Biology subjects

da Silva, A. M.

Publications and source records attributed to da Silva, A. M..

7 recordsLinked to original sources

bioRxiv2026

NLRP3 inflammasome signaling orchestrates hepatic granuloma organization and protective immunity

Granulomas are organized immune structures that contribute to host defense against persistent pathogens, yet the mechanisms that coordinate their assembly and protective function remain incompletely understood. Here, using experimental visceral leishmaniasis caused by Leishmania infantum and samples from patients with active disease, we identify the NLRP3 inflammasome as a regulator of protective hepatic granulomatous immunity. Inflammasome-associated mediators were elevated in patients and correlated with systemic inflammation. In mice, L. infantum induced NLRP3 inflammasome activation within hepatic granulomas, while single-cell and spatial transcriptomic analyses revealed enrichment of inflammasome-associated transcription in hepatic macrophages and granuloma-associated regions. NLRP3 deficiency did not prevent granuloma initiation but impaired granuloma expansion, cellular organization, and leukocyte accumulation. This response required Caspase-1/11 and IL-18, but was independent of IL-1{beta}. Loss of NLRP3 also impaired parasite control despite reducing hepatic inflammation and histopathological alterations. Together, our findings identify NLRP3-Caspase-1/11-IL-18 signaling as a mechanism that coordinates the cellular and spatial organization of protective hepatic granulomas, revealing granuloma architecture as a previously unrecognized function of inflammasome-mediated immunity and providing mechanistic insight into host resistance in visceral leishmaniasis, a potentially fatal neglected tropical disease.

immunology↗

bioRxiv2026

Cryo-EM structure analysis of phage {Phi}Xacm4-11 that infects the phytopathogen Xanthomonas citri

Very few bacteriophages that infect Xanthomonas species have been characterized genetically and only one 3D structure, the capsid of a siphovirus that infects the phytopathogen Xanthomonas citri, has been determined at high resolution. This study presents the annotated DNA sequence and detailed structural analysis of {Phi}Xacm4-11, a podovirus that infects Xanthomonas citri, shedding light on its unique architecture and functional attributes, providing insights into the molecular mechanisms underlying host recognition and infection. Annotation of the genome revealed conserved features among related phages, but also distinct genetic elements that may contribute to {Phi}Xacm4-11s specificity toward X. citri. Genes associated with host recognition and infection were identified, including the genes potentially coding for the receptor-binding proteins (RBPs) at the tail fibre tip, offering insights into their role in bacterial attachment. Using high-resolution cryo-electron microscopy, we resolved the architecture of the mature, pre-released virion, revealing a T7-like head-tail assembly with a well-defined portal-tail complex embedded at a unique fivefold vertex. Our findings provide a detailed view of the structural and functional components of {Phi}Xacm4-11, furthering our understanding of its molecular interactions with X. citri and its potential application in phage therapy against phytopathogens. SIGNIFICANCE STATEMENTBacteriophages are increasingly recognized as powerful tools to control bacterial pathogens in medicine and agriculture, yet the structural basis of host recognition and genome delivery remains poorly understood for most phages. Here, we present a comprehensive structural and functional analysis of {Phi}Xacm4-11, a podovirus that infects the plant pathogen Xanthomonas citri. By combining genome annotation, proteomics, and high-resolution cryo-electron microscopy, we reveal the complete architecture of the mature virion and its specialized portal-tail machinery. Our results show how this short-tailed phage deploys an internal injection device to penetrate the bacterial cell envelope and highlight structural features linked to type IV pilus-dependent infection. These findings provide insights into phage entry mechanisms and establish {Phi}Xacm4-11 as a model for engineering biocontrol strategies.

biochemistry↗

bioRxiv2025

Transcriptome profiling reveals differential expression of virulence genes in Xylella fastidiosa under nutrient-rich and xylem-like conditions

