Interaction between Pseudomonas aeruginosa and Aspergillus fumigatus in cystic fibrosis

Inhibitory effect of phenazines on the growth of A. fumigatus

Pseudomonas aeruginosa-secreted phenazines prevent the growth of A. fumigatus. It is thought that the toxic effects of phenazines on prokaryotes and diverse eukaryotic hosts result from their redox activities or inactivation of oxidative stress response proteins (Hassett et al., 1992; Muller, 2002; O’Malley et al., 2003). In target cells, reduced phenazines are oxidized by NAD(P)H and oxygen to generate reactive oxygen species (ROS), specifically O₂^·− Moreover, generation of reactive nitrogen species (RNS) is induced by overproduction of O₂^·− (Martínez & Andriantsitohaina, 2009). Nitric oxide (NO^·) is produced by mitochondrial processes, and highly toxic peroxynitrite radicals (ONOO⁻) are generated via reactions between NO^· with O₂^·− radicals (Martínez & Andriantsitohaina, 2009). Overall, mitochondria are the main target of phenazine-produced ROS and RNS, and phenazines have a significant impact on the mitochondrial ultrastructure of A. fumigatus hyphae. All four phenazines (PYO, PCA, PCN, 1-HP) show A. fumigatus growth inhibitory effects by inducing the production of ROS, specifically O₂^·−, and the RNS ONOO⁻ (pathway ① in Fig. 1) (Briard et al., 2015).

Another related study reported that phenazine-derived metabolites acting as interspecies signals can affect filamentous fungal development through oxidative stress regulation (Zheng et al., 2015b). In P. aeruginosa–A. fumigatus co-culture biofilms, development of the latter is differentially modulated by phenazine-derived metabolites of the former. With a decreasing phenazine gradient, A. fumigatus shifts from weak vegetative growth to an asexual sporulation phase (conidiation), and this shift in morphology is correlated with the production of phenazine radicals and concomitant ROS generation by phenazine redox cycling.

DiRhls induce A. fumigatus to produce an extracellular matrix that facilitates P. aeruginosa binding. A. fumigatus growth can be inhibited by diRhls, which blocks the β1,3 glucan synthase (GS) activity (pathway ① in Fig. 1) (Briard et al., 2017).

A recent study reported that a double phenazine mutant was similar to the wild-type organism in terms of its inhibitory power against A. fumigatus (Sass et al., 2018), with little difference caused by the complete lack of phenazine molecules due to mutation. The results of this study are different from those of previous studies. Several previous studies have emphasized the inhibitory power of phenazines on A. fumigatus (Briard et al., 2015, 2017; Zheng et al., 2015b), but a deficiency of these molecules via mutation appeared to cause little difference in this study. This finding suggests that the concentrations of phenazines that have been previously studied in vitro may be irrelevant to those in vivo. In addition, compensation for the loss of phenazine-mediated inhibitory activity by upregulation of other factors in the mutants could not be excluded in this study. Further research is needed to explore the effect of phenazines on the growth of A. fumigatus.

Effects of the inhibition of the QS system on the growth of A. fumigatus

The P. aeruginosa QS network plays a role in inhibiting A. fumigatus growth and biofilm formation (pathway ③ in Fig. 1) (Mowat et al., 2010). The las QS system is essential for the production of the diffusible signaling molecule acyl homoserine lactone (AHL) N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12 HSL) (Smith & Iglewski, 2003), resulting in the expression of specific target genes in P. aeruginosa. In addition, 3-oxo-C12 HSL is one of the AHLs frequently identified in extracts of respiratory secretions from CF patients infected with P. aeruginosa (Smith & Iglewski, 2003). By utilizing two P. aeruginosa QS knockout strains, PAO1:ΔLasI and PAO1:ΔLasR, one study illustrated that 3-oxo-C12 HSL inhibits A. fumigatus biofilm formation (Mowat et al., 2010). The PAO1:ΔLasI strain was unable to synthesize 3-oxo-C12 HSL, whereas PAO1:ΔLasR synthesized 3-oxo-C12 HSL but could not respond to it. Furthermore, A. fumigatus growth was significantly greater when in direct co-culture with PAO1:ΔLasI and PAO1:ΔLasR than in co-culture with wild-type PAO1. The indirect effect of the P. aeruginosa QS knockout strains on A. fumigatus biofilm development was assessed using the Transwell system, which showed significantly less inhibition of A. fumigatus biofilm development when in indirect co-culture with PAO1:ΔLasI and PAO1:ΔLasR than with the wild-type strain. Additionally, the cellular viability of A. fumigatus conidia and the biomass of A. fumigatus biofilms were reduced by diffusible and heat-stable soluble molecules, such as decanol, decanoic acid and dodecanol (structurally similar to the QS molecules produced by P. aeruginosa) in a concentration-dependent manner. At the molecular level, it is likely that these molecules lead to hyphal repression by affecting key transcription factors (Mowat et al., 2010).

