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1 Dual proteomics of Drosophila melanogaster hemolymph infected
2 with the heritable endosymbiont Spiroplasma poulsonii
† † 3 Florent Masson , Samuel Rommelaere , Alice Marra, Fanny Schüpfer, Bruno Lemaitre*
4 Global Health Institute, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL),
5 Lausanne, Switzerland
6
† 7 These authors contributed equally
8
9 *Corresponding author:
10 Phone number: +41 21 693 18 31
11 Email address: [email protected]
12
13 Running title: Drosophila/Spiroplasma proteomics
14
15 Key words: Spiroplasma, endosymbiosis, proteomics
16
17 ORCID numbers:
18 Florent Masson: 0000-0002-5828-2616
19 Samuel Rommelaere: 0000-0003-3201-0847
20 Bruno Lemaitre: 0000-0001-7970-1667 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
21 Abstract
22
23 Insects are frequently infected with heritable bacterial endosymbionts.
24 Endosymbionts have a dramatic impact on their host physiology and evolution. Their
25 tissue distribution is variable with some species being housed intracellularly, some
26 extracellularly and some having a mixed lifestyle. The impact of extracellular
27 endosymbionts on the biofluids they colonize (e.g. insect hemolymph) is however
28 difficult to appreciate because biofluids composition can depend on the contribution
29 of numerous tissues.
30 Here we address the question of Drosophila hemolymph proteome response to the
31 infection with the endosymbiont Spiroplasma poulsonii. S. poulsonii inhabits the fly
32 hemolymph and gets vertically transmitted over generations by hijacking the
33 oogenesis in females. Using dual proteomics on infected hemolymph, we uncovered
34 a low level and chronic activation of the Toll immune pathway by S. poulsonii that
35 was previously undetected by transcriptomics-based approaches. Using Drosophila
36 genetics, we also identified candidate proteins putatively involved in controlling S.
37 poulsonii growth. Last, we also provide a deep proteome of S. poulsonii, which, in
38 combination with previously published transcriptomics data, improves our
39 understanding of the post-transcriptional regulations operating in this bacterium.
40
41 Summary statement
42
43 We report the changes in Drosophila melanogaster hemolymph proteome upon
44 infection with the heritable bacterial endosymbiont Spiroplasma poulsonii.
1 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
45 Introduction
46
47 Insects frequently harbor vertically transmitted bacterial symbionts living within their
48 tissues, called endosymbionts (Douglas, 2015). Endosymbionts have a major impact
49 on the host physiology and evolution as they provide ecological benefits such as the
50 ability for the host to thrive on unbalanced diets (Douglas, 2016), protection against
51 viruses or parasites (Hedges et al., 2008; Oliver et al., 2003; Scarborough et al.,
52 2005; Teixeira et al., 2008) or tolerance to heat (Montllor et al., 2002). A peculiar
53 group of insect endosymbionts also directly affects their host reproduction. This
54 group is taxonomically diverse and includes bacteria from the Wolbachia,
55 Spiroplasma, Arsenophonus, Cardinium and Rickettsia genera (Duron et al., 2008;
56 Medina et al., 2019). Four reproduction-manipulative mechanisms have been
57 unraveled so far, namely cytoplasmic incompatibility, male-killing, parthenogenesis
58 and male feminization (Werren et al., 2008), all of them leading to an evolutionary
59 drive that favors infected individuals over non-infected ones and promotes the
60 endosymbiont spread into populations. Their ease of spread coupled with the virus-
61 protecting ability of some species (Hedges et al., 2008; Teixeira et al., 2008) make
62 them promising tools to control insect pest populations or to render them refractory to
63 human arboviruses (Hoffmann et al., 2011). Reproductive manipulators are however
64 fastidious bacteria that are difficult to manipulate, hence slowing down research on
65 the functional aspects of their interaction with their hosts (Masson and Lemaitre,
66 2020). Their real impact on host physiology thus remains largely elusive.
67
68 Spiroplasma are long helical bacteria belonging to the Mollicutes class, which
69 are devoid of cell wall (Gasparich, 2002). They infect a wide range of arthropods and
2 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
70 plants where they act as pathogens, commensals or vertically transmitted
71 endosymbionts depending on the species (Gasparich, 2002). S. poulsonii (hereafter
72 Spiroplasma) is a vertically transmitted endosymbiont infecting the fruit fly Drosophila
73 (Haselkorn, 2010; Mateos et al., 2006). It lives free in the host hemolymph where it
74 feeds on circulating lipids (Herren et al., 2014) and achieves vertical transmission by
75 infecting oocytes (Herren et al., 2013). Most strains cause male-killing, that is the
76 death of the male progeny during early embryogenesis through the action of a toxin
77 named Spaid (Harumoto and Lemaitre, 2018). Spiroplasma infection also confers
78 protection to the fly or its larvae against major natural enemies such as parasitoid
79 wasps (Ballinger and Perlman, 2017; Xie et al., 2014) and nematodes (Hamilton et
80 al., 2016; Jaenike et al., 2010).
81
82 Spiroplasma has long been considered as untractable, but recent technical
83 advances such as the development of in vitro culture (Masson et al., 2018) and
84 transformation (Masson et al., 2020b) make it an emergent model for studying the
85 functional aspects of insect-endosymbiont interactions. Some major steps have been
86 made in the understanding of the male killing (Harumoto et al., 2014; Harumoto et al.,
87 2016; Harumoto et al., 2018; Veneti et al., 2005) and protection phenotypes
88 (Ballinger and Perlman, 2017; Hamilton et al., 2016; Paredes et al., 2016) or on the
89 way the bacterial titer was kept under control in the adult hemolymph (Herren et al.,
90 2014; Paredes et al., 2015). Such studies, however, relied on single-gene studies
91 and did not capture the full picture of the impact of Spiroplasma on its host
92 physiology. We tackled this question using dual-proteomics on fly hemolymph
93 infected by Spiroplasma. This non-targeted approach allowed us to get an extensive
94 overview of the end-effect of Spiroplasma infection on the fly hemolymph protein
3 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
95 composition and to identify previously unsuspected groups of proteins that where
96 over- or under-represented in infected hemolymph. Using Drosophila genetics to
97 knock-down the corresponding genes, we identified new putative regulators of the
98 bacterial titer. This work also provides the most comprehensive Spiroplasma
99 proteome to date. By comparing this proteome to the existing transcriptomics data,
100 we draw conclusions about Spiroplasma post-transcriptional regulations.
