RESUMEN
The methyltransferase FliB posttranslationally modifies surface-exposed É-N-lysine residues of flagellin, the protomer of the flagellar filament in Salmonella enterica (S. enterica). Flagellin methylation, reported originally in 1959, was recently shown to enhance host cell adhesion and invasion by increasing the flagellar hydrophobicity. The role of FliB in this process, however, remained enigmatic. In this study, we investigated the properties and mechanisms of FliB from S. enterica in vivo and in vitro. We show that FliB is an S-adenosylmethionine (SAM) dependent methyltransferase, forming a membrane associated oligomer that modifies flagellin in the bacterial cytosol. Using X-band electron paramagnetic resonance (EPR) spectroscopy, zero-field 57Fe Mössbauer spectroscopy, methylation assays and chromatography coupled mass spectrometry (MS) analysis, we further found that FliB contains an oxygen sensitive [4Fe-4S] cluster that is essential for the methyl transfer reaction and might mediate a radical mechanism. Our data indicate that the [4Fe-4S] cluster is coordinated by a cysteine rich motif in FliB that is highly conserved among multiple genera of the Enterobacteriaceae family.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelina/metabolismo , Proteínas Hierro-Azufre/metabolismo , Lisina/metabolismo , Metiltransferasas/metabolismo , S-Adenosilmetionina/metabolismo , Salmonella typhi/enzimología , Proteínas Bacterianas/genética , Flagelina/química , Proteínas Hierro-Azufre/genética , Lisina/química , Metilación , Metiltransferasas/genéticaRESUMEN
DNA Gyrase is a type II topoisomerase that utilizes the energy of ATP hydrolysis for introducing negative supercoils in DNA. The protein comprises two subunits GyrA and GyrB that form a GyrA2GyrB2 heterotetramer. GyrB subunit contains the N-terminal domain (GBNTD) for ATPase activity and the C-terminal domain (GBCTD) for interaction with GyrA and DNA. Earlier structural studies have revealed three different conformational states for GBNTD during ATP hydrolysis defined as open, semi-open, and closed. Here we report, the three-dimensional structure of a new transient closed conformation of GBNTD from Salmonella Typhi (StGBNTD) at 1.94 Å resolution. Based on the structural analysis of this transient closed conformation, we propose the role of protein in the mechanism of ATP hydrolysis. We further explored the effect of pH on ATPase activity and structural stability of the GBNTD using CD and fluorescence spectroscopy at varying pH environment. Kinetic parameters obtained from the ATPase assay were correlated with its secondary and tertiary structure at their respective pH environment. The protein possessed maximum ATPase activity and structural stability at optimum pH 8. At acidic pH, a remarkable decrease in both enzymatic activity and structural stability was observed whereas at alkaline pH there was no significant change. The structural analysis of StGBNTD reveals the role of polar interactions in stabilizing the overall dimeric conformation of the protein.
Asunto(s)
Adenosina Trifosfatasas/química , Girasa de ADN/química , Salmonella typhi/enzimología , Adenosina Trifosfatasas/genética , Cristalografía por Rayos X , Girasa de ADN/genética , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Dominios Proteicos , Salmonella typhi/genéticaRESUMEN
Gram-negative bacteria have evolved numerous pathways to secrete proteins across their complex cell envelopes. Here, we describe a protein secretion system that uses a holin membrane protein in tandem with a cell wall-editing enzyme to mediate the secretion of substrate proteins from the periplasm to the cell exterior. The identity of the cell wall-editing enzymes involved was found to vary across biological systems. For instance, the chitinase secretion pathway of Serratia marcescens uses an endopeptidase to facilitate secretion, whereas the secretion of Typhoid toxin in Salmonella enterica serovar Typhi relies on a muramidase. Various families of holins are also predicted to be involved. Genomic analysis indicates that this pathway is conserved and implicated in the secretion of hydrolytic enzymes and toxins for a range of bacteria. The pairing of holins from different families with various types of peptidoglycan hydrolases suggests that this secretion pathway evolved multiple times. We suggest that the complementary bodies of evidence presented is sufficient to propose that the pathway be named the Type 10 Secretion System (TXSS).
