Regulación de la síntesis de ácidos grasos en micobacterias

Entre las enfermedades infecciosas, la tuberculosis (TB) continúa siendo una de las principales causas de muerte entre los adultos. Algunos años atrás se pensó que esta enfermedad estaba controlada y que sería erradicada a mediano plazo. Sin embargo, hoy en día la TB está restablecida debido a diversos factores, entre ellas la aparición del SIDA. Mycobacterium tuberculosis, el agente etiológico de la tuberculosis, presenta una pared celular inusual, característica de todas las micobacterias. Esta envoltura celular resulta esencial para la viabilidad y supervivencia de las mismas en ambientes hostiles y consiste de una capa altamente impermeable de ácidos micólicos de 70-90 átomos de carbono unidos covalentemente al peptidoglicano (PG) a través de un polisacárido conector, el arabinogalactano (AG). Los ácidos micólicos son los componentes mayoritarios de la envoltura celular de las micobacterias y juegan un rol crucial en su compleja arquitectura y en su impermeabilidad. La biosíntesis de los ácidos micólicos requiere de dos tipos de sintasas de ácidos grasos (FAS): la enzima multifuncional FAS-I, similar a la presente en eucariotas, y el sistema dependiente de la proteína transportadora de acilos (ACP), FAS-II, el cual consiste de una serie de enzimas donde cada una cataliza un paso en la vía de elongación de acil-CoAs de cadena mediana C12-C16, previamente sintetizados por FAS-I. Los componentes genéticos del sistema FAS-II han sido identificados en M. tuberculosis y se encuentran agrupados en tres unidades transcripcionales principales: fabD-acpM-kasA-kasB-accD6 (operón fasII), mabA-inhA y hadA-hadB-hadC. Análisis de microarreglos demostraron que el tratamiento de M. tuberculosis con diversos antibióticos que afectan la síntesis de ácidos micólicos, como isoniacida (INH), etionamida (ETH) o tiolactomicina (TLM), inducen la transcripción de los genes de operón fasII. A su vez fas, el gen que codifica para la enzima multifuncional FAS-I, también se induce tras el tratamiento de M. tuberculosis con INH, sugiriendo la existencia de señales regulatorias comunes a los dos sistemas FAS. Esta información junto al concepto general sobre la existencia de sistemas reguladores que controlan la homeostasis lipídica en la mayoría de los organismos, llevó a que nuestro grupo de investigación se propusiera estudiar quién y cómo se regulan a nivel transcripcional los sistemas de síntesis de ácidos grasos en micobacterias. Es así que fuimos capaces de identificar y caracterizar una proteína reguladora del operón fasII, a quien llamamos MabR (por sus siglas en inglés, Mycolic acid biosynthesis Regulator). Los resultados de esta investigación representaron la primera caracterización de un regulador clave para el metabolismo de ácidos grasos en M. tuberculosis y sentaron las bases para el desarrollo del trabajo de tesis aquí presentado. Los estudios de microarreglos y proteómica antes detallados, junto con la evidencia de que la transcripción del gen fas se veía afectada cuando alterábamos los niveles fisiológicos de MabR, sugirieron la existencia de un mecanismo de regulación coordinado entre los dos sistemas FAS mediado por MabR. La identificación de una repetición invertida en la secuencia promotora del gen fas, similar a la reconocida por MabR en la región promotora del operón fasII, nos llevó a pensar que MabR podría estar regulando de manera directa la transcripción del mismo. Sin embargo, la incapacidad de evidenciar esta interacción in vitro nos condujo a la búsqueda de un nuevo regulador transcripcional del sistema FAS-I. Para alcanzar los objetivos propuestos, en el presente trabajo de tesis se caracterizó la región promotora del gen fas de M. tuberculosis y Mycobacterium smegmatis, comprobando que este gen forma parte de un operón al que denominamos operón fas-acpS. Se identificó y purificó una proteína reguladora de dicho operón, denominada FasR (por sus siglas en inglés, Fatty acid synthase Regulator), la cual fue caracterizada mediante diversos análisis bioquímicos y genéticos. Pudimos determinar que FasR se une a tres repeticiones de una secuencia operadora conservada, en la región promotora del operón fas-acpS. Estudios in vitro e in vivo demostraron que FasR es un activador transcripcional esencial en M. smegmatis, cuya afinidad por la región promotora del operón fas-acpS es modulada por acil-CoAs de cadena larga, productos del sistema FAS-I. La mayoría de los experimentos realizados en este trabajo de tesis utilizaron a M. smegmatis como sistema modelo, elección que responde a razones prácticas. En conclusión, los resultados obtenidos en el presente trabajo de tesis, junto con aquellos previamente publicados por nuestro grupo, sugieren que los dos sistemas FAS deben estar estrictamente co-regulados a nivel transcripcional para mantener la homeostasis lipídica en las micobacterias, y que la disrupción o alteración de dicha comunicación conduce a un microorganismo altamente comprometido en su viabilidad y/o capacidad infectiva.Fil: Mondino, Sonia Soledad. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Transcriptional regulation of fatty acid biosynthesis in mycobacteria

