Biology subjects

Colque, C. A.

Publications and source records attributed to Colque, C. A..

8 recordsLinked to original sources

bioRxiv2026

Host-microbe-immune interactions in an air-liquid interface airway model

BackgroundAir-liquid interface (ALI) cell culture systems have improved the study of host-microbe interactions in respiratory infections. However, most ALI models lack immune components, limiting their ability to capture epithelial-immune crosstalk. To address this, we developed a dual-cell ALI model incorporating human peripheral blood monocyte-derived macrophages beneath differentiated airway epithelial cells. MethodologyMacrophages were seeded on the basolateral side of transwell inserts using fibronectin coating. Model characterization included transepithelial electrical resistance (TEER) to assess epithelial barrier integrity, IL-8 secretion as a marker of epithelial inflammatory signaling, and confocal microscopy to evaluate cellular architecture before and after infection. Mono-and dual-cell cultures were infected with the laboratory strain Pseudomonas aeruginosa PAO1. ResultsMacrophages adhered stably to the basolateral surface without compromising epithelial barrier integrity. Following infection, IL-8 secretion was elevated in epithelial monocultures compared to dual-cell cultures, suggesting early immune modulation in the presence of macrophages. While overall bacterial burden was comparable, confocal imaging revealed clustered bacterial growth in monocultures and a more dispersed spatial distribution in dual-cell cultures. ConclusionsThis dual-cell ALI model enables investigation of early epithelial-immune interactions, inflammatory modulation, and bacterial colonization dynamics during airway infection. The system provides a versatile and human-relevant platform for studying respiratory host-pathogen interactions.

cell biology↗

bioRxiv2025

In situ profiling of bacterial growth activity during the early stages of Pseudomonas aeruginosa infection in airway epithelia

Pseudomonas aeruginosa is an opportunistic pathogen of major clinical importance, which frequently gives rise to persistent antibiotic resilient infections. To investigate the connection between the bacterial physiological activity and the host environment during the early stages of bacterial infections, we have established a dual-fluorescent reporter system to monitor growth activity of P. aeruginosa during infection of a human airway epithelial model. This approach enables quantitative and spatially resolved analysis of bacterial growth within distinct infection micro-niches. Using this infection model, we compared the infection routes for the reference strain PAO1 as well as for two patho-adaptive mutants, PAO1{Delta}pscC ({Delta}pscC) and PAO1{Delta}mexZ ({Delta}mexZ). All three strains colonized apical-, intracellular-, interepithelial-, and epithelial barrier breach sites, but with strain-specific patterns of localisation. PAO1 was rarely observed intracellularly,{Delta} pscC was detected in few cases at epithelial-barrier breach- and interepithelial sites, and{Delta} mexZ colonized apical and intracellular niches in only few cases. Measurements of bacterial growth activities in these niches further revealed distinct hierarchies of bacterial growth activity among the tissue sites: PAO1 bacteria were most active at epithelial-barrier breach sites,{Delta} pscC at interepithelial sites, and{Delta} mexZ in intracellular niches. Taken together, these data support a model in which P. aeruginosa follows a progressive infection continuum from apical colonization to barrier breach, involving interepithelial spread, whereas the mutant strains represented truncated or altered versions of this infection program. More broadly, this study demonstrates the utility of unstable fluorescent reporters for capturing dynamic, niche-specific growth activity patterns during host-pathogen interactions, with implications for both basic pathogenesis research and therapeutic development. Author SummaryIn this study, we set out to better understand how bacterial infections develop and progress on human tissues. To do so, we created fluorescent "reporter" strains of Pseudomonas aeruginosa, a bacterium that commonly infects the lungs of people with cystic fibrosis and other chronic lung diseases. These genetically modified bacteria allow us to directly observe their growth activity in living human cell cultures using advanced microscopy. Here, we can see how the bacteria grow and spread at different locations within the tissue, and if there are any sites they prefer relative to others. We can also see if those patterns change, depending on any mutations the bacteria might have. In the future, the same genetic engineering can be applied to other bacteria, and the fluorescence can inform us of a range of important bacterial functions; for instance, at precisely what point of the infection process - and where in the tissues - the bacteria produce toxins, are stressed, produce antibiotic-resistance proteins or divide, for example. Our experiments revealed that some genetic mutations, that are often found in bacterial isolates from hospital infections, change the preference of colonization to distinct tissue sites and confer specialized and unique patterns - and peaks - of growth activity. Some bacterial strains tend to grow on the surface of the tissue, while others are more likely to move between or inside human cells. These behaviours reflect how bacteria evolve during long-term infections and adapt to different environments within the body tissues. The approach we present here provides a new tool for studying how bacterial infections unfold and may ultimately help identify more effective ways to treat or control them.

microbiology↗

bioRxiv2025

Carbapenem-resistance oprD mutations reshape Pseudomonas aeruginosa host-pathogen interactions during infection

