RESUMEN
Bacteriophage infection, a pivotal process in microbiology, initiates with the phage's tail recognizing and binding to the bacterial cell surface, which then mediates the injection of viral DNA. Although comprehensive studies on the interaction between bacteriophage lambda and its outer membrane receptor, LamB, have provided rich information about the system's biochemical properties, the precise molecular mechanism remains undetermined. This study revealed the high-resolution cryo-electron microscopy (cryo-EM) structures of the bacteriophage lambda tail complexed with its irreversible Shigella sonnei 3070 LamB receptor and the closed central tail fiber. These structures reveal the complex processes that trigger infection and demonstrate a substantial conformational change in the phage lambda tail tip upon LamB binding. Providing detailed structures of bacteriophage lambda infection initiation, this study contributes to the expanding knowledge of lambda-bacterial interaction, which holds significance in the fields of microbiology and therapeutic development.
Asunto(s)
Bacteriófago lambda , Microscopía por Crioelectrón , Shigella sonnei , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Bacteriófago lambda/fisiología , Shigella sonnei/virología , Shigella sonnei/metabolismo , Proteínas de la Cola de los Virus/metabolismo , Proteínas de la Cola de los Virus/química , Proteínas de la Cola de los Virus/genética , Porinas/metabolismo , Porinas/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/ultraestructura , Unión Proteica , Modelos Moleculares , Conformación Proteica , Receptores ViralesRESUMEN
Outer membrane vesicles (OMV) represent an innovative platform for the design of polysaccharide based vaccines. Generalized Modules for Membrane Antigens (GMMA), OMV released from engineered Gram-negative bacteria, have been proposed for the delivery of the O-Antigen, key target for protective immunity against several pathogens including Shigella. altSonflex1-2-3 is a GMMA based vaccine, including S. sonnei and S. flexneri 1b, 2a and 3a O-Antigens, with the aim to elicit broad protection against the most prevalent Shigella serotypes, especially affecting children in low-middle income countries. Here we developed an In Vitro Relative Potency assay, based on recognition of O-Antigen by functional monoclonal antibodies selected to bind the key epitopes of the different O-Antigen active ingredients, directly applied to our Alhydrogel-formulated vaccine. Heat-stressed altSonflex1-2-3 formulations were generated and extensively characterized. The impact of detected biochemical changes in in vivo and in vitro potency assays was assessed. The overall results showed how the in vitro assay can replace the use of animals, overcoming the inherently high variability of in vivo potency studies. The entire panel of physico-chemical methods developed will contribute to detect suboptimal batches and will be valuable to perform stability studies. The work on Shigella vaccine candidate can be easily extended to other O-Antigen based vaccines.
Asunto(s)
Vacunas contra la Shigella , Shigella , Animales , Antígenos O , Shigella sonnei/metabolismo , Vacunas contra la Shigella/metabolismoRESUMEN
The host factor for RNA phage Qß replicase (Hfq) is a crucial post-transcriptional regulator in many bacterial pathogens, facilitating the interaction between small non-coding RNAs (sRNAs) and their target mRNAs. Studies have suggested that Hfq plays a role in antibiotic resistance and virulence in bacteria, although its functions in Shigella are not fully understood. In this study, we investigated the functional roles of Hfq in Shigella sonnei (S. sonnei) by constructing an hfq deletion mutant. Our phenotypic assays showed that the hfq deletion mutant was more sensitivity to antibiotics and had impaired virulence. Transcriptome analyses supported the results concerning the phenotype of the hfq mutant and showed that differentially expressed genes were mainly enriched in the KEGG pathways two-component system, ABC transporters, ribosome, and Escherichia coli biofilm formation. Additionally, we predicted eleven novel Hfq-dependent sRNAs, which were potentially involved in the regulation of antibiotic resistance and/or virulence in S. sonnei. Our findings suggest that Hfq plays a post-transcriptional role in regulating antibiotic resistance and virulence in S. sonnei, and could provide a basis for future studies on Hfq-sRNA-mRNA regulatory networks in this important pathogen.
