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1.
Immunity ; 55(2): 324-340.e8, 2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35139353

RESUMO

The aryl hydrocarbon receptor (AhR) is a sensor of products of tryptophan metabolism and a potent modulator of immunity. Here, we examined the impact of AhR in tumor-associated macrophage (TAM) function in pancreatic ductal adenocarcinoma (PDAC). TAMs exhibited high AhR activity and Ahr-deficient macrophages developed an inflammatory phenotype. Deletion of Ahr in myeloid cells or pharmacologic inhibition of AhR reduced PDAC growth, improved efficacy of immune checkpoint blockade, and increased intra-tumoral frequencies of IFNγ+CD8+ T cells. Macrophage tryptophan metabolism was not required for this effect. Rather, macrophage AhR activity was dependent on Lactobacillus metabolization of dietary tryptophan to indoles. Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα+IFNγ+CD8+ T cells; provision of dietary indoles blocked this effect. In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype, suggesting conservation of this regulatory axis in human disease.


Assuntos
Tolerância Imunológica/imunologia , Receptores de Hidrocarboneto Arílico/imunologia , Triptofano/imunologia , Macrófagos Associados a Tumor/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Humanos , Indóis/imunologia , Indóis/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Microbiota/imunologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Prognóstico , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Triptofano/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/metabolismo
2.
Infect Immun ; 88(10)2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32719152

RESUMO

Itaconate is a dicarboxylic acid that inhibits the isocitrate lyase enzyme of the bacterial glyoxylate shunt. Activated macrophages have been shown to produce itaconate, suggesting that these immune cells may employ this metabolite as a weapon against invading bacteria. Here, we demonstrate that in vitro, itaconate can exhibit bactericidal effects under acidic conditions similar to the pH of a macrophage phagosome. In parallel, successful pathogens, including Salmonella, have acquired a genetic operon encoding itaconate degradation proteins, which are induced heavily in macrophages. We characterized the regulation of this operon by the neighboring gene ripR in specific response to itaconate. Moreover, we developed an itaconate biosensor based on the operon promoter that can detect itaconate in a semiquantitative manner and, when combined with the ripR gene, is sufficient for itaconate-regulated expression in Escherichia coli Using this biosensor with fluorescence microscopy, we observed bacteria responding to itaconate in the phagosomes of macrophages and provide additional evidence that gamma interferon stimulates macrophage itaconate synthesis and that J774 mouse macrophages produce substantially more itaconate than the human THP-1 monocyte cell line. In summary, we examined the role of itaconate as an antibacterial metabolite in mouse and human macrophages, characterized the regulation of Salmonella's defense against it, and developed it as a convenient itaconate biosensor and inducible promoter system.


Assuntos
Ilhas Genômicas/genética , Salmonella typhimurium/metabolismo , Succinatos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Óperon , Fagossomos/metabolismo , Fagossomos/microbiologia , Regiões Promotoras Genéticas , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Succinatos/farmacologia , Fatores de Transcrição/genética
3.
Nat Commun ; 11(1): 1802, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286276

RESUMO

Inflammatory bowel disease patients have a greatly increased risk of developing colitis-associated colon cancer (CAC); however, the basis for inflammation-induced genetic damage requisite for neoplasia is unclear. Using three models of CAC, we find that sustained inflammation triggers 8-oxoguanine DNA lesions. Strikingly, antioxidants or iNOS inhibitors reduce 8-oxoguanine and polyps in CAC models. Because the mismatch repair (MMR) system repairs 8-oxoguanine and is frequently defective in colorectal cancer (CRC), we test whether 8-oxoguanine mediates oncogenesis in a Lynch syndrome (MMR-deficient) model. We show that microbiota generates an accumulation of 8-oxoguanine lesions in MMR-deficient colons. Accordingly, we find that 8-oxoguanine is elevated in neoplastic tissue of Lynch syndrome patients compared to matched untransformed tissue or non-Lynch syndrome neoplastic tissue. While antioxidants reduce 8-oxoguanine, they do not reduce CRC in Lynch syndrome models. Hence, microbe-induced oxidative/nitrosative DNA damage play causative roles in inflammatory CRC models, but not in Lynch syndrome models.


