RESUMO
Upon DNA damage, cells activate the DNA damage response (DDR) to coordinate proliferation and DNA repair. Dietary, metabolic, and environmental inputs are emerging as modulators of how DNA surveillance and repair take place. Lipids hold potential to convey these cues, although little is known about how. We observed that lipid droplet (LD) number specifically increased in response to DNA breaks. Using Saccharomyces cerevisiae and cultured human cells, we show that the selective storage of sterols into these LD concomitantly stabilizes phosphatidylinositol-4-phosphate (PI(4)P) at the Golgi, where it binds the DDR kinase ATM. In turn, this titration attenuates the initial nuclear ATM-driven response to DNA breaks, thus allowing processive repair. Furthermore, manipulating this loop impacts the kinetics of DNA damage signaling and repair in a predictable manner. Thus, our findings have major implications for tackling genetic instability pathologies through dietary and pharmacological interventions.
Assuntos
Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Esteróis/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Dano ao DNA , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
BACKGROUND: Both phospholipid synthesis and the detection of DNA damage are coupled to cell cycle progression, yet whether these two aspects crosstalk to each other remains unassessed. We postulate here that shortage of phospholipids, which negatively affects proliferation, may reduce the need for checkpoint activation in response to DNA damage. RESULTS: To test this hypothesis, we explore here the DNA Damage Response activation in response to seven different genotoxins, in three distinct cell types, and manipulate phospholipid synthesis both pharmacologically and genetically. This allows us to point at the DNA damage response kinase ATR as responsible for the coordination between phospholipid levels and DNA damage sensing. CONCLUSIONS AND SIGNIFICANCE: ATR could combine its ability to sense DNA damage and phospholipid profiles in order to finetune the response to DNA lesions depending on metabolic cues. Further, our analysis reveals the functional significance of this crosstalk to keep genome homeostasis.
Assuntos
Fosfolipídeos , Proteínas Quinases , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Dano ao DNA , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismoRESUMO
Through the past decade, MALDI-TOF MS has been recognized as a fast and robust tool for identification of most bacteria in clinical microbiology. However, the accuracy of this method to identify Neisseria species is still debated, and few data are available about commensal Neisseria species identification. In this study, we assessed two MALDI-TOF MS systems (Bruker Biotyper and Andromas) for the identification of 88, 18, and 29 isolates of Neisseria gonorrhoeae, Neisseria meningitidis, and commensal Neisseria species, respectively. All 88 isolates of N. gonorrhoeae were correctly identified using both systems, and most N. meningitidis and commensal Neisseria species were well identified: only 1/18 isolates of N. meningitidis was misidentified using Bruker Biotyper, and 1 isolate of Neisseria polysaccharea was misidentified as N. meningitidis using both systems. These results strengthen the possibility to use MALDI-TOF MS as a single method for Neisseria identification in routine, with excellent performance for N. gonorrhoeae identification. However, results should be interpreted prudently for N. meningitdis and commensal Neisseria species when isolated from genital and oropharyngeal samples where these both species can coexist.
Assuntos
Neisseria/classificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Bancos de Espécimes Biológicos , Humanos , Neisseria/isolamento & purificação , Neisseria gonorrhoeae/isolamento & purificação , Neisseria meningitidis/isolamento & purificação , SimbioseRESUMO
We report a multidrug-resistant Neisseria gonorrhoeae urogenital and pharyngeal infection with ceftriaxone resistance and intermediate resistance to azithromycin in a heterosexual woman in her 20s in France. Treatment with ceftriaxone plus doxycycline failed for the pharyngeal localisation. Whole-genome sequencing of isolate F90 identified MLST1903, NG-MAST ST3435, NG-STAR233, and relevant resistance determinants. F90 showed phenotypic and genotypic similarities to an internationally spreading multidrug-resistant and ceftriaxone-resistant clone detected in Japan and subsequently in Australia, Canada and Denmark.
Assuntos
Antibacterianos/uso terapêutico , Ceftriaxona/uso terapêutico , Doxiciclina/uso terapêutico , Gonorreia/tratamento farmacológico , Neisseria gonorrhoeae/isolamento & purificação , Adulto , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Gonorreia/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Técnicas de Amplificação de Ácido Nucleico , Falha de Tratamento , Sequenciamento Completo do GenomaRESUMO
Two extended-spectrum cephalosporin-resistant Neisseria gonorrhoeae isolates were discovered among 6,340 (0.03%) French isolates between 2010 and 2014. One isolate corresponded to the F89 multidrug-resistant N. gonorrhoeae isolate harboring a penA mosaic; whole-genome sequencing highlighted an additional R251H substitution in the ftsX gene recently involved in cephalosporin resistance. The other, ceftriaxone-resistant isolate (MIC, 0.25 mg/liter) harbored the PBP2 pattern XXXVI plus a P551S substitution and belonged to sequence type ST1579 (multilocus sequence typing [MLST]).
