Intracellular Pseudomonas aeruginosa: An Overlooked Reservoir in the Lungs of People with Cystic Fibrosis?

People with the genetic disease cystic fibrosis (CF) are highly susceptible to chronic infections of the lower respiratory tract because of impaired clearance of viscous mucus associated with dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein (1). The CF lung microenvironment represents an ideal niche for the opportunistic pathogen Pseudomonas aeruginosa, which causes infections in more than 40% of adult patients (2) (Cystic Fibrosis Foundation, 2023) and often cannot be eradicated by available antimicrobials and the patient’s immune system (3). P. aeruginosa is considered a clinically important pathogen in CF because infections are associated with worse lung function, higher inflammation, and an enhanced risk of acute exacerbations and mortality (3). Despite the recent availability of highly effective CFTR modulator therapies, which improve host cell physiology at the level of CFTR protein function, chronic colonization of CF lungs by P. aeruginosa is not resolved in this patient population (4).

Since the first report of P. aeruginosa biofilms in the sputum of people with CF using electron microscopic imaging by Lam and colleagues over 40 years ago (5), most studies have confirmed an extracellular phenotype of this pathogen (either as planktonic cells or biofilm aggregates) after microscopic and biochemical investigation of CF lung explants and postmortem tissues (6–8). P. aeruginosa is nearly exclusively localized in airway luminal mucus rather than being associated with or invading airway tissue (9–11). Hence, this clinically important pathogen has historically been considered as an extracellular pathogen in the lungs of people with CF.

This paradigm is being challenged by intriguing novel insights presented by Malet and colleagues (pp. 1453–1462) in this issue of the Journal, where a comprehensive dataset provides evidence of intracellular presence of P. aeruginosa in airway epithelial cells of some people with CF (12) (main findings summarized in Figure 1). The breakthrough results of this study were generated using lung explants of seven people with CF who underwent lung transplant. All patients had a history of P. aeruginosa–positive sputum cultures, with at least one positive culture in the last 2 years before transplant. One of the strengths of this study is that complementary lines of evidence were collected to demonstrate the presence of P. aeruginosa intracellularly, including both culture-based methods for detecting viable and culturable bacteria inside airway epithelial cells and culture-independent (imaging) methods. Of particular interest is that thin-section immunohistochemistry (IHC) findings were validated with the tissue-clearing technique MiPACT (microbial identification after passive CLARITY technique) coupled with confocal scanning laser microscopy on thick tissue sections (1 mm³). The latter enabled in situ imaging of host–microbe interactions at the single-cell level in three dimensions, thereby maintaining spatial information of the native tissue.

Figure 1.

Main findings of the study by Malet and colleagues (11) describing the presence of intracellular Pseudomonas aeruginosa in airway epithelial cells of some people with cystic fibrosis (CF).

The authors found that three out of seven analyzed lung explants contained airway epithelial cells with intracellular P. aeruginosa using all employed methods. Notably, intracellular P. aeruginosa was rarely detected (an estimated 0.5–0.01% of epithelial cells were positive, on average), and single bacteria inside airway cells were typically found. On the basis of analysis of three lung lobes per patient using culture-based microbiological analysis, regional heterogeneity in the intracellular P. aeruginosa colony-forming units was observed. Although costaining of P. aeruginosa and markers of airway cell types was not performed to demonstrate whether invasion of this pathogen was cell type–specific, the authors report intracellular P. aeruginosa in both ciliated epithelial cells in the bronchi and multinucleated/polymorphonuclear cells in the lumen, based on IHC or MiPACT. Another interesting aspect of this study is that the airway perimeter was measured in all IHC tissue sections to understand if there is an association between airway size and the presence of intracellular P. aeruginosa. The measured perimeter ranged from 343 μm (small bronchioles) to 48,330 μm (bronchi). It was observed that airways which contained intracellular P. aeruginosa were 10-fold larger in perimeter than those that did not. Furthermore, airways with intracellular P. aeruginosa had a higher bacterial burden in the lumen but were not associated with higher levels of histopathological inflammation.

The results of this study are in line with previous observations in vitro using cell cultures and/or in animal models, where P. aeruginosa has been shown to invade epithelial cells of the lung, bladder, and cornea (13–15). Nevertheless, it remains to be determined whether insights from these model systems, such as those related to bacteria and host cell receptors/mediators involved in P. aeruginosa invasion, are applicable for the observations in this study. In this regard, it is important to note that mutations in genes encoding classical virulence factors involved in intracellular colonization, such as the type 3 secretion system, are frequently reported in P. aeruginosa CF isolates (16). Further investigation of P. aeruginosa phenotypes and genotypes that may be involved in the intracellular localization in CF airway epithelial cells is an exciting avenue of research, in particular because P. aeruginosa has been shown to diversify into variants during chronic infection that differ between spatially heterogeneous conditions in the lung (17). It is thus tempting to hypothesize that regional adaptation of P. aeruginosa may lead to variants with enhanced ability to invade the host (such as mutations in the gene encoding the type III secretion system negative regulator ExsD, which cause type 3 secretion hyperactivity) (17).

One of the limitations of this study is that lungs derived from patients with end-stage disease were analyzed, which questions the generalizability of the findings for people with CF who have milder disease or for patients without CF with chronic P. aeruginosa infections, such as bronchiectasis. Also, this study was conducted before the broad availability of highly effective CFTR modulator therapies. Hence, the question arises whether intracellular P. aeruginosa can be observed in the current CFTR modulator era, in particular because the intracellular phenotype was found to be associated with higher bacterial load, and P. aeruginosa load is often reduced with CFTR modulators (18), even though contradictory results have been reported (4).

Taken together, this breakthrough work opens up many new research questions and may influence the development of antimicrobials against P. aeruginosa. It goes without saying that strategies targeting planktonic and biofilm bacteria that are located extracellularly in the mucus layer are of major importance to tackle chronic infections in CF. Yet, the study by Malet and colleagues now also supports the importance of finding ways to easily detect and eradicate tissue-associated P. aeruginosa, because it may act as a reservoir for persistent infections.