Biology subjects

Sousa, F.

Publications and source records attributed to Sousa, F..

5 recordsLinked to original sources

bioRxiv2026

Integrated Resistome and Quantitative Proteomics Reveal Coordinated Resistance Architecture in MDR and XDR Gram-Negative ICU Pathogens

ObjectivesAntimicrobial resistance (AMR) in Gram-negative pathogens is driven by complex and coordinated molecular mechanisms that remain incompletely characterized. This study integrated phenotypic, genomic, and quantitative proteomic analyses to characterize multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria circulating in an intensive care unit (ICU) in Northeastern Brazil. MethodsA total of 259 Gram-negative isolates collected between 2019 and 2021 underwent species identification, antimicrobial susceptibility testing, and targeted qPCR for resistance genes. Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa representing susceptible, MDR, and XDR phenotypes were selected for whole-genome sequencing and label-free quantitative proteomics. Differential protein abundance was assessed using Limma with |log2FC| > 1 and p < 0.05. ResultsK. pneumoniae (47%), A. baumannii (24%), and P. aeruginosa (21%) predominated. Carbapenem resistance reached 44%, 93%, and 61%, respectively, and MDR/XDR phenotypes occurred in >30% of isolates. Genomic analyses revealed dense resistomes with coexisting {beta}-lactamases (blaKPC, blaNDM, blaCTX-M, OXA) and widespread efflux systems. Proteomic profiling demonstrated phenotype-associated differences in outer membrane proteins, transport systems, regulatory proteins, and metabolic pathways. XDR isolates showed additional enrichment of envelope remodeling proteins, stress response mechanisms, and proteostasis-associated factors. ConclusionsMDR and XDR Gram-negative ICU pathogens exhibit coordinated resistance architecture characterized by accumulation of resistance genes and adaptive proteomic remodeling. Integrated multi-omics approaches provide mechanistic insight into antimicrobial resistance and support improved surveillance and therapeutic strategies. What is known?O_LIAntimicrobial resistance is a priority and a serious problem in global health, resulting in high rates of morbidity and mortality. C_LIO_LIKlebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa are on the World Health Organizations (WHO) priority list as major causes of morbidity and mortality worldwide. C_LIO_LIClassical characterization of susceptibility and resistance phenotypes does not capture the complexity of antimicrobial resistance and hampers effective control measures and actions to minimize the evolutionary dynamics of resistance in these bacteria. C_LI What is new?O_LIThe study characterizes the phenotypic pattern of antimicrobial susceptibility, the presence and sequencing of the resistome and virulome, and analyzes the label-free quantitative proteome of susceptible, MDR, and XDR phenotypes in strains of K. pneumoniae, A. baumannii, and P. aeruginosa circulating in hospital ICUs in Brazil. C_LIO_LIMDR and XDR gram-negative phenotypes are associated with a dense resistome, with widespread dissemination of beta-lactamase genes (bla_KPC, bla_NDM, bla_CTX-M, and OXA) and RND-type (MEXs) and acrAB-tolC efflux pumps, without changes in virulence genes. C_LIO_LIProteomic analysis demonstrated increased production of beta-lactamases, components of efflux pump systems, outer membrane protein synthesis, protection for oxidative stress mechanisms, proteins for iron acquisition, and systemic regulators. XDR strains additionally showed enhanced remodeling of the cell envelope, activation of proteostasis, and metabolic adaptation. C_LI

microbiology↗

bioRxiv2025

Patient-derived organoids to study glycosylation dynamics during gastric disease

BACKGROUND AND AIMSAberrant cellular glycosylation remains a key event that accompanies and actively sustains gastric neoplastic transformation. Patient-derived organoids (PDOs) have recently emerged as a promising ex vivo model to study human gastric disorders. Since the PDOs glycosylation landscape remains unknown, this study aims to evaluate PDOs as potential avatars of in vivo tissue glycosylation profiles in the gastric context. METHODSFresh gastric mucosa samples derived from non-tumoral obese patients (n=11), adjacent tumor mucosa samples (n=29), and tumor tissue samples derived from gastric cancer (GC) patients (n=30) were used to establish a biobank of gastric PDOs (n=56). The N- and O-glycophenotypes of normal, adjacent, and tumor PDOs and respective in vivo tissues were thoroughly characterized by immunostaining. Additionally, a comparative glycan analysis was performed over time, upon PDO biobanking and xenografting in mice. The binding of two Helicobacter pylori (H. pylori) isogenic strains with distinct glycan-binding affinities was assessed in parental gastric mucosa tissues and compared with the respective PDOs before and after modulation of their glycan landscape. RESULTSOur results show that PDOs mimic different phenotypes of the carcinogenic cascade and recapitulate parental gastric tissues glycosylation profile. Tumor PDOs recapitulate the inter- and intra-heterogeneity features observed in GC, which is maintained over time, upon biobanking and xenografting. We demonstrated that the expression of type I and type II Lewis antigens is dynamically controlled by PDOs differentiation status, which results in differential binding to H. pylori strains displaying distinct glycan-binding adhesins, mirroring the gastric epithelium tissue interactions. CONCLUSIONSThis study established PDOs as invaluable ex vivo tools to study the complex glycan dynamics in both gastric physiological and pathological settings.

