , Animais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Morte Celular , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Células HeLa , Interações entre Hospedeiro e Microrganismos , Humanos , Lisossomos/metabolismo , Lisossomos/microbiologia , Lisossomos/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Vacúolos/metabolismo , Vacúolos/microbiologia , Vacúolos/ultraestrutura , Yersinia enterocolitica/crescimento & desenvolvimento , Yersinia enterocolitica/metabolismo
13.
Comp Immunol Microbiol Infect Dis ; 63: 131-135, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30961808

RESUMO

The aim of the study was to determine whether the presence of the Yersinia virulence plasmid could affect the production of enterotoxin YstA by Y. enterocolitica strains isolated from pigs which are the main source of infection for humans. The phenotypic features characteristic for the Yersinia virulence plasmid were detected on CRMOX agar in 8 out of 12 strains producing enterotoxin YstA, in 5 out of 12 doubtful strains, and in 11 out of 12 strains not producing YstA. Autoagglutination ability was detected in all 12 Y. enterocolitica strains that were positive in the suckling mice bioassay, in 11 doubtful strains and 10 negative strains. CRMOX+ colonies were generally ystA, myfA, virF and yadA positive, while CRMOX- colonies were only ystA and myfA positive. The amplicons of yadA were not detected in 2 (8.3%) out of 24 CRMOX+ and virF positive strains. The results of this study indicate that the presence of pYV does not affect the enterotoxin-producing ability of Y. enterocolitica strains.


Assuntos
Toxinas Bacterianas/biossíntese , Enterotoxinas/biossíntese , Plasmídeos/genética , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Adesinas Bacterianas/metabolismo , Animais , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Meios de Cultura/farmacologia , DNA Bacteriano/genética , Humanos , Camundongos , Suínos , Doenças dos Suínos/microbiologia , Yersinia enterocolitica/patogenicidade
14.
Artigo em Inglês | MEDLINE | ID: mdl-30294593

RESUMO

We show that Yersinia enterocolitica strain Ye9 (bio-serotype 2/O:9) utilizes heme-containing molecules as an iron source. The Ye9 genome contains two multigenic clusters, hemPRSTUV-1 and hemPRST-2, encoding putative heme receptors HemR1 and HemR2, that share 62% amino acid identity. Expression of these proteins in an Escherichia coli mutant defective in heme biosynthesis allowed this strain to use hemin and hemoglobin as a source of porphyrin. The hemPRSTUV-1 and hemPRST-2 clusters are organized as operons, expressed from the phem-1 and weaker phem-2 promoters, respectively. Expression of both operons is negatively regulated by iron and the iron-responsive transcriptional repressor Fur. In addition, OmpR, the response regulator of two component system (TCSs) EnvZ/OmpR, represses transcription of both operons through interaction with binding sequences overlapping the -35 region of their promoters. Western blot analysis of the level of HemR1 in ompR, fur, and ompRfur mutants, showed an additive effect of these mutations, indicating that OmpR may regulate HemR expression independently of Fur. However, the effect of OmpR on the activity of the phem-1 promoter and on HemR1 production was observed in both iron-depleted and iron-replete conditions, i.e., when Fur represses the iron-regulated promoter. In addition, a hairpin RNA thermometer, composed of four uracil residues (FourU) that pair with the ribosome-binding site in the 5'-untranslated region (5'-UTR) of hemR1 was predicted by in silico analysis. However, thermoregulated expression of HemR1 could not be demonstrated. Taken together, these data suggest that Fur and OmpR control iron/heme acquisition via a complex mechanism based on negative regulation of hemR1 and hemR2 at the transcriptional level. This interplay could fine-tune the level of heme receptor proteins to allow Y. enterocolitica to fulfill its iron/heme requirements without over-accumulation, which might be important for pathogenic growth within human hosts.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Receptores de Superfície Celular/metabolismo , Transativadores/metabolismo , Transcrição Genética , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Hemeproteínas/metabolismo , Ferro/metabolismo , Família Multigênica , Óperon , Proteínas Repressoras/metabolismo , Yersinia enterocolitica/classificação
15.
Integr Biol (Camb) ; 10(9): 502-515, 2018 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-30101242