Xylella fastidiosa is a xylem-limited, insect-vectored pathogen. To capture the metabolic changes that underlie its adaptation to both nutrient-rich and xylem-like environments, we performed transcriptome analyses on two reference strains, Temecula1 and 9a5c, sampled at early and late exponential phases in a complex laboratory medium (PWG) and a defined, xylem-mimicking medium (PIM6). Across eight conditions (24 transcriptomes), over 90% of annotated coding sequences were expressed, although 40-80% of transcripts exhibited low abundance (TPM < 100). In contrast, a core set of highly abundant transcripts was seen in both strains and media, including the non-coding RNAs 6S and RNase P, as well as multiple colicin- and microcin-like toxins, proteases, lipases, and stress-response factors. Comprehensive pathway reconstruction confirmed the activity of all essential metabolic pathways, while revealing conditional enrichment of stress-related and defense modules under nutrient limitation. We annotated 5' and 3' untranslated regions for thousands of transcripts and defined over 900 operons across both genomes. Differential expression analysis demonstrated that PIM6 medium confers a far greater transcriptional reprogramming between early and late phases than PWG. Strain-to-strain comparisons revealed that up to 12.6% of orthologous genes were differentially regulated, emphasizing divergent regulatory networks even under identical growth conditions. This multi-dimensional transcriptome atlas not only refines X. fastidiosa genome annotation but also pinpoints candidate regulators, non-coding RNAs, and operon variants for functional validation. By illuminating the dynamic gene expression landscapes that enable xylem colonization and environmental resilience, our study lays a foundation for targeted disruption of key pathways in this important plant pathogen.

genetics↗

bioRxiv2021

Isolation of a Bacillus safensis from mine tailings in Peru, genomic characterization and characterization of its cyanide- degrading enzyme CynD

Cyanide is widely used in industry as a potent lixiviant due to its capacity to tightly bind metals. This property imparts cyanide enormous toxicity to all known organisms. Thus, industries that utilize this compound must reduce its concentration in recycled or waste waters. Physical, chemical, and biological treatments have been used for cyanide remediation; however, none of them meet all the desired characteristics: efficiency, low cost and low environmental impact. A better understanding of metabolic pathways and biochemistry of enzymes involved in cyanide degradation is a necessary step to improve cyanide bioremediation efficacy to satisfy the industry requirements. Here, we used several approaches to explore this topic. We have isolated three cyanide-degrading Bacillus strains from water in contact with mine tailings from Lima, Peru, and classified them as Bacillus safensis PER-URP-08, Bacillus licheniformis PER-URP-12, and Bacillus subtilis PER-URP-17 based on 16S rRNA gene sequencing and core genome analyses. Additionally, core genome analyses of 132 publicly available genomes of Bacillus pumilus group including B. safensis and B. altitudinis allowed us to reclassify some strains and identify two strains that did not match with any known species of the Bacillus pumilus group. We searched for possible routes of cyanide-degradation in the genomes of these three strains and identified putative B. licheniformis PER-URP-12 and B. subtilis PER-URP-17 rhodaneses and B. safensis PER-URP-08 cyanide dihydratase (CynD) sequences possibly involved cyanide degradation. We identified characteristic C-terminal residues that differentiate CynD from B. pumilus and B. safensis, and showed that, differently from CynD from B. pumilus C1, recombinant CynD from the Bacillus safensis PER-URP-08 strain remains active up to pH 9 and presents a distinct oligomerization pattern at pH 8 and 9. Moreover, transcripts of B. safensis PER-URP-08 CynD (CynDPER-URP-08) are strongly induced in the presence of cyanide. Our results warrant further investigation of B. safensis PER-URP-08 and CynDPER-URP-08 as potential tools for cyanide-bioremediation.

microbiology↗

bioRxiv2021

Comparative genomics of Xylella fastidiosa suggests determinants of host-specificity and expands its mobile genetic elements repertoire

