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The S1A protease family members CG10764 and CG4793 regulate cellular immunity in Drosophila

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The S1A protease family members CG10764 and CG4793 regulate cellular immunity in Drosophila

Abstract

In nature, Drosophila melanogaster larvae are infected by parasitoid wasps and mount a cellular immune response to this infection. Several conserved signaling pathways have been implicated in coordinating this response, however our understanding of the integration and regulation of these pathways is incomplete. Members of the S1A serine protease family have been previously linked to immune functions, and our findings suggest roles for two S1A family members, CG10764 and CG4793 in the cellular immune response to parasitoid infection.
The S1A protease family members CG10764 and CG4793 regulate
cellular immunity in Drosophila
Pooja KR1, Jonathan Lee1 and Nathan T Mortimer
1School of Biological Sciences, Illinois State University
§To whom correspondence should be addressed: ntmorti@ilstu.edu
Abstract
In nature, Drosophila melanogaster larvae are infected by parasitoid wasps and mount a cellular immune response to this
infection. Several conserved signaling pathways have been implicated in coordinating this response, however our
understanding of the integration and regulation of these pathways is incomplete. Members of the S1A serine protease
family have been previously linked to immune functions, and our findings suggest roles for two S1A family members,
CG10764 and CG4793 in the cellular immune response to parasitoid infection.
Figure 1. Characterization of S1A serine protease family members in fly cellular immunity: Box plots showing the
proportion of wasp eggs encapsulated following infection by the avirulent wasp Leptopilina clavipes when the indicated
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genes are knocked down by RNA interference (RNAi) in (A) lamellocytes or (B) plasmatocytes. For these experiments,
we crossed immune cell GAL4 males to the indicated UAS-RNAi genotype females, and the resulting offspring were
infected and assayed for the proportion of parasitoid eggs that were encapsulated. Two independent UAS-RNAi lines were
used for each gene. (A) RNAi knock down in lamellocytes using msn-GAL4. Knocking down CG10764 in lamellocytes
significantly reduced the encapsulation rate. Conversely, knockdown of CG4793 in lamellocytes resulted in a significant
increase in the encapsulation rate. (B) RNAi knock down in plasmatocytes using eater-GAL4. Knocking down CG10764
in plasmatocytes resulted in a significant reduction in the encapsulation rate. RNAi knock down of CG4793 in
plasmatocytes had no effect. Plots show quartile data with the box giving the interquartile range and whiskers extending to
the minimum and maximum. The median is indicated by a solid line. *indicates p value < 0.05 compared to the control
genotypes, UAS-GAL4RNAi-1 for the first set of RNAi lines (UAS-CG10764GL01210,UAS-CG4793HMC03765) and UAS-
GAL4RNAi-2 for the second set of RNAi lines (UAS-CG10764NIG.10764R,UAS- CG4793NIG.4793R) by Dunnett’s test.
Description
Cellular immune responses are an important aspect of innate host defense against infection and are broadly conserved
from insects to mammals. The model organism Drosophila melanogaster uses the cellular encapsulation response to
protect against macroparasite infection (Carton et al., 2008; Mortimer, 2013). This response shows genetic conservation
with human immune responses (Howell et al., 2012), and may serve as a useful model to better understand human
immune cell functions. Drosophila larvae are commonly infected by parasitoid wasps and following infection mount a
cellular immune response to kill the parasite. This response is mediated by two cell types, circulating macrophage-like
immune cells known as plasmatocytes and infection-induced immune cells called lamellocytes (Honti et al., 2014; Rizki,
1957). Plasmatocytes operate as the first line responders to infection by recognizing and binding to the wasp egg
(Mortimer et al., 2012; Russo et al., 1996). This process is then followed by the production of lamellocytes that form a
consolidated multi-layered capsule, thereby killing the wasp (Kim-Jo et al., 2019; Russo et al., 1996). Recent findings
have begun to elucidate the regulation of the encapsulation response in Drosophila, including a role for the evolutionarily
conserved JAK-STAT signaling pathway (Sorrentino et al. 2004; Yang et al. 2015). The roles of JAK-STAT signaling are
not completely understood, but the pathway has been linked to the production of lamellocytes (Bausek and Zeidler, 2014;
Hanratty and Dearolf, 1993; Luo et al., 1995, 1997; Sorrentino et al., 2004).