RESUMO

In bacterial type 3 secretion, substrate proteins are actively transported from the bacterial cytoplasm into the host cell cytoplasm by a large membrane-embedded machinery called the injectisome. Injectisomes transport secretion substrates in response to specific environmental signals, but the molecular details by which the cytosolic secretion substrates are selected and transported through the type 3 secretion pathway remain unclear. Secretion activity and substrate selectivity are thought to be controlled by a sorting platform consisting of the proteins SctK, SctQ, SctL, and SctN, which together localize to the cytoplasmic side of membrane-embedded injectisomes. However, recent work revealed that sorting platform proteins additionally exhibit substantial cytosolic populations and that SctQ reversibly binds to and dissociates from the cytoplasmic side of membrane-embedded injectisomes. Based on these observations, we hypothesized that dynamic molecular turnover at the injectisome and cytosolic assembly among sorting platform proteins is a critical regulatory component of type 3 secretion. To determine whether sorting platform complexes exist in the cytosol, we measured the diffusive properties of the two central sorting platform proteins, SctQ and SctL, using live cell high-throughput 3D single-molecule tracking microscopy. Single-molecule trajectories, measured in wild-type and mutant Yersinia enterocolitica cells, reveal that both SctQ and SctL exist in several distinct diffusive states in the cytosol, indicating that these proteins form stable homo- and hetero-oligomeric complexes in their native environment. Our findings provide the first diffusive state-resolved insights into the dynamic regulatory network that interfaces stationary membrane-embedded injectisomes with the soluble cytosolic components of the type 3 secretion system.


Assuntos
Proteínas de Bactérias/metabolismo , Citosol/metabolismo , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos , Yersinia enterocolitica/metabolismo , Algoritmos , Membrana Celular/metabolismo , Flagelos , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência , Método de Monte Carlo , Plasmídeos/metabolismo , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Especificidade por Substrato , Virulência
16.
Zhonghua Yu Fang Yi Xue Za Zhi ; 52(6): 653-660, 2018 Jun 06.
Artigo em Chinês | MEDLINE | ID: mdl-29886689

RESUMO

Objective: In this study, we analyze the regulation mechanisms of the expression of ampD in AmpC ß-lactamase and the regulation mechanism of ß-N-acetylglucosaminidase (NagZ) in Yersinia enterocolitica. Methods: We construct the mutation strains of Yersinia enterocolitica AmpD (AmpD1-3) gene (ampD1-3), Low-Molecular-Mass Penicillin-Binding Proteins (LMM PBPs) gene (pbp4, pbp5a, pbp5b, pbp7), NagZ gene (nagZ), and ampR gene by deleting and complementing genes, and induce them by cefoxitin. We determined the activity of AmpC ß-lactamase activity (U) of mutant strains (basal level and induce level) by using cephalothiophene hydrolysis method, the promoter activity of AmpC ß-lactamase ((relative light unit (RLU)) was detected by the luxCDABEreporter system, and the activity of ß-N-acetylglucosaminidase (nmol/L) was determined by by using 4-nitrophenyl N-acetyl-ß-D-glucosaminide as the chromogenic substrate. Results: AmpD1 (Basal level: (3.29±1.58) U; Induced level: (4.08±1.75) U) was the most potent one. The YEΔ5b, YEΔ4Δ5b, YEΔ5aΔ5b and YEΔ5bΔ7 of ampC promoter activity increase significantly, whichYEΔ4Δ5b is the highest one (Basal level: (106 903.16±61 910.61) RLU; Induced level: (205 427.45±45 352.17) RLU). The YEΔ4Δ5bΔ7 of ampC promoter activity is the highest among triple mutant strain (Basal level: (304 108.04±99 274.53) RLU; Induced level: (531 440.21±68 891.02) RLU). Quadruple deletion strain YEΔ4Δ5aΔ5bΔ7 have the highest ampC promoter activity (Basal level: (1 013 810.99±260 955.96) RLU; Induced level: (1 230 214.59±205 526.79) RLU). After the deletion of nagZ gene, there is no significant change in ß-lactamase activity of YEΔD1D2D3ΔZ, while ß-lactamase activity of YEΔ4Δ5aΔ5bΔ7ΔZ shows a significant decrease (Basal level: (0.30±0.20) U; Induced level: (0.29±0.21) U), which basically drops to the wild strain level. Conclusion: This is the first report of ampC multi-step upregulation mechanism driven by three AmpD homologues in Yersinia enterocolitica. The AmpC regulation mode with the function of single PBP4, PBP5a or PBP7 is relatively low, which work in coordination with PBP5b. Yersinia enterocolitica have both NagZ-depend and NagZ-independent mechanisms for ß-lactamase expression.