The Gram-negative bacterium Xylella fastidiosa colonizes plant xylem vessels and is obligately vectored by xylem sap-feeding hemipteran insects. X. fastidiosa causes diseases in many plant species but in a variety of its plant hosts this bacterium behaves as a commensal endophyte. Originally confined to the Americas, infecting mainly grapevine, citrus and coffee plants, X. fastidiosa has spread to several plant species in Europe, causing devastating crop diseases. Although many pathogenicity and virulence factors have been identified in X. fastidiosa which enable the bacterium to successfully establish in the xylem tissue, the mechanisms by which distinct X. fastidiosa strains colonize and cause disease in specific plant hosts have not been fully elucidated. Here we present comparative analyses of 94 publicly available whole-genome sequences of X. fastidiosa strains with the goal of providing insights into plant host specificity determinants for this phytopathogen as well as of expanding the knowledge of its mobile genetic elements (MGE) content, mainly prophages. Our results revealed a pangenome of 4,549 protein coding sequences (CDSs) which is still open. The core- and accessory genomes comprise 954 and 2,219 CDSs, respectively. Phylogenetic tree construction using all core genome CDSs grouped the strains in three major clades of subspecies fastidiosa, multiplex and pauca, with subclades related to the strains sequence type (ST) obtained from multi-locus sequence typing (MLST). The geographic region where the strains were collected showed stronger association with the clades of X. fastidiosa strains rather than the plant species from which they were isolated. Among the CDS related to virulence and pathogenicity found in the core genome, those related to lipopolysaccharide (LPS) synthesis and trimeric autotransporter adhesins (TAA) are somewhat related with the plant host of a given strain according to phylogenetic inference. The X. fastidiosa accessory genome is represented by an abundant and heterogeneous mobilome, which includes a diversity of prophage regions. In summary, the genome comparisons reported here will enable a better understanding of the diversity of phylogenetically close genomes and warrant further investigation of LPS and TAAs as potential X. fastidiosa host-specificity determinants. Impact statementThe bacterium Xylella fastidiosa is a pathogen that infects many plant species and has caused devastating diseases in grapevine, citrus, coffee, and olive plants. This phytopathogen X. fastidiosa is original from the Americas and has emerged in Europe where it is causing severe economic losses for olive producers, mainly in Italy. Although many pathogenicity and virulence factors have been identified in X. fastidiosa, which enable this bacterium to successfully establish in the xylem vessels network, the mechanisms by which distinct X. fastidiosa strains colonize and cause disease in the different plant host species have not been fully elucidated. The comparative analyses of 94 whole-genome sequences from X. fastidiosa strains from diverse hosts and geographic regions provide insights into host specificity determinants for this phytopathogen as well as expand the knowledge of its mobile genetic elements (MGE) content, mainly prophages. Our results contribute for a better understanding of the diversity of phylogenetically close genomes and warrant further experimental investigation of lipopolysaccharide and trimeric autotransporter adhesins as potential host-specificity determinants for X. fastidiosa. Data summaryAll genomic sequences were accessed from publicly available GenBank RefSeq database at NCBI (National Center for Biotechnology Information). A full listing of NCBI accession numbers for X. fastidiosa strains described in this paper is available in Table S1 (available in the online version of this article).

genomics↗

bioRxiv2021

Controlled spatial organization of bacterial clusters reveals cell filamentation is vital for Xylella fastidiosa biofilm formation

The morphological plasticity of bacteria to form filamentous cells commonly represents an adaptive strategy induced by stresses. In contrast, for diverse pathogens filamentous cells have been observed during biofilm formation, with function yet to be elucidated. To identify prior hypothesized quorum sensing as trigger of such cell morphogenesis, spatially controlled cell adhesion is pivotal. Here, we demonstrate highly-selective cell adhesion of the biofilm-forming phytopathogen Xylella fastidiosa to gold-patterned SiO2 substrates with well-defined geometries and dimensions. The consequent control of both cell density and distances between cell clusters using these patterns provided evidence of quorum sensing governing filamentous cell formation. While cell morphogenesis is induced by cell cluster density, filamentous cell growth is oriented towards neighboring cell clusters and distance-dependent; large interconnected cell clusters create the early biofilm structural framework. Together, our findings and investigative platform could facilitate therapeutic developments targeting biofilm formation mechanisms of X. fastidiosa and other pathogens.

microbiology↗

bioRxiv2020

vHULK, a new tool for bacteriophage host prediction based on annotated genomic features and deep neural networks

The experimental determination of a bacteriophage host is a laborious procedure. For this reason, there is a pressing need for reliable computational predictions of bacteriophage hosts in phage research in general and in phage therapy in particular. Here, we present a new program called vHULK for phage host prediction based on 9,504 phage genome features. These features take into account alignment significance scores between predicted-protein sequences in the phage genomes and a curated database of viral protein families. The features were fed to a deep neural network, and four distinct models were trained to predict 61 different host genera and 52 host species. In random controlled test sets, the program obtained 99% and 98% accuracy values at the genus and species levels, respectively. On a validation dataset with 2,178 phage genomes, mean accuracies were 82% and 52% at the genus and species levels, respectively. When compared against other phage host prediction programs on the same validation dataset, vHULK achieved substantially better performance, therefore demonstrating that the program is an advance on the state-of-art in phage host prediction. vHULK is freely available at https://github.com/LaboratorioBioinformatica/vHULK.

bioinformatics↗