Biology subjects

Silva, C. S.

Publications and source records attributed to Silva, C. S..

7 recordsLinked to original sources

bioRxiv2026

PIM6 Induces Tolerance and Confers Protection against Mycobacterium tuberculosis in Primary Macrophages

Glycolipids are major components of the mycobacterial cell envelope and play critical roles in host-pathogen interactions. Here we investigate the capacity of the mycobacterial glycolipid phosphatidyl-myo-inositol hexamannosides (PIM6) to modulate innate responsiveness in naive primary human monocytes and monocyte-derived macrophages (MDMs). Monocytes and MDMs prestimulated with PIM6, followed by 5 days of rest, secreted attenuated levels of tumor necrosis factor alpha (TNF-), interleukin 6 (IL-6), and interleukin 10 (IL-10) following LPS challenge. PIM6 pretreatment significantly altered pattern recognition receptor expression through the experimental time course and programmed a tolerized phenotype in myeloid cells independent of TLR2 activity. In an infection challenge with H37Rv Mycobacterium tuberculosis (M.tb), MDMs pretreated with PIM6 secreted decreased levels of TNF- and IL-10 and exhibited early increased bacterial control. Together, these data reveal PIM6 as a complex immunomodulator that tolerizes myeloid cells to subsequent stimuli and exerts direct effects on the control of M.tb infection.

immunology↗

bioRxiv2026

A single methylation site regulates HRSV nucleocapsid architecture and replication

The nucleoprotein N of the human respiratory syncytial virus (HRSV) encases the viral genome, forming a flexible N-RNA nucleocapsid helix that serves as template for the viral polymerase L. Recent structural analysis revealed a non-canonical helical nucleocapsid arrangement that modulates RNA accessibility, yet its impact on polymerase function remains unknown. Here, we identified symmetric dimethylation of residue R27 of N as a critical modulator of nucleocapsid architecture and viral replication. We also showed that the methylase PRMT5 interacts with N and likely catalyses R27 methylation. Molecular dynamics simulations of RNA-free N dimers indicate that R27 methylation enhances opening and closing of the RNA-binding cavity, whereas a methylation-mimicking R27M substitution favours a closed state. Cryo-electron microscopy reveals a canonical, markedly straight R27M helix with increased rise and pitch. These findings demonstrate that post-translational modifications fine-tune interactions between N protomers, shaping nucleocapsid assembly, structure and dynamics, and thereby controlling HRSV replication.

microbiology↗

bioRxiv2024

IMMUNOPHENOTYPING AND FUNCTIONAL ANALYSIS OF NK CELL SUBSETS IN Mycobacterium tuberculosis-INFECTED INDIVIDUALS

Infection with Mycobacterium tuberculosis remains a global health challenge, with diverse clinical outcomes ranging from latent TB (LTB) infection to active TB disease (ATB). We conducted a comprehensive analysis of NK cell subsets and function in individuals with LTB, ATB, and healthy controls to elucidate their potential association with TB pathogenesis. Peripheral blood mononuclear cells (PBMCs) were isolated from Mtb-infected individuals and analyzed by mass cytometry and flow cytometry. We identified distinct NK cell subsets and evaluated their functional responses to stimulation. Our findings revealed comparable frequencies of total and NK cell subsets across LTB, ATB, and controls. Functional assays demonstrated similar degranulation, cytokine production, and proliferation capabilities among NK cell subsets across the three groups. This study provides insights into the heterogeneity of NK cell responses in TB and highlights the need for standardized methodologies with well-characterized cohorts controlling for donor background. Further investigations are warranted to delineate the specific roles of NK cells in TB immunity and pathogenesis.

immunology↗

bioRxiv2023

A previously-unrecognized motif of transcription factor RYBP, hotspot of cancer-related mutations, is essential for the integrity of Polycomb repressive complex 1

Polycomb repressive complex 1 (PRC1) catalyzes monoubiquitination of histone H2A on Lys119, promoting gene silencing. Cells at different developmental stages and in different tissues express different PRC1 isoforms. All isoforms share the same catalytic core (subunits RING1B and PCGF) and vary in the composition of regulatory subunits, clustering in two major classes. Canonical isoforms (cPRC1) are regulated by CBX-like subunits, while variant isoforms (vPRC1) are regulated by RYBP-like subunits. The molecular bases for how regulatory subunits affect the structural assembly of the complex and its catalytic activity are still largely unknown. To fill this knowledge gap, here we have specifically studied how RYBP regulates vPRC1 structure and function. Integrating the machine-learning algorithm AlphaFold2 and NMR, we have identified novel vPRC1 structural motifs in RING1B and RYBP. While the new RING1B motif is dispensable for vPRC1 assembly, the RYBP motif is essential for mediating inter-subunit interactions between RYBP and the catalytic RING1B-PCGF4 heterodimer. Importantly, the RYBP motif harbors cancer-related mutations systematically positioned on the same face of a putative transiently-forming -helix. Biochemical, biophysical and enzymatic characterization of purified cancer-related mutants confirm that this region is crucial for the structural stability of the complex. Overall, our data offer novel insights into the molecular architecture of vPRC1 and the effects of its regulatory subunit on the biochemical, structural, enzymatic, and physio-pathological properties of the complex.

