RESUMEN
Mycobacterium abscessus (Mabs) drives life-shortening mortality in cystic fibrosis (CF) patients, primarily because of its resistance to chemotherapeutic agents. To date, our knowledge on the host and bacterial determinants driving Mabs pathology in CF patient lung remains rudimentary. Here, we used human airway organoids (AOs) microinjected with smooth (S) or rough (R-)Mabs to evaluate bacteria fitness, host responses to infection, and new treatment efficacy. We show that S Mabs formed biofilm, and R Mabs formed cord serpentines and displayed a higher virulence. While Mabs infection triggers enhanced oxidative stress, pharmacological activation of antioxidant pathways resulted in better control of Mabs growth and reduced virulence. Genetic and pharmacological inhibition of the CFTR is associated with better growth and higher virulence of S and R Mabs. Finally, pharmacological activation of antioxidant pathways inhibited Mabs growth, at least in part through the quinone oxidoreductase NQO1, and improved efficacy in combination with cefoxitin, a first line antibiotic. In conclusion, we have established AOs as a suitable human system to decipher mechanisms of CF-driven respiratory infection by Mabs and propose boosting of the NRF2-NQO1 axis as a potential host-directed strategy to improve Mabs infection control.
Asunto(s)
Fibrosis Quística , Mycobacterium abscessus , Humanos , Fibrosis Quística/tratamiento farmacológico , Antioxidantes , Oxidación-Reducción , Estrés OxidativoRESUMEN
Mycobacterium abscessus is an emerging opportunistic pathogen responsible for chronic lung diseases, especially in patients with cystic fibrosis. Treatment failure of M. abscessus infections is primarily associated with intrinsic or acquired antibiotic resistance. However, there is growing evidence that antibiotic tolerance, i.e., the ability of bacteria to transiently survive exposure to bactericidal antibiotics through physiological adaptations, contributes to the relapse of chronic infections and the emergence of acquired drug resistance. Yet, our understanding of the molecular mechanisms that underlie antibiotic tolerance in M. abscessus remains limited. In the present work, a mutant with increased cross-tolerance to the first- and second-line antibiotics cefoxitin and moxifloxacin, respectively, has been isolated by experimental evolution. This mutant harbors a mutation in serB2, a gene involved in L-serine biosynthesis. Metabolic changes caused by this mutation alter the intracellular redox balance to a more reduced state that induces overexpression of the transcriptional regulator WhiB7 during the stationary phase, promoting tolerance through activation of a WhiB7-dependant adaptive stress response. These findings suggest that alteration of amino acid metabolism and, more generally, conditions that trigger whiB7 overexpression, makes M. abscessus more tolerant to antibiotic treatment.
RESUMEN
Respiratory infections remain a major global health concern. Tuberculosis is one of the top 10 causes of death worldwide, while infections with Non-Tuberculous Mycobacteria are rising globally. Recent advances in human tissue modeling offer a unique opportunity to grow different human "organs" in vitro, including the human airway, that faithfully recapitulates lung architecture and function. Here, we have explored the potential of human airway organoids (AOs) as a novel system in which to assess the very early steps of mycobacterial infection. We reveal that Mycobacterium tuberculosis (Mtb) and Mycobacterium abscessus (Mabs) mainly reside as extracellular bacteria and infect epithelial cells with very low efficiency. While the AO microenvironment was able to control, but not eliminate Mtb, Mabs thrives. We demonstrate that AOs responded to infection by modulating cytokine, antimicrobial peptide, and mucin gene expression. Given the importance of myeloid cells in mycobacterial infection, we co-cultured infected AOs with human monocyte-derived macrophages and found that these cells interact with the organoid epithelium. We conclude that adult stem cell (ASC)-derived AOs can be used to decipher very early events of mycobacteria infection in human settings thus offering new avenues for fundamental and therapeutic research.
Asunto(s)
Mycobacterium abscessus , Mycobacterium tuberculosis , Tuberculosis , Humanos , Macrófagos/microbiología , Micobacterias no Tuberculosas , Organoides , Tuberculosis/microbiologíaRESUMEN
Understanding the molecular strategies used by Mycobacterium tuberculosis to invade and persist within the host is of paramount importance to tackle the tuberculosis pandemic. Comparative genomic surveys have revealed that hadC, encoding a subunit of the HadBC dehydratase, is mutated in the avirulent M. tuberculosis H37Ra strain. We show here that mutation or deletion of hadC affects the biosynthesis of oxygenated mycolic acids, substantially reducing their production level. Additionally, it causes the loss of atypical extra-long mycolic acids, demonstrating the involvement of HadBC in the late elongation steps of mycolic acid biosynthesis. These events have an impact on the morphotype, cording capacity and biofilm growth of the bacilli as well as on their sensitivity to agents such as rifampicin. Furthermore, deletion of hadC leads to a dramatic loss of virulence: an almost 4-log drop of the bacterial load in the lungs and spleens of infected immunodeficient mice. Both its unique function and importance for M. tuberculosis virulence make HadBC an attractive therapeutic target for tuberculosis drug development.