cell biology↗

bioRxiv2025

Gut3Gel: A High Throughput Mucus Model for Culturing Human Intestinal Microbiota

The human intestinal microbiota plays a crucial role in health and disease, yet recreating its complex interactions in vitro remains a significant challenge. Gut3Gel introduced herein as a novel in vitro mucus model, designed for culturing complex microbial communities without the need for anaerobic conditions. Intestinal microbiota samples from five donors were individually inoculated in Gut3Gel and cultured for 72 hours. Taxonomic composition assessment revealed that Gut3Gel sustains diverse microbial species and particularly promotes the growth of mucus-associated bacteria including Bifidobacterium, Lactobacillus, and Faecalibacterium. Microbial metabolic activity within Gut3Gel was confirmed by the increased production of acetate and butyrate, as well as of exopolysaccharides. Gut3Gel reproduces physiological features of intestinal mucus, providing a reproducible and scalable culturing platform. These features make Gut3Gel a promising tool for advancing microbiota research with potential applications in drug screening, microbiome mining, and high throughput testing of microbiome-modulating molecules.

microbiology↗

bioRxiv2023

In Campylobacter jejuni a new type of chaperone receives heme b from ferrochelatase

Intracellular heme formation and trafficking are fundamental processes in living organisms. Three biogenesis pathways are used by bacteria and archaea to produce iron protoporphyrin IX (heme b) that diverge after the formation of the common intermediate uroporphyrinogen III (urogen III). In this work, we identify and provide a detailed characterization of the enzymes involved in the transformation of urogen III into heme. We show that in this organism operates the protoporphyrin-dependent pathway (PPD pathway), in which the last reaction is the incorporation of ferrous iron into the porphyrin ring by the ferrochelatase enzyme. In general, following this final reaction, little is known about how the formed heme b reaches the target proteins. In particular, the chaperons that are thought to be required to traffic heme for incorporation into hemeproteins to avoid the cytotoxicity associated to free heme, remain largely unidentified. We identified in C. jejuni a chaperon-like protein, named CgdH2, that binds heme with a dissociation constant of 4.9 {+/-} 1.0 {micro}M, a binding that is impaired upon mutation of residues histidine 45 and 133. We show that C. jejuni CgdH2 establishes protein-protein interactions with ferrochelatase, which should enable for the observed transfer of heme from ferrochelatase to CgdH2. Phylogenetic analysis revealed that C. jejuni CgdH2 is evolutionarily distinct from the currently known chaperones. Therefore, CgdH2 is a novel chaperone and the first protein identified as an acceptor of the intracellularly formed heme, thus enlarging our understanding of bacterial heme homeostasis.

biochemistry↗

bioRxiv2022

Targeted metagenomics using probe capture detects a larger diversity of nitrogen and methane cycling genes in complex microbial communities than traditional metagenomics

Microorganisms are key players in the global cycling of nitrogen (N) and carbon (C), controlling their availability and fluxes, including the emissions of the powerful greenhouse gases nitrous oxide (N2O) and methane (CH4). Characterizing the microbial functional guilds driving these processes is crucial for understanding ecosystem functioning and predicting their responses to environmental changes. Standard sequence-based characterization methods often reveal only a limited fraction of their diversity in nature because of their low relative abundance, the insufficient sequencing depth of traditional metagenomes of complex communities, and limitations in coverage of PCR-based assays. Here, we developed and tested a targeted metagenomics approach based on probe capture and hybridization to simultaneously characterize the diversity of multiple key metabolic genes involved in inorganic N and CH4 cycling. We designed comprehensive probe libraries for each of the 14 target marker genes comprising 264,000 unique probes. These probes were used to selectively enrich the target genes in shotgun metagenomic libraries. In validation experiments with the mock communities of known microorganisms, targeted metagenomics yielded gene profiles similar to those of the original communities. Only GC content had a small effect on probe efficiency, as low GC targets were less efficiently detected than those with high GC, within the mock communities. Furthermore, the relative abundances of the marker genes obtained using targeted or traditional shotgun metagenomics from agricultural and wetland soils were significantly correlated, indicating that the targeted approach did not introduce significant quantitative bias. In addition, using archaeal amoA genes as a case-study, targeted metagenomics identified substantially higher taxonomic diversity and a larger number of sequence reads per sample, yielding diversity estimates 28 or 1.24 times higher than shotgun metagenomics or amplicon sequencing, respectively. Notably, shotgun metagenomics detected only three out of the 84 amoA gene phylotypes detected using targeted metagenomics. Our results show that targeted metagenomics complements current approaches to characterize key microbial populations and functional guilds in biogeochemical cycles in different ecosystems, enabling more detailed, simultaneous characterization of multiple functional genes. Manuscript contribution to the fieldMetagenomic sequencing often yields limited numbers of sequences of rare microbial taxa or functional genes, preventing in-depth analyses of specific populations and functional groups. Amplicon-based approaches enable the higher diversity coverage of target populations, but the drawback is the difficulty in designing unbiased primers that cover the highest intra-group diversity. Targeted metagenomics overcomes these challenges and results in similar community structure as traditional amplicon sequencing, while expanding the sequence space in a less biased metagenomic-based approach. Therefore, targeted metagenomics is an invaluable tool for studying the diversity of specific populations within complex natural microbiomes. Here, we present and evaluate a probe library designed for targeted metagenomics of nitrogen and methane cycling genes in complex communities.

microbiology↗