Members of the S1A protease family are involved in many physiological processes, including the regulation of
invertebrate immune responses (Cao and Jiang, 2018). In Drosophila, the S1A family is composed of more than 200 genes
and includes the catalytically active serine proteases (SPs) and the serine protease homologs (SPHs), a group of SP-like
proteins that are enzymatically inactive (Cao and Jiang, 2018). Many S1A family members have been linked to the
antimicrobial immune response including the SP genes spirit, grass, psh and SPE, and the SPH genes sphe, sphinx1 and
sphinx2 (Buchon et al., 2009; El Chamy et al., 2008; Kambris et al., 2006; Ligoxygakis et al., 2002; Patrnogic and
Leclerc, 2017). However, the role of SP and SPH genes in regulating the fly antiparasitoid immune response is still not
well-defined.
A recent study of transcriptional targets of JAK-STAT pathway activity showed that the S1A family members, the SP gene
CG10764 (also known as SP77) and the SPH gene CG4793 (also known as cSPH128) are JAK-STAT pathway target
genes (Bina et al., 2010). The JAK-STAT pathway is important for the production of lamellocytes following parasitoid
infection (Sorrentino et al., 2004; Yang et al., 2015), and ectopic pathway activity leads to tumorigenesis as characterized
by the precocious accumulation of lamellocytes (Ekas et al., 2010; Harrison et al., 1995). RNA interference (RNAi)
mediated knock down of CG10764 and CG4793 in the JAK-STAT tumor model suggested that these genes may play
antagonistic roles in regulating JAK-STAT signaling and lamellocyte production (Bina et al., 2010).
To evaluate the functional roles of these JAK-STAT regulated S1A family members in fly cellular immunity, we used two
different RNAi lines with unique sequence targets to knock down each gene in both the plasmatocyte (using eater-GAL4)
(Tokusumi et al. 2009a) and lamellocyte (using msn-GAL4) (Lam, et al. 2010; Tokusumi et al. 2009b) immune cell types
and compared their ability to encapsulate parasitoid wasp eggs following infection. We find that knocking down CG10764
with either of the RNAi lines in lamellocytes (Figure 1A; UAS-CG10764GL01210: p= 0.00222, nEXP = 73, nCTRL = 72;
UAS-CG10764NIG.10764R: p=0.0112, nEXP = 71 , nCTRL = 67) or plasmatocytes (Figure 1B; UAS-CG10764GL01210: p=
0.000955, nEXP = 75, nCTRL = 81 ; UAS-CG10764NIG.10764R: p= 1.74e-06, nEXP = 57 , nCTRL = 62) results in a
significant reduction in the proportion of wasp eggs that are successfully encapsulated. These findings suggest that
CG10764 may act as a positive regulator of encapsulation in both fly immune cell types. Conversely, RNAi-mediated
knock down of CG4793 in lamellocytes with either of the RNAi lines results in a significant increase in encapsulation rate
(Figure 1A; UAS-CG4793HMC03765: p= 1.09e- 05, nEXP, = 80, nCTRL = 72 ; UAS- CG4793NIG.4793R : p= 0.0098, nEXP, =
60, nCTRL = 67), but has no effect when knocked down in plasmatocytes (Figure 1B; UAS-CG4793HMC03765 : p=
0.56975, nEXP,=57, nCTRL =62 ; UAS- CG4793NIG.4793R : p= 0.321 , nEXP, = 57 , nCTRL = 62). This suggests that
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CG4793 may act as a negative regulator of encapsulation specifically in lamellocytes, the immune cell subtype that is
induced following infection.
Based on our observations, we hypothesize that CG10764 and CG4793 play important and distinct roles in balancing
immune activation. CG10764 appears to regulate the initiation of pro-immune signaling which triggers the host immune
response against parasitoid infection. CG10764 likely encodes an active serine protease, and may influence immune
activation through the direct cleavage of target proteins. On the other hand, CG4793 appears to be responsible for limiting
the immune response when the defense mechanism is elicited. This is an important role which allows the host to avoid
self-directed immune damage due to an overreactive immune system. CG4793 is an SPH gene and encodes a protein that
is predicted to be catalytically inactive. However, these SPH proteins play regulatory roles in a variety of processes (Cao
and Jiang, 2018), and it is likely that CG4793 is acting through a similar mechanism to limit immune activity. Thus, these
S1A family members likely have cell-specific roles and regulate the cellular encapsulation process through distinct
mechanisms.