In CF patients, P. aeruginosa produces rhamnolipids (Rhls), which are controlled by the QS system. Rhls are largely composed of diRhls and monorhamnolipids, and the diRhls secreted by P. aeruginosa may affect A. fumigatus. For example, diRhls induce A. fumigatus to produce an extracellular matrix that facilitates binding by P. aeruginosa (Briard et al., 2017). As stated above, diRhls also inhibit A. fumigatus growth by blocking β1,3 GS activity and altering cell wall architecture. In the presence of diRhls, A. fumigatus displays multibranched hyphae and a thicker cell wall rich in chitin. This growth phenotype of A. fumigatus is similar to that following treatment with anti-fungal echinocandins. Although the two rhamnose moieties attached to fatty acyl chains are essential structures for the interaction of diRhl with β1,3 GS, the site of β1,3 GS action differs between diRhls and echinocandins. Overall, diRhls and azole anti-fungals exhibit a synergistic anti-fungal effect (Briard et al., 2017).

A recent study reported that alkylhydroxyquinolones (AHQs), autoinducers secreted by P. aeruginosa, could suppress biofilm formation in A. fumigatus. The AHQ interkingdom signaling molecules 2-heptyl-3-hydroxy-4-quinolone (PQS) and 2-heptyl-4-quinolone (HHQ), which are involved in QS in P. aeruginosa, were both able to alter A. fumigatus biofilm biomass and structure (Reen et al., 2016). Both pro- and anti-oxidant activities have been reported for PQS and HHQ. AHQ interkingdom signaling molecules can interact with lipopolysaccharides, cellular membranes, and membrane vesicles in several bacterial species (Häussler & Becker, 2008). Redox-active phenazines of P. aeruginosa, which exhibit inhibitory activity against A. fumigatus growth, are also controlled by AHQs (Moree et al., 2012). Overall, these small interkingdom signaling molecules of P. aeruginosa disrupt A. fumigatus biofilm formation and render A. fumigatus susceptible to clearance by drugs. As these bacterial molecules are selectively non-cytotoxic to host cell lines, they may be used as viable molecular therapeutics.

The anti-A. fumigatus capacity of P. aeruginosa pyoverdine mutants has been assessed in recent studies. Some residual inhibition of A. fumigatus can be detected in pyoverdine mutants, and in addition to the anti-Aspergillus effect of pyoverdine, other inhibitors may contribute to the total fungal inhibition by wild-type P. aeruginosa. Some of these residual inhibitors in pyoverdine mutants may be related to QS-regulated metabolites, such as Rhls. The anti-Aspergillus ability of QS mutants was also examined, with the results showing that QS-regulated metabolites have an important anti-Aspergillus function. Indeed, these metabolites are potential intermicrobial inhibitors. The decreased anti-A. fumigatus activity of QS mutants might be related to loss of the combined activity of many downstream products, and decreases in pyoverdine production in QS mutants may also lead to their reduced anti-Aspergillus capacity (Sass et al., 2018).

Inhibition of A. fumigatus growth by Fe metabolism

Pseudomonas aeruginosa inhibits A. fumigatus growth through Fe limitation, which can, in part, result from the modulation of siderophore production by the fungus due to metabolites from the bacterium (Phelan et al., 2014).

Recent research has shown that P. aeruginosa pyoverdine can suppress A. fumigatus growth and biofilm formation via the chelation of iron, reducing its availability to A. fumigatus (pathway ④ in Fig. 1) (Sass et al., 2018). In this study, pvdD pchE and pvdD mutants (loss of pyoverdine and siderophore), which are defective in inhibiting A. fumigatus growth and biofilm formation in various assays, were evaluated. The inhibitory effect of pyoverdine deletion mutants was restored with pure pyoverdine, and the A. fumigatus sidA mutant that is unable to produce siderophores was found to be hypersusceptible to P. aeruginosa metabolites and to pyoverdine. Thus, the siderophore-deficient A. fumigatus mutant was readily inhibited by P. aeruginosa. Clinical P. aeruginosa isolates derived from the lungs of CF patients have revealed a correlation between the amount of pyoverdine produced and the anti-fungal activity of clinical samples. The results suggest that the siderophore pyoverdine is an important inhibitory molecule (Sass et al., 2018).

