Drosophila melanogaster as an organism model for studying cystic fibrosis and its major associated microbial infections

ABSTRACT

Cystic fibrosis (CF) is a human genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene that encodes a chloride channel. The most severe clinical manifestation is associated with chronic pulmonary infections by pathogenic and opportunistic microbes. Drosophila melanogaster has become the invertebrate model of choice for modeling microbial infections and studying the induced innate immune response. Here, we review its contribution to the understanding of infections with six major pathogens associated with CF (Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia, Mycobacterium abscessus, Streptococcus pneumoniae, and Aspergillus fumigatus) together with the perspectives opened by the recent availability of two CF models in this model organism.

BACTERIAL INFECTIONS IN CYSTIC FIBROSIS

Cystic fibrosis (CF) is a human genetic disease with a recessive autosomal transmission. It is the most common genetic disease among Caucasians and affects approximately 7.97/100,000 persons in the USA and 7.37/100,000 in the European Union (1). Although the pulmonary form is the most severe clinical manifestation, other exocrine organs may also be affected (e.g., the pancreas and intestine). The disease is caused by loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene (2 – 4), which encodes a member of the adenosine triphosphate (ATP)-binding cassette (ABC) protein superfamily (3). CFTR is an ATP-gated ion channel that conducts chloride ions across epithelial cell membranes (5, 6), as well as glutathione thiocyanates and bicarbonates.

In addition to modulating the chloride transport, it regulates the activity of other ion channels such as the trimeric epithelial sodium channel (ENaC), which consists of the subunits α, β, and γ. How CFTR negatively regulates ENaC is still controversial. According to König and collaborators, this regulation occurs indirectly through the accumulation of intracellular chlorine (7). However, conflicting results have shown that inhibition of ENaC by CFTR is independent of the direction and extent of chloride transport (8). Studies have shown that CFTR inhibits ENaC through a direct physical interaction (9) or by regulating ENaC subunit quantities (10).

In any case, CFTR dysfunction leads to an excessive activity of the trimeric ENaC channel, causing uncontrolled sodium and excessive water entry into the epithelial cells following the osmotic gradient. This leads to dehydration of the intraluminal surface and an increase in the thickness of the mucus bordering the epithelium (11). In the lungs, the accumulation of thick viscous secretions causes obstruction and inflammation of the airways. These prevent the proper functioning of the mucociliary barrier, which is the primary protective barrier against many pathogens (12). In addition, this mucus has poor antibacterial activity owing to its reduction in acidity. Indeed, CFTR dysfunction prevents the exit of bicarbonate ions. This modified mucus constitutes the ideal environment for the accumulation, proliferation, and persistence of pathogenic and/or opportunistic microorganisms.

Chronic and recurrent infections and persistent inflammation cause airway damage that can lead to bronchiectasis and thus, a decline in respiratory function (13). The ensuing respiratory failure is the cause of more than 90% of the recorded deaths (14). According to the 2021 report of the French Cystic Fibrosis Registry, these infections are mainly due to Staphylococcus aureus (60.6%), Pseudomonas aeruginosa (34%), Aspergillus fumigatus (21.6%), Haemophilus influenzae (10.1%), Stenotrophomonas maltophilia (9.3%), Achromobacter xylosoxidans (6.4%), Streptococcus pneumoniae (1.7%), non-tuberculous mycobacteria (NTM; 3.7%), and Burkholderia cepacia (2%). The prevalence of these pathogens varies according to the geography. For comparison, in the USA, S. aureus, P. aeruginosa, and NTM have approximately 63%, 24%, and 9.6%, respectively, of the overall prevalence according to the 2021 report of the Cystic Fibrosis Foundation (CFF). The dynamics of the prevalence of the isolated pathogens also changes with time. For example, the respective trends in the prevalence of S. aureus and P. aeruginosa have taken opposite trajectories over time in the USA. While the former is becoming increasingly prevalent (29% in 1991 vs. 63% in 2021), the latter is less prevalent over time (61% in 1991 vs. 24% in 2021). The same trend is observed in Europe (15).