The main purpose of our study is to understand how mycobacteria exert control over the biosynthesis of their membrane lipids and find out the key components of the regulatory network that control fatty acid biosynthesis at the transcriptional level. In this article we describe the identification and purification of FasR, a transcriptional regulator from Mycobacterium sp. that controls the expression of the fatty acid synthase (fas) and the 4-phosphopantetheinyl transferase (acpS) encoding genes, whose products are involved in the fatty acid and mycolic acid biosynthesis pathways. In vitro studies demonstrated that fas and acpS genes are part of the same transcriptional unit and that FasR specifically binds to three conserved operator sequences present in the fas-acpS promoter region (Pfas). The construction and further characterization of a fasR conditional mutant confirmed that FasR is a transcriptional activator of the fas-acpS operon and that this protein is essential for mycobacteria viability. Furthermore, the combined used of Pfas–lacZ fusions in different fasR backgrounds and electrophoretic mobility shift assays experiments, strongly suggested that long-chain acyl-CoAs are the effector molecules that modulate the affinity of FasR for its DNA binding sequences and therefore the expression of the essential fas-acpS operon.Fil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Mondino, Sonia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Gago, Gabriela Marisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentin

FasR regulates fatty acid biosynthesis and is essential for virulence of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is the world’s leading cause of death from an infectious disease. One of the main features of this pathogen is the complex and dynamic lipid composition of the cell envelope, which adapts to the variable host environment and defines the fate of infection by actively interacting with and modulating immune responses. However, while much has been learned about the enzymes of the numerous lipid pathways, little knowledge is available regarding the proteins and metabolic signals regulating lipid metabolism during M. tuberculosis infection. In this work, we constructed and characterized a FasR-deficient mutant in M. tuberculosis and demonstrated that FasR positively regulates fas and acpS expression. Lipidomic analysis of the wild type and mutant strains revealed complete rearrangement of most lipid components of the cell envelope, with phospholipids, mycolic acids, sulfolipids, and phthiocerol dimycocerosates relative abundance severely altered. As a consequence, replication of the mutant strain was impaired in macrophages leading to reduced virulence in a mouse model of infection. Moreover, we show that the fasR mutant resides in acidified cellular compartments, suggesting that the lipid perturbation caused by the mutation prevented M. tuberculosis inhibition of phagolysosome maturation. This study identified FasR as a novel factor involved in regulation of mycobacterial virulence and provides evidence for the essential role that modulation of lipid homeostasis plays in the outcome of M. tuberculosis infection.Instituto de BiotecnologíaFil: Mondino, Sonia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario. Laboratory of Physiology and Genetics of Actinomycetes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vazquez, Cristina Lourdes. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cabruja, Matias. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario. Laboratory of Physiology and Genetics of Actinomycetes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sala, Claudia. Ecole Polytechnique Fédérale de Lausanne. Global Health Institute; SuizaFil: Cazenave-Gassiot, Amaury. National University of Singapore. Yong Loo Lin School of Medicine. Department of Biochemistry. Singapore Lipidomics Incubator; SingapurFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wenk, Markus R. National University of Singapore. Yong Loo Lin School of Medicine. Department of Biochemistry. Singapore Lipidomics Incubator; SingapurFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de investigaciones Científicas y Tecnológicas; ArgentinaFil: Cole, Stewart T. Ecole Polytechnique Fédérale de Lausanne. Global Health Institute; SuizaFil: Gramajo, Hugo. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario. Laboratory of Physiology and Genetics of Actinomycetes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gago, Gabriela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario. Laboratory of Physiology and Genetics of Actinomycetes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

