RESUMO
Downstream metabolic events can contribute to the lethality of drugs or agents that interact with a primary cellular target. In bacteria, the production of reactive oxygen species (ROS) has been associated with the lethal effects of a variety of stresses including bactericidal antibiotics, but the relative contribution of this oxidative component to cell death depends on a variety of factors. Experimental evidence has suggested that unresolvable DNA problems caused by incorporation of oxidized nucleotides into nascent DNA followed by incomplete base excision repair contribute to the ROS-dependent component of antibiotic lethality. Expression of the chimeric periplasmic-cytoplasmic MalE-LacZ72-47 protein is an historically important lethal stress originally identified during seminal genetic experiments that defined the SecY-dependent protein translocation system. Multiple, independent lines of evidence presented here indicate that the predominant mechanism for MalE-LacZ lethality shares attributes with the ROS-dependent component of antibiotic lethality. MalE-LacZ lethality requires molecular oxygen, and its expression induces ROS production. The increased susceptibility of mutants sensitive to oxidative stress to MalE-LacZ lethality indicates that ROS contribute causally to cell death rather than simply being produced by dying cells. Observations that support the proposed mechanism of cell death include MalE-LacZ expression being bacteriostatic rather than bactericidal in cells that overexpress MutT, a nucleotide sanitizer that hydrolyzes 8-oxo-dGTP to the monophosphate, or that lack MutM and MutY, DNA glycosylases that process base pairs involving 8-oxo-dGTP. Our studies suggest stress-induced physiological changes that favor this mode of ROS-dependent death.
RESUMO
The QseEF histidine kinase/response regulator system modulates expression of enterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica serovar Typhimurium virulence genes in response to the host neurotransmitters epinephrine and norepinephrine. qseG, which encodes an outer membrane lipoprotein, is cotranscribed with qseEF in these enteric pathogens, but there is little knowledge of its role in virulence. Here, we found that in EHEC QseG interacts with the type III secretion system (T3SS) gate protein SepL and modulates the kinetics of attaching and effacing (AE) lesion formation on tissue-cultured cells. Moreover, an EHEC ΔqseG mutant had reduced intestinal colonization in an infant rabbit model. Additionally, in Citrobacter rodentium, an AE lesion-forming pathogen like EHEC, QseG is required for full virulence in a mouse model. In S Typhimurium, we found that QseG regulates the phase switch between the two flagellin types, FliC and FljB. In an S Typhimurium ΔqseG mutant, the phase-variable promoter for fljB is preferentially switched into the "on" position, leading to overproduction of this phase two flagellin. In infection of tissue-cultured cells, the S Typhimurium ΔqseG mutant provokes increased inflammatory cytokine production versus the wild type; in vivo, in a murine infection model, the ΔqseG strain caused a more severe inflammatory response and was attenuated versus the wild-type strain. Collectively, our findings demonstrate that QseG is important for full virulence in several enteric pathogens and controls flagellar phase variation in S Typhimurium, and they highlight both the complexity and conservation of the regulatory networks that control the virulence of enteric pathogens.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Citrobacter rodentium/fisiologia , Escherichia coli Êntero-Hemorrágica/fisiologia , Proteínas de Escherichia coli/metabolismo , Flagelos/fisiologia , Salmonella typhimurium/fisiologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Flagelina/biossíntese , Regulação Bacteriana da Expressão Gênica , Camundongos , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Coelhos , Deleção de Sequência , Transcrição Gênica , VirulênciaRESUMO
The product of the human C21orf57 (huYBEY) gene is predicted to be a homologue of the highly conserved YbeY proteins found in nearly all bacteria. We show that, like its bacterial and chloroplast counterparts, the HuYbeY protein is an RNase and that it retains sufficient function in common with bacterial YbeY proteins to partially suppress numerous aspects of the complex phenotype of an Escherichia coli ΔybeY mutant. Expression of HuYbeY in Saccharomyces cerevisiae, which lacks a YbeY homologue, results in a severe growth phenotype. This observation suggests that the function of HuYbeY in human cells is likely regulated through specific interactions with partner proteins similarly to the way YbeY is regulated in bacteria.
