Biology subjects

Carvalho, A.

Publications and source records attributed to Carvalho, A..

29 records · Page 2Linked to original sources

bioRxiv2021

Deficiency in cytosine DNA methylation leads to high chaperonin expression and tolerance to aminoglycosides in Vibrio cholerae

Antibiotic resistance has become a major global issue. Understanding the molecular mechanisms underlying microbial adaptation to antibiotics is of keen importance to fight Antimicrobial Resistance (AMR). Aminoglycosides are a class of antibiotics that target the small subunit of the bacterial ribosome, disrupting translational fidelity and increasing the levels of misfolded proteins in the cell. In this work, we investigated the role of VchM, a DNA methyltransferase, in the response of the human pathogen Vibrio cholerae to aminoglycosides. VchM is a V. cholerae specific orphan m5C DNA methyltransferase that generates cytosine methylation at 5'-RCCGGY-3' motifs. We show that deletion of vchM, although causing a growth defect in absence of stress, allows V. cholerae cells to cope with aminoglycoside stress at both sub-lethal and lethal concentrations of these antibiotics. Through transcriptomic and genetic approaches, we show that groESL-2 (a specific set of chaperonin-encoding genes located on the second chromosome of V. cholerae), are upregulated in cells lacking vchM and are needed for the tolerance of vchM mutant to lethal aminoglycoside treatment, likely by fighting aminoglycoside-induced misfolded proteins. Interestingly, preventing VchM methylation of the four RCCGGY sites located in groESL-2 region, leads to a higher expression of these genes in WT cells, showing that VchM modulates the expression of these chaperonins in V. cholerae directly through DNA methylation. AUTHOR SUMMARYBacteria are organisms with a remarkable ability to adapt to several stress conditions, including to the presence of antibiotics. The molecular mechanisms underlying such adaptation lead, very often, to phenomena like antimicrobial tolerance and resistance, responsible for the frequent failure of antibiotic treatment. The study of these molecular mechanisms is thus an important tool to understand development of antimicrobial resistance in bacteria. In this work, we show that abrogating cytosine DNA methylation in Vibrio cholerae increases its tolerance to aminoglycosides, a class of antibiotics that cause protein misfolding. DNA methylation is known to affect gene expression and regulate several cellular processes in bacteria. Here we provide evidence that DNA methylation also has a more direct role in controlling antibiotic susceptibility in bacteria. Consequently, the study of bacterial DNA methyltransferases and DNA methylation should not be overlooked when addressing the problem of antimicrobial tolerance/resistance.

microbiology↗

bioRxiv2021

Aspergillus fumigatus acetate utilisation impacts virulence traits and pathogenicity

Aspergillus fumigatus is a major opportunistic fungal pathogen of immunocompromised and immunocompetent hosts. To successfully establish an infection, A. fumigatus needs to use host carbon sources, such as acetate, present in the body fluids and peripheral tissues. However, utilisation of acetate as a carbon source by fungi in the context of infection has not been investigated. This work shows that acetate is metabolised via different pathways in A. fumigatus and that acetate utilisation is under the regulatory control of a transcription factor (TF), FacB. A. fumigatus acetate utilisation is subject to carbon catabolite repression (CCR), although this is only partially dependent on the TF and main regulator of CCR CreA. The available extracellular carbon source, in this case glucose and acetate, significantly affected A. fumigatus virulence traits such as secondary metabolite secretion and cell wall composition, with the latter having consequences for resistance to oxidative stress, to anti-fungal drugs and to human neutrophil-mediated killing. Furthermore, deletion of facB significantly impaired the in vivo virulence of A. fumigatus in both insect and mammalian models of invasive aspergillosis. This is the first report on acetate utilisation in A. fumigatus and this work further highlights the importance of available host-specific carbon sources in shaping fungal virulence traits and subsequent disease outcome, and a potential target for the development of anti-fungal strategies. ImportanceAspergillus fumigatus is an opportunistic fungal pathogen in humans. During infection, A. fumigatus is predicted to use host carbon sources, such as acetate, present in body fluids and peripheral tissues, to sustain growth and promote colonisation and invasion. This work shows that A. fumigatus metabolises acetate via different pathways, a process that is dependent on the transcription factor FacB. Furthermore, the type and concentration of the extracellular available carbon source were determined to shape A. fumigatus virulence determinants such as secondary metabolite secretion and cell wall composition. Subsequently, interactions with immune cells are altered in a carbon source-specific manner. FacB is required for A. fumigatus in vivo virulence in both insect and mammalian models of invasive aspergillosis. This is the first report that characterises acetate utilisation in A. fumigatus and highlights the importance of available host-specific carbon sources in shaping virulence traits and potentially subsequent disease outcome.

