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1.
PLoS One ; 15(3): e0226395, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32150579

RESUMEN

Bacterial microcompartments (MCPs) are protein-based organelles that encapsulate metabolic pathways. Metabolic engineers have recently sought to repurpose MCPs to encapsulate heterologous pathways to increase flux through pathways of interest. As MCP engineering becomes more common, standardized methods for analyzing changes to MCPs and interpreting results across studies will become increasingly important. In this study, we demonstrate that different imaging techniques yield variations in the apparent size of purified MCPs from Salmonella enterica serovar Typhimurium LT2, likely due to variations in sample preparation methods. We provide guidelines for preparing samples for MCP imaging and outline expected variations in apparent size and morphology between methods. With this report we aim to establish an aid for comparing results across studies.


Asunto(s)
Regulación Bacteriana de la Expresión Génica/fisiología , Redes y Vías Metabólicas/fisiología , Salmonella typhimurium/metabolismo , Salmonella typhimurium/genética
2.
PLoS Genet ; 16(2): e1008607, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32053596

RESUMEN

RHS elements are components of conserved toxin-delivery systems, wide-spread within the bacterial kingdom and some of the most positively selected genes known. However, very little is known about how Rhs toxins affect bacterial biology. Salmonella Typhimurium contains a full-length rhs gene and an adjacent orphan rhs gene, which lacks the conserved delivery part of the Rhs protein. Here we show that, in addition to the conventional delivery, Rhs toxin-antitoxin pairs encode for functional type-II toxin-antitoxin (TA) loci that regulate S. Typhimurium proliferation within macrophages. Mutant S. Typhimurium cells lacking both Rhs toxins proliferate 2-times better within macrophages, mainly because of an increased growth rate. Thus, in addition to providing strong positive selection for the rhs loci under conditions when there is little or no toxin delivery, internal expression of the toxin-antitoxin system regulates growth in the stressful environment found inside macrophages.


Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Macrófagos/microbiología , Salmonella typhimurium/genética , Sistemas Toxina-Antitoxina/genética , Animales , Proteínas Bacterianas/metabolismo , Sitios Genéticos/genética , Ratones , Mutación , Sistemas de Lectura Abierta/genética , Regiones Promotoras Genéticas/genética , Células RAW 264.7 , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/patogenicidad , Selección Genética
3.
Nat Commun ; 11(1): 864, 2020 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-32054833

RESUMEN

Siroheme is the central cofactor in a conserved class of sulfite and nitrite reductases that catalyze the six-electron reduction of sulfite to sulfide and nitrite to ammonia. In Salmonella enterica serovar Typhimurium, siroheme is produced by a trifunctional enzyme, siroheme synthase (CysG). A bifunctional active site that is distinct from its methyltransferase activity catalyzes the final two steps, NAD+-dependent dehydrogenation and iron chelation. How this active site performs such different chemistries is unknown. Here, we report the structures of CysG bound to precorrin-2, the initial substrate; sirohydrochlorin, the dehydrogenation product/chelation substrate; and a cobalt-sirohydrochlorin product. We identified binding poses for all three tetrapyrroles and tested the roles of specific amino acids in both activities to give insights into how a bifunctional active site catalyzes two different chemistries and acts as an iron-specific chelatase in the final step of siroheme synthesis.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Hemo/análogos & derivados , Metiltransferasas/química , Metiltransferasas/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Dominio Catalítico/genética , Electroquímica , Ferroquelatasa/química , Ferroquelatasa/genética , Ferroquelatasa/metabolismo , Hemo/biosíntesis , Hemo/química , Metiltransferasas/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxidorreductasas/química , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Especificidad por Sustrato , Tetrapirroles/química , Tetrapirroles/metabolismo , Uroporfirinas/química , Uroporfirinas/metabolismo
5.
Mutat Res ; 849: 503137, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-32087853

RESUMEN

The International Workshop on Genotoxicity Testing (IWGT) meets every four years to obtain consensus on unresolved issues associated with genotoxicity testing. At the 2017 IWGT meeting in Tokyo, four sub-groups addressed issues associated with the Organization for Economic Cooperation and Development (OECD) Test Guideline TG471, which describes the use of bacterial reverse-mutation tests. The strains sub-group analyzed test data from >10,000 chemicals, tested additional chemicals, and concluded that some strains listed in TG471 are unnecessary because they detected fewer mutagens than other strains that the guideline describes as equivalent. Thus, they concluded that a smaller panel of strains would suffice to detect most mutagens. The laboratory proficiency sub-group recommended (a) establishing strain cell banks, (b) developing bacterial growth protocols that optimize assay sensitivity, and (c) testing "proficiency compounds" to gain assay experience and establish historical positive and control databases. The sub-group on criteria for assay evaluation recommended that laboratories (a) track positive and negative control data; (b) develop acceptability criteria for positive and negative controls; (c) optimize dose-spacing and the number of analyzable doses when there is evidence of toxicity; (d) use a combination of three criteria to evaluate results: a dose-related increase in revertants, a clear increase in revertants in at least one dose relative to the concurrent negative control, and at least one dose that produced an increase in revertants above control limits established by the laboratory from historical negative controls; and (e) establish experimental designs to resolve unclear results. The in silico sub-group summarized in silico utility as a tool in genotoxicity assessment but made no specific recommendations for TG471. Thus, the workgroup identified issues that could be addressed if TG471 is revised. The companion papers (a) provide evidence-based approaches, (b) recommend priorities, and (c) give examples of clearly defined terms to support revision of TG471.