Asunto(s)
Proteínas Bacterianas/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Ácidos Micólicos/química , Tuberculosis/microbiología , Animales , Antituberculosos/farmacología , Carga Bacteriana , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Eliminación de Gen , Pulmón/microbiología , Ratones , Mutación , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/enzimología , Ácidos Micólicos/metabolismo , Bazo/microbiología , Virulencia/genéticaRESUMEN
UNLABELLED: Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates. IMPORTANCE: Mycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many bacteria, adaptation to starvation relies partly on the stringent response. M. tuberculosis's unique outer membrane layer, the mycomembrane, is crucial for its viability and virulence. Despite its being the target of the major antituberculosis drugs, only scattered information exists on how the genes required for biosynthesis of the mycomembrane are expressed and regulated during starvation. This work has addressed this issue as a step toward the identification of new targets in the fight against M. tuberculosis.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/genética , Hidroliasas/genética , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/fisiología , Regulación hacia Abajo , Ácido Graso Sintasas/biosíntesis , Ácido Graso Sintasas/genética , Hidroliasas/biosíntesis , Mycobacterium tuberculosis/genética , Biosíntesis de Proteínas/genética , InaniciónRESUMEN
Escherichia coli can survive extreme acid stress for several hours. The most efficient acid resistance system is based on glutamate decarboxylation by the GadA and GadB decarboxylases and the import of glutamate via the GadC membrane protein. The expression of the corresponding genes is controlled by GadE, the central activator of glutamate-dependent acid resistance (GDAR). We have previously shown by genetic approaches that as well as GadE, the response regulator of the Rcs system, RcsB is absolutely required for control of gadA/BC transcription. In the presence of GadE, basal activity of RcsB stimulates the expression of gadA/BC, whereas activation of RcsB leads to general repression of the gad genes. We report here the results of various in vitro assays that show RcsB to regulate by direct binding to the gadA promoter region. Furthermore, activation of gadA transcription requires a GAD box and binding of an RcsB/GadE heterodimer. In addition, we have identified an RcsB box, which lies just upstream of the -10 element of gadA promoter and is involved in repression of this operon.
Asunto(s)
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Glutamato Descarboxilasa/genética , Proteínas de la Membrana/genética , Factores de Transcripción/metabolismo , Sitios de Unión , Dimerización , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biosíntesis , Glutamato Descarboxilasa/biosíntesis , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/biosíntesis , Mutación Puntual , Elementos Reguladores de la Transcripción , Estrés Fisiológico/genética , Transcripción GenéticaRESUMEN
Mycolic acids (MAs) have a strategic location within the mycobacterial envelope, deeply influencing its architecture and permeability, and play a determinant role in the pathogenicity of mycobacteria. The fatty acid synthase type II (FAS-II) multienzyme system is involved in their biosynthesis. A combination of pull-downs and proteomics analyses led to the discovery of a mycobacterial protein, HadD, displaying highly specific interactions with the dehydratase HadAB of FAS-II. In vitro activity assays and homology modeling showed that HadD is, like HadAB, a hot dog folded (R)-specific hydratase/dehydratase. A hadD knockout mutant of Mycobacterium smegmatis produced only the medium-size alpha'-MAs. Data strongly suggest that HadD is involved in building the third meromycolic segment during the late FAS-II elongation cycles, leading to the synthesis of the full-size alpha- and epoxy-MAs. The change in the envelope composition induced by hadD inactivation strongly altered the bacterial fitness and capacities to aggregate, assemble into colonies or biofilms and spread by sliding motility, and conferred a hypersensitivity to the firstline antimycobacterial drug rifampicin. This showed that the cell surface properties and the envelope integrity were greatly affected. With the alarmingly increasing case number of nontuberculous mycobacterial diseases, HadD appears as an attractive target for drug development.
