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1.
Mutat Res ; 863-864: 503299, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33678240

RESUMO

N-Acyloxy-N-alkoxyamides are direct-acting mutagens in S. typhimurium TA100 and TA98. A reliable QSAR for their activity in TA100 has been developed, which indicates reversible intercalation into the DNA helix through naphthalene substituents. In this paper, we show that fluorene as a substituent does not facilitate intercalation while fluorenone does, although the efficacy is determined by the position of substitution on the fluorenone as well as the N-acyloxy-N-alkoxyamide side chain. Where intercalation is evident, the increased binding to DNA is similar to that of naphthalene and is worth the equivalent of ca four LogP hydrophobicity units. 4-Substituted fluorenones, where the anomeric amide group is in the bay region do not intercalate, which is attributed to the requirement for a weaker edge-on, rather than an end-on intercalation. Mutagencity in S. typhimurium TA98, which detects frame shifts through intercalation, supports the findings. Fluorene appears not to intercalate, which points to the fact that the charge delocalised 2-fluorenylnitrenium ion, the ultimate metabolite from 2-aminofluorene (AF) and 2-acetylaminofluorene (AAF) is the itercalating agent responsible for frameshift mutations leading to their carcinogenicity.


Assuntos
Substâncias Intercalantes , Mutagênese , Mutagênicos , Salmonella typhimurium , Substâncias Intercalantes/química , Substâncias Intercalantes/farmacologia , Testes de Mutagenicidade , Mutagênicos/química , Mutagênicos/farmacologia , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo
3.
Nat Commun ; 12(1): 1546, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33750771

RESUMO

Many bacterial pathogens rely on virulent type III secretion systems (T3SSs) or injectisomes to translocate effector proteins in order to establish infection. The central component of the injectisome is the needle complex which assembles a continuous conduit crossing the bacterial envelope and the host cell membrane to mediate effector protein translocation. However, the molecular principles underlying type III secretion remain elusive. Here, we report a structure of an active Salmonella enterica serovar Typhimurium needle complex engaged with the effector protein SptP in two functional states, revealing the complete 800Å-long secretion conduit and unraveling the critical role of the export apparatus (EA) subcomplex in type III secretion. Unfolded substrates enter the EA through a hydrophilic constriction formed by SpaQ proteins, which enables side chain-independent substrate transport. Above, a methionine gasket formed by SpaP proteins functions as a gate that dilates to accommodate substrates while preventing leaky pore formation. Following gate penetration, a moveable SpaR loop first folds up to then support substrate transport. Together, these findings establish the molecular basis for substrate translocation through T3SSs and improve our understanding of bacterial pathogenicity and motility.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transporte Proteico/fisiologia , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo III/química , Sistemas de Secreção Tipo III/metabolismo , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Salmonella enterica/metabolismo , Salmonella typhimurium/genética , Sistemas de Secreção Tipo III/genética
4.
Nucleic Acids Res ; 49(4): 2357-2374, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33638994

RESUMO

RcsB is a transcriptional regulator that controls expression of numerous genes in enteric bacteria. RcsB accomplishes this role alone or in combination with auxiliary transcriptional factors independently or dependently of phosphorylation. To understand the mechanisms by which RcsB regulates such large number of genes, we performed structural studies as well as in vitro and in vivo functional studies with different RcsB variants. Our structural data reveal that RcsB binds promoters of target genes such as rprA and flhDC in a dimeric active conformation. In this state, the RcsB homodimer docks the DNA-binding domains into the major groove of the DNA, facilitating an initial weak read-out of the target sequence. Interestingly, comparative structural analyses also show that DNA binding may stabilize an active conformation in unphosphorylated RcsB. Furthermore, RNAseq performed in strains expressing wild-type or several RcsB variants provided new insights into the contribution of phosphorylation to gene regulation and assign a potential role of RcsB in controlling iron metabolism. Finally, we delimited the RcsB box for homodimeric active binding to DNA as the sequence TN(G/A)GAN4TC(T/C)NA. This RcsB box was found in promoter, intergenic and intragenic regions, facilitating both increased or decreased gene transcription.


