RESUMEN
Mono-O-glycosylation of target proteins by bacterial toxins or effector proteins is a well-known mechanism by which bacteria interfere with essential functions of host cells. The respective glycosyltransferases are important virulence factors such as the Clostridioides difficile toxins A and B. Here, we describe two glycosyltransferases of Yersinia species that have a high sequence identity: YeGT from the zoonotic pathogen Yersinia enterocolitica and YkGT from the murine pathogen Yersinia kristensenii. We show that both modify Rho family proteins by attachment of GlcNAc at tyrosine residues (Tyr-34 in RhoA). Notably, the enzymes differed in their target protein specificity. While YeGT modified RhoA, B, and C, YkGT possessed a broader substrate spectrum and glycosylated not only Rho but also Rac and Cdc42 subfamily proteins. Mutagenesis studies indicated that residue 177 is important for this broader target spectrum. We determined the crystal structure of YeGT shortened by 16 residues N terminally (sYeGT) in the ligand-free state and bound to UDP, the product of substrate hydrolysis. The structure assigns sYeGT to the GT-A family. It shares high structural similarity to glycosyltransferase domains from toxins. We also demonstrated that the 16 most N-terminal residues of YeGT and YkGT are important for the mediated translocation into the host cell using the pore-forming protective antigen of anthrax toxin. Mediated introduction into HeLa cells or ectopic expression of YeGT and YkGT caused morphological changes and redistribution of the actin cytoskeleton. The data suggest that YeGT and YkGT are likely bacterial effectors belonging to the family of tyrosine glycosylating bacterial glycosyltransferases.
Asunto(s)
Proteínas Bacterianas , Tirosina , Yersinia , Glicosilación , Humanos , Yersinia/metabolismo , Yersinia/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Tirosina/metabolismo , Tirosina/química , Glicosiltransferasas/metabolismo , Glicosiltransferasas/genética , Glicosiltransferasas/química , Proteína de Unión al GTP rhoA/metabolismo , Yersinia enterocolitica/metabolismo , Yersinia enterocolitica/genética , Animales , Células HeLa , Ratones , Cristalografía por Rayos X , Yersiniosis/metabolismo , Yersiniosis/microbiologíaRESUMEN
Bacteria use type III secretion injectisomes to inject effector proteins into eukaryotic target cells. Recruitment of effectors to the machinery and the resulting export hierarchy involve the sorting platform. These conserved proteins form pod structures at the cytosolic interface of the injectisome but are also mobile in the cytosol. Photoactivated localization microscopy in Yersinia enterocolitica revealed a direct interaction of the sorting platform proteins SctQ and SctL with effectors in the cytosol of live bacteria. These proteins form larger cytosolic protein complexes involving the ATPase SctN and the membrane connector SctK. The mobility and composition of these mobile pod structures are modulated in the presence of effectors and their chaperones, and upon initiation of secretion, which also increases the number of injectisomes from ~5 to ~18 per bacterium. Our quantitative data support an effector shuttling mechanism, in which sorting platform proteins bind to effectors in the cytosol and deliver the cargo to the export gate at the membrane-bound injectisome.
Asunto(s)
Sistemas de Secreción Tipo III , Yersinia enterocolitica , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Citosol/metabolismo , Transporte de Proteínas , Microscopía FluorescenteRESUMEN
Poly-proline II helices are secondary structure motifs frequently found in ligand-binding sites. They exhibit increased flexibility and solvent exposure compared to the strongly hydrogen-bonded α-helices or ß-strands and can therefore easily be misinterpreted as completely unstructured regions with an extremely high rotational freedom. Here, we show that the adhesin YadA of Yersinia enterocolitica serotype O:9 contains a poly-proline II helix interaction motif in the N-terminal region. The motif is involved in the interaction of YadAO:9 with heparin, a host glycosaminoglycan. We show that the basic residues within the N-terminal motif of YadA are required for electrostatic interactions with the sulfate groups of heparin. Biophysical methods including CD spectroscopy, solution-state NMR and SAXS all independently support the presence of a poly-proline helix allowing YadAO:9 binding to the rigid heparin. Lastly, we show that host cells deficient in sulfation of heparin and heparan sulfate are not targeted by YadAO:9 -mediated adhesion. We speculate that the YadAO:9 -heparin interaction plays an important and highly strain-specific role in the pathogenicity of Yersinia enterocolitica serotype O:9.
