Biology subjects

Azevedo, V.

Publications and source records attributed to Azevedo, V..

8 recordsLinked to original sources

bioRxiv2025

Staphylococcus aureus derived extracellular vesicles trigger multiple inflammatory pathways in host cells and deliver their RNA cargo following their internalization and lysis within late endosomes

Staphylococcus aureus is a major opportunistic Gram-positive pathogen whose virulence can be partly mediated by extracellular vesicles (EVs). These nanosized particles carry a diverse cargo and can play key roles in host-pathogen interactions. Here, we investigated the production, composition, and host cell interactions of EVs secreted by the methicillin-resistant S. aureus strain N315, one of the most widely studied clinical isolates. To mimic infection, EVs produced from RPMI supplemented with 10% LB were purified by size exclusion chromatography, and characterized by nanoparticle tracking analysis and electron microscopy. N315 EVs contained a diverse molecular cargo, selectively enriched, and including proteins, lipoproteins, DNA, RNA, lipoteichoic acid, and peptidoglycan. Protection assays and confocal microscopy confirmed the intravesicular localization of RNAs and proteins. N315 EVs were recognized by multiple host pattern recognition receptors (PPRs) in human MG-63 cells, located at the cell surface (TLR1, TLR2, TLR4, TLR6), within endosomes (TLR3, TLR7, TLR4) and in the cytoplasm (NOD2). EV exposure promoted the selective upregulation of these PRRs, activated the NF-{kappa}B and JAK/STAT signaling pathways, and induced the expression of pro-inflammatory cytokines such as IL1{beta}, IL6, and CXCL8. N315 EVs were internalized via a dynamin-dependent endocytosis mechanism and trafficked to late endosomes, where RNA cargo release occurred. Importantly, inhibition of EVs uptake altered cytokine gene expression, confirming that internalization is required for immunomodulation. This work provides a better understanding of the mechanisms of interaction of S. aureus EVs with host cells and sheds light on the fate of EVs and their cargo once internalized by cells. IMPORTANCEAntibiotic resistance, coupled with the limited discovery of new antimicrobial agents, poses a growing threat to public health. Staphylococcus aureus is one of the six highly virulent and antibiotic-resistant bacterial species responsible for both severe hospital- and community-acquired infections. This pathogen demonstrates a remarkable ability to adapt to diverse hosts and environments and to produce a broad arsenal of virulence factors. Gaining deeper insight into its pathogenic mechanisms and host interactions may reveal novel therapeutic targets and alternative treatment strategies. Over the past decade, a key advancement has been the identification of extracellular vesicle production by S. aureus and its critical role as a new secretion system for virulence factors. This study shed light on the immunogenic effect of EVs and their role as a vehicle for the delivery of their contents into host cells. These adjuvant-like properties open new avenues for research and potential intervention.

microbiology↗

bioRxiv2022

SARS-CoV-2 variants show a gradual declining pathogenicity and pro-inflammatory cytokine stimulation and an increasing antigenic and anti-inflammatory cytokine induction

Hyper-transmissibility with decreased disease severity are typical characteristics of Omicron variant. To understand this phenomenon, we used various bioinformatics approaches to analyze randomly selected genome sequences (one each) of the Gamma, Delta, and Omicron variants submitted to NCBI from 15 to 31 December 2021. We show that: (i) Pathogenicity of SARS-CoV-2 variants decreases in the order: Wuhan > Gamma > Delta > Omicron; however, the antigenic property follows the order: Omicron > Gamma > Wuhan > Delta. (ii) Omicron Spike RBD has lower pathogenicity but higher antigenicity than other variants. (iii) Decreased disease severity by Omicron variant may be due to its decreased pro-inflammatory and IL-6 stimulation and increased IFN-{gamma} and IL-4 induction efficacy. (iv) Mutations in N protein are associated with decreased IL-6 induction and human DDX21-mediated increased IL-4 production in Omicron. (v) Due to mutations, the stability of S, M, N, and E proteins decreases in the order: Omicron > Gamma > Delta > Wuhan. (vi) Stronger Spike RBD-hACE2 binding in Omicron is associated with increased transmissibility. However, the lowest stability of the Omicron Spike protein makes Spike RBD-hACE2 interaction weak for systemic infection and for causing severe disease. Finally (vii), the highest instability of Omicron E protein may also be associated with decreased viral maturation and low viral load leading to less severe disease and faster recovery. Our method may be used for other similar viruses, and these findings will contribute to the understanding of the dynamics of SARS-CoV-2 variants and the management of emerging variants.