Assuntos
Ceftriaxona/farmacologia , Resistência às Cefalosporinas/genética , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Cefixima/farmacologia , Proteínas de Ciclo Celular/genética , França , Genoma Bacteriano , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Mutação , Neisseria gonorrhoeae/isolamento & purificaçãoAssuntos
Gonorreia/epidemiologia , Gonorreia/microbiologia , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/isolamento & purificação , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Comores/epidemiologia , DNA Bacteriano/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Neisseria gonorrhoeae/classificação , Neisseria gonorrhoeae/efeitos dos fármacos , Filogenia , Estudos Retrospectivos , Reunião/epidemiologiaRESUMO
The nuclear envelope (NE) encloses the genetic material and functions in chromatin organization and stability. In Saccharomyces cerevisiae, the NE is bound to the ribosomal DNA (rDNA), highly repeated and transcribed, thus prone to genetic instability. While tethering limits instability, it simultaneously triggers notable NE remodeling. We posit here that NE remodeling may contribute to genome integrity maintenance. The NE importance in genome expression, structure, and integrity is well recognized, yet studies mostly focus on peripheral proteins and nuclear pores, not on the membrane itself. We recently characterized a NE invagination drastically obliterating the rDNA, which we propose here as a model to probe if and how membranes play an active role in genome stability preservation.
Assuntos
Membrana Nuclear , Poro Nuclear , Humanos , Membrana Nuclear/genética , Poro Nuclear/genética , Poro Nuclear/metabolismo , Instabilidade Genômica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismoRESUMO
The nuclear membrane defines the boundaries that confine, protect and shape the genome. As such, its blebbing, ruptures and deformations are known to compromise the integrity of genetic material. Yet, drastic transitions of the nuclear membrane such as its invagination towards the nucleoplasm or its capacity to emit nuclear lipid droplets (nLD) have not been evaluated with respect to their impact on genome dynamics. To begin assessing this, in this work we used Saccharomyces cerevisiae as a model to ask whether a selection of genotoxins can trigger the formation of nLD. We report that nLD formation is not a general feature of all genotoxins, but of those engendering replication stress. Exacerbation of endogenous replication stress by genetic tools also elicited nLD formation. When exploring the lipid features of the nuclear membrane at the base of this emission, we revealed a link with the unsaturation profile of its phospholipids and, for the first time, of its sterol content. We propose that stressed replication forks may stimulate nLD birth by anchoring to the inner nuclear membrane, provided that the lipid context is adequate. Further, we point to a transcriptional feed-back process that counteracts the membrane's proneness to emit nLD. With nLD representing platforms onto which genome-modifying reactions can occur, our findings highlight them as important players in the response to replication stress.
Assuntos
Gotículas Lipídicas , Metabolismo dos Lipídeos , Núcleo Celular/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Mutagênicos , Fosfolipídeos/metabolismo , Saccharomyces cerevisiaeRESUMO
Membrane contact sites are functional nodes at which organelles reorganize metabolic pathways and adapt to changing cues. In Saccharomyces cerevisiae, the nuclear envelope subdomain surrounding the nucleolus, very plastic and prone to expansion, can establish contacts with the vacuole and be remodeled in response to various metabolic stresses. While using genotoxins with unrelated purposes, we serendipitously discovered a fully new remodeling event at this nuclear subdomain: the nuclear envelope partitions into its regular contact with the vacuole and a dramatic internalization within the nucleus. This leads to the nuclear engulfment of a globular, cytoplasmic portion. In spite of how we discovered it, the phenomenon is likely DNA damage-independent. We define lipids supporting negative curvature, such as phosphatidic acid and sterols, as bona fide drivers of this event. Mechanistically, we suggest that the engulfment of the cytoplasm triggers a suction phenomenon that enhances the docking of proton pump-containing vesicles with the vacuolar membrane, which we show matches a boost in autophagy. Thus, our findings unveil an unprecedented remodeling of the nucleolus-surrounding membranes with impact on metabolic adaptation.