microbiology↗

bioRxiv2021

Microbiota instruct IL-17A-producing innate lymphoid cells to promote skin inflammation in cutaneous leishmaniasis

Innate lymphoid cells (ILCs) comprise a heterogeneous population of immune cells that maintain barrier function and can initiate a protective or pathological immune response upon infection. Here we show the involvement of IL-17A-producing ILCs in microbiota-driven immunopathology in cutaneous leishmaniasis. IL-17A-producing ILCs were ROR{gamma}t+ and were enriched in Leishmania major infected skin, and topical colonization with Staphylococcus epidermidis before L. major infection exacerbated the skin inflammatory responses and IL-17A-producing ROR{gamma}t+ ILC accumulation without impacting type 1 immune responses. IL-17A responses in ILCs were directed by Batf3 dependent CD103+ dendritic cells, and experiments using ILC deficient Rag1-/- mice established that IL-17A+ ILCs were sufficient in driving the inflammatory responses. As depletion of ILCs or neutralization of IL-17A diminished the microbiota mediated immunopathology. Taken together, this study indicates that the skin microbiota promotes ROR{gamma}t+ IL-17A-producing ILCs, which augment the skin inflammation in cutaneous leishmaniasis.

immunology↗

bioRxiv2021

Microbiota and stress: a loop that impacts memory

Chronic stress and the gut microbiota appear to comprise a feed-forward loop, which contributes to the development of depressive disorders. Evidence suggests that memory can also be impaired by either chronic stress or microbiota imbalance. However, it remains to be established whether these could be a part of an integrated loop model and be responsible for memory impairments. To shed light on this, we used a two-pronged approach in Japanese quail: first stress-induced alterations in gut microbiota were characterized, then we tested whether this altered microbiota could affect brain and memory function when transferred to a germ-free host. The cecal microbiota of chronically stressed quails was found to be significantly different from that of unstressed individuals with lower and {beta} diversities and increased Bacteroidetes abundance largely represented by the Alistipes genus, a well-known stress target in rodents and humans. The transfer of this altered microbiota into germ-free quails decreased their spatial and cue-based memory abilities as previously demonstrated in the stressed donors. The recipients also displayed increased anxiety-like behavior, reduced basal plasma corticosterone levels and differential gene expression in the brain. Furthermore, cecal microbiota transfer from a chronically stressed individual was sufficient to mimic the adverse impact of chronic stress on memory in recipient hosts and this action may be related to the Alistipes genus. Our results provide evidence of a feed-forward loop system linking the microbiota-gut-brain axis to stress and memory function and suggest that maintaining a healthy microbiota could help alleviate memory impairments linked to chronic stress.

animal behavior and cognition↗

bioRxiv2020

Dynein-dynactin segregate meiotic chromosomes in C. elegans spermatocytes

The dynactin complex is an essential co-factor of the microtubule-based motor dynein. Dynein-dynactin have well-documented roles in spindle assembly and positioning during C. elegans female meiosis and embryonic mitosis, while dynein-dynactins contribution to male meiosis has not been investigated. Here, we characterize the G33S mutation in DNC-1s N-terminal microtubule binding domain, which corresponds to G59S in the human dynactin subunit p150/Glued that causes motor neuron disease. In spermatocytes, dnc-1(G33S) delays spindle assembly and penetrantly inhibits anaphase spindle elongation in meiosis I, which prevents homologous chromosome segregation and generates aneuploid sperm with an extra centrosome. Consequently, embryos produced by dnc-1(G33S) hermaphrodites exhibit a high incidence of tetrapolar mitotic spindles, yet dnc-1(G33S) embryos with bipolar spindles proceed through early mitotic divisions without errors in chromosome segregation. Deletion of the DNC-1 N-terminus shows that defective meiosis in dnc-1(G33S) spermatocytes is not due to DNC-1s inability to interact with microtubules. Rather, our results suggest that the DNC-1(G33S) protein, which is aggregation-prone in vitro, is less stable in spermatocytes than the early embryo, resulting in different phenotypic severity in the two dividing tissues. Thus, the unusual hypomorphic nature of the dnc-1(G33S) mutant reveals that dynein-dynactin drive meiotic chromosome segregation in spermatocytes and illustrates that the extent to which protein misfolding leads to loss of function can vary significantly between cell types.

