Biology subjects

Chavez, F. P.

Publications and source records attributed to Chavez, F. P..

11 recordsLinked to original sources

bioRxiv2026

Linking immunity and nutrition: Vital DNA extracellular trap formation in Dictyostelium discoideum reveals an ancestral strategy for microbial management

Extracellular traps (ETs) were originally described in neutrophils as DNA-based structures that immobilize microbes and contribute to innate immunity. Subsequent studies revealed that ET formation occurs across diverse immune cell types and can proceed through non-lytic mechanisms involving mitochondrial DNA release. Whether ETosis also operates outside classical immune contexts and what its ancestral functions may be remain incompletely understood. Here, we show that vegetative, phagocytic cells of the social amoeba Dictyostelium discoideum, a professional bacterial predator with phagocytic mechanisms conserved with those of mammalian innate immune cells, deploy extracellular DNA traps in response to bacterial cues. ET formation is selectively induced by specific lipopolysaccharide variants, is not triggered by canonical neutrophil NET inducers, and occurs through a vital ETosis mechanism that preserves membrane integrity and feeding capacity. Ultrastructural analyses provide the first visualization of extracellular traps in Amoebozoa, revealing extracellular filamentous networks that physically capture bacteria. Molecular characterization demonstrates that amoeboid ETs are enriched in mitochondrial DNA and harbor a dynamic proteomic repertoire dominated by mitochondrial components, DNA-associated proteins, and multiple antibacterial effectors. Notably, ET composition varies with the bacterial stimulus, indicating that ETs are not static structures but rather responsive extracellular assemblies. Together, these findings establish ET formation as a regulated response in a unicellular phagocyte and suggest that extracellular traps may have originally functioned in microbial management during feeding, prior to their elaboration as immune effectors in multicellular organisms. HighlightsO_LIVegetative Dictyostelium discoideum cells deploy mitochondrial DNA-based extracellular traps in response to bacterial cues. C_LIO_LIET formation occurs through a vital, non-lytic mechanism that preserves membrane integrity and feeding capacity. C_LIO_LIExtracellular traps exhibit stimulus-dependent composition and are enriched in mitochondrial and antimicrobial proteins. C_LIO_LIETosis functions as an ancestral strategy for microbial containment and management beyond canonical immune contexts. C_LI

microbiology↗

bioRxiv2026

Intrinsic class C extended-spectrum β-lactamases mediate resistance to oxyimino-cephalosporins in Antarctic Pseudomonas fluorescens complex bacteria

From a One Health perspective, identifying environmental reservoirs of antibiotic resistance determinants with potential clinical relevance is increasingly important. Among resistance mechanisms, {beta}-lactamases are of particular concern because they compromise the efficacy of {beta}-lactam antibiotics, the most widely used class in clinical practice. Antarctic soils harbor thousands of putative {beta}-lactamase genes, yet the functional properties and resistance contributions of these enzymes remain largely unexplored. Here, we investigated the distribution, diversity, and functional impact of intrinsic class C {beta}-lactamases (AmpC-type) in Antarctic members of the Pseudomonas fluorescens species complex, a lineage widely distributed in Antarctic soils and increasingly recognized as an opportunistic pathogen of humans, animals, and plants. Genome-scale analyses revealed that class C {beta}-lactamases are intrinsic, widespread, and highly diverse within this lineage. Phenotypic assays demonstrated that Antarctic isolates exhibit elevated resistance to {beta}-lactam antibiotics, particularly oxyimino-cephalosporins such as cefotaxime and ceftazidime, and that this phenotype is largely attenuated by {beta}-lactamase inhibition. Heterologous expression of selected Antarctic AmpC variants in a susceptible Escherichia coli host confirmed their ability to increase minimum inhibitory concentrations to oxyimino-cephalosporins. Notably, several Antarctic {beta}-lactamases harbor amino acid substitutions previously associated with extended-spectrum AmpC (ESAC) variants, including M174L and N346I, while others display broader substrate profiles despite lacking known ESAC-associated signatures. Comparative analyses further showed that the sequence diversity of Antarctic class C {beta}-lactamases exceeds that reported for clinical PDC variants from Pseudomonas aeruginosa. Together, these findings provide functional evidence that pristine Antarctic environments constitute reservoirs of naturally occurring class C {beta}-lactamases with extended-spectrum potential. Our results highlight the evolutionary depth and functional diversity of environmental {beta}-lactamases and underscore the importance of incorporating remote ecosystems into One Health-oriented antimicrobial resistance surveillance frameworks.