bioinformatics↗

bioRxiv2021

Machine learning models exploring characteristic single-nucleotide signatures in Yellow Fever Virus

Yellow fever virus (YFV) is the agent of the most severe mosquito-borne disease in the tropics. Recently, Brazil suffered major YFV outbreaks with a high fatality rate affecting areas where the virus has not been reported for decades, consisting of urban areas where a large number of unvaccinated people live. We developed a machine learning framework combining three different algorithms (XGBoost, random forest and regularized logistic regression). This method was applied to 56 YFV sequences from human infections and 27 from non-human primate (NHPs) infections to investigate the presence of genetic signatures possibly related to disease severity (in human related sequences) and differences in the PCR cycle threshold (Ct) values (in NHP related sequences). Our analyses reveal four non-synonymous single nucleotide variations (SNVs) on sequences from human infections, in proteins NS3 (E614D), NS4a (I69V), NS5 (R727G, V643A) and six non-synonymous SNVs on NHP sequences, in proteins E (L385F), NS1 (A171V), NS3 (I184V) and NS5 (N11S, I374V, E641D). We performed comparative protein structural analysis on these SNVs, describing possible impacts on protein function. Despite the fact that the dataset is limited in size and that this study does not consider virus-host interactions, our work highlights the use of machine learning as a versatile and fast initial approach to genomic data exploration. ImportanceYellow fever is responsible for 29-60 thousand deaths annually in South America and Africa and is the most severe mosquito-borne disease in the tropics. Given the range of clinical outcomes and the availability of YFV genomic data, the use of machine learning analysis promises to be a powerful tool in the investigation of genetic signatures that could impact disease severity and its potential of being reintroduced in an urban transmission cycle. This can assist in the search for biomarkers of severity as well as help elucidating variations in hosts Ct value. This work aims to propose a relatively fast and inexpensive computational analysis framework, which can be used as a real-time, innitial strategy associated with genomic surveillance to identify a set of single nucleotide variants putatively related to biological and clinical characteristics being observed.

microbiology↗

bioRxiv2021

Chikungunya virus ECSA lineage reintroduction in the northeasternmost region of Brazil

The Northeast region of Brazil registered the second highest incidence proportion of chikungunya fever in 2019. In that year an outbreak consisting of patients presented with febrile disease associated with joint pain were reported by the public primary health care service in the city of Natal, Rio Grande do Norte state, in March 2019. At first, the aetiological agent of the disease was undetermined. Since much is still unknown about chikungunya virus (CHIKV) genomic diversity and evolutionary history in this northeasternmost state, we used a combination of portable whole genome sequencing, molecular clock, and epidemiological analyses that revealed the re-introduction of the CHIKV East-Central-South-African (ECSA) lineage into Rio Grande do Norte. We estimated CHIKV ECSA lineage was first introduced into Rio Grande do Norte in early June 2014, while the 2019 outbreak clade diverged around April 2018 during a period of increased chikungunya incidence in the Southeast region, which might have acted as a source of virus dispersion towards the Northeast region. Together, these results confirm the ECSA lineage continues to spread across the country through interregional importation events likely mediated by human mobility. HIGHLIGHTSCHIKV ECSA lineage introduction into Rio Grande do Norte state, Northeast Brazil, was estimated to early June 2014 At least two CHIKV importation events occurred in Rio Grande do Norte state, Brazil The 2019 chikungunya outbreak in Rio Grande do Norte was likely caused by a second event of CHIKV introduction imported from Rio de Janeiro state.

