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1.
Commun Biol ; 4(1): 448, 2021 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-33837253

RESUMEN

In Pseudomonas aeruginosa, Ttg2D is the soluble periplasmic phospholipid-binding component of an ABC transport system thought to be involved in maintaining the asymmetry of the outer membrane. Here we use the crystallographic structure of Ttg2D at 2.5 Å resolution to reveal that this protein can accommodate four acyl chains. Analysis of the available structures of Ttg2D orthologs shows that they conform a new substrate-binding-protein structural cluster. Native and denaturing mass spectrometry experiments confirm that Ttg2D, produced both heterologously and homologously and isolated from the periplasm, can carry two diacyl glycerophospholipids as well as one cardiolipin. Binding is notably promiscuous, allowing the transport of various molecular species. In vitro binding assays coupled to native mass spectrometry show that binding of cardiolipin is spontaneous. Gene knockout experiments in P. aeruginosa multidrug-resistant strains reveal that the Ttg2 system is involved in low-level intrinsic resistance against certain antibiotics that use a lipid-mediated pathway to permeate through membranes.


Asunto(s)
Proteínas Bacterianas/genética , Glicerofosfolípidos/metabolismo , Proteínas de Transporte de Membrana/genética , Periplasma/metabolismo , Pseudomonas aeruginosa/genética , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana/genética , Proteínas de Transporte de Membrana/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo
2.
Front Microbiol ; 9: 928, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29867838

RESUMEN

Infections by Streptococcus pneumoniae are a major cause of morbidity and mortality worldwide, often causing community-acquired pneumonia, otitis media and also bacteremia and meningitis. Studies on S. pneumoniae are mainly focused on its virulence or capacity to evade the host immune system, but little is known about the injury caused in lungs during a pneumococcal infection. Herein we investigated this issue comparing the proteome profile of lungs from S. pneumoniae-infected mice with control mice by means of difference gel electrophoresis (DIGE) technology. In order to obtain reliable results three biological replicas were used, and four technical replicas were carried out in each biological replica. Proteomic comparison was performed at two time points: 24 and 48 h post infection. A total of 91 proteins were identified with different abundance. We found important changes in the protein profiles during pneumococcal infection mainly associated with regulation of vesicle-mediated transport, wound healing, and cytoskeleton organization. In conclusion, the results obtained show that the cytoskeleton of the host cell is modified in S. pneumoniae infection.

3.
Sci Rep ; 7(1): 15291, 2017 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-29127378

RESUMEN

Salmonella possesses virulence determinants that allow replication under extreme conditions and invasion of host cells, causing disease. Here, we examined four putative genes predicted to encode membrane proteins (ydiY, ybdJ, STM1441 and ynaJ) and a putative transcriptional factor (yedF). These genes were identified in a previous study of a S. Typhimurium clinical isolate and its multidrug-resistant counterpart. For STM1441 and yedF a reduced ability to interact with HeLa cells was observed in the knock-out mutants, but an increase in this ability was absent when these genes were overexpressed, except for yedF which phenotype was rescued when yedF was restored. In the absence of yedF, decreased expression was seen for: i) virulence-related genes involved in motility, chemotaxis, attachment and survival inside the host cell; ii) global regulators of the invasion process (hilA, hilC and hilD); and iii) factors involved in LPS biosynthesis. In contrast, an increased expression was observed for anaerobic metabolism genes. We propose yedF is involved in the regulation of Salmonella pathogenesis and contributes to the activation of the virulence machinery. Moreover, we propose that, when oxygen is available, yedF contributes sustained repression of the anaerobic pathway. Therefore, we recommend this gene be named vrf, for virulence-related factor.


Asunto(s)
Proteínas Bacterianas , Farmacorresistencia Bacteriana Múltiple , Salmonella typhimurium , Factores de Transcripción , Factores de Virulencia , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células HeLa , Humanos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidad , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo
4.
Pediatr Infect Dis J ; 36(11): 1079-1086, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28650938