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101 Results
102 1. Drosophila hemolymph protein profiling
103 We investigated the effect of Spiroplasma infection on Drosophila hemolymph protein
104 content using Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS). To
105 this end, we extracted total hemolymph from uninfected and infected 10 days old
106 females. At this age, Spiroplasma is already present at high titers in the hemolymph
107 but does not cause major deleterious phenotypes to the fly (Herren and Lemaitre,
108 2011; Masson et al., 2020a). Extraction was achieved by puncturing the thorax and
109 drawing out with a microinjector. This procedure induces some tissue damage but
110 recovers very few hemocytes (circulating immunes cells).
111 Peptides were mapped to both the Drosophila and Spiroplasma predicted proteomes,
112 hence allowing having an overview of i) differentially represented Drosophila proteins
113 in infected versus uninfected hemolymph and ii) an in-depth Spiroplasma proteome
114 in the infected hemolymph samples. These two datasets will be analyzed in separate
115 sections.
116 The mapping on Drosophila proteome allowed the identification of 909 quantified
117 protein groups (a protein group contains proteins that cannot be distinguished based
118 on the identified peptides). The complete list of quantified Drosophila proteins and
119 their fold-change upon Spiroplasma infection is available in Supplementary Table S1.
120 The hemolymph extraction process involves tissue damage (e.g. the subcuticular
121 epithelium, muscles and fat body) that leads to the release of intracellular proteins in
122 the sample. Hence we first sorted protein groups depending on whether the main
123 protein is predicted to be extracellular or not. Proteins were defined as extracellular if
124 they bore a predicted signal peptide or were annotated with a Gene Ontology (GO)
125 term “extracellular”, or intracellular if no signal peptide nor any “extracellular” GO
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126 term was predicted. Based on this localization criteria the Drosophila dataset could
127 be split in 555 intracellular protein groups representing 61% of the total proteome and
128 354 extracellular proteins representing the remaining 39% of the total proteome
129 (Figure 1A).
130
131 We then compared the relative amounts of protein groups between infected and
132 uninfected hemolymph. Of the intracellular protein groups, 35 were differentially
133 represented in the Spiroplasma infected samples, including 8 overrepresented and
134 27 depleted groups compared to the uninfected control (Figure 1B). A functional GO
135 analysis on the differentially represented protein groups revealed that a significant
136 part of these protein groups were related to the proteasome (subunits α2, 3, 4 and 7
137 and subunits β3 and 6) and the Toll immune pathway (Figure 1C). The Toll pathway
138 GO enrichment comprises only proteasome subunits, probably because of their
139 involvement in the degradation of Toll pathway intermediates (e.g. Cactus
140 (Palombella et al., 1994)). While differentially abundant intracellular protein groups
141 were mostly underrepresented in Spiroplasma infected hemolymph, analysis of the
142 extracellular protein subset revealed an opposite trend. Among the 71 extracellular
143 protein groups having a significantly different abundance, 4 were depleted and 67
144 were overrepresented in the infected samples compared to the uninfected ones
145 (Figure 1D). Surprisingly, the functional GO annotation highlighted an
146 overrepresentation of serine-endopeptidase molecular function. Serine proteases are
147 involved in the regulation of the melanization and the Toll pathways and indeed the
148 GO terms are enriched for these biological processes as well (Figure 1E).
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149 2. Immune-related protein enrichment is a consequence of a mild
150 transcriptional activation
151
152 Since Spiroplasma is devoid of cell wall, it lacks microbe-associated molecular
153 patterns such as peptidoglycan and was not expected to interact with pattern-
154 recognition receptors regulating the fly immune system. This idea was supported by
155 previous work showing that Spiroplasma do not trigger a strong level of Toll and Imd
156 pathway, the two main immune pathways in Drosophila (Herren and Lemaitre, 2011;
157 Hurst et al., 2003). Previous work also showed that flies defective for the inducible
158 humoral immune response have normal Spiroplasma titer, suggesting that immune
159 pathways are not required to control Spiroplasma growth (Herren and Lemaitre,
160 2011). Our observation that several immune-related proteins are more abundant in
161 the hemolymph of infected flies (Figure 2A and Supplementary Table S1) led us to
162 investigate further on this point. We first tested if the changes in immune-related
163 protein abundance were a consequence of altered gene expression. We measured
164 the corresponding mRNA levels of several differentially represented proteins (Figure
165 2B). Immune-related genes were slightly upregulated in infected flies, although their
166 induction levels did not compare with those observed after systemic infection by a
167 pathogenic bacteria (De Gregorio et al., 2001; Troha et al., 2018). These results
168 confirm that Spiroplasma does not trigger a strong immune response (Herren and
169 Lemaitre, 2011; Hurst et al., 2003). But our data reveals that the presence of
170 Spiroplama still provokes a mild and chronic production of immunity proteins.
171
172 3. A majority of enriched hemolymphatic proteins are not involved in
173 Spiroplasma growth control
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174
175 A systemic infection or a genetic induction of the humoral immune response
176 promotes Spiroplasma growth in flies (Herren and Lemaitre, 2011). We therefore
177 wondered if the presence of small amounts of antimicrobial peptides (AMPs) could
178 be beneficial for Spiroplasma growth. To test this hypothesis, we measured
179 Spiroplasma titer in flies over-expressing different AMP genes (Tzou et al., 2002).
180 We found that AMP gene overexpression did not increase Spiroplasma titer (Figure
181 3A). Similarly, flies lacking ten AMP coding genes (Hanson et al., 2019) had a
182 Spiroplasma titer comparable to that of control flies (Figure 3B). These results
183 suggest that AMPs released during the humoral immune response do not promote
184 Spiroplasma growth.
185 We then tested if other immune-related proteins found more abundant in infected
186 hemolymph could alter Spiroplasma growth. We first compared the Spiroplasma
187 titers of control OregonR flies with that of hayan, attD and tep2 mutants. We observed
188 no difference in bacterial titer, indicating that these genes are not required neither
189 detrimental to Spiroplasma growth (Figure 3B). We then used an in vivo RNAi
190 approach to silence immune genes coding for the most enriched proteins in
191 Spiroplasma-infected flies using the ubiquitously expressed Act5C-GAL4 driver.
192 Silencing these genes did not provoke an increase in Spiroplasma titer, further
193 reinforcing the idea that immune proteins do not prevent Spiroplasma growth (Figure
194 3C).