Asunto(s)
Escherichia coli/efectos de los fármacos , Mutagénesis , Pruebas de Mutagenicidad/normas , Mutágenos/toxicidad , Salmonella typhimurium/efectos de los fármacos , Animales , Bancos de Muestras Biológicas/organización & administración , Bases de Datos de Compuestos Químicos/provisión & distribución , Escherichia coli/genética , Guías como Asunto , Humanos , Cooperación Internacional , Mutágenos/clasificación , Salmonella typhimurium/genética , Tokio
6.
PLoS Negl Trop Dis ; 14(2): e0007875, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32084128

RESUMEN

BACKGROUND: Clinical observations and animal studies have suggested that Salmonella intestinal carriage is promoted by concurrent Schistosoma infection. The present study assessed association of Salmonella intestinal carriage and Schistosoma mansoni infection among individuals in a Schistosoma endemic area in sub-Saharan Africa. METHODS: From November 2015 to March 2016, a cross-sectional community-wide study was conducted in Kifua II, a rural village in Kongo Central Province, Democratic Republic of Congo. Stool samples were collected and analyzed for Salmonella intestinal carriage (culture) and Schistosoma mansoni infection (Kato Katz microscopy with determination of egg load). Salmonella Typhimurium and Enteritidis isolates were assessed for genetic similarity with blood culture isolates obtained during the same period in a neighboring hospital using multi-locus variable-numbers tandem repeat analysis (MLVA). RESULTS: A total of 1,108 participants were included (median age 15 years (IQR: 7-36), male-to-female ratio of 1:1.1). The overall prevalence of Schistosoma mansoni infection and non-typhoidal Salmonella carriage was 51.2% (95% CI: 48.2-54.1) and 3.4% (95% CI: 2.5-4.7) respectively, with 2.2% (95% CI: 1.5-3.2) of participants coinfected. The proportion of Salmonella carriage tended to be higher among Schistosoma mansoni infected participants compared to non-infected participants but this difference did not reach statistical significance (4.2% versus 2.6%, p = 0.132). However, the proportion of Salmonella carriage among participants with a heavy Schistosoma mansoni infection was significantly higher compared to those with a light and moderate infection (8.7% versus 3.2%, p = 0.012) and compared to Schistosoma mansoni negatives (8.7% versus 2.6%, p = 0.002). The 38 Salmonella isolates comprised five and four Enteritidis and Typhimurium serotypes respectively, the majority of them had MLVA types identical or similar to those observed among blood culture isolates. CONCLUSION: Salmonella intestinal carriage was associated with a heavy intensity of Schistosoma mansoni infection. Further studies are needed to address causation.


Asunto(s)
Portador Sano/microbiología , Intestinos/microbiología , Salmonella typhimurium/aislamiento & purificación , Esquistosomiasis mansoni/parasitología , Adolescente , Adulto , Animales , Portador Sano/epidemiología , Niño , Coinfección/epidemiología , Coinfección/microbiología , Coinfección/parasitología , Estudios Transversales , República Democrática del Congo/epidemiología , Femenino , Humanos , Masculino , Población Rural , Salmonella typhimurium/genética , Schistosoma mansoni/genética , Schistosoma mansoni/aislamiento & purificación , Schistosoma mansoni/fisiología , Esquistosomiasis mansoni/epidemiología , Adulto Joven
7.
Nat Commun ; 11(1): 107, 2020 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-31919364

RESUMEN

Bacteria commonly form dense biofilms encased in extracellular polymeric substances (EPS). Biofilms are often extremely tolerant to antimicrobials but their reliance on shared EPS may also be a weakness as social evolution theory predicts that inhibiting shared traits can select against resistance. Here we show that EPS of Salmonella biofilms is a cooperative trait whose benefit is shared among cells, and that EPS inhibition reduces both cell attachment and antimicrobial tolerance. We then compare an EPS inhibitor to conventional antimicrobials in an evolutionary experiment. While resistance against conventional antimicrobials rapidly evolves, we see no evolution of resistance to EPS inhibition. We further show that a resistant strain is outcompeted by a susceptible strain under EPS inhibitor treatment, explaining why resistance does not evolve. Our work suggests that targeting cooperative traits is a viable solution to the problem of antimicrobial resistance.