Asunto(s)
ARN Pequeño no Traducido , Shigella sonnei , Virulencia/genética , Shigella sonnei/genética , Shigella sonnei/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Farmacorresistencia Microbiana , Escherichia coli/metabolismo , ARN/metabolismo , Proteína de Factor 1 del Huésped/genética , Proteína de Factor 1 del Huésped/metabolismo , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismoRESUMEN
There is a continuously increasing pressure associated with the appearance of Salmonella enterica Serovar typhimurium (S. typhimurium) and Shigella sonnei (S. sonnei) that have developed pathogenic multiple antibiotic resistance and the cost of cure and control of these enterobacteriaceae infections increases annually. The current report for first time demonstrated the distinguished antimicrobial action of camel lactoferrin (cLf) obtained from the milk of different clans of camel in Saudi Arabia against S. typhimurium and S. sonnei. These cLf subtypes showed comparable antimicrobial potential when tested against the two bacterial strains but were superior to either bovine (bLf) or human lactoferrin (hLf). The synergism between lactoferrins and antibiotics concerning their antibacterial efficacies against the two bacterial strains was evident. Exploring mechanisms by which camel lactoferrin can kill S. typhimurium and S. sonnei revealed that cLf affects bacterial protein profile. Besides, it interacts with bacterial lipopolysaccharides (LPS) and numerous membrane proteins of S. typhimurium and S. sonnei, with each bacterial strain possessing distinctive binding membrane proteins for lactoferrin. Furthermore, as evidenced by electron microscopy analysis, cLf induces extracellular and intracellular morphological changes in the test bacterial strains when used alone or in combination treatment with antibiotics. Lactoferrin and antibiotics combination strongly disrupts the integrity of the bacterial cells and their membranes. Therefore, cLf can kill S. typhimurium and S. sonnei by four different mechanisms, such as iron chelation, affecting some bacterial proteins, binding to bacterial LPS and membrane proteins, and impairing the integrity of the bacterial cells and their membranes.
Asunto(s)
Antiinfecciosos , Salmonella typhimurium , Animales , Bovinos , Humanos , Salmonella typhimurium/metabolismo , Lactoferrina/farmacología , Shigella sonnei/metabolismo , Camelus/metabolismo , Lipopolisacáridos/farmacología , Serogrupo , Antibacterianos/farmacología , Proteínas de la Membrana/metabolismoRESUMEN
Climate change is expected to affect not only availability and quality of water, the valuable resource of human life on Earth, but also ultimately public health issue. A six-year monitoring (total 20 times) of Escherichia coli O157, Salmonella enterica, Legionella pneumophila, Shigella sonnei, Campylobacter jejuni, and Vibrio cholerae was conducted at five raw water sampling sites including two lakes, Hyundo region (Geum River) and two locations near Water Intake Plants of Han River (Guui region) and Nakdong River (Moolgeum region). A total 100 samples of 40 L water were tested. Most of the targeted bacteria were found in 77% of the samples and at least one of the target bacteria was detected (65%). Among all the detected bacteria, E. coli O157 were the most prevalent with a detection frequency of 22%, while S. sonnei was the least prevalent with a detection frequency of 2%. Nearly all the bacteria (except for S. sonnei) were present in samples from Lake Soyang, Lake Juam, and the Moolgeum region in Nakdong River, while C. jejuni was detected in those from the Guui region in Han River. During the six-year sampling period, individual targeted noxious bacteria in water samples exhibited seasonal patterns in their occurrence that were different from the indicator bacteria levels in the water samples. The fact that they were detected in the five Korea's representative water environments make it necessary to establish the chemical and biological analysis for noxious bacteria and sophisticated management systems in response to climate change.
