A truncated mutation of MucA in Pseudomonas aeruginosa from a bronchiectasis patient affects T3SS expression and inflammasome activation

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic airway infection in bronchiectasis patients and is closely associated with poor prognosis. Strains isolated from chronically infected patients typically have a mucoid phenotype due to the overproduction of alginate. In this study, we isolate a P. aeruginosa strain from the sputum of a patient with bronchiectasis and find that a truncated mutation occurred in mucA, which is named mucA117. mucA117 causes the strain to transform into a mucoid phenotype, downregulates the expression of T3SS and inflammasome ligands such as fliC and allows it to avoid inflammasome activation. The truncated mutation of the MucA protein may help P. aeruginosa escape clearance by the immune system, enabling long-term colonization.

Introduction

Pseudomonas aeruginosa is an opportunistic pathogen that can cause acute and chronic infections in humans, particularly in patients with compromised immune defense. Acute infections usually include respiratory and urinary tract infections, such as ventilator-associated pneumonia [1]. Chronic infections usually occur in patients with cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and bronchiectasis [ 2, 3] . During chronic infection, P. aeruginosa is difficult to eliminate, which will cause irreversible and destructive changes in the respiratory system and have a serious influence on the patient’s quality of life and survival time [4].

Bronchiectasis is a chronic endobronchial suppurative disease that is characterized by damage to the bronchi due to repeated infections of various microorganisms and inflammation of the airways, resulting in irreversible expansion of the bronchi [ 5, 6] . In the Asia-Pacific region, the prevalence of bronchiectasis is high, and the potential genetic predisposition may be one of the reasons for its high prevalence. P. aeruginosa is one of the most common pathogens isolated from the sputum of patients with bronchiectasis, regardless of whether it is in stable or acutely worsening periods [7]. At present, chronic colonization by P. aeruginosa is an important risk factor for the severity and prognosis of bronchiectasis and has been included in various bronchiectasis severity scoring systems, such as BSI [8] and FACED score [9].

During chronic lung infections, common genetic adaptations and advantageous phenotypic switches occur in P. aeruginosa to ensure its persistence in the lung [10]. These adaptation processes include conversion to the mucoid phenotype [11], avoidance of inflammasome activation [12], loss of motility [13], inactivation of quorum sensing function [14], and so on. Mucoid colonies, which are caused by overproduction of the polysaccharide alginate, are widely considered to be a marker for the transition to chronic infection [15] and can be used as an independent predictor to identify poor prognosis with P. aeruginosa pneumonia [16]. P. aeruginosa transforms into a mucoid phenotype, usually mediated by the destruction of mucA. Mutations of mucA are found in 82% of mucoid isolates of CF patients [17]. However, there are few reports on mucA mutations in bronchiectasis. The binding between MucA and AlgU prevents AlgU from encoding the enzymes required for alginate synthesis. Loss-of-function mutations in MucA lead to elevated transcription of the alg operon and result in alginate overproduction. P. aeruginosa triggers robust inflammatory responses during acute infection, which usually leads to pathogen clearance and resolution of infection [18]. However, during chronic pulmonary infections, these phenotypic switches help the bacteria avoid inflammasome activation and phagocytic clearance, therefore enhancing their ability to persist [19].

We currently know little about mucA mutations of P. aeruginosa isolated from patients with bronchiectasis. In this study, we isolated P. aeruginosa strain L012 from the respiratory tract of a patient with bronchiectasis and found that it showed mucoid colonies and weakened cytotoxicity compared with the wild-type strain PAO1. Through comparative analysis of the genome, we found a truncated mutation located in mucA of this strain, which was named mucA117. It can cause the production of mucus, reduce the expression of T3SS, and avoid the activation of inflammasomes. We speculate that this mutation favors the long-term presence of P. aeruginosa in the airway.