DROSOPHILA, AN ESTABLISHED ORGANISM MODEL FOR THE STUDY OF PATHOGENS

Drosophila melanogaster is a century-old organism model that is used in various aspects of life sciences such as genetics, developmental biology, cellular biology, neurobiology, and immunity. The constant development and availability of different genetic tools have facilitated its genetic manipulation, making Drosophila central to the study of responses to infection and host-pathogen interactions in the last three decades. In their own natural environment, fruit flies face a panel of viruses, bacteria, fungi, and parasites [e.g., wasp (16)]. In the laboratory, Drosophila is used as an experimental host to study infection with its natural pathogens as well as human ones. Indeed, Drosophila has become an attractive and emergent model for studying host response, virulence factors, and pathophysiology of pathogens associated with human infectious diseases, such as those caused by Zika Virus, Mycobacterium marinum, Listeria monocytogenes, and Candida albicans (17 – 21).

Drosophila is a dipteran with three larval stages and a complete metamorphosis. In laboratory, third instar larvae and adults are usually infected either orally, locally by wounding or systemically by injecting the microorganism.

Drosophila lacks an adaptive immune response but has innate immunity involving conserved signaling pathways. In both mammals and flies, the JNK, JAK-STAT, and NFκB signaling pathways are critical for immune response regulation (22). To note, the response mediated by Toll-like receptors was discovered in this organism (23). Drosophila pattern recognition receptors (PRRs) recognize the pathogen-associated molecular patterns (PAMPs) of microbes, such as peptidoglycan (PGN) or lipoteichoic acid (LTA) (24). They induce an adequate immune response involving both cellular and humoral response (25).

The cellular response is based on blood cells (hemocytes) which are equivalent to mammalian monocytes and macrophages. Until recently, three morphologically distinct types of hemocytes have been identified: plasmatocytes, crystal cells [involved in wound healing, reactive oxygen species (ROS) production, and hypoxic response], and lamellocytes (involved in response to wasp parasitization) (26). The most abundant hemocytes are plasmatocytes that respond to wound signals and control the coagulation response. They also phagocytose and encapsulate invading pathogens and clear apoptotic bodies (27). However, this simplistic classification of hemocytes into three subtypes has been reviewed thanks to recent studies based on single-cell sequencing on either larval (28 – 31), adult (32), or pupal (33) hemocytes. Collectively, these studies have identified at least eight distinct specialized hemocyte subpopulations waiting to be functionally characterized.

The existence of an inducible humoral response in fruit flies was first reported in 1972 (34). This response is mainly based on the production of antimicrobial peptides (AMPs), primarily by the fat body, which is functionally homologous to mammalian adipocytes and liver. AMPs can also be produced locally by epithelial cells or hemocytes. Two conserved NFκB signaling pathways, Toll and Immune deficiency (Imd), mediate AMP production. The former is implicated in response to both bacteria with Lys-type PGN (mainly Gram-positive) and fungal infections, while the second is involved in responses to infection by bacteria with DAP-type PGN (mainly Gram-negative) (22, 23, 35, 36). In addition to AMPs production, humoral response also includes the generation of ROS by DUOX proteins locally at the epithelial level (37, 38).

LESSONS FROM DROSOPHILA INFECTIONS WITH SOME CF MAJOR PATHOGENS

Drosophila is commonly used to study infections with a single pathogen associated with CF or co-infection. Here, we review the contributions of this model organism to the identification of host receptors, in vivo validation of virulence factors, and to the screening of effective drugs. We will follow the prevalence of these pathogens as reported by the French Cystic Fibrosis Registry in 2021, and the number of relevant publications. An overview is provided by Table 1.

TABLE 1.

Modeling the infections by major CF pathogens in Drosophila

Pathogen	Host immune response					Host manipulation by the pathogen	In vivo validated antimicrobial
	Cellular response		Humoral response		Other response(s)
	Phagocytosis	ROS production	Toll	IMD
S. aureus	Receptors: Croquemort, Draper, Eater				?	Neutralization of host oxidative and antimicrobial responses by catalase Reduction of PGN detection and humoral response by producing D-alanylated teichoic acid	Nisin NAI-107 Plumbagin
P. aeruginosa	Receptors: ?	?			Activation of JNK pathway in enterocytes during oral infection Nutritional immunity: iron sequestration from the hemolymph and relocation to the fat body	Prevention of phagocytosis by hemocytes by RhIR and the exotoxin ExoS Induction of apoptosis of S2 cells by ExoS and Exotoxin A Suppression of AMP production	Lytic phage MPK1 Lytic phage MPK6 Baicalin
B. cepacia	? Receptors: ?	?			Activation of TOR pathway for tolerance and resistance	?	?
M. abscessus	Receptors: ?	?			Granzyme-mediated cytotoxic response by thanacytes	?	Tigecycline Linezolid
S. pneumoniae	Receptors: ?	?			Activation of adenosine signaling for metabolic switch	Loss of circadian regulation of locomotor activity	?
A. fumigatus	? Receptors: ?	?			?	?	Voriconazole Posaconazole Terbinafine