Assuntos
Proteínas de Bactérias/química , Regiões Promotoras Genéticas , Regulon , Salmonella typhimurium/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Modelos Moleculares , Mutação , Fosforilação , Conformação Proteica , Salmonella typhimurium/metabolismo , Transcrição Genética
5.
J Vis Exp ; (167)2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33586704

RESUMO

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia coli and Salmonella Typhimurium, could repress the expression of outer membrane proteins. To further investigate the regulation function of micC in Salmonella Enteritidis, we cloned the micC gene in the Salmonella Enteritidis strain 50336, and then constructed the mutant 50336ΔmicC by the λ Red-based recombination system and the complemented mutant 50336ΔmicC/pmicC carrying recombinant plasmid pBR322 expressing micC. qRT-PCR results demonstrated that transcription of ompD in 50336ΔmicC was 1.3-fold higher than that in the wild type strain, while the transcription of ompA and ompC in 50336ΔmicC were 2.2-fold and 3-fold higher than those in the wild type strain. These indicated that micC represses the expression of ompA and ompC. In the following study, the pathogenicity of 50336ΔmicC was detected by both infecting 6-week-old Balb/c mice and 1-day-old chickens. The result showed that the LD50 of the wild type strain 50336, the mutants 50336ΔmicC and 50336ΔmicC/pmicC for 6-week-old Balb/c mice were 12.59 CFU, 5.01 CFU, and 19.95 CFU, respectively. The LD50 of the strains for 1-day-old chickens were 1.13 x 109 CFU, 1.55 x 108 CFU, and 2.54 x 108 CFU, respectively. It indicated that deletion of micC enhanced virulence of S. Enteritidis in mice and chickens by regulating expression of outer membrane proteins.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , RNA não Traduzido/genética , Salmonella enteritidis/genética , Salmonella enteritidis/patogenicidade , Animais , Proteínas de Bactérias/metabolismo , Galinhas/microbiologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mutação/genética , Plasmídeos/genética , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Recombinação Genética/genética , Salmonelose Animal/microbiologia , Salmonella typhimurium/genética , Virulência/genética
6.
Nat Commun ; 12(1): 348, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441540

RESUMO

In the enteric pathogen Salmonella enterica serovar Typhimurium, invasion and motility are coordinated by the master regulator HilD, which induces expression of the type III secretion system 1 (T3SS1) and motility genes. Methyl-accepting chemotaxis proteins (MCPs) detect specific ligands and control the direction of the flagellar motor, promoting tumbling and changes in direction (if a repellent is detected) or smooth swimming (in the presence of an attractant). Here, we show that HilD induces smooth swimming by upregulating an uncharacterized MCP (McpC), and this is important for invasion of epithelial cells. Remarkably, in vitro assays show that McpC can suppress tumbling and increase smooth swimming in the absence of exogenous ligands. Expression of mcpC is repressed by the universal regulator H-NS, which can be displaced by HilD. Our results highlight the importance of smooth swimming for Salmonella Typhimurium invasiveness and indicate that McpC can act via a ligand-independent mechanism when incorporated into the chemotactic receptor array.


Assuntos
Proteínas de Bactérias/metabolismo , Quimiotaxia/fisiologia , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Salmonella typhimurium/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias/genética , Células CACO-2 , Bovinos , Células Cultivadas , Quimiotaxia/genética , Regulação Bacteriana da Expressão Gênica , Células HeLa , Humanos , Proteínas Quimiotáticas Aceptoras de Metil/genética , Camundongos Endogâmicos C57BL , Movimento/fisiologia , Mutação , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologia , Fatores de Transcrição/genética
7.
Nat Commun ; 12(1): 328, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436566

RESUMO

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Genoma Bacteriano , RNA de Transferência/genética , Salmonella typhimurium/genética , Aminoácidos/metabolismo , Aminoacilação , Anticódon/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Códon/genética , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Guanosina Trifosfato/metabolismo , Hidrólise , Metilação , Modelos Moleculares , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo
8.
Biosens Bioelectron ; 178: 113001, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33493900

