Biology subjects

La Rosa, R.

Publications and source records attributed to La Rosa, R..

9 recordsLinked to original sources

bioRxiv2026

Human airway model reveals host-pathogen co-infection dynamics of Staphylococcus aureus and Pseudomonas aeruginosa

Polymicrobial airway infections, particularly those involving Staphylococcus aureus and Pseudomonas aeruginosa, are a hallmark of severe chronic respiratory diseases such as those in people with cystic fibrosis (pwCF). Yet, the mechanisms enabling their co-existence remain poorly understood. Using a human air-liquid interface (ALI) airway model, we dissected host-pathogen and interspecies dynamics during mono- and co-infections with clinically relevant bacterial pathogens. We found that S. aureus strains exhibited distinct colonization patterns and virulence profiles, with the deletion of the agr quorum sensing locus leading to attenuated epithelial disruption. Additionally, the H1 component of P. aeruginosas type VI secretion system was critical for competitive exclusion of S. aureus, its deletion allowing for occasional co-localization of both species. Importantly, co-infection timing strongly influenced pathogen dominance and epithelial damage, with pre-colonization of ALI models with S. aureus prior to P. aeruginosa infection, mimicking clinical observations in CF. Our results reveal that strain-specific traits and secretion systems influence microbial competition and persistence in the airway and underscore the importance of physiologically relevant models for studying chronic infections. Moreover, they provide the foundation for exploring therapeutic strategies for polymicrobial infections that account for the colonization sequence and microbial evolution for treatment design.

microbiology↗

bioRxiv2025

Immune protection and disease tolerance in Pseudomonas aeruginosa infections by pyruvate cross-feeding

Chronic infections by Pseudomonas aeruginosa in people with cystic fibrosis are characterized by persistent inflammation and oxidative stress, yet the mechanisms enabling bacterial persistence are not fully understood. Here, we identify a novel persistence mechanism mediated by pyruvate secretion resulting from mutations in the pyruvate dehydrogenase complex in clinical isolates of P. aeruginosa, with analogous mutations also identified in Staphylococcus aureus, Haemophilus influenzae and Stenotrophomonas maltophilia. These mutations lead to elevated extracellular pyruvate which helps the establishment a more tolerogenic infection microenvironment and favor bacterial persistence. Pyruvate exerts multiple roles: scavenges reactive oxygen species such as H2O2, suppresses host immune activation both in airway epithelial cells and macrophages, and increases bacterial survival during phagocytosis. This metabolic crosstalk promotes bacterial persistence while limiting host tissue damage, aligning with the concept of disease tolerance. Our findings reveal pyruvate as a bacterial immunometabolite that mimics host antioxidant defenses, reshaping the infection niche to favor long-term colonization. This work highlights the broader role of secreted metabolites in host-pathogen interactions and suggests new strategies targeting metabolic pathways to manage chronic infections.

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↗

bioRxiv2024

Macrolide resistance through uL4 and uL22 ribosomal mutations in Pseudomonas aeruginosa

Macrolides are widely used antibiotics for the treatment of bacterial airway infections. Due to its elevated minimum inhibitory concentration in standardized culture media, Pseudomonas aeruginosa is considered intrinsically resistant and, therefore, antibiotic susceptibility testing against macrolides is not performed. Nevertheless, due to macrolides immunomodulatory effect and suppression of virulence factors, they are used for the treatment of persistent P. aeruginosa infections. Here, we demonstrate that macrolides are, instead, effective antibiotics against P. aeruginosa airway infections in an air-liquid interface (ALI) infection model system resembling the human airways. Importantly, macrolide treatment in both people with cystic fibrosis and primary ciliary dyskinesia patients leads to the accumulation of uL4 and uL22 ribosomal protein mutations in P. aeruginosa which causes antibiotic resistance. Consequently, higher concentrations of antibiotics are needed to modulate the macrolide-dependent suppression of virulence. Surprisingly, even in the absence of antibiotics, these mutations also lead to a collateral reduction in growth rate, virulence and pathogenicity in airway ALI infections which are pivotal for the establishment of a persistent infection. Altogether, these results lend further support to the consideration of macrolides as de facto antibiotics against P. aeruginosa and the need for resistance monitoring upon prolonged macrolide treatment.

microbiology↗

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↗

bioRxiv2023

Detuning of the Ribosome Conformational Landscape Promotes Antibiotic Resistance and Collateral Sensitivity

Around 50% of the current antibiotic arsenal targets the ribosome, thus resistance to ribosome-targeting antibiotics poses severe challenges to antimicrobial treatments. Here, we characterize a 12-nucleotide deletion in the rplF gene encoding the uL6 ribosomal protein, which was identified in a tobramycin-resistant strain of Pseudomonas aeruginosa isolated from a cystic fibrosis patient. To understand this resistance, we determined 87 cryo-EM structures of wild-type and mutant ribosomes characterizing their conformational landscape. Our analysis reveals how detuning of the ribosome dynamics alters its rotational movement circumventing tobramycin inhibition. The mutation compromises the 50S assembly, triggering structural instability and inducing a different rotational dynamic of the 70S. We found 4 new binding sites of tobramycin, one of them exclusive of the mutant, where the binding of the antibiotic acts as an allosteric activator skipping inhibition. Our data also illustrate how chloramphenicol stabilizes the mutant ribosome, enhancing inhibition and thereby leading to collateral sensitivity.

biophysics↗

bioRxiv2023

Metabolic specialization drives reduced pathogenicity in Pseudomonas aeruginosa CF clinical isolates