195 Among the most differentially represented proteins in Spiroplasma infected
196 hemolymph, we also detected several proteins of unknown function. To test their role
197 in Spiroplasma growth control, we used the same RNAi-mediated knockdown
198 approach and quantified Spiroplasma titer in these flies. Most of the RNAi lines
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199 tested showed no change in Spiroplasma titer as compared to control lines
200 (Supplementary Figure S1). Two RNAi lines targeting CG18067 and CG14762,
201 respectively, showed a slight yet not significant reduction in Spiroplasma titer. These
202 results suggest that all the tested genes do not facilitate Spiroplasma growth nor
203 control its titer. Instead, these proteins are likely to be induced as a consequence of
204 Spiroplasma infection and may participate in the host adaptation to the bacteria. A
205 mutant line was available for only one of the most enriched uncharacterized proteins
206 (CG15293). We found that this mutation leads to a significant increase in bacteria
207 titer (Supplementary Figure S1). This gene codes for a 37 kDa secreted protein with
208 no known function. Our results raised the hypothesis that CG15293 participates in
209 the control of Spiroplasma titer in vivo.
210
211 4. Transcriptome-proteome correlation in Spiroplasma poulsonii
212
213 The proteomics analysis of infected hemolymph samples also allowed the detection
214 and quantification of Spiroplasma proteins. With a total of 503 proteins, this is the
215 most comprehensive Spiroplasma proteome to date. The complete list of
216 Spiroplasma quantified proteins is available in Supplementary Table S2.
217 We took advantage of a previously published transcriptomics dataset of Spiroplasma
218 (Masson et al., 2018) to compare the expression level of Spiroplasma genes to their
219 corresponding protein abundance by building a linear model of the proteomics signal
220 as a function of the transcript level (Figure 4). The two measures are poorly
221 correlated (Kendall’s τ = 0.40). Analyzing the normalized residuals of the model also
222 allowed us to identify proteins that are particularly discrepant with the model, that is
223 with absolute standardized residuals greater that 2. This approach uncovered 27
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224 proteins, of which 11 have a significantly higher abundance and 16 a lower
225 abundance in the proteome that what was predicted from the transcriptomics data
226 (Supplementary Table S3). Over-represented proteins with a reliable annotation
227 include the membrane lectin Spiralin B, the cytoskeletal protein Fibrilin, the glucose
228 transporter Crr, the potassium channel KtrA, a ferritin-like protein Ftn, the
229 chromosome partitioning protein ParA and the DNA polymerase subunit DnaN.
230 Under-represented proteins include the transporters SteT and YdcV, the
231 serine/threonine phosphatase Stp, the helicase PcrA, the ATP synthase subunit
232 AtpH, the GMP reductase GuaC and the tRNA-(guanine-N(7)-)-methyltransferase
233 TrmB. Other proteins have no predicted function.
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234 Discussion
235 This study provides an extensive characterization of the proteome of
236 Spiroplasma-infected Drosophila hemolymph. This dual-proteomics approach
237 provides a deep proteome of Spiroplasma in its natural environment and pinpoints
238 host proteins which abundance is altered by the presence of the bacteria. The power
239 of Drosophila genetics allowed us to test the involvement of these candidate genes in
240 regulating endosymbiosis. A targeted genetic screen revealed that most of the
241 differentially abundant proteins upon Spiroplasma infection do not participate in the
242 control of symbiont growth but may rather be involved in host adaptation to
243 Spiroplasma.
244 Spiroplasma is devoid of cell wall, hence devoid of peptidoglycan, which is the
245 main immune elicitor for the insect immune system (Lemaitre and Hoffmann, 2007).
246 This led to the assumption that Spiroplasma was undetectable by the canonical
247 innate immune pathways. Furthermore, the elicitation of the fly immune system (by
248 an infection or using genetics) has no impact on Spiroplasma titer, suggesting that
249 the bacteria are not only invisible by also resistant to the fly immune effectors (Herren
250 and Lemaitre, 2011). Flies that over-express AMPs and the ΔAMP10 mutant line
251 have normal bacterial titer that confirms the host immune system does not affect
252 Spiroplasma growth. However, numerous immune-related proteins (mostly
253 associated to the Toll pathway) were enriched in Spiroplasma infected hemolymph,
254 including soluble receptors (GNBP1, GNBP-like3), signal transduction intermediates
255 (Spätzle-Processing Enzyme and Hayan) and effectors or putative effectors (Attacin,
256 Bomanin Bc3 and CG33470). The Spiroplasma titer was not altered in several flies
257 carrying loss-of function alleles of these genes, indicating that the immune elicitation
258 is a consequence of the infection but not a control mechanism. It is worth noting that
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259 the expression levels of the genes were extremely low as compared to a fully-fledged
260 immune response to pathogenic bacteria (De Gregorio et al., 2001). Such low
261 induction of the immune response has been reported in flies experiencing stress,
262 such as cold or heat stress (MacMillan et al., 2016; Telonis-Scott et al., 2013).
263 Therefore, the mild induction of immune proteins in infected hemolymph may be an
264 unspecific stress response associated to the presence of bacteria. Another attractive
265 hypothesis would be that proteases released by Spiroplasma could trigger the
266 soluble sensor Persephone and activate the Toll pathway in a peptidoglycan-
267 independent fashion (Gottar et al., 2006; Issa et al., 2018).