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Microbiología del Agua , Recursos Hídricos , Campylobacter jejuni/aislamiento & purificación , Campylobacter jejuni/metabolismo , Fenómenos Químicos , Escherichia coli O157/aislamiento & purificación , Escherichia coli O157/metabolismo , Legionella pneumophila/aislamiento & purificación , Legionella pneumophila/metabolismo , República de Corea , Ríos/microbiología , Salmonella enterica/aislamiento & purificación , Salmonella enterica/metabolismo , Shigella sonnei/aislamiento & purificación , Shigella sonnei/metabolismo , Vibrio cholerae/aislamiento & purificación , Vibrio cholerae/metabolismo , Calidad del AguaAsunto(s)
Disentería Bacilar/patología , Shigella sonnei , Disentería Bacilar/epidemiología , Genoma Bacteriano/genética , Humanos , Shigella sonnei/clasificación , Shigella sonnei/genética , Shigella sonnei/metabolismo , Shigella sonnei/patogenicidad , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/fisiología , Sistemas de Secreción Tipo VI/genética , Sistemas de Secreción Tipo VI/fisiologíaRESUMEN
Although multiprotein membrane complexes play crucial roles in bacterial physiology and virulence, the mechanisms governing their quality control remain incompletely understood. In particular, it is not known how unincorporated, orphan components of protein complexes are recognised and eliminated from membranes. Rhomboids, the most widespread and largest superfamily of intramembrane proteases, are known to play key roles in eukaryotes. In contrast, the function of prokaryotic rhomboids has remained enigmatic. Here, we show that the Shigella sonnei rhomboid proteases GlpG and the newly identified Rhom7 are involved in membrane protein quality control by specifically targeting components of respiratory complexes, with the metastable transmembrane domains (TMDs) of rhomboid substrates protected when they are incorporated into a functional complex. Initial cleavage by GlpG or Rhom7 allows subsequent degradation of the orphan substrate. Given the occurrence of this strategy in an evolutionary ancient organism and the presence of rhomboids in all domains of life, it is likely that this form of quality control also mediates critical events in eukaryotes and protects cells from the damaging effects of orphan proteins.
Asunto(s)
Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Shigella sonnei/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Transporte de Electrón , Endopeptidasas/química , Dominios Proteicos , Proteolisis , Shigella sonnei/metabolismo , Especificidad por SustratoRESUMEN
Lactobacillus species possesses surface exposed Mucin Binding Protein (MucBP) which plays a role in adhesion to gastrointestinal mucin. MucBP contains one or more mucin binding domain (MBD), the functionality of which has yet not been characterized thoroughly. Here, we have characterized a 93-amino acid MBD (MBD93) of MucBP (LAF_0673) from Lactobacillus fermentum. Multiple sequence alignment of L. fermentum MBD93 exhibited â¼60% sequence homology with MBDs from other Lactobacillus species. Further, we cloned, expressed and purified MBD93 from Escherichia coli as N-terminal histidine-tagged protein (6X His-MBD93). The purified MBD93 was able to bind to mucin and showed strong affinity towards the terminally expressed mucin glycans viz. N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), Galactose (Gal), and Sialic acid (N-acetylneuraminic acid; Neu5Ac). In silico experiments further confirmed the interaction between homology modeled MBD93 to mucin glycans through hydrogen-bonding with its surface amino acid residues Ser57, Pro58, Ile60, Tyr63 and Ala65. We also have demonstrated that MBD93 was able to inhibit the adhesion of enteric pathogens, including E. coli, Salmonella Paratyphi A, Shigella sonnei and Proteus vulgaris to mucin. Our results suggested that L. fermentum MBD93 is a functionally sufficient unit to act as an adhesin and to protect from invading enteric pathogens.