cell biology↗

bioRxiv2020

Regulation of Wolbachia proliferation by the amplification and deletion of an addictive genomic island

Wolbachia is one of the most prevalent bacterial endosymbionts, infecting approximately 40% of terrestrial arthropod species. Wolbachia is often a reproductive parasite but can also provide fitness benefits to its host, as, for example, protection against viral pathogens. This protective effect is currently being applied to fight arboviruses transmission by releasing Wolbachia-transinfected mosquitoes. Titre regulation is a crucial aspect of Wolbachia biology. Higher titres can lead to stronger phenotypes and fidelity of transmission but can have a higher cost to the host. Since Wolbachia is maternally transmitted, its fitness depends on host fitness, and, therefore, its cost to the host may be under selection. Understanding how Wolbachia titres are regulated and other aspects of Wolbachia biology has been hampered by the lack of genetic tools. Here we developed a forward genetic screen to identify new Wolbachia over-proliferative mutant variants. We characterized in detail two new mutants, wMelPop2 and wMelOctoless, and show that the amplification or loss of the Octomom genomic region lead to over-proliferation. These results confirm previous data and expand on the complex role of this genomic region in the control of Wolbachia proliferation. Both new mutants shorten the host lifespan and increase antiviral protection. Moreover, we show that Wolbachia proliferation rate in Drosophila melanogaster depends on the interaction between Octomom copy number, the host developmental stage, and temperature. Our analysis also suggests that the life shortening and antiviral protection phenotypes of Wolbachia are dependent on different, but related, properties of the endosymbiont; the rate of proliferation and the titres near the time of infection, respectively. Altogether, we demonstrate the feasibility of a novel and unbiased experimental approach to study Wolbachia biology, which can be further adapted to characterize other genetically intractable bacterial endosymbionts.

microbiology↗

bioRxiv2020

PIG-1 MELK-dependent phosphorylation of nonmuscle myosin II promotes apoptosis through CES-1 Snail partitioning

The mechanism(s) through which mammalian kinase MELK promotes tumorigenesis is not understood. We find that the C. elegans orthologue of MELK, PIG-1, promotes apoptosis by partitioning an anti-apoptotic factor. The C. elegans NSM neuroblast divides to produce a larger cell that differentiates into a neuron and a smaller cell that dies. We find that in this context, PIG-1 is required for partitioning of CES-1 Snail, a transcriptional repressor of the pro-apoptotic gene egl-1 BH3-only. pig-1 MELK is controlled by both a ces-1 Snail- and par-4 LKB1-dependent pathway, and may act through phosphorylation and cortical enrichment of nonmuscle myosin II prior to neuroblast division. We propose that pig-1 MELK-induced local contractility of the actomyosin network plays a conserved role in the acquisition of the apoptotic fate. Our work also uncovers an auto-regulatory loop through which ces-1 Snail controls its own activity through the formation of a gradient of CES-1 Snail protein. Significance StatementApoptosis is critical for the elimination of unwanted cells. What distinguishes wanted from unwanted cells in developing animals is poorly understood. We report that in the C. elegans NSM neuroblast lineage, the level of CES-1, a Snail-family member and transcriptional repressor of the pro-apoptotic gene egl-1, contributes to this process. In addition, we demonstrate that C. elegans PIG-1, the orthologue of mammalian proto-oncoprotein MELK, plays a critical role in controlling CES-1Snail levels. Specifically, during NSM neuroblast division, PIG-1MELK controls partitioning of CES-1Snail into one but not the other daughter cell thereby promoting the making of one wanted and one unwanted cell. Furthermore, we present evidence that PIG-1MELK acts prior to NSM neuroblast division by locally activating the actomyosin network.