As pyoverdine can capture iron from the environment, it can deprive A. fumigatus of the iron that is essential for its growth and metabolism. Iron sequestration by pyoverdine leads to iron starvation and increased siderophore secretion by A. fumigatus (Sass et al., 2018). In a shared microenvironment, P. aeruginosa and A. fumigatus compete for iron to promote their own survival, and high pyoverdine expression antagonizes A. fumigatus metabolism and growth, which might support anti-fungal treatment. Key aspects of the competition between P. aeruginosa and A. fumigatus include the relative amounts of siderophores produced, the speed of siderophore production, and the relative affinity for Fe.

Bacteriophage Pf4 inhibits the metabolic activity of A. fumigatus biofilms

In a recent study, it was reported that the Pf4 phagosome can inhibit A. fumigatus metabolism and growth by binding iron and causing iron deficiency (pathway ④ Fig. 1) (Penner et al., 2016). Pf4 inhibition of A. fumigatus is caused by iron binding and the sequestration of A. fumigatus iron resources (pathway ① Fig. 2), and inhibition of A. fumigatus metabolism by Pf4 can be overcomed with ferric iron supplementation. Moreover, inhibition of A. fumigatus biofilm formation by phages is reversed by low doses of iron, indicating that A. fumigatus is more sensitive to iron inhibition by the Pf4 phage than it is to other elements in P. aeruginosa supernatants (Penner et al., 2016). This Pf4 phage-mediated inhibition was found to be dose dependent and could be alleviated by phage denaturation. This inhibition of Pf4 was more significant in preformed A. fumigatus biofilms than during biofilm formation. In contrast, Pf4 had no effect on planktonic conidia (Penner et al., 2016). These findings suggest that the site of phage action is specific to the extracellular matrix or hyphae (Reichhardt et al., 2015). Another two phages, Pf1 and fd, showed no inhibitory action against A. fumigatus. Pf4 attaches to A. fumigatus hyphae, and fungal inhibition may occur at the biofilm surface. The shorter phage Pf1 did not bind as extensively to A. fumigatus biofilms as did Pf4 and exhibited less efficient inhibition (Penner et al., 2016).

Acute infection of P. aeruginosa by Pf bacteriophage can decrease the production of pyoverdine and the inhibitory capacity toward A. fumigatus biofilms. Thus, the reduced production of anti-microbials by P. aeruginosa infected by Pf bacteriophage may promote co-infection with A. fumigatus in CF airways (Secor et al., 2017).

SCVs in A. fumigatus intermicrobial competition

In a study of SCVs in intermicrobial competition with A. fumigatus (Anand et al., 2017b), the SCVs isolated from P. aeruginosa was shown to inhibit A. fumigatus biofilm formation, and this inhibitory capacity toward A. fumigatus biofilm was found to be related to pyoverdine (pathway ④ in Fig. 1) (Anand et al., 2017b). Indeed, isolated SCVs with high pyoverdine production had the highest inhibitory capacity in every co-culture method evaluated. Correspondingly, the two SCV isolates with the lowest inhibitory activities did not produce pyoverdine, suggesting that pyoverdine is the key P. aeruginosa inhibitor of A. fumigatus (Anand et al., 2017b).

Pseudomonas aeruginosa SCVs exhibit heterogeneity in inhibiting A. fumigatus biofilms. For instance, the inhibitory abilities of clinical SCVs isolates and reference CF non-mucoid isolates of P. aeruginosa or filtrates from P. aeruginosa planktonic or biofilm cultures were compared by coincubation with A. fumigatus during biofilm formation or in preformed biofilm. The metabolic activities of A. fumigatus biofilms were measured by different assays, with pyoverdine in filtrates being measured by spectrophotometry. The results showed that SCVs inhibited A. fumigatus biofilm formation, although the inhibitory effects of different SCVs were quite different. By adjusting planktonic culture filtrates, differences in SCV inhibition were related to SCV growth or deficient inhibitor production. Overall, the ability of SCVs to inhibit A. fumigatus biofilm was related to pyoverdine (Anand et al., 2017b). Thus, SCVs isolated from P. aeruginosa may be important in CF because they are capable of inhibiting A. fumigatus biofilm.