Asunto(s)
Antibacterianos/farmacología , Biopelículas/crecimiento & desarrollo , Matriz Extracelular de Sustancias Poliméricas/metabolismo , Imidazoles/farmacología , Salmonella typhimurium/crecimiento & desarrollo , Farmacorresistencia Bacteriana/genética , Farmacorresistencia Bacteriana/fisiología , Matriz Extracelular de Sustancias Poliméricas/efectos de los fármacos , Salmonella typhimurium/genética
8.
Emerg Microbes Infect ; 9(1): 1-4, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31859589

RESUMEN

CRISPR-based typing was performed to subtype isolates of S. Typhimurium and its monophasic variant Salmonella 4,[5],12:i:- from humans and animals between 2009 and 2017 in China. CRISPR typing classified all isolates into two lineages and four sub-lineages. All isolates from Lineage II and Lineage IB-1 were Salmonella Typhimurium. All of Salmonella 4,[5],12:i: - isolates were distributed in Lineage IA and Lineage IB-2, which all belonged to ST34 by MLST typing. Only Lineage IB-2 contained ST34 isolates from both Salmonella Typhimurium and Salmonella 4,[5],12:i:-. Among the isolates of ST34, TST4 was identified as the most common CRISPR type representing 86.5% of Salmonella 4,[5],12:i:- and 14.5 % of Salmonella Typhimurium mainly from pigs and humans. This study demonstrated that TST4-ST34 isolates were predominant in Salmonella 4,[5],12:i:-, and pig was the main reservoir for Salmonella 4,[5],12:i:- in China, which might have the potential to transmit to humans by pig production.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Reservorios de Enfermedades , Técnicas de Genotipaje/métodos , Carne/microbiología , Salmonella typhimurium/clasificación , Salmonella typhimurium/aislamiento & purificación , Sus scrofa , Animales , China , Diarrea/microbiología , Electroforesis en Gel de Campo Pulsado , Humanos , Tipificación de Secuencias Multilocus , Filogenia , Infecciones por Salmonella/microbiología , Salmonella typhimurium/genética
9.
PLoS Negl Trop Dis ; 13(12): e0007490, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31790394

RESUMEN

Schistosoma mansoni threatens hundreds of millions of people in >50 countries. Schistosomulae migrate through the lung and adult worms reside in blood vessels adjacent to the intestinal mucosa. Current candidate vaccines aren't designed to elicit a mucosal response. We have repurposed an attenuated Salmonella enterica Typhimurium strain (YS1646) to produce such a vaccine targeting Cathepsin B (CatB), a digestive enzyme important for parasite survival. Promoter-Type 3 secretory signal pairs were screened for protein expression in vitro and transfected into YS1646 to generate candidate vaccine strains. Two strains were selected for in vivo evaluation (nirB_SspH1 and SspH1_SspH1). Female C57BL/6 mice were immunized twice, 3 weeks apart, using six strategies: i) saline gavage (control), ii) the 'empty' YS1646 vector orally (PO) followed by intramuscular (IM) recombinant CatB (20µg IM rCatB), iii) two doses of IM rCatB, iv) two PO doses of YS1646-CatB, v) IM rCatB then PO YS1646-CatB and vi) PO YS1646-CatB then IM rCatB. Serum IgG responses to CatB were monitored by ELISA. Three weeks after the second dose, mice were challenged with 150 cercariae and sacrificed 7 weeks later to assess adult worm and egg burden (liver and intestine), granuloma size and egg morphology. CatB-specific IgG antibodies were low/absent in the control and PO only groups but rose substantially in other groups (5898-6766ng/mL). The highest response was in animals that received nirB_SspH1 YS1646 PO then IM rCatB. In this group, reductions in worm and intestine/liver egg burden (vs. control) were 93.1% and 79.5%/90.3% respectively (all P < .0001). Granuloma size was reduced in all vaccinated groups (range 32.9-52.8 x103µm2) and most significantly in the nirB_SspH1 + CatB IM group (34.7±3.4 x103µm2vs. 62.2±6.1 x103µm2: vs. control P < .01). Many eggs in the vaccinated animals had abnormal morphology. Targeting CatB using a multi-modality approach can provide almost complete protection against S. mansoni challenge.