A role for CG10764 and CG4793 in modulating JAK-STAT pathway activity has been previously demonstrated (Bina et
al., 2010). Interestingly, these S1A family members were also shown to have opposing effects on the phenotype seen in
hopTum flies, which display a melanotic phenotype due to ectopic JAK-STAT signaling (Bina et al., 2010; Hanratty and
Dearolf, 1993; Luo et al., 1995). Here we show that CG10764 and CG4793 may also act antagonistically to maintain a
balanced immune response and based on these previous studies, we hypothesize that this could potentially be via
regulation of JAK-STAT signaling. However, a detailed mechanistic understanding of how these S1A family genes
regulate cellular immunity and how their activity may be linked to JAK-STAT pathway signaling remain to be established.
Additionally, further research into the human homologs of CG10764 and CG4793 may reveal conserved functions in
human immunity and JAK-STAT mediated disease.
Methods
Request a detailed protocol
Drosophila genetics. Tissue-specific modulation of gene expression can be achieved in D. melanogaster using the yeast-
derived UAS-GAL4 system. GAL4 is a transcription factor that binds to the UAS enhancer sequence present in the
promoter region controlling expression of the gene of interest (Brand and Perrimon, 1993). We used hemocyte specific
GAL4 lines and two UAS-RNAi lines with distinct target sequences to knock down the genes of interest in each hemocyte
type. UAS-GAL4RNAi was used as the control genotype. Independent control experiments were run with each UAS-RNAi
experiment; UAS-GAL4RNAi-1 refers to the control replicates for experiments with the UAS-CG4793HMC03765 and UAS-
CG10764GL01210 constructs and UAS-GAL4RNAi-2 refers to the control replicates for experiments with the UAS-
CG10764NIG.10764R and UAS- CG4793NIG.4793R constructs. All Drosophila crosses were maintained on standard
Drosophila medium (Molasses Formulation, Genesee Scientific) at 25C° on a 12 hour light:dark cycle.
Parasitoid wasp infection. For each genotype tested, approximately 25 virgin female GAL4 flies were mated with 10
UAS-RNAi line males. These crosses were transferred to egg lay chambers containing grape-juice plates (Genesee
Scientific) supplemented with yeast paste and allowed to lay for 72 hours. For infection experiments, 25 F1 second instar
larvae were picked from the egg lay plates and transferred into small petri dishes with standard Drosophila medium
(Molasses Formulation, Genesee Scientific) together with 3 female LcNet wasps. All of the surviving larvae
(~25/infection plate) were dissected 72 hours post infection and the number of encapsulated wasp eggs and live wasp
larvae were counted. Each genotype for each experiment was performed in triplicate. All experimental crosses and
infections were carried out at 25°C.
Encapsulation rate. After a 72 hour wasp exposure, larvae from each plate were dissected and scored for the presence of
an encapsulated wasp egg or live wasp larva, to assay the encapsulation rate.
Data analysis and statistics. To analyze the effect of knockdown of proteases on wasp egg encapsulation rate, we used
generalized linear models with quasibinomial errors to test for an effect of genotype, and then we performed Dunnett’s
post hoc tests to compare each of the experimental genotypes to the control genotype. All statistics were done in the R
statistical computing environment (R Core Team, 2020) using the “multcomp” (Hothorn et al., 2008), “plyr” package
(Wickham, 2011). Graphs were produced using the “ggplot2” package (Wickham, 2009).
Reagents
The following Drosophila melanogaster stocks were used in this experiment:
UAS-RNAi lines:
Short Genotype Full Genotype Stock ID
UAS-CG4793HMC03765 y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.GL01210}attP40 BDSC:41628
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UAS-CG10764GL01210 y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]= TRiP. HMC03765}attP40 BDSC:41628
UAS-GAL4RNAi y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=VALIUM20-GAL4.1}attP2 BDSC:35784
UAS-CG10764NIG.10764R P{NIG.10764R} NIG:10764R-1
UAS- CG4793NIG.4793R P{NIG.4793R} NIG:4793R-1
GAL4 lines (provided by Robert Schulz, University of Notre Dame):
Genotype (FlyBase ID) Expression Reference
eater217-GAL4 (FBtp0057112) Plasmatocytes (Tokusumi et al. 2009a)
msn-GAL4 (FBtp0083721) Lamellocytes (Lam et al. 2010; Tokusumi et al. 2009b)
We additionally used the figitid parasitoid wasp species Leptopilina clavipes (strain LcNet) (Mortimer et al. 2012) reared
in the lab on Drosophila virilis.