Antibiotic resistance is a major global health threat. While its role in reducing drug susceptibility is well established, the broader consequences of resistance mutations on bacterial physiology and behavior during infection remain poorly understood. Carbapenem-resistant Pseudomonas aeruginosa is considered among the highest- priority bacterial threats, with resistance commonly driven by loss-of-function mutations in the carbapenem uptake porin OprD. Here we show that such mutations can arise in clinical isolates even without prior carbapenem treatment, indicating that their effects during infection extend beyond antibiotic resistance. Consistent with this, we found that oprD mutants exhibit enhanced early attachment to and translocation across airway epithelial barriers in infection models, an effect maintained across strains with distinct clinical genomic backgrounds and infection dynamics. Our findings indicate that loss of OprD alters the bacterial outer membrane charge and reduces mucus entrapment, thereby facilitating epithelial barrier colonization. Overall, these results illustrate how antibiotic resistance mutations can directly shape infection dynamics, extending their impact well beyond antimicrobial susceptibility.

microbiology↗

bioRxiv2025

Infection dynamics and virulence potential of clinical Pseudomonas aeruginosa isolates in a human airway epithelium model system

Persistent bacterial infections constitute an increasing health problem, often associated with antibiotic resistance. However, despite extensive antibiotic treatment, Pseudomonas aeruginosa persists for decades in people with cystic fibrosis (pwCF), remaining susceptible. Host-pathogen interactions during infection may, therefore, be a major contributing factor to treatment failure. Using an infection model based on human airway epithelial cells, cultured at the air-liquid interface (ALI), we simulated the infection process of P. aeruginosa to investigate the colonization dynamics and virulence potential during infection in ALI models from non-CF and CF donors, and the BCi-NS1.1 cell line. Infections by reference strains and clinical isolates from pwCF revealed four infection clusters based on virulence, epithelial damage, and localization within the epithelium, in a strain-specific manner regardless of the type of ALI model or clonal lineage. Modulator treatment to restore CFTR channel function did not change infection patterns in CF ALI models. Dual RNA-seq revealed that bacterial colonization of ALI models significantly upregulated host inflammatory pathways, dependent on the strains virulence. Simultaneously, while bacterial gene expression was similar in non-CF and BCi-NS1.1 ALI models, CF models promoted differential regulation in a type III secretion mutant. Altogether, our results profile key infection dynamics occurring in pwCF and provide ground knowledge on the interplay between the airway epithelium and P. aeruginosa.

microbiology↗

bioRxiv2023

Cystic fibrosis systemic immune profile is associated with lung microbes and characterized by widespread alterations in the innate and adaptive immune compartments

Polymicrobial airway infections and detrimental inflammation characterize patients with cystic fibrosis (CF), a disease with heterogeneous clinical outcomes. How the overall immune response is affected in CF, its relationships with the lung microbiome, and the source of clinical heterogeneity are unclear. Our work identifies a specific CF immune profile characterized by widespread hyperactivation, enrichment of CD35+/CD49d+ neutrophils, and reduction in dendritic cells. Further, our data indicate signs of immune dysregulation due to alterations in Tregs homeostasis, which, together with an impaired B-cell immune function, are linked with patients lung function and are potentially the source of clinical heterogeneity. Indeed, clinical heterogeneity does not stem from a specific lung microbiome; yet, commensal bacteria correlate with higher concentrations of circulating immune cells and lower expression of leukocyte activation markers, a condition reversed by pathogenic microorganisms. Overall, our findings provide unique markers and immunomodulatory targets for improving the treatment of CF.

immunology↗

bioRxiv2023

Adaptive evolution of P. aeruginosa in human airways shows phenotypic convergence despite diverse patterns of genomic changes

Selective forces in the environment drive bacterial adaptation to novel niches, choosing the fitter variants in the population. However, in dynamic and changing environments, the evolutionary processes controlling bacterial adaptation are difficult to monitor. Here, we follow 9 cystic fibrosis patients chronically infected with Pseudomonas aeruginosa, as a proxy for bacterial adaptation. We identify and describe the bacterial changes and evolution occurring between 15 and 35 years of within host evolution. We combine whole genome sequencing, RNAseq and metabolomics, and compare the evolutionary trajectories directed by the adaptation of four different P. aeruginosa lineages to the lung. Our data suggest divergent evolution at the genomic level for most of the genes, with signs of convergent evolution with respect to acquisition of mutations in regulatory genes, which drive the transcriptional and metabolomic program at late time of evolution. Metabolomics further confirmed convergent adaptive phenotypic evolution as documented by reduction of the quorum sensing molecules acyl-homoserine lactone, phenazines and rhamnolipids (except for quinolones). The modulation of the quorum sensing repertoire suggests that similar selective forces characterize at late times of evolution independent of the patient. Collectively, our data suggest that similar environments and similar P. aeruginosa populations in the patients at prolonged time of infection are associated with an overall reduction of virulence-associated features and phenotypic convergence. SummarySelective forces in the human environment drive bacterial adaptation to novel niches, choosing the fitter variants in the population. We have investigated the evolutionary processes in 9 CF patients infected with Pseudomonas aeruginosa in the airways for several decades. To describe the within host evolution and trajectories of four different P. aeruginosa lineages to the lung environment we have combined whole genome sequencing, RNAseq and metabolomics. In this patient cohorte with persistent bacterial infections our data suggest that similar environments and similar P. aeruginosa populations in the patients at prolonged time of infection are associated with an overall reduction of virulence-associated features and phenotypic convergence

microbiology↗

bioRxiv2021

β-lactamase remodeling and evolution of collateral resistance in hypermutator Pseudomonas aeruginosa upon long-term antibiotic therapy