Assuntos
Acetilglucosaminidase/metabolismo , Proteínas de Bactérias/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Yersinia enterocolitica/metabolismo , beta-Lactamases/metabolismo , Humanos
17.
Microbiol Res ; 207: 211-225, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29458857

RESUMO

Cysteine proteases of the papain family, including mammalian cathepsins, play important physiological roles, however, their excessive activity may contribute to the development of various pathologies. Therefore, cysteine cathepsin inhibitors are being considered as promising drugs to treat cathepsin-driven diseases. Diverse saprophytic and parasitic microbes produce such inhibitors, which target the host's proteases playing pivotal roles in immune responses, thus leading to the survival of microbes within their host. Yersinia enterocolitica is a Gram-negative zoopathogenic coccobacillus, which has developed several mechanisms to evade the host's immune system. Nevertheless, the bacterium has not yet been shown to produce any cysteine protease inhibitors. Here we demonstrate that Y. enterocolitica strains of different bioserotypes and genotypes synthesize papain and human cathepsin L inhibitors, but not bovine cathepsin B inhibitors. By employing fluorimetry and zymography, the cell-surface inhibitors were shown to associate peripherally with the outer membrane, while the inhibitors present in cell-free extracts proved to: interact reversibly with their target enzymes, exhibit thermolability and stability in a range of pH values (5-9), and have high molecular weights. Batch affinity chromatography on papain-agarose resin was then undertaken to isolate putative inhibitors of cysteine proteases from the bacterial extract. The isolated 18 kDa protein was identified by LC-MS/MS as the periplasmic chaperone Skp. The Skp-containing eluate inhibited the activity of cysteine cathepsins produced by human dermal fibroblasts. The homologous Skp protein was also isolated from the extract of Escherichia coli. Our results point to a possible new biological role of the bacterial chaperone Skp.


Assuntos
Catepsinas/metabolismo , Extratos Celulares/química , Inibidores de Cisteína Proteinase/metabolismo , Papaína/antagonistas & inibidores , Yersinia enterocolitica/metabolismo , Animais , Bovinos , Cisteína Proteases/metabolismo , Proteínas de Ligação a DNA , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Humanos , Chaperonas Moleculares , Papaína/metabolismo
18.
Cell Mol Life Sci ; 75(12): 2273-2289, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29285573

RESUMO

Effector proteins are key virulence factors of pathogenic bacteria that target and subvert the functions of essential host defense mechanisms. Typically, these proteins are delivered into infected host cells via the type III secretion system (T3SS). Recently, however, several effector proteins have been found to enter host cells in a T3SS-independent manner thereby widening the potential range of these virulence factors. Prototypes of such bacteria-derived cell-penetrating effectors (CPEs) are the Yersinia enterocolitica-derived YopM as well as the Salmonella typhimurium effector SspH1. Here, we investigated specifically the group of bacterial LPX effector proteins comprising the Shigella IpaH proteins, which constitute a subtype of the leucine-rich repeat protein family and share significant homologies in sequence and structure. With particular emphasis on the Shigella-effector IpaH9.8, uptake into eukaryotic cell lines was shown. Recombinant IpaH9.8 (rIpaH9.8) is internalized via endocytic mechanisms and follows the endo-lysosomal pathway before escaping into the cytosol. The N-terminal alpha-helical domain of IpaH9.8 was identified as the protein transduction domain required for its CPE ability as well as for being able to deliver other proteinaceous cargo. rIpaH9.8 is functional as an ubiquitin E3 ligase and targets NEMO for poly-ubiquitination upon cell penetration. Strikingly, we could also detect other recombinant LPX effector proteins from Shigella and Salmonella intracellularly when applied to eukaryotic cells. In this study, we provide further evidence for the general concept of T3SS-independent translocation by identifying novel cell-penetrating features of these LPX effectors revealing an abundant species-spanning family of CPE.