Materials and Methods

Bacterial strains and growth conditions

The bacterial strains used in this study are listed in Table 1. The P. aeruginosa strain PAO1 is the wild-type strain used in this study. P. aeruginosa strain L012 was isolated in 2021 from the sputum of a 74-year-old male bronchiectasis patient at Ruijin Hospital. All strains were grown on Luria-Bertani (LB) agar or in LB broth at 37ºC. LB supplemented with 5 mM EGTA and 20 mM MgCl ₂ was used as a T3SS inducing condition. Antibiotics were used at the following final concentrations: gentamicin (Gm) at 30 μg/mL, carbenicillin (Cb) at 150 μg/mL and tetracycline (Tc) at 100 μg/mL. In the strains containing the plasmids with an arabinose-inducible promoter, 10 mM arabinose was added to induce the expression of plasmid-borne genes. When observing the mucus formation of the strains, no additional arabinose was added.

Table 1 Bacterial strains and plasmids used in this study

Strain or plasmid	Description	Source or reference
P. aeruginosa strains
PAO1	Wild-type strain	Stored in our laboratory
L012	Clinically isolated strain	Isolated from Ruijin Hospital
PAO1Δ mucA	PAO1 with mucA deleted; Gm r	This study
Plasmids
pCasPA	Bacterial expression of Cas9 nuclease and λ-Red recombination system; Tc r	[1]
pACRISPR	A sgRNA expression plasmid for targeting a specific sequence; Cb r	[1]
pACRISPR- mucA-spacer	pACRISPR derivative with the spacer of the mucA	This study
pACRISPR- mucA-spacer-Up-Gm-Downstream	pACRISPR derivative with the repair arms of the mucA	This study
pHERD20T (EV)	Shuttle vector with P BAD promoter; Cb r	From Dr. Yufeng Yao’s lab
pPAO1 mucA	PAO1 mucA carried on pHERD20T	This study
pL012 mucA	L012 mucA carried on pHERD20T	This study

Gm ^r, gentamicin resistance; Tc ^r, tetracycline resistance; Cb ^r, carbenicillin resistance.

Genome sequencing and assemblies

DNA extracted from the P. aeruginosa strain L012 was subjected to whole genome sequencing using Novoseq 6000 (Novogene, Beijing, China) following the supplier’s protocol (Illumina, San Diego, USA). Surviving reads were assembled by a de novo approach using SPAdes [20]. To perform the pan-genome analyses, genome sequences were first annotated with Prokka [21]. Pan-genomes were then analyzed with Roary [22], and Snippy was used for variant detection. For KEGG functional annotation and pathway analysis, BlastKoala [23] was used with mutated genes.

Construction of deletion mutants and plasmids

The plasmids used in this work are listed in Table 1. The construction of the strain PAO1Δ mucA was as described in a previous study [24]. The primers used are shown in Supplementary Table S1. After pACRISPR was digested with BsaΙ (NEB, Massachusetts, USA), the spacers were ligated to the plasmid. Then, the pACRISPR- mucAspacer was digested with XbaΙ (Thermo Scientific, Waltham, USA) and XhoΙ (Thermo Scientific). The 500 bp fragment upstream of the start codon, the 500 bp fragment downstream of the stop codon of mucA and the Gm fragment were amplified by PCR, and then these three fragments were ligated into the digested pACRISPR- mucA-spacer. pCasPA was electroporated into the P. aeruginosa strain PAO1, and then pACRISPR- mucA-spacer-Up-Gm-Downstream was electroporated into this strain. Successful deletions were verified by sequencing.

To complement the PAO1Δ mucA mutant, the coding regions of the mucA locus were PCR amplified from P. aeruginosa strain PAO1 genomic DNA using the primers listed in Supplementary Table S1, which were designed to include HindΙΙΙ (Takara, Kyoto, Japan) and EcoRΙ (Takara) sites. The amplified product was cloned into pHERD20T. The plasmid pPAO1 mucA was tested for the correct insert by sequencing. Then, pPAO1 mucA was electroporated into the P. aeruginosa strain PAO1Δ mucA, and this strain was named PAO1Δ mucA/PAO1 mucA. pPAO1 mucA was also electroporated into P. aeruginosa strain L012 to construct L012/PAO1 mucA. The coding regions of the L012 mucA locus were obtained by PCR amplification from P. aeruginosa strain L012 genomic DNA and then used a similar strategy to construct P. aeruginosa strain PAO1Δ mucA/L012 mucA. pHERD20T (empty vector, EV) was electroporated into the P. aeruginosa strains PAO1Δ mucA and L012 to construct the P. aeruginosa strains PAO1Δ mucA/EV and L012/EV.