microbiology↗

bioRxiv2025

Expanding the toolbox for hypervirulent Klebsiella pneumoniae using the social amoeba Dictyostelium discoideum as a virulence biosensor

Hypervirulent Klebsiella pneumoniae (hvKp) represents a critical global threat due to its capacity to cause severe, community-acquired infections in healthy individuals and its convergence with multidrug resistance. However, current methods to evaluate hvKp virulence are limited by their reliance on mammalian models, which are costly, ethically constrained, and unsuitable for high-throughput applications. Here, we introduce a biosensing approach using the social amoeba Dictyostelium discoideum as a living phenotypic sensor for hvKp virulence. Through a suite of quantitative assays integrating predation resistance, social development, and real-time single-cell motility tracking, we show that D. discoideum can discriminate avirulent, virulent, and hypervirulent K. pneumoniae strains. Using the model hvKp strain SGH10 and its capsule-null mutant {Delta}wcaJ, we demonstrate that capsule production is strongly associated with phagocytosis resistance, developmental arrest, and amoebae paralysis, all quantifiable through imaging and motility-based readouts. Multivariate data integration revealed a clear segregation of virulence phenotypes, validating D. discoideum as a sensitive, scalable, and genetically tractable biosensor for functional virulence detection. This work establishes a conceptual and methodological framework for developing cell-based biosensors that report virulence levels through measurable host behavioral responses and outlines a calibration path to vertebrate endpoints. These systems provide a low-cost alternative to mammalian systems, especially for early-stage evaluation, and a potential foundation for automated high-throughput screening and surveillance of virulence in critical priority bacterial pathogens.

microbiology↗

bioRxiv2025

A structure-guided pipeline to uncover the underexplored beta-lactamases from Antarctic and Subantarctic soil microbiota

As the One Health approach points out, the environment can significantly affect human health, particularly having diverse and profound implications in the ongoing antimicrobial resistance crisis. One central aspect is the potential role of environmental microorganisms as a source of resistance genes that could emerge among pathogens aided by horizontal transfer. In this context, previous reports showed that the Antarctic soil microbiota hosts a rich resistome, including putative beta-lactamases conferring resistance to beta-lactams, the most widely used antibiotics for treating bacterial infections worldwide. However, the diversity of beta-lactamases across different areas of Antarctica and Subantarctic islands and their associated microbiota remains largely unexplored. In this study, we analyzed an extensive collection of Antarctic soil metagenomes, applying a novel bioinformatic pipeline based on combining sequence identity and structural similarity criteria to search for bona fide distant beta-lactamase orthologs. We found several classes and families, with a notable predominance of class-B metallo-beta-lactamases, including proposed novel families and variants of known families. The beta-lactamase diversity and dominant classes varied across sites following changes in the microbial community, observing three main sample groups: 1) Subantarctic islands, 2) Antarctic Peninsula and surrounding islands; and 3) Cold-desert environments. The most prevalent beta-lactamases corresponded to subclass B3. We reconstructed more than 3500 metagenome-assembled genomes (MAGs) and searched for beta-lactamases among them. The main taxa encoding the beta-lactamases were Pseudomonadota and Bacteroidota. Moreover, a certain proportion of beta-lactamases was associated with mobile genetic elements, especially integrons and insertion sequences, suggesting their potential dissemination by horizontal transference. This evidence reinforces the role of the environment, and especially (sub)Antarctic soils, as a reservoir of resistance genes, in particular, beta-lactamases.

bioinformatics↗

bioRxiv2024

Life on the edge: microbial diversity, resistome, and virulome in soils from the Union Glacier cold desert