RESUMEN

BACKGROUND: Escherichia coli is one of the most frequent causes of late-onset neonatal sepsis. The aim of this study was to characterize an outbreak of neonatal sepsis occurring in the neonatal intensive care unit of the Hospital Clinic of Barcelona from April to August 2013. METHODS: After presentation of the index case, all E. coli isolates from previously hospitalized neonates, health-care workers and neonates admitted to the neonatal intensive care unit from April to October 2013 were tested for K1 antigen positivity and epidemiologically compared by pulse-field gel electrophoresis. Furthermore, the E. coli K1 strains collected from neonates during this period were analyzed by different methods (serotyping, phylotyping, polymerase chain reaction of virulence factors, antimicrobial resistance and "in vitro" assays in Human Brain Microvascular Endothelial Cells (HBMEC)). RESULTS: An E. coli O18:K1:H7 sequence type 95 and phylogenetic group B2 strain was the cause of the outbreak involving 6 preterm neonates: 1 with late septicemia because of a urinary focus and 5 with late-onset septicemia and meningitis, 3 of whom died. All showed the same pulsotype, full resistance to ampicillin and intermediate resistance to gentamicin. The outbreak strain carried the pathogenicity island (PAI) IIJ96-like domain that could explain the high-grade bacteremia necessary to develop meningitis. CONCLUSIONS: All the E. coli isolates responsible for this outbreak belonged to a single clone suggesting a common source of infection, and it was categorized as O18:K1:H7. Despite the bacteria's pathogenicity has an important role in the severity of infection, the host-associated factors were crucial for the fatal outcomes.


Asunto(s)
Brotes de Enfermedades/estadística & datos numéricos , Infecciones por Escherichia coli/microbiología , Escherichia coli/genética , Escherichia coli/patogenicidad , Unidades de Cuidado Intensivo Neonatal , Bacteriemia/epidemiología , Bacteriemia/microbiología , Estudios de Cohortes , Infecciones por Escherichia coli/epidemiología , Femenino , Humanos , Lactante , Recién Nacido , Masculino , España/epidemiología , Factores de Virulencia/genética
5.
Proteomics ; 17(15-16)2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28643898

RESUMEN

Exposure to cow's milk constitutes one of the most common causes of food allergy. In addition, exposure to soy proteins has become relevant in a restricted proportion of milk allergic pediatric patients treated with soy formulae as a dairy substitute, because of the cross-allergenicity described between soy and milk proteins. We have previously identified several cross-reactive allergens between milk and soy that may explain this intolerance. The purpose of the present work was to identify epitopes in the purified αS1-casein and the recombinant soy allergen Gly m 5.0101 (Gly m 5) using an α-casein-specific monoclonal antibody (1D5 mAb) through two different approaches for epitope mapping, to understand cross-reactivity between milk and soy. The 1D5 mAb was immobilized onto magnetic beads, incubated with the peptide mixture previously obtained by enzymatic digestion of the allergens, and the captured peptides were identified by MALDI-TOF MS analysis. On a second approach, the peptide mixture was resolved by RP-HPLC and immunodominant peptides were identified by dot blot with the mAb. Finally, recognized peptides were sequenced by MALDI-TOF MS. This novel MS based approach led us to identify and characterize four peptides on α-casein and three peptides on Gly m 5 with a common core motif. Information obtained from these cross-reactive epitopes allows us to gain valuable insight into the molecular mechanisms of cross-reactivity, to further develop new and more effective vaccines for food allergy.


Asunto(s)
Alérgenos/inmunología , Reacciones Cruzadas , Mapeo Epitopo/métodos , Epítopos de Linfocito B/inmunología , Glycine max/química , Leche/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Animales , Caseínas/análisis , Bovinos , Epítopos de Linfocito B/análisis , Femenino , Humanos , Lactante , Proteínas de la Leche/análisis , Proteínas de la Leche/inmunología , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/inmunología , Proteínas de Soja/análisis
6.
J Proteomics ; 142: 122-9, 2016 06 16.
Artículo en Inglés | MEDLINE | ID: mdl-27185551

RESUMEN

UNLABELLED: Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence that displays a high genomic diversity, complicating the study of its pathogenicity, virulence and resistance factors. The interaction of bacterial pathogens with host cells is largely mediated by outer membrane proteins (OMPs). Indeed, several OMPs of Gram-negative bacteria have been recognized as important virulence factors and targets for host immune recognition or to be involved in mechanisms of resistance to antimicrobials. OMPs are also present in outer membrane vesicles (OMVs), which bacteria constitutively secrete to the extracellular milieu and are essential for bacterial survival and pathogenesis. Here, we report the characterization of the OMP and native OMV subproteomes of a clinical isolate (M30) and a collection strain (ATCC13637) of S. maltophilia. We had previously shown that the ATCC13637 strain has an attenuated phenotype in a zebrafish model of infection, as well as a distinct susceptibility profile against a panel of antimicrobials. The protein profiles of the OMP and OMV subproteomes of these two strains and their differences consequently point at pathogenesis, virulence or resistance proteins, such as two variants of the quorum-sensing factor Ax21 that are found to be highly abundant in the OMP fraction and exported to OMVs. BIOLOGICAL SIGNIFICANCE: Stenotrophomonas maltophilia is rapidly climbing positions in the ranking of multidrug-resistant pathogens that are frequently isolated in hospital environments. Being an emerging human pathogen, the knowledge on the factors determining the pathogenicity, virulence and resistance traits of this microorganism is still scarce. Outer membrane proteins (OMPs) and vesicles (OMVs) are key elements for the interaction of Gram-negative bacteria with their environment -including the host-and have fundamental roles in both infection and resistance processes. The present study sets a first basis for a phenotype-dependent characterisation of the OMP subproteome of S. maltophilia and complements very recent work on the OMV subproteome of this species. The variability found among even two strains demonstrates once more that the analysis of genotypically and phenotypically distinct isolates under various conditions will be required before we can draw a significant picture of the OMP and OMV subproteomes of S. maltophilia.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/análisis , Proteómica/métodos , Stenotrophomonas maltophilia/patogenicidad , Factores de Virulencia/análisis , Animales , Electroforesis en Gel de Poliacrilamida , Interacciones Huésped-Patógeno/inmunología , Humanos , Stenotrophomonas maltophilia/química , Stenotrophomonas maltophilia/aislamiento & purificación , Espectrometría de Masas en Tándem , Pez Cebra
7.
Sci Rep ; 6: 26221, 2016 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-27193696