Assuntos
Colite/complicações , Colite/patologia , Neoplasias Colorretais/complicações , Neoplasias Colorretais/patologia , Dano ao DNA , Helicobacter pylori/fisiologia , Estresse Oxidativo , Polipose Adenomatosa do Colo/complicações , Polipose Adenomatosa do Colo/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antioxidantes/farmacologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Colite/induzido quimicamente , Colite/microbiologia , Colo/efeitos dos fármacos , Colo/patologia , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo do DNA/efeitos dos fármacos , Sulfato de Dextrana , Modelos Animais de Doenças , Disbiose/complicações , Disbiose/patologia , Escherichia coli/metabolismo , Feminino , Guanosina/análogos & derivados , Guanosina/metabolismo , Infecções por Helicobacter/complicações , Helicobacter pylori/efeitos dos fármacos , Humanos , Inflamação/complicações , Inflamação/patologia , Interleucina-10/deficiência , Interleucina-10/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mutação/genética , Estresse Oxidativo/efeitos dos fármacos
4.
Sci Immunol ; 4(42)2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31862865

RESUMO

Redundant mechanisms support immunoglobulin A (IgA) responses to intestinal antigens. These include multiple priming sites [mesenteric lymph nodes (MLNs), Peyer's patches, and isolated lymphoid follicles] and various cytokines that promote class switch to IgA, even in the absence of T cells. Despite these backup mechanisms, vaccination against enteric pathogens such as rotavirus has limited success in some populations. Genetic and environmental signals experienced during early life are known to influence mucosal immunity, yet the mechanisms for how these exposures operate remain unclear. Here, we used rotavirus infection to follow antigen-specific IgA responses through time and in different gut compartments. Using genetic and pharmacological approaches, we tested the role of the lymphotoxin (LT) pathway-known to support IgA responses-at different developmental stages. We found that LT-ß receptor (LTßR) signaling in early life programs intestinal IgA responses in adulthood by affecting antibody class switch recombination to IgA and subsequent generation of IgA antibody-secreting cells within an intact MLN. In addition, early-life LTßR signaling dictates the phenotype and function of MLN stromal cells to support IgA responses in the adult. Collectively, our studies uncover new mechanistic insights into how early-life LTßR signaling affects mucosal immune responses during adulthood.


Assuntos
Imunoglobulina A/imunologia , Linfonodos/imunologia , Receptor beta de Linfotoxina/imunologia , Linfotoxina-alfa/imunologia , Mesentério/imunologia , Células Estromais/imunologia , Animais , Fezes/microbiologia , Feminino , Imunidade nas Mucosas , Linfonodos/citologia , Receptor beta de Linfotoxina/genética , Linfotoxina-alfa/genética , Masculino , Mesentério/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout
5.
Anesth Analg ; 120(1): 60-65, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25185592

RESUMO

BACKGROUND: Many factors affect the accuracy of hemoglobin concentration values. In this study, we evaluated whether the hemoglobin concentration obtained by means of arterial blood gas (ABG) co-oximetry and complete blood count (CBC) central laboratory techniques clinically correlate when using simultaneous measurements of hemoglobin concentration obtained during complex spine fusion surgery. METHODS: Three hundred forty-eight patients who underwent spinal fusion of >3 bony levels between September 2006 and September 2010, with concurrent ABG and CBC samples, were identified. The mean difference between pairs of measured hemoglobin values was determined using limits of agreement analysis. Error grid analysis was used to delineate correlation of samples in relation to hemoglobin values within the range considered for transfusion. RESULTS: The median difference (ABG-CBC) between the measured hemoglobin values was 0.4 g/dL (95% confidence interval [CI], 0.35-0.40 g/dL; P < 0.0001). Limits of agreement analysis correcting for repeated observations in multiple patients demonstrated that the mean difference between measured hemoglobin values (i.e., bias) was 0.4 g/dL (95% CI, 0.36-0.41 g/dL), and the 95% limits of agreement of the difference between paired measurements were -0.70 to 1.47 g/dL. The magnitude of the difference between the measured hemoglobin values was >0.5 g/dL in 44.5% of patients (95% CI, 42.2%-46.8%); however, 6.8% (95% CI, 5.8%-8.1%) of paired measurements had a difference of >1.0 g/dL. There was only fair-to-moderate agreement between the CBC and ABG values within the clinically significant range of hemoglobin values of 7 to 10 g/dL (Cohen κ = 0.39; 95% CI, 0.33-0.45). CONCLUSIONS: The hemoglobin values obtained from ABG and CBC cannot be used interchangeably when verifying accuracy of novel point-of-care hemoglobin measurement modalities or when managing a patient with critical blood loss.


