RESUMEN
Alternative transcription start site (TSS) usage regulation has been identified as a major means of gene expression regulation in metazoans. However, in fungi, its impact remains elusive as its study has thus far been restricted to model yeasts. Here, we first re-analyzed TSS-seq data to define genuine TSS clusters in 2 species of pathogenic Cryptococcus. We identified 2 types of TSS clusters associated with specific DNA sequence motifs. Our analysis also revealed that alternative TSS usage regulation in response to environmental cues is widespread in Cryptococcus, altering gene expression and protein targeting. Importantly, we performed a forward genetic screen to identify a unique transcription factor (TF) named Tur1, which regulates alternative TSS (altTSS) usage genome-wide when cells switch from exponential phase to stationary phase. ChiP-Seq and DamID-Seq analyses suggest that at some loci, the role of Tur1 might be direct. Tur1 has been previously shown to be essential for virulence in C. neoformans. We demonstrated here that a tur1Δ mutant strain is more sensitive to superoxide stress and phagocytosed more efficiently by macrophages than the wild-type (WT) strain.
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Proteínas Fúngicas , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Factores de Transcripción , Sitio de Iniciación de la Transcripción , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Cryptococcus/genética , Cryptococcus/patogenicidad , Cryptococcus/metabolismo , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidad , Cryptococcus neoformans/metabolismo , Macrófagos/microbiología , Macrófagos/metabolismo , Animales , Ratones , Virulencia/genética , Fagocitosis/genéticaRESUMEN
Resistance to fluconazole (FLC), the most widely used antifungal drug, is typically achieved by altering the azole drug target and/or drug efflux pumps. Recent reports have suggested a link between vesicular trafficking and antifungal resistance. Here, we identified novel Cryptococcus neoformans regulators of extracellular vesicle (EV) biogenesis that impact FLC resistance. In particular, the transcription factor Hap2 does not affect the expression of the drug target or efflux pumps, yet it impacts the cellular sterol profile. Subinhibitory FLC concentrations also downregulate EV production. Moreover, in vitro spontaneous FLC-resistant colonies showed altered EV production, and the acquisition of FLC resistance was associated with decreased EV production in clinical isolates. Finally, the reversion of FLC resistance was associated with increased EV production. These data suggest a model in which fungal cells can regulate EV production in place of regulating the drug target gene expression as a first line of defense against antifungal assault in this fungal pathogen. IMPORTANCE Extracellular vesicles (EVs) are membrane-enveloped particles that are released by cells into the extracellular space. Fungal EVs can mediate community interactions and biofilm formation, but their functions remain poorly understood. Here, we report the identification of the first regulators of EV production in the major fungal pathogen Cryptococcus neoformans. Surprisingly, we uncover a novel role of EVs in modulating antifungal drug resistance. Disruption of EV production was associated with altered lipid composition and changes in fluconazole susceptibility. Spontaneous azole-resistant mutants were deficient in EV production, while loss of resistance restored initial EV production levels. These findings were recapitulated in C. neoformans clinical isolates, indicating that azole resistance and EV production are coregulated in diverse strains. Our study reveals a new mechanism of drug resistance in which cells adapt to azole stress by modulating EV production.
Asunto(s)
Criptococosis , Cryptococcus neoformans , Vesículas Extracelulares , Fluconazol/farmacología , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Criptococosis/microbiología , Azoles , Farmacorresistencia Fúngica/genética , Pruebas de Sensibilidad MicrobianaRESUMEN
Vibrio cholerae N16961 genome encodes 18 type II Toxin/Antitoxin (TA) systems, all but one located inside gene cassettes of its chromosomal superintegron (SI). This study aims to investigate additional TA systems in this genome. We screened for all two-genes operons of uncharacterized function by analyzing previous RNAseq data. Assays on nine candidates, revealed one additional functional type II TA encoded by the VCA0497-0498 operon, carried inside a SI cassette. We showed that VCA0498 antitoxin alone and in complex with VCA0497 represses its own operon promoter. VCA0497-0498 is the second element of the recently identified dhiT/dhiA superfamily uncharacterized type II TA system. RNAseq analysis revealed that another SI cassette encodes a novel type I TA system: VCA0495 gene and its two associated antisense non-coding RNAs, ncRNA495 and ncRNA496. Silencing of both antisense ncRNAs lead to cell death, demonstrating the type I TA function. Both VCA0497 and VCA0495 toxins do not show any homology to functionally characterized toxins, however our preliminary data suggest that their activity may end up in mRNA degradation, directly or indirectly. Our findings increase the TA systems number carried in this SI to 19, preferentially located in its distal end, confirming their importance in this large cassette array.