Inhibition of A. fumigatus by P. aeruginosa under conditions of hypoxia

Most studies examining the inhibition of A. fumigatus by P. aeruginosa have been performed under normoxic conditions. However, patients with acute exacerbation or progression of CF may exhibit hypoxia in focal lung sites (Cowley et al., 2015; Lambiase, Catania & Rossano, 2010; Worlitzsch et al., 2002). A. fumigatus inhibition by P. aeruginosa was recently evaluated under hypoxic conditions (Anand, Clemons & Stevens, 2017a), and the results showed that although the inhibitory activities of P. aeruginosa were effective under aerobic, hypoxic, or anaerobic conditions, P. aeruginosa growth was slow under hypoxic or anaerobic conditions, thus decreasing the ability of P. aeruginosa filtrates to inhibit A. fumigatus growth and biofilm formation. Regardless of the planktonic or biofilm state, the extracellular molecules produced by P. aeruginosa under anaerobic conditions were less inhibitory toward A. fumigatus growth and biofilm formation than were those under aerobic conditions. Therefore, the inhibitory power of P. aeruginosa against both A. fumigatus preformed biofilm and biofilm formation was decreased under hypoxic conditions (Anand, Clemons & Stevens, 2017a).

During the course of CF progression, P. aeruginosa mutants with a low level of pyoverdine production often appear, and the ratio of Fe³⁺ to Fe²⁺ decreases under hypoxic conditions. Intracellular iron acquisition by P. aeruginosa occurs mainly through the ingestion of low-activity Fe²⁺ via the production of phenazines and membrane permease (Cartron et al., 2006; Cornelis & Dingemans, 2013; Nguyen & Oglesby-Sherrouse, 2015). P. aeruginosa in the airway of CF patients typically shows low pyoverdine expression, and as mentioned above, pyoverdine can inhibit A. fumigatus growth by depriving A. fumigatus of iron. Under hypoxic conditions, the inhibitory effect of P. aeruginosa on A. fumigatus declines; thus, the growth of A. fumigatus is promoted (Sass et al., 2018). These findings explain why A. fumigatus is able to colonize CF airways following P. aeruginosa colonization and why A. fumigatus may persist during CF disease progression or chronic lung infection (Amin et al., 2010; Baxter et al., 2013a; Fillaux et al., 2012; Forsyth et al., 1988; Mirković et al., 2016; Nicolai et al., 1990; Speirs, Van Der Ent & Beekman, 2012).

After a long period of hypoxia, the interaction between P. aeruginosa and A. fumigatus appears to be similar to that under normoxic conditions (Anand, Clemons & Stevens, 2017a). Other factors may also affect P. aeruginosa and A. fumigatus interactions, such as prolonged use of antibiotics or inhaled corticosteroids (Noni et al., 2014).

The different inhibitory capacities of P. aeruginosa on A. fumigatus between planktonic and biofilm states

Pseudomonas aeruginosa and A. fumigatus are commonly found in the airways of patients with CF in the form of biofilms, and their pathogenicity and resistance in the biofilm state differ from those in the planktonic state. The inhibitory capacity of P. aeruginosa toward A. fumigatus in the biofilm state is also different from that in the planktonic state (Mowat et al., 2010). As an example, the supernatant extracted from P. aeruginosa biofilm was more effective than that extracted from planktonic cells (Ferreira et al., 2015). Pyoverdine plays an important role in P. aeruginosa biofilm formation, and its production is higher in biofilm than in planktonic cells (Visaggio et al., 2015). Thus, the P. aeruginosa biofilm-mediated suppression of A. fumigatus growth and biofilm formation is greater than planktonic P. aeruginosa-mediated suppression. It is also possible that other inhibitors may play an important role in this process (Anand et al., 2017b).