Asunto(s)
Sistemas de Secreción Bacterianos/fisiología , Bacterias Gramnegativas/enzimología , Bacterias Gramnegativas/fisiología , N-Acetil Muramoil-L-Alanina Amidasa/fisiología , Peptidoglicano/metabolismo , Transporte de Proteínas , Proteínas Virales/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/fisiología , Pared Celular/metabolismo , Quitinasas/metabolismo , Endopeptidasas/metabolismo , Endotoxinas/metabolismo , Humanos , Muramidasa/metabolismo , Salmonella typhi/enzimología , Salmonella typhi/fisiología , Serratia marcescens/enzimología , Serratia marcescens/fisiologíaRESUMEN
The emerged resistance in Typhoidal Salmonella has limited the treatment options for typhoid fever. In this scenario, there is a need to find alternate treatment modalities against this pathogen. Amongst the therapeutic agents currently being used to treat enteric fever, quinolones have enjoyed considerable success since past three decades. These drugs act upon DNA gyrase and the acquired resistance is due to mutations at Ser83 and Asp87 of gyrase A subunit. In the present study DNA gyrase enzyme was targeted to seek out potential new inhibitors which are not affected by these mutations. Molecular modelling and docking studies were performed in Schrödinger's molecular modelling software. Homology model of DNA gyrase-DNA complex was built using templates 1AB4 and 3LTN. Molecular dynamic simulations were performed in SPC solvent for 100 ns. Total 17,900,742 drug like molecules were downloaded from ZINC library of chemical compounds. The Glide XP score of the compounds ranged from -5.285 to -13.692. All the ligands bound at the four base pair staggered nick in the DNA binding groove of DNA gyrase enzyme with their aromatic rings intercalating between the bases of two successive nucleotides stabilized by π - π stacking interactions. The binding pocket of DNA gyrase B comprising conserved residues Lys 447, Gly 448, Lys 449, Ile 450, Leu 451, Gln 465 and Val 467 interacts with the ligand molecules through van der Waals interactions. The MIC (minimum inhibitory concentration), MBC (minimum bactericidal concentration) and IC50 of the tested compounds ranged from 500 to 125 mg/L, 750 to 500 mg/L and 100 to 12.5 mg/L, respectively. The selected hits bind to quinolone binding pocket, but their mode of binding and conformation is different to fluoroquinolones, and hence, their binding is not affected by mutations at Ser83 or Asp87 positions. These lead compounds can be further explored as a scaffold to design inhibitors against DNA gyrase to bypass quinolone resistance.
Asunto(s)
Antibacterianos/farmacología , Girasa de ADN/metabolismo , Descubrimiento de Drogas , Salmonella typhi/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Salmonella typhi/enzimología , Relación Estructura-ActividadRESUMEN
Drug repurposing is an efficient alternative approach to counter the increasing drug-resistant pathogens to treat infectious diseases. FtsZ is an essential bacterial cytokinesis protein involved in the formation of cell-division complex and targeting FtsZ using FDA approved drugs is a promising strategy to identify and develop a new antibacterial drug. Using in silico pharmacophore-based screening of drug bank, molecular docking and molecular dynamics simulations, we identified six drugs inhibiting the function of stFtsZ from Salmonella Typhi. The selected drugs target stFtsZ at the hydrophobic cleft formed between the C-terminal domain and helix α7 with binding energy better than -8 kcal/mol. Out of these six drugs, benzethonium chloride showed promising results at 8 µM concentration where it inhibits stFtsZ GTPase activity by 80% and prevents polymerization. Benzethonium chloride also possesses an excellent antibacterial activity against the bacterial culture of Salmonella Typhi (ATCC 19430), Staphylococcus aureus (ATCC 43300) and Escherichia coli (ATCC 25922) with the MIC values of 8 µg/mL, 1 µg/mL and 12 µg/mL, respectively. Based on our current study, the scaffold of benzethonium chloride can be used for the development of broad-spectrum antibacterial agents against drug-resistant pathogens.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Bencetonio/farmacología , Proteínas del Citoesqueleto/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Salmonella typhi/efectos de los fármacos , Antibacterianos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Bencetonio/química , Sitios de Unión , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Inhibidores Enzimáticos/química , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Salmonella typhi/enzimologíaRESUMEN
The essentiality of DNA Gyrase in basic cellular processes in bacterial pathogens makes it an ideal drug target. Though the Gyrase has a conserved mechanism of action, the complete DNA wrapping and binding process is still unknown. In this study, we have identified six arginine residues R556, R612, R667, R716, R766, and R817 in the DNA GyraseA - C-terminal domain from Salmonella enterica serovar Typhi (StGyrA-CTD) to be essential for DNA wrapping and sliding by a sequence and structure analysis. Through site-directed mutagenesis and EMSA studies, we observed that the substitution of R667 (blade 3) and R716 (blade 4) in StGyrA-CTD led to loss of DNA binding. Whereas, upon mutation of residue R612 (blade2), R766 (blade5) and R817 (blade6) along with supporting residue R712 (blade 4) a decrease in binding affinity was seen. Our results indicate that R667 and R716 act as a pivot point in DNA wrapping and sliding during gyrase catalytic activity. In this study, we propose that the DNA wrapping mechanism commences with DNA binding at blade3 and blade4 followed by other blades to facilitate the DNA sliding during supercoiling activity. This study provides a better understanding of the DNA binding and wrapping mechanism of GyrA-CTD in DNA Gyrase.