Acknowledgments: We would like to thank members of the Mortimer lab for discussion of the project, and Robert Schulz
and Tsuyoshi Tokusumi for providing Drosophila stocks. Stocks obtained from the Bloomington Drosophila Stock Center
(Bloomington, IN, USA) (NIH P40OD018537) and the National Institute of Genetics Fly Stock Center (Mishima, Japan)
were used in this study.
References
Bausek N, Zeidler MP. 2014. Gα73B is a downstream effector of JAK/STAT signalling and a regulator of Rho1 in
Drosophila haematopoiesis. J Cell Sci 127: 101-10. PMID: 24163435.
Bina S, Wright VM, Fisher KH, Milo M, Zeidler MP. 2010. Transcriptional targets of Drosophila JAK/STAT pathway
signalling as effectors of haematopoietic tumour formation. EMBO Rep 11: 201-7. PMID: 20168330.
Brand AH, Perrimon N. 1993. Targeted gene expression as a means of altering cell fates and generating dominant
phenotypes. Development 118: 401-15. PMID: 8223268.
Buchon N, Poidevin M, Kwon HM, Guillou A, Sottas V, Lee BL, Lemaitre B. 2009. A single modular serine protease
integrates signals from pattern-recognition receptors upstream of the Drosophila Toll pathway. Proc Natl Acad Sci U S A
106: 12442-7. PMID: 19590012.
Cao X, Jiang H. 2018. Building a platform for predicting functions of serine protease-related proteins in Drosophila
melanogaster and other insects. Insect Biochem Mol Biol 103: 53-69. PMID: 30367934.
Carton Y, Poirié M, Nappi AJ. 2008. Insect immune resistance to parasitoids. Insect Sci 15: 67–87. DOI: 10.1111/j.1744-
7917.2008.00188.x
Ekas LA, Cardozo TJ, Flaherty MS, McMillan EA, Gonsalves FC, Bach EA. 2010. Characterization of a dominant-active
STAT that promotes tumorigenesis in Drosophila. Dev Biol 344: 621-36. PMID: 20501334.
El Chamy L, Leclerc V, Caldelari I, Reichhart JM. 2008. Sensing of 'danger signals' and pathogen-associated molecular
patterns defines binary signaling pathways 'upstream' of Toll. Nat Immunol 9: 1165-70. PMID: 18724373.
Hanratty WP, Dearolf CR. 1993. The Drosophila Tumorous-lethal hematopoietic oncogene is a dominant mutation in the
hopscotch locus. Mol Gen Genet 238: 33-7. PMID: 8479437.
Harrison DA, Binari R, Nahreini TS, Gilman M, Perrimon N. 1995. Activation of a Drosophila Janus kinase (JAK) causes
hematopoietic neoplasia and developmental defects. EMBO J 14: 2857-65. PMID: 7796812.
Honti V, Csordás G, Kurucz É, Márkus R, Andó I. 2014. The cell-mediated immunity of Drosophila melanogaster:
hemocyte lineages, immune compartments, microanatomy and regulation. Dev Comp Immunol 42: 47-56. PMID:
23800719.
Hothorn T, Bretz F, Westfall P. 2008. Simultaneous inference in general parametric models. Biom J 50: 346-63. PMID:
18481363.
Howell L, Sampson CJ, Xavier MJ, Bolukbasi E, Heck MM, Williams MJ. 2012. A directed miniscreen for genes
involved in the Drosophila anti-parasitoid immune response. Immunogenetics 64: 155-61. PMID: 21947570.
Kambris Z, Brun S, Jang IH, Nam HJ, Romeo Y, Takahashi K, Lee WJ, Ueda R, Lemaitre B. 2006. Drosophila immunity:
a large-scale in vivo RNAi screen identifies five serine proteases required for Toll activation. Curr Biol 16: 808-13. PMID:
16631589.
2/22/2021 - Open Access
Kim-Jo C, Gatti JL, Poirié M. 2019. Drosophila Cellular Immunity Against Parasitoid Wasps: A Complex and Time-
Dependent Process. Front Physiol 10: 603. PMID: 31156469.
Lam VK, Tokusumi T, Cerabona D, Schulz RA. 2010. Specific cell ablation in Drosophila using the toxic viral protein
M2(H37A). Fly (Austin) 4: 338-43. PMID: 20798602.
Ligoxygakis P, Pelte N, Hoffmann JA, Reichhart JM. 2002. Activation of Drosophila Toll during fungal infection by a
blood serine protease. Science 297: 114-6. PMID: 12098703.