Asunto(s)
Adhesinas Bacterianas , Yersinia enterocolitica , Adhesinas Bacterianas/química , Heparina/metabolismo , Dispersión del Ángulo Pequeño , Serogrupo , Electricidad Estática , Difracción de Rayos X , Yersinia enterocolitica/química , Yersinia enterocolitica/metabolismoRESUMEN
The fast envelope stress responses play a key role in the transmission and pathogenesis of Yersinia enterocolitica, one of the most common foodborne pathogens. Our previous study showed that deletion of the waaF gene, essential for the biosynthesis of lipopolysaccharide (LPS) core polysaccharides, led to the formation of a truncated LPS structure and induced cell envelope stress. This envelope stress may disturb the intracellular signal transduction, thereby affecting the physiological functions of Y. enterocolitica. In this study, truncated LPS caused by waaF deletion was used as a model of envelope stress in Y. enterocolitica. We investigated the mechanisms of envelope stress responses and the cellular functions affected by truncated LPS. Transcriptome analysis and phenotypic validation showed that LPS truncation reduced flagellar assembly, bacterial chemotaxis, and inositol phosphate metabolism, presenting lower pathogenicity and viability both in vivo and in vitro environments. Further 4D label-free phosphorylation analysis confirmed that truncated LPS perturbed multiple intracellular signal transduction pathways. Specifically, a comprehensive discussion was conducted on the mechanisms by which chemotactic signal transduction and Rcs system contribute to the inhibition of chemotaxis. Finally, the pathogenicity of Y. enterocolitica with truncated LPS was evaluated in vitro using IPEC-J2 cells as models, and it was found that truncated LPS exhibited reduced adhesion, invasion, and toxicity of Y. enterocolitica to IPEC-J2 cells. Our research provides an understanding of LPS in the regulation of Y. enterocolitica viability and pathogenicity and, thus, opening new avenues to develop novel food safety strategies or drugs to prevent and control Y. enterocolitica infections. KEY POINTS: ⢠Truncated LPS reduces flagellar assembly, chemotaxis, and inositol phosphate metabolism in Y. enterocolitica. ⢠Truncated LPS reduces adhesion, invasion, and toxicity of Y. enterocolitica to IPEC-J2 cells. ⢠Truncated LPS regulates intracellular signal transduction of Y. enterocolitica.
Asunto(s)
Yersiniosis , Yersinia enterocolitica , Humanos , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Lipopolisacáridos/metabolismo , Virulencia , Perfilación de la Expresión Génica , Fosfatos de Inositol/metabolismo , Yersiniosis/microbiologíaRESUMEN
DksA is a proteobacterial regulator that binds directly to the secondary channel of RNA polymerase with (p)ppGpp and is responsible for various bacterial physiological activities. While (p)ppGpp is known to be involved in the regulation and response of fatty acid metabolism pathways in many foodborne pathogens, the role of DksA in this process has yet to be clarified. This study aimed to characterize the function of DksA on fatty acid metabolism and cell membrane structure in Yersinia enterocolitica. Therefore, comparison analysis of gene expression, growth conditions, and membrane permeabilization among the wide-type (WT), DksA-deficient mutant (YEND), and the complemented strain was carried out. It confirmed that deletion of DksA led to a more than four-fold decrease in the expression of fatty acid degradation genes, including fadADEIJ. Additionally, YEND exhibited a smaller growth gap compared to the WT strain at low temperatures, indicating that DksA is not required for the growth of Y. enterocolitica in cold environments. Given that polymyxin B is a cationic antimicrobial peptide that targets the cell membrane, the roles of DksA under polymyxin B exposure were also characterized. It was found that DksA positively regulates the integrity of the inner and outer membranes of Y. enterocolitica under polymyxin B, preventing the leakage of intracellular nucleic acids and proteins and ultimately reducing the sensitivity of Y. enterocolitica to polymyxin B. Taken together, this study provides insights into the functions of DksA and paves the way for novel fungicide development.