As mentioned above, the inhibitory effects of P. aeruginosa on A. fumigatus biofilm formation and preformed biofilms are different; it has been reported that P. aeruginosa can inhibit A. fumigatus biofilm formation but has almost no effect on preformed biofilms. The mature filamentous biofilms of A. fumigatus clearly restricted the inhibitory capacity of P. aeruginosa (Mowat et al., 2010), and another study showed that preformed A. fumigatus biofilm was more resistant to P. aeruginosa (Ferreira et al., 2015). According to a recent study, preformed A. fumigatus biofilms are inhibited by biofilm filtrates of P. aeruginosa strains isolated from CF patients via apoptosis, an effect that is related to mitochondrial membrane damage caused by metacaspase activation (Shirazi et al., 2016). In contrast, the inhibitory capacity of P. aeruginosa Pf4 phage toward A. fumigatus preformed biofilm is higher than that during biofilm formation. The P. aeruginosa phage Pf4 had little effect on planktonic conidial growth (Penner et al., 2016), suggesting that hyphae or the extracellular matrix is the specific site of phage action (Reichhardt et al., 2015).

The different inhibitory abilities of P. aeruginosa on A. fumigatus conidia and hyphae

After A. fumigatus conidia colonization in the CF patient airway, A. fumigatus gradually forms a biofilm that is rich in hyphae, and P. aeruginosa exhibits different inhibitory capacities toward A. fumigatus conidia and hyphae. In simultaneous static co-cultures, P. aeruginosa cells can effectively kill A. fumigatus conidia cells, but P. aeruginosa cells show only a minor inhibitory effect on sporelings grown for 12 h or longer as well as hyphae (Manavathu, Vager & Vazquez, 2014). Indeed, during co-cultivation with P. aeruginosa, A. fumigatus sporelings grown for 12 h or longer and young hyphae were stronger than ungerminated conidia with respect to the formation of P. aeruginosa–A. fumigatus biofilm.

Aspergillus fumigatus hyphae can withstand the fungicidal effect of P. aeruginosa and can produce the cytotoxic compound gliotoxin, which has anti-bacterial activity. Production of mycotoxin increases during mycelial growth and biofilm formation in A. fumigatus (Manavathu, Vager & Vazquez, 2014), and P. aeruginosa growth and its ability to kill A. fumigatus are suppressed with increasing levels of gliotoxin. In addition, virulence factor production and the inhibitory action of P. aeruginosa are strengthened by this increased metabolic activity in cells. Overall, the metabolic activity of germinating conidia and young sporelings is strong, whereas that of mature hyphae is limited in the apical regions of filaments. The apex, which has high metabolic activity, is the site at which P. aeruginosa binds to A. fumigatus hyphae and acquires nutrients (Toljander et al., 2007); hyphae, which has low metabolic activity, are not sensitive to the toxic molecules of P. aeruginosa. In fact, the cell walls of mature hyphae are poorly permeable to the toxic molecules of P. aeruginosa. Hence, mature hyphae are not easily killed by P. aeruginosa (Manavathu, Vager & Vazquez, 2014).

Comparison of the inhibitory effect between different P. aeruginosa strains from CF patients and non-CF patients

In addition to CF patients, P. aeruginosa and A. fumigatus also co-exist in the airways of patients with conditions such as chronic obstructive pulmonary disease, bronchiectasis, and hospital-acquired pneumonia. P. aeruginosa in CF patient airways can be divided into two types: mucoid and non-mucoid. A recent survey showed that both non-CF and CF A. fumigatus strains are inhibited by P. aeruginosa metabolic products (Nazik et al., 2017), and another study reported that both P. aeruginosa cells and filtrates isolated from CF patients had greater inhibitory effects on A. fumigatus growth and biofilm formation in vitro than did materials isolated from non-CF patients (Ferreira et al., 2015). Furthermore, non-mucoid P. aeruginosa exerted greater inhibitory effects on A. fumigatus than did mucoid P. aeruginosa in CF patients (Shirazi et al., 2016).

Pseudomonas aeruginosa isolated from the airway of CF patients has a greater chance of contact and longer duration of co-existence with A. fumigatus than isolates from non-CF patients. In addition, P. aeruginosa isolated from CF patients produces more toxic products and inhibitors than that from non-CF patients. Thus, CF patient-derived P. aeruginosa has a greater inhibitory effect against A. fumigatus growth and biofilm formation than strains isolated from non-CF patients. Mucoid P. aeruginosa usually exists in the deep and hypoxic zone of the lung (Gaspar et al., 2013; Pressler et al., 2006; Tramper-Stranders et al., 2012), and the synthesis of toxic products and inhibitors of P. aeruginosa is reduced under hypoxic conditions. Overall, the inhibitory capacity of mucoid CF P. aeruginosa filtrates is less than that of non-mucoid CF filtrates.