RESUMEN

A key issue towards developing new chemotherapeutic approaches to fight Mycobacterium tuberculosis is to understand the mechanisms underlying drug resistance. Previous studies have shown that genes Rv1686c-Rv1687c and Rv3161c, predicted to encode an ATP-binding cassette transporter and a dioxygenase respectively, are induced in the presence of triclosan and other antimicrobial compounds. Therefore a possible role in drug resistance has been suggested for the products of these genes although no functional studies have been done. The aim of the present study was to clarify the role of Rv1686c-Rv1687c and Rv3161c in M. tuberculosis resistance to triclosan and other drugs. To this end, deficient mutants and overproducing strains for both systems were constructed and their minimal inhibitory concentration (MIC) against over 20 compounds, including triclosan, was evaluated. Unexpectedly, no differences between the MIC of these strains and the wild-type H37Rv were observed for any of the compounds tested. Moreover the MIC of triclosan was not affected by efflux pump inhibitors that inhibit the activity of transporters similar to the one encoded by Rv1686c-Rv1687c. These results suggest that none of the two systems is directly involved in M. tuberculosis resistance to triclosan or to any of the antimicrobials tested.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/biosíntesis , Antiinfecciosos Locales/metabolismo , Dioxigenasas/biosíntesis , Farmacorresistencia Bacteriana , Mycobacterium tuberculosis/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Triclosán/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Dioxigenasas/genética , Eliminación de Gen , Expresión Génica , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética
8.
J Antimicrob Chemother ; 71(1): 111-22, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26446080

RESUMEN

OBJECTIVES: The relationship between quinolone resistance acquisition and invasion impairment has been studied in some Salmonella enterica serovars. However, little information has been reported regarding the invasive human-restricted pathogen Salmonella Typhi. The aim of this study was to investigate the molecular mechanisms of quinolone resistance acquisition and its impact on virulence in this serovar. METHODS: Two antibiotic-resistant mutants (Ty_c1 and Ty_c2) were generated from a Salmonella Typhi clinical isolate (Ty_wt). The three strains were compared in terms of antimicrobial susceptibility, molecular mechanisms of resistance, gene expression of virulence-related factors, ability to invade eukaryotic cells (human epithelial cells and macrophages) and cytokine production. RESULTS: Multidrug resistance in Ty_c2 was attributed to AcrAB/TolC overproduction, decreased OmpF (both mediated by the mar regulon) and decreased OmpC. The two mutants showed a gradually reduced expression of virulence-related genes (invA, hilA, hilD, fliC and fimA), correlating with decreased motility, reduced infection of HeLa cells and impaired uptake by and intracellular survival in human macrophages. Moreover, Ty_c2 also showed reduced tviA expression. Additionally, we revealed a significant reduction in TNF-α and IL-1ß production and decreased NF-κB activation. CONCLUSIONS: In this study, we provide an in-depth characterization of the molecular mechanisms of antibiotic resistance in the Salmonella Typhi serovar and evidence that acquisition of antimicrobial resistance is concomitantly detected with a loss of virulence (epithelial cell invasion, macrophage phagocytosis and cytokine production). We suggest that the low prevalence of clinical isolates of Salmonella Typhi highly resistant to ciprofloxacin is due to poor immunogenicity and impaired dissemination ability of these isolates.


