A conserved, immune-regulated peritrophin promotes Vibrio cholerae colonization of the arthropod intestine

Domain: biology
Published: 2026-03-13
Editorial outcome: minor_revision

Abstract
Vibrio cholerae is a human diarrheal pathogen and an estuarine organism that associates
with both terrestrial and aquatic arthropods. Using the model terrestrial arthropod
Drosophila melanogaster, we previously showed that V. cholerae adheres to the
arthropod intestine and activates the enteroendocrine cell innate immune response to
augment expression of the enteroendocrine peptide tachykinin (Tk). Here we show that
enteroendocrine innate immune signaling and Tk promote V. cholerae colonization of the
arthropod intestine. To investigate this, we measure the impact of TkRNAi on intestinal
gene expression. In addition to decreasing expression of antimicrobial peptides and
lipases, TkRNAi reduced the expression of chitinases and chitin-binding proteins including
the small, secreted chitin-binding protein Perit rophin-15a (Peri-15a). Peri-15a interacts
with chitin fibrils in the peritrophic matrix, a protective coating that overlies the arthropod
intestinal epithelium. We uncover that Peri-15a is essential for robust V. cholerae
colonization of the gut. Homologs of Peri-15a are widespread in both terrestrial and
aquatic organisms including marine non-biting midges, marine copepods, rotifers, and
cyanobacteria. We propose that V. cholerae activation of the enteroendocrine cell innate
immune response and Peri-15a expression represents a strategy to maximize
colonization of the arthropod host intestine.

Introduction
Vibrio cholerae is best known as a human pathogen that causes the diarrheal
disease cholera due to expression of the principal virulence factors, the toxin co-regulated
pilus and cholera toxin (1). However, both toxigenic and non-toxigenic strains of V.
cholerae are found in estuarine aquatic environments (2-5). In these environments, V.
cholerae is found in association with large and small marine arthropods including midges,
rotifers, copepods, and cladocerans (4-7). The exoskeletons of these organisms are rich
in chitin, and it has been suggested that the ability of V. cholerae to attach to, degrade,
and grow on chitin contributes to this environmental association (8-12). Chitin also
activates natural competence in V. cholerae, thus promoting horizontal transfer of genetic
material, which is likely important to environmental adaptation and survival (13, 14).

Drosophila melanogaster, the common fruit fly and model arthropod, has revealed
novel aspects of the interaction of V. cholerae with both the arthropod and mammalian
intestinal epithelia (15-23). The Drosophila intestine consists of a foregut, midgut, hindgut
and rectum (Fig 1AA). The midgut is the principal site of digestion and absorption of
nutrients, much like the mammalian small intestine. The midgut epithelium is separated
from the intestinal contents by a porous peritrophic matrix (PM) (24). The PM consists of
a lumen-exposed, web-like structure comprised of chitin and chitin-binding proteins, some
of which are termed peritrophins, and an inner layer of mucins that are anchored to the
chitin (25). The midgut is divided into the cardia and anterior midgut (AMG), where most
of the microbiota reside, the acidic middle midgut, and the posterior midgut (PMG), which
is relatively microbe-free (26). Because microbes entering the midgut first encounter the
AMG, the innate immune response in this region is critical for intestinal defense. Cell
types in the midgut include enterocytes with interspersed intestinal stem cells and
enteroendocrine cells. Enterocytes absorb nutrients from the diet, stem cells replenish
the intestinal epithelium as needed, and enteroendocrine cells release packets of small
regulatory peptides known as enteroendocrine peptides in response to luminal and
systemic host signals. Enteroendocrine cells in the AMG are specialized for responding
to microbial metabolites such which may signal the presence of viable microbes in the gut
(27-29).

Each cell type in the intestinal epithelium encodes components of the innate immune
immunodeficiency (IMD) pathway (Fig 1B) (30). The IMD pathway responds to microbial
signals such as peptidoglycan, by phosphorylating and cleaving the transcription factor
Relish (Rel). The N-terminus of Rel then translocates to the nucleus where it activates
transcription of antimicrobial peptides (AMPs) such as Diptericin A (DptA), Attacin A
(AttA), and Cecropin A1 (CecA1) (31). Enteroendocrine cells of the AMG that express
the enteroendocrine peptide Tachykinin (Tk) also activate IMD signaling in response to
small molecules such as acetate and tryptophan (19, 28, 29). In these cells, IMD signaling
activates transcription of Tk, which promotes intestinal lipid utilization in addition to AMP
expression (29, 32). In the absence of an invasive intestinal infection, the response of
the intestine to microbes is principally under the control of enteroendocrine cells and Tk
(27).