Asunto(s)
Proteínas Bacterianas/metabolismo , Acido Graso Sintasa Tipo II/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium smegmatis/fisiología , Ácidos Micólicos/metabolismo , Proteínas Bacterianas/genética , Biopelículas , Vías Biosintéticas , Acido Graso Sintasa Tipo II/genética , Eliminación de Gen , Genes Esenciales , Humanos , Mycobacterium smegmatis/genéticaRESUMEN
Gram positive mycobacteria with a high GC content, such as the etiological agent of tuberculosis Mycobacterium tuberculosis, possess an outer membrane mainly composed of mycolic acids (MAs), the so-called mycomembrane, which is essential for the cell. About thirty genes are involved in the biosynthesis of MAs, which include the hadA, hadB and hadC genes that encode the dehydratases Fatty Acid Synthase type II (FAS-II) known to function as the heterodimers HadA-HadB and HadB-HadC. The present study shows that M. smegmatis cells remain viable in the absence of either HadA and HadC or both. Inactivation of HadC has a dramatic effect on the physiology and fitness of the mutant strains whereas that of HadA exacerbates the phenotype of a hadC deletion. The hadC mutants exhibit a novel MA profile, display a distinct colony morphology, are less aggregated, are impaired for sliding motility and biofilm development and are more resistant to detergent. Conversely, the hadC mutants are significantly more susceptible to low- and high-temperature and to selective toxic compounds, including several current anti-tubercular drugs.
Asunto(s)
Proteínas Bacterianas/metabolismo , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Mycobacterium smegmatis/fisiología , Ácidos Micólicos/metabolismo , Proteínas Bacterianas/genética , Supervivencia Celular , Pruebas de Sensibilidad Microbiana , Infecciones por Mycobacterium no Tuberculosas/microbiologíaRESUMEN
The RcsCDB signal transduction system is an atypical His-Asp phosphorelay. Notably, the response regulator RcsB can be activated either by phosphorylation through the RcsCD pathway or by an accessory cofactor RcsA. Although conserved in Enterobacteriaceae, the role of this system in adaptation to environmental stress conditions is largely unknown. This study reveals that the response regulator RcsB is essential to glutamate-dependent acid resistance, a condition pertinent to the lifestyle of Escherichia coli. The requirement for RcsB is independent of its activation by either the RcsCD or the RcsA pathway. The basal activity of RcsB appears to be necessary and sufficient for acid resistance. The sensitivity of the rcsB strain to low pH is correlated to a strong reduction of the expression of the glutamate decarboxylase genes, gadA and gadB, during the stationary phase of growth. This effect on gadA/B expression is not mediated by the general stress sigma factor RpoS, but does require a functional gadE allele and the previously identified GadE box. Therefore activation of gadAB expression and acid resistance absolutely requires both GadE and RcsB. In contrast, an increase in RcsB activity through the activation of the RcsCD phosphorelay or the RcsA pathway or through overproduction of the protein leads to general repression of the expression of the gad genes and a corresponding reduction in acid resistance.
Asunto(s)
Ácidos/farmacología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiología , Escherichia coli/efectos de los fármacos , Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Complejos Multienzimáticos/genética , Fosfoproteínas Fosfatasas/genética , Fosfotransferasas/genética , Proteínas Quinasas/genética , Factores de Transcripción/fisiología , Adaptación Fisiológica , Ácido Aspártico/metabolismo , Farmacorresistencia Bacteriana , Glutamato Descarboxilasa/genética , Histidina/metabolismo , Fosforilación , Factores de Transcripción/genéticaRESUMEN
The RcsCDB signal transduction system is an atypical His-Asp phosphorelay conserved in gamma-proteobacteria. Besides the three proteins directly involved in the phosphorelay, two proteins modulate the activity of the system. One is RcsA, which can stimulate the activity of the response regulator RcsB independently of the phosphorelay to regulate a subset of RcsB targets. The other is RcsF, a putative outer membrane lipoprotein mediating the signaling to the sensor RcsC. How RcsF transduces the signal to RcsC is unknown. Although the molecular and physiological signals remain to be identified, the common feature among the reported Rcs-activating conditions is perturbation of the envelope. As an initial step to explore the RcsF-RcsC functional relationship, we demonstrate that RcsF is an outer membrane lipoprotein oriented towards the periplasm. We also report that a null mutation in surA, a gene required for correct folding of periplasmic proteins, activates the Rcs pathway through RcsF. In contrast, activation of this pathway by overproduction of the membrane chaperone-like protein DjlA does not require RcsF. Conversely, activation of the pathway by RcsF overproduction does not require DjlA either, indicating the existence of two independent signaling pathways toward RcsC.