Asunto(s)
Antibacterianos/farmacología , Interacciones Huésped-Patógeno , Mutación , Quinolonas/farmacología , Salmonella typhi/efectos de los fármacos , Salmonella typhi/patogenicidad , Citocinas/metabolismo , Farmacorresistencia Bacteriana , Endocitosis , Células Epiteliales/microbiología , Perfilación de la Expresión Génica , Humanos , Macrófagos/microbiología , Pruebas de Sensibilidad Microbiana , Salmonella typhi/genética , Virulencia , Factores de Virulencia/biosíntesis
9.
J Antimicrob Chemother ; 70(11): 3004-13, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26260130

RESUMEN

OBJECTIVES: The main objective of this study was to investigate the relationship among the in vivo acquisition of antimicrobial resistance in Pseudomonas aeruginosa clinical isolates, the underlying molecular mechanisms and previous exposure to antipseudomonal agents. METHODS: PFGE was used to study the molecular relatedness of the strains. The MICs of ceftazidime, cefepime, piperacillin/tazobactam, imipenem, meropenem, ciprofloxacin and amikacin were determined. Outer membrane protein profiles were assessed to study OprD expression. RT-PCR was performed to analyse ampC, mexB, mexD, mexF and mexY expression. The presence of mutations was analysed through DNA sequencing. RESULTS: We collected 17 clonally related paired isolates [including first positive samples (A) and those with MICs increased ≥4-fold (B)]. Most B isolates with increased MICs of imipenem, meropenem and ceftazidime became resistant to these drugs. The most prevalent resistance mechanisms detected were OprD loss (65%), mexB overexpression (53%), ampC derepression (29%), quinolone target gene mutations (24%) and increased mexY expression (24%). Five (29%) B isolates developed multidrug resistance. Meropenem was the most frequently (71%) received treatment, explaining the high prevalence of oprD mutations and likely mexB overexpression. Previous exposure to ceftazidime showed a higher impact on selection of increased MICs than previous exposure to piperacillin/tazobactam. CONCLUSIONS: Stepwise acquisition of resistance has a critical impact on the resistance phenotypes of P. aeruginosa, leading to a complex scenario for finding effective antimicrobial regimens. In the clinical setting, meropenem seems to be the most frequent driver of multidrug resistance development, while piperacillin/tazobactam, in contrast to ceftazidime, seems to be the ß-lactam least associated with the selection of resistance mechanisms.


Asunto(s)
Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana , Evolución Molecular , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Electroforesis en Gel de Campo Pulsado , Perfilación de la Expresión Génica , Humanos , Unidades de Cuidados Intensivos , Pruebas de Sensibilidad Microbiana , Tipificación Molecular , Pseudomonas aeruginosa/clasificación , Reacción en Cadena en Tiempo Real de la Polimerasa , beta-Lactamasas/genética
10.
J Antimicrob Chemother ; 70(11): 2981-6, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26311838

RESUMEN

OBJECTIVES: Acinetobacter nosocomialis has increasingly been reported as an opportunistic pathogen causing nosocomial infections. Although it is more susceptible to all antimicrobial agents than Acinetobacter baumannii, MDR clinical isolates have also been described. In addition, several studies have shown a high percentage of resistance to colistin. Therefore, in the present study we investigated the mechanism of resistance to colistin in this microorganism. METHODS: Colistin-resistant strains were selected from the original colistin-susceptible A. nosocomialis strain following multi-step mutant selection. Comparative genomic and proteomic analyses of both colistin-susceptible and colistin-resistant A. nosocomialis strains were performed. In addition, virulence was investigated using the Caenorhabditis elegans assay. RESULTS: The colistin-resistant mutants selected showed a lower resistance profile for other types of antibacterial agents together with a significant decrease in virulence. The LT50 (i.e. time required to kill 50% of the nematodes) for the colistin-susceptible strain (WT) was 7 days compared with 9 days for the colistin-resistant strain (256) (P < 0.0001). In the genomic studies, several mutations were observed in the lpxD genes, leading to the loss of LPS in the colistin-resistant strains. The proteomic studies showed several up- and down-regulated proteins that may be involved in colistin resistance or in a decrease in the resistance profile for several antibiotics. CONCLUSIONS: This study shows that the mechanism of resistance to colistin by A. nosocomialis is mainly associated with the loss of LPS due to mutations in the lpxD gene, although changes in the expression of some proteins cannot be ruled out. In addition, the acquisition of colistin resistance is related to a decrease in virulence.