Drosophila ingestion of V. cholerae results in the formation of a biofilm in the PMG
without further invasion and activates IMD signaling in enteroendocrine cells leading to
an increase in intestinal Tk and AMP expression (17, 19, 28). Here we report that, despite
the susceptibility of V. cholerae to AMPs, innate immune signaling and Tk expression in
enteroendocrine cells increases V. cholerae colonization of the arthropod intestine. To
understand how Tk regulates V. cholerae colonization, we compared gene expression in
the intestines of control and Tk knockdown flies using RNA-seq analysis. In addition to
the known impacts on genes encoding AMPs and lipid catabolism enzymes, we found
that Tk activates transcription of genes encoding chitin modification and chitin-binding
proteins. We demonstrate that expression of a small, abundant chitin-binding protein,
Peritrophin-15a (Peri-15a), which consists of a secretion peptide and one type 2 chitin-
binding domain, is induced by V. cholerae infection and increases V. cholerae adhesion
to the arthropod intestine. Underscoring the environmental relevance of our findings, the
literature suggests that association of Vibrio species with zooplankton activates both the
innate immune response and transcription of chitin-binding proteins (33). Furthermore,
AlphaFold predictions show that close structural homologs of Peri-15a are present in
many organisms that associate with V. cholera e in the environment including copepods,
rotifers, cyanobacteria, non-biting midges, and house flies (34). Based on these findings,
we propose that V. cholerae activation of the host enteroendocrine innate immune
response may augment colonization of the intestinal surface of environmental arthropod
hosts.

Results
The enteroendocrine innate immune response enhances V. cholerae colonization
of the arthropod intestine. We previously showed that V. cholerae infection activates
intestinal IMD signaling in enteroendocrine cells leading to increased AMP transcription
in the arthropod intestine (19, 29). We hypothesized that this response to V. cholerae
infection would limit intestinal colonization. To test this, we used a Gal4/UAS binary
system to generate flies carrying the yeast transcriptional activator Gal4 fused to a
sequence that drives gene expression to Tk-expressing enteroendocrine cells (Tk>) and
RNAi’s targeting various genes fused to the upstream activating sequence to which Gal4
binds (GeneRNAi) (35). As a control for background, we used Tk> flies crossed to the
parental stock into which the RNAi constructs were inserted (36). We first fed V. cholerae
to Tk> and Tk>RelRNAi flies for 48 hours and then washed out non-adherent bacteria by
transferring the flies to phosphate-buffered saline (PBS) for 24 hours. Unexpectedly,
RelRNAi in Tk-expressing cells decreased V. cholerae colonization, suggesting that IMD
signaling in Tk-expressing enteroendocrine cells promotes intestinal colonization (Fig
1C). IMD signaling in enteroendocrine cells is essential for Tk expression in the AMG
(29). To test the role of Tk in this process, we measured V. cholerae colonization in
Tk>TkRNAi flies and again observed a decrease in colonization (Fig 1D). Tk communicates
with enterocytes via the TkR99D receptor (28, 32). To test whether colonization was
brought about by an enterocyte-specific product, we used the enterocyte driver Myo31DF-
Gal4 (NP1>) and TkR99D-specific RNAi to knock down TkR99D in enterocytes and
compared colonization of NP1> and NP1>TkR99DRNAi flies. TkR99DRNAi in enterocytes
increased V. cholerae colonization, as did RelRNAi (Figs. 1E and F). Since enterocytes
are an important source of AMPs, we reasoned that decreased IMD signaling in these
cells and, therefore, AMP production might explain the observed increase in V. cholerae
colonization of NP1>TkR99DRNAi and NP1>RelRNAi flies. We compared V. cholerae
colonization of flies with 10 AMPs deleted (∆AMP10, DefSK3, AttCMi, DroAttABSK2, MtkR1,
DptABSK1; DrsR1, AttDSK1) with the parental control (Fig 1G) (37). As predicted, the
∆AMP10 flies displayed increased colonization. We conclude that AMPs diminish V.
cholerae intestinal colonization, that a Tk-regulated product of enteroendocrine cells
promotes V. cholerae intestinal colonization, and that V. cholerae intestinal colonization
of the arthropod intestine is a composite of these two opposing processes.