Assuntos
Cloroplastos/química , Cloroplastos/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Evolução Molecular , Metaloproteínas/química , Metaloproteínas/genética , Ribonucleases/química , Ribonucleases/genética , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada/genética , Dados de Sequência MolecularRESUMO
Enterohemorrhagic Escherichia coli (EHEC) is a significant human pathogen and is the cause of bloody diarrhea and hemolytic-uremic syndrome. The virulence repertoire of EHEC includes the genes within the locus of enterocyte effacement (LEE) that are largely organized in five operons, LEE1 to LEE5, which encode a type III secretion system, several effectors, chaperones, and regulatory proteins. In addition, EHEC also encodes several non-LEE-encoded effectors and fimbrial operons. The virulence genes of this pathogen are under a large amount of posttranscriptional regulation. The small RNAs (sRNAs) GlmY and GlmZ activate the translation of glucosamine synthase (GlmS) in E. coli K-12, and in EHEC they destabilize the 3' fragments of the LEE4 and LEE5 operons and promote translation of the non-LEE-encoded effector EspFu. We investigated the global changes of EHEC gene expression governed by GlmY and GlmZ using RNA sequencing and gene arrays. This study extends the known effects of GlmY and GlmZ regulation to show that they promote expression of the curli adhesin, repress the expression of tryptophan metabolism genes, and promote the expression of acid resistance genes and the non-LEE-encoded effector NleA. In addition, seven novel EHEC-specific sRNAs were identified using RNA sequencing, and three of them--sRNA56, sRNA103, and sRNA350--were shown to regulate urease, fimbria, and the LEE, respectively. These findings expand the knowledge of posttranscriptional regulation in EHEC.
Assuntos
Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Fosfoproteínas/genética , Processamento Pós-Transcricional do RNA/genética , Pequeno RNA não Traduzido/metabolismo , Adesinas Bacterianas/biossíntese , Aderência Bacteriana/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos , Linhagem Celular Tumoral , Escherichia coli O157/genética , Escherichia coli O157/imunologia , Proteínas de Escherichia coli/biossíntese , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Células HeLa , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Análise em Microsséries , Fosfoproteínas/biossíntese , Pequeno RNA não Traduzido/genética , Triptofano/metabolismo , Urease/biossíntese , Virulência/genética , Fatores de Virulência/biossínteseRESUMO
The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 codes for two interacting DNA binding proteins, Cra and KdpE, that coregulate expression of the locus of enterocyte effacement (LEE) genes in a metabolite-dependent manner. Cra is a transcription factor that uses fluctuations in the concentration of carbon metabolism intermediates to positively regulate virulence of EHEC. KdpE is a response regulator that activates the transcription of homeostasis genes in response to salt-induced osmolarity and virulence genes in response to changes in metabolite concentrations. Here, we probed the transcriptional profiles of the Δcra, ΔkdpE, and Δcra ΔkdpE mutant strains and show that Cra and KdpE share several targets besides the LEE, but both Cra and KdpE also have independent targets. Several genes within O-islands (genomic islands present in EHEC but absent from E. coli K-12), such as Z0639, Z0640, Z3388, Z4267, and espFu (encoding an effector necessary for formation of attaching and effacing lesions on epithelial cells), were directly regulated by both Cra and KdpE, while Z2077 was only regulated by Cra. These studies identified and confirmed new direct targets for Cra and KdpE that included putative virulence factors as well as characterized virulence factors, such as EspFu and EspG. These results map out the role of the two interacting regulators, Cra and KdpE, in EHEC pathogenesis and global gene regulation.
Assuntos
Proteínas de Bactérias/genética , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Proteínas Repressoras/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Regulação para Baixo , Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Genéticos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Deleção de Sequência , Transativadores/genética , Transativadores/metabolismo , Transcriptoma , Regulação para Cima , Virulência , Fatores de Virulência/metabolismoRESUMO
ß-Lactam antibiotics disrupt the assembly of peptidoglycan (PG) within the bacterial cell wall by inhibiting the enzymatic activity of penicillin-binding proteins (PBPs). It was recently shown that ß-lactam treatment initializes a futile cycle of PG synthesis and degradation, highlighting major gaps in our understanding of the lethal effects of PBP inhibition by ß-lactam antibiotics. Here, we assess the downstream metabolic consequences of treatment of Escherichia coli with the ß-lactam mecillinam and show that lethality from PBP2 inhibition is a specific consequence of toxic metabolic shifts induced by energy demand from multiple catabolic and anabolic processes, including accelerated protein synthesis downstream of PG futile cycling. Resource allocation into these processes is coincident with alterations in ATP synthesis and utilization, as well as a broadly dysregulated cellular redox environment. These results indicate that the disruption of normal anabolic-catabolic homeostasis by PBP inhibition is an essential factor for ß-lactam antibiotic lethality.