Asunto(s)
Cápsulas Bacterianas/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiología , Escherichia coli/metabolismo , Complejos Multienzimáticos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosfotransferasas/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Western Blotting , Proteínas Portadoras , Cisteína , Proteínas de Escherichia coli/genética , Datos de Secuencia Molecular , Isomerasa de Peptidilprolil , Periplasma/metabolismo , SerinaRESUMEN
Transcription of the Escherichia coli osmB gene is induced by several stress conditions. osmB is expressed from two promoters, osmBp1 and osmBp2. The downstream promoter, osmBp2, is induced after osmotic shock or upon entry into stationary phase in a sigma(S)-dependent manner. The upstream promoter, osmBp1, is independent of sigma(S) and is activated by RcsB, the response regulator of the His-Asp phosphorelay signal transduction system RcsCDB. RcsB is responsible for the induction of osmBp1 following treatment with chlorpromazine. Activation of osmBp1 by RcsB requires a sequence upstream of its -35 element similar to the RcsB binding site consensus, suggesting a direct regulatory role. osmB appears as another example of a multistress-responsive gene whose transcription involves both a sigma(S)-dependent promoter and a second one independent of sigma(S) but controlled by stress-specific transcription factors.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Lipoproteínas/genética , Proteínas Periplasmáticas/genética , Regiones Promotoras Genéticas , Factor sigma/metabolismo , Factores de Transcripción/metabolismo , Adaptación Fisiológica , Ácido Aspártico/metabolismo , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Secuencia de Bases , ADN Bacteriano , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/fisiología , Histidina/metabolismo , Lipoproteínas/biosíntesis , Datos de Secuencia Molecular , Proteínas Periplasmáticas/biosíntesis , Transducción de SeñalRESUMEN
The RcsCDB His-Asp phosphorelay is shown to positively regulate the bdm (biofilm-dependent modulation) and sra (stationary-phase-induced ribosome-associated protein) genes in Escherichia coli. The regulation is direct and requires an RcsB box next to the bdm -35 element. In addition, bdm is shown to be activated by osmotic shock in an Rcs-dependent way.
Asunto(s)
Biopelículas , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Complejos Multienzimáticos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Quinasas/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Ácido Aspártico/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Codón Iniciador , Proteínas de Escherichia coli/genética , Histamina/metabolismo , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Operón/fisiología , Fosfoproteínas Fosfatasas/genética , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Proteínas Quinasas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.
Asunto(s)
Proteínas Bacterianas/fisiología , Clorpromazina/farmacología , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Complejos Multienzimáticos/fisiología , Fosfoproteínas Fosfatasas/fisiología , Proteínas Quinasas/fisiología , Factores de Transcripción , Escherichia coli/crecimiento & desarrolloRESUMEN
Transcription of the Escherichia coli osmC gene is induced by several stress conditions. osmC is expressed from two overlapping promoters, osmCp1 and osmCp2. The proximal promoter, osmCp2, is transcribed at the entry into the stationary phase by the sigma(s) sigma factor. The distal promoter, osmCp1, is activated by NhaR and RcsB. NhaR is a positive regulator of the LysR family and is known to be an activator of the nhaA gene encoding an Na(+)/H(+) antiporter. RcsB is the response regulator of the RcsCDB His-Asp phosphorelay signal transduction system. Genetic data indicated that activation of osmCp1 by both NhaR and RcsB requires the same short sequences upstream of the -35 region of the promoter. Accordingly, DNase I footprint analysis indicated that both activators protect an overlapping region close to the -35 box of the promoter and suggested that the regulatory effect is direct. Despite the overlap of the binding sites, each activator acts independent of the other and is specific for a particular stress. NhaR can stimulate osmCp1 in response to an osmotic signal even in the absence of RcsB. RcsB is responsible for the induction of osmCp1 by alteration of the cell envelope, even in the absence of NhaR. osmCp1 as an example of multiple-stress-responsive promoter is discussed in light of a comparison of the NhaR and RcsB target regions in the Enterobacteriaceae.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Huella de ADN , Desoxirribonucleasas/metabolismo , Enterobacteriaceae/química , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Respuesta al Choque Térmico , Datos de Secuencia Molecular , Alineación de Secuencia , Transactivadores/metabolismoRESUMEN
The genes involved in flagellum synthesis, motility and chemotaxis in Escherichia coli are expressed in a hierarchical fashion. At the top of the hierarchy lies the master regulator FlhDC, required for the expression of the whole set of genes. The operon flhDC is controlled by numerous regulators including H-NS, CRP, EnvZ/OmpR, QseBC and LrhA. In the present work, we report that the flhDC operon is also negatively regulated by the His-Asp phosphorelay system RcsCDB. The regulation is potentiated by the RcsB cofactor RcsA. Genetic analysis indicates that an RcsAB box, located downstream of the promoter, is required for the regulation. The binding of RcsB and RcsA to this site was demonstrated by gel retardation and DNase I protection assays. In addition, mutation analysis suggests that RcsA-specific determinants lie in the right part of the 'RcsAB box'.