Stenotrophomonas maltophilia and Achromobacter xylosoxidans were excluded for the following reasons. Stenotrophomonas maltophilia has been isolated at the surface and in the gut of wild female Drosophila captured in Puerto Rico (39). Its intestinal presence was confirmed in laboratory strains (40). Achromobacter xylosoxidans, has been reported to be pathogenic for Drosophila, as its injection in adult males leads to rapid dose-dependent death (41).

Staphylococcus aureus

Infections with the Gram-positive bacterium Staphylococcus aureus (S. aureus) are among the most prevalent in CF patients. Injection of live S. aureus into Drosophila leads to an important transcriptional response and a systemic infection resulting in a reduction in fly life expectancy (42, 43). Phagocytosis plays a major role in the response to S. aureus infection as flies devoid of plasmatocytes succumb more rapidly (44, 45). Drosophila Schneider 2 (S2) cells were used as a surrogate for hemocytes. Indeed, this widely used cell line, derived from late embryos, is phagocytic. Genetic screening of S2 cells identified Eater and Croquemort, as S. aureus scavenger receptors (44). This was confirmed in adult hemocytes (44) and mammalian macrophages (46). Croquemort is the first CD36 family member to be described as being involved in bacterial recognition. Eater does not recognize LTA, a cell wall polymer found in Gram-positive bacteria. Indeed, the ltaS mutant strain (deficient in LTA synthesis) was phagocytosed less by wild-type hemocytes than the wild-type S. aureus strain. Moreover, the ltaS mutant strain was equally phagocytosed by wild-type and Eater-lacking larval hemocytes (47). However, this was not the case for hemocytes lacking the receptor Draper, whose extracellular region binds LTA, strongly suggesting that this cell wall component is its ligand, contrary to Eater (47).

The integrin βυ is also involved in S. aureus recognition by the hemocytes but through peptidoglycan. Indeed, a mutant bacterial strain that produces reduced levels of PGN, due to defective UDP-N-acetylenolpyruvylglucosamine reductase, was less efficiently phagocytosed by integrin βυ-deficient hemocytes (48).

PGN recognition proteins (PGRPs), such as PGRP-SA and PGRP-SC1a, are also important for the recognition and phagocytosis of S. aureus (49). However, wall teichoic acids (WTAs), which are covalently linked to PGN, mitigate S. aureus recognition by these Drosophila immune receptors. Indeed, infection with strains with defective WTA production led to a reduction of S. aureus virulence. This loss of pathogenicity is due to increased PGN binding and detection by PGRP-A (50). Complementary to inducing a cellular response, S. aureus PGRP-SA-mediated recognition systemically activates the Toll pathway leading to AMPs production (e.g., Drosomycin, Defensin, and Metchnikowin) (51). Although Imd-related AMPs are not induced, this pathway is required for effective clearance of the infection (52).

Moreover, fly infections have been used to validate known virulence factors, such as hemolysin α (53), as well as to identify new ones. An example is the production of D-alanylated teichoic acid, which reduces PGN recognition by host receptors and thus interferes with the host humoral response to S. aureus infection (51).

Drosophila infections have confirmed that methicillin-resistant S. aureus (MRSA) isolates, notably the USA300 and PFGE strains, were less virulent than non-MRSA isolates (54). Correlations with clinical observations were found for the community-associated MRSA strains USA300, USA400, and CMRSA2. Indeed, the latter are more virulent than the hospital-associated strain CMRSA6 (53). Recently, a model of oral USA300 infection in Drosophila larvae showed that bacterial catalase neutralizes a DUOX-mediated oxidative response that promotes AMPs production through Toll pathway activation (55).