Assuntos
Aderência Bacteriana/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência Conservada/genética , Família Multigênica , Domínios e Motivos de Interação entre Proteínas/fisiologia , Fatores de Virulência/química , Animais , Proteínas de Bactérias/metabolismo , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Camundongos , Domínios e Motivos de Interação entre Proteínas/genética , Células RAW 264.7 , Especificidade da Espécie , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Yersinia enterocolitica/química , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo
19.
Sci Rep ; 7(1): 14934, 2017 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-29097778

RESUMO

The carbon storage regulator A (CsrA) and its homologs play an important role in coordinating the expression of bacterial virulence factors required for successful host infection. In addition, bacterial pathogens with deficiency of CsrA are typically attenuated for virulence. In 2016, the first series of small-molecule inhibitors of CsrA-RNA interaction were identified, which were found to achieve the CsrA-RNA inhibition by binding to the CsrA, without interfering with the RNA. However, the binding mechanism of these inhibitors of CsrA is not known. Herein, we applied molecular docking, molecular dynamics and binding free energy calculations to investigate the binding mode of inhibitors to CsrA. We found that the G11(RNA)-binding site is the most important binding site for CsrA inhibitors. An inhibitor with the proper size range can bind to that site and form a stable complex. We also found that inhibitors with larger size ranges bind to the entire CsrA-RNA interface, but have loose binding. However, this loose binding still resulted in inhibitory activity. The calculated binding free energy from MM/GBSA has a good correlation with the derived experimental binding energy, which might be used as a tool to further select CsrA inhibitors after a first-round of high-throughput virtual screening.


Assuntos
Proteínas de Bactérias/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Yersinia enterocolitica/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Sítios de Ligação/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , RNA/química , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Bibliotecas de Moléculas Pequenas/química , Termodinâmica , Yersiniose/tratamento farmacológico , Yersiniose/metabolismo , Yersiniose/microbiologia , Yersinia enterocolitica/química , Yersinia enterocolitica/efeitos dos fármacos
20.
FEMS Microbiol Lett ; 364(20)2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29044402

RESUMO

This study investigated how carbon storage regulator A (CsrA) affects expression of the Ysa and Ysc type 3 secretion (T3S) system genetic regulatory cascades that control Ysps (Yersinia secreted proteins) and Yops (Yersinia outer proteins) export, respectively. Given that most often CsrA acts as a mediator of mRNA stability, an activity that can be monitored using lacZ transcriptional fusions, we employed a collection of reporter strains to assess Ysa and Ysc gene expression. To this end, bacteria were cultivated to induce either the Ysa or the Ysc T3S system. Comparison of csrA mutants to the wild-type strain revealed that, in response to the respective inducing conditions, genes spanning the Ysa and Ysc gene cascades displayed increased expressions. Then, the possibility that CsrA affects secretion of Ysps and Yops was tested and the profiles of secreted proteins by wild-type and csrA mutant strains were compared by proteomic analysis. Ysps were over-secreted and Yops were under-secreted, for the csrA mutant. These results support the hypothesis that CsrA affects both the Ysa and Ysc T3S systems in Yersinia enterocolitica. They further support the conclusion that CsrA plays an important role in controlling adaptation of this pathogenic bacterium during its lifecycle shift between a terrestrial and parasitic existence.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Yersinia enterocolitica/metabolismo , Proteínas de Bactérias/genética , Carbono/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Reporter/genética , Proteômica , Sistemas de Secreção Tipo III/genética , Yersinia enterocolitica/genética , beta-Galactosidase/genética
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