developmental biology↗

bioRxiv2019

Bayesian modelling to assess differential expression from single-cell data

Disease profiling, treatment development, and the identification of new cell populations are some of the most relevant applications relying on differentially expressed genes (DEG) analysis. In this context, three leading technologies emerged; namely, DNA microarrays, bulk RNA sequencing (RNA-seq), and single-cell RNA sequencing (scRNA-seq), the main focus of this work. Although scRNA-seq tends to offer more accurate data, it is still limited by many confounding factors. We introduce two novel approaches to assess DEG: extended Bayesian zero-inflated negative binomial factorization (ext-ZINBayes) and single-cell differential analysis (SIENA). In addition, we benchmark the proposed methods with known DEG analysis tools for single-cell and bulk RNA data, using two real public datasets. One contains house mouse cells of two different types, while the other gathers human peripheral blood mononuclear cells divided into four types. The results show that the two procedures can be very competitive with existing methods (scVI, SCDE, MAST, and DEseq) in identifying relevant putative biomarkers. In terms of scalability and correctness, SIENA stands out from ext-ZINBayes and some of the existing methods. As single-cell datasets become increasingly larger, SIENA may emerge as a powerful tool to discover functional differences between two conditions. Both methods are publicly available (https://github.com/JoanaGodinho/SIENA, https://github.com/JoanaGodinho/ext-ZINBayes).

bioinformatics↗

bioRxiv2019

Flotillin-dependent lipid-raft microdomains are required for functional phagolysosomes against fungal infections

Lipid rafts form signaling platforms on biological membranes with incompletely characterized role in immune response to infection. Here we report that lipid raft microdomains are essential components of the phagolysosomal membrane of macrophages. Genetic deletion of the lipidraft chaperons flotillin-1 and flotillin-2 demonstrate that the assembly of both major defense complexes vATPase and NADPH oxidase on the phagolysosomal membrane requires lipid rafts. Furthermore, we discovered a new virulence mechanism leading to the dysregulation of lipid-raft formation by melanized wild-type conidia of the important human-pathogenic fungus Aspergillus fumigatus. This results in reduced phagolysosomal acidification. Phagolysosomes with ingested melanized conidia contain a reduced amount of free Ca2+ ions as compared to phagolysosomes with melanin-free conidia. In agreement with a role of Ca2+ for generation of functional lipid rafts, we show that Ca2+-dependent calmodulin activity is required for lipid-raft formation on the phagolysosome. We identified a single nucleotide polymorphism in the human FLOT1 gene that results in heightened susceptibility for invasive aspergillosis in hematopoietic stem-cell transplant recipients. Collectively, flotillin-dependent lipid rafts on the phagolysosomal membrane play an essential role in protective antifungal immunity in humans.

microbiology↗

bioRxiv2019

Paracoccin overexpression in Paracoccidioides brasiliensis reveals the influence of chitin hydrolysis on fungal virulence and host immune response