268 Another uncovering of this study is the depletion of proteasome
269 components in the hemolymph upon Spiroplasma infection. As the ubiquitin-
270 proteasome is a major degradation pathway for intracellular proteins (Kleiger and
271 Mayor, 2014), the components that we detected in the hemolymph are presumably
272 released from cells that broke upon hemolymph collection (epithelial, fat or muscular
273 cells, but also possibly hemocytes). However, functionally active 20S proteasome
274 units have been discovered circulating in extracellular fluids in mammals, including in
275 serum (Sixt and Dahlmann, 2008) hence we cannot exclude the existence of
276 circulating proteasome units in Drosophila hemolymph. Host ubiquitin-proteasome
277 systems have long been suspected to be a key element in symbiotic homeostasis. It
278 is upregulated in cells harboring intracellular symbionts in weevils, presumably to
279 increase protein turnover and provide free amino-acids to the bacteria (Heddi et al.,
280 2005). In vitro work on cell cultures infected by the facultative endosymbiont
281 Wolbachia also revealed that it induces the host proteasome presumably also to
282 support its own growth (Fallon and Witthuhn, 2009; He et al., 2019; White et al.,
283 2017). Remarkably, one proteasome subunit was detected as enriched upon
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284 Spiroplasma infection in the aphid Aphis citricidus when the insect was feeding on a
285 suboptimum but not on an optimum host plant, suggesting an interaction between
286 symbiosis, nutrition and the proteasome-ubiquitin degradation system (Guidolin et al.,
287 2018). In the case of Drosophila-Spiroplasma interaction, the depletion of
288 proteasome subunits upon infection could thus be a titer regulation mechanism: by
289 decreasing its proteolysis, the host would limit the release of amino acids in the
290 extracellular space that would be usable by Spiroplasma.
291 Our approach also produced an in-depth Spiroplasma proteome on total
292 proteins regardless of their cell localization. This gives a quantitatively unbiased
293 overview of each protein abundance which was not achieved by previous data based
294 on detergent extractions (Paredes et al., 2015). Such quantitative approach allowed
295 us to make a comparison between the transcript and protein levels on about one
296 fourth of the total number of predicted coding sequences (Masson et al., 2018).
297 Although the correlation between transcriptomics and proteomics data greatly varies
298 among models, tissues and experimental set-ups (Haider and Pal, 2013), our data
299 indicate a rather low correlation in the case of Spiroplasma. The correlations between
300 transcript and protein levels depends on the interaction between transcript stability
301 and protein stability (Schwanhäusser et al., 2011). It also depends on the intrinsic
302 chemical properties of each transcript that affect the ribosome binding or the
303 translation speed, for example the Shine-Dalgarno sequence in prokaryotes
304 (Bingham et al., 1986) or more importantly the codon adaptation index of the coding
305 sequence (Lithwick and Margalit, 2003). An explanation for the overall lack of
306 correlation between transcripts and proteins regardless of the model could be that
307 selection operates at the protein level, hence changes in mRNA levels would be
308 offset by post-transcriptional mechanisms to maintain stable protein levels over
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309 evolutionary times (Schrimpf et al., 2009). This hypothesis entails that genes having
310 a low mRNA level compared to their protein levels are likely to be under strong
311 selective pressure to maintain high protein abundancy through post-transcriptional
312 regulations. Intriguingly, this is the case for S. poulsonii Spiralin B, a protein
313 homologous to the S. citri Spiralin A, a membrane lectin suspected to be essential for
314 insect to plant transmission (Duret et al., 2003; Killiny et al., 2005). Spiralin B has
315 been identified as a putative virulence factor in S. poulsonii as it is upregulated when
316 the bacteria are in the fly hemolymph compared to in vitro culture (Masson et al.,
317 2018) and could be involved in oocyte infection for vertical transmission. Similarly,
318 the Crr glucose transporter has an unexpectedly high protein/mRNA ratio, possibly in
319 connection with its role in bacteria survival in the hemolymph. Spiroplasma has a
320 pseudogenized transporter for trehalose, the most abundant sugar in the hemolymph
321 (Paredes et al., 2015). This could have been selected over host-symbiont coevolution
322 to prevent Spiroplasma overgrowth, hence increasing the stability of the interaction
323 by limiting the metabolic cost of harboring the bacteria. Maintaining high Crr levels
324 could thus be an offset response to maintain bacteria proliferation despite trehalose
325 inaccessibility. Last, the ferritin-like protein Ftn has also a bias towards high protein
326 abundancy, suggesting that iron could be a key metabolite in Spiroplasma-
327 Drosophila symbiotic homeostasis.
328 All tissues bathed by hemolymph contribute to its composition. As a
329 consequence, studying the impact of symbionts on this biofluid is unapproachable by
330 transcriptomic methods only. Dual proteomics proved to be an efficient approach to
331 overcome this hurdle and gain novel insights into the Drosophila-Spiroplasma
332 symbiosis. This method is also promising for the study of other symbioses,
333 particularly those where symbionts inhabit biofluids rather than cells.
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334 Material and Methods
335 Spiroplasma and Drosophila stocks
336 Flies were kept at 25°C on cornmeal medium (35.28 g of cornmeal, 35.28 g of
337 inactivated yeast, 3.72 g of agar, 36 ml of fruits juice, 2.9 ml of propionic acid and
338 15.9 ml of Moldex for 600 ml of medium). The complete list of fly stocks is available
339 in Supplementary Table S4.
340 Spiroplasma poulsonii strain Uganda-1 (Pool et al., 2006) was used for all infections.
341 Fly stocks were infected as previously described (Herren and Lemaitre, 2011).
342 Briefly, 9 nL of Spiroplasma-infected hemolymph was injected in their thorax. The
343 progeny of these flies was collected after 5-7 days using male killing as a proxy to
344 assess the infection (100% female progeny). Flies from at least the 3rd generation
345 post-injection were used experimentally.
346
347 Hemolymph extraction procedure
348 Embryos were collected from conventionally reared flies and dechorionated using a
349 bleach-based previously published method (Broderick et al., 2014) to ensure that
350 they were devoid of horizontally transmitted pathogens. One generation was then left
351 to develop in conventional rearing conditions to allow for gut microbiota
352 recontamination. Hemolymph was extracted from 10 days-old flies from the second
353 generation after bleach treatment using a Nanoject II (Drummond) as previously
354 described (Herren et al., 2014). 1 µL of hemolymph was collected for each sample
355 and frozen at -80°C until further use. Hemolymph was then diluted 10 times with PBS
356 containing Protease Inhibitor Cocktail 1X (Roche). 1 µl was used for protein
357 quantification with the Pierce BCA Protein Assay Kit (Thermofisher). The remaining 9
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358 µl were mixed with SDS (0.2% final), DTT (2.5mM final) and PMSF (10µM final).