Antibiotic resistance development has been studied using approaches that range from laboratory experimental evolution, surveillance and epidemiology, to clinical isolate sequencing. However, evolutionary trajectories depend on the environment in which selection takes place, compelling to address evolutionary analyses in antibiotic-treated patients, to embrace the whole inherent environmental complexities as well as their dynamics over time. Herein, we address the complexity of the bacterial adaptive response to changing antibiotic selective pressures by studying the long-term in-patient evolution of a broad diversity of {beta}-lactam resistant Pseudomonas aeruginosa clones. By using mutational and ultra-deep amplicon sequencing, we analyzed multiple generations of a P. aeruginosa hypermutator strain persisting for more than 26 years of chronic infection in the airways of a cystic fibrosis (CF) patient. We identified the accumulation of multiple alterations targeting the chromosomally encoded class C {beta}-lactamase (blaPDC), providing structural and functional protein changes that resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance. This evolution was linked to the persistent treatment with ceftazidime, which we demonstrate selected for variants with robust catalytic activity against this expanded-spectrum cephalosporin. Surprisingly, "a gain of function" of collateral resistance towards ceftolozane, a more recently introduced cephalosporin that was not prescribed to this patient, was also observed and the biochemical basis of this cross-resistance phenomenon was elucidated. This work unveils the diversity of evolutionary trajectories driven by bacteria in the natural CF environmental setting, towards a multidrug resistant phenotype after years of antibiotic treatment against a formidable pathogen.

microbiology↗

bioRxiv2019

Hypermutator Pseudomonas aeruginosa exploits multiple genetic pathways to develop multidrug resistance during long term infections in the airways of cystic fibrosis patients

Pseudomonas aeruginosa exploits intrinsic and acquired resistance mechanisms to resist almost every antibiotic used in chemotherapy. Antimicrobial resistance in P. aeruginosa isolated from cystic fibrosis (CF) patients is further enhanced by the occurrence of hypermutator strains, a hallmark of chronic CF infections. However, the within-patient genetic diversity of P. aeruginosa populations related to antibiotic resistance remains unexplored. Here, we show the evolution of the mutational resistome profile of a P. aeruginosa hypermutator lineage by performing longitudinal and transversal analyses of isolates collected from a CF patient throughout 20 years of chronic infection. Our results show the accumulation of thousands of mutations with an overall evolutionary history characterized by purifying selection. However, mutations in antibiotic resistance genes appear to be positively selected, driven by antibiotic treatment. Antibiotic resistance increased as infection progressed towards the establishment of a population constituted by genotypically diversified coexisting sub-lineages, all of which converged to multi-drug resistance. These sub-lineages emerged by parallel evolution through distinct evolutionary pathways, which affected genes of the same functional categories. Interestingly, ampC and fstI, encoding the {beta}-lactamase and penicillin-binding protein 3, respectively, were found among the most frequently mutated genes. In fact, both genes were targeted by multiple independent mutational events, which led to a wide diversity of coexisting alleles underlying {beta}-lactam resistance. Our findings indicate that hypermutators, apart from boosting antibiotic resistance evolution by simultaneously targeting several genes, favor the emergence of adaptive innovative alleles by clustering beneficial/compensatory mutations in the same gene, hence expanding P. aeruginosa strategies for persistence.\n\nIMPORTANCEBy increasing mutation rates, hypermutators boost antibiotic resistance evolution by enabling bacterial pathogens to fully exploit their genetic potential and achieve resistance mechanisms for almost every known antimicrobial agent. Here, we show how co-existing clones from a P. aeruginosa hypermutator lineage that evolved during 20 years of chronic infection and antibiotic chemotherapy, converged to multidrug resistance by targeting genes from alternative genetic pathways that are part of the broad P. aeruginosa resistome. Within this complex assembly of combinatorial genetic changes, in some specific cases, multiple mutations are needed in the same gene to reach a fine tuned resistance phenotype. Hypermutability enables this genetic edition towards higher resistance profiles by recurrently targeting these genes, thus promoting new epistatic relationships and the emergence of innovative resistance-conferring alleles. Our findings help to understand this link between hypermutability and antibiotic resistance, a key challenge for the design of new therapeutic strategies.

microbiology↗