Asunto(s)
Arginina/genética , Girasa de ADN/genética , Conformación Proteica en Lámina beta/genética , Salmonella typhi/genética , Secuencia de Aminoácidos/genética , Girasa de ADN/ultraestructura , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación/genética , Unión Proteica/genética , Dominios Proteicos/genética , Salmonella typhi/enzimología , Salmonella typhi/patogenicidadRESUMEN
INTRODUCTION: The multidrug-resistant (MDR) Salmonella enterica serovar Typhi isolates have been increasingly reported from the Asian and African countries. The emergence of isolates with decreased susceptibility to fluoroquinolones and cephalosporins has worsened the situation. Recently, an outbreak from Sindh, Pakistan was reported caused by extensively drug-resistant (XDR) S. Typhi strains. METHODOLOGY: In the present study, a total of 82 cases of typhoid have been investigated during 2018 from the febrile children referred to a tertiary care hospital in the population-wise largest province (Punjab) of Pakistan. S. Typhi was identified by standard microbiological techniques and isolates were characterized for antimicrobial resistance profiling and minimum inhibitory concentrations were determined. The presence of various ESBL genes in S. Typhi was confirmed by the PCR. RESULTS: Out of the 82 isolates tested, 35 (43%) were found to be XDR; resistant to the first-line drugs. The resistance to third-generation cephalosporins was mainly mediated by extended-spectrum beta-lactamases i.e. blaTEM and blaCTX-M genes. CONCLUSIONS: The higher prevalence of ESBL producing Salmonella typhi clinical strains raises the concern about transmission prevention and infection management in the community as well as clinical settings. Moreover, the study highlights the problem concerning the declining antibiotic arsenal for the therapeutic management of typhoid fever and the emergence and spread of XDR strains in Pakistan.
Asunto(s)
Antibacterianos/farmacología , Salmonella typhi/efectos de los fármacos , Fiebre Tifoidea/microbiología , Adolescente , Niño , Preescolar , Farmacorresistencia Bacteriana Múltiple , Femenino , Humanos , Lactante , Masculino , Pruebas de Sensibilidad Microbiana , Pakistán , Reacción en Cadena de la Polimerasa , Salmonella typhi/enzimología , Salmonella typhi/aislamiento & purificación , Centros de Atención Terciaria , Resistencia betalactámica , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
MurE ligase is known to play a significant role in peptidoglycan biosynthesis. It catalyzes the addition of meso-diaminopimelic acid to nucleotide precursor. The protein can adopt different conformations for its proper functioning. Different environmental conditions can alter the stability and function of enzyme due to their ability to disrupt interactions between different domains. We have explored the pH and temperature dependent conformational changes in MurE ligase from Salmonella Typhi and estimated the protein stability. The study enabled us to decipher the effect of different milieu condition in the enzyme activity. At acidic pH 3.0, StMurE ligase forms molten globule (MG) state and at alkaline pH it is in unfolded state. The different states of StMurE ligase were characterized using various spectroscopic techniques. These techniques including near-UV CD, far-UV CD, ANS fluorescence, differential scanning calorimetry and fluorescence spectroscopy helped to determine the secondary structural changes and detect local conformational modifications. The structural analysis using StMurE ligase homology model revealed the variations in ionization states of catalytic amino acid residues involved in substrate binding. This study provides an insight into the dynamics states of StMurE ligase at different environmental conditions during bacterial pathogenesis.