biochemistry↗

bioRxiv2021

Stimulation with mycobacterial glycolipids and PPD reveals different innate immune response profiles in active and latent TB

Upon infection with Mycobacterium tuberculosis (Mtb) the host immune response might clear the bacteria, control its growth leading to latent tuberculosis (LTB), or fail to control its growth resulting in active TB (ATB). There is however no clear understanding of the features underlying a more or less effective response. Mtb glycolipids are abundant in the bacterial cell envelope and modulate the immune response to Mtb, but the patterns of response to glycolipids are still underexplored. To identify the CD45+ leukocyte activation landscape induced by Mtb glycolipids in peripheral blood of ATB and LTB, we performed a detailed assessment of the immune response of PBMCs to the Mtb glycolipids lipoarabinomannan (LAM) and its biosynthetic precursor phosphatidyl-inositol mannoside (PIM), and PPD. At 24 h and 5 days of stimulation, cell profiling and secretome analysis was done using mass cytometry and high-multiplex immunoassay. PIM mainly affected antigen-presenting cells to produce both proinflammatory (IL-2, IL-6, IL-17A, TNF- and GM-CSF), and IL-4 and IL-10 cytokines, but not IFN-{gamma}. LAM triggered a similar, albeit weaker, response. By contrast, PPD induced an increase in IFN-{gamma}-producing cells. Moreover, PPD also led to increased numbers of IL-2, IL-6, IL-10, IL-17A, TNF- and GrzB-producing cells. Treatment with an anti-TLR2 antibody led to partial inhibition of PIM-induced IL-6 production in myeloid cells, suggesting that PIM induces IL-6 production through TLR2. Expansion of monocyte subsets in response to PIM or LAM was reduced in both ATB and LTB as compared to healthy controls, suggesting a hyporesponsive/tolerance pattern in Mtb-infected individuals.

immunology↗

bioRxiv2021

The Intervening Domain Is Required For DNA-binding and Functional Identity of Plant MADS Transcription Factors

The MADS transcription factors (TF) are an ancient protein family with a high degree of sequence identity that bind almost identical DNA sequences across all eukaryotic kingdoms of life, yet fulfill dramatically different physiological roles. In plants, the family is divided into two main lineages, type I and II, based on sequence conservation of the DNA-binding MADS-box domain (M domain) with yeast and animal M domains. Here, we demonstrate that DNA binding in both lineages absolutely requires a short amino acid sequence C-terminal to the M domain called the Intervening domain (I domain) in type II MADS. Structural elucidation of the MI domains from the floral regulator, SEPALLATA3 (SEP3), shows a highly conserved MADS-box fold with the I domain forming an alpha helix and acting to stabilize the M domain. Based on secondary structure prediction, sequences fulfilling the same function as the SEP3 I domain can be found in both lineages of plant MADS TFs, suggesting the I domain is a conserved and required part of the DNA-binding domain. Using the floral organ identity MADS TFs, SEP3, APETALA1 (AP1) and AGAMOUS (AG), domain swapping demonstrate that the I domain alters DNA-binding specificity based on seq-DAP-seq experiments. Yeast 2-hybrid experiments further revealed the role of the I domain in dimerization specificity. Surprisingly, introducing AG carrying the I domain of AP1 in the Arabidopsis ap1 mutant, resulted in a high degree of complementation and restoration of first and second whorl organs. Taken together, these data demonstrate that the I domain acts both as an integral part of the DNA-binding domain and strongly contributes to the functional identity of the MADS TF.

plant biology↗

bioRxiv2020

Humic acids enrich the plant microbiota with bacterial candidates for the suppression of pathogens

Humic acids (HAs) stimulate the growth of several plant species by regulating their hormonal and redox metabolisms. Nevertheless, studies on the relationship of these substances with the plant-associated microbiota are almost nonexistent. Here, we hypothesized that the effect of HAs occurs in parallel with the regulation of the plant-associated bacterial community. Our results show the positive influence of HAs on the growth of rice and its stimulation of the root system. Metataxonomics revealed that the structure and composition of root bacterial communities were affected upon the application of HAs. Chitinophaga and Mucilaginibacter were the predominant genera in HA-treated roots. These bacteria produce enzymes that degrade compounds like those present in the wall of fungi, oomycetes, and nematode eggs. Pseudomonas and the Gp 1 group of Acidobacteria, both siderophore-producers and plant-growth promoters were also enriched, although with lower abundances. Given these results, we suggest that plants recruit these microorganisms in response to the stress caused by the HA-root interaction. For the first time, our findings indicate that HA-stimulated plants adopt the ecological strategy of recruiting members of the bacterial community that are candidates for the suppression of pathogens and, therefore, involved in plant defense.

microbiology↗