The high-latitude regions of Antarctica remain among the most remote, extreme, and least explored areas on Earth. Despite the highly restrictive conditions, microbial life has been found in these environments, although with limited information on their genetic properties and functional capabilities. Moreover, the accelerated melting of the Antarctic permafrost, the increasing exposure of soils, and the growing human transit pose the question of whether these environments could be a source of microbes or genes that could emerge and cause global health problems. In this line, although a high bacterial diversity and autochthonous multidrug-resistant bacteria have been found in soils of the Antarctic Peninsula, we still lack information regarding the resistome of areas closer to the South Pole. Moreover, no previous studies have evaluated the pathogenic potential of microbes inhabiting Antarctic soils. In this work, we combined metagenomic and culture-dependent approaches to investigate the microbial diversity, resistome, virulome, and mobile genetic elements (MGEs) in soils from Union Glacier, a high-latitude cold desert in West Antarctica. Despite the low organic matter content, diverse bacterial lineages were found, predominating Actinomycetota and Pseudomonadota, with limited archaeal and fungal taxa. We recovered more than 80 species-level representative genomes (SRGs) of predominant bacterial taxa and the archaeon Nitrosocosmicus sp. Diverse putative resistance and virulence genes were predicted among the SRGs, metagenomic reads, and contigs. Furthermore, we characterized bacterial isolates resistant to up to 24 clinical antibiotics, mainly Pseudomonas, Arthrobacter, Plantibacter, and Flavobacterium. Moreover, some isolates produced putative virulence factors, including siderophores, pyocyanins, and exoenzymes with hemolytic, lecithinase, protease, and DNAse activity. This evidence uncovers a largely unexplored resistome and virulome hosted by deep Antarcticas soil microbial communities and the presence of bacteria with pathogenic potential, highlighting the relevance of One Health approaches for environmental surveillance in the white continent. HIGHLIGHTS-Union Glacier soils host a microbial community dominated by bacteria, mainly from the phylum Actinomycetota. -Archaea from the Nitrosocosmicus genus (family Nitrosphaeraceae) were ubiquitously detected. -Although extreme and remote, these soils host multidrug-resistant and potentially pathogenic bacteria. Some were cultured and tested in vitro. -Metagenomes and species-level representative genomes revealed diverse putative resistance and virulence genes. -Part of the putative antimicrobial resistance genes and virulence factors could be associated with mobile elements in bacterial genomes.

microbiology↗

bioRxiv2023

Molecular Forecasting of Domoic Acid during a Pervasive Toxic Diatom Bloom

In 2015, the largest recorded harmful algal bloom (HAB) occurred in the Northeast Pacific, causing nearly 100 million dollars in damages to fisheries and killing many protected marine mammals. Dominated by the toxic diatom Pseudo-nitzschia australis, this bloom produced high levels of the neurotoxin domoic acid (DA). Through molecular and transcriptional characterization of 52 near-weekly phytoplankton net-tow samples collected at a bloom hotspot in Monterey Bay, California, we identified active transcription of known DA biosynthesis (dab) genes from the three identified toxigenic species, including P. australis as the primary origin of toxicity. Elevated expression of silicon transporters (sit1) during the bloom supports the previously hypothesized role of dissolved silica (Si) exhaustion in contributing to bloom physiology and toxicity. We find that co-expression of the dabA and sit1 genes serves as a robust predictor of DA one week in advance, potentially enabling the forecasting of DA-producing HABs. We additionally present evidence that low levels of iron could have co-limited the diatom population along with low Si. Iron limitation represents a previously unrecognized driver of both toxin production and ecological success of the low iron adapted Pseudo-nitzschia genus during the 2015 bloom, and increasing pervasiveness of iron limitation may fuel the escalating magnitude and frequency of toxic Pseudo-nitzschia blooms globally. Our results advance understanding of bloom physiology underlying toxin production, bloom prediction, and the impact of global change on toxic blooms. SignificancePseudo-nitzschia diatoms form oceanic harmful algal blooms that threaten human health through production of the neurotoxin domoic acid (DA). DA biosynthetic gene expression is hypothesized to control DA production in the environment, yet what regulates expression of these genes is yet to be discovered. In this study, we uncovered expression of DA biosynthesis genes by multiple toxigenic Pseudo-nitzschia species during an economically impactful bloom along the North American West Coast, and identified genes that predict DA in advance of its production. We discovered that iron and silica co-limitation restrained the bloom and likely promoted toxin production. This work suggests that increasing iron limitation due to global change may play a previously unrecognized role in driving bloom frequency and toxicity.

ecology↗

bioRxiv2023

Dictyostelium discoideum-assisted pharmacognosy of plant resources for discovering antivirulence molecules targeting Klebsiella pneumoniae