To identify drugs effective against these MRSAs, a panel of antibacterial peptides was screened in vivo. Two antibiotics, nisin and NAI-107, have been shown to have the ability to rescue adult flies from fatal infections with the USA300 strain. NAI-107 presented an efficacy equivalent to that of vancomycin, a widely applied antibiotic for the treatment of serious MRSA infections (56).

The antimicrobial activity of plumbagin, a phytochemical, was also validated with the Drosophila systemic infection model, whether with S. aureus alone or in co-infection with C. albicans, as is often observed in the urinary tract in humans. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) has been identified in vitro as a potent antimicrobial agent against S. aureus and C. albicans (57).

Pseudomonas aeruginosa

Drosophila is susceptible to both oral and systemic infections by the Gram-negative bacterium P. aeruginosa. This leads to the invasion of host tissues, then their degradation and ultimately death through the bacterial spread in the hemolymph (34, 58 – 60).

P. aeruginosa infections induce systemic AMP production mediated by both the Toll and Imd pathways (60, 61), a local epithelial Imd-dependent one and a cellular response (59). More recently, a novel and evolutionarily conserved defense mechanism has been reported (62). P. aeruginosa infection induces the overexpression of the iron transferrin 1-encoding gene in the fat body. The consequence is sequestration of iron from the hemolymph and its relocation to the fat body. The importance of the competition for iron between P. aeruginosa and its host is further supported by the reduced pathogenicity of a siderophore-defective strain of P. aeruginosa in Drosophila (62).

Fruit flies have been used to screen P. aeruginosa mutants and thus to validate (63) and identify new virulence factors (e.g., relA) (64). Similarly, the contribution of certain virulence factors has been characterized in vivo in fruit flies. Examples include the oxylipins involved in biofilm formation and virulence (65), glutathione biosynthesis genes gshA and gshB (66), transcriptional regulators PA1226 and PA1413, which modulate the virulence (67), reactive chlorine species resistance factor RcsA (68), glucose transport regulator GltB (69), and the nitrite reductase NirA (70). The essential role of the P. aeruginosa respiratory chain in virulence and pathogenicity has also been demonstrated in Drosophila. Indeed, a PA4427-PA4431 operon mutant strain, defective for respiratory chain complex III (cytobc1), induces less mortality in Drosophila than the PAO1 reference strain (71).

Many pathogenic Gram-negative bacteria, including P. aeruginosa, possess a type III secretion system (T3SS), which injects virulence factors into their host (72). The presence and activation of T3SS are required in P. aeruginosa to induce fly death (73). The exotoxin ExoS, whose injection into the host cell cytoplasm is mediated by T3SS, interferes with bacterial phagocytosis by hemocytes (74). ExoS is known to target host Rho GTPases and the contribution of different fly Rho GTPases to P. aeruginosa infection resistance has been assessed in vivo, revealing that Rac2 is the main target of ExoS to prevent engulfment (75). ExoS can also induce apoptosis at least in Drosophila S2 cells (76), similar to Exotoxin A (77).

P. aeruginosa uses quorum sensing (QS) to regulate and adapt its gene expression. During infection, the QS signaling molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) is essential for the bacterial virulence in flies. Drosophila lacks Paraoxonases (PONs) which are able to degrade 3OC12-HSL in vitro. Transgenic expression of human PON1 protects flies against P. aeruginosa infection lethality by interfering with 3OC12-HSL-dependent QS (78). The QS transcription factor RhIR interferes with the host’s cellular immune response during the early stages of infection (59). P. aeruginosa can also inhibit the host response by suppressing AMP production (61).

Chronic P. aeruginosa infection in patients with CF is associated with the formation of mucoid micro-colonies called biofilms. These are observed in the Drosophila crop, the functional equivalent of the mammalian stomach, after oral infection. Bacteria recovered from this in vivo biofilm present an increased antibiotic resistance and less virulence than the planktonic bacteria (79). Transcriptional regulator PA3898 controls biofilm formation and virulence in Drosophila (80). Furthermore, oral infection with P. aeruginosa leads to midgut hyperplasia. This is due to activation of the stress response JNK pathway in enterocytes, leading to their apoptosis and indirectly to the overproliferation of intestinal stem cells (81).