Asunto(s)
Antitoxinas , Toxinas Bacterianas , Vibrio cholerae , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Escherichia coli/metabolismo , Antitoxinas/genética , Antitoxinas/metabolismo , Regiones Promotoras GenéticasRESUMEN
Abundance and diversity of bacteria and their viral predators, bacteriophages (phages), in the digestive tract are associated with human health. Particularly intriguing is the long-term coexistence of these two antagonistic populations. We performed genome-wide RNA sequencing on a human enteroaggregative Escherichia coli isolate to identify genes differentially expressed between in vitro conditions and in murine intestines. We experimentally demonstrated that four of these differentially expressed genes modified the interactions between E. coli and three virulent phages by either increasing or decreasing its susceptibility/resistance pattern and also by interfering with biofilm formation. Therefore, the regulation of bacterial genes expression during the colonization of the digestive tract influences the coexistence of phages and bacteria, highlighting the intricacy of tripartite relationships between phages, bacteria, and the animal host in intestinal homeostasis.
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Bacteriófagos , Animales , Bacterias/genética , Bacteriófagos/fisiología , Escherichia coli/genética , Expresión Génica , Genes Bacterianos , RatonesRESUMEN
Entamoeba is a genus of Amoebozoa that includes the intestine-colonizing pathogenic species Entamoeba histolytica. To understand the basis of gene regulation in E. histolytica from an evolutionary perspective, we have profiled the transcriptomes of its closely related species E. dispar, E. moshkovskii and E. invadens. Genome-wide identification of transcription start sites (TSS) and polyadenylation sites (PAS) revealed the similarities and differences of their gene regulatory sequences. In particular, we found the widespread initiation of antisense transcription from within the gene coding sequences is a common feature among all Entamoeba species. Interestingly, we observed the enrichment of antisense transcription in genes involved in several processes that are common to species infecting the human intestine, e.g., the metabolism of phospholipids. These results suggest a potentially conserved and compact gene regulatory system in Entamoeba.
RESUMEN
Group 3 innate lymphoid cells (ILC3s) are innate immune effectors that contribute to host defense. Whether ILC3 functions are stably modified after pathogen encounter is unknown. Here, we assess the impact of a time-restricted enterobacterial challenge to long-term ILC3 activation in mice. We found that intestinal ILC3s persist for months in an activated state after exposure to Citrobacter rodentium. Upon rechallenge, these "trained" ILC3s proliferate, display enhanced interleukin-22 (IL-22) responses, and have a superior capacity to control infection compared with naïve ILC3s. Metabolic changes occur in C. rodentium-exposed ILC3s, but only trained ILC3s have an enhanced proliferative capacity that contributes to increased IL-22 production. Accordingly, a limited encounter with a pathogen can promote durable phenotypic and functional changes in intestinal ILC3s that contribute to long-term mucosal defense.