Asunto(s)
Antígenos Helmínticos/inmunología , Catepsina B/inmunología , Portadores de Fármacos , Salmonella typhimurium/genética , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/prevención & control , Vacunas Sintéticas/inmunología , Animales , Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/genética , Catepsina B/genética , Modelos Animales de Enfermedad , Femenino , Inmunoglobulina G/sangre , Inyecciones Intramusculares , Ratones Endogámicos C57BL , Schistosoma mansoni/genética , Esquistosomiasis mansoni/patología , Resultado del Tratamiento , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/administración & dosificación
10.
Pol J Microbiol ; 68(4): 439-447, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31880888

RESUMEN

Salmonella infection is most common in patients with infected aortic aneurysm, especially in Asia. When the aortic wall is heavily atherosclerotic, the intima is vulnerable to invasion by Salmonella, leading to the development of infected aortic aneurysm. By using THP-1 macrophage-derived foam cells to mimic atherosclerosis, we investigated the role of three Salmonella enterica serotypes - Typhimurium, Enteritidis, and Choleraesuis - in foam cell autophagy and inflammasome formation. Herein, we provide possible pathogenesis of Salmonella-associated infected aortic aneurysms. Three S. enterica serotypes with or without virulence plasmid were studied. Through Western blotting, we investigated cell autophagy induction and inflammasome formation in Salmonella-infected THP-1 macrophage-derived foam cells, detected CD36 expression after Salmonella infection through flow cytometry, and measured interleukin (IL)-1ß, IL-12, and interferon (IFN)-α levels through enzyme-linked immunosorbent assay. At 0.5 h after infection, plasmid-bearing S. Enteritidis OU7130 induced the highest foam cell autophagy - significantly higher than that induced by plasmid-less OU7067. However, plasmid-bearing S. Choleraesuis induced less foam cell autophagy than did its plasmid-less strain. In foam cells, plasmid-less Salmonella infection (particularly S. Choleraesuis OU7266 infection) led to higher CD36 expression than did plasmid-bearing strains infection. OU7130 and OU7266 infection induced the highest IL-1ß secretion. OU7067-infected foam cells secreted the highest IL-12p35 level. Plasmid-bearing S. Typhimurium OU5045 induced a higher IFN-α level than did other Salmonella serotypes. Salmonella serotypes are correlated with foam cell autophagy and IL-1ß secretion. Salmonella may affect the course of foam cells formation, or even aortic aneurysm, through autophagy.Salmonella infection is most common in patients with infected aortic aneurysm, especially in Asia. When the aortic wall is heavily atherosclerotic, the intima is vulnerable to invasion by Salmonella, leading to the development of infected aortic aneurysm. By using THP-1 macrophage-derived foam cells to mimic atherosclerosis, we investigated the role of three Salmonella enterica serotypes ­ Typhimurium, Enteritidis, and Choleraesuis ­ in foam cell autophagy and inflammasome formation. Herein, we provide possible pathogenesis of Salmonella-associated infected aortic aneurysms. Three S. enterica serotypes with or without virulence plasmid were studied. Through Western blotting, we investigated cell autophagy induction and inflammasome formation in Salmonella-infected THP-1 macrophage-derived foam cells, detected CD36 expression after Salmonella infection through flow cytometry, and measured interleukin (IL)-1ß, IL-12, and interferon (IFN)-α levels through enzyme-linked immunosorbent assay. At 0.5 h after infection, plasmid-bearing S. Enteritidis OU7130 induced the highest foam cell autophagy ­ significantly higher than that induced by plasmid-less OU7067. However, plasmid-bearing S. Choleraesuis induced less foam cell autophagy than did its plasmid-less strain. In foam cells, plasmid-less Salmonella infection (particularly S. Choleraesuis OU7266 infection) led to higher CD36 expression than did plasmid-bearing strains infection. OU7130 and OU7266 infection induced the highest IL-1ß secretion. OU7067-infected foam cells secreted the highest IL-12p35 level. Plasmid-bearing S. Typhimurium OU5045 induced a higher IFN-α level than did other Salmonella serotypes. Salmonella serotypes are correlated with foam cell autophagy and IL-1ß secretion. Salmonella may affect the course of foam cells formation, or even aortic aneurysm, through autophagy.


Asunto(s)
Aneurisma de la Aorta/microbiología , Infecciones por Salmonella/microbiología , Salmonella typhimurium/patogenicidad , Animales , Aneurisma de la Aorta/genética , Aneurisma de la Aorta/inmunología , Línea Celular , Humanos , Interferón-alfa/genética , Interferón-alfa/inmunología , Interleucina-12/genética , Interleucina-12/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Antígeno Ki-1/genética , Antígeno Ki-1/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Monocitos/inmunología , Monocitos/microbiología , Plásmidos/genética , Plásmidos/metabolismo , Infecciones por Salmonella/genética , Infecciones por Salmonella/inmunología , Salmonella typhimurium/genética , Salmonella typhimurium/fisiología , Serogrupo , Virulencia
11.
Pol J Microbiol ; 68(4): 559-563, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31880899