Asunto(s)
Concentración de Iones de Hidrógeno , Modelos Moleculares , Péptido Sintasas/química , Conformación Proteica , Salmonella typhi/enzimología , Temperatura , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Cinética , Péptido Sintasas/metabolismo , Desnaturalización Proteica , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
Typhoid toxin is a virulence factor for the bacterial pathogen Salmonella Typhi, which causes typhoid fever in humans. After its synthesis by intracellular bacteria, typhoid toxin is secreted into the lumen of the Salmonella-containing vacuole by a secretion mechanism strictly dependent on TtsA, a specific muramidase that facilitates toxin transport through the peptidoglycan layer. Here we show that substrate recognition by TtsA depends on a discrete domain within its carboxy terminus, which targets the enzyme to the bacterial poles to recognize YcbB-edited peptidoglycan. Comparison of the atomic structures of TtsA bound to its substrate and that of a close homolog with different specificity identified specific determinants involved in substrate recognition. Combined with structure-guided mutagenesis and in vitro and in vivo crosslinking experiments, this study provides an unprecedented view of the mechanisms by which a muramidase recognizes its peptidoglycan substrate to facilitate protein secretion.
Asunto(s)
Toxinas Bacterianas/metabolismo , Muramidasa/metabolismo , Salmonella typhi/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Modelos Moleculares , Estructura Molecular , Mutación , Peptidoglicano/metabolismo , Transporte de Proteínas , Salmonella typhi/enzimología , Especificidad por Sustrato , Factores de Virulencia/metabolismoRESUMEN
A homology model of ferrochelatase (HemH), the heme biosynthesis terminal step enzyme from Salmonella Typhi was generated to understand the mechanism of metal insertion into protoporphyrin IX for heme biosynthesis. The overall fold of membrane associated ferrochelatase (StFc) from S. Typhi is similar to human and Yeast ferrochelatase than Bacillus subtilis, and Bacillus anthracis. An insertion of 16 amino acid residues in helical switch having hydrophobic patch proposed to interact with membrane lipids and in opening and closing of heme binding cleft. The sequence analysis and the docking study revealed that the protoporphyrin binding site in StFc has a crucial replacement of Tyr/Met to Leu13 unique in comparison to other known structures, where Tyr13 observed in B. subtilis/B. anthracis while Met76 in human/yeast play important role in holding protoporphyrin in optimal orientation for metalation. A sitting-a-top (SAT) complex mechanism for metalation is proposed with His194 and Glu264 lie at the bottom and Leu13 on the top of the porphyrin ring. In addition, an entry and exit mechanism is also proposed for protoporphyrin binding into cavity by opening and closing of helical switch using molecular dynamics simulation studies of Apo and heme complexed model structure of S. Typhi HemH.
Asunto(s)
Proteínas Bacterianas/química , Ferroquelatasa/química , Simulación de Dinámica Molecular , Salmonella typhi/enzimología , Bacillus anthracis/enzimología , Bacillus subtilis/enzimología , Humanos , Protoporfirinas/químicaRESUMEN
PURPOSE: Ceftriaxone is the drug of choice for typhoid fever and the emergence of resistant Salmonella Typhi raises major concerns for treatment. There are an increasing number of sporadic reports of ceftriaxone-resistant S. Typhi and limiting the risk of treatment failure in the patient and outbreaks in the community must be prioritized. This study describes the use of whole genome sequencing to guide outbreak identification and case management. METHODOLOGY: An isolate of ceftriaxone-resistant S. Typhi from the blood of a child taken in 2000 at the Popular Diagnostic Center, Dhaka, Bangladesh was subjected to whole genome sequencing, using an Illumina NextSeq 500 and analysis using Geneious software.Results/Key findings. Comparison with other ceftriaxone-resistant S. Typhi revealed an isolate from the Democratic Republic of the Congo in 2015 as the closest relative but no evidence of an outbreak. A plasmid belonging to incompatibility group I1 (IncI1-ST31) which included blaCTX-M-15 (ceftriaxone resistance) associated with ISEcp-1 was identified. High similarity (90â%) was seen with pS115, an IncI1 plasmid from S. Enteritidis, and with pESBL-EA11, an incI1 plasmid from E. coli (99â%) showing that S. Typhi has access to ceftriaxone resistance through the acquisition of common plasmids. CONCLUSIONS: The transmission of ceftriaxone resistance from E. coli to S. Typhi is of concern because of clinical resistance to ceftriaxone, the main stay of typhoid treatment. Whole genome sequencing, albeit several years after the isolation, demonstrated the success of containment but clinical trials with alternative agents are urgently required.