Asunto(s)
Adhesinas Bacterianas , Adhesión Bacteriana/efectos de los fármacos , Limosilactobacillus fermentum , Mucinas/metabolismo , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Adhesinas Bacterianas/farmacología , Animales , Escherichia coli Enteropatógena/metabolismo , Escherichia coli Enteropatógena/patogenicidad , Limosilactobacillus fermentum/química , Limosilactobacillus fermentum/genética , Limosilactobacillus fermentum/metabolismo , Dominios Proteicos , Proteus vulgaris/metabolismo , Proteus vulgaris/patogenicidad , Salmonella paratyphi A/metabolismo , Salmonella paratyphi A/patogenicidad , Shigella sonnei/metabolismo , Shigella sonnei/patogenicidad , PorcinosRESUMEN
Bacillary dysentery caused by infection with Shigella spp. remains as serious and common health problem throughout the world. It is a highly multi drug resistant organism and rarely identified from the patient at the early stage of infection. S. sonnei is the most frequently isolated species causing shigellosis in industrialized countries. The antigenicity of outer membrane protein of this pathogen expressed during human infection has not been identified to date. We have studied the antigenic outer membrane proteins expressed by S. sonnei, with the aim of identifying presence of specific IgA and IgG in human serum against the candidate protein biomarkers. Three antigenic OMPs sized 33.3, 43.8 and 100.3 kDa were uniquely recognized by IgA and IgG from patients with S. sonnei infection, and did not cross-react with sera from patients with other types of infection. The antigenic proteome data generated in this study are a first for OMPs of S. sonnei, and they provide important insights of human immune responses. Furthermore, numerous prime candidate proteins were identified which will aid the development of new diagnostic tools for the detection of S. sonnei.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Disentería Bacilar/microbiología , Shigella sonnei/inmunología , Adolescente , Adulto , Proteínas de la Membrana Bacteriana Externa/metabolismo , Niño , Femenino , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Shigella sonnei/aislamiento & purificación , Shigella sonnei/metabolismo , Adulto JovenRESUMEN
Shigella sonnei is one of the major causes of shigellosis in technically advanced countries and reports of its unprecedented increase are published from the Middle East, Latin America, and Asia. The pathogen exhibits resistance against first and second line antibiotics which highlights the need for the development of an effective broad-spectrum vaccine. A computational based approach comprising subtractive reverse vaccinology was used for the identification of potential peptide-based vaccine candidates in the proteome of S. sonnei reference strain (53G). The protocol revealed three essential, host non-homologous, highly virulent, antigenic, conserved and adhesive vaccine proteins: TolC, PhoE, and outer membrane porin protein. The cellular interactome of these proteins supports their direct and indirect involvement in biologically significant pathways, essential for pathogen survival. Epitope mapping of these candidates reveals the presence of surface exposed 9-mer B-cell-derived T-cell epitopes of an antigenic, virulent, non-allergen nature and have broad-spectrum potency. In addition, molecular docking studies demonstrated the deep binding of the epitopes in the binding groove and the stability of the complex with the most common binding allele in the human population, DRB1*0101. Future characterization of the screened epitopes in order to further investigate the immune protection efficacy in animal models is highly desirable.
Asunto(s)
Vacunas Bacterianas/inmunología , Disentería Bacilar/inmunología , Shigella sonnei/inmunología , Vacunas de Subunidad/inmunología , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Biología Computacional/métodos , Disentería Bacilar/microbiología , Disentería Bacilar/patología , Mapeo Epitopo , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Humanos , Porinas/inmunología , Porinas/metabolismo , Unión Proteica/inmunología , Proteoma/inmunología , Proteoma/metabolismo , Proteómica/métodos , Shigella sonnei/metabolismo , Shigella sonnei/fisiologíaRESUMEN
Shigella is a leading cause of dysentery worldwide, with the majority of infections caused by two subgroups, S. flexneri and S. sonnei. Although S. flexneri has been highly prevalent in low-income countries, global development has brought an increase in S. sonnei at the expense of S. flexneri. However, the mechanisms behind this shift are not understood. Here we report that S. sonnei, but not S. flexneri, encodes a type VI secretion system (T6SS) that provides a competitive advantage in the gut. S. sonnei competes against E. coli and S. flexneri in mixed cultures, but this advantage is reduced in T6SS mutant strains. In addition, S. sonnei can persist as well as outcompete E. coli and S. flexneri in mice in a T6SS-dependent manner. These findings suggest that S. sonnei has a competitive advantage over S. flexneri and potentially explain the increasing global prevalence of S. sonnei.