Cell culture and in vitro infection assay

Peritoneal macrophages (PMs) were harvested from C57BL/6 female mice after intraperitoneal injection of 1 mL of 5% thioglycollate broth, as previously described [25]. Lavage the peritoneal cavity with 10 mL of cold PBS followed by centrifugation at 200 g for 5 min to obtain cells. PMs were cultured in 6-well cell culture clusters overnight at 37ºC with 5% CO ₂ and then infected with mid-log phase P. aeruginosa cells. The supernatants were collected 6 hours postinfection (hpi) for LDH detection and 24 hpi for ELISA detection. The supernatants and cell lysates were collected 6 hpi for caspase-1 detection. All animal experiment procedures were approved by Animal Ethics Review Committee of Shanghai Jiao Tong University (project number A-2018-021).

Cell viability assay

The supernatants of PMs were collected 6 hpi, and cell death was evaluated by detecting the amount of LDH released. LDH release was measured by using a CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, USA). A microplate reader was used to detect the absorbance at 490 nm.

Inflammasome activation assay

PMs were infected with P. aeruginosa for 6 h, and the supernatants were treated with methanol and chloroform with vortexing and centrifugation at 10,000 g for 5 min to obtain total proteins. Cell lysates were obtained by lysing PMs in RIPA buffer (Sangon Biotech, Shanghai, China). Equal amounts of total proteins extracted from supernatants and lysates of PMs were diluted in 5× SDS-PAGE loading buffer and boiled for 5 min. Proteins were separated on 12% polyacrylamide gels. Following electrophoretic transfer of protein onto PVDF membranes (Millipore, Billerica, USA), membranes were blocked in 5% skim milk. The membranes were incubated with mouse anti-caspase-1 monoclonal antibody (Adipogen, San Diego, USA) at 4ºC overnight. Then, the membranes were incubated with HRP-conjugated mouse antibody (Beyotime, Shanghai, China) for 1 h at room temperature, followed by visualization using a chemiluminescent substrate (Thermo Scientific).

T3SS protein secretion assay

Overnight P. aeruginosa cultures were subcultured in fresh LB containing 5 mM EGTA and 20 mM MgCl ₂ and grown to log-phase (OD ₆₀₀=1.0). Whole-cell lysates were obtained by sonication, and the supernatants were obtained by centrifugation. Bacterial supernatants were treated with 15% trichloroacetic acid (TCA) at 4ºC overnight. The precipitated proteins were collected by centrifugation and washed with acetone. Equal amounts of protein samples were diluted in 5× SDS-PAGE loading buffer and boiled for 5 min. Proteins were separated on 12% polyacrylamide gels and transferred onto PVDF membranes. The membranes were incubated with rabbit anti-PcrV polyclonal antibody at 4ºC overnight. Rabbit anti-PcrV polyclonal antibody was prepared by our laboratory. The PcrV protein of the P. aeruginosa strain PAO1 was expressed in E. coli BL21, and then the rabbit was immunized subcutaneously with the purified protein in Freund’s complete adjuvant. The membranes were incubated with HRP-conjugated rabbit antibody (Sigma, St Louis, USA) for 1 h at room temperature, followed by visualization using a chemiluminescent substrate.

Total RNA isolation and quantitative real-time PCR

Total bacterial RNA was isolated from planktonic cells. The planktonic cells were prepared from the log-phase cells subcultured at 37ºC in LB broth (OD ₆₀₀=0.5). The RNA samples were prepared using TRIzol LS Reagent (Thermo Scientific) and then reverse transcribed into cDNA. The reverse transcription reaction was conducted using a SuperScript ΙΙΙ First-Strand Synthesis SuperMix Kit (Thermo Scientific). Primer sequences are shown in Supplementary Table S1. Then, real-time quantitative PCR (qPCR) was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The 30S ribosomal protein gene rpsL was used as an internal control. The following thermal cycler conditions were used: 30 s at 95ºC, followed by 40 cycles of 10 s at 95ºC and 30 s at 60ºC.