Assuntos
Andinocilina/farmacologia , Antibacterianos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Andinocilina/química , Antibacterianos/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Homeostase/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/metabolismoRESUMO
Enterohemorrhagic Escherichia coli O157:H7 (EHEC) causes bloody diarrhea and hemolytic-uremic syndrome. EHEC encodes the sRNA chaperone Hfq, which is important in posttranscriptional regulation. In EHEC strain EDL933, Hfq acts as a negative regulator of the locus of enterocyte effacement (LEE), which encodes most of the proteins involved in type III secretion and attaching and effacing (AE) lesions. Here, we deleted hfq in the EHEC strain 86-24 and compared global transcription profiles of the hfq mutant and wild-type (WT) strains in exponential growth phase. Deletion of hfq affected transcription of genes common to nonpathogenic and pathogenic strains of E. coli as well as pathogen-specific genes. Downregulated genes in the hfq mutant included ler, the transcriptional activator of all the LEE genes, as well as genes encoded in the LEE2 to -5 operons. Decreased expression of the LEE genes in the hfq mutant occurred at middle, late, and stationary growth phases. We also confirmed decreased regulation of the LEE genes by examining the proteins secreted and AE lesion formation by the hfq mutant and WT strains. Deletion of hfq also caused decreased expression of the two-component system qseBC, which is involved in interkingdom signaling and virulence gene regulation in EHEC, as well as an increase in expression of stx(2AB), which encodes the deadly Shiga toxin. Altogether, these data indicate that Hfq plays a regulatory role in EHEC 86-24 that is different from what has been reported for EHEC strain EDL933 and that the role of Hfq in EHEC virulence regulation extends beyond the LEE.
Assuntos
Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/metabolismo , Fatores de Virulência/genética , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Fator Proteico 1 do Hospedeiro/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fatores de Virulência/metabolismoRESUMO
Various lethal stresses, including bactericidal antibiotics, can trigger the production of reactive oxygen species (ROS) that contribute to killing. Incomplete base excision repair (BER) of oxidized nucleotides, especially 8-oxo-dG, has been identified as a major component of ROS-induced lethality. However, the relative contributions of this pathway to death vary widely between stresses, due in part to poorly understood complex differences in the physiological changes caused by these stresses. To identify new lethal stresses that kill cells through this pathway, we screened an essential protein degradation library and found that depletion of either DapB or Dxr leads to cell death through incomplete BER; the contribution of this pathway to overall cell death is greater for DapB than for Dxr. Depletion of either protein generates oxidative stress, which increases incorporation of 8-oxo-dG into the genome. This oxidative stress is causally related to cell death, as plating on an antioxidant provided a protective effect. Moreover, incomplete BER was central to this cell death, as mutants lacking the key BER DNA glycosylases MutM and MutY were less susceptible, while overexpression of the nucleotide sanitizer MutT, which degrades 8-oxo-dGTP to prevent its incorporation, was protective. RNA sequencing of cells depleted of these proteins revealed widely different transcriptional responses to these stresses. Our discovery that oxidative stress-induced incomplete BER is highly dependent on the exact physiological changes that the cell experiences helps explain the past confusion that arose concerning the role of ROS in antibiotic lethality. IMPORTANCE Bacterial cell death is a poorly understood process. The generation of reactive oxygen species (ROS) is an apparently common response to challenges by a wide variety of lethal stresses, including bactericidal antibiotics. Incomplete BER of nucleotides damaged by these ROS, especially 8-oxo-dG, is a significant contributing factor to this lethality, but the levels of its contribution vary widely between different lethal stresses. A better understanding of the conditions that cause cells to die because of incomplete BER may lead to improved strategies for targeting this mode of death as an adjunct to antimicrobial therapy.
Assuntos
Reparo do DNA , Proteínas de Escherichia coli , Escherichia coli , Estresse Oxidativo , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , 8-Hidroxi-2'-Desoxiguanosina/farmacologia , Antibacterianos/farmacologia , Dano ao DNA , Reparo do DNA/genética , Reparo do DNA/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Nucleotídeos/metabolismo , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Pirofosfatases/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Numerous lethal stresses in bacteria including antibiotics, thymineless death, and MalE-LacZ expression trigger an increase in the production of reactive oxygen species. This results in the oxidation of the nucleotide pool by radicals produced by Fenton chemistry. Following the incorporation of these oxidized nucleotides into the genome, the cell's unsuccessful attempt to repair these lesions through base excision repair (BER) contributes causally to the lethality of these stresses. We review the evidence for this phenomenon of incomplete BER-mediated cell death and discuss how better understanding this pathway could contribute to the development of new antibiotics.