Asunto(s)
Antibacterianos/farmacología , Ceftriaxona/farmacología , Plásmidos/genética , Salmonella typhi/enzimología , Fiebre Tifoidea/microbiología , beta-Lactamasas/genética , Resistencia a las Cefalosporinas/genética , Niño , Brotes de Enfermedades , Farmacorresistencia Bacteriana/genética , Humanos , Filogenia , Salmonella typhi/efectos de los fármacos , Salmonella typhi/genética , Salmonella typhi/aislamiento & purificación , Secuenciación Completa del GenomaRESUMEN
MurE ligase catalyzes the assembly of peptide moiety, an essential component of bacterial cell wall. We have explored the conformational stability and unfolding equilibrium behaviour of the protein MurE ligase by determining the conformational free energy, entropy and enthalpy parameters under stress conditions. MurE from Salmonella enterica Serovar Typhi was cloned, expressed and purified. Conformational changes associated with increasing concentration of GdmCl- and urea-induced denaturation of MurE were monitored using Circular Dichroism (CD) and fluorescence spectroscopies. The secondary structural content of protein estimated by CD experiment is in close agreement with the predicted MurE ligase structure by homology modeling. Denaturant-induced transition curve was analyzed for thermodynamic parameters. Average values for MurE ligase of ΔGD0â¯=â¯3.13â¯kcalâ¯mol-1, mâ¯=â¯1.52â¯kcalâ¯mol-1â¯M-1 and Cm (=ΔGD0/m)â¯=â¯2.05â¯M were calculated in the presence of GdmCl whereas in the case of urea these were ΔGD0â¯=â¯3.04â¯kcalâ¯mol-1, mâ¯=â¯1.20â¯kcalâ¯mol-1â¯M-1 and Cm (=ΔGD0/m)â¯=â¯2.53â¯M. The observed superposition of normalized transition curve of two independent optical properties suggested that GdmCl- and urea-induced denaturation follow a two-state process.
Asunto(s)
Estructura Molecular , Péptido Sintasas/química , Péptido Sintasas/metabolismo , Salmonella typhi/enzimología , Dicroismo Circular , Activación Enzimática , Estabilidad de Enzimas , Modelos Moleculares , Péptido Sintasas/genética , Péptido Sintasas/aislamiento & purificación , Conformación Proteica , Desnaturalización Proteica , Salmonella typhi/genética , Análisis Espectral , Relación Estructura-ActividadRESUMEN
We report a typhoid fever case with a Salmonella enterica serovar Typhi isolate showing extended spectrum ß-lactamase (ESBL) production in the Democratic Republic of the Congo. Whole genome sequencing revealed that the strain carried a plasmid-mediated CTX-M-15 ESBL gene and did not belong to the dominant H58 Salmonella Typhi clade.
Asunto(s)
Salmonella typhi/enzimología , beta-Lactamasas/metabolismo , Niño , República Democrática del Congo/epidemiología , Genoma Bacteriano/genética , Humanos , Masculino , Filogenia , Salmonella typhi/genética , Fiebre Tifoidea/epidemiología , Fiebre Tifoidea/microbiología , beta-Lactamasas/genéticaRESUMEN
STM2360 is a gene located in a small operon of undetermined function in Salmonella enterica serovar Typhimurium LT2. The amino acid sequence of STM2360 shows significant similarity (â¼30% identity) to diaminopimelate decarboxylase (DapDC), a Fold III pyridoxal-5'-phosphate (PLP) dependent enzyme involved in l-lysine biosynthesis. We have found that the protein coded by STM2360 has a previously undocumented catalytic activity, d-ornithine/d-lysine decarboxylase (DOKDC). The reaction products, cadaverine and putrescine, respectively, were identified by NMR and mass spectrometry. The substrate specificity of DOKDC is d-Lysine > d-Ornithine. This is the first pyridoxal-5'-phosphate dependent decarboxylase identified to act on d-amino acids. STM2358, located in the same operon, has ornithine racemase activity. This suggests that the physiological substrate of the decarboxylase and the operon is ornithine. Homologs of STM2360 with high sequence identity (>80%) are found in other common enterobacteria, including species of Klebsiella, Citrobacter, Vibrio and Hafnia, as well as Clostridium in the Firmicutes, and Pseudomonas.