RESUMEN

We demonstrate here for the first time the use of an IncP-1ß plasmid, R751, as a gene capture vehicle for recombineering/conjugation strategies to clone large segments of bacterial genomes (20 - 100 + Kb). We designed R751 derivatives containing alternative markers for greater flexibility when using the R751 vehicle across different bacteria. These markers are removable if desired as part of the cloning procedure (with no extra steps needed). We demonstrated utility via cloning of 38 and 22 kb genomic segments from Salmonella enterica serovar Typhimurium and Escherichia coli, respectively. The plasmids expand the options available for use in recombineering/conjugation-based cloning applications.We demonstrate here for the first time the use of an IncP-1ß plasmid, R751, as a gene capture vehicle for recombineering/conjugation strategies to clone large segments of bacterial genomes (20 ­ 100 + Kb). We designed R751 derivatives containing alternative markers for greater flexibility when using the R751 vehicle across different bacteria. These markers are removable if desired as part of the cloning procedure (with no extra steps needed). We demonstrated utility via cloning of 38 and 22 kb genomic segments from Salmonella enterica serovar Typhimurium and Escherichia coli, respectively. The plasmids expand the options available for use in recombineering/conjugation-based cloning applications.


Asunto(s)
Clonación Molecular , Conjugación Genética , Escherichia coli/genética , Plásmidos/genética , Salmonella typhimurium/genética , ADN Bacteriano/genética , Recombinación Genética
12.
PLoS Negl Trop Dis ; 13(10): e0007782, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31609964

RESUMEN

BACKGROUND: Salmonella Typhimurium and Enteritidis are major causes of bloodstream infection in children in sub-Saharan Africa. This study assessed evidence for their zoonotic versus human reservoir. METHODS: Index patients were children with blood culture confirmed Salmonella infection recruited during a microbiological surveillance study in Nanoro, rural Burkina between May 2013 and August 2014. After consent, their households were visited. Stool from household members and livestock (pooled samples per species) as well as drinking water were cultured for Salmonella. Isolates with identical serotype obtained from index patient and any household sample were defined as "paired isolates" and assessed for genetic relatedness by multilocus variable number tandem-repeat analysis (MLVA) and whole-genome sequencing (WGS). RESULTS: Twenty-nine households were visited for 32/42 (76.2%) eligible index patients: two households comprised two index patients each, and in a third household the index patient had a recurrent infection. Among the 32 index patients, serotypes were Salmonella Typhimurium (n = 26), Salmonella Enteritidis (n = 5) and Salmonella Freetown (n = 1). All Typhimurium isolates were sequence type (ST)313. Median delay between blood culture sampling and household visits was 13 days (range 6-26). Salmonella was obtained from 16/186 (8.6%) livestock samples (13 serotypes) and 18/290 (6.2%) household members (9 serotypes). None of the water samples yielded Salmonella. Paired Salmonella Typhimurium isolates were obtained from three households representing four index patients. MLVA types were identical in two pairs and similar in the third (consisting of two index patients and one household member). WGS showed a strong genetic relatedness with 0 to 2 core genome SNPs difference between pairs on a household level. Livestock samples did not yield any Salmonella Typhimurium or Salmonella Enteritidis, and the latter was exclusively obtained from blood culture. Other serotypes shared by human and/or livestock carriers in the same household were Salmonella Derby, Drac, Tennessee and Muenster. CONCLUSIONS/SIGNIFICANCE: The current study provides further evidence of a human reservoir for invasive non-Typhoidal Salmonella (iNTS) in sub-Saharan Africa.


Asunto(s)
Reservorios de Enfermedades/microbiología , Composición Familiar , Infecciones por Salmonella/microbiología , Salmonella/clasificación , Salmonella/aislamiento & purificación , Adolescente , Animales , Burkina Faso/epidemiología , Niño , Preescolar , Monitoreo del Ambiente , Heces/microbiología , Femenino , Humanos , Lactante , Ganado , Masculino , Tipificación de Secuencias Multilocus , Filogenia , Salmonella/genética , Infecciones por Salmonella/epidemiología , Salmonella enterica/genética , Salmonella enterica/aislamiento & purificación , Salmonella enteritidis/genética , Salmonella enteritidis/aislamiento & purificación , Salmonella typhimurium/genética , Salmonella typhimurium/aislamiento & purificación , Serogrupo , Microbiología del Agua , Secuenciación Completa del Genoma
13.
PLoS Genet ; 15(10): e1008425, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31589608