More than 18,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells.

The genus Legionella comprises 65 species, among which Legionella pneumophila is a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80 Legionella genomes spanning 58 species. Uniquely, an immense repository of 18,000 secreted proteins encoding 137 different eukaryotic-like domains and over 200 eukaryotic-like proteins is paired with a highly conserved type IV secretion system (T4SS). Specifically, we show that eukaryotic Rho- and Rab-GTPase domains are found nearly exclusively in eukaryotes and Legionella Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F-box, U-box, and SET domains were present in >70% of all species, suggesting that manipulation of host signal transduction, protein turnover, and chromatin modification pathways are fundamental intracellular replication strategies for legionellae. In contrast, the Sec-7 domain was restricted to L. pneumophila and seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly, this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis, indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by interdomain gene transfer. Furthermore, we revealed the surprising extent to which legionellae have coopted genes and thus cellular functions from their eukaryotic hosts, providing an understanding of how dynamic reshuffling and gene acquisition have led to the emergence of major human pathogens

Molecular Mimicry: a Paradigm of Host-Microbe Coevolution Illustrated by Legionella

International audienceThrough coevolution with host cells, microorganisms have acquired mechanisms to avoid the detection by the host surveillance system and to use the cell's supplies to establish themselves. Indeed, certain pathogens have evolved proteins that imitate specific eukaryotic cell proteins, allowing them to manipulate host pathways, a phenomenon termed molecular mimicry. Bacterial "eukaryotic-like proteins" are a remarkable example of molecular mimicry. They are defined as proteins that strongly resemble eukaryotic proteins or that carry domains that are predominantly present in eukaryotes and that are generally absent from prokaryotes. The widest diversity of eukaryotic-like proteins known to date can be found in members of the bacterial genus Legionella, some of which cause a severe pneumonia in humans. The characterization of a number of these proteins shed light on their importance during infection. The subsequent identification of eukaryotic-like genes in the genomes of other amoeba-associated bacteria and bacterial symbionts suggested that eukaryotic-like proteins are a common means of bacterial evasion and communication, shaped by the continuous interactions between bacteria and their protozoan hosts. In this review, we discuss the concept of molecular mimicry using Legionella as an example and show that eukaryotic-like proteins effectively manipulate host cell pathways. The study of the function and evolution of such proteins is an exciting field of research that is leading us toward a better understanding of the complex world of bacterium-host interactions. Ultimately, this knowledge will teach us how host pathways are manipulated and how infections may possibly be tackled

3D cryo-EM imaging of bacterial flagella: Novel structural and mechanistic insights into cell motility