Asunto(s)
Disentería Bacilar/microbiología , Shigella sonnei/metabolismo , Sistemas de Secreción Tipo VI/genética , Sistemas de Secreción Tipo VI/fisiología , Animales , Antibiosis/fisiología , Técnicas de Cocultivo , Colon/microbiología , Colon/patología , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Femenino , Cobayas , Lactobacillus/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , Interacciones Microbianas , Mutación , Shigella flexneri/efectos de los fármacos , Shigella flexneri/genética , Shigella flexneri/crecimiento & desarrollo , Shigella sonnei/genética , Shigella sonnei/crecimiento & desarrollo , Sistemas de Secreción Tipo VI/farmacologíaRESUMEN
In this paper, a Whole-Bacteria SELEX (WB-SELEX) strategy was adopted to isolate specific aptamers against Shigella sonnei. Real-time PCR amplification and post-SELEX experiment revealed that the selected aptmers possessed a high binding affinity and specificity for S. sonnei. Of the 21 aptamers tested, the C(t) values of the SS-3 and SS-4 aptamers (Ct = 13.89 and Ct = 12.23, respectively) had the lowest value compared to other aptamer candidates. The SS-3 and SS-4 aptamers also displayed a binding affinity (KD) of 39.32 ± 5.02 nM and 15.89 ± 1.77 nM, respectively. An aptamer-based fluorescent biosensor assay was designed to detect and discriminate S. sonnei cells using a sandwich complex pair of SS-3 and SS-4. The detection of S. sonnei by the aptamer based fluorescent biosensor platform consisted of three elements: (1) 5'amine-SS-4 modification in a 96-well type microtiter plate surface (N-oxysuccinimide, NOS) as capture probes; (2) the incubation with S. sonnei and test microbes in functionalized 96 assay wells in parallel; (3) the readout of fluorescent activity using a Cy5-labeled SS-3 aptamer as the detector. Our platform showed a significant ability to detect and discriminate S. sonnei from other enteric species such as E. coli, Salmonella typhimurium and other Shigella species (S. flexneri, S. boydii). In this study, we demonstrated the feasibility of an aptamer sensor platform to detect S. sonnei in a variety of foods and pave the way for its use in diagnosing shigellosis through multiple, portable designs.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Técnicas Biosensibles/métodos , Shigella sonnei/clasificación , Shigella sonnei/aislamiento & purificación , Aptámeros de Nucleótidos/aislamiento & purificación , Escherichia coli/clasificación , Escherichia coli/aislamiento & purificación , Fluorescencia , Reacción en Cadena en Tiempo Real de la Polimerasa , Técnica SELEX de Producción de Aptámeros , Salmonella typhimurium/clasificación , Salmonella typhimurium/aislamiento & purificación , Sensibilidad y Especificidad , Shigella sonnei/metabolismoRESUMEN
A major limitation in using bacteriophage-based applications is their narrow host range. Approaches for extending the host range have focused primarily on lytic phages in hosts supporting their propagation rather than approaches for extending the ability of DNA transduction into phage-restrictive hosts. To extend the host range of T7 phage for DNA transduction, we have designed hybrid particles displaying various phage tail/tail fiber proteins. These modular particles were programmed to package and transduce DNA into hosts that restrict T7 phage propagation. We have also developed an innovative generalizable platform that considerably enhances DNA transfer into new hosts by artificially selecting tails that efficiently transduce DNA. In addition, we have demonstrated that the hybrid particles transduce desired DNA into desired hosts. This study thus critically extends and improves the ability of the particles to transduce DNA into novel phage-restrictive hosts, providing a platform for myriad applications that require this ability.
Asunto(s)
Bacteriófago T7/genética , ADN Bacteriano/genética , ADN Viral/genética , Escherichia coli/genética , Vectores Genéticos , Klebsiella pneumoniae/genética , Shigella sonnei/genética , Transducción Genética/métodos , Virión , ADN Bacteriano/biosíntesis , ADN Viral/biosíntesis , Escherichia coli/metabolismo , Escherichia coli/virología , Regulación Bacteriana de la Expresión Génica , Regulación Viral de la Expresión Génica , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/virología , Shigella sonnei/metabolismo , Shigella sonnei/virologíaRESUMEN
Serum Bactericidal Activity (SBA) assay is the method of choice to evaluate the complement-mediated functional activity of both infection- and vaccine-induced antibodies. To perform a typical SBA assay, serial dilutions of sera are incubated with target bacterial strains and complement. The conventional SBA assay is based on plating on agar the SBA reaction mix and counting the surviving bacterial colony forming units (CFU) at each serum dilution. Even with automated colony counting, it is labor-intensive, time-consuming and not amenable for large-scale studies. Here, we have developed a luminescence-based SBA (L-SBA) method able to detect surviving bacteria by measuring their ATP. At the end of the SBA reaction, a single commercially available reagent is added to each well of the SBA plate, and the resulting luminescence signal is measured in a microplate reader. The signal obtained is proportional to the ATP present, which is directly proportional to the number of viable bacteria. Bactericidal activity is subsequently calculated. We demonstrated the applicability of L-SBA with multiple bacterial serovars, from 5 species: Citrobacter freundii, Salmonella enterica serovars Typhimurium and Enteritidis, Shigella flexneri serovars 2a and 3a, Shigella sonnei and Neisseria meningitidis. Serum bactericidal titers obtained by the luminescence readout method strongly correlate with the data obtained by the conventional agar plate-based assay, and the new assay is highly reproducible. L-SBA considerably shortens assay time, facilitates data acquisition and analysis and reduces the operator dependency, avoiding the plating and counting of CFUs. Our results demonstrate that L-SBA is a useful high-throughput bactericidal assay.
Asunto(s)
Adenosina Trifosfato/inmunología , Bacterias/inmunología , Actividad Bactericida de la Sangre/inmunología , Ensayos Analíticos de Alto Rendimiento/métodos , Sueros Inmunes/inmunología , Adenosina Trifosfato/metabolismo , Animales , Bacterias/metabolismo , Citrobacter freundii/inmunología , Citrobacter freundii/metabolismo , Sueros Inmunes/sangre , Modelos Lineales , Ratones , Viabilidad Microbiana/inmunología , Neisseria meningitidis/inmunología , Neisseria meningitidis/metabolismo , Reproducibilidad de los Resultados , Salmonella enterica/inmunología , Salmonella enterica/metabolismo , Shigella flexneri/inmunología , Shigella flexneri/metabolismo , Shigella sonnei/inmunología , Shigella sonnei/metabolismo , Especificidad de la EspecieRESUMEN
The chb operon of Escherichia coli is involved in the utilization of chitooligosaccharides. While acquisition of two classes of mutations leading to altered regulation of the chb operon is necessary to confer the ability to utilize the glucose disaccharide cellobiose to wild-type strains of E. coli, in the closely related organism Shigella sonnei, Cel+ mutants arise relatively faster, requiring only a single mutational event. In Type I mutants, the insertion of IS600 at -21 leads to ChbR regulator-independent, constitutive expression of the operon. In Type II mutants, the insertion of IS2/600 within the distal binding site of the negative regulator NagC leads to ChbR-dependent cellobiose-inducible expression of the operon. These studies underscore the significance of strain background, specifically the diversity of transposable elements, in the evolution of novel metabolic functions. Constitutive expression of the chb operon also enables utilization of the aromatic ß-glucosides arbutin and salicin, implying that the chb structural genes are inherently promiscuous.