Paracoccidioides brasiliensis and P. lutzii, etiological agents of paracoccidioidomycosis (PCM), develop as mycelia at 25-30 {degrees}C and as yeast at 35-37 {degrees}C. Only a few Paracoccidioides spp. proteins are well characterized. Thus, we studied paracoccin (PCN) from P. brasiliensis, its role in the fungus biology, and its relationship with the host innate immune cells. Cloning and heterologous expression analysis revealed its lectin, enzymatic, and immunomodulatory properties. Recently, we employed a system based on Agrobacterium tumefaciens-mediated transformation to manipulate P. brasiliensis yeast genes to obtain clones knocked-down for PCN, which after all, are unable to transit from yeast to mycelium forms, causing a mild pulmonary disease. Herein, we generate P. brasiliensis overexpressing PCN (ov-PCN). To date, it was not explored the overexpressing of endogenous components in Paracoccidioides spp. Therefore, we investigate the role of PCN in fungal biology and pathogenesis. Augmented levels of PCN mRNA and protein, and N-acetylglucosaminidase activity confirmed PCN overexpression in ov-PCN of P. brasiliensis yeasts. Interestingly, PCN overexpression did not affect the yeasts growth or viability and favored cell separation. The ov-PCN clones transitioned faster to the mycelium form than the wt-PCN yeasts. Concerning infection, while most of mice infected with the wt-yeasts (90%) survive at least until the 70th day, all mice infected with ov-PCN yeasts were already died at the 35th day post-infection. In vitro assays showed that ov-PCN were more susceptible to phagocytosis by macrophages. Finally, it was verified that the chitin particles isolated from the ov-PCN cells were smaller than those obtained from the wt-PCN yeasts. Macrophages stimulated with the chitin isolated from ov-PCN produce IL-10, whereas the particles with a wider size range harvested from wt-PCN yeasts induced TNF- and IL-1{beta} secretion. The anti-inflammatory microenvironment from macrophage stimulation with small chitin particles hampers the development of a protective immune response against the fungus. We postulated that the high grade of chitin cleavage, as the results of augmented PCN expression, favors pathogenesis following P. brasiliensis infection. Thus, PCN is a relevant virulence fungal factor.\n\nAUTHOR SUMMARYParacoccidioides spp. are pathogenic fungi that cause paracoccidioidomycosis (PCM) in humans, the main deep mycosis of Latin America. Recently, by knocking down the paracoccin gene, our group showed that this lectin is necessary for the morphological transition from yeast to hyphae, and that this decrease results in low P. brasiliensis virulence. Here, after overexpress PCN, we revealed the importance of the yeast chitin hydrolysis to the host response. Infection of mice with ov-PCN yeasts causes severe lung disease compared to moderate disease caused by wt-PCN yeasts. The release of smaller chitin particles was as a result of an accelerated chitin hydrolysis provided by ov-PCN yeasts. Interestingly, these smallest chitin particles are able to modulate host response by increasing IL-10 in the meantime that decrease TNF- secretion, thus hampering Th1 immune response that is crucial in the fight against this fungi. These findings represent a significant advance in the knowledge about the role of PCN chitinase in P. brasiliensis.

microbiology↗

bioRxiv2018

A new schizophrenia model: immune activation is associated with induction of thetryptophan catabolite pathway and increased eotaxin levels which together determinememory impairments and schizophrenia symptom dimensions.

AbstractO_ST_ABSObjectiveC_ST_ABSRecently, we reported that stable-phase schizophrenia is characterized by two interrelated symptom dimensions: PHEMN (psychotic, hostility, excitation, mannerism and negative symptoms); and DAPS (depressive, anxiety and physio-somatic symptoms) and that Major Neuro-Cognitive psychosis (MNP) is the full blown phenotype of schizophrenia (largely overlapping with deficit schizophrenia). Herein we examined the effects of immune activation in association with tryptophan catabolite (TRYCAT) patterning and memory disorders on PHEMN/DAPS dimensions and MNP.\n\nMethodSerum levels of macrophage inflammatory protein-1 (MIP-1), soluble interleukin (IL)-1 receptor antagonist (sIL-1RA), IL-10, eotaxin, IgA/IgM responses to TRYCATs, and Consortium to Establish a Registry for Alzheimers disease (CERAD) tests were assessed in 40 controls and 80 schizophrenia patients.\n\nResultsSchizophrenia and MNP were predicted by significantly increased levels of IL-10, eotaxin and TRYCATs. A large part of the variance in both PHEMN/DAPS symptom dimensions (42.8%) was explained by cytokine levels and TRYCATs combined. The MTP+sTL-1R A+IL-10 composite score and eotaxin explained each around 19% of the variance in symptom dimensions, and approximately 18% of memory deficits. Moreover, MIP+sIL-1RA+IL-10 was significantly associated with elevations in picolinic acid, xanthurenic acid and 3-OH-kynurenine. Partial Least Squares path modeling shows that the highly significant effects of MIP+sIL-1RA+IL-10 on symptomatology are mediated by the effects of noxious TRYCATs on memory deficits.\n\nConclusionsCurrent findings indicate that in schizophrenia, immune activation may underpin activation of indoleamine-2,3-dioxygenase and kynurenine monooxygenase, while impairments in episodic and semantic memory may be caused by the neurotoxic effects of TRYCATs and eotaxin. The combined effects of immune activation, eotaxin and memory defects determine to a large extent PHEMN/DAPS symptoms and the MNP phenotype. These findings indicate that schizophrenia phenomenology is largely mediated by multiple neuro-immune pathways and that immune activation, increased production of eotaxin and neurotoxic TRYCATs (picolinic acid, xanthurenic acid and 3-HO-kynurenine) are new drug targets in schizophrenia and MNP.

neuroscience↗