The PM protein Peritrophin-15a is activated by Tk and the enteroendocrine innate
immune response. To identify host colonization factors regulated by Tk, we performed
RNA-seq on the intestines of Tk>TkRNAi and Tk> driver-only flies (Fig 2A and
Supplementary Data 1). Using a pa dj ≤0.05 and 2-fold change as thresholds, we
identified 136 differentially regulated genes of which transcription of 69 were increased
and transcription of 67 were decreased. Tk is known to regulate both innate immune
signaling and lipid catabolism (29, 32). RNA-seq analysis showed 20 differentially
regulated genes involved in innate immunity (dark green circles, Fig 2A and
Supplementary Data 1) and 9 involved in lipid catabolism (brown circles, Fig 2A and
Supplementary Data 1) (38). To independently confirm a subset of these, we measured
transcription of the genes encoding the AMP Defensin (Def) and the lipases CG17192
and Lipase 3 (Lip3) by qRT-PCR, which corroborated the RNA-seq results (Fig 2B).
Opposite regulation of the two lipase genes CG17192 and Lip3 was unexpected, so we
compared lipid accumulation in the intestines of Tk> and Tk>TkRNAi flies. As previously
reported, lipid accumulation was observed in the intestines of Tk>TkRNAi flies (Figs. 2C
and D) (32). Importantly, this was only observed in the AMG but not in the PMG. Since
Tk is present in both the AMG and PMG, we conclude that control of lipid accumulation
is specific to the AMG Tk (29, 32).

In addition to genes encoding metabolic, regulatory, and unknown functions, we noted
four genes (CG10725, Peritrophin-15a, Peritrophin-15b, and Idgf3) annotated as
encoding chitin-binding proteins, three of which were significantly activated by Tk and one
of which was repressed (Supplementary Data 1). Chitin-binding proteins are structural
components of the PM, the protective layer of the intestine closest to the lumen where V.
cholerae is located during infection (39). Previous publications suggested that disruption
of the PM increased the susceptibility of Drosophila to intestinal bacteria and yet we
observed a decreased burden of V. cholerae with TkRNAi (40). Nevertheless, we
hypothesized, that a chitin-binding protein might enhance pathogen colonization if it
promoted adhesion to the PM surface. A previous study in the tsetse fly showed that the
peritrophins Pro 1 and 2, which are homologs of the Drosophila Peritrophins-15a and b
(Peri-15a and b), have little impact on the structure of the PM and do not affect
colonization of the intestine by the Gram-negative rods Enterobacter and Serratia (41).
Because the transcript abundance of Peri-15a and Peri-15b was significantly decreased
in the Tk>TkRNAi RNA-seq analysis as compared with the Tk> control, we reasoned they
might be the Tk-regulated V. cholerae adhesion factor we sought. Peri-15a and 15b are
homologous proteins consisting of 92 aa and 93 aa, respectively. They are encoded in
neighboring regions of the second chromosome and, therefore, may be the product of a
gene duplication. An alignment using Clustal Omega showed that they are 45% identical
and 58% similar (Fig 3A) (42). AlphaFold predicts very similar structures for the two
proteins, which consist of an N-terminal signal peptide and one type 2 chitin binding
domain (Fig 3B) (34, 43). The Peri-15a transcript is most highly expressed in
enteroendocrine cells of the AMG, and is approximately 40X and 750X more highly
expressed, respectively, than Peri-15b and Peritrophin A, the other two named
peritrophins in the Drosophila genome (Fig 3C) (30). Therefore, we focused our studies
on Peri-15a. We first confirmed our RNA-seq results with an independent qRT-PCR
experiment comparing Peri-15a transcription in the intestines of Tk>TkRNAi flies and Tk>
flies. As expected, knockdown of Tk resulted in decreased transcript abundance of Peri-
15a (Fig 3D). Tk>RelRNAi also decreased Peri-15a transcript abundance, demonstrating
that Peri-15a transcription is activated by IMD signaling (Fig 3E).

Peri-15a expression is increased by V. cholerae infection and promotes
colonization. We next measured transcription of Tk and Peri-15a in the setting of V.
cholerae infection. This significantly increased transcription of both Tk and Peri-15a (Fig
4A). We then assessed the impact of Peri-15a on V. cholerae colonization. Considering
that other enteroendocrine cells might produce Peri-15a in addition to Tk-expressing
ones, we knocked down Peri-15a transcription using the pan-enteroendocrine cell driver
prospero (pros>) and Peri-15aRNAi. Peri-15aRNAi reduced V. cholerae colonization of the
Drosophila intestine by approximately 100-fold (Fig 4C) and decreased intestinal Peri-
15a transcription 2-fold but did not alter transcription of Tk or the gene encoding the AMP
Def (Fig 4D). To further rule out an effect of Peri-15a on Tk expression, we performed Tk
and lipid staining on the intestines of pros> and pros>Peri-15aRNAi flies. pros>Peri-15aRNAi
had no impact on intestinal lipid accumulation (Fig 4E and F and Fig S1). A minimal
decrease in numbers of Tk+ cells was observed in the AMG only. This suggests Tk is
upstream of Peri-15a and that decreased colonization of the pros>Peri-15aRNAi intestine
is not the result of increased AMP transcription.

