RESUMEN
Metabolic pathways are now considered as intrinsic virulence attributes of pathogenic bacteria and thus represent potential targets for antibacterial strategies. Here we focused on the role of the pentose phosphate pathway (PPP) and its connections with other metabolic pathways in the pathophysiology of Francisella novicida. The involvement of the PPP in the intracellular life cycle of Francisella was first demonstrated by studying PPP inactivating mutants. Indeed, we observed that inactivation of the tktA, rpiA or rpe genes severely impaired intramacrophage multiplication during the first 24 hours. However, time-lapse video microscopy demonstrated that rpiA and rpe mutants were able to resume late intracellular multiplication. To better understand the links between PPP and other metabolic networks in the bacterium, we also performed an extensive proteo-metabolomic analysis of these mutants. We show that the PPP constitutes a major bacterial metabolic hub with multiple connections to glycolysis, the tricarboxylic acid cycle and other pathways, such as fatty acid degradation and sulfur metabolism. Altogether our study highlights how PPP plays a key role in the pathogenesis and growth of Francisella in its intracellular niche.
Asunto(s)
Proteínas Bacterianas/metabolismo , Drosophila melanogaster/metabolismo , Francisella/patogenicidad , Infecciones por Bacterias Gramnegativas/microbiología , Metaboloma , Vía de Pentosa Fosfato , Proteoma , Animales , Proteínas Bacterianas/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/microbiología , Francisella/metabolismo , Regulación Bacteriana de la Expresión Génica , Glucólisis , Macrófagos/metabolismo , Macrófagos/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , MutaciónRESUMEN
The bacterial pathogen Francisella tularensis possesses a noncanonical type VI secretion system (T6SS) that is required for phagosomal escape in infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the T6SS in culture. By differential proteomics, we found here that the amounts of the T6SS proteins remained unchanged upon KCl stimulation, suggesting involvement of post-translational modifications in T6SS assembly. A phosphoproteomic analysis indeed identified a unique phosphorylation site on IglB, a key component of the T6SS sheath. Substitutions of Y139 with alanine or phosphomimetics prevented T6SS formation and abolished phagosomal escape whereas substitution with phenylalanine delayed but did not abolish phagosomal escape in J774-1 macrophages. Altogether our data demonstrated that the Y139 site of IglB plays a critical role in T6SS biogenesis, suggesting that sheath phosphorylation could participate to T6SS dynamics.Data are available via ProteomeXchange with identifier PXD013619; and on MS-Viewer, key lkaqkllxwx.
Asunto(s)
Francisella tularensis/metabolismo , Sistemas de Secreción Tipo VI/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Procesamiento Automatizado de Datos , Francisella tularensis/genética , Francisella tularensis/ultraestructura , Cromatografía de Gases y Espectrometría de Masas , Humanos , Macrófagos/microbiología , Estructura Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Cloruro de Potasio/farmacología , Procesamiento Proteico-Postraduccional , Proteómica , Espectrometría de Masas en Tándem , Sistemas de Secreción Tipo VI/química , Sistemas de Secreción Tipo VI/efectos de los fármacos , Sistemas de Secreción Tipo VI/genéticaRESUMEN
During an infection, the detection of pathogens is mediated through the interactions between pathogen-associated molecular patterns (PAMPs) and pathogen recognition receptors. ß-Heptose 1,7-bisphosphate (ßHBP), an intermediate of the lipopolysaccharide (LPS) biosynthesis pathway, was recently identified as a bacterial PAMP. It was reported that ßHBP sensing leads to oligomerization of TIFA proteins, a mechanism controlling NF-κB activation and pro-inflammatory gene expression. Here, we compare the ability of chemically synthesized ßHBP and Shigella flexneri lysate to induce TIFA oligomerization in epithelial cells. We find that, unlike bacterial lysate, ßHBP fails to initiate rapid TIFA oligomerization. It only induces delayed signaling, suggesting that ßHBP must be processed intracellularly to trigger inflammation. Gene deletion and complementation analysis of the LPS biosynthesis pathway revealed that ADP-heptose is the bacterial metabolite responsible for rapid TIFA oligomerization. ADP-heptose sensing occurs down to 10-10 M. During S. flexneri infection, it results in cytokine production, a process dependent on the kinase ALPK1. Altogether, our results rule out a major role of ßHBP in S. flexneri infection and identify ADP-heptose as a new bacterial PAMP.
Asunto(s)
Adenosina Difosfato/metabolismo , Heptosas/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Shigella flexneri/metabolismo , Células HeLa , Heptosas/síntesis química , Heptosas/química , Humanos , Neisseria , Proteínas Quinasas/metabolismo , Multimerización de Proteína , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
Bacterial pathogens adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of the facultative intracellular pathogen, Francisella novicida. More than 900 Francisella proteins were identified in infected macrophages after a 10-h infection. Biotin biosynthesis-related proteins were upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, proteins encoded by the Francisella pathogenicity island (FPI) were downregulated, supporting the importance of the F. tularensis Type VI Secretion System for vacuole escape, not cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella's intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. Over-representation analysis highlighted cell-cell contact and actin polymerization in macrophage deregulated proteins. Using flow cytometry and live cell imaging, we demonstrated that merocytophagy involves diverse cell-to-cell contacts and actin polymerization-dependent processes. These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in future research.Data are available via ProteomeXchange with identifier PXD035145.