Assuntos
Antibacterianos/farmacologia , Morte Celular/efeitos dos fármacos , Dano ao DNA , Reparo do DNA , Espécies Reativas de Oxigênio/metabolismo , Animais , Antibacterianos/toxicidade , DNA/metabolismo , Eucariotos/efeitos dos fármacos , Eucariotos/genética , Eucariotos/metabolismo , Humanos , Estresse OxidativoRESUMO
UNLABELLED: Remodeling of the host cytoskeleton is a common strategy employed by bacterial pathogens. Although there is vigorous investigation of the cell biology underlying these bacterially mediated cytoskeleton modifications, knowledge of the plasticity and dynamics of the bacterial signaling networks that regulate the expression of genes necessary for these phenotypes is lacking. Enterohemorrhagic Escherichia coli attaches to enterocytes, forming pedestal-like structures. Pedestal formation requires the expression of the locus-of-enterocyte-effacement (LEE) and espFu genes. The LEE encodes a molecular syringe, a type III secretion system (T3SS) used by pathogens to translocate effectors such as EspFu into the host cell. By using a combination of genetic, biochemical, and cell biology approaches, we show that pedestal formation relies on posttranscriptional regulation by two small RNAs (sRNAs), GlmY and GlmZ. The GlmY and GlmZ sRNAs are unique; they have extensive secondary structures and work in concert. Although these sRNAs may offer unique insights into RNA and posttranscriptional biology, thus far, only one target and one mechanism of action (exposure of the ribosome binding site from the glmS gene to promote its translation) has been described. Here we uncovered new targets and two different molecular mechanisms of action of these sRNAs. In the case of EspFu expression, they promote translation by cleavage of the transcript, while in regard to the LEE, they promote destabilization of the mRNA. Our findings reveal that two unique sRNAs act in concert through different molecular mechanisms to coordinate bacterial attachment to mammalian cells. IMPORTANCE: Pathogens evolve by horizontal acquisition of pathogenicity islands. We describe here how two sRNAs, GlmY and GlmZ, involved in cellular metabolism and cellular architecture, through the posttranscriptional control of GlmS (the previously only known target of GlmY and GlmZ), which controls amino sugar synthesis, have been coopted to modulate the expression of virulence. These sRNAs quickly allow for plasticity in gene expression in order for enterohemorrhagic Escherichia coli to fine-tune the expression of its complex type III secretion machinery and its effectors to promote bacterial attachment and subsequent actin rearrangement on host cells. Pedestal formation is a very dynamic process. Many of the genes necessary for pedestal formation are located within the same operon to evolutionarily guarantee that they are inherited together. However, it is worth noting that within these operons, several genes need to yield more proteins than others and that these differences cannot be efficiently regulated at the transcriptional level.
Assuntos
Actinas/metabolismo , Aderência Bacteriana , Escherichia coli Êntero-Hemorrágica/genética , Regulação Bacteriana da Expressão Gênica , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Pequeno RNA não Traduzido/genética , Animais , Enterócitos/microbiologia , Escherichia coli Êntero-Hemorrágica/metabolismo , Humanos , Pequeno RNA não Traduzido/metabolismoRESUMO
UNLABELLED: Bacterial pathogens must be able to both recognize suitable niches within the host for colonization and successfully compete with commensal flora for nutrients in order to establish infection. Ethanolamine (EA) is a major component of mammalian and bacterial membranes and is used by pathogens as a carbon and/or nitrogen source in the gastrointestinal tract. The deadly human pathogen enterohemorrhagic Escherichia coli O157:H7 (EHEC) uses EA in the intestine as a nitrogen source as a competitive advantage for colonization over the microbial flora. Here we show that EA is not only important for nitrogen metabolism but that it is also used as a signaling molecule in cell-to-cell signaling to activate virulence gene expression in EHEC. EA in concentrations that cannot promote growth as a nitrogen source can activate expression of EHEC's repertoire of virulence genes. The EutR transcription factor, known to be the receptor of EA, is only partially responsible for this regulation, suggesting that yet another EA receptor exists. This important link of EA with metabolism, cell-to-cell signaling, and pathogenesis, highlights the fact that a fundamental means of communication within microbial communities relies on energy production and processing of metabolites. Here we show for the first time that bacterial pathogens not only exploit EA as a metabolite but also coopt EA as a signaling molecule to recognize the gastrointestinal environment and promote virulence expression. IMPORTANCE: In order to successfully cause disease, a pathogen must be able to sense a host environment and modulate expression of its virulence genes as well as compete with the indigenous microbiota for nutrients. Ethanolamine (EA) is present in the large intestine due to the turnover of intestinal cells. Here, we show that the human pathogen Escherichia coli O157:H7, which causes bloody diarrhea and hemolytic-uremic syndrome, regulates virulence gene expression through EA metabolism and by responding to EA as a signal. These findings provide the first information directly linking EA with bacterial pathogenesis.