Asunto(s)
Proteínas de Escherichia coli , Yersinia enterocolitica , Polimixina B/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Guanosina Pentafosfato/metabolismo , Membrana Celular/metabolismo , Ácidos Grasos , Regulación Bacteriana de la Expresión Génica , Proteínas de Escherichia coli/metabolismoRESUMEN
Iron is both essential for and potentially toxic to bacteria, so the precise maintenance of iron homeostasis is necessary for their survival. Our previous study indicated that in the human enteropathogen Yersinia enterocolitica, the regulator OmpR directly controls the transcription of the fur, fecA and fepA genes, encoding the ferric uptake repressor and two transporters of ferric siderophores, respectively. This study was undertaken to determine the significance of the RNA chaperone Hfq and the small RNAs OmrA and RyhB1 in the post-transcriptional control of the expression of these OmpR targets. We show that Hfq silences fur, fecA and fepA expression post-transcriptionally and negatively affects the production of FLAG-tagged Fur, FecA and FepA proteins. In addition, we found that the fur gene is under the negative control of the sRNA RyhB1, while fecA and fepA are negatively regulated by the sRNA OmrA. Finally, our data revealed that the role of OmrA results from a complex interplay of transcriptional and post-transcriptional effects in the feedback circuit between the regulator OmpR and the sRNA OmrA. Thus, the expression of fur, fecA and fepA is subject to complex transcriptional and post-transcriptional regulation in order to maintain iron homeostasis in Y. enterocolitica.
Asunto(s)
ARN Pequeño no Traducido , Yersinia enterocolitica , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Hierro/metabolismo , Homeostasis/genética , Regulación Bacteriana de la Expresión GénicaRESUMEN
The stringent response is a rapid response system that is ubiquitous in bacteria, allowing them to sense changes in the external environment and undergo extensive physiological transformations. However, the regulators (p)ppGpp and DksA have extensive and complex regulatory patterns. Our previous studies demonstrated that (p)ppGpp and DksA in Yersinia enterocolitica positively co-regulated motility, antibiotic resistance, and environmental tolerance but had opposite roles in biofilm formation. To reveal the cellular functions regulated by (p)ppGpp and DksA comprehensively, the gene expression profiles of wild-type, ΔrelA, ΔrelAΔspoT, and ΔdksAΔrelAΔspoT strains were compared using RNA-Seq. Results showed that (p)ppGpp and DksA repressed the expression of ribosomal synthesis genes and enhanced the expression of genes involved in intracellular energy and material metabolism, amino acid transport and synthesis, flagella formation, and the phosphate transfer system. Additionally, (p)ppGpp and DksA inhibited amino acid utilization (such as arginine and cystine) and chemotaxis in Y. enterocolitica. Overall, the results of this study unraveled the link between (p)ppGpp and DksA in the metabolic networks, amino acid utilization, and chemotaxis in Y. enterocolitica and enhanced the understanding of stringent responses in Enterobacteriaceae.
Asunto(s)
Proteínas de Escherichia coli , Yersinia enterocolitica , Guanosina Pentafosfato/metabolismo , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Transcriptoma , Quimiotaxis/genética , Aminoácidos/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Escherichia coli/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
The membrane-embedded injectisome, the structural component of the virulence-associated type III secretion system (T3SS), is used by Gram-negative bacterial pathogens to inject species-specific effector proteins into eukaryotic host cells. The cytosolic injectisome proteins are required for export of effectors and display both stationary, injectisome-bound populations and freely diffusing cytosolic populations. How the cytosolic injectisome proteins interact with each other in the cytosol and associate with membrane-embedded injectisomes remains unclear. Here, we utilized three-dimensional (3D) single-molecule tracking to resolve distinct cytosolic complexes of injectisome proteins in living Yersinia enterocolitica cells. Tracking of the enhanced yellow fluorescent protein (eYFP)-labeled ATPase YeSctN and its regulator, YeSctL, revealed that these proteins form a cytosolic complex with each other and then further with YeSctQ. YeSctNL and YeSctNLQ complexes can be observed both in wild-type cells and in ΔsctD mutants, which cannot assemble injectisomes. In ΔsctQ mutants, the relative abundance of the YeSctNL complex is considerably increased. These data indicate that distinct cytosolic complexes of injectisome proteins can form prior to injectisome binding, which has important implications for how injectisomes are functionally regulated. IMPORTANCE Injectisomes are membrane-embedded, multiprotein assemblies used by bacterial pathogens to inject virulent effector proteins into eukaryotic host cells. Protein secretion is regulated by cytosolic proteins that dynamically bind and unbind at injectisomes. However, how these regulatory proteins interact with each other remains unknown. By measuring the diffusion rates of single molecules in living cells, we show that cytosolic injectisome proteins form distinct oligomeric complexes with each other prior to binding to injectisomes. We additionally identify the molecular compositions of these complexes and quantify their relative abundances. Quantifying to what extent cytosolic proteins exist as part of larger complexes in living cells has important implications for deciphering the complexity of biomolecular mechanisms. The results and methods reported here are thus relevant for advancing our understanding of how injectisomes and related multiprotein assemblies, such as bacterial flagellar motors, are functionally regulated.
Asunto(s)
Sistemas de Secreción Tipo III , Yersinia enterocolitica , Sistemas de Secreción Tipo III/metabolismo , Citosol , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte de ProteínasRESUMEN
Stringent response plays an important role in the response of Enterobacteriaceae pathogens to rapid environmental changes. It has been shown that synergistic and antagonistic actions exist between the signaling molecules (p)ppGpp and DksA in several foodborne pathogens; however, the biological function of these molecules and their interactions in Yersinia are still unclear. This study systematically investigated the role of stringent response in Yersinia enterocolitica, a typical foodborne Enterobacteriaceae pathogen, by deleting the (p)ppGpp and DksA biosynthesis genes. (p)ppGpp and DksA copositively regulated most phenotypes, such as motility, antibiotic resistance, and tolerance to oxidative stress, whereas they exhibited independent and/or divergent roles in the growth and biofilm synthesis of Y. enterocolitica. Gene expression analysis revealed that (p)ppGpp- and DksA-deficiency reduced the transcription of flagellar synthesis genes (fliC and flgD) and biofilm synthesis genes (bssS and hmsHFRS), which could potentially contribute to changes in motility and biofilm formation. These results indicate that stringent response regulators (p)ppGpp and DksA have a synergistic role and independent or even completely opposite biological functions in regulating genes and phenotypes of Y. enterocolitica. Our findings revealed the biofunctional relationships between (p)ppGpp and DksA and the underlying molecular mechanisms in the regulation of the pathogenic phenotype of Y. enterocolitica. IMPORTANCE The synergetic actions between the stringent response signaling molecules, (p)ppGpp and DksA, have been widely reported. However, recent transcriptomic and phenotypic studies have suggested that independent or even opposite actions exist between them. In this study, we demonstrated that the knockout of (p)ppGpp and DksA affects the polymorphic phenotype of Yersinia enterocolitica. Although most of the tested phenotypes, such as motility, antibiotic resistance, and tolerance to oxidative stress, were copositively regulated by (p)ppGpp and DksA, it also showed inconsistencies in biofilm formation ability as well as some independent phenotypes. This study deepens our understanding of the strategies of foodborne pathogens to survive in complex environments, so as to provide theoretical basis for the control and treatment of these microorganisms.
Asunto(s)
Proteínas Bacterianas , Yersinia enterocolitica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Guanosina Pentafosfato , Fenotipo , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismoRESUMEN
New point-of-care (POC) diagnosis of bacterial infections are imperative to overcome the deficiencies of conventional methods, such as culture and molecular methods. In this study, we identified new aptamers that bind to the virulence factor Yersinia adhesin A (YadA) of Yersinia enterocolitica using cell-systematic evolution of ligands by exponential enrichment (cell-SELEX). Escherichia coli expressing YadA on the cell surface was used as a target cell. After eight cycles of selection, the final aptamer pool was sequenced by high throughput sequencing using the Illumina Novaseq platform. The sequencing data, analyzed using the Geneious software, was aligned, filtered and demultiplexed to obtain the key nucleotides possibly involved in the target binding. The most promising aptamer candidate, Apt1, bound specifically to YadA with a dissociation constant (Kd) of 11 nM. Apt1 was used to develop a simple electrochemical biosensor with a two-step, label-free design towards the detection of YadA. The sensor surface modifications and its ability to bind successfully and stably to YadA were confirmed by cyclic voltammetry, impedance spectroscopy and square wave voltammetry. The biosensor enabled the detection of YadA in a linear range between 7.0 × 104 and 7.0 × 107 CFU mL−1 and showed a square correlation coefficient >0.99. The standard deviation and the limit of detection was ~2.5% and 7.0 × 104 CFU mL−1, respectively. Overall, the results suggest that this novel biosensor incorporating Apt1 can potentially be used as a sensitive POC detection system to aid the diagnosis of Y. enterocolitica infections. Furthermore, this simple yet innovative approach could be replicated to select aptamers for other (bacterial) targets and to develop the corresponding biosensors for their detection.
Asunto(s)
Técnicas Biosensibles , Yersinia enterocolitica , Espectroscopía Dieléctrica , Factores de Virulencia/metabolismo , Yersinia enterocolitica/metabolismoRESUMEN
Lawsonia intracellularis is the causative agent of proliferative enteropathy. While it harbors genes encoding the entire apparatus required for the type III secretion system (T3SS) and the expression of some of these components has been detected during experimental infection, the identification of L. intracellularis T3SS substrates (effector proteins) has been hampered. The Yersinia T3SS and yeast growth inhibition assays are two important heterologous systems used for the characterization of effector proteins. Bacterial EPIYA effectors are a distinct class of bacterial effectors defined by the presence of EPIYA or the EPIYA-related motif. When delivered into host cells via a T3SS or type IV secretion system, these effectors undergo tyrosine phosphorylation of the EPIYA motif, which enables them to manipulate host cell signaling by promiscuously interacting with multiple SH2 domain-containing proteins. A previous study showed that L. intracellularis LI0666 contains two EPIYA motifs and speculated that this protein could be a T3SS effector. In this study, we show that LI0666 is secreted by Yersinia in a T3SS-dependent manner and inhibits yeast growth. LI0666 is phosphorylated at tyrosine residues in porcine intestinal epithelial cells and in human epithelial cells. Like the archetypal EPIYA effector CagA, the EPIYA-containing region is not required for LI0666 association with yeast and mammalian cell membranes. Our results indicate that LI0666 is an authentic bacterial EPIYA effector. Identification of the tyrosine kinases that are responsible for LI0666 phosphorylation and the SH2 domain-containing host proteins that LI0666 interacts with will help to explore the molecular mechanisms of LI0666 in disease development.
