Evaluating Bacterial Pathogenesis Using a Model of Human Airway Organoids Infected with Pseudomonas aeruginosa Biofilms

Tang, Mingfang; Liao, Shumin; Qu, Jing; Liu, Yixin; Han, Shuhong; Cai, Zhao; Fan, Yunping; Yang, Liang; Li, Shuo; Li, Liang

doi:10.1128/spectrum.02408-22

Cited by 15 publications

(9 citation statements)

References 84 publications

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“…Still, further analyses are required to determine the cross-regulation between ribosome mutations and virulence and this can only be revealed by the use of complex model systems taking into account the complexity of an infection and the host-pathogen interactions. ALI airway models can fulfil such role and allow more in-depth investigations of antibiotic resistance, virulence and persistence 46,47 . For future advances, the integration of an active immune system will be beneficial for even closer to patient investigations.…”

Section: Discussionmentioning

confidence: 99%

Macrolide resistance through uL4 and uL22 ribosomal mutations inPseudomonas aeruginosa

Goltermann,

Laborda,

Irazoqui

et al. 2024

Preprint

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Macrolides are widely used antibiotics for the treatment of bacterial airway infections. Due to its elevated minimum inhibitory concentration in standardized culture media,Pseudomonasaeruginosa 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 persistentP. aeruginosainfections. Here, we demonstrate that macrolides are, instead, effective antibiotics againstP. aeruginosaairway 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 inP. aeruginosawhich 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 againstP. aeruginosaand the need for resistance monitoring upon prolonged macrolide treatment.

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Section: Discussionmentioning

confidence: 99%

Macrolide resistance through uL4 and uL22 ribosomal mutations inPseudomonas aeruginosa

Goltermann,

Laborda,

Irazoqui

et al. 2024

Preprint

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“…Previous attempts to co-culture P. aeruginosa in 2D have been performed using cancer cell lines [32][33][34][35][36]78,79 or primary human airway cultures 34,37 . The latter offers clear advantages over the former, because of the non-cancerous nature of the primary cultures.…”

Section: Discussionmentioning

confidence: 99%

“…To date, P. aeruginosa CF infection models vary from the study of P. aeruginosa isolates from CF subjects [19][20][21] to growing P. aeruginosa in vitro in artificial CF sputum and other bacterial media [22][23][24][25][26] , using various CF animal models [27][28][29] , working with ex vivo CF lungs 30,31 , or co-culture systems using cancer cell lines and primary cells [32][33][34][35][36][37] . Each of these models present distinct advantages and disadvantages 38 .…”

Section: Introductionmentioning

confidence: 99%

Establishment and characterization of a newPseudomonas aeruginosainfection model using 2D airway organoids and dual RNA sequencing

Pleguezuelos-Manzano

Beenker

Son

et al. 2023

Preprint

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Pseudomonas aeruginosa is a Gram-negative bacterium that is notorious for infections in the airway of cystic fibrosis (CF) subjects. Often, these infections become chronic, leading to higher morbidity and mortality rates. Bacterial quorum sensing (QS) coordinates the expression of virulence factors and the formation of biofilms at a population level. QS has become the focus of attention for development of alternatives to antimicrobials targeting P. aeruginosa infections. However, a better understanding of the bacteria-host interaction, and the role of QS in infection, is required. In this study, we set up a new P. aeruginosa infection model, using 2D airway organoids derived from healthy and CF individuals. Using dual RNA-sequencing, we dissected their interaction, focusing on the role of QS. As expected, P. aeruginosa induced epithelial inflammation. However, QS signaling did not affect the epithelial airway cells. The epithelium influenced several infection-related processes of P. aeruginosa, including metabolic changes, induction of type 3 and type 6 secretion systems (T3SS and T6SS), and increased expression of antibiotic resistance genes, including mexXY efflux pump and several porins. Interestingly, the epithelium influenced the regulation by QS of the type 2 (T2SS) and T6SS. Finally, we compared our model with in vivo P. aeruginosa transcriptomic datasets, from samples directly isolated from the airways of CF subjects. This shows that our model recapitulates important aspects of in vivo infection, like enhanced denitrification, betaine/choline metabolism, increased antibiotic resistance, as well as an overall decrease of motility-related genes. This relevant infection model is interesting for future investigations, helping to reduce the burden of P. aeruginosa infections in CF.

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“…In-frame deletion of target genes in PAO1 was performed using the suicide pK18 plasmid, as previously described 15,74 . Brie y, the upstream and downstream DNA fragments anking the targeted single nucleotide substitution, or the full-length gene of interest were PCR-ampli ed and cloned into the pK18 vector (linearized with BamHI and HindIII) containing a gentamycin (Gm) resistance gene using Gibson assembly (NEB, E5510S).…”

Section: Genetic Manipulationmentioning

confidence: 99%

Divergent molecular strategies drive convergent evolutionary adaptation to kin competition in biofilms

Tang,

Yang,

Han

et al. 2024

Preprint

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Biofilms are a hotspot of bacterial social interactions that are characterized by cooperation and competition. Even a mono-species biofilm would evolve and diversify into polymorphic subpopulations so that bacterial kins of different genotypes would compete for limited nutrient and space. However, the specific biological functions underlying biofilm diversification and competition adaptation are poorly demonstrated. Here, we launched and monitored the experimental evolution of Pseudomonas aeruginosa biofilms, finding that two competition-adaptive derivatives rapidly emerged with variable capacities in forming biofilm. Further investigations identified that two novel divergent molecular trajectories were adopted for convergent adaptation to kin competition: one involved hijacking bacteriophage superinfection to aggressively inhibit kin competitors, while the other induced a subtle change in c-di-GMP signaling to gain a positional advantage via enhanced early biofilm adhesion. Bioinformatics analyses implicated that similar evolutionary strategies were prevalent among clinical P. aeruginosa strains, indicative of parallelism between natural and experimental evolution. Divergence in the molecular bases illustrated the adaptive values of genomic plasticity for gaining competitive fitness in the biofilm. Finally, we demonstrated that these competition-adaptive mutations reduced bacterial virulence. Our findings showed how kin competition shaped the P. aeruginosa biofilm evolution. More importantly, it revealed new insights into molecular targets for the treatment of recalcitrant biofilm infections formed by this clinically relevant pathogen.

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Evaluating Bacterial Pathogenesis Using a Model of Human Airway Organoids Infected with Pseudomonas aeruginosa Biofilms

Abstract: Human airway organoids (HAOs) are an organotypic model of human airway mucociliary epithelium. The HAOs can closely resemble their origin organ in terms of epithelium architecture and physiological function.

Cited by 15 publications

References 84 publications

Macrolide resistance through uL4 and uL22 ribosomal mutations inPseudomonas aeruginosa

Macrolide resistance through uL4 and uL22 ribosomal mutations inPseudomonas aeruginosa

Establishment and characterization of a newPseudomonas aeruginosainfection model using 2D airway organoids and dual RNA sequencing

Divergent molecular strategies drive convergent evolutionary adaptation to kin competition in biofilms

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