Asunto(s)
Acinetobacter/efectos de los fármacos , Acinetobacter/patogenicidad , Antibacterianos/farmacología , Colistina/farmacología , Farmacorresistencia Bacteriana , Lipopolisacáridos/metabolismo , Factores de Virulencia/metabolismo , Acinetobacter/aislamiento & purificación , Infecciones por Acinetobacter/microbiología , Animales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Caenorhabditis elegans , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Genoma Bacteriano , Mutación , Proteoma , Pase Seriado , Virulencia
11.
PLoS Negl Trop Dis ; 9(7): e0003917, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26222657

RESUMEN

Burkholderia pseudomallei is a Gram-negative bacterium responsible for melioidosis, a serious and often fatal infectious disease that is poorly controlled by existing treatments. Due to its inherent resistance to the major antibiotic classes and its facultative intracellular pathogenicity, an effective vaccine would be extremely desirable, along with appropriate prevention and therapeutic management. One of the main subunit vaccine candidates is flagellin of Burkholderia pseudomallei (FliCBp). Here, we present the high resolution crystal structure of FliCBp and report the synthesis and characterization of three peptides predicted to be both B and T cell FliCBp epitopes, by both structure-based in silico methods, and sequence-based epitope prediction tools. All three epitopes were shown to be immunoreactive against human IgG antibodies and to elicit cytokine production from human peripheral blood mononuclear cells. Furthermore, two of the peptides (F51-69 and F270-288) were found to be dominant immunoreactive epitopes, and their antibodies enhanced the bactericidal activities of purified human neutrophils. The epitopes derived from this study may represent potential melioidosis vaccine components.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Burkholderia pseudomallei/inmunología , Flagelina/química , Flagelina/inmunología , Melioidosis/microbiología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Burkholderia pseudomallei/química , Burkholderia pseudomallei/genética , Mapeo Epitopo , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Flagelina/genética , Humanos , Leucocitos Mononucleares/inmunología , Melioidosis/inmunología , Datos de Secuencia Molecular , Fagocitosis , Difracción de Rayos X
12.
Artículo en Inglés | MEDLINE | ID: mdl-26029670

RESUMEN

Quorum Sensing (QS) mediated by Acyl Homoserine Lactone (AHL) molecules are probably the most widespread and studied among Gram-negative bacteria. Canonical AHL systems are composed by a synthase (LuxI family) and a regulator element (LuxR family), whose genes are usually adjacent in the genome. However, incomplete AHL-QS machinery lacking the synthase LuxI is frequently observed in Proteobacteria, and the regulator element is then referred as LuxR solo. It has been shown that certain LuxR solos participate in interspecific communication by detecting signals produced by different organisms. In the case of Stenotrophomonas maltophilia, a preliminary genome sequence analysis revealed numerous putative luxR genes, none of them associated to a luxI gene. From these, the hypothetical LuxR solo Smlt1839, here designated SmoR, presents a conserved AHL binding domain and a helix-turn-helix DNA binding motif. Its genomic organization-adjacent to hchA gene-indicate that SmoR belongs to the new family "LuxR regulator chaperone HchA-associated." AHL-binding assays revealed that SmoR binds to AHLs in-vitro, at least to oxo-C8-homoserine lactone, and it regulates operon transcription, likely by recognizing a conserved palindromic regulatory box in the hchA upstream region. Supplementation with concentrated supernatants from Pseudomonas aeruginosa, which contain significant amounts of AHLs, promoted swarming motility in S. maltophilia. Contrarily, no swarming stimulation was observed when the P. aeruginosa supernatant was treated with the lactonase AiiA from Bacillus subtilis, confirming that AHL contributes to enhance the swarming ability of S. maltophilia. Finally, mutation of smoR resulted in a swarming alteration and an apparent insensitivity to the exogenous AHLs provided by P. aeruginosa. In conclusion, our results demonstrate that S. maltophilia senses AHLs produced by neighboring bacteria through the LuxR solo SmoR, regulating population behaviors such as swarming motility.


Asunto(s)
Acil-Butirolactonas/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , Locomoción , Datos de Secuencia Molecular , Pseudomonas aeruginosa/química , Análisis de Secuencia de ADN , Stenotrophomonas maltophilia/fisiología
13.
FEBS J ; 282(7): 1319-33, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25645451

RESUMEN

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a potentially fatal infection that is endemic in Southeast Asia and Northern Australia that is poorly controlled by antibiotics. Research efforts to identify antigenic components for a melioidosis vaccine have led to the identification of several proteins, including subunits forming the flagella that mediate bacterial motility, host colonization, and virulence. This study focuses on the B. pseudomallei flagellar hook-associated protein (FlgK(Bp)), and provides the first insights into the 3D structure of FlgK proteins as targets for structure-based antigen engineering. The FlgK(Bp) crystal structure (presented here at 1.8-Å resolution) reveals a multidomain fold, comprising two small ß-domains protruding from a large elongated α-helical bundle core. The evident structural similarity to flagellin, the flagellar filament subunit protein, suggests that, depending on the bacterial species, flagellar hook-associated proteins are likely to show a conserved, elongated α-helical bundle scaffold coupled to a variable number of smaller domains. Furthermore, we present immune serum recognition data confirming, in agreement with previous findings, that recovered melioidosis patients produce elevated levels of antibodies against FlgK(Bp), in comparison with seronegative and seropositive healthy subjects. Moreover, we show that FlgK(Bp) has cytotoxic effects on cultured murine macrophages, suggesting an important role in bacterial pathogenesis. Finally, computational epitope prediction methods applied to the FlgK(Bp) crystal structure, coupled with in vitro mapping, allowed us to predict three antigenic regions that locate to discrete protein domains. Taken together, our results point to FlgK(Bp) as a candidate for the design and production of epitope-containing subunits/domains as potential vaccine components.