Asunto(s)
Lawsonia (Bacteria) , Yersinia enterocolitica , Secuencias de Aminoácidos , Animales , Bacterias , Proteínas Bacterianas/metabolismo , Mamíferos , Saccharomyces cerevisiae , Porcinos , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Tirosina/metabolismo , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismoRESUMEN
In enteropathogen, Yersinia enterocolitica, the genes encoding phage shock proteins are organized in an operon (pspA-E), which is activated at the various types of cellular stress (i.e., extracytoplasmic or envelop stress) whereas, PspA negatively regulates PspF, a transcriptional activator of pspA-E and pspG, and is also involved in other cellular machinery maintenance processes. The exact mechanism of association and dissociation of PspA and PspF during the stress response is not entirely clear. In this concern, we address conformational change of PspA in different pH conditions using various in-silico and biophysical methods. At the near-neutral pH, CD and FTIR measurements reveal a ß-like conformational change of PspA; however, AFM measurement indicates the lower oligomeric form at the above-mentioned pH. Additionally, the results of the MD simulation also support the conformational changes which indicate salt-bridge strength takes an intermediate position compared to other pHs. Furthermore, the bio-layer interferometry study confirms the stable complex formation that takes place between PspA and PspF at the near-neutral pH. It, thus, appears that PspA conformational change in adverse pH conditions abandons PspF from having a stable complex with it, and thus, the latter can act as a trans-activator. Taken together, it seems that PspA alone can transduce adverse signals by changing its conformation.
Asunto(s)
Proteínas de Escherichia coli , Yersinia enterocolitica , Proteínas Bacterianas/química , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/genética , Transactivadores/genética , Transactivadores/metabolismo , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismoRESUMEN
Although the use of the probiotic bacterium Lactobacillus for the treatment and prevention of diseases caused by various pathogenic bacteria has received increasing attention in recent years, its mechanism remains incompletely understood. Levilactobacillus brevis 23017 is a select probiotic strain that can regulate the immunity of host animals and resist pathogen infections. In this study, we analyzed the effect of L. brevis 23017 on Yersinia enterocolitica intestinal infection in a BALB/c mouse model and discussed its underlying mechanism. We found that in the mouse model, L. brevis 23017 prevented the damage of villi in the small intestine and decelerated weight loss after Y. enterocolitica infection. Moreover, we focused on the mechanism of the protective effect of L. brevis 23017 from the perspective of the damage and repair of the intestinal mucosal barrier. We observed that L. brevis 23017 maintained a normal mucosal barrier by altering the expression of tight junction proteins. Notably, our results indicated that L. brevis 23017 effectively promoted the secretion of the intestine-specific secretory immunoglobulin A (SIgA) by B cells via regulating cytokines and oxidative damage levels. This mechanism may be the reason for its protective role in Y. enterocolitica infection. In addition, our results demonstrated that the mechanism of L. brevis 23017 was related to antibacterial colonization and inflammation regulation and closely related to antioxidative stress and SIgA promotion. The protective effect of L. brevis 23017 on mice was related to the signaling pathway protein p38 MAPK and the phosphorylation levels of NF-κB. Our study provided novel insight into the mechanism of Lactobacillus against pathogenic bacterial infections. Such insight is of great importance for the prevention, diagnosis, and treatment of related diseases.
Asunto(s)
Yersiniosis , Yersinia enterocolitica , Animales , Modelos Animales de Enfermedad , Inmunoglobulina A Secretora , Ratones , Ratones Endogámicos BALB C , FN-kappa B/genética , FN-kappa B/metabolismo , Yersinia enterocolitica/metabolismoRESUMEN
In a previous study, differential proteomic analysis was used to identify membrane proteins of the human enteropathogen Yersinia enterocolitica, whose levels are influenced by OmpR, the transcriptional regulator in the two-component EnvZ/OmpR system. Interestingly, this analysis demonstrated that at 37 °C, OmpR negatively affects the level of over a dozen Ysc-Yop proteins, which constitute a type III secretion system (T3SS) that is essential for the pathogenicity of Y. enterocolitica. Here, we focused our analysis on the role of OmpR in the expression and secretion of Yops (translocators and effectors). Western blotting with anti-Yops antiserum and specific anti-YopD, -YopE and -YopH antibodies, confirmed that the production of Yops is down-regulated by OmpR with the greatest negative effect on YopD. The RT-qPCR analysis demonstrated that, while OmpR had a negligible effect on the activity of regulatory genes virF and yscM1, it highly repressed the expression of yopD. OmpR was found to bind to the promoter of the lcrGVsycD-yopBD operon, suggesting a direct regulatory effect. In addition, we demonstrated that the negative regulatory influence of OmpR on the Ysc-Yop T3SS correlated with its positive role in the expression of flhDC, the master regulator of the flagellar-associated T3SS.