International audienceBacterial flagella are nanomachines that enable cells to move at high speeds. Comprising 25 and more different types of proteins, the flagellum is a large supramolecular assembly organized into three widely conserved substructures: a basal body including the rotary motor, a connecting hook, and a long filament. The whole flagellum from Escherichia coli weighs ∼20 MDa, without considering its filament portion, which is by itself a ∼1.6 GDa structure arranged as a multimer of ∼30,000 flagellin protomers. Breakthroughs regarding flagellar structure and function have been achieved in the last few years, mainly because of the revolutionary improvements in 3D cryo-EM methods. This review discusses novel structures and mechanistic insights derived from such high-resolution studies, advancing our understanding of each one of the three major flagellar segments. The rotation mechanism of the motor has been unveiled with unprecedented detail, showing a two-cogwheel machine propelled by a Brownian ratchet device. In addition, by imaging the flagellin-like protomers that make up the hook in its native bent configuration, their unexpected conformational plasticity challenges the paradigm of a two-state conformational rearrangement mechanism for flagellin-fold proteins. Finally, imaging of the filaments of periplasmic flagella, which endow Spirochete bacteria with their singular motility style, uncovered a strikingly asymmetric protein sheath that coats the flagellin core, challenging the view of filaments as simple homopolymeric structures that work as freely whirling whips. Further research will shed more light on the functional details of this amazing nanomachine, but our current understanding has definitely come a long way

Targeting of host organelles by pathogenic bacteria: a sophisticated subversion strategy.

International audienceMany bacterial pathogens have evolved the ability to subvert and exploit host functions in order to enter and replicate in eukaryotic cells. For example, bacteria have developed specific mechanisms to target eukaryotic organelles such as the nucleus, the mitochondria, the endoplasmic reticulum and the Golgi apparatus. In this Review, we highlight the most recent advances in our understanding of the mechanisms that bacterial pathogens use to target these organelles. We also discuss how these strategies allow bacteria to manipulate host functions and to ultimately enable bacterial infection

Legionnaires’ Disease: State of the Art Knowledge of Pathogenesis Mechanisms of Legionella

International audienceLegionella species are environmental gram-negative bacteria able to cause a severe form of pneumonia in humans known as Legionnaires' disease. Since the identification of Legionella pneumophila in 1977, four decades of research on Legionella biology and Legionnaires' disease have brought important insights into the biology of the bacteria and the molecular mechanisms that these intracellular pathogens use to cause disease in humans. Nowadays, Legionella species constitute a remarkable model of bacterial adaptation, with a genus genome shaped by their close coevolution with amoebae and an ability to exploit many hosts and signaling pathways through the secretion of a myriad of effector proteins, many of which have a eukaryotic origin. This review aims to discuss current knowledge of Legionella infection mechanisms and future research directions to be taken that might answer the many remaining open questions. This research will without a doubt be a terrific scientific journey worth taking

A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism

Unlike most bacteria, mycobacteria rely on the multi-domain enzyme eukaryote-like fatty acid synthase I (FAS I) to make fatty acids de novo. These metabolites are precursors of the biosynthesis of most of the lipids present both in the complex mycobacteria cell wall and in the storage lipids inside the cell. In order to study the role of the type I FAS system in Mycobacterium lipid metabolism in vivo, we constructed a conditional mutant in the fas-acpS operon of Mycobacterium smegmatis and analysed in detail the impact of reduced de novo fatty acid biosynthesis on the global architecture of the cell envelope. As expected, the mutant exhibited growth defect in the non-permissive condition that correlated well with the lower expression of fas-acpS and the concomitant reduction of FAS I, confirming that FAS I is essential for survival. The reduction observed in FAS I provoked an accumulation of its substrates, acetyl-CoA and malonyl-CoA, and a strong reduction of C12 to C18 acyl-CoAs, but not of long-chain acyl-CoAs (C19 to C24). The most intriguing result was the ability of the mutant to keep synthesizing mycolic acids when fatty acid biosynthesis was impaired. A detailed comparative lipidomic analysis showed that although reduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis, mycolic acids were still being synthesized in the mutant, although with a different relative species distribution. However, when triacylglycerol degradation was inhibited, mycolic acid biosynthesis was significantly reduced, suggesting that storage lipids could be an intracellular reservoir of fatty acids for the biosynthesis of complex lipids in mycobacteria. Understanding the interaction between FAS I and the metabolic pathways that rely on FAS I products is a key step to better understand how lipid homeostasis is regulated in this microorganism and how this regulation could play a role during infection in pathogenic mycobacteria.Fil: Cabruja, Matias Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Mondino, Sonia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Tsai, Yi Ting. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Lara, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Gago, Gabriela Marisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin