Identification of Drosophila Mutants Altering Defense of and Endurance to Listeria monocytogenes Infection
Janelle S. Ayres, Nancy Freitag, David S. Schneider


We extended the use of Drosophila beyond being a model for signaling pathways required for pattern recognition immune signaling and show that the fly can be used to identify genes required for pathogenesis and host–pathogen interactions. We performed a forward genetic screen to identify Drosophila mutations altering sensitivity to the intracellular pathogen Listeria monocytogenes. We recovered 18 mutants with increased susceptibility to infection, none of which were previously shown to function in a Drosophila immune response. Using secondary screens, we divided these mutants into two groups: In the first group, mutants have reduced endurance to infections but show no change in bacterial growth. This is a new fly immunity phenotype that is not commonly studied. In the second group, mutants have a typical defense defect in which bacterial growth is increased and survival is decreased. By further challenging mutant flies with L. monocytogenes mutants, we identified subgroups of fly mutants that affect specific stages of the L. monocytogenes life cycle, exit from the vacuole, or actin-based movement. There is no overlap between our genes and the hundreds of genes identified in Drosophila S2 cells fighting L. monocytogenes infection, using genomewide RNAi screens in vitro. By using a whole-animal model and screening for host survival, we revealed genes involved in physiologies different from those that were found in previous screens, which all had defects in defensive immune signaling.

INTRACELLULAR pathogens are responsible for a large group of infectious diseases; for example, >500 million people worldwide suffer from tuberculosis, AIDS, and malaria each year ( By residing in a host cell, these pathogens protect themselves from some host immune responses and drug therapies. The ability to enter and survive within a host cell requires a close and intricate interaction between pathogen and host; by manipulating host processes, pathogens can prevent immune responses or subvert host processes to aid in infection.

Listeria monocytogenes is a gram-positive, facultative intracellular bacterium and is the cause of listeriosis, a serious food-borne disease. L. monocytogenes has been widely used as a model pathogen to better understand the molecular and cellular aspects of intracellular pathogenesis and mammalian cell-mediated immunity. In vitro studies using cultured cells as a model host defined the L. monocytogenes intracellular life cycle and virulence factors (Cossart and Lecuit 1998; Portnoy et al. 1988; Kreft and Vazquez-Boland 2001; Vazquez-Boland et al. 2001) and demonstrated that L. monocytogenes can enter professional phagocytes or nonphagocytic cells (Cossart et al. 2003). Entry into nonphagocytic cells is dependent on surface proteins called internalins (Cossart and Lecuit 1998; Lecuit et al. 1999, 2001). Upon entry, a single-membrane vacuole forms around the bacterium (Cossart and Lecuit 1998). Secretion of a pore-forming cytotoxin, listeriolysin O (LLO), disrupts the phagosome membrane, freeing the bacterium into the cytosol where it can grow and divide (Kuhn et al. 1988; Portnoy et al. 1988; Gedde et al. 2000; O'Riordan and Portnoy 2002). Expression of the actin-nucleating protein ActA facilitates polymerization of host actin at one pole of the bacterium, resulting in directional movement through the cytosol. Bacteria are able to spread to neighboring cells and escape from a second double-membrane phagosome into the cytosol of a new host cell (Tilney and Portnoy 1989; Tilney et al. 1990; Domann et al. 1992; Kocks et al. 1992, 1993).

The development of RNA interference (RNAi) technology provided researchers with a new approach to studying host factors involved in host–pathogen interactions in the fly (Ramet et al. 2002; Foley and O'Farrell 2004; Ayres and Schneider 2006). Two recent reports describe the use of RNAi in Drosophila S2 cells (embryo-derived phagocytic cells from Drosophila) to identify host factors involved in L. monocytogenes infection; these screens helped clarify the cell biology of the interactions of L. monocytogenes with professional phagocytes (Agaisse et al. 2005; Cheng et al. 2005). The assays used in these in vitro screens measured bacterial loads or qualitative changes in bacterial patterns. This limited the scope of the screens to the identification of fly genes regulating the bacterial life cycle. Surprisingly, these screens did not identify genes affecting the well-characterized pattern recognition pathways, Toll and Imd. Moreover, these in vitro systems are obviously limited in their ability to explore how whole-animal physiologies interact with an infecting microbe and how L. monocytogenes can enter and survive in a wide variety of cell types.

To overcome these limitations, we performed a forward genetic screen in whole Drosophila to identify host genes required to survive infections. We had three goals. First, we wanted to cast a wider net than has been previously used to measure immunity in Drosophila. Much of what we know about fly immunity has been deciphered using extracellular microbes in immunocompromised flies. By using L. monocytogenes in our study we were able to identify host genes important for survival of an intracellular infection. These genes may be specific to a L. monocytogenes infection, to intracellular pathogens in general, or to a variety of pathogens. By coupling survival and bacterial proliferation as our output phenotypes, we were able to identify mutants with defense defects as well as lines with pathological defects—those that die from the disease faster without altered bacterial load. We hypothesize that this second class of mutants is less able to endure the stress of an infection. Second, we wanted to determine whether genes required to survive infection in whole flies were the same as those identified in cultured cells by RNAi screens. By doing this, our screen would reveal differences in the host genes involved in the interactions between whole-animal physiology and the microbe, as well as those host genes involved in the interactions between one cell type and the infecting microbe. Third, we wanted to probe the host contributions to the infection because most studies involving host–pathogen interactions with an intracellular pathogen have focused on identifying microbial factors required for infection.

The Drosophila innate immune response has three arms: First, the cellular immune response depends on circulating phagocytic cells that can engulf and clear foreign microbes. Second, the melanization response produces melanin and toxic reactive oxygen species at wounds and sites of infection. The third branch is the humoral immune response, which involves the production of antimicrobial peptides (AMP) by the fat body. These AMPs are produced largely under the control of the Toll and Imd pathways. Both of these pathways are activated by microbial elicitors and disruption of these pathways immunocompromises the fly such that the fly becomes sensitive to normally nonpathogenic bacteria like Escherichia coli (Lemaitre et al. 1995, 1996, 1997; Kopp and Medzhitov 1999, 2003; Khush et al. 2001). Genetic screens in whole flies have primarily concentrated on AMP signaling as an output, strengthening our understanding of signaling through the Toll and Imd pathways (Meng et al. 1999; Leulier et al. 2000; Rutschmann et al. 2000; Wu et al. 2001; Khush et al. 2002; Gesellchen et al. 2005; Yagi and Ip 2005; Kambris et al. 2006). In our screen we monitored fly survival as well as bacterial growth in mutant flies. By focusing on phenotypes other than AMP transcription, we anticipated that we would identify host genes affecting a wider array of immunological and pathological functions.

We identified 18 mutants with increased sensitivity to L. monocytogenes infection. None of these genes were described previously to have immune or pathogenesis functions. Secondary screens grouped these mutants into phenotypic classes. The flies can be split into classes on the basis of their ability to control the growth of L. monocytogenes. We define one group as having defects in endurance and suspect that it has difficulties controlling pathogenesis because mutant flies die faster than wild-type flies even though the bacterial load is the same as in wild-type flies. A second class appears to lack defense functions. We define this group as immunocompromised because it shows increased fly death that is correlated with rapid growth of L. monocytogenes. The mutants can also be split into either sensitive or wild-type classes on the basis of their response to Staphylococcus aureus or Salmonella typhimurium. Secondary screens that used L. monocytogenes mutants allowed us to determine that three of our fly mutants may be acting at the vacuole and cytoplasmic stages of the bacteria life cycle. Thus, by screening for survival as an endpoint instead of monitoring the transcriptional output of Toll or imd signaling, we identified host genes involved in immunity and pathogenesis that have not been identified previously.


Flies, bacterial strains, and media:

The Exelixis Drosophila melanogaster piggyBac insertion mutant collection was used for the survival screen. To serve as a wild-type control the w1118 line used to generate the isogenic collection was used. All viable lines available at the Bloomington Drosophila Stock Center at the beginning of the screen were obtained (1231 lines in total). Flies were kept in standard fly bottles containing dextrose medium. L. monocytogenes strains 10403S, DP-L2161 (Δhly), and DP-L3078 (ΔactA) were stored at −80° in brain–heart infusion (BHI) broth containing 15% glycerol. The L. monocytogenes mutant strains were derived from the 10403S parent strain and generated in the laboratory of Daniel A. Portnoy (University of California, Berkeley, CA). S. typhimurium SL1344 was stored at −80° in LB media containing 15% glycerol. S. aureus clinical isolate was from the Clinical Microbiological Laboratory at Stanford University and was stored at −80° in BHI media containing 15% glycerol.

Survival screen:

For infection of adult flies, L. monocytogenes was grown overnight in BHI medium at 37° without shaking. A total of 10 adult flies, aged 5–7 days old, per mutant line were injected with 50 nl of culture using a picospritzer and pulled glass needle. Flies were injected in the anterior abdomen on the ventrolateral surface with ∼1000 colony-forming units (CFUs). Mutant lines were tested in groups of a minimum of 20 lines, in addition to a wild-type control. Once infected, flies were transferred to vials containing dextrose medium and incubated at 29° and the number of dead flies for each line was counted every 24 hr postinfection until all flies were dead. The median time to death (MTD), the time postinfection when 50% of infected flies have succumbed to the infection, for each line was determined and those exhibiting the most extreme phenotypes were flagged for further testing. Mutant lines that failed to exhibit a phenotype in the initial test were not further tested. Candidate lines were retested in three independent experiments to eliminate any false positives. Thirty-five 7-day-old male flies per line were infected as before and the death rate of the mutant flies for each line was compared directly to the death rate of wild-type control flies, using Graphpad Prism software. Using log-rank analysis, the P-value was determined. Lines that exhibited a death rate with a P-value <0.05 for all three retests were considered positive mutants.

Secondary screens:

S. aureus and S. typhimurium secondary screens:

For infection of adult flies with S. aureus, bacteria were grown overnight in BHI medium at 37° with shaking. Approximately 100 CFUs were injected into each fly. For infection with S. typhimurium, bacteria were grown in LB medium at 37° and ∼10,000 CFUs were injected into each fly. For all infections, 35 7-day-old male flies per mutant line were infected as mentioned above and the death rate for each line was directly compared to that of wild-type flies and analyzed as previously mentioned.

Quantification of CFUs in flies:

Infected flies were homogenized in BHI media supplemented with 1% Triton X-100. Homogenates were serially diluted and plated on BHI agar and incubated overnight at 37°. The number of CFUs per mutant line was compared to that of wild-type flies using Graphpad Prism software for three independent experiments. Using an unpaired t-test, the P-value was determined. Mutant lines that exhibited a P-value <0.05 for all three retests were considered to have significantly different bacterial growth compared to the wild-type control.

L. monocytogenes mutants:

For infection of adult flies with both DP-L2161 and DP-L3078, bacteria were grown at 37° overnight without shaking. Thirty-five 7-day-old male flies per mutant line were infected for three independent replicates for each mutant fly line and flies were injected with ∼1000 CFUs. The death rate for each line was directly compared to a wild-type control and analyzed using Graphpad Prism software as described above.

Verification of the PiggyBac insertion site:

Inverse PCR was done to determine the insertion site for the PiggyBac (PBac) element following a protocol provided by Exelixis. Briefly, Genomic DNA from each mutant fly line was isolated using the QIAGEN (Valencia, CA) DNeasy kit. 5′- and 3′-end digestions were done using Sau3AI and HinPI, respectively, and incubated at 37° for 3 hr. Ligations were done using T4 DNA ligase at 4° overnight. PCR reactions were performed using the primer sequences and reaction conditions provided by Exelixis (


Screening for host genes important for survival:

To search for host genes that are important for surviving an infection, we conducted a forward genetic screen in a mutant population of Drosophila and monitored death rates following challenge with L. monocytogenes. L. monocytogenes establishes a lethal infection upon injection into Drosophila (Figure 1A) (Mansfield et al. 2003). Wild-type flies injected with 103 CFUs exhibited a MTD of 4–5 days postinfection. The predictable death kinetics allowed us to use survival as an output phenotype for our screen. We hypothesized that screening flies for their ability to survive L. monocytogenes infections would allow us to determine immune mechanisms used to fight this microbe, host mechanisms exploited by the pathogen, and physiologies that drive pathogenesis in the fly.

Figure 1.—

L. monocytogenes monocytogenes infection in Drosophila melanogaster. Wild-type flies were injected with 103 CFUs of either wild-type or mutant L. monocytogenes. Survival and growth (growth of wild-type bacteria only) were monitored over the course of the infection. (A) L. monocytogenes strain 10403S (wild type) infection. Wild-type flies exhibit a median time to death (MTD) of ∼5 days postinfection. (B) Growth of 10403S in flies. Bacteria reach levels >104 CFUs by 48 hr postinfection. **P < 0.005 (t-test). (C) Δhly infection in flies. Wild-type flies exhibit a MTD of ∼20 days postinfection. (D) ΔactA infection in flies. Infected flies exhibit a MTD of ∼10 days postinfection. Statistical analysis on survival curves was done using log-rank analysis and lines with P < 0.05 were considered statistically significant.

For our initial screen we tested 1231 viable mutants from the publicly available collection of Exelixis PBac homozygous transposon insertion lines. We tested only homozygous viable lines and this number represents ∼8% coverage of the Drosophila genome. Age-matched male flies from each mutant line were injected with 103 CFUs of L. monocytogenes and survival was monitored. Approximately half of the mutants tested exhibited a MTD of 4 days postinfection (Table 1). The mutant lines that exhibited the most extreme phenotypes with MTDs of day 1 and day 7 (∼100 mutants) were tested further to eliminate any false positives. The candidate mutants were tested in three independent experiments and the death curves for each were compared to the wild-type control and statistical analysis was done. After three rounds of retesting >80% of these mutants were considered false positives and a total of 18 mutants, all with increased sensitivity to infection, were identified as positive mutants that affect host susceptibility to L. monocytogenes infection (Table 2). For our study, we define any fly that exhibits a reproducible, significantly different faster death rate compared to wild-type flies as sensitive and any fly with a slower death rate compared to wild type as resistant. We injected the positive mutants with media alone to confirm that the increased mortality was infection dependent (data not shown).

View this table:

Distribution of mutant fly lines

View this table:

Screening for genes affecting defense and endurance

Identification of genes:

A benefit of using the Exelixis PBac collection is that the insertion sites of the transposons for the mutants in the collection are publicly available. The Bloomington Drosophila Stock Center ( has verified the reported insertion sites. For additional verification, we identified the PBac-element insertion site in each of our positive mutants using iPCR and by BLAST sequence analysis of the flanking DNA sequences. Our gene identities agree with those reported by Exelixis (Table 3). We also outcrossed our 18 mutants to the parental strain and challenged the F4 generation with L. monocytogenes to reduce the possibility of background effects on host survival. Using the computed gene (CG) numbers for the genes we determined the gene ontology (GO) terms from FlyBase (, which are listed in Table 3.

View this table:

Summary of positive Drosophila lines

Ubiquitination components:

From our screen we identified three mutant lines whose candidate genes can be categorized as genes involved in the ubiquitination process. These are crossbronx, CG15120, and CG2247. This was not surprising as previous studies demonstrated the importance of ubiquitin-mediated protein degradation in the regulation of the Toll and Imd pathways. Activation of the Toll pathway requires the ubiquitination and degradation of cactus, the inhibitor of the NF-kb-like transcription factor DIF. Lys-63 polyubiquitination of TRAF6 is necessary for signaling through the Imd pathway (Zhou et al. 2005). Additionally, ubiquitination is important for controlling the cytotoxicity of LLO after release of L. monocytogenes from the vacuole during infection.

cbx is characterized as a ubiquitin-conjugating E2 enzyme. It has been studied in processes including axon pruning, sperm development, and sperm individualization. In the context of fly immunity, cbx has been identified from a genetic screen to possibly have a role in crystal cell development, a type of immune cell in the fly (Milchanowski et al. 2004). The identification of this gene may reveal a role for crystal cells in the host defense against bacterial infection.

CG15120 is a homolog of the Parkin coregulated gene protein, PACRG, which is involved in ubiquitination; moreover, polymorphisms in PACRG and a related gene, PARK2, are linked to increased susceptibility to the intracellular pathogen Mycobacterium leprae in humans (Lorenzetti et al. 2004; Schurr et al. 2006). Polymorphisms in PACRG and PARK2 have been linked to the susceptibility of humans to the pathogens S. typhi and S. paratyphi (Ali et al. 2006). The third ubiquitin-related gene, CG2247 represented in line 18050, contains an F-box domain that facilitates the interactions between proteins during polyubiquitination. Our identification of these genes may reveal new processes in host defense and pathogenesis in which ubiquitin-mediated protein degradation is involved.

RNA binding/processing components:

The second main class of mutants we identified from our screen includes proteins involved in various processes involving RNA. CG3527 is involved in rRNA processing, specifically processing of the small ribosomal subunit. CG32706 and CG3056 are reported to have mRNA-binding activity. Expression and mutation analyses have not been published for these genes.

Additional interesting genes recovered:

Two additional genes we recovered that we find of great interest are ets21C and gr28b. ets21C is a DNA-binding protein with transcription factor activity. Previous studies have demonstrated that ets21C is immune regulated: Its expression is upregulated in Drosophila S2 cells in response to an LPS challenge and regulation is dependent on activation of JNK signaling via the Imd pathway (Boutros et al. 2002; Park et al. 2004). No immune function for this gene has been demonstrated.

gr28b is a seven-pass transmembrane gustatory receptor important for taste perception. Expression studies have shown this protein is expressed in the bitter taste cells of the sensilla in the fly taste organs. Identifying a gene involved in taste perception in the context of immunity and pathogenesis was somewhat surprising. Future experiments will reveal if the feeding behavior of the fly is affected, thus affecting the immune response in the fly. Alternatively, gr28b may be serving a function other than taste perception in the fly.

Phenotypic characterization of mutants—defense defects vs. endurance defects:

To distinguish which of our mutants had defects in immunity vs. pathogenesis, we performed three secondary screens. First, we measured L. monocytogenes growth to determine whether flies had a defect in preventing growth of this microbe. Second, we measured survival when infected with S. aureus to determine whether the mutants had a general defect in preventing gram-positive bacterial growth (Toll mutants are very sensitive to both these microbes). Third, we measured survival when infected with S. typhimurium to determine whether the mutants were sensitive to gram-negative bacteria (as might be expected for Imd pathway mutants) (Table 4).

View this table:

Characterization of Drosophila mutants—sensitivity to S. aureus and S. typhimurium and growth of L. monocytogenes

To monitor L. monocytogenes growth, flies were infected, homogenized at 0, 24, and 48 hr postinfection, and plated to count viable bacteria. Twelve mutants had increased L. monocytogenes growth when compared to wild-type flies. We consider the nature of these mutations to be immunocompromising to the fly because they allow increased growth of L. monocytogenes. An alternative explanation for some of these mutants could be that the environment of the pathogen in the mutant fly is more favorable to L. monocytogenes growth and the mutation affects physiology outside of what would normally be considered the immune system. The remaining mutants exhibited no significant difference in L. monocytogenes levels compared with wild type. This is a new class of mutant phenotype in the sense that it is not commonly studied in the field of fly immunity. Since microbial proliferation is unchanged in this group, these mutants should not be regarded as immunocompromised; instead, we suggest that these flies have an altered physiology that makes them more sensitive to the pathology induced by the L. monocytogenes infection. These flies are less able to endure the infection than wild-type flies even though bacterial levels are similar.

Often, studies examining the Drosophila immune response to gram-positive bacteria involve challenges with nonpathogens and have used specific molecular readouts such as AMP production to assess immune activity (Michel et al. 2001; Degregorio et al. 2002; Rutschmann et al. 2002; Tauszig-Delamasure et al. 2002). Defects in signaling resulted in an inability to mount an immune response to all tested gram-positive microbes. On the basis of our examination of our mutants, it is clear that the Drosophila response to gram-positive bacteria is more complex than has been previously appreciated. Not all of our mutants are sensitive to a second gram-positive pathogen, S. aureus. The presence of the two mutant lines that are clearly immunocompromised with respect to L. monocytogenes but show no change in S. aureus susceptibility indicates that there are multiple mechanisms and pathways used to fight gram-positive pathogenic bacteria.

To determine whether mutations causing increased sensitivity to L. monocytogenes infection could also affect infections with gram-negative bacteria, we challenged flies with S. typhimurium. Only one gene, CG11293, shows increased sensitivity to S. typhimurium. This mutant exhibits increased sensitivity to L. monocytogenes and S. aureus infection but unaltered L. monocytogenes growth. We suggest that these flies have a general pathogenesis defect because they are sensitive to three different pathogenic bacteria even though the bacterial load is comparable to that of wild-type flies. gr28b exhibits increased resistance to S. typhimurium infection while being more sensitive to S. aureus. This increased resistance was an unexpected phenotype; we are not aware of any other fly mutations that cause resistance to gram-negative bacteria but increased sensitivity to gram-positive bacteria.

Characterization of L. monocytogenes mutants in Drosophila mutants:

We chose L. monocytogenes as the pathogen for our screen because the life cycle and virulence factors have been well characterized, and, by using bacterial mutants, we can test for changes in pathogenesis in our mutant flies. After being taken up by a host cell, L. monocytogenes secretes LLO to lyse the phagosome membrane and release the bacteria into the cytoplasm. The protein ActA is important for nucleating host actin at one pole of the bacterium. Actin polymerization drives motility of L. monocytogenes in the cytoplasm of an infected cell and enables the bacteria to move from one host cell to another without entering the extracellular space, avoiding immune defenses of the host. We found that Δhly (LLO deletion) and ΔactA mutants are strongly attenuated in wild-type Drosophila, with a MTD of 18–20 days and 11–12 days, respectively (Figure 1, C and D). We examined susceptibility of our fly mutants to infection with the L. monocytogenes mutants Δhly and ΔactA to determine if any of our Drosophila mutants are involved in specific steps of the bacterial life cycle. We hypothesized that if any of our fly mutations are involved in specific stages of the life cycle, then the fly susceptibility to mutant bacterial infections will be different compared to that to a wild-type bacterial infection.

In 14 mutant fly lines, we found that the mutant L. monocytogenes strains acted as they do in wild-type flies, killing flies more slowly than do wild-type L. monocytogenes (Table 5) (individual death curves are provided in the supplemental materials). For 4 of our fly mutants (CG11489, CG4857, CG32706, and gr28b) the flies were equally sensitive to at least one L. monocytogenes mutant and wild-type L. monocytogenes. Two mutants, CG11489 and CG4857, are just as sensitive to infection with ΔactA as they are to infection with wild-type bacteria while being much less sensitive to Δhly mutants. In other words, even though the ΔactA L. monocytogenes are defective in cell-to-cell spread, these mutant fly lines are just as sensitive as if they had been infected with wild-type bacteria. However, these two mutant fly lines respond like wild-type flies to an infection with L. monocytogenes lacking LLO—they are more resistant. On the basis of their RNAi screen results, Cheng et al. (2005) suggested that mutants like this might be more sensitive to LLO and that the knocked-down genes were involved in controlling LLO toxicity. This model could also apply to our mutants; however, we find that our mutants are also sensitive to S. aureus and presumably sensitivity to S. aureus is not caused by the same perturbed process that might result in sensitivity to LLO. Perhaps this mutant affects a more general process involved in sensing bacteria.

View this table:

Characterization of L. monocytogenes mutants in Drosophila lines

CG32706 is sensitive to Δhly mutants but is comparatively insensitive to ΔactA infection. That is, even though the Δhly strain is defective in vacuole escape, it kills this mutant fly line with similar kinetics as wild-type L. monocytogenes. Yet this fly line is resistant to L. monocytogenes that lack ActA and are defective in cell-to-cell spread. Perhaps in these flies LLO is not required for L. monocytogenes to be released into the cytosol. Similar phenotypes have been observed in in vitro screens in which genes involved in later stages of vesicular trafficking had been knocked down (Cheng et al. 2005).

gr28b is sensitive to Δhly and ΔactA L. monocytogenes. As shown above, gr28b is sensitive to L. monocytogenes and S. aureus, but has increased resistance to S. typhimurium. Because the mutant strains of L. monocytogenes are not attenuated in this fly line, we suggest that LLO and ActA are not required for the bacteria to elicit pathogenic effects on this fly line. In other words, L. monocytogenes lacking factors required for normal intracellular pathogenesis are not attenuated, indicating that L. monocytogenes in the gr28b mutant may be an extracellular population.

Members of immunity pathways were not identified:

Two known immunity genes were present in the collection of mutant flies we tested (imd and kenny). Because previous studies of fly immunity have primarily focused on the Toll and Imd pathways, we anticipated that these two genes would be identified from our screen. These mutants had median times to death of 2 days, but were not tested further because they were outside of our cutoff of a median time to death of 1 day. As we screened a relatively small number of mutants, it is possible that we did not hit any members of the Toll signaling pathway because of our small sample size. In addition, some Toll pathway mutants would not meet our viability requirement and would be excluded. Regardless, this screen demonstrates that fighting an infection and the interactions that occur between host and pathogen are complex, and many processes in addition to the pathways regulating AMP transcription are involved.

The importance of performing both in vivo and in vitro screens:

We compared our list of mutant genes with the hundreds of genes identified in RNAi screens monitoring L. monocytogenes growth in cultured Drosophila cells. Because many mutations in essential functions would have a lethal phenotype in the whole fly, we anticipated that we would not find some genes in the whole fly that were identified in vitro. Conversely, we also expected to find genes that were important in the whole animal but not in tissue culture. Nonetheless, we expected significant overlap between the sets of genes isolated in these two screens; instead, we found no overlap. The screen by Agaisse et al. (2005) was a genomewide screen and theoretically all of our mutants should have been tested in that screen. The dsRNA library used by Cheng et al. (2005) contained eight of our positive mutants, none of which were identified from their in vitro screen (see Table 3). Both of these in vitro screens selected for bacterial phenotypes, including growth and vacuole escape, and utilized one cell type. The majority of genes identified from these in vitro screens are, not surprisingly, involved in vesicular trafficking and phagocytosis. By screening for host survival in a whole-animal model, we were able to identify different host genes involved in a variety of processes during infection, including the immune response and pathogenesis.


In this study we demonstrate the power of combining two genetically tractable organisms, one host and one pathogen, to reveal a previously uncharacterized group of genes involved in immunity and pathogenesis. Previous genetic studies using each organism separately greatly limited the scope of genes and processes that can be identified. The use of Drosophila to study the innate immune response to infection facilitated our understanding of signaling events that lead to the production of AMPs. Previous screens in the fly and in S2 cells have focused on identifying genes involved in these signaling pathways, but they did not look beyond this process. In vitro studies using L. monocytogenes have expanded our knowledge to better understand pathogenic mechanisms used by intracellular pathogens. Yet these genetic studies have focused more on the bacteria than on the host genes involved in the infection. Attempts to overcome these limitations have been made with RNAi screens in S2 cells infected with L. monocytogenes. These screens have revealed important host factors required for internalization and growth of L. monocytogenes in host cells but were not capable of identifying immunity- or pathogenesis-related genes. By screening for host survival in a whole-fly model, we could probe the host contributions to infection that extend to processes beyond those found by other screens and uncovered genes that were not discovered by the other methods.


We thank Marc Dionne, Linh Pham, Eric Mabery, and Madeleine Moule for critical review of the manuscript. DP-L3078 (ΔactA) and DP-L2161 (Δhly) were kindly provided by Julie Theriot (Stanford University). The S. aureus strain was provided by Ellen Jo Baron. We thank Stanley Falkow, Michael Simon, Julie Theriot, and all members of the Schneider lab for helpful discussions. We especially thank Adam, Baxter, and Buster Ayres for their support. This work was supported by National Institutes of Health grants RO1 A1053080-01 (D.S.S.) and A1055651 (D.S.S.) and by the National Science Foundation graduate research program (J.S.A.).


  • Communicating editor: K. V. Anderson

  • Received October 24, 2007.
  • Accepted December 19, 2007.


View Abstract