Assuntos
Hemoglobinometria/métodos , Monitorização Intraoperatória/métodos , Oximetria/métodos , Fusão Vertebral/métodos , Gasometria , Humanos , Laboratórios Hospitalares , Tamanho da Amostra
6.
J Biol Chem ; 289(41): 28160-71, 2014 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-25148683

RESUMO

Ribosome stalling during translation can be caused by a number of characterized mechanisms. However, the impact of elongation stalls on protein levels is variable, and the reasons for this are often unclear. To investigate this relationship, we examined the bacterial translation elongation factor P (EF-P), which plays a critical role in rescuing ribosomes stalled at specific amino acid sequences including polyproline motifs. In previous proteomic analyses of both Salmonella and Escherichia coli efp mutants, it was evident that not all proteins containing a polyproline motif were dependent on EF-P for efficient expression in vivo. The α- and ß-subunits of ATP synthase, AtpA and AtpD, are translated from the same mRNA transcript, and both contain a PPG motif; however, proteomic analysis revealed that AtpD levels are strongly dependent on EF-P, whereas AtpA levels are independent of EF-P. Using these model proteins, we systematically determined that EF-P dependence is strongly influenced by elements in the 5'-untranslated region of the mRNA. By mutating either the Shine-Dalgarno sequence or the start codon, we find that EF-P dependence correlates directly with the rate of translation initiation where strongly expressed proteins show the greatest dependence on EF-P. Our findings demonstrate that polyproline-induced stalls exert a net effect on protein levels only if they limit translation significantly more than initiation. This model can be generalized to explain why sequences that induce pauses in translation elongation to, for example, facilitate folding do not necessarily exact a penalty on the overall production of the protein.


Assuntos
Escherichia coli/genética , Elongação Traducional da Cadeia Peptídica/genética , Iniciação Traducional da Cadeia Peptídica/genética , Ribossomos/genética , Salmonella typhimurium/genética , Regiões 5' não Traduzidas , Sequência de Bases , Escherichia coli/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Modelos Genéticos , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Ribossomos/metabolismo , Salmonella typhimurium/metabolismo
7.
Cell ; 158(2): 288-299, 2014 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-25036629

RESUMO

The etiology of colorectal cancer (CRC) has been linked to deficiencies in mismatch repair and adenomatous polyposis coli (APC) proteins, diet, inflammatory processes, and gut microbiota. However, the mechanism through which the microbiota synergizes with these etiologic factors to promote CRC is not clear. We report that altering the microbiota composition reduces CRC in APC(Min/+)MSH2(-/-) mice, and that a diet reduced in carbohydrates phenocopies this effect. Gut microbes did not induce CRC in these mice through an inflammatory response or the production of DNA mutagens but rather by providing carbohydrate-derived metabolites such as butyrate that fuel hyperproliferation of MSH2(-/-) colon epithelial cells. Further, we provide evidence that the mismatch repair pathway has a role in regulating ß-catenin activity and modulating the differentiation of transit-amplifying cells in the colon. These data thereby provide an explanation for the interaction between microbiota, diet, and mismatch repair deficiency in CRC induction. PAPERCLIP:


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Carboidratos da Dieta/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Butiratos/metabolismo , Proliferação de Células , Transformação Celular Neoplásica , Pólipos do Colo/metabolismo , Pólipos do Colo/microbiologia , Pólipos do Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/microbiologia , Reparo de Erro de Pareamento de DNA , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Inflamação/genética , Inflamação/metabolismo , Inflamação/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/metabolismo , Organismos Livres de Patógenos Específicos , beta Catenina/metabolismo
8.
Nucleic Acids Res ; 42(5): 3261-71, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24335280

RESUMO

Elongation factor P (EF-P) is a conserved ribosome-binding protein that structurally mimics tRNA to enable the synthesis of peptides containing motifs that otherwise would induce translational stalling, including polyproline. In many bacteria, EF-P function requires post-translational modification with (R)-ß-lysine by the lysyl-tRNA synthetase paralog PoxA. To investigate how recognition of EF-P by PoxA evolved from tRNA recognition by aminoacyl-tRNA synthetases, we compared the roles of EF-P/PoxA polar contacts with analogous interactions in a closely related tRNA/synthetase complex. PoxA was found to recognize EF-P solely via identity elements in the acceptor loop, the domain of the protein that interacts with the ribosome peptidyl transferase center and mimics the 3'-acceptor stem of tRNA. Although the EF-P acceptor loop residues required for PoxA recognition are highly conserved, their conservation was found to be independent of the phylogenetic distribution of PoxA. This suggests EF-P first evolved tRNA mimicry to optimize interactions with the ribosome, with PoxA-catalyzed aminoacylation evolving later as a secondary mechanism to further improve ribosome binding and translation control.


Assuntos
Evolução Molecular , Lisina-tRNA Ligase/química , Mimetismo Molecular , Fatores de Alongamento de Peptídeos/química , Biossíntese de Proteínas , Aspartato-tRNA Ligase/química , Aspartato-tRNA Ligase/metabolismo , Sítios de Ligação , Domínio Catalítico , Modelos Moleculares , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Aminoacilação de RNA de Transferência
9.
mBio ; 4(2): e00180-13, 2013 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-23611909

RESUMO

UNLABELLED: Elongation factor P (EF-P) is a universally conserved bacterial translation factor homologous to eukaryotic/archaeal initiation factor 5A. In Salmonella, deletion of the efp gene results in pleiotropic phenotypes, including increased susceptibility to numerous cellular stressors. Only a limited number of proteins are affected by the loss of EF-P, and it has recently been determined that EF-P plays a critical role in rescuing ribosomes stalled at PPP and PPG peptide sequences. Here we present an unbiased in vivo investigation of the specific targets of EF-P by employing stable isotope labeling of amino acids in cell culture (SILAC) to compare the proteomes of wild-type and efp mutant Salmonella. We found that metabolic and motility genes are prominent among the subset of proteins with decreased production in the Δefp mutant. Furthermore, particular tripeptide motifs are statistically overrepresented among the proteins downregulated in efp mutant strains. These include both PPP and PPG but also additional motifs, such as APP and YIRYIR, which were confirmed to induce EF-P dependence by a translational fusion assay. Notably, we found that many proteins containing polyproline motifs are not misregulated in an EF-P-deficient background, suggesting that the factors that govern EF-P-mediated regulation are complex. Finally, we analyzed the specific region of the PoxB protein that is modulated by EF-P and found that mutation of any residue within a specific GSCGPG sequence eliminates the requirement for EF-P. This work expands the known repertoire of EF-P target motifs and implicates factors beyond polyproline motifs that are required for EF-P-mediated regulation. IMPORTANCE: Bacterial cells regulate gene expression at several points during and after transcription. During protein synthesis, for example, factors can interact with the ribosome to influence the production of specific proteins. Bacterial elongation factor P (EF-P) is a protein that facilitates the synthesis of proteins that contain polyproline motifs by preventing the ribosome from stalling. Bacterial cells that lack EF-P are viable but are sensitive to a large number of stress conditions. In this study, a global analysis of protein synthesis revealed that EF-P regulates many more proteins in the cell than predicted based solely on the prevalence of polyproline motifs. Several new EF-P-regulated motifs were uncovered, thereby providing a more complete picture of how this critical factor influences the cell's response to stress at the level of protein synthesis.