Asunto(s)
Citrobacter rodentium/inmunología , Infecciones por Enterobacteriaceae/inmunología , Inmunidad Mucosa , Mucosa Intestinal/inmunología , Activación de Linfocitos , Linfocitos/inmunología , Inmunidad Adaptativa , Animales , Proliferación Celular , Femenino , Inmunidad Innata , Memoria Inmunológica , Interleucinas/metabolismo , Intestinos/inmunología , Listeria monocytogenes , Listeriosis/inmunología , Linfocitos/metabolismo , Masculino , Redes y Vías Metabólicas , Ratones , Ratones Endogámicos C57BL , Consumo de Oxígeno , RNA-Seq , Reinfección/inmunología , Interleucina-22RESUMEN
Pathogenic Leptospira are the causative agents of leptospirosis, the most widespread zoonotic infectious disease. Leptospirosis is a potentially severe and life-threatening emerging disease with highest burden in sub-tropical areas and impoverished populations. Mechanisms allowing pathogenic Leptospira to survive inside a host and induce acute leptospirosis are not fully understood. The ability to resist deadly oxidants produced by the host during infection is pivotal for Leptospira virulence. We have previously shown that genes encoding defenses against oxidants in L. interrogans are repressed by PerRA (encoded by LIMLP_10155), a peroxide stress regulator of the Fur family. In this study, we describe the identification and characterization of another putative PerR-like regulator (LIMLP_05620) in L. interrogans. Protein sequence and phylogenetic analyses indicated that LIMLP_05620 displayed all the canonical PerR amino acid residues and is restricted to pathogenic Leptospira clades. We therefore named this PerR-like regulator PerRB. In L. interrogans, the PerRB regulon is distinct from that of PerRA. While a perRA mutant had a greater tolerance to peroxide, inactivating perRB led to a higher tolerance to superoxide, suggesting that these two regulators have a distinct function in the adaptation of L. interrogans to oxidative stress. The concomitant inactivation of perRA and perRB resulted in a higher tolerance to both peroxide and superoxide and, unlike the single mutants, a double perRAperRB mutant was avirulent. Interestingly, this correlated with major changes in gene and non-coding RNA expression. Notably, several virulence-associated genes (clpB, ligA/B, and lvrAB) were repressed. By obtaining a double mutant in a pathogenic Leptospira strain, our study has uncovered an interplay of two PerRs in the adaptation of Leptospira to oxidative stress with a putative role in virulence and pathogenicity, most likely through the transcriptional control of a complex regulatory network.
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Proteínas Bacterianas/metabolismo , Redes Reguladoras de Genes/genética , Leptospira/genética , Leptospirosis/microbiología , Adaptación Fisiológica , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Leptospira/patogenicidad , Leptospira/fisiología , Modelos Moleculares , Mutación , Estrés Oxidativo , Filogenia , Regulón/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Alineación de Secuencia , VirulenciaRESUMEN
Indole is a molecule proposed to be involved in bacterial signaling. We find that indole secretion is induced by sublethal tobramycin concentrations and increases persistence to aminoglycosides in V. cholerae. Indole transcriptomics showed increased expression of raiA, a ribosome associated factor. Deletion of raiA abolishes the appearance of indole dependent persisters to aminoglycosides, although its overexpression leads to 100-fold increase of persisters, and a reduction in lag phase, evocative of increased active 70S ribosome content, confirmed by sucrose gradient analysis. We propose that, under stress conditions, RaiA-bound inactive 70S ribosomes are stored as "sleeping ribosomes", and are rapidly reactivated upon stress relief. Our results point to an active process of persister formation through ribosome protection during translational stress (e.g., aminoglycoside treatment) and reactivation upon antibiotic removal. Translation is a universal process, and these results could help elucidate a mechanism of persistence formation in a controlled, thus inducible way.