RESUMEN

Evolutionarily conserved NusG protein enhances bacterial RNA polymerase processivity but can also promote transcription termination by binding to, and stimulating the activity of, Rho factor. Rho terminates transcription upon anchoring to cytidine-rich motifs, the so-called Rho utilization sites (Rut) in nascent RNA. Both NusG and Rho have been implicated in the silencing of horizontally-acquired A/T-rich DNA by nucleoid structuring protein H-NS. However, the relative roles of the two proteins in H-NS-mediated gene silencing remain incompletely defined. In the present study, a Salmonella strain carrying the nusG gene under the control of an arabinose-inducible repressor was used to assess the genome-wide response to NusG depletion. Results from two complementary approaches, i) screening lacZ protein fusions generated by random transposition and ii) transcriptomic analysis, converged to show that loss of NusG causes massive upregulation of Salmonella pathogenicity islands (SPIs) and other H-NS-silenced loci. A similar, although not identical, SPI-upregulated profile was observed in a strain with a mutation in the rho gene, Rho K130Q. Surprisingly, Rho mutation Y80C, which affects Rho's primary RNA binding domain, had either no effect or made H-NS-mediated silencing of SPIs even tighter. Thus, while corroborating the notion that bound H-NS can trigger Rho-dependent transcription termination in vivo, these data suggest that H-NS-elicited termination occurs entirely through a NusG-dependent pathway and is less dependent on Rut site binding by Rho. We provide evidence that through Rho recruitment, and possibly through other still unidentified mechanisms, NusG prevents pervasive transcripts from elongating into H-NS-silenced regions. Failure to perform this function causes the feedforward activation of the entire Salmonella virulence program. These findings provide further insight into NusG/Rho contribution in H-NS-mediated gene silencing and underscore the importance of this contribution for the proper functioning of a global regulatory response in growing bacteria. The complete set of transcriptomic data is freely available for viewing through a user-friendly genome browser interface.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Factores de Elongación de Péptidos/metabolismo , Salmonella typhimurium/genética , Factores de Transcripción/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Sitios Genéticos , Factores de Elongación de Péptidos/genética , ARN Bacteriano/metabolismo , Factor Rho/genética , Factor Rho/metabolismo , Salmonella typhimurium/patogenicidad , Factores de Transcripción/genética , Terminación de la Transcripción Genética , Regulación hacia Arriba , Factores de Virulencia/genética
14.
BMC Genomics ; 20(1): 731, 2019 Oct 12.
Artículo en Inglés | MEDLINE | ID: mdl-31606035

RESUMEN

BACKGROUND: TetR-family transcriptional regulators (TFTRs) are DNA binding factors that regulate gene expression in bacteria. Well-studied TFTRs, such as AcrR, which regulates efflux pump expression, are usually encoded alongside target operons. Recently, it has emerged that there are many TFTRs which act as global multi-target regulators. Our classical view of TFTRs as simple, single-target regulators therefore needs to be reconsidered. As some TFTRs regulate essential processes (e.g. metabolism) or processes which are important determinants of resistance and virulence (e.g. biofilm formation and efflux gene expression) and as TFTRs are present throughout pathogenic bacteria, they may be good drug discovery targets for tackling antimicrobial resistant infections. However, the prevalence and conservation of individual TFTR genes in Gram-negative species, has to our knowledge, not yet been studied. RESULTS: Here, a wide-scale search for TFTRs in available proteomes of clinically relevant pathogens Salmonella and Escherichia species was performed and these regulators further characterised. The majority of identified TFTRs are involved in efflux regulation in both Escherichia and Salmonella. The percentage variance in TFTR genes of these genera was found to be higher in those regulating genes involved in efflux, bleach survival or biofilm formation than those regulating more constrained processes. Some TFTRs were found to be present in all strains and species of these two genera, whereas others (i.e. TetR) are only present in some strains and some (i.e. RamR) are genera-specific. Two further pathogens on the WHO priority pathogen list (K. pneumoniae and P. aeruginosa) were then searched for the presence of the TFTRs conserved in Escherichia and Salmonella. CONCLUSIONS: Through bioinformatics and literature analyses, we present that TFTRs are a varied and heterogeneous family of proteins required for the regulation of numerous important processes, with consequences to antimicrobial resistance and virulence, and that the roles and responses of these proteins are frequently underestimated.


Asunto(s)
Biología Computacional/métodos , Farmacorresistencia Bacteriana , Bacterias Gramnegativas/clasificación , Factores de Transcripción/genética , Proteínas Bacterianas/genética , Secuencia Conservada , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Bacterias Gramnegativas/genética , Klebsiella pneumoniae/genética , Funciones de Verosimilitud , Familia de Multigenes , Filogenia , Salmonella typhimurium/genética
15.
J Basic Microbiol ; 59(11): 1143-1153, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31577373

RESUMEN

Salmonella enterica serovar Typhimurium (S. Tm) is a major intracellular pathogen that infects humans and animals, and its survival and growth in macrophages is essential for its pathogenicity. More than 50 putative regulatory proteins are encoded by the S. Tm genome, but the functions of these regulatory proteins in mediating S. Tm pathogenicity are largely unknown. In this study, we investigated the biological function of the STM0030 gene, which encodes a putative LysR-type transcriptional regulator. We found that STM0030 is upregulated 2.8-5.7-fold during S. Tm growth in macrophages. Further, mutating this gene decreased bacterial growth in macrophages and attenuated virulence in mice. RNA-sequencing to investigate the regulatory function of STM0030 in S. Tm revealed that 447 genes were differentially expressed between the mutant and the wild-type strains; 429 of these genes were downregulated, suggesting that STM0030 mainly acts as a transcriptional activator. Moreover, the expression of gluconate, maltose, and hexose-p transport genes, as well as allantoin utilization genes were downregulated in the STM0030 mutant; this might be associated with the observed decrease in intracellular replication and pathogenicity of the mutant. Our findings suggest that STM0030 is a new pathogenicity-associated regulatory protein that broadens our understanding of the virulence regulatory network of S. Tm.


