Pseudomonas aeruginosa Can Degrade Interferon λ, Thereby Repressing the Antiviral Response of Bronchial Epithelial Cells

To the Editor:

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in defects in chloride and bicarbonate transport. Epithelial cells are the main cell type affected by this dysfunction with symptoms ranging from pancreatic malabsorption and biliary cirrhosis to bronchiectasis and small airway obstruction. The dominant pathology in the lung is a progressive respiratory impairment caused by thickened mucus secretion, failure to clear microorganisms, and generation of a toxic proinflammatory environment (Ratjen and others 2015). Respiratory disease due to chronic bacterial infection leads to early morbidity and mortality in 85% of CF patients (Barto and Flume 2010).

Pseudomonas aeruginosa is the most prevalent pathogenic bacterium in the lung of adult CF patients (Sherrard and others 2014). Infections caused by this organism begin as recurrent intermittent colonization of the airway that can initially be resolved by antibiotic therapy. However, these bacteria progressively adapt to the CF lung environment and establish chronic infections that eventually lead to respiratory failure and death. During the transition from intermittent to chronic infection, P. aeruginosa undergoes mutations and transcriptional alterations, including upregulation of antibiotic resistance factors and downregulation of virulence factors such as toxins and proteases (Nguyen and Singh 2006). Those proteases are known to target host proinflammatory cytokines such as interleukin (IL)-6 and IL-8, consequently, reducing host damage and cell recruitment. Therefore, their downregulation during establishment of persistent infection worsens inflammation and contributes to pathogenesis of chronic lung disease (LaFayette and others 2015).

Interferons (IFNs) are innate host mediators initially described as factors capable of “interfering” with viral replication. Although these cytokines play a role in viral infections, they also participate in immune responses to bacteria, fungi, and parasites, and display regulatory functions in cancer and autoimmunity. Three classes of IFNs have been identified: type I, type II, and type III. The latter, and most recently discovered category, is composed of 4 members in humans: IFN-λ1, IFN-λ2, IFN-λ3, and IFN-λ4. Their production is triggered after sensing of damage or pathogen-associated molecular patterns by pattern recognition receptors (PRRs). All 4 members of the IFN-λ family signal through a common receptor, IFN-λ receptor (IFNLR), mostly localized at mucosal barriers. Ligand binding activates a JAK/STAT-dependent pathway leading to the transcription of numerous interferon-stimulated genes (ISGs) that act as forward and feedback loop mediators as well as antiviral effectors (Syedbasha and Egli 2017).

Modulation of the type I IFN response has been demonstrated in the context of various infections. Examples include the NS1 protein from the influenza A virus, able to block IFN production through PRR inhibition and mRNA targeting (Wang and Fish 2017) and NSP4, a protease of the equine arteritis virus, that has been shown to antagonize type I IFN production by cleavage of the nuclear factor-κB (NF-κB) essential modulator (Chen and others 2019). In bacteria, Salmonella enterica can inhibit autophagy that also negatively regulates type I IFN induction (Owen and others 2016). Furthermore, Bacillus anthracis is known to alter IFN signaling by preventing lipopolysaccharide-mediated IFN regulatory factor3 (IRF3) production, a requirement for type I IFN production (Pellizzari and others 1999). Viruses have also been shown to modulate type III IFN response by blocking the nuclear translocation of IRF1 (Zhang and others 2018). However, little is known about the role of bacteria in counteracting the effects of type III IFN. A recent study provides evidence that bacteria are also capable of manipulating the type III IFN response. Sörensen et al. were able to demonstrate that AprA, a protease of P. aeruginosa, is capable of degrading IFN λ produced by virally infected airway epithelial cells (Fig. 1) (Sorensen and others 2020).

FIG. 1.

Pseudomonas aeruginosa secreted protease AprA degrades IFN-λ and reduces the antiviral response to RSV and hRV infection. Sörensen and others (2020) report that the protease AprA can inhibit the antiviral response of bronchial epithelial cells. When RSV or hRV infect the airway, they are sensed by endosomal PRRs of bronchial epithelial cells. This recognition triggers the activation and nuclear translocation of transcription factors such as IRF1, IRF3, IRF7, and NF-κB, leading to transcription, translation, and secretion of IFN-λ. The binding of this cytokine to its receptor (IFNLR) activates a JAK/STAT-dependent pathway that promotes the transcription of ISGs through binding to an ISRE. Those ISGs, which include MX1 and OAS1, have antiviral properties and decrease virus spread and cytopathic effects. However, specific strains of the bacterium P. aeruginosa, such as PAO1, can alter this mechanism. This microorganism produces the alkaline protease AprA, whose expression depends on the quorum sensing regulator LasR. After secretion through the T1SS, this enzyme degrades IFN-λ, thereby inhibiting downstream signaling through IFNLR, production of ISGs, and antiviral effects. hRV, human rhinovirus; IFN, interferon; IFNLR, IFN-λ receptor; IRF, IFN regulatory factor; ISG, interferon stimulated gene; ISRE, interferon stimulated response element; NF-κB, nuclear factor-κB; PRR, pattern recognition receptor; RSV, respiratory syncytial virus; T1SS, type I secretion system. Color images are available online.