RESUMEN
Spirochetes can be distinguished from other bacteria by their spiral-shaped morphology and subpolar periplasmic flagella. This study focused on FlhF and FlhG, which control the spatial and numerical regulation of flagella in many exoflagellated bacteria, in the spirochete Leptospira. In contrast to flhF which seems to be essential in Leptospira, we demonstrated that flhG- mutants in both the saprophyte L. biflexa and the pathogen L. interrogans were less motile than the wild-type strains in gel-like environments but not hyperflagellated as reported previously in other bacteria. Cryo-electron tomography revealed that the distance between the flagellar basal body and the tip of the cell decreased significantly in the flhG- mutant in comparison to wild-type and complemented strains. Additionally, comparative transcriptome analyses of L. biflexa flhG- and wild-type strains showed that FlhG acts as a negative regulator of transcription of some flagellar genes. We found that the L. interrogans flhG- mutant was attenuated for virulence in the hamster model. Cross-species complementation also showed that flhG is not interchangeable between species. Our results indicate that FlhF and FlhG in Leptospira contribute to governing cell motility but our data support the hypothesis that FlhF and FlhG function differently in each bacterial species, including among spirochetes.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Flagelos/genética , Flagelos/metabolismo , Leptospira/genética , Leptospira/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Microscopía por Crioelectrón , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Humanos , Leptospira/citología , Leptospirosis/microbiología , Mutación , Spirochaetales/genética , Spirochaetales/metabolismo , VirulenciaRESUMEN
Acid mine drainages (AMDs), metal-rich acidic effluents generated by mining activities, are colonized by prokaryotic and eukaryotic microorganisms widely distributed among different phyla. We compared metatranscriptomic data from two sampling stations in the Carnoulès AMD and from a third station in the nearby Amous River, focussing on processes involved in primary production and litter decomposition. A synergistic relationship between the green and brown food webs was favoured in the AMD sediments by the low carbon content and the availability of mineral nutrients: primary production of organic matter would benefit C-limited decomposers whose activity of organic matter mineralization would in turn profit primary producers. This balance could be locally disturbed by heterogeneous factors such as an input of plant debris from the riparian vegetation, strongly boosting the growth of Tremellales which would then outcompete primary producers. In the unpolluted Amous River on the contrary, the competition for limited mineral nutrients was dominated by the green food web, fish and bacterivorous protists having a positive effect on phytoplankton. These results suggest that in addition to direct effects of low pH and metal contamination, trophic conditions like carbon or mineral nutrient limitations also have a strong impact on assembly and activities of AMDs' microbial communities.
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Cadena Alimentaria , Fitoplancton , Animales , Eucariontes , Células Procariotas , RíosRESUMEN
Queuosine is a naturally occurring modified ribonucleoside found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His, and Tyr. Eukaryotes lack pathways to synthesize queuine, the nucleobase precursor to queuosine, and must obtain it from diet or gut microbiota. Here, we describe the effects of queuine on the physiology of the eukaryotic parasite Entamoeba histolytica, the causative agent of amebic dysentery. Queuine is efficiently incorporated into E. histolytica tRNAs by a tRNA-guanine transglycosylase (EhTGT) and this incorporation stimulates the methylation of C38 in [Formula: see text] Queuine protects the parasite against oxidative stress (OS) and antagonizes the negative effect that oxidation has on translation by inducing the expression of genes involved in the OS response, such as heat shock protein 70 (Hsp70), antioxidant enzymes, and enzymes involved in DNA repair. On the other hand, queuine impairs E. histolytica virulence by downregulating the expression of genes previously associated with virulence, including cysteine proteases, cytoskeletal proteins, and small GTPases. Silencing of EhTGT prevents incorporation of queuine into tRNAs and strongly impairs methylation of C38 in [Formula: see text], parasite growth, resistance to OS, and cytopathic activity. Overall, our data reveal that queuine plays a dual role in promoting OS resistance and reducing parasite virulence.IMPORTANCEEntamoeba histolytica is a unicellular parasite that causes amebiasis. The parasite resides in the colon and feeds on the colonic microbiota. The gut flora is implicated in the onset of symptomatic amebiasis due to alterations in the composition of bacteria. These bacteria modulate the physiology of the parasite and affect the virulence of the parasite through unknown mechanisms. Queuine, a modified nucleobase of queuosine, is exclusively produced by the gut bacteria and leads to tRNA modification at the anticodon loops of specific tRNAs. We found that queuine induces mild oxidative stress resistance in the parasite and attenuates its virulence. Our study highlights the importance of bacterially derived products in shaping the physiology of the parasite. The fact that queuine inhibits the virulence of E. histolytica may lead to new strategies for preventing and/or treating amebiasis by providing to the host queuine directly or via probiotics.