Statistical analysis

The results were analyzed by GraphPad Prism version 8, and statistical significance was evaluated by t test. Differences were considered to be significant at P<0.05.

Results

A truncated mutation occurs in the MucA protein of P. aeruginosa strain L012, which can transform the strain into mucoid

The P. aeruginosa strain L012 used in this study was isolated from the sputum of a bronchiectasis patient at Ruijin Hospital. We found that P. aeruginosa strain L012 was phenotypically mucoid on LB agar ( Figure 1A). To explore the adaptive changes in P. aeruginosa strain L012 during chronic infection, we performed comparative analysis of the genome between P. aeruginosa strain L012 and the laboratory strain PAO1. We screened the genes unique to P. aeruginosa strain PAO1 and the genes with insertions, deletions, frameshifts and early terminations in P. aeruginosa strain L012, and compared to P. aeruginosa strain PAO1. Compared with P. aeruginosa strain PAO1, the gene mutations of P. aeruginosa strain L012 occurred in the bacterial secretion system, quorum sensing system, biofilm formation, regulatory genes and so on ( Supplementary Table S2). Among them, a truncated mutation was found in the mucA of P. aeruginosa strain L012, c. 349C>T (p. Gln117*), which was different from common mutation sites. We named this mutation mucA117 in our research. The most common mutation in CF and bronchiectasis is mucA22 [26], known as a deletion of a G residue in a string of five G residues located between positions 429–433 of the mucA coding region [27]. MucA is a protein located in the cytoplasmic membrane with 194 residues and contains three domains. The N-terminal domain (residues 0–78) is the AlgU binding domain (AlgU BD), residues 84–103 are the transmembrane domain (TM) and the C-terminal domain (residues 146–194) is the MucB binding domain (MucB BD) [ 28, 29] ( Figure 1B). mucA117 led to MucA with a truncated C-terminal periplasmic domain, which might make it unable to bind with the periplasmic MucB protein and cause the dysregulation of downstream genes, such as alginate synthesis-related genes.

Figure 1 .

P. aeruginosa strain L012 is mucoid colonies and a truncated mutation of mucA occurs in strain L012

(A) P. aeruginosa strains PAO1 and L012 were grown on LB agar and imaged after 24 h to observe mucus formation. (B) Schematic diagram of the MucA structure. Green, AlgU binding domain (AlgU BD); yellow, the transmembrane domain (TM); and blue, the MucB binding domain (MucB BD). The red line indicates the mutation position of P. aeruginosa strain L012, and the green line indicates the mutation position of mucA22.

mucA117 can lead the strain to mucoid and reduce pyroptosis and inflammasome activation of PMs

We used the arabinose-inducible pHERD20T plasmid to express truncated L012 mucA in the P. aeruginosa strain PAO1Δ mucA (PAO1Δ mucA/L012 mucA) to explore whether mucA117 has an effect on alginate synthesis. We found that after mucA was deleted, the strain changed from nonmucoid to mucoid. The PAO1Δ mucA mutant transformed into the nonmucoid phenotype after expressing wild-type mucA ( Figure 2A). However, the strain still maintained the mucoid phenotype after expressing L012 mucA ( Figure 2A). We also complemented wild-type mucA in P. aeruginosa strain L012 (L012/PAO1 mucA) with the pHERD20T plasmid. After expressing wild-type mucA, the P. aeruginosa strain L012/PAO1 mucA transformed into nonmucoid cells ( Figure 2A). These results showed that mucA117 causes the strain to become mucoid.