Metabolism provides the foundation for all cellular functions. During persistent infections, in adapted pathogenic bacteria metabolism functions radically differently compared with more naive strains. Whether this is simply a necessary accommodation to the persistence phenotype or if metabolism plays a direct role in achieving persistence in the host is still unclear. Here, we characterize a convergent shift in metabolic function(s) linked with the persistence phenotype during Pseudomonas aeruginosa colonization in the airways of people with cystic fibrosis. We show that clinically-relevant mutations in the key metabolic enzyme, pyruvate dehydrogenase, lead to a host-specialized metabolism together with a lower virulence and immune response recruitment. These changes in infection phenotype are mediated by impaired type III secretion system activity and by secretion of the antioxidant metabolite, pyruvate, respectively. Our results show how metabolic adaptations directly impinge on persistence and pathogenicity in this organism.

microbiology↗

bioRxiv2022

Macrolide therapy in Pseudomonas aeruginosa infections causes uL4 ribosomal protein mutations leading to high-level resistance

BackgroundCystic fibrosis (CF) patients have reduced mucociliary clearance resulting in recurring and chronic bacterial lung infections. Pseudomonas aeruginosa is one of the most common pathogens to colonize the airways of CF patients and can persist in the lungs for decades. CF patients infected with P. aeruginosa are treated with macrolides to inhibit quorum sensing, mucoidity and has additional immunomodulatory effects. However, according to the EUCAST committee, P. aeruginosa is not susceptible to macrolides leaving resistance mechanisms largely overlooked. MethodsUsing a modified susceptibility testing protocol, P. aeruginosa isolates harbouring a mutated uL4 ribosomal protein were tested for resistance against macrolide antibiotics. Quorum sensing related properties, alterations in proteome composition and ribosome subunits distribution were further analysed to characterize the effect of the uL4 mutations on the physiology of the bacteria. FindingsSeveral uL4 mutations were identified in isolates from P. aeruginosa collections from various sources and geographical locations. Most of them mapped to the conserved loop region of uL4 and resulted in increased survival upon macrolide exposure. uL4 mutations did not negatively impact the physiology of the bacteria and greater concentrations of antibiotic were needed to inhibit the growth, reduce swimming motility, and induce redox sensitivity. Proteome analysis revealed that pathways involved in ribosome adaptation displayed altered expression levels possibly to compensate for the uL4 mutations, which changed the subunit distribution of the ribosome. InterpretationMacrolides used against P. aeruginosa cause selection of macrolide resistant mutants which is a widespread - but uncharacterized - phenomenon. Using a modified susceptibility test, revealed that macrolides are indeed effective bacteriostatic antibiotics against P. aeruginosa and that this effect - along with macrolide-induced quorum sensing modulation - are drastically reduced in uL4 mutants. Macrolide antibiotics should, therefore, be considered as active antimicrobial agents against P. aeruginosa and resistance development should be contemplated especially when patients are treated with prolonged courses of macrolides. FundingCystic Fibrosis Foundation, Independent Research Fund Denmark, Novo Nordisk Foundation Research in contextO_ST_ABSEvidence before this studyC_ST_ABSMacrolide antibiotics are readily prescribed as anti-inflammatory therapy against Pseudomonas aeruginosa infections in cystic fibrosis patients. This treatment strategy has largely overlooked the direct antimicrobial effect of this drug class on this pathogen, which is considered non-susceptible according to the European Committee on Antimicrobial Susceptibility Testing. This is because standardized antimicrobial susceptibility testing is sub-optimal for the quantification of macrolide minimum inhibitory concentrations due to the interference of the growth medium. Mutations in ribosomal RNA and increased efflux has been shown to reduce the effect of macrolides on P. aeruginosa, however, the involvement of ribosomal proteins has not been investigated in P. aeruginosa. Work done in other bacterial species such as Escherichia coli, Streptococcus pneumoniae, Legionella pneumophila and Neisseria gonorrhoeae has identified the ribosomal proteins uL4 and uL22 as targets for evolved macrolide resistance in vitro and in vivo. Especially the extended loop region of uL4 has been identified as important for macrolide susceptibility. We found no other studies published addressing the emergence of macrolide resistance through uL4 or uL22 mutations in P. aeruginosa. Added value of this studyWe present evidence that macrolide antibiotics used as anti-inflammatory agents and bacterial modulators against P. aeruginosa infection in cystic fibrosis (CF) patients cause the emergence of resistant strains through mutations in the ribosomal protein uL4. The importance of this finding is underlined by the identification of uL4 mutations in multiple strains not only within our strain collection comprised of CF associated isolates but throughout all available sequences of clinical P. aeruginosa isolates spanning several continents and different infection types. uL4 mutations resulted in significantly reduced susceptibility towards macrolide antibiotics with respect to bacteriostatic and quorum sensing modulation effects. The generation time in presence of macrolide antibiotics was unaffected in strains harbouring uL4 mutations, while it was reduced in wild type strains indicating a fitness advantage of the mutation. Implications of all the available evidenceThe uL4 mutants identified in this study were significantly more resistant towards macrolide antibiotics than strains harbouring a wild type uL4 hereby revealing that not only are macrolides effective as antimicrobial agents against P. aeruginosa but also that the imposed selective pressure causes resistant mutants to arise through mutations in the ribosomal uL4 protein. These mutations are also present but uncharacterized in other collections of clinical P. aeruginosa isolates from patient groups who typically receive long courses of macrolide treatment. The effect of the uL4 and possibly other hitherto unknown mutations on macrolide susceptibility can be determined via modification of the standard susceptibility testing protocol. Along with evidence describing other types of macrolide resistance mechanisms in P. aeruginosa such as rRNA modification and increased efflux activity, our results demonstrate that macrolide resistance development is widespread in P. aeruginosa and macrolide antibiotics should be considered as antimicrobial agents against P. aeruginosa with the same precautions being taken to avoid resistance development as for any other antimicrobial agent.

microbiology↗