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Entamoeba histolytica/efectos de los fármacos , Entamoeba histolytica/patogenicidad , Guanina/análogos & derivados , Estrés Oxidativo/efectos de los fármacos , Animales , Entamoeba histolytica/genética , Femenino , Guanina/metabolismo , Guanina/farmacología , Células HeLa , Humanos , Metilación , Ratones , Ratones Endogámicos BALB C , ARN de Transferencia/metabolismoRESUMEN
In bacteria, DNA methylation can be facilitated by 'orphan' DNA methyltransferases lacking cognate restriction endonucleases, but whether and how these enzymes control key cellular processes are poorly understood. The effects of a specific modification, 4-methylcytosine (4mC), are even less clear, as this epigenetic marker is unique to bacteria and archaea, whereas the bulk of epigenetic research is currently performed on eukaryotes. Here, we characterize a 4mC methyltransferase from the understudied pathogen Leptospira spp. Inactivating this enzyme resulted in complete abrogation of CTAG motif methylation, leading to genome-wide dysregulation of gene expression. Mutants exhibited growth defects, decreased adhesion to host cells, higher susceptibility to LPS-targeting antibiotics, and, importantly, were no longer virulent in an acute infection model. Further investigation resulted in the discovery of at least one gene, that of an ECF sigma factor, whose transcription was altered in the methylase mutant and, subsequently, by mutation of the CTAG motifs in the promoter of the gene. The genes that comprise the regulon of this sigma factor were, accordingly, dysregulated in the methylase mutant and in a strain overexpressing the sigma factor. Our results highlight the importance of 4mC in Leptospira physiology, and suggest the same of other understudied species.
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Proteínas Bacterianas/genética , Citosina/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Bacteriano/metabolismo , Epigénesis Genética , Genoma Bacteriano , Leptospira interrogans/genética , Animales , Proteínas Bacterianas/metabolismo , Citosina/análogos & derivados , ADN (Citosina-5-)-Metiltransferasas/deficiencia , Metilación de ADN , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Leptospira interrogans/metabolismo , Leptospira interrogans/patogenicidad , Leptospirosis/microbiología , Leptospirosis/mortalidad , Leptospirosis/patología , Mesocricetus , Regiones Promotoras Genéticas , Factor sigma/genética , Factor sigma/metabolismo , Análisis de Supervivencia , Transcripción Genética , VirulenciaRESUMEN
Natural antisense transcripts (NAT) have been reported in prokaryotes and eukaryotes. While the functions of most reported NATs remain unknown, their potentials in regulating the transcription of their counterparts have been speculated. Entamoeba histolytica, which is a unicellular eukaryotic parasite, has a compact protein-coding genome with very short intronic and intergenic regions. The regulatory mechanisms of gene expression in this compact genome are under-described. In this study, by genome-wide mapping of RNA-Seq data in the genome of E. histolytica, we show that a substantial fraction of its protein-coding genes (28%) has significant transcription on their opposite strand (i.e. NAT). Intriguingly, we found the location of transcription start sites or polyadenylation sites of NAT are determined by the specific motifs encoded on the opposite strand of the gene coding sequences, thereby providing a compact regulatory system for gene transcription. Moreover, we demonstrated that NATs are globally up-regulated under various environmental conditions including temperature stress and pathogenicity. While NATs do not appear to be consequences of spurious transcription, they may play a role in regulating gene expression in E. histolytica, a hypothesis which needs to be tested.