Figure 2 .

mucA117 turns the strain into mucoid and reduces the its ability to induce pyroptosis and the activation of inflammasomes in PMs

(A) The P. aeruginosa strains PAO1Δ mucA/EV, PAO1Δ mucA/PAO1 mucA, PAO1Δ mucA/L012 mucA, L012/EV and L012/PAO1 mucA were grown on LB agar without arabinose and imaged after 24 h to observe mucus formation. (B) PMs were infected with PAO1, PAO1Δ mucA/EV, PAO1Δ mucA/PAO1 mucA and PAO1Δ mucA/L012 mucA (MOI=10) for 6 h. Cell death was determined by measuring the LDH released into the supernatants. (C) Caspase-1 cleavage in the supernatants and cell lysates of PMs infected with PAO1, PAO1Δ mucA/EV, PAO1Δ mucA/PAO1 mucA and PAO1Δ mucA/L012 mucA for 6 h. * P<0.05, ** P<0.01 (unpaired t test).

P. aeruginosa strain PAO1 infection is able to activate NLRC4 and NLRP3 inflammasomes and initiate a type of rapid inflammatory cell death termed pyroptosis in macrophages [30]. PMs were infected with the P. aeruginosa strains PAO1, PAO1Δ mucA/EV, PAO1Δ mucA/PAO1 mucA and PAO1Δ mucA/L012 mucA to evaluate the ability to induce cell death, which was measured by the amount of LDH released into the supernatants. The amount of LDH released into the supernatants of cells infected with PAO1Δ mucA/EV and PAO1Δ mucA/L012 mucA was not significantly different from that in the uninfected group ( Figure 2B). However, they were both significantly lower than those in cells infected with the P. aeruginosa strain PAO1Δ mucA/PAO1 mucA ( Figure 2B). Then, we detected the activation of caspase-1. After PMs were infected with the P. aeruginosa strains PAO1 and PAO1Δ mucA/PAO1 mucA, caspase-1 was cleaved into the active form p20 ( Figure 2C). In contrast, caspase-1 cleavage could not be detected after infection with the P. aeruginosa strains PAO1Δ mucA/EV and PAO1Δ mucA/L012 mucA ( Figure 2C). These data indicated that both mucA deletion and mucA117 will affect its function of inducing cell pyroptosis and activating inflammasomes.

We also infected PMs with P. aeruginosa strains L012, L012/EV and L012/PAO1 mucA. Unfortunately, neither L012/EV nor L012/PAO1 mucA induced cell death ( Supplementary Figure S1A) or inflammasome activation ( Supplementary Figure S1B) in PMs. We speculate that the reason why the P. aeruginosa strain L012 cannot cause inflammasome activation is complex and that mucA mutation may be one of them.

mucA117 downregulates the expression of T3SS and inflammasome ligand fliC, which may be related to the increased expression of algU and algR

Transfer of inflammasome ligands into host cells via functional T3SS in P. aeruginosa is critical for inflammasome activation, and infection with T3SS-knockout P. aeruginosa cannot activate the inflammasome [31]. PcrV is an important structural translocation component which is necessary for T3SS secretion [32]. The protein FliC can be recognized by the NLRC4 inflammasome and secreted by T3SS [33]. As an important inflammasome ligand, it can induce inflammasome activation. In our study, we found that mucA117 failed to induce pyroptosis or inflammasome activation in PMs. We speculated that this effect might be mediated by the reduced expression of T3SS and the key inflammasome ligands. Therefore, we detected the T3SS protein PcrV by western blot analysis and the mRNA expressions of pcrV and flagellin monomer fliC by qPCR analysis. As predicted, compared with those in the P. aeruginosa strains PAO1 and PAO1Δ mucA/PAO1 mucA, the T3SSs in the stains PAO1Δ mucA/EV and PAO1Δ mucA/L012 mucA were defective, and the expression of PcrV could not be detected in the whole-cell extracts or culture supernatants ( Figure 3A). Similarly, in the P. aeruginosa strains PAO1Δ mucA/EV and PAO1Δ mucA/L012 mucA, the expressions of pcrV and fliC were significantly lower than those in the P. aeruginosa strains PAO1/EV and PAO1Δ mucA/PAO1 mucA ( Figure 3B). These results showed that mucA117 can downregulate the expression of T3SS protein and inflammasome ligands such as fliC, which might be the reason for avoiding the activation of inflammasomes and pyroptosis in PMs.