359 Aliquots of 15µg were used for proteomics analysis.
360
361 Proteomics sample preparation and LC-MS/MS
362 Each sample was digested by Filter Aided Sample Preparation (FASP) (Wiśniewski
363 et al., 2009) with minor modifications. Dithiothreitol (DTT) was replaced by Tris (2-
364 carboxyethyl)phosphine (TCEP) as reducing agent and iodoacetamide by
365 chloracetamide as alkylating agent. A proteolytic digestion was performed using
366 Endoproteinase Lys-C and Trypsin. Peptides were desalted on C18 StageTips
367 (Rappsilber et al., 2007) and dried down by vacuum centrifugation. For LC-MS/MS
368 analysis, peptides were resuspended and separated by reversed-phase
369 chromatography on a Dionex Ultimate 3000 RSLC nanoUPLC system in-line
370 connected with an Orbitrap Q Exactive HF Mass Spectrometer (Thermo Fischer
371 Scientific). Database search was performed using MaxQuant 1.5.1.2 (Cox and Mann,
372 2008) against a concatenated database consisting of the Ensemble Drosophila
373 melanogaster protein database (BDGP5.25) and a custom Spiroplama poulsonii
374 proteome predicted from the reference genome (Genbank accession number
375 JTLV00000000.2). Carbamidomethylation was set as fixed modification, whereas
376 oxidation (M) and acetylation (Protein N-term) were considered as variable
377 modifications. Label-free quantification was performed by MaxQuant using the
378 standard settings. Hits were considered significant when the fold-change between
379 infected and uninfected hemolymph was >2 or <-2 and the FDR<0.05.
380 Complete proteomics data are available on the ProteomExchange repository with the
381 accession number #[data under submission].
382
16 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
383 Spiroplasma quantification
384 Spiroplasma quantification was performed by qPCR as previously described (Masson
385 et al., 2020a). Briefly, the DNA of pools of 5 whole flies was extracted and the copy
386 number of a single-copy bacterial gene (dnaA or dnaK) was quantified and
387 normalized by that of the host gene rsp17. Primers sequences are available in
388 Supplementary Table S4. Each experiment has been repeated 2 or 3 independent
389 times with at least 3 technical replicates each. Data were analyzed by one-way
390 ANOVA.
391
392 RT-qPCR
393 Gene expression measurement were performed by RT-qPCR as previously
394 described (Iatsenko et al., 2020; Romeo and Lemaitre, 2008). Briefly, 10 whole flies
395 were crushed and their RNA extracted with the Trizol method. Reverse transcription
396 was carried out using a PrimeScript RT kit (Takara) and a mix of random hexamers
397 and oligo-dTs. qPCR was performed on a QuantStudio 3 (Applied Biosystems) with
398 PowerUp SYBR Green Master Mix using primers sequences available in
399 Supplementary Table S4. The expression of the target gene was normalized by that
400 of the housekeeping gene rp49 (rpL32) using the 2-∆∆CT method (Pfaffl, 2001).
401 Each experiment has been repeated 2 or 3 independent times with at least 3
402 technical replicates each. Data were analyzed by Mann-Whitney tests.
403
404 Acknowledgments
405 We are grateful to Florence Armand, Romain Hamelin and the EPFL Proteomics
406 Core Facility for sharing their expertise on proteomics.
407
17 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
408 Competing interests
409 The authors declare no competing interests.
410
411 Funding
412 This work was funded by the Swiss National Science Foundation grant
413 N°310030_185295.
18 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
414 References
415 Ballinger, M. J. and Perlman, S. J. (2017). Generality of toxins in defensive symbiosis:
416 Ribosome-inactivating proteins and defense against parasitic wasps in Drosophila.
417 PLoS Pathog. 13, e1006431.
418 Bingham, A. H., Ponnambalam, S., Chan, B. and Busby, S. (1986). Mutations that reduce
419 expression from the P2 promoter of the Escherichia coli galactose operon. Gene 41,
420 67–74.
421 Broderick, N. a., Buchon, N. and Lemaitre, B. (2014). Microbiota-induced changes in
422 Drosophila melanogaster host gene expression and gut morphology. MBio 5, 1–13.
423 Cox, J. and Mann, M. (2008). MaxQuant enables high peptide identification rates,
424 individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
425 Nat. Biotechnol. 26, 1367–1372.
426 De Gregorio, E., Spellman, P. T., Rubin, G. M. and Lemaitre, B. (2001). Genome-wide
427 analysis of the Drosophila immune response by using oligonucleotide microarrays.
428 Proc. Nat. Acad. Sci. 98, 12590–12595.
429 Douglas, A. E. (2015). Multiorganismal Insects: Diversity and Function of Resident
430 Microorganisms. Annu. Rev. Entomol. 60, 17–34.
431 Douglas, A. E. (2016). How multi-partner endosymbioses function. Nat. Rev. Microbiol. 14,
432 731–743.
433 Duret, S., Berho, N., Danet, J. L., Garnier, M. and Renaudin, J. (2003). Spiralin is not
434 essential for helicity, motility, or pathogenicity but is required for efficient transmission of
435 Spiroplasma citri by its leafhopper vector Circulifer haematoceps. Appl. Environ.
436 Microbiol. 69, 6225–6234.
437 Duron, O., Bouchon, D., Boutin, S., Bellamy, L., Zhou, L., Engelstadter, J. and Hurst, G.
438 D. (2008). The diversity of reproductive parasites among arthropods: Wolbachia do not
439 walk alone. BMC Biol. 6, 27.
440 Fallon, A. M. and Witthuhn, B. A. (2009). Proteasome activity in a naïve mosquito cell line
19 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
441 infected with Wolbachia pipientis wAlbB. In Vitro Cell. Dev. Biol. Anim. 45, 460–466.
442 Gasparich, G. E. (2002). Spiroplasmas: evolution, adaptation and diversity. Front. Biosci. 7,
443 619–640.
444 Gottar, M., Gobert, V., Matskevich, A. A., Reichhart, J.-M., Wang, C., Butt, T. M., Belvin,
445 M., Hoffmann, J. A. and Ferrandon, D. (2006). Dual detection of fungal infections in
446 Drosophila via recognition of glucans and sensing of virulence factors. Cell 127, 1425–
447 1437.
448 Guidolin, A. S., Cataldi, T. R., Labate, C. A., Francis, F. and Cônsoli, F. L. (2018).
449 Spiroplasma affects host aphid proteomics feeding on two nutritional resources. Sci.
450 Rep. 8, 2466.
451 Haider, S. and Pal, R. (2013). Integrated analysis of transcriptomic and proteomic data.
452 Curr. Genomics 14, 91–110.
453 Hamilton, P. T., Peng, F., Boulanger, M. J. and Perlman, S. J. (2016). A ribosome-
454 inactivating protein in a Drosophila defensive symbiont. Proc. Natl. Acad. Sci. U. S. A.
455 113, 350–355.
456 Hanson, M. A., Dostálová, A., Ceroni, C., Poidevin, M., Kondo, S. and Lemaitre, B.
457 (2019). Synergy and remarkable specificity of antimicrobial peptides in vivo using a
458 systematic knockout approach. Elife 8, e44341.
459 Harumoto, T. and Lemaitre, B. (2018). Male-killing toxin in a bacterial symbiont of
460 Drosophila. Nature 557, 252–255.
461 Harumoto, T., Anbutsu, H. and Fukatsu, T. (2014). Male-killing Spiroplasma induces sex-
462 specific cell death via host apoptotic pathway. PLoS Pathog. 10, e1003956.
463 Harumoto, T., Anbutsu, H., Lemaitre, B. and Fukatsu, T. (2016). Male-killing symbiont
464 damages host’s dosage-compensated sex chromosome to induce embryonic apoptosis.
465 Nat. Commun. 7, 12781.
466 Harumoto, T., Fukatsu, T. and Lemaitre, B. (2018). Common and unique strategies of
467 male killing evolved in two distinct Drosophila symbionts. Proc. R. Soc. B Biol. Sci. 285,.
468 Haselkorn, T. S. (2010). The Spiroplasma heritable bacterial endosymbiont of Drosophila.
20 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
469 Fly (Austin). 4, 80–87.
470 He, Z., Zheng, Y., Yu, W.-J., Fang, Y., Mao, B. and Wang, Y.-F. (2019). How do Wolbachia
471 modify the Drosophila ovary? New evidences support the “titration-restitution” model for
472 the mechanisms of Wolbachia-induced CI. BMC Genomics 20, 608.
473 Heddi, A., Vallier, A., Anselme, C., Xin, H., Rahbe, Y. and Wackers, F. (2005). Molecular
474 and cellular profiles of insect bacteriocytes: mutualism and harm at the initial
475 evolutionary step of symbiogenesis. Cell Microbiol 7, 293–305.
476 Hedges, L. M., Brownlie, J. C., O’Neill, S. L. and Johnson, K. N. (2008). Wolbachia and
477 virus protection in insects. Science (80-. ). 322, 702.
478 Herren, J. K. and Lemaitre, B. (2011). Spiroplasma and host immunity: Activation of
479 humoral immune responses increases endosymbiont load and susceptibility to certain
480 Gram-negative bacterial pathogens in Drosophila melanogaster. Cell. Microbiol. 13,
481 1385–1396.
482 Herren, J. K., Paredes, J. C., Schüpfer, F. and Lemaitre, B. (2013). Vertical Transmission
483 of a Drosophila Endosymbiont Via Cooption of the Yolk Transport and Internalization
484 Machinery. MBio 4 (2), e00532-12.
485 Herren, J. K., Paredes, J. C., Schüpfer, F., Arafah, K., Bulet, P. and Lemaitre, B. (2014).
486 Insect endosymbiont proliferation is limited by lipid availability. Elife 3, e02964.
487 Hoffmann, A. A., Montgomery, B. L., Popovici, J., Iturbe-Ormaetxe, I., Johnson, P. H.,
488 Muzzi, F., Greenfield, M., Durkan, M., Leong, Y. S., Dong, Y., et al. (2011).
489 Successful establishment of Wolbachia in Aedes populations to suppress dengue
490 transmission. Nature 476, 454.
491 Hurst, G. D. D., Anbutsu, H., Kutsukake, M. and Fukatsu, T. (2003). Hidden from the host:
492 Spiroplasma bacteria infecting Drosophila do not cause an immune response, but are
493 suppressed by ectopic immune activation. Insect Mol. Biol. 12, 93–97.
494 Iatsenko, I., Marra, A., Boquete, J.-P., Peña, J. and Lemaitre, B. (2020). Iron
495 sequestration by transferrin 1 mediates nutritional immunity in Drosophila melanogaster.
496 Proc. Natl. Acad. Sci. 117, 7317 LP-7325.
21 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
497 Issa, N., Guillaumot, N., Lauret, E., Matt, N., Schaeffer-Reiss, C., Van Dorsselaer, A.,
498 Reichhart, J.-M. and Veillard, F. (2018). The Circulating Protease Persephone Is an
499 Immune Sensor for Microbial Proteolytic Activities Upstream of the Drosophila Toll
500 Pathway. Mol. Cell 69, 539–550.e6.
501 Jaenike, J., Unckless, R., Cockburn, S. N., Boelio, L. M. and Perlman, S. J. (2010).
502 Adaptation via symbiosis: recent spread of a Drosophila defensive symbiont. Science
503 (80-. ). 329, 212–215.
504 Killiny, N., Castroviejo, M. and Saillard, C. (2005). Spiroplasma citri Spiralin acts in vitro as
505 a lectin binding to glycoproteins from its insect vector Circulifer haematoceps.
506 Phytopathology 95, 541–548.
507 Kleiger, G. and Mayor, T. (2014). Perilous journey: a tour of the ubiquitin-proteasome
508 system. Trends Cell Biol. 24, 352–359.
509 Lemaitre, B. and Hoffmann, J. (2007). The host defense of Drosophila melanogaster. Annu
510 Rev Immunol 25, 697–743.
511 Lithwick, G. and Margalit, H. (2003). Hierarchy of sequence-dependent features associated
512 with prokaryotic translation. Genome Res. 13, 2665–2673.
513 MacMillan, H. A., Knee, J. M., Dennis, A. B., Udaka, H., Marshall, K. E., Merritt, T. J. S.
514 and Sinclair, B. J. (2016). Cold acclimation wholly reorganizes the Drosophila
515 melanogaster transcriptome and metabolome. Sci. Rep. 6, 28999.