The rise of antibiotic-resistant bacterial strains poses a significant global health challenge, underscoring the critical need for innovative strategies to address this threat. Natural products and their derivatives have emerged as a promising reservoir for drug discovery. The social amoeba Dictyostelium discoideum is an advantageous model organism in this effort. Using this invertebrate model, we introduce a novel perspective to screen natural plant extracts for molecules with potential antivirulence activity. As a proof of concept, we established a simple high-throughput assay to screen for antivirulence molecules targeting Klebsiella pneumoniae among extracts of Helenium aromaticum. Thus, we aimed to identify compounds attenuating K. pneumoniae virulence without inducing cytotoxic effects on amoeba cells. Notably, the methanolic root extract of H. aromaticum but not other extracts fulfilled these prerequisites. Further analysis via UHPLC-ESI-MS/MS led to the identification of 24 chemical compounds boasting potential antivirulence attributes. This research underscores the potential of employing D. discoideum-assisted pharmacognosy for unearthing novel antivirulence agents against multidrug-resistant pathogens. Table of Content Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/564015v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@17530faorg.highwire.dtl.DTLVardef@e28259org.highwire.dtl.DTLVardef@dbc87eorg.highwire.dtl.DTLVardef@147de2d_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Effects of Polyphosphate metabolism Mutations on Biofilm, Capsule Formation and Virulence Traits in Hypervirulent ST23 Klebsiella pneumoniae SGH10

The emergence of hypervirulent Klebsiella pneumoniae (hvKP) strains poses a significant threat to public health due to their high mortality rates and propensity to cause severe community-acquired infections in otherwise healthy individuals. The ability of hvKP to form biofilms and produce a protective capsule contributes to its enhanced virulence and is a significant challenge to effective antibiotic treatment. Therefore, understanding the molecular mechanisms underlying hvKP virulence and biofilm formation is crucial for developing new therapeutic strategies. Polyphosphate Kinase 1 (PPK1) is an enzyme responsible for inorganic polyphosphate synthesis and plays a vital role in regulating various physiological processes in bacteria. In this study, we investigated the impact of polyP metabolism on the biofilm and capsule formation and virulence traits in hvKP using Dictyostelium discoideum amoeba as a model host. We found that the PPK1 null-mutant was impaired in biofilm and capsule formation and showed attenuated virulence in D. discoideum compared to the wild-type strain. We performed a shotgun proteomic analysis of the PPK1 mutant and wild-type strain to gain further insight into the underlying molecular mechanism. The results revealed that the PPK1 mutant had a differential expression of proteins (DEP) involved in capsule synthesis (Wzi - Ugd), biofilm formation (MrkC-D-H), synthesis of the colibactin genotoxin precursor (ClbB), as well as proteins associated with the synthesis and modification of lipid A (ArnB -LpxC - PagP). These proteomic findings corroborate the phenotypic observations and indicate that the PPK1 mutation is associated with impaired biofilm and capsule formation and attenuated virulence in hypervirulent K. pneumoniae. Overall, our study highlights the importance of polyP synthesis in regulating extracellular biomolecules and virulence in K. pneumoniae and provides insights into potential therapeutic targets for treating K. pneumoniae infections.

microbiology↗

bioRxiv2022

Population genomics, resistance, pathogenic potential, and mobile genetic elements of carbapenem-resistant Klebsiella pneumoniae causing infections in Chile

Multidrug and carbapenem-resistant K. pneumoniae (CR-Kp) are considered critical threats to global health and key traffickers of resistance genes to other pathogens. In Chile, although a sustained increase in CR-Kp infections has been observed, few strains have been described at the genomic level, lacking molecular details of their resistance and virulence determinants and the mobile elements mediating their dissemination. In this work, we studied the antimicrobial resistance and performed a comparative genomics analysis of ten CR-Kp isolates from the Chilean surveillance of carbapenem-resistant Enterobacteriaceae. High resistance to most of the antibiotics tested was observed among the isolates, five ST25, three ST11, one ST45, and one ST505, which harbored a total of 44 plasmids, many of them predicted to be conjugative and carrying genes conferring resistance to a variety of antibiotic, metals, and disinfectants. Ten plasmids encoding either KPC-2, NDM-1, or NDM-7 carbapenemases were characterized, including novel plasmids with increased resistance gene load and a novel genetic environment for blaKPC-2 gene. This corresponds to the first report of ST25 and ST45 Kp producing NDM-7 in South America, and of an ST505 CR-Kp worldwide, producing both NDM-7 and KPC-2. Moreover, we characterized a variety of genomic islands carrying virulence and fitness factors. These results provide baseline knowledge for the detailed understanding of molecular and genetic determinants behind antibiotic resistance and virulence of K. pneumoniae in Chile and South America.