Asunto(s)
Proteínas Bacterianas/química , Burkholderia pseudomallei/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/química , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/fisiología , Línea Celular , Simulación por Computador , Cristalografía por Rayos X , Epítopos/química , Humanos , Macrófagos/inmunología , Macrófagos/microbiología , Melioidosis/sangre , Melioidosis/inmunología , Melioidosis/microbiología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular
14.
ACS Chem Biol ; 10(3): 803-12, 2015 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-25495888

RESUMEN

Burkholderia pseudomallei is the etiological agent of melioidosis, a severe endemic disease in South-East Asia, causing septicemia and organ failure with high mortality rates. Current treatments and diagnostic approaches are largely ineffective. The development of new diagnostic tools and vaccines toward effective therapeutic opportunities against B. pseudomallei is therefore an urgent priority. In the framework of a multidisciplinary project tackling melioidosis through reverse and structural vaccinology, BPSL1050 was identified as a candidate for immunodiagnostic and vaccine development based on its reactivity against the sera of melioidosis patients. We determined its NMR solution structure and dynamics, and by novel computational methods we predicted immunogenic epitopes that once synthesized were able to elicit the production of antibodies inducing the agglutination of the bacterium and recognizing both BPSL1050 and B. pseudomallei crude extracts. Overall, these results hold promise for novel chemical biology approaches in the discovery of new diagnostic and prophylactic tools against melioidosis.


Asunto(s)
Anticuerpos Antibacterianos/química , Antígenos Bacterianos/química , Burkholderia pseudomallei/efectos de los fármacos , Epítopos/química , Melioidosis/inmunología , Pruebas de Aglutinación , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/farmacología , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Linfocitos B/inmunología , Linfocitos B/microbiología , Linfocitos B/patología , Burkholderia pseudomallei/química , Burkholderia pseudomallei/inmunología , Mapeo Epitopo , Epítopos/genética , Epítopos/inmunología , Humanos , Sueros Inmunes/química , Sueros Inmunes/inmunología , Melioidosis/sangre , Melioidosis/microbiología , Melioidosis/patología , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Relación Estructura-Actividad
15.
Rev Esp Quimioter ; 27(2): 87-92, 2014 Jun.
Artículo en Español | MEDLINE | ID: mdl-24940887

RESUMEN

In the last decade we have witnessed a remarkable increase in the number of strains isolated in hospitals that are producing extended spectrum ß-lactamases (ESBL) or, more recently, carbapenemases. This makes clear the need for a system for rapid detection of these resistance mechanisms that allow the selection of the most suitable antibiotic treatment in order to improve patient care. Recent data support the possibility of using mass spectrometry (MS), specifically MALDI -TOF (Matrix-Assisted Laser Desorption / Ionization, Time-of-Flight ) systems to identify specific resistance mechanisms and their use offers several advantages. First, the economic cost of each determination is clearly inferior to the classical molecular techniques for detection of resistance genes. Second, detection of resistance by MALDI -TOF reduces the time for obtaining results compared to the routine methods currently employed. Finally, the possibility that this method allows us to detect enzymes not previously characterized, that there is no information about the genes that encode them. Therefore, we believe that this may be a good tool to implement in clinical microbiology laboratories. This review aims to present the latest developments in this field.


Asunto(s)
Proteínas Bacterianas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Resistencia betalactámica , beta-Lactamasas/metabolismo , Estabilidad de Medicamentos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/enzimología , Hidrólisis , Pruebas de Sensibilidad Microbiana/métodos , Factores de Tiempo , beta-Lactamas/metabolismo
16.
Rev. esp. quimioter ; 27(2): 87-92, jun. 2014.
Artículo en Español | IBECS | ID: ibc-123825