RESUMO

Amplification-based nucleic acid detection is widely employed in food safety, medical diagnosis and environment monitoring. However, conventional nucleic acid analysis has to be carried out in laboratories because of requiring expensive instruments and trained personnel. If people could do nucleic acid detection at home by themselves, the application of nucleic acid detection would be greatly accelerated. We herein reported a polypropylene (PP) bag-based method for convenient detection of nucleic acids in the oil-sealed space. The PP bag has three chambers which are responsible for lysis, washing and amplification/detection, respectively. After adding sample, nucleic acids are adsorbed on magnetic particles (MPs) and moved into these three chambers successively through immiscible oil channel by an external magnet. Combined with isothermal amplification, the PP bag can be incubated in a water bath or milk warmer and acted as a reaction tube. With highly specific CRISPR technology, Salmonella typhimurium (St) and SARS-CoV-2 can be visually detected in these PP bags within 1 h, indicating its potential household application. To further improve the reliability of nucleic acid testing at home, a logic decision method is introduced by detecting both target and endogenous reference gene. Positive/negative/invalid detection result can be obtained by chronologically adding the CRISPR reagents of target and endogenous reference gene. We anticipate that this PP bag can provide a novel toolkit for nucleic acid detection in people's daily life.


Assuntos
/métodos , /virologia , Sistemas CRISPR-Cas , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , /isolamento & purificação , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Microbiologia de Alimentos , Humanos , Magnetismo , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Polipropilenos , RNA Viral/genética , RNA Viral/isolamento & purificação , Salmonella typhimurium/genética , Salmonella typhimurium/isolamento & purificação
9.
Science ; 371(6527): 400-405, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33479153

RESUMO

Key to the success of intracellular pathogens is the ability to sense and respond to a changing host cell environment. Macrophages exposed to microbial products undergo metabolic changes that drive inflammatory responses. However, the role of macrophage metabolic reprogramming in bacterial adaptation to the intracellular environment has not been explored. Here, using metabolic profiling and dual RNA sequencing, we show that succinate accumulation in macrophages is sensed by intracellular Salmonella Typhimurium (S. Tm) to promote antimicrobial resistance and type III secretion. S Tm lacking the succinate uptake transporter DcuB displays impaired survival in macrophages and in mice. Thus, S Tm co-opts the metabolic reprogramming of infected macrophages as a signal that induces its own virulence and survival, providing an additional perspective on metabolic host-pathogen cross-talk.


Assuntos
Interações Hospedeiro-Patógeno , Macrófagos/metabolismo , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Ácido Succínico/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sobrevivência Celular , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Modelos Animais de Doenças , Feminino , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , RNA-Seq , Salmonella typhimurium/genética , Virulência
11.
Nucleic Acids Res ; 49(2): 832-846, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33406256

RESUMO

The Salmonella genomic island 1 (SGI1) and its variants are mobilized by IncA and IncC conjugative plasmids. SGI1-family elements and their helper plasmids are effective transporters of multidrug resistance determinants. SGI1 exploits the transfer apparatus of the helper plasmid and hijacks its activator complex, AcaCD, to trigger the expression of several SGI1 genes. In this way, SGI1 times its excision from the chromosome to the helper entry and expresses mating pore components that enhance SGI1 transfer. The SGI1-encoded T4SS components and the FlhDC-family activator proved to be interchangeable with their IncC-encoded homologs, indicating multiple interactions between SGI1 and its helpers. As a new aspect of this crosstalk, we report here the helper-induced replication of SGI1, which requires both activators, AcaCD and FlhDCSGI1, and significantly increases the stability of SGI1 when coexists with the helper plasmid. We have identified the oriVSGI1 and shown that S004-repA operon encodes for a translationally coupled leader protein and an IncN2/N3-related RepA that are expressed under the control of the AcaCD-responsive promoter PS004. This replicon transiently maintains SGI1 as a 4-8-copy plasmid, not only stabilizing the island but also contributing to the fast displacement of the helper plasmid.