Promotion effect of P. aeruginosa on A. fumigatus colonization and growth

Under certain conditions, P. aeruginosa promotes the growth of A. fumigatus. Therefore, many CF patients are susceptible to infection with A. fumigatus after infection with P. aeruginosa (Paugam et al., 2010). One study hypothesized that P. aeruginosa infection promotes the evolution of A. fumigatus sensitization (Kraemer et al., 2006).

Another study reported reduced colonization of A. fumigatus after anti-infective treatment of P. aeruginosa in patients with acute exacerbation of CF, potentially because the bacterium protects A. fumigatus via immune factors and growth conditions (Baxter et al., 2013b). During acute exacerbation in CF patients, the colonization and growth of A. fumigatus may be related to the negative effects of P. aeruginosa on host lung function and immune defense. Resistance of P. aeruginosa biofilms to host immune responses contributes to the growth and multiplication of A. fumigatus (pathway ⑤ in Fig. 1) (Baxter et al., 2013a). By analyzing pre- and post-antibiotic sputum samples from adult CF patients, a study showed that intravenous antibiotics targeting P. aeruginosa during CF pulmonary exacerbations had a negative impact on the colonization and growth of A. fumigatus (Baxter et al., 2013b). Because P. aeruginosa contributes to the colonization and growth of A. fumigatus in CF patients, it is also possible that both microbes work together to combat host immune factors, resulting in increased infection and decreased pulmonary function. Nonetheless, their relationship may become competitive with growth, and P. aeruginosa inhibits A. fumigatus in various ways, as described above. Further research is required to verify the interdependence between these two organisms for survival within the airways of CF patients.

Sub-bacteriostatic concentrations of phenazines induce A. fumigatus growth

Regarding P. aeruginosa and A. fumigatus competitive growth, the former produces phenazines to inhibit the growth of the latter (Zheng, Keller & Wang, 2015a), yet one study demonstrated that sub-bacteriostatic concentrations of phenazines can induce A. fumigatus growth in specific situations. For instance, it was reported that sub-bacteriostatic concentrations of PYO, PCA, and PCN can induce A. fumigatus growth by promoting iron uptake. The redox function of 1-HP, which is capable of chelating iron ions and inducing iron starvation in A. fumigatus, can also promote A. fumigatus growth (pathway ① in Fig. 1) (Briard et al., 2015).

Aspergillus fumigatus can obtain iron resources through low-affinity ferrous iron uptake, high-affinity reductive iron uptake and siderophore-mediated iron uptake (Schrettl et al., 2004). Three sub-bacteriostatic phenazines, PYO, PCA, and PCN, can induce the growth of an A. fumigatus mutant lacking SidAp and siderophore biosynthesis under iron starvation conditions, and it was suggested that phenazines reduce Fe³⁺ to Fe²⁺ and promote the ferroxidase FetCp/permease FtrAp complex of the A. fumigatus mutant to take up Fe²⁺ (pathway ③ in Fig. 2) (Briard et al., 2015). Fusarinine C (FsC) and triacetylfusarinine C (TAFC) are two extracellular siderophores of A. fumigatus, while ferricrocin and hydroxyferricrocin are two intracellular siderophores of A. fumigatus (Haas, 2012). Sub-bacteriostatic 1-HP produced by P. aeruginosa promotes A. fumigatus growth by iron chelation and stimulation of TAFC secretion (pathway ④ in Fig. 2) (Briard et al., 2015).

VOCs promote A. fumigatus growth

A recent study showed that VOCs released by P. aeruginosa can promote A. fumigatus growth (pathway ⑥ in Fig. 1) (Briard, Heddergott & Latgé, 2016), and dimethyl sulfide was found to be a VOC with an enhancement effect on A. fumigatus, which is mediated by the gas phase. During sulfur starvation, A. fumigatus utilizes exogenous VOCs to promote growth, and in patients with CF, it is possible that P. aeruginosa promotes the colonization and growth of A. fumigatus by releasing VOCs, causing a rapid decline in lung function (Briard, Heddergott & Latgé, 2016). This result is consistent with the results of past clinical studies showing that P. aeruginosa and A. fumigatus co-infection resulted in decreased lung function (Amin et al., 2010; Baxter et al., 2013b).