Figure 3 .

mucA117 downregulates the expression of T3SS and inflammasome ligand, which may be related to the increased expressions of algU and algR

(A) Under T3SS induction conditions, the PcrV levels in the P. aeruginosa strains PAO1, PAO1Δ mucA/EV, PAO1Δ mucA/PAO1 mucA, PAO1Δ mucA/L012 mucA, L012/EV and L012/PAO1 mucA were detected in culture supernatants and whole-cell extracts. (B) Relative expression of pcrV, fliC, algU and algR in these strains. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 (unpaired t test).

We also detected the expression of T3SS and inflammasome ligand fliC in P. aeruginosa strains L012/EV and L012/PAO1 mucA. After expressing wild-type mucA in L012, the ability of the strain to synthesize and secrete PcrV was restored ( Figure 3A). The relative expressions of pcrV and fliC were significantly higher in P. aeruginosa strains L012/EV and L012/PAO1 mucA than in L012/EV ( Figure 3B). The above results indicated that in P. aeruginosa strain L012, the decreased expressions of T3SS and inflammasome ligands are due to the truncated mucA. In P. aeruginosa strain L012, in addition to the truncated mutation of mucA, various types of mutations also occur in many important virulence genes, which may be the reason why P. aeruginosa strain L012/PAO1 mucA cannot induce pyroptosis or inflammasome activation in PMs.

Resent research shows that AlgU and the response regulator AlgR are required for mucA-mediated inhibition of T3SS gene expression [ 34‒ 36] . AlgU can also repress flagellum biosynthesis by inhibiting the expression of fleQ [37]. Therefore, we detected the relative expression of algU and algR in the strains by qPCR ( Figure 3B). In the P. aeruginosa strains PAO1Δ mucA/EV and PAO1Δ mucA/L012 mucA, the expressions of algU and algR were significantly higher than those in PAO1/EV and PAO1Δ mucA/PAO1 mucA. Consistent with these findings, the expressions of these two genes were significantly reduced in the P. aeruginosa strain L012/PAO1 mucA. Our results indicated that mucA117 can increase the expressions of algU and algR, which is similar to a previous study on the mucA22 mutant [ 34‒ 36] . We speculate that mucA117 downregulates the expression of T3SS genes and flagellin gene fliC through increasing the products of algU and algR, thereby not activating the inflammasome.

Discussion

A hallmark of P. aeruginosa in airways of chronic infections is their mucoid colony morphology, which results in the inactivation of MucA. The anti-sigma factor MucA is a transmembrane protein. Its N-terminal domain is located in the cytoplasm and can bind with AlgU, preventing the transcription of AlgU-dependent genes [38]. Its C-terminal domain is located in the periplasm and can bind with MucB, thereby preventing the cleavage of MucA by the periplasmic AlgW protease [39]. The loss-of-function mutation of mucA leads to increased transcription of algU. AlgU is an extracytoplasmic sigma factor (ECFσ) that can regulate at least 350 genes, including those responsible for the production of itself, algR and alginate biosynthetic enzymes [40]. The mutation types of mucA are very diverse. The most common mutation is mucA22, and this mutation makes the MucA protein unable to bind with the periplasmic MucB protein, making it less stable than wild-type MucA [41], which unbalances the MucA/AlgU ratio and leads to increased alginate synthesis [42]. In our research, we isolated P. aeruginosa strain L012 from a bronchiectasis patient and found a truncated mutation in mucA (p. Gln117*) named mucA117. Similar to the mucA22 mutant, the MucA protein of P. aeruginosa strain L012 is truncated between the transmembrane domain and the periplasmic domain that interacts with MucB. Therefore, we speculate that the MucA of P. aeruginosa strain L012 cannot bind with the periplasmic MucB protein either, thereby releasing AlgU. As a result, P. aeruginosa strain L012 transforms into a mucoid colony.