516 Masson, F. and Lemaitre, B. (2020). Growing Ungrowable Bacteria: Overview and
517 Perspectives on Insect Symbiont Culturability. Microbiol. Mol. Biol. Rev. 84, e00089-20.
518 Masson, F., Calderon Copete, S., Schüpfer, F., Garcia-Arraez, G. and Lemaitre, B.
519 (2018). In Vitro Culture of the Insect Endosymbiont Spiroplasma poulsonii Highlights
520 Bacterial Genes Involved in Host-Symbiont Interaction. MBio 9(2), e00024-18.
521 Masson, F., Calderon-Copete, S., Schüpfer, F., Vigneron, A., Rommelaere, S., Garcia-
522 Arraez, M. G., Paredes, J. C. and Lemaitre, B. (2020a). Blind killing of both male and
523 female Drosophila embryos by a natural variant of the endosymbiotic bacterium
524 Spiroplasma poulsonii. Cell. Microbiol. 22(5), e13156.
22 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
525 Masson, F., Schüpfer, F., Jollivet, C. and Lemaitre, B. (2020b). Transformation of the
526 Drosophila sex-manipulative endosymbiont Spiroplasma poulsonii and persisting
527 hurdles for functional genetics studies. Appl. Environ. Microbiol. AEM.00835-20.
528 Mateos, M., Castrezana, S. J., Nankivell, B. J., Estes, A. M., Markow, T. A. and Moran,
529 N. A. (2006). Heritable Endosymbionts of Drosophila. Genetics 174, 363–376.
530 Medina, P., Russell, S. L. and Corbett-Detig, R. (2019). Deep data mining reveals variable
531 abundance and distribution of microbial reproductive manipulators within and among
532 diverse host species. bioRxiv 679837.
533 Montllor, C. B., Maxmen, A. and Purcell, A. H. (2002). Facultative bacterial endosymbionts
534 benefit pea aphids Acyrthosiphon pisum under heat stress. Ecol. Entomol. 27, 189–195.
535 Oliver, K. M., Russell, J. A., Moran, N. A. and Hunter, M. S. (2003). Facultative bacterial
536 symbionts in aphids confer resistance to parasitic wasps. Proc Natl Acad Sci U S A 100,
537 1803–1807.
538 Palombella, V. J., Rando, O. J., Goldberg, A. L. and Maniatis, T. (1994). The ubiquitin-
539 proteasome pathway is required for processing the NF-kappa B1 precursor protein and
540 the activation of NF-kappa B. Cell 78, 773–785.
541 Paredes, J. C., Herren, J. K., Schüpfer, F., Marin, R., Claverol, S., Kuo, C.-H., Lemaitre,
542 B. and Béven, L. (2015). Genome sequence of the Drosophila melanogaster male-
543 killing Spiroplasma strain MSRO endosymbiont. MBio 6(2), e02437-14.
544 Paredes, J. C., Herren, J. K., Schüpfer, F. and Lemaitre, B. (2016). The Role of Lipid
545 Competition for Endosymbiont-Mediated Protection against Parasitoid Wasps in
546 Drosophila. MBio 7(4), e01006-16.
547 Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-
548 PCR. Nucleic Acids Res. 29, e45–e45.
549 Pool, J. E., Wong, a and Aquadro, C. F. (2006). Finding of male-killing Spiroplasma
550 infecting Drosophila melanogaster in Africa implies transatlantic migration of this
551 endosymbiont. Heredity (Edinb). 97, 27–32.
552 Rappsilber, J., Mann, M. and Ishihama, Y. (2007). Protocol for micro-purification,
23 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
553 enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.
554 Nat. Protoc. 2, 1896–1906.
555 Romeo, Y. and Lemaitre, B. (2008). Drosophila immunity: methods for monitoring the
556 activity of Toll and Imd signaling pathways. Methods Mol. Biol. 415, 379–394.
557 Scarborough, C. L., Ferrari, J. and Godfray, H. C. (2005). Aphid protected from pathogen
558 by endosymbiont. Science (80-. ). 310, 1781.
559 Schrimpf, S. P., Weiss, M., Reiter, L., Ahrens, C. H., Jovanovic, M., Malmström, J.,
560 Brunner, E., Mohanty, S., Lercher, M. J., Hunziker, P. E., et al. (2009). Comparative
561 Functional Analysis of the Caenorhabditis elegans and Drosophila melanogaster
562 Proteomes. PLOS Biol. 7, e1000048.
563 Schwanhäusser, B., Busse, D., Li, N., Dittmar, G., Schuchhardt, J., Wolf, J., Chen, W.
564 and Selbach, M. (2011). Global quantification of mammalian gene expression control.
565 Nature 473, 337–342.
566 Sixt, S. U. and Dahlmann, B. (2008). Extracellular, circulating proteasomes and ubiquitin —
567 Incidence and relevance. Biochim. Biophys. Acta - Mol. Basis Dis. 1782, 817–823.
568 Teixeira, L., Ferreira, A. and Ashburner, M. (2008). The bacterial symbiont Wolbachia
569 induces resistance to RNA viral infections in Drosophila melanogaster. PLoS Biol 6, e2.
570 Telonis-Scott, M., van Heerwaarden, B., Johnson, T. K., Hoffmann, A. A. and Sgrò, C.
571 M. (2013). New Levels of Transcriptome Complexity at Upper Thermal Limits in Wild
572 Drosophila Revealed by Exon Expression Analysis. Genetics 195, 809–830.
573 Troha, K., Im, J. H., Revah, J., Lazzaro, B. P. and Buchon, N. (2018). Comparative
574 transcriptomics reveals CrebA as a novel regulator of infection tolerance in D.
575 melanogaster. PLoS Pathog. 14, e1006847.
576 Tzou, P., Reichhart, J.-M. and Lemaitre, B. (2002). Constitutive expression of a single
577 antimicrobial peptide can restore wild-type resistance to infection in immunodeficient
578 Drosophila mutants. Proc. Natl. Acad. Sci. U. S. A. 99, 2152–2157.
579 Veneti, Z., Bentley, J. K., Koana, T., Braig, H. R. and Hurst, G. D. D. (2005). A Functional
580 Dosage Compensation Complex Required for Male Killing in Drosophila. Science (80-.
24 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
581 ). 307, 1461–1463.