Assuntos
Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , Conjugação Genética/genética , Farmacorresistência Bacteriana Múltipla/genética , Sequências Repetitivas Dispersas/genética , Salmonella typhimurium/genética , Proteínas de Bactérias/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA , Dosagem de Genes , Regulação Bacteriana da Expressão Gênica/genética , Genes Reporter , Integrases/metabolismo , Óperon/genética , Filogenia , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas , Recombinases/metabolismo , Replicon/genética , Alinhamento de Sequência , Transativadores/genética , Transativadores/metabolismo
12.
Methods Mol Biol ; 2225: 39-61, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33108656

RESUMO

Vaccines are the most effective means to prevent infectious diseases, especially for viral infection. The key to an excellent antiviral vaccine is the ability to induce long-term protective immunity against a specific virus. Bacterial vaccine vectors have been used to impart protection against self, as well as heterologous antigens. One significant benefit of using live bacterial vaccine vectors is their ability to invade and colonize deep effector lymphoid tissues after mucosal delivery. The bacterium Salmonella is considered the best at this deep colonization. This is critically essential for inducing protective immunity. This chapter describes the methodology for developing genetically modified self-destructing Salmonella (GMS) vaccine delivery systems targeting influenza infection. Specifically, the methods covered include the procedures for the development of GMSs for protective antigen delivery to induce cellular immune responses and DNA vaccine delivery to induce systemic immunity against the influenza virus. These self-destructing GMS could be modified to provide effective biological containment for genetically engineered bacteria used for a diversity of purposes in addition to vaccines.


Assuntos
Engenharia Genética/métodos , Imunização/métodos , Vacinas contra Influenza/genética , Influenza Humana/prevenção & controle , Salmonella typhimurium/genética , Vacinas de DNA/genética , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Antígenos Virais/genética , Antígenos Virais/imunologia , Feminino , Genes Letais , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade nas Mucosas/efeitos dos fármacos , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos BALB C , Nucleoproteínas/genética , Nucleoproteínas/imunologia , Organismos Geneticamente Modificados , Plasmídeos/química , Plasmídeos/metabolismo , Salmonella typhimurium/imunologia , Transgenes
13.
Biochim Biophys Acta Gen Subj ; 1865(1): 129748, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32980501

RESUMO

BACKGROUND: Structural studies of a Salmonella Typhimurium flagellin protein indicated that four polar or charged C-terminal amino acid residues line the inner channel of the flagellum. The hydrophilic character of these putative channel-lining residues was predicted to be essential to facilitate the transport of unfolded flagellin monomers during flagellar assembly. The structure-function relationship of these putative channel-lining residues was investigated by site-directed mutagenesis to examine effects of side chain polarity and size on flagella assembly and function. METHODS: Channel-lining residue variants were generated using site-directed mutagenesis to substitute alanine and other residues to examine the effects of altered side-chain polarity on export and assembly. The export, in vivo motility function, and flagellar structure of variants was characterized by agar motility, video microscopy, immunofluorescence, and SDS-PAGE. RESULTS: Alanine substitution yielded decreased motility and flagellar assembly for three of the four residues. However, alanine substitution of residue Arg 494 did not alter export, although substitution with negatively charged glutamate decreased motility and flagellar filament length. Furthermore, many of the C-terminal mutations affected flagellar filament morphology and stability, often resulting in more tightly coiled and/or more brittle flagella than the wild type. CONCLUSIONS: The four channel-lining C-terminal residues may facilitate monomer protein transport but also have structural roles in determining the stability and morphology of the flagellum. GENERAL SIGNIFICANCE: These results provide further insight into the complex process of bacterial flagellin export and flagellar assembly and provide evidence of previously unknown structural functions for the four putative channel-lining residues.