Asunto(s)
Francisella tularensis , Tularemia , Animales , Francisella tularensis/genética , Actinas/metabolismo , Biotina/metabolismo , Proteómica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Macrófagos/metabolismo , Estadios del Ciclo de Vida , Tularemia/microbiología , Islas GenómicasRESUMEN
Tryptophan (Trp) is an essential amino acid, whose metabolism is a key gatekeeper of intestinal homeostasis. Yet, its systemic effects, particularly on atherosclerosis, remain unknown. Here we show that high-fat diet (HFD) increases the activity of intestinal indoleamine 2, 3-dioxygenase 1 (IDO), which shifts Trp metabolism from the production of microbiota-derived indole metabolites towards kynurenine production. Under HFD, the specific deletion of IDO in intestinal epithelial cells leads to intestinal inflammation, impaired intestinal barrier, augmented lesional T lymphocytes and atherosclerosis. This is associated with an increase in serotonin production and a decrease in indole metabolites, thus hijacking Trp for the serotonin pathway. Inhibition of intestinal serotonin production or supplementation with indole derivatives alleviates plaque inflammation and atherosclerosis. In summary, we uncover a pivotal role of intestinal IDO in the fine-tuning of Trp metabolism with systemic effects on atherosclerosis, paving the way for new therapeutic strategies to relieve gut-associated inflammatory diseases.
Asunto(s)
Aterosclerosis , Dieta Alta en Grasa , Indolamina-Pirrol 2,3,-Dioxigenasa , Mucosa Intestinal , Ratones Endogámicos C57BL , Serotonina , Triptófano , Animales , Triptófano/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/genética , Aterosclerosis/tratamiento farmacológico , Dieta Alta en Grasa/efectos adversos , Ratones , Serotonina/metabolismo , Mucosa Intestinal/metabolismo , Quinurenina/metabolismo , Masculino , Microbioma Gastrointestinal , Indoles/farmacología , Inflamación/metabolismo , Ratones Noqueados , Intestinos/patología , Linfocitos T/metabolismo , Linfocitos T/inmunología , Modelos Animales de EnfermedadRESUMEN
Discovered at the beginning of the 20th century by Nobel laureate Élie Metchnikoff, probiotics have more recently emerged as a potential noninvasive therapeutic approach for the treatment of various chronic diseases. However, recent population-based clinical studies suggest that probiotics are often ineffective and may even exhibit potential deleterious effects. Hence, a deeper molecular understanding of strain-specific beneficial effects, together with the identification of endogenous/exogenous factors modulating probiotic efficacy, is needed. The lack of consistency in probiotic efficacy, together with the observation that numerous preclinical findings on probiotics are not translating once applied to humans through clinical trials, suggests a central role for environmental factors, such as dietary patterns, in probiotic efficacy. Two recent studies have been instrumental in filling this knowledge gap, defining the role played by diet in probiotic efficacy on metabolic deregulations in both mouse models and humans .
Asunto(s)
Dieta , Probióticos , Humanos , Animales , Ratones , Probióticos/administración & dosificación , Probióticos/efectos adversos , Microbiota , Enfermedades Metabólicas/terapia , Inflamación/terapia , Enfermedad Crónica/terapiaRESUMEN
Staphylococcus aureus is a predominant cause of chronic lung infections. While the airway environment is rich in highly sialylated mucins, the interaction of S. aureus with sialic acid is poorly characterized. Using S. aureus USA300 as well as clinical isolates, we demonstrate that quorum-sensing dysfunction, a hallmark of S. aureus adaptation, correlates with a greater ability to consume free sialic acid, providing a growth advantage in an air-liquid interface model and in vivo. Furthermore, RNA-seq experiment reveals that free sialic acid triggers transcriptional reprogramming promoting S. aureus chronic lifestyle. To support the clinical relevance of our results, we show the co-occurrence of S. aureus, sialidase-producing microbiota and free sialic acid in the airway of patients with cystic fibrosis. Our findings suggest a dual role for sialic acid in S. aureus airway infection, triggering virulence reprogramming and driving S. aureus adaptive strategies through the selection of quorum-sensing dysfunctional strains.
Asunto(s)
Infecciones Estafilocócicas , Staphylococcus aureus , Humanos , Percepción de Quorum/genética , Ácido N-Acetilneuramínico , Sistema Respiratorio , Proteínas BacterianasRESUMEN
Bacterial acute pneumonia is responsible for an extremely large burden of death worldwide and diagnosis is paramount in the management of patients. While multidrug-resistant bacteria is one of the biggest health threats in the coming decades, clinicians urgently need access to novel diagnostic technologies. In this review, we will first present the already existing and largely used techniques that allow identifying pathogen-associated pneumonia. Then, we will discuss the latest and most promising technological advances that are based on connected technologies (artificial intelligence-based and Omics-based) or rapid tests, to improve the management of lung infections caused by pathogenic bacteria. We also aim to highlight the mutual benefits of fundamental and clinical studies for a better understanding of lung infections and their more efficient diagnostic management.