Asunto(s)
Carboxiliasas/genética , Carboxiliasas/metabolismo , Ornitina Descarboxilasa/genética , Ornitina Descarboxilasa/metabolismo , Salmonella typhi/enzimología , Salmonella typhi/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
We report a traveler who acquired a Salmonella enterica subspecies enterica serovar Typhi strain with resistance against ß-lactams, cephalosporins (extended-spectrum ß-lactamase-producing type SHV-12), and quinolones (plasmid-mediated quinolone resistance gene qnrB7). After clinical deterioration using meropenem monotherapy, treatment success was achieved after commencement of fosfomycin in conjunction with high-dose meropenem. The case illustrates clinical challenges of multidrug-resistant S. Typhi.
Asunto(s)
Antibacterianos/uso terapéutico , Fosfomicina/uso terapéutico , Salmonella typhi , Tienamicinas/uso terapéutico , Fiebre Tifoidea , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple , Fluoroquinolonas/farmacología , Humanos , Masculino , Meropenem , Salmonella typhi/efectos de los fármacos , Salmonella typhi/enzimología , Fiebre Tifoidea/tratamiento farmacológico , Fiebre Tifoidea/microbiología , beta-LactamasasRESUMEN
DNA gyrase, a type II topoisomerase maintains the topology of DNA by introducing negative supercoils using energy generated by ATP hydrolysis. It is composed of two subunits, GyrA and GyrB (GyrA2GyrB2 hetero-tetramer). GyrB comprises two domains, a 43kDa amino N-terminus (GBNTD) and 47kDa carboxyl C- terminus (GBCTD). Till now no study has been reported in terms of stability of Gyrase B and its domains using chemical denaturants related to its function. To understand the role of each domain in GyrB subunit, we estimated the thermodynamic stability of GBF and its individual domains using urea and GdmCl. Changes in secondary and tertiary structures were monitored using circular dichroism and fluorescence spectroscopy. The Cm values for GBNTD, GBCTD and GBF proteins were found to be 2.25, 1.65 and 1.82M during GdmCl-induced denaturation and 2.95, 2.25 and 2.67M for urea-induced denaturation. It is observed that GBNTD is more stable than GBCTD and it contributes to overall stability of GyrB. The lower Cm and ΔG values reflect the flexibility of GBCTD to form the catalytic site along with GANTD for cleavage or religation reaction. Both GdmCl- and urea-induced denaturation of GyrB domains were reversible over the entire range of concentration.
Asunto(s)
Girasa de ADN/química , Desnaturalización Proteica/efectos de los fármacos , Subunidades de Proteína/química , Salmonella typhi/enzimología , Relación Dosis-Respuesta a Droga , Estabilidad de Enzimas/efectos de los fármacos , Guanidina/farmacología , Dominios Proteicos , Urea/farmacologíaRESUMEN
Complex glycans cover the gut epithelial surface to protect the cell from the environment. Invasive pathogens must breach the glycan layer before initiating infection. While glycan degradation is crucial for infection, this process is inadequately understood. Salmonella contains 47 glycosyl hydrolases (GHs) that may degrade the glycan. We hypothesized that keystone genes from the entire GH complement of Salmonella are required to degrade glycans to change infection. This study determined that GHs recognize the terminal monosaccharides (N-acetylneuraminic acid (Neu5Ac), galactose, mannose, and fucose) and significantly (p < 0.05) alter infection. During infection, Salmonella used its two GHs sialidase nanH and amylase malS for internalization by targeting different glycan structures. The host glycans were altered during Salmonella association via the induction of N-glycan biosynthesis pathways leading to modification of host glycans by increasing fucosylation and mannose content, while decreasing sialylation. Gene expression analysis indicated that the host cell responded by regulating more than 50 genes resulting in remodeled glycans in response to Salmonella treatment. This study established the glycan structures on colonic epithelial cells, determined that Salmonella required two keystone GHs for internalization, and left remodeled host glycans as a result of infection. These data indicate that microbial GHs are undiscovered virulence factors.