Inflammasome activation occurs in response to a notably high number of pathogenic microbes and is critical for host defense against various microbial infections. During P. aeruginosa infection, the NLRC4 inflammasome is activated in response to recognition of bacterial flagellin by Naip5 [43], T3SS needle proteins EprI, MxiH, BsaL and PscF by murine Naip1 or by human NAIP (hNAIP) [44], and T3SS rod protein PscI by Naip2 [45]. Then, they trigger pyroptotic membrane rupture with subsequent secretion of IL-1β and IL-18. Recently, the lipid A motif of lipopolysaccharide (LPS) was shown to induce caspase-11-dependent pyroptosis [30]. When the inflammasome is activated, it can produce proinflammatory cytokines, allowing the recruitment of inflammatory and phagocytic cells to release proinflammatory mediators and elastase [46], which is necessary for bacterial clearance. However, this process also increases injury and pathological responses of the lung and is associated with poor host outcomes [47]. Current studies have shown that the expressions of T3SS genes, including the T3SS needle protein PscF, is downregulated in the mucA22 mutant [34]. This regulation is AlgU- and AlgR-dependent. The study by Tart et al. [37] showed that the mucA22 mutant can downregulate the expression of flagellin genes such as fliC through AlgU. Our results showed that mucA117 led to increased expressions of AlgU and AlgR and downregulated the expressions of T3SS and inflammasome ligands, such as the flagellin monomer FliC. Therefore, we speculate that mucA117 may have a similar function to the mucA22 mutant in downregulating T3SS and flagellin genes, which might be a strategy to avoid inflammasome activation under selective pressure by the innate immune system during chronic infection. Failure to activate the inflammasome may make it difficult for the host’s immune system to clear P. aeruginosa, thereby allowing long-term colonization of the strain in the respiratory tract.

However, in the P. aeruginosa strain L012/PAO1 mucA, the expression of the T3SS protein PcrV and fliC was increased, and the expressions of algU and algR were decreased, but the ability to induce cell death and inflammasome activation of PMs was not restored. In the process of chronic infection, strains have undergone a rich and complex evolutionary process to adapt to the respiratory environment. In addition to the truncated mutation of mucA, various types of mutations also occur in other key virulence factors of P. aeruginosa strain L012. For example, we found a deletion mutation (p. Arg168_Leu169del) in the T3SS gene pscK and an insertion mutation (p. Pro4del insGlnAla) in the T3SS gene pcrH. PscK is a member of the SctK protein family, which can act as an adaptor protein interfacing SctQ with the 24-fold symmetric SctD inner membrane ring [48]. Its main function is to identify the secretion substrate and drive the secretion process. PcrH is the chaperone protein of PopB and PopD, which is responsible for the presecretory stabilization and efficient secretion of PopB and PopD translocators [49]. Compared with the wild-type strain, Δ pcrH cannot induce morphological changes in monolayer HeLa cells because it cannot maintain effective PopB and PopD secretion and therefore cannot transfer cytotoxins [50]. The mutations in these genes may also lead to partial loss of T3SS function so that the inflammasome is not activated. We speculate that the attenuation of inflammasome activation may be caused by a combination of many factors, and mucA117 is one of them, even though the expressions of the T3SS protein PcrV and some inflammasome ligands are increased.

In summary, we isolated a P. aeruginosa strain with a truncated mutation of mucA from a patient with bronchiectasis. This truncated MucA protein turned the strain into mucoid and downregulated the expression of T3SS and inflammasome ligands such as fliC and reduced inflammasome activation. These changes might make the strain difficult to clear, favoring its long-term colonization.

Supplementary Data

Supplementary data is available at Acta Biochimica et Biophysica Sinica online.

COMPETING INTERESTS

The authors declare that they have no conflict of interest.

Funding Statement

This work was supported by the grants from the National Natural Science Foundation of China (Nos. 81971896, 81800190, and 81471908), the Natural Science Foundation of Shanghai Program (No. 19ZR1428600), and the National Innovative Research Team of High-Level Local Universities in Shanghai.