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Entamoeba histolytica/genética , ARN sin Sentido/genética , Transcripción Genética , Entamoeba histolytica/metabolismo , Perfilación de la Expresión Génica , ARN sin Sentido/metabolismoRESUMEN
Pathogenic Leptospira spp. are the causative agents of the waterborne zoonotic disease leptospirosis. Leptospira are challenged by numerous adverse conditions, including deadly reactive oxygen species (ROS), when infecting their hosts. Withstanding ROS produced by the host innate immunity is an important strategy evolved by pathogenic Leptospira for persisting in and colonizing hosts. In L. interrogans, genes encoding defenses against ROS are repressed by the peroxide stress regulator, PerR. In this study, RNA sequencing was performed to characterize both the L. interrogans response to low and high concentrations of hydrogen peroxide and the PerR regulon. We showed that Leptospira solicit three main peroxidase machineries (catalase, cytochrome C peroxidase and peroxiredoxin) and heme to detoxify oxidants produced during peroxide stress. In addition, canonical molecular chaperones of the heat shock response and DNA repair proteins from the SOS response were required for Leptospira recovering from oxidative damage. Identification of the PerR regulon upon exposure to H2O2 allowed to define the contribution of this regulator in the oxidative stress response. This study has revealed a PerR-independent regulatory network involving other transcriptional regulators, two-component systems and sigma factors as well as non-coding RNAs that putatively orchestrate, in concert with PerR, the oxidative stress response. We have shown that PerR-regulated genes encoding a TonB-dependent transporter and a two-component system (VicKR) are involved in Leptospira tolerance to superoxide. This could represent the first defense mechanism against superoxide in L. interrogans, a bacterium lacking canonical superoxide dismutase. Our findings provide an insight into the mechanisms required by pathogenic Leptospira to overcome oxidative damage during infection-related conditions. This will participate in framing future hypothesis-driven studies to identify and decipher novel virulence mechanisms in this life-threatening pathogen.
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Peróxido de Hidrógeno/farmacología , Leptospira/patogenicidad , Estrés Oxidativo/efectos de los fármacos , Peróxidos/metabolismo , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/genética , Hierro/metabolismo , Leptospira/efectos de los fármacos , Leptospira interrogans/efectos de los fármacos , Leptospira interrogans/genética , Leptospirosis/genética , Chaperonas Moleculares/metabolismo , Estrés Oxidativo/fisiología , Virulencia/efectos de los fármacos , Virulencia/fisiologíaRESUMEN
Thiomonas bacteria are ubiquitous at acid mine drainage sites and play key roles in the remediation of water at these locations by oxidizing arsenite to arsenate, favouring the sorption of arsenic by iron oxides and their coprecipitation. Understanding the adaptive capacities of these bacteria is crucial to revealing how they persist and remain active in such extreme conditions. Interestingly, it was previously observed that after exposure to arsenite, when grown in a biofilm, some strains of Thiomonas bacteria develop variants that are more resistant to arsenic. Here, we identified the mechanisms involved in the emergence of such variants in biofilms. We found that the percentage of variants generated increased in the presence of high concentrations of arsenite (5.33 mM), especially in the detached cells after growth under biofilm-forming conditions. Analysis of gene expression in the parent strain CB2 revealed that genes involved in DNA repair were upregulated in the conditions where variants were observed. Finally, we assessed the phenotypes and genomes of the subsequent variants generated to evaluate the number of mutations compared to the parent strain. We determined that multiple point mutations accumulated after exposure to arsenite when cells were grown under biofilm conditions. Some of these mutations were found in what is referred to as ICE19, a genomic island (GI) carrying arsenic-resistance genes, also harbouring characteristics of an integrative and conjugative element (ICE). The mutations likely favoured the excision and duplication of this GI. This research aids in understanding how Thiomonas bacteria adapt to highly toxic environments, and, more generally, provides a window to bacterial genome evolution in extreme environments.