RESUMEN

En la última década hemos asistido a un notable incremento en el número de cepas aisladas en el medio hospitalario que son productoras de β-lactamasas de espectro extendido (BLEE) o, más recientemente, de carbapenemasas. Esta situación hace patente la necesidad de disponer de un sistema de detección rápida de estos mecanismos de resistencia que permita seleccionar de forma precoz el antibiótico más adecuado para poder mejorar la asistencia al paciente. Estudios recientes avalan la posibilidad de usar sistemas de espectrometría de masas (MS), específicamente sistemas MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization, Time-Of-Flight) para identificar determinados mecanismos de resistencia ya que su empleo ofrece diversas ventajas. En primer lugar, el coste económico de cada determinación es claramente inferior al de las técnicas moleculares clásicas de detección de genes de resistencia. En segundo lugar, la detección de resistencias mediante MALDI-TOF permite reducir el tiempo de obtención de resultados en comparación con los métodos de rutina actualmente empleados. Por último, cabe la posibilidad de que nos permita detectar enzimas no descritas previamente, de las que no se dispone de información acerca de los genes que las codifican. Por todo ello, creemos que ésta puede ser una metodología muy útil para implementar en los laboratorios de microbiología clínica. En esta revisión se pretende exponer los últimos avances en esta área (AU)


In the last decade we have witnessed a remarkable increase in the number of strains isolated in hospitals that are producing extended spectrum β-lactamases (ESBL) or, more recently, carbapenemases. This makes clear the need for a system for rapid detection of these resistance mechanisms that allow the selection of the most suitable antibiotic treatment in order to improve patient care. Recent data support the possibility of using mass spectrometry (MS), specifically MALDI -TOF (Matrix-Assisted Laser Desorption / Ionization, Time-of-Flight ) systems to identify specific resistance mechanisms and their use offers several advantages. First, the economic cost of each determination is clearly inferior to the classical molecular techniques for detection of resistance genes. Second, detection of resistance by MALDI -TOF reduces the time for obtaining results compared to the routine methods currently employed. Finally, the possibility that this method allows us to detect enzymes not previously characterized, that there is no information about the genes that encode them. Therefore, we believe that this may be a good tool to implement in clinical microbiology laboratories. This review aims to present the latest developments in this field (AU)


Asunto(s)
Humanos , Farmacorresistencia Microbiana , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Infecciones/microbiología , Pruebas de Sensibilidad Microbiana/métodos
17.
BMC Struct Biol ; 13: 19, 2013 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-24099525

RESUMEN

BACKGROUND: Increasing rates of antimicrobial resistance among uropathogens led, among other efforts, to the application of subtractive reverse vaccinology for the identification of antigens present in extraintestinal pathogenic E. coli (ExPEC) strains but absent or variable in non-pathogenic strains, in a quest for a broadly protective Escherichia coli vaccine. The protein coded by locus c5321 from CFT073 E. coli was identified as one of nine potential vaccine candidates against ExPEC and was able to confer protection with an efficacy of 33% in a mouse model of sepsis. c5321 (known also as EsiB) lacks functional annotation and structurally belongs to the Sel1-like repeat (SLR) family. Herein, as part of the general characterization of this potential antigen, we have focused on its structural properties. RESULTS: We report the 1.74 Å-resolution crystal structure of c5321 from CFT073 E. coli determined by Se-Met SAD phasing. The structure is composed of 11 SLR units in a topological organisation that highly resembles that found in HcpC from Helicobacter pylori, with the main difference residing in how the super-helical fold is stabilised. The stabilising effect of disulfide bridges in HcpC is replaced in c5321 by a strengthening of the inter-repeat hydrophobic core. A metal-ion binding site, uncharacteristic of SLR proteins, is detected between SLR units 3 and 4 in the region of the inter-repeat hydrophobic core. Crystal contacts are observed between the C-terminal tail of one molecule and the C-terminal amphipathic groove of a neighbouring one, resembling interactions between ligand and proteins containing tetratricopeptide-like repeats. CONCLUSIONS: The structure of antigen c5321 presents a mode of stabilization of the SLR fold different from that observed in close homologs of known structure. The location of the metal-ion binding site and the observed crystal contacts suggest a potential role in regulation of conformational flexibility and interaction with yet unidentified target proteins, respectively. These findings open new perspectives in both antigen design and for the identification of a functional role for this protective antigen.


Asunto(s)
Antígenos Bacterianos/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli Uropatógena/química , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Antígenos CD1/inmunología , Sitios de Unión , Secuencia de Consenso , Cristalografía por Rayos X , Mapeo Epitopo , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Vacunas contra Escherichia coli/metabolismo , Helicobacter pylori/química , Helicobacter pylori/inmunología , Helicobacter pylori/metabolismo , Magnesio/metabolismo , Ratones , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología Estructural de Proteína , Escherichia coli Uropatógena/inmunología
18.
Chem Biol ; 20(9): 1147-56, 2013 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-23993463

RESUMEN

We solved the crystal structure of Burkholderia pseudomallei acute phase antigen BPSL2765 in the context of a structural vaccinology study, in the area of melioidosis vaccine development. Based on the structure, we applied a recently developed method for epitope design that combines computational epitope predictions with in vitro mapping experiments and successfully identified a consensus sequence within the antigen that, when engineered as a synthetic peptide, was selectively immunorecognized to the same extent as the recombinant protein in sera from melioidosis-affected subjects. Antibodies raised against the consensus peptide were successfully tested in opsonization bacterial killing experiments and antibody-dependent agglutination tests of B. pseudomallei. Our strategy represents a step in the development of immunodiagnostics, in the production of specific antibodies and in the optimization of antigens for vaccine development, starting from structural and physicochemical principles.