Fruit flies can help to find alternative effective therapeutic strategies against P. aeruginosa infections, in addition to antibiotics. Indeed, the in vivo antibacterial efficacy of P. aeruginosa-targeting lytic phages, such as MPK1 and MPK6, has been assessed and proven in Drosophila (82, 83). Moreover, Baicalin, has been validated in vivo in Drosophila (84). This extract from the Chinese herb Scutellariae radix has been proposed as an alternative anti-P. aeruginosa compound targeting bacterial T3SS.

Aspergillus fumigatus

Immuno-compromised patients as well as those living with CF are prone to invasive aspergillosis. In order to examine the conserved Toll pathway associated with the response to fungal infection in Drosophila, including A. fumigatus (85), the virulence of different strains of the cosmopolitan filamentous fungus A. fumigatus was assessed using Toll-deficient flies (86). Infections were induced by injecting, feeding, or rolling flies with conidia (87). Concordance with results obtained in mammalian models was observed with either the hypovirulent strain ∆alb1 (88) or other A. fumigatus mutant strains defective in siderophore biosynthesis, starvation stress response (89), or Glicotoxin production (90).

Toll-deficient Drosophila have also been used to assess the in vivo efficacy of orally absorbed antifungal agents such as voriconazole and posaconazole, which are commonly used as prophylaxis and treatment for the fungus (88, 91). An in vitro pre-exposure of A. fumigatus to these molecules was performed before Drosophila infection with A. fumigatus. This pre-treatment of A. fumigatus did not affect the fungal virulence or the efficacy of the same molecules to clear the infection in vivo (88, 91). Synergistic effects have been observed when voriconazole was combined with terbinafine (87, 91, 92).

In vivo toxicity of volatile organic compounds (VOCs) produced by filamentous fungi (e.g., alcohols, aldehydes, thiols, esters) has been explored in flies. Exposition of Drosophila larvae to VOCs emitted by living fungi delayed metamorphosis toward the pupae stage and subsequently to the adult stage. In addition, this exposure was detrimental to both larval and adult survival (93 – 95). This toxigenic effect suggests that VOCs may contribute to the fungal pathogenesis, at least in flies.

Burkholderia cepacia complex

Drosophila is an established model for studying systemic infections caused by species of opportunistic Gram-negative bacteria belonging to the Burkholderia cepacia complex (Bcc). It has been used to characterize the virulence of different strains (96), the phenotype of some mutants (97 – 99), and also to identify virulence factors of strains isolated from CF patients (100).

In response to B. cepacia infection, fruit flies produce AMPs, such as Drosomycin and Diptericin, via both the Toll and Imd pathways (101). We recently demonstrated that the induced AMPs are crucial for Drosophila survival against B. cepacia infection (102).

Drosophila mutants for the period gene, whose circadian rhythm is altered, are more tolerant to Bcc infection (101). This study also revealed that both glucose and amino-acid intake improved host tolerance to infection and that the TOR pathway mediates both resistance and tolerance to Bcc infections (101).

Mycobacterium abscessus and the NTM

Drosophila is also a validated model for studying mycobacterial infections. As recently reviewed, most studies have focused on the pathogenic slow-growing Mycobacterium marinum to model tuberculosis (103). The most frequently isolated NTM in patients with CF are species of M. abscessus and M. avium complexes, M. fortuitum being rarely found (104, 105). In a study including French patients, M. abscessus accounted for more than half of the NTM isolated (104). This bacterium causes the most deleterious pulmonary infections in patients with CF (106). M. abscessus belongs to the group of fast-growing mycobacteria which are predominantly saprophytic. It is considered the most pathogenic species within this group (107).

After systemic injection, M. abscessus can proliferate within Drosophila, leading to severe tissue damage and, ultimately, death (108). It is recognized by PGRP-SA and activates the production of Drosomycin, a Toll-mediated AMP (108). Recently, we confirmed and extended this observation. Indeed, M. abscessus injection induced the expression of AMPs encoding genes, either Toll- or Imd-regulated and showed that these AMPs did not seem to play a major role for Drosophila survival during M. abscessus infection, as indicated by the similar survivals of wild-type and AMP-deficient flies (102). We therefore hypothesized and demonstrated that the intracellular localization of M. abscessus protects it from AMPs, particularly Defensin, which we have shown to have a direct bactericidal action against extracellular M. abscessus (109). Indeed, after its injection, M. abscessus is rapidly internalized by Drosophila plasmatocytes in which it grows (102), as observed during fly infection with M. marinum (18).

Fly infections have been used to validate mutants for genes encoding known virulence factors, such as the ∆0855 and ∆4532 c strains, both defective for intracellular growth (110, 111), as well as to identify some new genes such as MAB_0471, MAB_0472, and MAB_3317c (112).

Drosophila have also highlighted M. abscessus resistance to host innate cytotoxic responses. Indeed, thanacytes, a newly described hemocyte subpopulation identified by single-cell sequencing (29), induce caspase-dependent apoptosis in M. abscessus-infected plasmatocytes through the action of two serine proteases, encoded by CG30088 and CG30090. However, M. abscessus resists this lysis and spreads systemically, leading to bacteremia and subsequent death of infected flies. The resistance of M. abscessus to cytotoxic lysis of phagocytes was validated in a mammalian model after contact of infected murine primary macrophages with autologous natural killer cells. This propensity of M. abscessus to resist the host cytotoxic innate response, typical of strict pathogenic mycobacteria such as M. tuberculosis, could partially explain its superior pathogenicity among fast-growing mycobacteria.

M. abscessus is also multi-resistant to antibiotics, including most of the anti-tuberculosis drugs (113), making it difficult to treat its infections in patients with CF (114). Drosophila have been used to test the effectiveness of antibiotics against M. abscessus in vivo. Tigecycline treatment was the most efficient and its potency was increased when combined with linezolid (115).

Streptococcus pneumoniae

Injection of the Gram-positive bacterium S. pneumoniae in Drosophila causes lethal infections. Fly exposure to sublethal doses primes resistance to subsequent infections by S. pneumoniae (116). Phagocytosis by plasmatocytes is crucial for resistance to streptococcal infections (116 – 118). It is activated by Eiger, a Drosophila homolog of humans TNFα (119). Hemocyte activation requires increased consumption of energy, which is obtained by a systemic metabolic switch involving the release of glucose from glycogen. This is mediated by adenosine signaling and is modulated by adenosine deaminase ADGF-A to prevent the loss of energy reserves during chronic infection (118). Interestingly, this effect of adenosine has also been observed in a mice lung streptococcal infection model in which it regulates pulmonary neutrophil recruitment (120).

The Drosophila response to a systemic infection with S. pneumoniae is not limited to the immune cellular response because it also includes the production of AMPs, mediated by both Toll and Imd pathways (118).

S. pneumoniae infections have been used to assess whether interactions between circadian rhythm and immunity exist in flies, as observed in mammals (121). Infected wild-type flies lose circadian regulation of locomotor activity, whereas mutant flies for timeless or period, which encode components of the central circadian clock, were more sensitive than wild-type flies to S. pneumoniae infection (122).

CO-INFECTION MODELS

Most patients with CF are prone to polymicrobial infections. Drosophila has been used to study such interactions between pathogens as well as those with the host microbiota. Indeed, flies were orally infected with a combination of P. aeruginosa and strains isolated from the oral flora of patients with CF to compare bacterial virulence genes and host AMP gene expression with mono-infections. Thus, it was observed that co-infection with Streptococcus sp. and P. aeruginosa increased the production of the flagellar filament protein fliC in P. aeruginosa, most likely to increase its motility (123). Upon co-infection with Gram-positive bacteria, P. aeruginosa also presents an increased virulence, due to the production of antimicrobials and toxins that kill the other bacteria as well as the host cells. The latter is induced by the detection by P. aeruginosa of Gram-positive bacteria PGN (124).

Conversely, Streptococcus parasanguinis, a Gram-positive colonizer of the airway of patient with CF, hijacks P. aeruginosa exopolysaccharide alginate production to form a biofilm that limits P. aeruginosa growth. This biofilm contains streptococcal adhesins, which are also key factors for fly colonization and mortality (125). Nitrite reductase production is crucial for P. aeruginosa virulence (126).

A more recent model of co-infection with two common pathogens found in patients with CF was based on the co-injection with S. aureus and P. aeruginosa in adult Drosophila (127).