genomics↗

bioRxiv2020

Genomic diversity, population structure and accessory genome analysis of Pasteurella multocida: New Insights into host adaptation and disease specialization

Pasteurella multocida is a multi-host pathogen that infects a wide spectrum of domestic and wild animals including humans. Despite its impact on health and economics, P. multocida is considered an enigmatic pathogen and the genetic basis of its pathogenicity and host adaptation still remains unclear. Here we present a detailed genomic framework based on 336 whole-genome sequences of P. multocida isolates from different animal species and countries. Our data provide genomic support of the existence of two very divergent phylogroups (PmI and PmII), which present a barrier to homologous recombination suggesting genetic isolation. Additionally, a torCAD operon, which reduces TMAO (trimethylamine N-oxide) to produce energy during bacterial anaerobic respiration, is present only in PmI and can act as a hypothetical driver of niche segregation between phylogroups. The PmI phylogroup harbors strains that infect a wider range of hosts than PmII, and shows a highly diverse phylogeny and accessory genome. We identified nine clonal lineages for PmI, seven of which are associated with specific hosts or diseases and contain distinct accessory gene pools that can confer ecologically relevant phenotypes. We found differential presence of a trehalose metabolism operon in the bovine lineage associated with pneumonic pasteurellosis; while, citrate, L-arabinose, L-fucose, and D-allose operons are only present in avian lineages. These findings suggest that alternative metabolic pathways may facilitate the establishment of P. multocida during host colonization in the early stages of infection promoting the adaptation of P. multocida lineages to certain hosts.

microbiology↗

bioRxiv2020

Taxonomic classification of strain PO100/5 shows a broader geographic distribution and genetic markers of the recently described Corynebacterium silvaticum

The bacterial strain PO100/5 was isolated from a skin abscess of a pig (Sus scrofa domesticus) in the Alentejo region of southern Portugal. It was identified as Corynebacterium pseudotuberculosis using biochemical tests, multiplex PCR and Pulsed Field Gel Electrophoresis. After genome sequencing and rpoB phylogeny, the strain was classified as C. ulcerans. To better understand the taxonomy of this strain and improve identification methods, we compared strain PO100/5 to other publicly available genomes from the C. diphtheriae group. Taxonomic analysis reclassified it and three others strains as belonging to the recently described C. silvaticum, which have been isolated from wild boar and roe deer in Germany and Austria. The results showed that PO100/5 is the first sequenced genome of a C. silvaticum strain from a domestic animal and a different geographical region, is a putative producer of the diphtheriae toxin, and has a unique sequence type. Genomic analysis of PO100/5 showed four prophages and eight conserved genomic islands when compared to C. ulcerans. Pangenome analysis of 38 C. silvaticum and 76 C. ulcerans samples suggest that C. silvaticum is a clonal species, with 73.6% of conserved genes and a pangenome near to being closed ( > 0.952). 172 conserved genes are unique to C. silvaticum when compared to C. ulcerans, with most related to nutrient uptake and metabolism, prophages or immune evasion. These unique genes could be used as genetic markers for species identification. This information can be useful for identification and surveillance of this pathogen, especially in regard to the possibility of zoonotic transmission.

genomics↗

bioRxiv2018

Chikungunya virus outbreak in the Amazon region: replacement of the Asian genotype by an ECSA lineage