Assuntos
Proteínas de Saccharomyces cerevisiae , Autofagia/fisiologia , Citoplasma/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismoRESUMO
Lipid Droplets (LD) are dynamic organelles that originate in the Endoplasmic Reticulum and mostly bud off toward the cytoplasm, where they store neutral lipids for energy and protection purposes. LD also have diverse proteins on their surface, many of which are necessary for the their correct homeostasis. However, these organelles also act as reservoirs of proteins that can be made available elsewhere in the cell. In this sense, they act as sinks that titrate key regulators of many cellular processes. Among the specialized factors that reside on cytoplasmic LD are proteins destined for functions in the nucleus, but little is known about them and their impact on nuclear processes. By screening for nuclear proteins in publicly available LD proteomes, we found that they contain a subset of nucleoporins from the Nuclear Pore Complex (NPC). Exploring this, we demonstrate that LD act as a physiological reservoir, for nucleoporins, that impacts the conformation of NPCs and hence their function in nucleo-cytoplasmic transport, chromatin configuration, and genome stability. Furthermore, our in silico modeling predicts a role for LD-released fatty acids in regulating the transit of nucleoporins from LD through the cytoplasm and to nuclear pores.
Assuntos
Ácidos Graxos/metabolismo , Gotículas Lipídicas/metabolismo , Poro Nuclear/metabolismo , HumanosRESUMO
BACKGROUND: Postsurgical infections due to Mycobacterium chimaera appeared as a novel nosocomial threat in 2015, with a worldwide outbreak due to contaminated heater-cooler units used in open chest surgery. We report the results of investigations conducted in France including whole-genome sequencing comparison of patient and heater-cooler unit isolates. METHODS: We sought M. chimaera infection cases from 2010 onwards through national epidemiological investigations in health care facilities performing cardiopulmonary bypass, together with a survey on good practices and systematic heater-cooler unit microbial analyses. Clinical and heater-cooler unit isolates were subjected to whole-genome sequencing analyzed with regard to the reference outbreak strain Zuerich-1. RESULTS: Only 2 clinical cases were shown to be related to the outbreak, although 23% (41/175) of heater-cooler units were declared positive for M. avium complex. Specific measures to prevent infection were applied in 89% (50/56) of health care facilities, although only 14% (8/56) of them followed the manufacturer maintenance recommendations. Whole-genome sequencing comparison showed that the clinical isolates and 72% (26/36) of heater-cooler unit isolates belonged to the epidemic cluster. Within clinical isolates, 5-9 nonsynonymous single nucleotide polymorphisms were observed, among which an in vivo mutation in a putative efflux pump gene was observed in a clinical isolate obtained for 1 patient on antimicrobial treatment. CONCLUSIONS: Cases of postsurgical M. chimaera infections have been declared to be rare in France, although heater-cooler units were contaminated, as in other countries. Genomic analyses confirmed the connection to the outbreak and identified specific single nucleotide polymorphisms, including 1 suggesting fitness evolution in vivo.
RESUMO
OBJECTIVES: To characterise the genotypes of multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB) isolated in Algeria, where there is a low MDR-MTB incidence rate. METHODS: Ten MDR isolates and one resistant to isoniazid were investigated by PCR-Sanger sequencing for 10 loci involved in resistance. Amplicon-based next generation sequencing (NGS) of 15 loci was additionally performed on isolates harbouring novel mutations. RESULTS: Sanger and amplicon-NGS provided the same results as with GenoType kits. Mutations known to be associated with resistance were described for most isolates: rpoB S531L in seven of 10 rifampicin-R MTB isolates, katG S315T in nine of 11 isoniazid-R, and promoter inhA c-15t in three of 11, embB M306V or M306I in two of two ethambutol-R, rpsL K43R in four of eight or rrs a514c associated with gidB L16R in streptomycin-R, gyrA A90V in the ofloxacin-R pre-XDR isolate. New and rare mutations were also described in rpoB (deletion 512-513-514), katG (S315R, M126I/ R496L), gidB (V124G, E92A, V139A, G37V), and gyrA (P8A). Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) profiles were similar for three isolates (lineage Cameroon), indicating a possible clonal diffusion in epidemiologically unrelated patients. CONCLUSIONS: Resistant MTB isolates in Algeria harbour resistance genotypes similar to other countries, but some rare patterns may result from selection and transmission processes inherent to the country.