MODELING CF IN DROSOPHILA

Two CF-like models have been proposed in Drosophila. The first consists of mutant flies for the bereft gene which encodes miR-263a, a microRNA negatively regulating the quantity of transcripts encoding the α and β subunits of ENaC (ppk4 and ppk28, respectively). Thus, these flies are a model of ENaC hyperactivity. Indeed, phenotypes in their midgut are similar to those observed in epithelia of patients with CF. It was observed that there was excessive sodium entry within enterocytes, the most abundant intestinal cells, leading to an incoming flow of water following the osmotic gradient and to a dehydration of the intraluminal area bordering the epithelium (128).

These phenotypes are also observed in the second gastro-intestinal Drosophila CF, which has been more recently reported (129). It is a CFTR mutant model obtained by depleting in enterocytes the transcripts of CG5789/Cftr. This gene encodes the Drosophila structural and functional equivalent of human CTFR. Indeed, the expression of human CFTR in this CF model rescued gastro-intestinal phenotypes. Partial suppression of these phenotypes was also observed upon overexpression of miR-263a, suggesting that ENaC may act downstream of CFTR, as in humans (129).

Both models exhibit increased levels of antimicrobial peptides due to the activation of the Imd pathway in response to increased bacterial accumulation in the midgut. Moreover, they are more susceptible to oral infections with Pseudomonas aeruginosa (128, 129). Here again, human CFTR expression rescued this phenotype in flies depleted of Cftr transcripts, establishing a new model to study CF pathophysiology, particularly in respect to the susceptibility to pathogen infections (129).

It would be interesting to determine the susceptibility of both models to other major pathogens found in CF. To note, ENaC has been proposed to be involved in airway liquid clearance (130). One may wonder whether the CF phenotypes observed in miR-263a mutant flies are only restricted to the midgut and whether this model is more susceptible to systemic infections.

CONCLUDING REMARKS

The recent use of certain CFTR modulators has brought relief to many CF patients; but unfortunately, not to all. The development of relevant models is crucial for understanding CF pathophysiology and consequently for searching for effective molecules that can be beneficial in all kinds of cftr mutations leading to CF. Drosophila can meet this need, all the more so as CFTR and ENaC channels are present and their deregulation leads to a CF phenotype. As we have shown in this review, fruit flies have already allowed the identification of many virulence factors of the most common pathogens in patients with CF, as well as numerous host factors required to counter these infections. Drosophila use should make it possible to study and understand host resistance factors that are modulated in the context of CF. In the long term, treatments based on the modulation of the evolutionarily conserved susceptibility and predisposition factors could reduce CF-associated infections.

Biographies

Hamadoun Touré is holder of a master's degree in parasitology followed by a master's degree in host-pathogen interactions in 2019. Having always been interested in the latter field, he completed his PhD on the mechanisms of resistance of Mycobacterium abscessus to the host innate immune response using Drosophila as a model in Prof. Jean-Louis Herrmann's laboratory at Université Paris-Saclay. After defending his thesis in May 2023, he will join Dr. Meghan Koch's laboratory at the Fred Hutchinson Cancer Center in October 2023 as a postdoctoral fellow to study how the acquisition of maternal factors through breast milk contributes to shaping offspring immunity.

Jean-Louis Herrmann is University Professor and Hospital Practitioner, Head of the Bacteriology-Hygiene Laboratories at the R. Poincaré and A Paré hospitals, University Hospital Group of Paris-Saclay and Director of the Mixed Research Unit UMR1173 (Université de Versailles St Quentin en Yvelines and INSERM). His field of research concerns host-pathogen interactions, with a particular focus on mycobacteria, and currently Mycobacterium abscessus. He is currently involved in the development of diagnostic and therapeutic tests to tackle infectious diseases on the basis of this research.

Sébastien Szuplewski is Assistant Professor at the University of Versailles-Saint-Quentin-en-Yvelines (UVSQ). He received his Ph.D. in Developmental Biology from University of Paris XI, known nowadays as University Paris-Saclay, under the supervision of Dr. Régine Terracol at Institut Jacques Monod (IJM). Following his postdoctoral research in the laboratories of Pr. Stephen M. Cohen at EMBL, Temasek Life Sciences Laboratory (TLL) and Institute of Molecular and Cell Biology (IMCB), he joined the UVSQ-Laboratory of Genetics and Biology of the Cell (LGBC) working in part on host/pathogen interactions between Drosophila and Mycobacterium abscessus.