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Arsenitos/metabolismo , Biopelículas/crecimiento & desarrollo , Burkholderiales , Genoma Bacteriano/genética , Adaptación Fisiológica/genética , Arseniatos/metabolismo , Arsénico/metabolismo , Burkholderiales/genética , Burkholderiales/crecimiento & desarrollo , Burkholderiales/metabolismo , Reparación del ADN/genética , Elementos Transponibles de ADN/genética , Evolución Molecular , Perfilación de la Expresión Génica , Variación Genética/genética , Islas Genómicas/genética , Minería , Secuenciación Completa del GenomaRESUMEN
Leishmania parasites are the causative agents of human leishmaniases. They infect professional phagocytes of their mammalian hosts, including dendritic cells (DCs) that are essential for the initiation of adaptive immune responses. These immune functions strictly depend on the DC's capacity to differentiate from immature, antigen-capturing cells to mature, antigen-presenting cells-a process accompanied by profound changes in cellular phenotype and expression profile. Only little is known on how intracellular Leishmania affects this important process and DC transcriptional regulation. Here, we investigate these important open questions analyzing phenotypic, cytokine profile and transcriptomic changes in murine, immature bone marrow-derived DCs (iBMDCs) infected with antibody-opsonized and non-opsonized Leishmania amazonensis (L.am) amastigotes. DCs infected by non-opsonized amastigotes remained phenotypically immature whereas those infected by opsonized parasites displayed a semi-mature phenotype. The low frequency of infected DCs in culture led us to use DsRed2-transgenic parasites allowing for the enrichment of infected BMDCs by FACS. Sorted infected DCs were then subjected to transcriptomic analyses using Affymetrix GeneChip technology. Independent of parasite opsonization, Leishmania infection induced expression of genes related to key DC processes involved in MHC Class I-restricted antigen presentation and alternative NF-κB activation. DCs infected by non-opsonized parasites maintained an immature phenotype and showed a small but significant down-regulation of gene expression related to pro-inflammatory TLR signaling, the canonical NF-kB pathway and the NLRP3 inflammasome. This transcriptomic profile was further enhanced in DCs infected with opsonized parasites that displayed a semi-mature phenotype despite absence of inflammasome activation. This paradoxical DC phenotype represents a Leishmania-specific signature, which to our knowledge has not been observed with other opsonized infectious agents. In conclusion, systems-analyses of our transcriptomics data uncovered important and previously unappreciated changes in the DC transcription factor landscape, thus revealing a novel Leishmania immune subversion strategy directly acting on transcriptional control of gene expression. Our data raise important questions on the dynamic and reciprocal interplay between trans-acting and epigenetic regulators in establishing permissive conditions for intracellular Leishmania infection and polarization of the immune response.
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Células Dendríticas/inmunología , Células Dendríticas/parasitología , Interacciones Huésped-Parásitos/inmunología , Inflamasomas/inmunología , Leishmaniasis/inmunología , Animales , Femenino , Leishmania mexicana/inmunología , Ratones , Ratones Endogámicos BALB C , Transcriptoma/inmunologíaRESUMEN
Bacterial persister cells are phenotypic variants that exhibit a transient non-growing state and antibiotic tolerance. Here, we provide in vitro evidence of Staphylococcus aureus persisters within infected host cells. We show that the bacteria surviving antibiotic treatment within host cells are persisters, displaying biphasic killing and reaching a uniformly non-responsive, non-dividing state when followed at the single-cell level. This phenotype is stable but reversible upon antibiotic removal. Intracellular S. aureus persisters remain metabolically active but display an altered transcriptomic profile consistent with activation of stress responses, including the stringent response as well as cell wall stress, SOS and heat shock responses. These changes are associated with multidrug tolerance after exposure to a single antibiotic. We hypothesize that intracellular S. aureus persisters may constitute a reservoir for relapsing infection and could contribute to therapeutic failures.