582 Werren, J. H., Baldo, L. and Clark, M. E. (2008). Wolbachia: master manipulators of
583 invertebrate biology. Nat. Rev. Microbiol. 6, 741–751.
584 White, P. M., Serbus, L. R., Debec, A., Codina, A., Bray, W., Guichet, A., Lokey, R. S.
585 and Sullivan, W. (2017). Reliance of Wolbachia on High Rates of Host Proteolysis
586 Revealed by a Genome-Wide RNAi Screen of Drosophila Cells. Genetics 205, 1473–
587 1488.
588 Wiśniewski, J. R., Zougman, A., Nagaraj, N. and Mann, M. (2009). Universal sample
589 preparation method for proteome analysis. Nat. Methods 6, 359–362.
590 Xie, J., Butler, S., Sanchez, G. and Mateos, M. (2014). Male killing Spiroplasma protects
591 Drosophila melanogaster against two parasitoid wasps. Heredity (Edinb). 112, 399–408.
592
593
25 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
594 Figure Legends
595 Figure 1. Drosophila hemolymph protein profiling upon Spiroplasma infection 596 (A) Representation of the dataset repartition between intracellular and 597 extracellular protein groups. Extracellular groups are detected either by the 598 presence of a predicted signal peptide using the SignalP algorithm or the 599 annotation with a GO term referring to the extracellular space. The overlap 600 category corresponds to protein groups with both a predicted signal peptide 601 and a GO term “extracellular”. Numbers in brackets indicate the number of 602 protein groups in each category. 603 (B) Volcano plot of the intracellular protein group subset. The log fold-change 604 indicates the differential representation of the protein group in the infected 605 samples versus the uninfected samples. Bold purple dots indicate significance 606 as defined by an absolute fold change value over 2 or under -2 and a p-value 607 below 0.05. 608 (C) Functional GO annotation of the intracellular protein groups. Only significant 609 (p-value < 0.05) GO terms of hierarchy 4 or less are displayed. CC: Cell 610 Component; MF: Molecular Function; BP: Biological Process. 611 (D) Volcano plot of the extracellular protein group subset. The log fold-change 612 indicates the differential representation of the protein group in the infected 613 samples versus the uninfected samples. Bold purple dots indicate significance 614 as defined by an absolute fold change value over 2 or under -2 and a p-value 615 below 0.05. 616 (E) Functional GO annotation of the extracellular protein groups. Only significant 617 (p-value < 0.05) GO terms of hierarchy 4 or less are displayed. CC: Cell 618 Component; MF: Molecular Function; BP: Biological Process. 619 620 621 Figure 2. Spiroplasma infection induces a mild transcriptional immune 622 response 623 624 (A) Changes in a selection of immune-related protein group abundances 625 quantified by LC-MS/MS. Data are expressed as fold change of the 626 Label-Free Quantification (LFQ) values of Spiroplasma infected
26 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
627 hemolymph over the uninfected control (log2 scale). *, p<0.05; ** 628 p<0.005, ***; p<0.005, ****, p<0.0005 upon Student t-test. 629 (B) mRNA quantification of a selection of candidate genes in uninfected and 630 Spiroplasma-infected flies. Results are expressed as the fold change of 631 target mRNA normalized by rpL32 mRNA in Spiroplasma-infected flies as 632 compared to uninfected flies (log2 scale). *, p<0.05; ** p<0.005, ***; 633 p<0.0005 upon Mann-Whitney test on ΔCt values. 634 635 Figure 3. Targeted genetic screening of candidate proteins 636 (A-C) Quantification of Spiroplasma titer in two weeks old flies in various 637 genetic backgounds. Titer is expressed as the fold-change over the 638 appropriate control line. All graphs represent the mean +/- standard deviation 639 of a pool of at least 2 independent experiments. (A) Control flies (Act5C- 640 GAL4 driver crossed w1118, white bars) are compared to flies overexpressing 641 a single antimicrobial peptide gene driven by the ubiquitous Act5C-GAL4 642 driver (black bars). Titer is expressed as the fold-change over control. (B) 643 Quantification of Spiroplasma titer in wild-type flies (OregonR, white bar) and 644 hayan, attD and tep2 null mutants (black). Spiroplasma titer in AMP10 iso 645 flies were compared to that of their isogenic wild-type counterpart (w1118 iso, 646 white bar). (C) Quantification of the impact of RNAi-mediated knockdown on 647 Spiroplasma titer with the Act5C-GAL4 driver. Titer is expressed as the fold- 648 change over the control (Act5C-GAL4 driver crossed to w1118). 649 650 651 Figure 4. Spiroplasma transcription-translation correlation
652 Each dot represents a protein positioned according to its log2(LFQ; Label-free 653 Quantification) value in the proteome versus its log2(CPM ; Count Per Million) 654 in the transcriptomics dataset from (Masson et al., 2018). The black line 655 represents the linear model log2(LFQ) = 0.57892 x log2(CPM) + 21.38325, 656 with an adjusted R2 = 0.2909. Dot size and color are adjusted to the residuals 657 of the model, with bigger bluer dots indicating a higher residual hence a 658 stronger deviation from the linear model for the considered protein. 659
27 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
660 Supplementary Figure 1.
661 (A) Quantification of Spiroplasma titer in two weeks old flies. Titer is expressed 662 as the fold-change of Act5C-GAL4>UAS-RNAi over Act5C-GAL4> w1118. Bars 663 represent the mean +/- standard deviation of a pool of at least 2 independent 664 experiments. (B) Quantification of Spiroplasma titer in CG15293 mutant flies 665 compared to control yw flies. Bars represent the mean +/- standard deviation 666 of a pool of at least 3 independent experiments. ***; p<0.0005 upon Mann- 667 Whitney test on ΔΔCt values. 668 669
670 Supplementary Table 1.
671 Drosophila genes quantified by LC-MS/MS.
672
673 Supplementary Table 2.
674 Spiroplasma genes quantified by LC-MS/MS.
675
676 Supplementary Table 3.
677 Spiroplasma genes outlying the linear model between mRNA and protein
678 levels.
28 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432267; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.