Asunto(s)
Yersinia enterocolitica , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación hacia Abajo/genética , Regulación Bacteriana de la Expresión Génica , Proteómica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismoRESUMEN
Yersinia (Y.) enterocolitica, an etiological agent of yersiniosis, is a bacterium whose pathogenicity is determined, among other things, by its ability to produce toxins. The aim of this article was to present the most important toxins that are produced by biotype 1A strains of Y. enterocolitica, and to discuss their role in the pathogenesis of yersiniosis. Y. enterocolitica biotype 1A strains are able to synthesize variants of thermostable YST enterotoxin and play a key role in the pathogenesis of yersiniosis. Biotype 1A strains of Y. enterocolitica also produce Y. enterocolitica pore-forming toxins, YaxA and YaxB. These toxins form pores in the cell membrane of host target cells and cause osmotic lysis, which is of particular importance in systemic infections. Insecticidal toxin complex genes have been detected in some clinical biotype 1A strains of Y. enterocolitica. However, their role has not yet been fully elucidated. Strains belonging to biotype 1A have long been considered non-pathogenic. This view is beginning to change due to the emerging knowledge about the toxigenic potential of these bacteria and their ability to overcome the defense barriers of the host organism.
Asunto(s)
Yersinia enterocolitica , Animales , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/toxicidad , Enterotoxinas/biosíntesis , Enterotoxinas/toxicidad , Humanos , Virulencia , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Yersinia enterocolitica/patogenicidadRESUMEN
Protein tyrosine phosphatases (PTPs) are key virulence factors in pathogenic bacteria, consequently, they have become important targets for new approaches against these pathogens, especially in the fight against antibiotic resistance. Among these targets of interest YopH (Yersinia outer protein H) from virulent species of Yersinia is an example. PTPs can be reversibly inhibited by nitric oxide (NO) since the oxidative modification of cysteine residues may influence the protein structure and catalytic activity. We therefore investigated the effects of NO on the structure and enzymatic activity of Yersinia enterocolitica YopH in vitro. Through phosphatase activity assays, we observe that in the presence of NO YopH activity was inhibited by 50%, and that this oxidative modification is partially reversible in the presence of DTT. Furthermore, YopH S-nitrosylation was clearly confirmed by a biotin switch assay, high resolution mass spectrometry (MS) and X-ray crystallography approaches. The crystal structure confirmed the S-nitrosylation of the catalytic cysteine residue, Cys403, while the MS data provide evidence that Cys221 and Cys234 might also be modified by NO. Interestingly, circular dichroism spectroscopy shows that the S-nitrosylation affects secondary structure of wild type YopH, though to a lesser extent on the catalytic cysteine to serine YopH mutant. The data obtained demonstrate that S-nitrosylation inhibits the catalytic activity of YopH, with effects beyond the catalytic cysteine. These findings are helpful for designing effective YopH inhibitors and potential therapeutic strategies to fight this pathogen or others that use similar mechanisms to interfere in the signal transduction pathways of their hosts.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Cisteína/química , Óxido Nítrico/química , Proteínas Tirosina Fosfatasas/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Biotina/metabolismo , Catálisis , Cristalografía por Rayos X/métodos , Cisteína/metabolismo , Humanos , Espectrometría de Masas/métodos , Estructura Molecular , Óxido Nítrico/metabolismo , Oxidación-Reducción , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal , Yersinia enterocolitica/metabolismoRESUMEN