Asunto(s)
Antígenos Bacterianos/química , Proteínas Bacterianas/química , Vacunas Bacterianas/inmunología , Burkholderia pseudomallei/inmunología , Epítopos/química , Anticuerpos/sangre , Anticuerpos/inmunología , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Burkholderia pseudomallei/metabolismo , Cristalografía por Rayos X , Mapeo Epitopo , Epítopos/inmunología , Epítopos/metabolismo , Humanos , Simulación de Dinámica Molecular , Neutrófilos/citología , Neutrófilos/inmunología , Fagocitosis , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología
19.
PLoS Comput Biol ; 9(6): e1003115, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23825940

RESUMEN

The pilus 2a backbone protein (BP-2a) is one of the most structurally and functionally characterized components of a potential vaccine formulation against Group B Streptococcus. It is characterized by six main immunologically distinct allelic variants, each inducing variant-specific protection. To investigate the molecular determinants driving the variant immunogenic specificity of BP-2a, in terms of single residue contributions, we generated six monoclonal antibodies against a specific protein variant based on their capability to recognize the polymerized pili structure on the bacterial surface. Three mAbs were also able to induce complement-dependent opsonophagocytosis killing of live GBS and target the same linear epitope present in the structurally defined and immunodominant domain D3 of the protein. Molecular docking between the modelled scFv antibody sequences and the BP-2a crystal structure revealed the potential role at the binding interface of some non-conserved antigen residues. Mutagenesis analysis confirmed the necessity of a perfect balance between charges, size and polarity at the binding interface to obtain specific binding of mAbs to the protein antigen for a neutralizing response.


Asunto(s)
Proteínas Bacterianas/metabolismo , Streptococcus agalactiae/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Mapeo Epitopo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fagocitosis , Homología de Secuencia de Aminoácido , Streptococcus agalactiae/inmunología
20.
mBio ; 4(4)2013 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-23882011

RESUMEN

UNLABELLED: In this study, we have characterized the functional properties of a novel Escherichia coli antigen named EsiB (E. coli secretory immunoglobulin A-binding protein), recently reported to protect mice from sepsis. Gene distribution analysis of a panel of 267 strains representative of different E. coli pathotypes revealed that esiB is preferentially associated with extraintestinal strains, while the gene is rarely found in either intestinal or nonpathogenic strains. These findings were supported by the presence of anti-EsiB antibodies in the sera of patients affected by urinary tract infections (UTIs). By solving its crystal structure, we observed that EsiB adopts a superhelical fold composed of Sel1-like repeats (SLRs), a feature often associated with bacterial proteins possessing immunomodulatory functions. Indeed, we found that EsiB interacts with secretory immunoglobulin A (SIgA) through a specific motif identified by an immunocapturing approach. Functional assays showed that EsiB binding to SIgA is likely to interfere with productive FcαRI signaling, by inhibiting both SIgA-induced neutrophil chemotaxis and respiratory burst. Indeed, EsiB hampers SIgA-mediated signaling events by reducing the phosphorylation status of key signal-transducer cytosolic proteins, including mitogen-activated kinases. We propose that the interference with such immune events could contribute to the capacity of the bacterium to avoid clearance by neutrophils, as well as reducing the recruitment of immune cells to the infection site. IMPORTANCE: Pathogenic Escherichia coli infections have recently been exacerbated by increasing antibiotic resistance and the number of recurrent contagions. Attempts to develop preventive strategies against E. coli have not been successful, mainly due to the large antigenic and genetic variability of virulence factors, but also due to the complexity of the mechanisms used by the pathogen to evade the immune system. In this work, we elucidated the function of a recently discovered protective antigen, named EsiB, and described its capacity to interact with secretory immunoglobulin A (SIgA) and impair effector functions. This work unravels a novel strategy used by E. coli to subvert the host immune response and avoid neutrophil-dependent clearance.


Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/inmunología , Escherichia coli/patogenicidad , Inmunoglobulina A Secretora/metabolismo , Activación Neutrófila , Factores de Virulencia/metabolismo , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Técnicas de Inactivación de Genes , Humanos , Evasión Inmune , Ratones , Modelos Moleculares , Conformación Proteica , Factores de Virulencia/química , Factores de Virulencia/genética
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