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Celobiosa/metabolismo , Escherichia coli/genética , Operón , Shigella sonnei/genética , Arbutina/metabolismo , Alcoholes Bencílicos/metabolismo , Elementos Transponibles de ADN , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Evolución Molecular , Glucósidos/metabolismo , Mutación , Proteínas Represoras/genética , Shigella sonnei/metabolismoRESUMEN
To cope with hyperosmotic stress encountered in the environments and in the host, the pathogenic as well as non-pathogenic microbes use diverse transport systems to obtain osmoprotectants. To study the role of Shigella sonnei ProU system in response to hyperosmotic stress and virulence, we constructed deletion and complementation strains of proV and used an RNAi approach to silence the whole ProU operon. We compared the response between wild type and the mutants to the hyperosmotic pressure in vitro, and assessed virulence properties of the mutants using gentamicin protection assay as well as Galleria mellonella moth larvae model. In response to osmotic stress by either NaCl or KCl, S. sonnei highly up-regulates transcription of proVWX genes. Supplementation of betaine greatly elevates the growth of the wild type S. sonnei but not the proV mutants in M9 medium containing 0.2 M NaCl or 0.2 M KCl. The proV mutants are also defective in intracellular growth compared with the wild type. The moth larvae model of G. mellonella shows that either deletion of proV gene or knockdown of proVWX transcripts by RNAi significantly attenuates virulence. ProU system in S. sonnei is required to cope with osmotic stress for survival and multiplication in vitro, and for infection.
Asunto(s)
Proteínas Bacterianas/metabolismo , Osmorregulación , Shigella sonnei/fisiología , Shigella sonnei/patogenicidad , Animales , Proteínas Bacterianas/genética , Betaína/metabolismo , Bioensayo , Medios de Cultivo/química , Eliminación de Gen , Prueba de Complementación Genética , Células HEK293 , Humanos , Larva/microbiología , Larva/fisiología , Lepidópteros , Presión Osmótica , Cloruro de Potasio/metabolismo , Shigella sonnei/genética , Shigella sonnei/metabolismo , Cloruro de Sodio/metabolismo , Análisis de Supervivencia , VirulenciaRESUMEN
AIM: Determine features of epidemic process (EP) of Sonnei dysentery in Khabarovsk Region in 2012 - 2014 due to atypical causative agent. MATERIALS AND METHODS: Detailed characteristics of 161 cultures of Shigella sonnei isolated from 81 patients from epidemic focus in children boarding school in Bikin as well as from 22 patients from sporadic and group foci of dysenteryin Khabarovsk (biochemical type, colicin-genotype, spectrum of drug resistance) is given. Molecular-biologic subtyping was carried out for 11 strains by Pulsed Field Gel Electrophoresis method (PFGE). RESULTS: Materials of observation of a prolonged foci of Sonnei dysentery with contact-domestic transmission route of the infection in children boarding house for disabled (October 2012 - September 2014) are presented. The diseases are etiologically connected with atypical mannitol- negative types of shigella isolated for the first time in 40 years of observation in Khabarovsk region. Epidemic process of shigellosis was supported by prolonged carriership of the causative agent in patients and special contingent ofthe nursing home. Shigella cultures isolated in the focus belonged to the same colicin-genotype and 2 distinct drug resistance clones, but a single genotype established by PFGE method. CONCLUSION: Results of the studies give evidence on the importance of determi- nation of traditional phenotypic and contemporary genotypic variants of shigella and the neces- sity of search for arguments, additional methodic approaches for establishing similarities and differences of shigella isolates from within the same outbreak of the diseases as well as for com- parison of strains circulating in different territories.