In their study, Sörensen et al. used the conditioned medium (CM) from 2 different P. aeruginosa strains to treat airway epithelial cells infected with respiratory syncytial virus (RSV) or human rhinovirus (hRV). They observed that the CM from strain PA01 (PA01-CM), but not strain Boston, was able to inhibit the antiviral response of these cells, illustrated by a decrease in MX1 and OAS1 induction and an increase in viral load and viral-specific cytopathic effects. As MX1 and OAS1 are ISGs and IFN-λ was the main IFN induced by RSV, they investigated the effect of P. aeruginosa CM on IFN-λ induction and production. CM was shown to contain a secreted bacterial factor that could target and alter the IFN response. Incubation of PA01-CM with virally infected cells did not initially alter IFNL induction, yet, no IFN-λ protein was detectable in the supernatant of PA01-CM–treated cells, suggesting that inhibition could occur during translation, secretion, or directly target the IFN-λ protein. Downstream signaling was also impaired as STAT-1 phosphorylation was absent in cells incubated with PA01-CM. Addition of recombinant IFN-λ to cells incubated with PA01-CM did not alter downstream ISG expression, indicating that blockade of the IFN-λ receptor or destabilization of the cytokine were likely mechanisms.

Since P. aeruginosa is known to secrete a variety of proteases, they investigated its ability to degrade IFN-λ. Both strains of P. aerguginosa were able to degrade IFN-λ but PA01 displayed an increased degradation capacity with complete cytokine destruction after only 1 h of incubation. Casein degradation assays and zymography both indicated that PA01 had higher protease activity than the Boston strain, explaining the differential capacity of these 2 strains to inhibit the IFN response. Many proteases of P. aeruginosa are under the regulation of the quorum sensing regulator LasR, which is known to acquire mutations over time in CF patients. Sequencing of LasR from longitudinal CF isolates identified loss-of-function mutations in isolates unable to inhibit ISG transcription. The connection between LasR and protease production was confirmed using genetically defined mutations. The most important proteases regulated by LasR are AprA, LasA, LasB, and PrpL. Among all these protease mutants, only the aprA mutant was found to not suppress ISG induction.

Finally, to examine the biological significance of these findings, a collection of P. aeruginosa isolates from CF patients at various stages of infection were analyzed. A strong correlation between protease activity and IFN-λ degradation was observed, consistent with their earlier observations. They also noticed that P. aeruginosa isolates from intermittent infections had a higher propensity for IFN-λ degradation than strains from chronic infections, implying that CF patients with intermittent P. aeruginosa infection could be more susceptible to respiratory viral infections. Characterization of viruses from respiratory samples identified hRV as the most common virus present and it was found to be significantly higher in the sputum of CF patients intermittently infected with P. aeruginosa than in those of CF patients chronically infected by the bacterium. Furthermore, the hRV viral load was increased in samples of CF patients positive for P. aeruginosa at the time of sampling, indicating a possible symbiotic relationship between these organisms.

Although the modulation of type I IFN by viruses and bacteria has been reported, the ability to interfere with type III IFN by bacteria is not as well characterized. Prior studies had established that P. aeruginosa was capable of interfering with type III IFN production. The study by Chattoraj and others (2011) demonstrated inhibition of IFN-λ by P. aeruginosa CM in epithelial cells infected with hRV, but only in the context of primary CF cells. This study used a cell line that does not present mutations in the CFTR gene. CFTR mutations have been shown to impact Toll-like receptor (TLR) trafficking and inflammatory response (Bruscia and others 2011), therefore, IFN signaling could also be distinctly induced in CF cells. Several studies illustrate this point: for example, in Parker and others (2012), type I IFN signaling was found to be reduced in CF cells compared with wild-type cells. Nevertheless, the direct targeting and degradation of IFN-λ demonstrated in this article have not been described before, suggesting a unique and novel mechanism of action for AprA. However, AprA's proteolytic activity does not appear to be specific for IFN-λ. Several articles have linked it to the degradation of a variety cytokines, including IFN-γ and TNF-α (Kharazmi 1991), as well as complement inhibition (Laarman and others 2012) and modulation of TLR-mediated recognition of bacterial components (Casilag and others 2016).

The physiological relevance of the described mechanism remains to be shown. Indeed, no live bacteria were used in this model and physical interactions between bacteria and the epithelium could have an effect on host response (DiMango and others 1995). In addition, the CM used for the experiments is obtained by culturing bacteria for 5 days in closed-batch conditions. These might aid the production of quorum-sensing products, but the concentration of secreted and toxic factors at this late stationary phase stage is expected to be much higher than in the context of an in vivo infection. Sörensen et al. demonstrated that the AprA protease of P. aeruginosa can degrade IFN-λ, thereby downregulating ISG induction and the antiviral response. This study provides further evidence of the interaction between viruses and bacteria in the host and of their potential role in the lung of CF patients.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

Study in the laboratory was supported by NIH grant R01HL134870.