1

Title:

Development of the cellular immune system of Drosophila requires the Membrane

Attack Complex/Perforin-like Protein Torso-like

Authors:

Lauren Forbes-Beadle*, †, ‡, Tova Crossman‡, Travis K. Johnson*, †, ‡, Richard Burke‡,

Coral G. Warr‡, 1 and James C. Whisstock*,†, 1

Affiliations:

*Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria

3800, Australia.

†Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash

University, Clayton, Victoria 3800, Australia.

‡School of Biological Sciences, Monash University, Clayton, Victoria 3800, Australia.

1Corresponding authors. Coral Warr, School of Biological Sciences, Monash University,

Clayton, Victoria 3800, Australia. E-mail: coral.warr@monash.edu; James Whisstock,

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria

3800, Australia. E-mail james.whisstock@monash.edu. These authors contributed equally to

the work and are joint senior and corresponding authors.

Genetics: Early Online, published on August 17, 2016 as 10.1534/genetics.115.185462

Copyright 2016.

2

Running title:

Torso-like in Drosophila immunity

Keywords:

Membrane attack complex/perforin-like proteins

Torso-like

Cellular immunity

Drosophila melanogaster

Phagocytosis

Corresponding authors:

James Whisstock. Department of Biochemistry and Molecular Biology, Monash University,

Wellington Road, Clayton, Victoria 3800, Australia. E-mail: james.whisstock@monash.edu;

Phone number: +61 3 9902 9312

Coral Warr: School of Biological Sciences, Monash University, Wellington Road, Clayton,

Victoria 3800, Australia. E-mail: coral.warr@monash.edu; Phone number +61 3 9905 5504

3

ABSTRACT

Pore-forming members of the Membrane attack complex/perforin-like (MACPF)

protein superfamily perform well-characterised roles as mammalian immune effectors. For

example, complement component 9 and perforin function to directly form pores in the

membrane of Gram-negative pathogens or virally infected / transformed cells respectively. In

contrast, the only known MACPF protein in Drosophila melanogaster, Torso-like, plays

crucial roles during development in embryo patterning and larval growth. Here, we report that

in addition to these functions, Torso-like plays an important role in Drosophila immunity.

However, in contrast to a hypothesised effector function in, for example, elimination of Gram-

negative pathogens, we find that torso-like null mutants instead show increased susceptibility

to certain Gram-positive pathogens such as Staphylococcus aureus and Enterococcus faecalis.

We further show that this deficit is due to a severely reduced number of circulating immune

cells and, as a consequence, an impaired ability to phagocytose bacterial particles. Together

these data suggest that Torso-like plays an important role in controlling the development of the

Drosophila cellular immune system.

4

INTRODUCTION

Membrane attack complex/perforin-like (MACPF) proteins comprise a large,

functionally diverse superfamily of molecules that include the mammalian immunity proteins

complement component 9 (C9) and perforin. Both of these proteins function in immunity via

forming pores in the membrane of target cells (Rosado et al. 2008; Law et al. 2010; Hadders

et al. 2012). Structural studies have further revealed that MACPF proteins are related to an

ancient family of bacterial pore forming virulence factors, the cholesterol-dependent cytolysins

(Rosado et al. 2007). These data suggest an ancestral role for pore-forming MACPF proteins

in immune defence or attack. However, it is important to note that certain MACPF proteins

perform roles outside immunity or pathogenicity. For example a group of MACPF proteins in

mammals is important for brain development (Zheng et al. 1996; Adams et al. 2002; Kobayashi

et al. 2014), and the subject of this study, Torso-like (Tsl), is crucial for embryonic and larval

development in Drosophila (Stevens et al. 1990; Martin et al. 1994; Johnson et al. 2013). It is

currently unclear whether pore formation is central to the function of these molecules.

Many components of both cellular and humoral immunity share an ancient origin in

metazoan evolution, and as such studies in Drosophila have provided many crucial insights

into the mechanisms of mammalian immunity. The Drosophila cellular immune response acts

to eliminate pathogens and apoptotic cells using processes including phagocytosis,

encapsulation and melanisation. Plasmatocytes (hereon referred to as hemocytes) are the major

haematopoietic cell type present in adult Drosophila (Lanot et al. 2001) and are responsible

for the phagocytic immune response. The other immune cell type found in healthy adults are

the crystal cells, which enable melanisation of wound sites to prevent further invasion of

pathogens (Galko and Krasnow 2004; Ghosh et al. 2015). The Drosophila humoral immune

response involves recognition of foreign cell surface molecules by pattern recognition receptors

5

on fat body cells. These receptors can distinguish between different classes of microorganisms

and signal via the Toll and Imd pathways to induce production and secretion of specific

antimicrobial peptides (AMPs) into the hemolymph (reviewed in Lemaitre and Hoffmann

2007).

The only known MACPF protein in Drosophila is encoded by tsl. This molecule

performs several critical roles in development, and is best known for its maternal role in

embryonic patterning, where it controls activation of the Torso receptor tyrosine kinase to

specify the embryo termini (Stevens et al. 1990; Savant-Bhonsale and Montell 1993; Martin et

al. 1994). Tsl also plays a role in controlling developmental timing and overall body size (Grillo

et al. 2012; Johnson et al. 2013). However, it remains unresolved how Tsl functions in these

developmental contexts, and whether this involves pore formation and membrane disruption.

Here, we show that Tsl is also required for defence against certain microbial pathogens. We

further find that the immune defect observed in tsl mutants is likely due to a significant

reduction in phagocytic hemocytes, suggesting that Tsl is required for hemocyte development.

These data thus reveal a third role for Tsl in Drosophila development and raise the possibility

that certain mammalian MACPF proteins, traditionally associated with immune killing, may

also be integral to immune cell development.

6

MATERIALS AND METHODS

Drosophila Stocks: The following stocks from the Bloomington Drosophila Stock Centre

were used: w1118 (BL3605), Rele20 es (BL9457), da-Gal4 (BL12429) and Df(3R)cakiX-313

(BL6784; called Dfcaki here). The spz4 mutant was a gift from Bruno Lemaitre (Anderson and

Nusslein-Volhard 1984; Lemaitre et al. 1996). The tsl null mutant (tslΔ) and UAS-tsl are as

previously described (Johnson et al. 2013). All experiments were performed at 25 ˚ in non-

crowded conditions (40 individual larvae were picked into vials). Flies were raised and

maintained on standard fly media. In all experiments with adults the flies were 5-7 days old

unless otherwise indicated.

Infection and survival assays: Adult male flies were injected with bacteria using the septic

injury technique by pricking flies in the thorax (Apidianakis and Rahme 2009) using an ultra

micro tungsten 5μM needle (ProSciTech) dipped into concentrated bacterial solution. Bacterial

strains were obtained from Micromon Monash University. Bacterial cultures were pelleted via

centrifugation and resuspended in 0.1M MgSO4 to final concentrations of OD600 of 0.2 (S.

aureus), 200 (E. coli), 50 (E. carotovora) 200 (M. luteus) and 60 (E. faecalis). Each survival

experiment represents ≥30 individual adults per genotype and is representative of three

independent experiments. Survival following infection was measured each day for a period of

up to seven days, with measurements ceasing when the weakest genotypes no longer declined.

Differences in survival curves between pairs of genotypes were determined using log-rank

analysis in Graphpad Prism (version 6.02 for windows).

7

In vivo phagocytosis assays and analysis: Adult flies were injected into the upper abdomen

using a micro-injector (Eppendorf) with a solution of 0.1mg/mL S. aureus (#A10010) or E.

coli (#P35366) pHrodo bioparticles (Invitrogen) resuspended in live-imaging solution

according to the manufacturer’s protocol. The bio-particles are heat or chemically killed

bacterial particles of the E. coli (K-12 strain) or S. aureus (Wood strain without protein A)

fused to the pH sensitive dye pHrodo that fluoresces under acidic conditions (phagosomes or

lysosomes) to indicate phagocytosis. Flies were allowed to phagocytose the particles for 1.5

hours at 25˚. Flies were then anesthetised, their wings removed, and they were then mounted

onto modelling clay for imaging with a Leica DMLB compound fluorescent microscope.

Images were captured using Leica Application Suite (LAS) software and the exposure was set

such that the brightest images had a small number of saturating pixels. A phagocytic index was

determined by calculating the area of each adult abdomen and multiplying by the total

fluorescence in ImageJ, as previously described (Kocks et al. 2005). Each experiment was

repeated at least two times with at least 10 flies per genotype per treatment.

Antimicrobial gene transcript quantification: RNA was extracted from infected flies prior

to (0 hours) and following infection (6, 12 and 24 hours) with S. aureus or E. coli using the

septic injury assay. For each biological replicate, 5-10 adult flies were homogenised in Trisure

reagent (Bioline) and RNA purified according to the manufacturer’s protocol before DNase

(Promega) treatment. Complementary DNA was synthesised using the Tetro cDNA synthesis

kit (Bioline). Quantitative PCRs were performed in triplicate on a Rotor Gene 6000 (Corbett)

using SensiMix Sybr (Bioline). The following primers were used based on (Caroff et al. 2003):

Drs, sense 5’-CGTGAGAACCTTTTCCAATATGA-3’, antisense 5’-

TCCCAGGACCACCAGCAT-3’; Dpt, sense 5’-GCTGCGCAATCGCTTCTACT-3’,

antisense 5’- TGGTGGAGTGGGCTTCATG-3’; Rp49, sense 5’-

8

GCCGCTTCAAGGGACAGTATCT-3’, antisense 5’-AAACGCGGTTCTGCATGAG-3’.

Fold changes relative to Rp49 were determined using the delta CT method and means and

standard errors calculated from 3-4 biological replicates per genotype. Two way ANOVA was

used to determine whether significant differences existed between genotypes or time points.

When this was the case, the significant terms were dissected using Sidak’s multiple

comparisons tests (Graphpad Prism).

Bacterial load quantification: Adult male tslΔ null mutants and w1118 control flies were

infected with bacteria and collected at various time point post infection. Flies were

homogenised in 0.1M MgSO4, then the homogenate was serially diluted and plated onto Luria

Broth agar plates. The plates were then incubated at 37˚ for 18 hours. Data is represented as

the mean number of colonies ±1 standard error present in 6 individual flies at each time point.

Statistical analyses were performed as described for quantitative PCR.

Blood-cell quantifications: Larval hemocyte numbers were obtained by bleeding third instar

larvae onto a microscope slide in a 30μL drop of PBS. The drop was transferred to a

hemocytometer and cell numbers quantified per larva. Crystal cell numbers were determined

after heat treatment of larvae at 60˚C for 10 minutes in a water bath and quantification of the

black puncta representing the number of crystal cells, as previously described (Rizki et al.

1980). Data is represented as the mean and standard error of the number of cells present in 14-

20 individual larvae. Statistical significance was determined using unpaired two-tailed t-tests.

Data and reagent availability statement: Data and strains are available upon request.

9

RESULTS/DISCUSSION

We initially hypothesised that Tsl may, like its mammalian counterparts perforin and

C9, function as a terminal immune effector (i.e. through forming pores in the membrane of

target cells). If this is the case we reasoned that Tsl would be required for defence against

Gram-negative pathogens, but not against Gram-positive pathogens, which lack an outer cell

membrane. To investigate this a previously described null mutant, tslΔ (Johnson et al. 2013),

was challenged with a number of different bacterial species. Known mutants of the Toll and

Imd pathways, spz4 and Rele20 respectively (Anderson and Nusslein-Volhard 1984; Lemaitre et

al. 1996; Hedengren et al. 1999), were used as positive controls for the assays.

In our initial experiments we saw no increased susceptibility of tslΔ null mutants to

infection with the Gram-negative bacterial species Escherichia coli (log-rank, p=0.168, Figures

1A, S1), and E. carotovora (p=0.309, Figures 1B, S1), in contrast to the reduced survival of

the Imd pathway mutant Rele20 (p<0.001). When we tested survival to three different Gram-

positive bacterial species we found that tslΔ mutants showed no increased susceptibility to one

of them, Micrococcus luteus (tslΔ vs. tslΔ/+, p=0.974, Figures 1C, S1). In contrast, we found

that following infection with the other two Gram-positive microbes, S. aureus and E. faecalis,

tslΔ mutants showed a significantly increased susceptibility compared to controls. For S. aureus,

50% of the tslΔ mutant flies were alive seven days post infection (Figures 1D, S1), compared

to 76% of the tslΔ/+ heterozygous controls (p=0.003) or 86% of the w1118 controls (p<0.001).

When we used the ubiquitous da-Gal4 driver to drive Tsl expression in a tslΔ mutant

background, survival to S. aureus infection was rescued to wild type levels (tslΔ vs. da > tsl;

tslΔ, p<0.001), showing that this increased susceptibility was due to a role for Tsl (Figure 1D).

For E. faecalis (Figures 1E, S1) 77% of the tslΔ mutant flies were alive four days post infection

compared to 97% of the tslΔ/+ or the w1118 controls (p=0.022 in both cases). In both cases tslΔ

10

mutants were not as susceptible as the spz4 mutant flies (S. aureus infection survival: tslΔ = 50%

vs. spz4 = 0%, p<0.001; E. faecalis: tslΔ = 77% vs. spz4 = 47% survival, p=0.021).

To dissect this phenotype further we first asked whether loss of tsl affected the

induction of the AMP genes Drosomycin (Drs) and Diptericin (Dpt), as would be expected if

tsl functioned in either the Toll or Imd pathways respectively (Lemaitre et al. 1995; Lemaitre

et al. 1996). Adult tslΔ/+ heterozygous controls and tslΔ null mutant flies were infected with

either S. aureus or E. coli and collected at 0, 6, 12 and 24 hours post infection. We found that

expression of Drs was markedly induced in response to S. aureus infection, as expected (two-

way ANOVA; time effect: F3,24=119.1, p˂0.001, Figure 2A). tslΔ mutants showed a similar

overall Drs profile (genotype effect: F1,24=1.943, p=0.176), however Drs reached a higher level

than in the controls at 24 hours post infection (interaction effect: F3, 24=6.651, p=0.002; Sidak’s

multiple comparison test, 24 h, p<0.001; other time points p>0.05). Expression of Dpt was also

strongly induced in response to E.coli infection (time effect: F3,24=50.83, p<0.001, Figure 2B),

with tslΔ mutants expressing a similar level of Dpt to the controls across all time points

(genotype effect: F1, 24=0.808, p=0.378; interaction: F3, 24=2.349, p=0.098). We also asked if

tsl expression is induced following bacterial infection, as is commonly the case for components

of the systemic immunity pathways. No significant difference in tsl expression was observed

12 hours after infection with either S. aureus or E. coli (Figure S2). Taken together, these data

suggest that tsl does not participate in the systemic response to infection.

Since resistance to infection in Drosophila occurs via a combination of AMP

production, clotting and pathogen removal (reviewed in Lemaitre and Hoffmann 2007), we

next tracked bacterial numbers over time in tsl mutant flies to see whether the pathogens were

being effectively cleared from the animal, or were persisting and proliferating. For this

experiment we infected flies with either a pathogen that causes disease in healthy flies (S.

aureus) or one that only causes disease in flies mutant for components of the Toll or Imd

11

pathways, E.coli (Schneider et al. 2007). We found that the more virulent S. aureus remained

abundant for the infection period in both control and tslΔ mutants. Higher levels of S. aureus

were evident in tsl mutants across the infection period compared to controls (two-way ANOVA,

genotype effect: F1,26=32.63, p<0.001, Figure 2C). While there were differences between time

points (time effect: F2,26=25.23, p<0.001), a striking and significant increase was observed in

S. aureus numbers in tslΔ mutants compared to controls at 48 hours post infection (interaction

effect: F2,26=25.68, p<0.001, Sidak’s multiple comparison test, 48 h, p<0.001; other time points

p>0.05). We note that this time point coincides with the time at which survival after infection

by S. aureus rapidly declines in tslΔ mutants (Figure 1D). By contrast, E. coli was efficiently

cleared from the flies (time effect: F2,29=12.76, p<0.001, Figure 2D) in both tslΔ mutants and

controls (genotype effect: F1,29=3.410, p=0.075) across all time points examined (interaction:

F2,29=2.822, p=0.076). Together these data suggest that tsl may be required for fighting

infection by a more virulent pathogen such as S. aureus by suppressing its proliferation

independently of Drs induction.

Given these observations, we hypothesised that Tsl may play a role in the cellular

immune response. One of the main functions of the cellular immune response is to remove

pathogens from the circulation and destroy them via phagocytosis by hemocytes. To investigate

whether Tsl functions in phagocytosis, we tested the ability of tslΔ mutants to phagocytose

killed E. coli or S. aureus conjugated to the pH sensitive fluorophore pHrodo. Female flies

were tested initially as we wanted to avoid any interference with the detection of fluorescence

emission due to the additional body pigmentation in males. A significant decrease in

phagocytosis was observed in tsl mutant females compared to tslΔ/+ heterozygous controls after

infection with either E. coli (unpaired t-test, p<0.001) or S. aureus (p=0.038) pHrodo bacterial

particles (Figure 3). To confirm that this defect was due to loss of tsl, we also measured

phagocytic ability in female flies trans-heterozygous for tslΔ and a deficiency line that removes

12

tsl, Dfcaki (Martin and Ollo 1996; Grillo et al. 2012). The tslΔ/Dfcaki flies also phagocytosed

significantly fewer E. coli and S. aureus than the controls (p<0.001; Figure 3). There does not

appear to be any sexual dimorphism in this phenotype, as tslΔ mutant males showed a similar

decrease in phagocytosis of S. aureus particles (p=0.029, Figure S3). Taken together, these

data suggest that Tsl is required for phagocytosis during the cellular immune response.

The reduction in phagocytosis in tslΔ mutants could be due to either an effect on

hemocyte function or due to a decrease in their numbers. Adult hemocytes are produced during

both embryonic and larval development (Holz et al. 2003; Crozatier and Meister 2007),

however are technically challenging to isolate from adults. We therefore examined larval

hemocytes. Strikingly, we found that tslΔ mutants have less than half the number of hemocytes

than the controls (unpaired t-test, p=0.005, Figure 4A). It is therefore highly likely that the

defect in phagocytic ability we observe in tslΔ mutants is due to a severely reduced number of

hemocytes. As crystal cells are derived from the same embryonic lineage as the hemocytes

(Honti et al. 2010) we also investigated crystal cell numbers. These were also significantly

reduced in tslΔ mutants (p<0.001, Figure 4B), with tslΔ mutants having approximately 40% of

the number of cells as the controls.

Taken together, our data suggests that Tsl is required for development or survival of

hemocytes, and also crystal cells. This reduced number of immune cells likely underpins the

increased susceptibility we observed in tsl mutants to infection with the pathogenic microbes

S. aureus and E. faecalis. The fact that tsl mutants do not show increased susceptibility to two

other microbes, E. coli and M. luteus, can be explained by previous studies that have shown

that phagocytosis by hemocytes is not essential for normal survival of infection by these two

microbes. Specifically, blocking hemocytes by injection of polystyrene beads, which renders

them non-functional, has been shown to have no effect on susceptibility to infection by E. coli

(Elrod-Erickson et al. 2000). Similarly flies mutant for the eater gene, which is critical for

13

phagocytosis, exhibit no susceptibility to infection by M. luteus (Nehme et al. 2011). Overall,

the immune defects observed in tsl mutants support the idea that Tsl is required for normal

survival of infection by pathogenic bacteria for which clearance by phagocytosis is essential.

To date, the vast majority of MACPF family members have been shown to perform a

pore-forming function in immunity, as bacterial virulence factors or as the lethal factor in

certain venoms. We have not been able to produce Tsl protein recombinantly, and it is thus

unknown whether Tsl is a bona fide pore-forming protein. In embryonic terminal patterning

the role of Tsl is to control extracellular activation of a growth factor pathway (Stevens et al.

1990; Savant-Bhonsale and Montell 1993; Martin et al. 1994), although how it does so remains

unresolved. Tsl further performs an important role in larval growth (Johnson et al. 2013), and

it is possible that both this function, and the function of Tsl in immune cell development

described here, also relate to the extracellular control of signalling via certain growth factors.

A candidate growth factor signalling pathway is the platelet-derived growth factor and vascular

endothelial growth factor receptor (PVR) pathway, which plays a crucial role in Drosophila

hemocyte survival and differentiation (Heino et al. 2001; Munier et al. 2002; Bruckner et al.

2004). We further note that a developmental role has been ascribed for a number of neural

MACPF proteins in mammals (Zheng et al. 1996; Adams et al. 2002; Kobayashi et al. 2014).

Thus, given our findings in Drosophila, it is possible that a role for MACPF proteins in

mammalian immune cell development has been overlooked.

14

ACKNOWLEDGEMENTS

We thank the Australian Drosophila Biomedical Research Facility (OzDros) for technical

support, and also acknowledge the support from Monash Micro Imaging and the Australian

Research Council (ARC) Centre of Excellence in Advanced Molecular Imaging and Micromon.

We further thank the Bloomington Drosophila Stock Centre and Bruno Lemaitre for fly stocks.

T.K.J. is an ARC Discovery Early Career Researcher Award Fellow. J.C.W. is a National

Health and Medical Research Council of Australia Senior Principal Research Fellow, and he

further acknowledges the support from an ARC Federation Fellowship.

15

LITERATURE CITED

Adams, N. C., T. Tomoda, M. Cooper, G. Dietz and M. E. Hatten, 2002 Mice that lack

astrotactin have slowed neuronal migration. Development 1294: 965-972.

Anderson, K. V. and C. Nusslein-Volhard, 1984 Information for the dorsal-ventral pattern of

the Drosophila embryo is stored as maternal mRNA. Nature 3115983: 223-227.

Apidianakis, Y. and L. G. Rahme, 2009 Drosophila melanogaster as a model host for studying

Pseudomonas aeruginosa infection. Nat. Protoc. 49: 1285-1294.

Bruckner, K., L. Kockel, P. Duchek, C. M. Luque, P. Rorth, et al., 2004 The PDGF/VEGF

receptor controls blood cell survival in Drosophila. Dev. Cell 71: 73-84.

Caroff, M., W.-J. Lee, B. Lemaitre, F. Leulier, D. Mengin-Lecreulx, et al., 2003 The

Drosophila immune system detects bacteria through specific peptidoglycan recognition.

Nat. Immunol. 45: 478+.

Crozatier, M. and M. Meister, 2007 Drosophila haematopoiesis. Cell Microbiol. 95: 1117-1126.

Elrod-Erickson, M., S. Mishra and D. Schneider, 2000 Interactions between the cellular and

humoral immune responses in Drosophila. Curr. Biol. 1013: 781-784.

Galko, M. J. and M. A. Krasnow, 2004 Cellular and Genetic Analysis of Wound Healing in

Drosophila Larvae. PLoS Biol. 28: e239.

Ghosh, S., A. Singh, S. Mandal and L. Mandal, 2015 Active Hematopoietic Hubs in Drosophila

Adults Generate Hemocytes and Contribute to Immune Response. Dev. Cell 334: 478-

488.

Grillo, M., M. Furriols, C. de Miguel, X. Franch-Marro and J. Casanova, 2012 Conserved and

divergent elements in Torso RTK activation in Drosophila development. Sci. Rep. 2:

762.

16

Hadders, Michael A., D. Bubeck, P. Roversi, S. Hakobyan, F. Forneris, et al., 2012 Assembly

and Regulation of the Membrane Attack Complex Based on Structures of C5b6 and

sC5b9. Cell Rep. 13: 200-207.

Hedengren, M., Bengt Åsling, M. S. Dushay, I. Ando, S. Ekengren, et al., 1999 Relish, a

Central Factor in the Control of Humoral but Not Cellular Immunity in Drosophila.

Mol. Cell 45: 827-837.

Heino, T. I., T. Kärpänen, G. Wahlström, M. Pulkkinen, U. Eriksson, et al., 2001 The

Drosophila VEGF receptor homolog is expressed in hemocytes. Mech. Dev. 1091: 69-

77.

Holz, A., B. Bossinger, T. Strasser, W. Janning and R. Klapper, 2003 The two origins of

hemocytes in Drosophila. Development 13020: 4955-4962.

Honti, V., G. Csordás, R. Márkus, É. Kurucz, F. Jankovics, et al., 2010 Cell lineage tracing

reveals the plasticity of the hemocyte lineages and of the hematopoietic compartments

in Drosophila melanogaster. Mol. Immunol. 4711–12: 1997-2004.

Johnson, T. K., T. Crossman, K. A. Foote, M. A. Henstridge, M. J. Saligari, et al., 2013 Torso-

like functions independently of Torso to regulate Drosophila growth and

developmental timing. Proc. Natl. Acad. Sci. USA 11036: 14688-14692.

Kobayashi, M., T. Nakatani, T. Koda, K.-i. Matsumoto, R. Ozaki, et al., 2014 Absence of

BRINP1 in mice causes increase of hippocampal neurogenesis and behavioral

alterations relevant to human psychiatric disorders. Mol. Brain 71: 1-16.

Kocks, C., J. H. Cho, N. Nehme, J. Ulvila, A. M. Pearson, et al., 2005 Eater, a Transmembrane

Protein Mediating Phagocytosis of Bacterial Pathogens in Drosophila. Cell 1232: 335-

346.

Lanot, R., D. Zachary, F. Holder and M. Meister, 2001 Postembryonic Hematopoiesis in

Drosophila. Dev. Biol. 2302: 243-257.

17

Law, R. H. P., N. Lukoyanova, I. Voskoboinik, T. T. Caradoc-Davies, K. Baran, et al., 2010

The structural basis for membrane binding and pore formation by lymphocyte perforin.

Nature 4687322: 447-451.

Lemaitre, B. and J. Hoffmann, 2007 The Host Defense of Drosophila melanogaster. Annu.

Rev. Immunol. 251: 697-743.

Lemaitre, B., E. Kromer-Metzger, L. Michaut, E. Nicolas, M. Meister, et al., 1995 A recessive

mutation, immune deficiency (imd), defines two distinct control pathways in the

Drosophila host defense. Proc. Natl. Acad. Sci. USA 9221: 9465-9469.

Lemaitre, B., E. Nicolas, L. Michaut, J.-M. Reichhart and J. A. Hoffmann, 1996 The

Dorsoventral Regulatory Gene Cassette spätzle/Toll/cactus Controls the Potent

Antifungal Response in Drosophila Adults. Cell 866: 973-983.

Martin, J.-R., A. Raibaud and R. Ollo, 1994 Terminal pattern elements in Drosophila embryo

induced by the torso-like protein. Nature 3676465: 741-745.

Martin, J. R. and R. Ollo, 1996 A new Drosophila Ca2+/calmodulin-dependent protein kinase

(Caki) is localized in the central nervous system and implicated in walking speed.

EMBO J 158: 1865-1876.

Munier, A. I., D. Doucet, E. Perrodou, D. Zachary, M. Meister, et al., 2002 PVF2, a

PDGF/VEGF‐like growth factor, induces hemocyte proliferation in Drosophila larvae.

EMBO Rep. 312: 1195-1200.

Nehme, N. T., J. Quintin, J. H. Cho, J. Lee, M.-C. Lafarge, et al., 2011 Relative Roles of the

Cellular and Humoral Responses in the Drosophila Host Defense against Three Gram-

Positive Bacterial Infections. PLoS ONE 63: e14743.

Rizki, T. M., R. M. Rizki and E. H. Grell, 1980 A mutant affecting the crystal cells in

Drosophila melanogaster. Rouxs Arch. Dev. Biol. 1882: 91-99.

18

Rosado, C. J., A. M. Buckle, R. H. P. Law, R. E. Butcher, W.-T. Kan, et al., 2007 A Common

Fold Mediates Vertebrate Defense and Bacterial Attack. Science 3175844: 1548-1551.

Rosado, C. J., S. Kondos, T. E. Bull, M. J. Kuiper, R. H. P. Law, et al., 2008 The MACPF/CDC

family of pore-forming toxins. Cell. Microbiol. 109: 1765-1774.

Savant-Bhonsale, S. and D. J. Montell, 1993 torso-like encodes the localized determinant of

Drosophila terminal pattern formation. Genes Dev. 712b: 2548-2555.

Schneider, D. S., J. S. Ayres, S. M. Brandt, A. Costa, M. S. Dionne, et al., 2007 Drosophila

eiger Mutants Are Sensitive to Extracellular Pathogens. PLoS Pathog. 33: e41.

Stevens, L. M., H. G. Frohnhofer, M. Klingler and C. Nusslein-Volhard, 1990 Localized

requirement for torso-like expression in follicle cells for development of terminal

anlagen of the Drosophila embryo. Nature 3466285: 660-663.

Zheng, C., N. Heintz and M. E. Hatten, 1996 CNS Gene Encoding Astrotactin, Which Supports

Neuronal Migration Along Glial Fibers. Science 2725260: 417-419.

19

FIGURE LEGENDS

Figure 1. tsl mutants show increased susceptibility to infection by S. aureus and

E. faecalis.

(A) No significant decrease in survival was observed in tslΔ mutants compared to the

heterozygous tslΔ/+ control flies when infected with E. coli (log-rank, p=0.168), (B) E.

carotovora (p=0.309) or (C) M. luteus (p=0.974). (D) tslΔ mutants are more susceptible

to S. aureus infection than the heterozygous tslΔ/+ (p=0.003) or w1118 control flies

(p<0.001). The susceptibility of tslΔ mutants to S. aureus infection can be rescued by

ubiquitously expressing tsl using da-Gal4 (tslΔ vs. da > tsl, tslΔ, p=0.002). (E) tslΔ

mutants are more susceptible to E. faecalis infection than the heterozygous tslΔ/+ or

w1118 controls (p=0.022 in both cases). Significant differences were determined

between pairs of genotypes to either their corresponding heterozygous controls or as

otherwise indicated using log-rank analysis (*p<0.05, **p<0.01, ***p<0.001). Each

experiment represents the mean survival of ≥ 30 flies for each genotype and is

representative of three independent experiments (refer to Figure S1 for replicate

experiments).

20

Figure 2. Tsl is required for limiting S. aureus infection independently of Drs and

Dpt induction.

(A and B) tslΔ mutants and heterozygous tslΔ/+ control flies were infected with S.

aureus (A) or E. coli (B) and sampled at 0, 6, 12, and 24 hours following infection.

Expression levels of the antimicrobial peptides Drs (A) and Dpt (B) were quantified

relative to Rp49. Drs and Dpt were significantly induced following infection in tslΔ

mutants and tslΔ/+ control flies (two-way ANOVA, time effect, Drs: F3,24=119.1,

p˂0.001; Dpt: F3,24=50.83, p<0.001). No difference was observed between the

induction profiles of the tsl mutants and heterozygous tslΔ/+ control flies (genotype

effect, Drs: F1,24=1.943, p=0.176; Dpt: F1,24=0.808, p=0.378). However, tsl mutants did

display higher Drs levels at 24 hours post S. aureus infection (interaction effect, F3,

24=6.651, p=0.002; Sidak’s multiple comparison test, 24 h, p<0.001; other time points

p>0.05). (C and D) Male tslΔ homozygotes and w1118 control flies were infected with S.

aureus (C) or E. coli (D) and collected at 0, 6, 24 and 48 hours post infection. Flies

were homogenised and plated to quantify colony forming units. (C) The number of S.

aureus colonies was significantly higher in the tslΔ mutants compared to control flies

(genotype effect, F1, 26=32.63, p<0.001) and at 48 hours post infection a marked

increase was evident (interaction effect, F2,26=25.68, p<0.001, Sidak’s multiple

comparison test, 48 h, p<0.001; other time points p>0.05). (D) tslΔ mutants could

successfully clear E. coli infection similarly to controls (time effect, F2,29=12.76,

p<0.001; genotype effect, F1,29=3.410, p=0.075). Data are presented as the mean ±1

standard error of three to six biological replicates.

21

Figure 3. Tsl is required for phagocytosis.

Adult female flies were infected with killed E. coli or S. aureus conjugated to the pH

sensitive fluorophore pHrodo. Hemocytes that have phagocytosed the bacteria were

identified by fluorescence. (A) In vivo phagocytosis of killed E. coli bacteria in tslΔ/+

controls and tslΔ mutants. Areas where phagocytosis is occurring are indicated by

arrows. A mean fluorescent signal was measured from the entire adult abdomen of

each fly (dotted outline) and multiplied by the area to give a phagocytic index

measurement in arbitrary units (Kocks et al. 2005). (B) A significant reduction in E. coli

phagocytosis was observed in tslΔ mutants compared to tslΔ/+ controls (unpaired t-test,

p<0.001). (C) Flies with an independent deficiency mutation of tsl, Dfcaki, placed in

trans with tslΔ (tslΔ/Dfcaki) also showed a reduction in phagocytic ability compared to

the heterozygous Dfcaki/+control (p=0.001). (D) In vivo phagocytosis of S. aureus

pHrodo bacteria in tslΔ/+ and tslΔ. (E) tslΔ mutants phagocytosed significantly fewer S.

aureus bacteria than the tslΔ/+ control flies (p=0.038). (E) The tslΔ/Dfcaki flies also

displayed significantly reduced phagocytosis of S. aureus (p<0.001 compared to both

heterozygous controls). Each datapoint represents the phagocytic index measured

from an individual infected fly abdomen. Horizontal lines and error bars on each

scatterplot represent the mean phagocytic index values ±1 standard error (n≥11).

(*p<0.05, ***p<0.001).

22

Figure 4. tsl mutants have a reduced number of hemocytes and crystal cells.

(A) Quantification of circulating hemocytes in tslΔ larvae showed a significantly

reduced number of cells (7.27 ×104 cells mL-1) compared to w1118 control larvae (1.62

×105 cells mL-1) (unpaired t-test, p=0.005, n=20). (B and C) Third instar larvae heated

to 60˚C for 10 minutes show the presence of black puncta corresponding to sessile

crystal cells under the cuticle. tslΔ larvae have significantly fewer crystal cells

compared to wild type w1118 control larvae (p<0.001). Each point represents the

number of crystal cells per larva measured from the three posterior-most larval

abdominal segments. Horizontal lines and error bars on each scatterplot represent the

mean ±1 standard error (n≥14). Statistically significant differences (unpaired two-tailed

t-tests, **p<0.01, ***p<0.001) are indicated above the plots. Scale bar is 0.5mm.

23

Figure 1

24

Figure 2

25

Figure 3

26

Figure 4