Luo H, Hanratty WP, Dearolf CR. 1995. An amino acid substitution in the Drosophila hopTum-l Jak kinase causes
leukemia-like hematopoietic defects. EMBO J 14: 1412-20. PMID: 7729418.
Luo H, Rose P, Barber D, Hanratty WP, Lee S, Roberts TM, D'Andrea AD, Dearolf CR. 1997. Mutation in the Jak kinase
JH2 domain hyperactivates Drosophila and mammalian Jak-Stat pathways. Mol Cell Biol 17: 1562-71. PMID: 9032284.
Mortimer NT. 2013. Parasitoid wasp virulence: A window into fly immunity. Fly (Austin) 7: 242-8. PMID: 24088661.
Mortimer NT, Kacsoh BZ, Keebaugh ES, Schlenke TA. 2012. Mgat1-dependent N-glycosylation of membrane
components primes Drosophila melanogaster blood cells for the cellular encapsulation response. PLoS Pathog 8:
e1002819. PMID: 22829770.
Patrnogic J, Leclerc V. 2017. The serine protease homolog spheroide is involved in sensing of pathogenic Gram-positive
bacteria. PLoS One 12: e0188339. PMID: 29211760.
R Core Team. 2020. R: A language and environment for statistical computing.
Rizki MTM. 1957. Alterations in the haemocyte population of Drosophila melanogaster. J Morphol 100: 437–458. DOI:
10.1002/jmor.1051000303
Russo J, Dupas S, Frey F, Carton Y, Brehelin M. 1996. Insect immunity: early events in the encapsulation process of
parasitoid (Leptopilina boulardi) eggs in resistant and susceptible strains of Drosophila. Parasitology 112 ( Pt 1): 135-42.
PMID: 8587797.
Sorrentino RP, Melk JP, Govind S. 2004. Genetic analysis of contributions of dorsal group and JAK-Stat92E pathway
genes to larval hemocyte concentration and the egg encapsulation response in Drosophila. Genetics 166: 1343-56. PMID:
15082553.
Tokusumi T, Shoue DA, Tokusumi Y, Stoller JR, Schulz RA. 2009a. New hemocyte-specific enhancer-reporter transgenes
for the analysis of hematopoiesis in Drosophila. Genesis 47: 771-4. PMID: 19830816.
Tokusumi T, Sorrentino RP, Russell M, Ferrarese R, Govind S, Schulz RA. 2009b. Characterization of a lamellocyte
transcriptional enhancer located within the misshapen gene of Drosophila melanogaster. PLoS One 4: e6429. PMID:
19641625.
Wickham H. 2009. ggplot2: Elegant graphics for data analysis (New York: Springer-Verlag). DOI: 10.1007/978-0-387-
98141-3
Wickham H. 2011. The split-apply-combine strategy for data analysis. J Stat Softw 40: 1–29. DOI: 10.18637/jss.v040.i01
Yang H, Kronhamn J, Ekström JO, Korkut GG, Hultmark D. 2015. JAK/STAT signaling in Drosophila muscles controls
the cellular immune response against parasitoid infection. EMBO Rep 16: 1664-72. PMID: 26412855.
Funding: Research reported in this publication was supported by the National Institute Of General Medical Sciences of
the National Institutes of Health under Award Number R35GM133760 to NTM, and a Grant-in-Aid of Research from the
National Academy of Sciences administered by Sigma Xi, The Scientific Research Society to PKR.
Author Contributions: Pooja KR: Conceptualization, Formal analysis, Writing - original draft, Visualization,
Methodology, Investigation, Writing - review and editing, Funding acquisition. Jonathan Lee: Investigation, Validation.
Nathan T Mortimer: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing -
review and editing.
Reviewed By: Anonymous
History: Received August 26, 2020 Revision received January 14, 2021 Accepted February 11, 2021 Published
February 22, 2021
Copyright: © 2021 by the authors. This is an open-access article distributed under the terms of the Creative Commons
Attribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited.
2/22/2021 - Open Access
Citation: KR, P; Lee, J; Mortimer, NT (2021). The S1A protease family members CG10764 and CG4793 regulate
cellular immunity in Drosophila. microPublication Biology. https://doi.org/10.17912/micropub.biology.000370
2/22/2021 - Open Access
... The S1A family is comprised of more than 200 genes and includes both active proteases and catalytically inactive protease homologs [102]. S1A family members have been previously linked to immune responses against a variety of pathogens [84,94,[103][104][105]. Due to the wide array of encoded protein activities, our GO term analysis did not identify any significant enrichment for molecular function. ...
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