Asunto(s)
Proteínas Bacterianas/metabolismo , Salmonella typhimurium/patogenicidad , Factores de Transcripción/metabolismo , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Femenino , Regulación Bacteriana de la Expresión Génica , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Mutación , Células RAW 264.7 , Salmonelosis Animal/microbiología , Salmonella typhimurium/genética , Salmonella typhimurium/crecimiento & desarrollo , Factores de Transcripción/genética , Virulencia , Factores de Virulencia/genética
16.
Ecotoxicol Environ Saf ; 185: 109733, 2019 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-31580980

RESUMEN

Presence of missing data points in datasets is among main challenges in handling the toxicological data for nanomaterials. As the processing of missing data is an important part of data analysis, we have introduced a read-across approach that uses a combination of supervised and unsupervised machine learning techniques to fill the missing values. A series of classification models (supervised learning) was developed to predict class label, and self-organizing map approach (unsupervised learning) was used to estimate relative distances between nanoparticles and refine results obtained during supervised learning. In this study, genotoxicity of 49 silicon and metal oxide nanoparticles in Ames and Comet tests. Collected literature data did not demonstrate significant variations related to the change of size including selected bulk materials. Genotoxicity-related features of nanomaterials were represented by ionic characteristics. General tendencies found in the current study were convincingly linked to known theories of genotoxic action at nano-level. Mechanisms of primary and secondary genotoxic effects were discussed in the context of developed models.


Asunto(s)
Daño del ADN , Nanopartículas del Metal/toxicidad , Modelos Teóricos , Mutágenos/toxicidad , Aprendizaje Automático no Supervisado , Línea Celular , Ensayo Cometa , Humanos , Nanopartículas del Metal/clasificación , Mutágenos/clasificación , Óxidos/clasificación , Óxidos/toxicidad , Relación Estructura-Actividad Cuantitativa , Salmonella typhimurium/genética
17.
Ecotoxicol Environ Saf ; 186: 109822, 2019 Dec 30.
Artículo en Inglés | MEDLINE | ID: mdl-31634658

RESUMEN

Nitroaromatic compounds (NACs) are an important type of environmental organic pollutants. However, it is lack of sufficient information relating to their potential adverse effects on human health and the environment due to the limited resources. Thus, using in silico technologies to assess their potential hazardous effects is urgent and promising. In this study, quantitative structure activity relationship (QSAR) and classification models were constructed using a set of NACs based on their mutagenicity against Salmonella typhimurium TA100 strain. For QSAR studies, DRAGON descriptors together with quantum chemistry descriptors were calculated for characterizing the detailed molecular information. Based on genetic algorithm (GA) and multiple linear regression (MLR) analyses, we screened descriptors and developed QSAR models. For classification studies, seven machine learning methods along with six molecular fingerprints were applied to develop qualitative classification models. The goodness of fitting, reliability, robustness and predictive performance of all developed models were measured by rigorous statistical validation criteria, then the best QSAR and classification models were chosen. Moreover, the QSAR models with quantum chemistry descriptors were compared to that without quantum chemistry descriptors and previously reported models. Notably, we also obtained some specific molecular properties or privileged substructures responsible for the high mutagenicity of NACs. Overall, the developed QSAR and classification models can be utilized as potential tools for rapidly predicting the mutagenicity of new or untested NACs for environmental hazard assessment and regulatory purposes, and may provide insights into the in vivo toxicity mechanisms of NACs and related compounds.


Asunto(s)
Contaminantes Ambientales , Hidrocarburos Aromáticos , Mutágenos , Nitrocompuestos , Algoritmos , Simulación por Computador , Contaminantes Ambientales/química , Contaminantes Ambientales/toxicidad , Hidrocarburos Aromáticos/química , Hidrocarburos Aromáticos/toxicidad , Aprendizaje Automático , Mutágenos/química , Mutágenos/toxicidad , Nitrocompuestos/química , Nitrocompuestos/toxicidad , Relación Estructura-Actividad Cuantitativa , Reproducibilidad de los Resultados , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética
18.
Infect Immun ; 88(1)2019 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-31611277