Non-steroidal anti-inflammatory drugs (NSAIDs) can induce small-intestinal injuries through inhibition of prostaglandin synthesis. Gut has an important role in building and maintaining the barriers to avoid the luminal gut microbiota from invading the host, and cytoskeleton plays a crucial role in the maintenance of cellular barrier. The recent advances suggest a bi-directional interaction between the drugs and gut microbiota, where gut microbes can metabolize the drugs, and in response drugs can alter the composition of gut microbiota. In the present study, we evaluated the effect of diclofenac on rat gut, when co-administrated with either Yersinia enterocolitica strain 8081 (an enteropathogen) or Lactobacillus fermentum strain 9338 (a probiotic). The LC-MS/MS based label-free quantitation of rat gut proteins revealed 51.38% up-regulated, 48.62% down-regulated in diclofenac-Y. enterocolitica strain 8081 (D*Y), and 74.31% up-regulated, 25.69% down-regulated in diclofenac-L. fermentum strain 9338 (D*L) experiments. The identified proteins belonged to cytoskeleton, metabolism, heme biosynthesis and binding, stress response, apoptosis and redox homeostasis, immune and inflammatory response, and detoxification and antioxidant defence. Further, the histopathological and biochemical analysis indicated more pronounced histological alterations and oxidative stress (enhanced malonaldehyde and altered antioxidant levels) in D*Y rats than D*L rats, compared to control rats. Elevated plus maze (EPM) test performed to determine the behavioral changes, suggested increased anxiety in D*Y rats than D*L rats, compared to control rats. These results together suggest the differential role of either bacterium in biotransformation of diclofenac, and inflammatory and cellular redox response.
Asunto(s)
Antiinflamatorios no Esteroideos/toxicidad , Conducta Animal/efectos de los fármacos , Diclofenaco/toxicidad , Microbioma Gastrointestinal , Intestinos/efectos de los fármacos , Limosilactobacillus fermentum/metabolismo , Probióticos , Proteoma/efectos de los fármacos , Yersinia enterocolitica/metabolismo , Animales , Antiinflamatorios no Esteroideos/metabolismo , Biotransformación , Diclofenaco/metabolismo , Disbiosis , Prueba de Laberinto Elevado , Mediadores de Inflamación/metabolismo , Intestinos/metabolismo , Intestinos/microbiología , Intestinos/patología , Masculino , Estrés Oxidativo/efectos de los fármacos , Mapas de Interacción de Proteínas , Proteómica , Ratas Wistar , Transducción de SeñalRESUMEN
Yersinia outer protein M (YopM) is one of the effector proteins and essential for virulence. YopM is delivered by the Yersinia type III secretion system (T3SS) into the host cell, where it shows immunosuppressive effect through interaction with host proteins. Therefore, protein-protein interactions of YopM is significant to understand its molecular mechanism. In this study, we aimed to explore protein-protein interactions of YopM with the two components of T3SS, namely LcrV and LcrG. We used bimolecular fluorescence complementation (BiFC) assay and monitored the reassembly of green fluorescence protein in Escherichia coli. As an indicator of the protein-protein interaction, we monitored the in vivo reconstitution of fluorescence by measuring fluorescence intensity and imaging the cells under fluorescence microscope. We showed, for the first time, that YopM interacts with LcrG, but not with LcrV. Here, we propose BiFC assay as a simple method to screen novel interaction partners of YopM.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Bioensayo , Escherichia coli/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Mapeo de Interacción de Proteínas/métodos , Yersinia pseudotuberculosis/genética , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Fluorescencia , Expresión Génica , Genes Reporteros , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Unión Proteica , Transformación Genética , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Yersinia pestis/genética , Yersinia pestis/metabolismo , Yersinia pseudotuberculosis/metabolismoRESUMEN
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.
Asunto(s)
Antibacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Yersinia enterocolitica/metabolismo , beta-Lactamas/metabolismo , Antibacterianos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Transporte Biológico/genética , Biotecnología/métodos , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Modelos Moleculares , Estructura Molecular , Mutación , Nanotecnología/métodos , Unión Proteica , Dominios Proteicos , Ingeniería de Proteínas/métodos , Protones , Yersinia enterocolitica/genética , beta-Lactamas/químicaRESUMEN
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.