Without direct contact, P. aeruginosa and A. fumigatus interact through signaling molecules such as VOCs. With direct contact, P. aeruginosa can release the corresponding signaling molecules to promote A. fumigatus colonization and growth. However, when these pathogenic microorganisms grow and contact one another, they exert a mutual inhibitory effect with regard to nutrient competition (Briard, Heddergott & Latgé, 2016).

Metabolic transformation of phenazines produced by P. aeruginosa

Phenazines inhibit A. fumigatus by inducing the production of ROS and RNS. Sod2p of A. fumigatus can resist the injury caused by ROS and RNS and antagonize inhibition by P. aeruginosa (Briard et al., 2015). Another study demonstrated that the phenazines produced by P. aeruginosa can be metabolically converted by A. fumigatus to reduce their inhibitory effect. As an example, PCA can be converted to 1-HP,1-methoxyphenazine and phenazine-1-sulfate. Although 1-HP has an inhibitory effect on A. fumigatus, 1-HP was also able to induce the production of the A. fumigatus siderophores TAFC and FsC (Moree et al., 2012). Regardless, previous experiments have shown that 1-HP inhibits bacterial siderophore biosynthesis (Dietrich et al., 2008). PCA induces the reduction of Fe³⁺ to Fe²⁺ for P. aeruginosa biofilm formation (Wang et al., 2011), and conversion of PCA by A. fumigatus may also decrease P. aeruginosa iron acquisition for metabolism and biofilm formation. Phenazine is converted by A. fumigatus into metabolic products with potentially altered redox potentials, and these products may inhibit Fe³⁺ reduction in P. aeruginosa (pathway ② Fig. 2). The phenazines PYO and PCA produced by P. aeruginosa can be converted to phenazine dimers by A. fumigatus, which have a decreased inhibitory effect on A. fumigatus. The QS signaling molecule PYO of P. aeruginosa affects transcriptional regulation and induces biofilm formation. Metabolic conversion of PYO by A. fumigatus might have an effect on QS system regulation. Thus, A. fumigatus can transform P. aeruginosa metabolites and radically alter the effects on their interaction, including the degree of inhibition (pathway ② in Fig. 1) (Moree et al., 2012).

Gliotoxin produced by A. fumigatus interferes with the metabolic growth of P. aeruginosa

During co-culture, A. fumigatus can also invoke its own metabolism and signaling molecules to disrupt P. aeruginosa growth. Gliotoxin produced by A. fumigatus is also a major immunoevasive toxin that is important in mediating A. fumigatus-associated colonization within the context of CF (Chotirmall et al., 2014). Overall, the gliotoxin secreted by A. fumigatus suppresses the inhibitory ability and growth of P. aeruginosa (pathway ⑦ in Fig. 1) (Manavathu, Vager & Vazquez, 2014).

Reducing the sensitivity of P. aeruginosa to antibiotics and promoting chronic infection

Under specific conditions, A. fumigatus can maintain the growth of P. aeruginosa in a co-existence scenario. One case-control study showed that CF patients infected by A. fumigatus could easily develop chronic P. aeruginosa infections despite receiving anti-microbial therapy (pathway ⑧ in Fig. 1) (Pressler et al., 2006).

The sensitivity of P. aeruginosa to antibiotics also changes when it co-exists with A. fumigatus. One in vitro study showed that P. aeruginosa in a polymicrobial biofilm with A. fumigatus was less susceptible to cefepime than P. aeruginosa in a monomicrobial biofilm state (pathway ⑧ in Fig. 1) (Manavathu, Vager & Vazquez, 2014). The extracellular matrix of polymicrobial biofilms differs for P. aeruginosa monomicrobial biofilms, and the sensitivity of P. aeruginosa in polymicrobial biofilm to some antibiotics decreases as a result of the change in the biofilm extracellular matrix. Indeed, some antibiotics cannot kill P. aeruginosa in biofilm due to altered permeability of the polymicrobial biofilm extracellular matrix. In contrast, the change in extracellular matrix between polymicrobial biofilms and A. fumigatus monomicrobial biofilms is not obvious and may not be sufficient to cause changes in the sensitivity of A. fumigatus to anti-fungal agents. For example, the sensitivity of A. fumigatus to anti-fungal drugs, such as voriconazole and posaconazole, did not change in polymicrobial or monomicrobial biofilms (Manavathu, Vager & Vazquez, 2014).

At present, only a few studies have examined the effect of A. fumigatus on P. aeruginosa, and the underlying mechanism should be the focus of further research.