RESUMEN

To survive and replicate during infection, pathogens utilize different carbon and energy sources depending on the nutritional landscape of their host microenvironment. Salmonella enterica serovar Typhimurium is an intracellular bacterial pathogen that occupies diverse cellular niches. While it is clear that Salmonella Typhimurium requires access to glucose during systemic infection, data on the need for lipid metabolism are mixed. We report that Salmonella Typhimurium strains lacking lipid metabolism genes were defective for systemic infection of mice. Bacterial lipid import, ß-oxidation, and glyoxylate shunt genes were required for tissue colonization upon oral or intraperitoneal inoculation. In cultured macrophages, lipid import and ß-oxidation genes were required for bacterial replication and/or survival only when the cell culture medium was supplemented with nonessential amino acids. Removal of glucose from tissue culture medium further enhanced these phenotypes and, in addition, conferred a requirement for glyoxylate shunt genes. We also observed that Salmonella Typhimurium needs lipid metabolism genes in proinflammatory but not anti-inflammatory macrophages. These results suggest that during systemic infection, the Salmonella Typhimurium that relies upon host lipids to replicate is within proinflammatory macrophages that have access to amino acids but not glucose. An improved understanding of the host microenvironments in which pathogens have specific metabolic requirements may facilitate the development of targeted approaches to treatment.


Asunto(s)
Metabolismo de los Lípidos , Macrófagos/microbiología , Redes y Vías Metabólicas/genética , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/metabolismo , Aminoácidos/metabolismo , Animales , Glucosa/metabolismo , Ratones , Viabilidad Microbiana , Salmonelosis Animal/microbiología , Salmonelosis Animal/patología , Salmonella typhimurium/genética
19.
Vet Res ; 50(1): 71, 2019 Sep 24.
Artículo en Inglés | MEDLINE | ID: mdl-31551081

RESUMEN

Eggs and raw or undercooked egg-containing food items are frequently identified as the bacterial source during epidemiolocal investigation of Salmonella outbreaks. Multi-locus variable number of tandem repeats analysis (MLVA) is a widely used Salmonella typing method enabling the study of diversity within populations of the same serotype. In vivo passage, however, has been linked with changes in MLVA type and more broadly the Salmonella genome. We sought to investigate whether in vivo passage through layer hens had an effect on MLVA type as well as the bacterial genome and whether any mutations affected bacterial virulence. Layer hens were infected with either Salmonella Typhimurium DT9 (03-24-11-11-523) as part of a single infection or were co-infected with an equal amount of Salmonella Mbandaka. Salmonella shedding in both single and co-infected birds was variable over the course of the 16-week experiment. Salmonella Typhimurium and Salmonella Mbandaka were identified in feces of co-infected birds. Salmonella colonies isolated from fecal samples were subtyped using MLVA. A single change in SSTR-6 was observed in Salmonella Typhimurium strains isolated from co-infected birds. Isolates of Salmonella Typhimurium of both the parent (03-24-11-11-523) and modified (03-24-12-11-523) MLVA type were sequenced and compared with the genome of the parent strain. Sequence analysis revealed that in vivo passaging resulted in minor mutation events. Passaged isolates exhibited significantly higher invasiveness in cultured human intestinal epithelial cells than the parent strain. The microevolution observed in this study suggests that changes in MLVA may arise more commonly and may have clinical significance.


Asunto(s)
Pollos , Genoma Bacteriano/genética , Mutación/fisiología , Enfermedades de las Aves de Corral/fisiopatología , Salmonelosis Animal/fisiopatología , Salmonella typhimurium/fisiología , Animales , Células CACO-2 , Coinfección/microbiología , Coinfección/fisiopatología , Coinfección/veterinaria , Heces/microbiología , Femenino , Humanos , Repeticiones de Minisatélite , Tipificación de Secuencias Multilocus/veterinaria , Enfermedades de las Aves de Corral/microbiología , Salmonelosis Animal/microbiología , Salmonella enterica/fisiología , Salmonella typhimurium/genética , Pase Seriado , Virulencia
20.
Biochimie ; 167: 152-161, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31563538

RESUMEN

The intracellular pathogen Salmonella is an important cause of human foodborne diseases worldwide. Salmonella takes advantage of the phosphorelay regulatory systems to survive in the hostile environment of the host's gastrointestinal tract. It has been reported that the nitrate reductase Z (NR-Z), encoded by the narUZYV operon, is required during Salmonella transition to anaerobic environments and is constitutively produced at low levels, but little is known about the regulatory mechanism involved in the operon gene expression. In this work, we found that the RcsCDB system is activated by high concentrations of specific sugars as a carbon source. In this activation state, the RcsCDB system participates in the negative control of narUZYWV expression. This control strategy occurs during exponential growth when the carbon source is high, allowing for normal aerobic respiration. The RcsCDB system's participation in aerobic respiration is necessary to ensure efficient metabolism and optimal energy consumption when the bacteria are growing exponentially.


Asunto(s)
Proteínas Bacterianas/genética , Nitrato-Reductasa/genética , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/genética , Factores de Transcripción/fisiología , Regulación Bacteriana de la Expresión Génica , Operón , Regiones Promotoras Genéticas , Transcripción Genética
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