Asunto(s)
Técnicas de Tipificación Bacteriana , Brotes de Enfermedades , Farmacorresistencia Bacteriana , Disentería Bacilar , Genotipo , Shigella sonnei , Disentería Bacilar/epidemiología , Disentería Bacilar/microbiología , Femenino , Humanos , Masculino , Manitol/metabolismo , Shigella sonnei/clasificación , Shigella sonnei/genética , Shigella sonnei/aislamiento & purificación , Shigella sonnei/metabolismo , Siberia/epidemiologíaRESUMEN
Atypical enteropathogenic Escherichia coli (aEPEC) are heterogeneous strains in terms of serotypes, adherence patterns and the presence of novel virulence factors. This heterogeneity is intriguing, promoting studies trying to characterize these novel proteins and to better comprehend this pathotype group. In a previous study analyzing low-molecular mass proteomes of four representative aEPEC strains of three different adhesion phenotypes, we classified proteins according to their annotated function, with most of them being involved in metabolism and transport; while some of them were classified as hypothetical proteins. The majority of the hypothetical proteins were homologue products of genes identified in the genome of enterohemorrhagic E. coli. One of the hypothetical proteins was annotated as Z2335, with orthologue in EPEC, and by bioinformatics analysis, this protein was revealed to be the universal stress protein F (UspF). Thus, herein we successfully obtained a recombinant UspF protein from aEPEC, which is a α/ß, ATP-binding protein involved in stress response, with comparable protein production among the four studied strains, but showing noteworthy differences when cultivated in different stress conditions, also present in other enterobacterial species, such as Shigella sonnei and Citrobacter freundii. Furthermore, our results confirm that the Usp protein superfamily encompasses a conserved group of proteins involved in stress resistance in aEPEC and other Enterobacteriaceae.
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Citrobacter freundii , Escherichia coli Enteropatógena , Proteínas de Escherichia coli , Proteínas de Choque Térmico , Shigella sonnei , Citrobacter freundii/genética , Citrobacter freundii/metabolismo , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Shigella sonnei/genética , Shigella sonnei/metabolismoRESUMEN
Shigella sonnei has become predominant species causing shigellosis in Shanghai. Two hundred ninety-three S. sonnei were isolated in sentinel hospitals of Shanghai in 2011. We found an emergence of 8 strains of S. sonnei with negative phenotype for o-nitrophenyl-ß-d-galactopyranoside in late August, which showed distinct pulsed-field gel electrophoresis patterns from the other 285 S. sonnei and had genes deletion in lac and mhp operons.
Asunto(s)
Disentería Bacilar/microbiología , Nitrofenilgalactósidos/metabolismo , Shigella sonnei/clasificación , Shigella sonnei/aislamiento & purificación , Adulto , Técnicas de Tipificación Bacteriana , China , Electroforesis en Gel de Campo Pulsado , Femenino , Eliminación de Gen , Genotipo , Hospitales , Humanos , Masculino , Persona de Mediana Edad , Tipificación Molecular , Fenotipo , Shigella sonnei/genética , Shigella sonnei/metabolismo , Adulto JovenRESUMEN
Recently, we developed a high yield production process for outer membrane particles from genetically modified bacteria, called Generalized Modules of Membrane Antigens (GMMA), and the corresponding simple two step filtration purification, enabling economic manufacture of these particles for use as vaccines. Using a Shigella sonnei strain that was genetically modified to produce penta-acylated lipopolysaccharide (LPS) with reduced endotoxicity and to maintain the virulence plasmid encoding for the immunodominant O antigen component of the LPS, scale up of the process to GMP pilot scale was straightforward and gave high yields of GMMA with required purity and consistent results. GMMA were formulated with Alhydrogel and were highly immunogenic in mice and rabbits. In mice, a single immunization containing 29 ng protein and 1.75 ng of O antigen elicited substantial anti-LPS antibody levels. As GMMA contain LPS and lipoproteins, assessing potential reactogenicity was a key aspect of vaccine development. In an in vitro monocyte activation test, GMMA from the production strain showed a 600-fold lower stimulatory activity than GMMA with unmodified LPS. Two in vivo tests confirmed the low potential for reactogenicity. We established a modified rabbit pyrogenicity test based on the European Pharmacopoeia pyrogens method but using intramuscular administration of the full human dose (100 µg of protein). The vaccine elicited an average temperature rise of 0.5°C within four hours after administration, which was considered acceptable and showed that the test is able to detect a pyrogenic response. Furthermore, a repeat dose toxicology study in rabbits using intramuscular (100 µg/dose), intranasal (80 µg/dose), and intradermal (10 µg/dose) administration routes showed good tolerability of the vaccine by all routes and supported its suitability for use in humans. The S. sonnei GMMA vaccine is now in Phase 1 dose-escalation clinical trials.