Assuntos
Flagelina/metabolismo , Salmonella typhimurium/citologia , Flagelos/química , Flagelos/genética , Flagelos/metabolismo , Flagelos/ultraestrutura , Flagelina/química , Flagelina/genética , Humanos , Movimento (Física) , Mutagênese Sítio-Dirigida , Conformação Proteica , Infecções por Salmonella/microbiologia , Salmonella typhimurium/química , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo
14.
Nucleic Acids Res ; 48(19): 10832-10847, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33045730

RESUMO

Horizontally acquired genes are typically regulated by ancestral regulators. This regulation enables expression of horizontally acquired genes to be coordinated with that of preexisting genes. Here, we report a singular example of the opposite regulation: a horizontally acquired gene that controls an ancestral regulator, thereby promoting bacterial virulence. We establish that the horizontally acquired regulatory gene ssrB is necessary to activate the ancestral regulatory system PhoP/PhoQ of Salmonella enterica serovar Typhimurium (S. Typhimurium) in mildly acidic pH, which S. Typhimurium experiences inside macrophages. SsrB promotes phoP transcription by binding upstream of the phoP promoter. SsrB also increases ugtL transcription by binding to the ugtL promoter region, where it overcomes gene silencing by the heat-stable nucleoid structuring protein H-NS, enhancing virulence. The largely non-pathogenic species S. bongori failed to activate PhoP/PhoQ in mildly acidic pH because it lacks both the ssrB gene and the SsrB binding site in the target promoter. Low Mg2+ activated PhoP/PhoQ in both S. bongori and ssrB-lacking S. Typhimurium, indicating that the SsrB requirement for PhoP/PhoQ activation is signal-dependent. By controlling the ancestral genome, horizontally acquired genes are responsible for more crucial abilities, including virulence, than currently thought.


Assuntos
Proteínas de Bactérias/genética , Transferência Genética Horizontal , Proteínas de Membrana/genética , Salmonella typhimurium/genética , Fatores de Transcrição/genética , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular , Evolução Molecular , Feminino , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas , Salmonella typhimurium/patogenicidade , Fatores de Transcrição/metabolismo , Ativação Transcricional , Virulência/genética
15.
Chem Res Toxicol ; 33(10): 2668-2674, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-32894672

RESUMO

Inflammation is an immune response to protect against various types of infections. When unchecked, acute inflammation can be life-threatening, as seen with the current coronavirus pandemic. Strong oxidants, such as peroxynitrite produced by immune cells, are major mediators of the inflammation-associated pathogenesis. Cellular thiols play important roles in mitigating inflammation-associated macromolecular damage including DNA. Herein, we have demonstrated a role of glutathione (GSH) and other thiols in neutralizing the effect of peroxynitrite-mediated DNA damage through stable GSH-DNA adduct formation. Our observation supports the use of thiol supplements as a potential therapeutic strategy against severe COVID-19 cases and a Phase II (NCT04374461) open-label clinical trial launched in early May 2020 by the Memorial Sloan Kettering Cancer Center.


Assuntos
Adutos de DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Glutationa/farmacologia , Inflamação/fisiopatologia , Ácido Peroxinitroso/efeitos adversos , Doença Aguda , Animais , Betacoronavirus , Bovinos , Infecções por Coronavirus/tratamento farmacológico , DNA/química , Adutos de DNA/química , Dano ao DNA , Glutationa/química , Células HEK293 , Humanos , Mutagênicos/química , Mutagênicos/farmacologia , Pandemias , Ácido Peroxinitroso/química , Pneumonia Viral/tratamento farmacológico , Salmonella typhimurium/genética
16.
PLoS One ; 15(8): e0237886, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32810191

RESUMO

Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) causes gastroenteritis in many countries. However, in Brazil there are few studies that have conducted a virulence characterization of this serovar. The aim of this study was to evaluate the virulence potential of S. Typhimurium strains isolated in Brazil. Forty S. Typhimurium strains isolated from humans (n = 20) and food (n = 20) from Brazil were studied regarding their invasion and survival in human epithelial cells (Caco-2) and macrophages (U937). Their virulence potential was determined using the Galleria mellonella larvae model combined with the analysis of virulence genes by whole genome sequencing (WGS). A total of 67.5% of the S. Typhimurium studied (32.5% isolated from humans and 35% isolated from food) invaded Caco-2 epithelial cells at levels similar to or greater than the S. Typhimurium SL1344 prototype strain. In addition, 37.5% of the studied strains (25% isolated from humans and 12.5% isolated from food) survived in U937 human macrophages at levels similar to or greater than SL1344. S. Typhimurium strains isolated from humans (40%) and food (25%) showed high or intermediate virulence in G. mellonella larvae after seven days exposure. Approximately, 153 virulence genes of chromosomal and plasmidial origin were detected in the strains studied. In conclusion, the ability of the S. Typhimurium to invade Caco-2 epithelial cells was strain dependent and was not related to the source or the year of isolation. However, S. Typhimurium strains isolated from humans showed greater survival rates in U937 human macrophages, and presented higher proportion of isolates with a virulent profile in G. mellonella in comparison to strains isolated from food suggesting that this difference may be related to the higher frequency of human isolates which contained plasmid genes, such as spvABCDR operon, pefABCD operon, rck and mig-5.


Assuntos
Microbiologia de Alimentos , Salmonella typhimurium/genética , Salmonella typhimurium/isolamento & purificação , Animais , Brasil , Células CACO-2 , Sobrevivência Celular , Células Epiteliais/microbiologia , Genes Bacterianos , Genótipo , Humanos , Macrófagos/microbiologia , Mariposas/microbiologia , Fenótipo , Plasmídeos/genética , Salmonella typhimurium/patogenicidade , Células U937 , Virulência/genética
17.
Proc Natl Acad Sci U S A ; 117(34): 20717-20728, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32788367

RESUMO

Mucosal-associated invariant T (MAIT) cells are innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defense. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with selective absence of MAIT cells have not been identified. We report that typhoidal and nontyphoidal Salmonella enterica strains activate MAIT cells. However, S. Typhimurium sequence type 313 (ST313) lineage 2 strains, which are responsible for the burden of multidrug-resistant nontyphoidal invasive disease in Africa, escape MAIT cell recognition through overexpression of ribB This bacterial gene encodes the 4-dihydroxy-2-butanone-4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. The MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that ribB overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of S. Typhimurium ST313 lineage 2.


Assuntos
Células T Invariáveis Associadas à Mucosa/imunologia , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , África ao Sul do Saara , Antibacterianos , Diarreia/microbiologia , Diarreia/mortalidade , Humanos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/fisiologia , Células T Invariáveis Associadas à Mucosa/metabolismo , Infecções por Salmonella/imunologia , Salmonella typhimurium/patogenicidade
18.
Proc Natl Acad Sci U S A ; 117(33): 20235-20243, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32753384

RESUMO

All cells require Mg2+ to replicate and proliferate. The macrophage protein Slc11a1 is proposed to protect mice from invading microbes by causing Mg2+ starvation in host tissues. However, the Mg2+ transporter MgtB enables the facultative intracellular pathogen Salmonella enterica serovar Typhimurium to cause disease in mice harboring a functional Slc11a1 protein. Here, we report that, unexpectedly, the Salmonella small protein MgtR promotes MgtB degradation by the protease FtsH, which raises the question: How does Salmonella preserve MgtB to promote survival inside macrophages? We establish that the Salmonella small protein MgtU prevents MgtB proteolysis, even when MgtR is absent. Like MgtB, MgtU is necessary for survival in Slc11a1 +/+ macrophages, resistance to oxidative stress, and growth under Mg2+ limitation conditions. The Salmonella Mg2+ transporter MgtA is not protected by MgtU despite sharing 50% amino acid identity with MgtB and being degraded in an MgtR- and FtsH-dependent manner. Surprisingly, the mgtB, mgtR, and mgtU genes are part of the same transcript, providing a singular example of transcript-specifying proteins that promote and hinder degradation of the same target. Our findings demonstrate that small proteins can confer pathogen survival inside macrophages by altering the abundance of related transporters, thereby furthering homeostasis.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Macrófagos/microbiologia , Magnésio/metabolismo , Salmonella typhimurium/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Macrófagos/fisiologia , Camundongos , Plasmídeos/genética , Salmonella typhimurium/genética , Virulência
19.
PLoS Genet ; 16(7): e1008610, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716926

RESUMO

Two-component systems and phosphorelays play central roles in the ability of bacteria to rapidly respond to changing environments. In E. coli and related enterobacteria, the complex Rcs phosphorelay is a critical player in the bacterial response to antimicrobial peptides, beta-lactam antibiotics, and other disruptions at the cell surface. The Rcs system is unusual in that an inner membrane protein, IgaA, is essential due to its negative regulation of the RcsC/RcsD/RcsB phosphorelay. While it is known that IgaA transduces signals from the outer membrane lipoprotein RcsF, how it interacts with the phosphorelay has remained unknown. Here we performed in vivo interaction assays and genetic dissection of the critical proteins and found that IgaA interacts with the phosphorelay protein RcsD, and that this interaction is necessary for regulation. Interactions between IgaA and RcsD within their respective periplasmic domains of these two proteins anchor repression of signaling. However, the signaling response depends on a second interaction between cytoplasmic loop 1 of IgaA and a truncated Per-Arndt-Sim (PAS-like) domain in RcsD. A single point mutation in the PAS-like domain increased interactions between the two proteins and blocked induction of the phosphorelay. IgaA may regulate RcsC, the histidine kinase that initiates phosphotransfer through the phosphorelay, indirectly, via its contacts with RcsD. Unlike RcsD, and unlike many other histidine kinases, the periplasmic domain of RcsC is dispensable for the response to signals that induce the Rcs phosphorelay system. The multiple contacts between IgaA and RcsD constitute a poised sensing system, preventing potentially toxic over-activation of this phosphorelay while enabling it to rapidly and quantitatively respond to signals.


Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Fosfoproteínas/genética , Fosfotransferases/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Complexos Multienzimáticos/genética , Fosforilação/genética , Transporte Proteico/genética , Salmonella typhimurium/genética , Transdução de Sinais/genética
20.
PLoS One ; 15(7): e0236436, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32716946

RESUMO

Salmonella 4,[5],12:i:- are monophasic S. Typhimurium variants incapable of producing the second-phase flagellar antigen. They have emerged since the mid-1990s to become one of the most prevalent Salmonella serotypes causing human disease world-wide. Multiple genetic events associated with different genetic elements can result in the monophasic phenotype. Several jurisdictions have reported the emergence of a Salmonella 4,[5],12:i:- clone with SGI-4 and a genetic element (MREL) encoding a mercury resistance operon and antibiotic resistance loci that disrupts the second phase antigen region near the iroB locus in the Salmonella genome. We have sequenced 810 human and animal Canadian Salmonella 4,[5],12:i:- isolates and determined that isolates with SGI-4 and the mercury resistance element (MREL; also known as RR1&RR2) constitute several global clades containing various proportions of Canadian, US, and European isolates. Detailed analysis of the data provides a clearer picture of how these heavy metal elements interact with bacteria within the Salmonella population to produce the monophasic phenotype. Insertion of the MREL near iroB is associated with several deletions and rearrangements of the adjacent flaAB hin region, which may be useful for defining human case clusters that could represent outbreaks. Plasmids carrying genes encoding silver, copper, mercury, and antimicrobial resistance appear to be derived from IS26 mediated acquisition of these genes from genomes carrying SGI-4 and the MREL. Animal isolates with the mercury and As/Cu/Ag resistance elements are strongly associated with porcine sources in Canada as has been shown previously for other jurisdictions. The data acquired in these investigations, as well as from the extensive literature on the subject, may aid source attribution in outbreaks of the organism and interventions to decrease the prevalence of this clone and reduce its impact on human disease.


Assuntos
Metais Pesados/toxicidade , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Animais , Antígenos de Bactérias/genética , Sequência de Bases , Canadá , Variação Genética , Genoma Bacteriano , Genótipo , Humanos , Sequências Repetitivas Dispersas/genética , Fenótipo , Filogenia , Plasmídeos/genética , Salmonella typhimurium/isolamento & purificação , Suínos , Sintenia/genética
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