microbiology↗

bioRxiv2021

Mapping the microbial diversity and natural resistome of north Antarctica soils

The rising of multiresistant bacterial pathogens is currently one of the most critical threats to global health, demanding a better understanding of the origin and spread of antibiotic resistance. In this regard, the resistome hosted by the microbiota from natural and remote environments remains poorly explored. Moreover, little is known about the availability of antimicrobial resistance genes (ARGs) from these environments to be disseminated through horizontal transfer, potentially mediating the rise of novel resistance factors among clinically relevant pathogens. In this context, the North Antarctica soils are attractive ecosystems to study due to the presence of a microbiota naturally adapted to thrive in harsh conditions, including potential factors to resist natural toxic substances. In this work, we evaluated the antibiotic resistance of bacteria isolated from soils collected in humanized and non-intervened areas of North Antarctica. We identified resistance to a wide array of antibiotics, with isolates harboring up to 10 simultaneous resistances, mainly native Pseudomonas. Genomic analysis revealed the presence of a wide array of genes encoding efflux pumps but the lack of genes explaining some of the resistance phenotypes, suggesting novel uncharacterized mechanisms. Also, using 16S rRNA amplicon and shotgun metagenome sequencing, we explored the microbial diversity in the sampled soils and evaluated the presence of ARGs and their host microbiota. High microbial diversity was found in all the sites, with Proteobacteria, Bacteroidota, Acidobacteriota, and Verrucomicrobiota being the most abundant Phyla, while Candidatus Udaeobacter, RB41, Polaromonas, and Ferruginibacter the most abundant genera. We identified hundreds of genes potentially conferring resistance to more than 15 drug classes, both by short reads analyses and ARG detection among assembled contigs and MAGs obtained combining short and long-read sequence data. Polaromonas, Pseudomonas, Streptomyces, Variovorax, Bhurkolderia, and Gemmatimonas were the main host taxa of the identified ARGs. Part of these ARGs was found inside predicted plasmids, including a putative OXA-like beta-lactamase from Polaromonas harboring the key conserved residues of this kind of enzyme and a conserved predicted protein structure. All this evidence indicates that microbial communities from North Antarctica soil have a highly diverse natural resistome, part of it located inside mobile genetic elements, which would act as a source of novel ARGs.

microbiology↗

bioRxiv2017

In Vivo Host-Pathogen Interaction As Revealed By Global Proteomic Profiling Of Zebrafish Larvae

The outcome of a host-pathogen interaction is determined by the conditions of the host, the pathogen, and the environment. Although numerous proteomic studies of in vitro-grown microbial pathogens have been performed, in vivo proteomic approaches are still rare. In addition, increasing evidence supports that in vitro studies inadequately reflect in vivo conditions. Choosing the proper host is essential to detect the expression of proteins from the pathogen in vivo. Numerous studies have demonstrated the suitability of zebrafish (Danio rerio) embryos as a model to in vivo studies of Pseudomonas aeruginosa infection. In most zebrafish-pathogen studies, infection is achieved by microinjection of bacteria into the larvae. However, few reports using static immersion of bacterial pathogens have been published. In this study we infected 3 days post-fertilization (DPF) zebrafish larvae with P. aeruginosa PAO1 by immersion and injection and tracked the in vivo immune response by the zebrafish. Additionally, by using non-isotopic (Q-exactive) metaproteomics we simultaneously evaluated the proteomic response of the pathogen (P. aeruginosa PAO1) and the host (zebrafish). We found some zebrafish metabolic pathways, such as hypoxia response via HIF activation pathway, exclusively enriched in the larvae exposed by static immersion. In contrast, we found that inflammation mediated by chemokine and cytokine signaling pathways was exclusively enriched in the larvae exposed by injection, while the integrin signaling pathway and angiogenesis were solely enriched in the larvae exposed by immersion. We also found important virulence factors from P. aeruginosa that were enriched only after exposure by injection, such as the Type-III secretion system and flagella-associated proteins. On the other hand, P. aeruginosa proteins involved in processes like biofilm formation, cellular responses to antibiotic and starvation were enriched exclusively after an exposure by immersion.\n\nWe demonstrated the suitability of zebrafish embryos as a model for in vivo host-pathogen based proteomic studies in P. aeruginosa. Our global proteomic profiling identifies novel molecular signatures that give systematic insight into zebrafish-Pseudomonas interaction.

microbiology↗