BackgroundSince its first detection in the Caribbean in late 2013, chikungunya virus (CHIKV) has affected 51 countries in the Americas. The CHIKV epidemic in the Americas was caused by the CHIKV-Asian genotype. In August 2014, local transmission of the CHIKV-Asian genotype was detected in the Brazilian Amazon region. However, a distinct lineage, the CHIKV-East-Central-South-America (ECSA)-genotype, was detected nearly simultaneously in Feira de Santana, Bahia state, northeast Brazil. The genomic diversity and the dynamics of CHIKV in the Brazilian Amazon region remains poorly understood despite its importance to better understand the epidemiological spread and public health impact of CHIKV in the country.\n\nMethodology/Principal FindingsWe report a large CHIKV outbreak (5,928 notified cases between August 2014 and August 2018) in Boa vista municipality, capital city of Roraimas state, located in the Brazilian Amazon region. In just 48 hours, we generated 20 novel CHIKV-ECSA genomes from the Brazilian Amazon region using MinION portable genome sequencing. Phylogenetic analyses revealed that despite an early introduction of the Asian genotype in 2015 in Roraima, the large CHIKV outbreak in 2017 in Boa Vista was caused by an ECSA-lineage most likely introduced from northeastern Brazil.\n\nEpidemiological analyses suggest a basic reproductive number of R0 of 1.66, which translates in an estimated 39 (95% CI: 36 to 45) % of Roraimas population infected with CHIKV-ECSA. Finally, we find a strong association between Google search activity and the local laboratory-confirmed CHIKV cases in Roraima.\n\nConclusions/SignificanceThis study highlights the potential of combining traditional surveillance with portable genome sequencing technologies and digital epidemiology to inform public health surveillance in the Amazon region. Our data reveal a large CHIKV-ECSA outbreak in Boa Vista, limited potential for future CHIKV outbreaks, and indicate a replacement of the Asian genotype by the ECSA genotype in the Amazon region.\n\nAuthor SummaryUntil the end of 2017, Brazil notified the highest number of infections caused by chikungunya virus (CHIKV) in the Americas. We investigated a large CHIKV outbreak in Boa vista municipality in the Brazilian Amazon region. Rapid portable genome sequencing of 20 novel isolates and subsequent genetic analysis revealed that ECSA lineage was introduced from northeastern Brazil to Roraima around July 2016. Epidemiological analyses suggest a basic reproductive number of R0 of 1.66, which suggests that approximately 39% of Roraimas population was infected with CHIKV-ECSA. Given the dominance of the CHIKV-Asian genotype in the Americas, our data highlights the rapid spread of a less understood and poorly characterized CHIKV-ECSA genotype in Brazil. Investigations on potential associations between public health impact of CHIKV and genetic diversity of circulating strains are warranted to better evaluate its impact in Brazil and beyond.

epidemiology↗

bioRxiv2017

A potential link between tuberculosis and lung cancer through non-coding RNAs

Pulmonary tuberculosis caused by Mycobacterium and lung cancer are two major causes of deaths worldwide and the former increases the risk of developing lung cancer. However, the precise molecular mechanism of Mycobacterium associated increased risk of lung cancer is not entirely understood. Here, using in silico approaches, we show that hsa-mir-21 and M. tuberculosis sRNA_1096 and sRNA_1414 could play important roles in the pathogenesis of both these diseases. Further, we postulated a \"Genetic remittance\" hypothesis where these sRNAs may play important roles. The sRNA_1096 could be involved in tuberculosis through multiple infectious processes, and if transferred to the host, it may activate the TLR8 mediated pro-metastatic inflammatory pathway by acting as a ligand to TLR8 similar to the mir-21 leading to lung tumorigenesis and chemo-resistance. Analogous to SH3GL1, it may also regulate cell cycle. On the other hand, sRNA_1414 is probably involved in survivability and drug response of the pathogen. However, it may be a metastatic factor for lung cancer providing EPS8L1 and SORBS1 like functions upon remittance. Further, all these three non-coding RNAs are predicted to act in rifampicin resistance in Mycobacterium. Currently, we are applying robust bioinformatics strategies and conducting experimental validations to confirm our in-silico findings and hypothesis.

cancer biology↗