Asunto(s)
Glicocálix/química , Glicósido Hidrolasas/genética , Mucosa Intestinal/microbiología , Polisacáridos/química , Salmonella typhi/patogenicidad , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células CACO-2 , Línea Celular , Eliminación de Gen , Regulación de la Expresión Génica , Glicósido Hidrolasas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Técnicas In Vitro , Mucosa Intestinal/química , Polisacáridos/metabolismo , Proteolisis , Salmonella typhi/enzimología , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
The thiol-disulfide oxidoreductase CXXC catalytic domain of thioredoxin contributes to antioxidant defense in phylogenetically diverse organisms. We find that although the oxidoreductase activity of thioredoxin-1 protects Salmonella enterica serovar Typhimurium from hydrogen peroxide in vitro, it does not appear to contribute to Salmonella's antioxidant defenses in vivo. Nonetheless, thioredoxin-1 defends Salmonella from oxidative stress resulting from NADPH phagocyte oxidase macrophage expression during the innate immune response in mice. Thioredoxin-1 binds to the flexible linker, which connects the receiver and effector domains of SsrB, thereby keeping this response regulator in the soluble fraction. Thioredoxin-1, independently of thiol-disulfide exchange, activates intracellular SPI2 gene transcription required for Salmonella resistance to both reactive species generated by NADPH phagocyte oxidase and oxygen-independent lysosomal host defenses. These findings suggest that the horizontally acquired virulence determinant SsrB is regulated post-translationally by ancestrally present thioredoxin.
Asunto(s)
Antioxidantes/metabolismo , Proteínas Bacterianas/metabolismo , Tiorredoxinas/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Western Blotting , Dominio Catalítico , Línea Celular , Células HeLa , Humanos , Peróxido de Hidrógeno/toxicidad , Inmunidad Innata , Hígado/microbiología , Macrófagos/citología , Macrófagos/enzimología , Macrófagos/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Modelos Animales , NADPH Oxidasas/metabolismo , Fagocitosis/efectos de los fármacos , Unión Proteica , Proteína Disulfuro Reductasa (Glutatión)/metabolismo , Células RAW 264.7 , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Salmonella typhi/efectos de los fármacos , Salmonella typhi/enzimología , Salmonella typhi/crecimiento & desarrollo , Tiorredoxinas/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Cell-autonomous defense mechanisms are potent strategies that protect individual cells against intracellular pathogens. The Rab-family GTPase Rab32 was previously shown to restrict the intracellular human pathogen Salmonella Typhi, but its potential broader role in antimicrobial defense remains unknown. We show that Rab32 represents a general cell-autonomous, antimicrobial defense that is counteracted by two Salmonella effectors. Mice lacking Rab-32 or its nucleotide exchange factor BLOC-3 are permissive to S. Typhi infection and exhibit increased susceptibility to S. Typhimurium. S. Typhimurium counters this defense pathway by delivering two type III secretion effectors, SopD2, a Rab32 GAP, and GtgE, a specific Rab32 protease. An S. Typhimurium mutant strain lacking these two effectors exhibits markedly reduced virulence, which is fully restored in BLOC-3-deficient mice. These results demonstrate that a cell-autonomous, Rab32-dependent host defense pathway plays a central role in the defense against vacuolar pathogens and describe a mechanism evolved by a bacterial pathogen to counter it.
Asunto(s)
Infecciones por Salmonella/enzimología , Salmonella typhi/fisiología , Salmonella typhimurium/fisiología , Proteínas de Unión al GTP rab/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Ratones , Proteolisis , Infecciones por Salmonella/genética , Infecciones por Salmonella/microbiología , Salmonella typhi/enzimología , Salmonella typhi/genética , Salmonella typhi/patogenicidad , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Virulencia , Proteínas de Unión al GTP rab/genéticaRESUMEN
Typhoid presents a major health concern in developing countries with an estimated annual infection rate of 21 million. The disease is caused by Salmonella typhi, a pathogenic bacterium acquiring multiple drug resistance. We aim to identify proteins that could prove to be putative drug targets in the genome of S. typhi str. Ty2. We employed comparative and subtractive genomics to identify targets that are absent in humans and are essential to S. typhi Ty2. We concluded that 46 proteins essential to pathogen are absent in the host genome. Filtration on the basis of drug target prioritization singled out 20 potentially therapeutic targets. Their absence in the host and specificity to S. typhi Ty2 makes them ideal targets for treating typhoid in Homo sapiens. 3D structures of two of the final target enzymes, MurA and MurB have been predicted via homology modeling which are then used for a docking study.