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Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Células A549 , Animales , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Farmacorresistencia Bacteriana Múltiple/genética , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Perfilación de la Expresión Génica/métodos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Ratones , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/genética , Microscopía Confocal , Staphylococcus aureus/genética , Staphylococcus aureus/fisiología , Células THP-1RESUMEN
BAHD1 is a heterochomatinization factor recently described as a component of a multiprotein complex associated with histone deacetylases HDAC1/2. The physiological and patho-physiological functions of BAHD1 are not yet well characterized. Here, we examined the consequences of BAHD1 deficiency in the brains of male mice. While Bahd1 knockout mice had no detectable defects in brain anatomy, RNA sequencing profiling revealed about 2500 deregulated genes in Bahd1-/- brains compared to Bahd1+/+ brains. A majority of these genes were involved in nervous system development and function, behavior, metabolism and immunity. Exploration of the Allen Brain Atlas and Dropviz databases, assessing gene expression in the brain, revealed that expression of the Bahd1 gene was limited to a few territories and cell subtypes, particularly in the hippocampal formation, the isocortex and the olfactory regions. The effect of partial BAHD1 deficiency on behavior was then evaluated on Bahd1 heterozygous male mice, which have no lethal or metabolic phenotypes. Bahd1+/- mice showed anxiety-like behavior and reduced prepulse inhibition (PPI) of the startle response. Altogether, these results suggest that BAHD1 plays a role in chromatin-dependent gene regulation in a subset of brain cells and support recent evidence linking genetic alteration of BAHD1 to psychiatric disorders in a human patient.
Asunto(s)
Ansiedad/genética , Encéfalo/metabolismo , Proteínas Cromosómicas no Histona/genética , Reflejo de Sobresalto/genética , Animales , Ansiedad/fisiopatología , Encéfalo/patología , Cromatina/genética , Regulación de la Expresión Génica/genética , Haploinsuficiencia/genética , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , Humanos , Ratones , Ratones Noqueados , Fenotipo , Análisis de Secuencia de ARNRESUMEN
Microfold (M) cell host-pathogen interaction studies would benefit from the visual analysis of dynamic cellular and microbial interplays. We adapted a human in vitro M cell model to physiological bacterial infections, expression of fluorescent localization reporters and long-term three-dimensional time-lapse microscopy. This approach allows following key steps of M cell infection dynamics at subcellular resolution, from the apical onset to basolateral epithelial dissemination. We focused on the intracellular pathogen Shigella flexneri, classically reported to transcytose through M cells to initiate bacillary dysentery in humans, while eliciting poorly protective immune responses. Our workflow was critical to reveal that S. flexneri develops a bimodal lifestyle within M cells leading to rapid transcytosis or delayed vacuolar rupture, followed by direct actin motility-based propagation to neighboring enterocytes. Moreover, we show that Listeria monocytogenes, another intracellular pathogen sharing a tropism for M cells, disseminates in a similar manner and evades M cell transcytosis completely. We established that actin-based M cell-to-enterocyte spread is the major dissemination pathway for both pathogens and avoids their exposure to basolateral compartments in our system. Our results challenge the notion that intracellular pathogens are readily transcytosed by M cells to inductive immune compartments in vivo, providing a potential mechanism for their ability to evade adaptive immunity.
Asunto(s)
Disentería Bacilar/microbiología , Enterocitos/microbiología , Células Epiteliales/microbiología , Listeria monocytogenes/fisiología , Listeriosis/microbiología , Shigella flexneri/fisiología , Células CACO-2 , Humanos , Listeria monocytogenes/genética , Shigella flexneri/genéticaRESUMEN
Entamoeba histolytica is the causative agent of amebiasis, an infectious disease targeting the intestine and the liver in humans. Two types of intestinal infection are caused by this parasite: silent infection, which occurs in the majority of cases, and invasive disease, which affects 10% of infected persons. To understand the intestinal pathogenic process, several in vitro models, such as cell cultures, human tissue explants or human intestine xenografts in mice, have been employed. Nevertheless, our knowledge on the early steps of amebic intestinal infection and the molecules involved during human-parasite interaction is scarce, in part due to limitations in the experimental settings. In the present work, we took advantage of tissue engineering approaches to build a three-dimensional (3D)-intestinal model that is able to replicate the general characteristics of the human colon. This system consists of an epithelial layer that develops tight and adherens junctions, a mucus layer and a lamina propria-like compartment made up of collagen containing macrophages and fibroblast. By means of microscopy imaging, omics assays and the evaluation of immune responses, we show a very dynamic interaction between E. histolytica and the 3D-intestinal model. Our data highlight the importance of several virulence markers occurring in patients or in experimental models, but they also demonstrate the involvement of under described molecules and regulatory factors in the amoebic invasive process.