Assuntos
Motivos de Aminoácidos , Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Biossíntese de Proteínas , Salmonella enterica/metabolismo , Proteínas de Bactérias/análise , Escherichia coli/genética , Deleção de Genes , Marcação por Isótopo , Fatores de Alongamento de Peptídeos/genética , Proteoma/análise , Salmonella enterica/genética
10.
J Biol Chem ; 288(6): 4416-23, 2013 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-23277358

RESUMO

Post-translational modification of bacterial elongation factor P (EF-P) with (R)-ß-lysine at a conserved lysine residue activates the protein in vivo and increases puromycin reactivity of the ribosome in vitro. The additional hydroxylation of EF-P at the same lysine residue by the YfcM protein has also recently been described. The roles of modified and unmodified EF-P during different steps in translation, and how this correlates to its physiological role in the cell, have recently been linked to the synthesis of polyproline stretches in proteins. Polysome analysis indicated that EF-P functions in translation elongation, rather than initiation as proposed previously. This was further supported by the inability of EF-P to enhance the rate of formation of fMet-Lys or fMet-Phe, indicating that the role of EF-P is not to specifically stimulate formation of the first peptide bond. Investigation of hydroxyl-(ß)-lysyl-EF-P showed 30% increased puromycin reactivity but no differences in dipeptide synthesis rates when compared with the ß-lysylated form. Unlike disruption of the other genes required for EF-P modification, deletion of yfcM had no phenotypic consequences in Salmonella. Taken together, our findings indicate that EF-P functions in translation elongation, a role critically dependent on post-translational ß-lysylation but not hydroxylation.


Assuntos
Proteínas de Bactérias/metabolismo , Lisina/metabolismo , Elongação Traducional da Cadeia Peptídica/fisiologia , Fatores de Alongamento de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Salmonella enterica/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroxilação/fisiologia , Lisina/genética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Fatores de Alongamento de Peptídeos/genética , Salmonella enterica/genética
11.
Mol Microbiol ; 65(2): 477-93, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17630976

RESUMO

The two-component system SsrA-SsrB activates expression of a type III secretion system required for replication in macrophages and systemic infection in mice. Here we characterize the SsrB-dependent regulation of genes within Salmonella pathogenicity island 2 (SPI-2). Primer extension and DNase I footprinting identified multiple SsrB-regulated promoters within SPI-2 located upstream of ssaB, sseA, ssaG and ssaM. We previously demonstrated that ssrA and ssrB transcription is uncoupled. Overexpression of SsrB in the absence of its cognate kinase, SsrA, is sufficient to activate SPI-2 transcription. Because SsrB requires phosphorylation to relieve inhibitory contacts that occlude its DNA-binding domain, additional components must phosphorylate SsrB. SPI-2 promoters examined in single copy were highly SsrB-dependent, activated during growth in macrophages and induced by acidic pH. The nucleoid structuring protein H-NS represses horizontally acquired genes; we confirmed that H-NS is a negative regulator of SPI-2 gene expression. In the absence of H-NS, the requirement for SsrB in activating SPI-2 genes is substantially reduced, suggesting a role for SsrB in countering H-NS silencing. SsrB activates transcription of multiple operons within SPI-2 by binding to degenerate DNA targets at diversely organized promoters. SsrB appears to possess dual activities to promote SPI-2 gene expression: activation of transcription and relief of H-NS-mediated repression.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Ilhas Genômicas/genética , Salmonella typhimurium/patogenicidade , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Macrófagos/microbiologia , Proteínas de Membrana/genética , Camundongos , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Salmonella typhimurium/genética , Ativação Transcricional
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