Dietary ecdysone increases V. cholerae colonization of the arthropod gut. We
previously showed that the fly steroid hormone 20-hydroxyecdysone (20E) activates the
intestinal innate immune response and Tk expression (28). We hypothesized that
intestinal Peri-15a expression would, therefore, be activated by dietary supplementation
with 20E. To test this, we compared Tk and Peri-15a transcription in the intestines of flies
fed LB alone or supplemented with 20E. As shown in Figure 5A, administration of 20E to
Drosophila increased expression of Tk and Peri-15a. Furthermore, 20E administration
increased intestinal colonization by V. cholerae (Fig 5B). To demonstrate that this
increase in colonization was correlated with Peri-15a expression, we assessed the impact
of 20E on V. cholerae colonization in pros> and pros>Peri-15aRNAi flies. Supplementation
with 20E increased colonization of pros> flies but did not significantly increase V. cholerae
colonization in pros>Peri-15a flies (Fig 5C). This demonstrates that the increased
colonization observed with 20E supplementation is dependent on Peri-15a and further
supports our contention that Peri-15a directly promotes host colonization.

Decreasing the porosity of the PM chitin layer does not increase V. cholerae
colonization. Peri-15a has a single chitin-binding domain and is, therefore, hypothesized
to be a capping protein that limits the length of chitin strands in the PM (44). We reasoned
that Peri-15aRNAi might alter the porosity of the PM. To independently test the impact of
increasing the porosity of the PM surface on V. cholerae colonization, we examined
intestinal chitinases. In the cotton bollworm, silencing of the Cht4 homolog results in a
less porous PM with a higher chitin content (45). Cht4 and Cht9, which are the most
highly expressed chitinases in the midgut, are principally expressed in enteroblasts and
enterocytes of the Drosophila midgut (46) (Fig 6A). To determine how they impact V.
cholerae colonization, we compared V. cholerae colonization of NP1>Cht4RNAi and
NP1>Cht9RNAi flies with NP1> flies. Cht9RNAi had no effect on V. cholerae colonization,
while Cht4RNAi unexpectedly decreased colonization (Fig 6B). It was surprising that
decreased PM porosity and increased chitin content would inhibit V. cholerae colonization
directly, since chitin has been shown to promote V. cholerae surface adhesion (12, 47,
48). Studies on Tsetse flies have suggested that decreased PM porosity diminishes
access of microbial products to the intestinal epithelium and, thus, reduces IMD signaling
(41). Based on this precedent, we hypothesized that decreased PM porosity in
NP1>Cht4RNAi flies might decrease IMD signaling and, consequently, Tk and Peri-15A
expression. To test this, we quantified transcription of Cht4, Tk, Def, and Peri-15a in the
intestines of NP1>Cht4RNAi and NP1> flies (Fig 6C) and found that all four transcripts were
decreased by Cht4RNAi. These findings are consistent with a model in which Cht4RNAi
reduces access of microbial products to the intestinal epithelium leading to decreased
IMD signaling and Peri-15a transcription and does not support a model in which continuity
of the PM promotes V. cholerae colonization.

Peri-15a is not essential to maintain PM integrity. We questioned whether Peri-
15aRNAi might alter the permeability of the PM. Penetration of ingested FITC-labelled
dextran into intestinal cells has previously been used to demonstrate the essential role of
the chitin-binding protein Crystallin (Crys) in PM integrity (40). We, therefore, used 40kDa
and 500kDa FITC-labelled dextran to assess the permeability of the PMs of pros> and
pros>Peri-15aRNAi flies, including the CrysMB08319 mutant as a positive control, and
NP1>Cht4RNAi flies as a negative control (Fig 7). As proof of principle, no leakage of either
the 40 kDa- and 500 kDa FITC-labelled dextran was observable for NP1> and
NP1>Cht4RNAi flies, while clear leakage of both the 40 kDa- and 500 kDa FITC-labelled
dextran was observed with the CrysMB08319 mutant. As compared with the positive control,
there was no leakage in either pros> or pros>Peri-15aRNAi fly intestines with either the 40
kDa- and 500 kDa FITC-labelled dextran. We conclude that, unlike Crys, Peri-15a is not
essential for the integrity of the PM.

Interestingly, we noted a difference in the distribution of the 40 kDa FITC-labelled dextran
in the PMG of several of the pros>Peri-15aRNAi intestines consistent with a filling defect
(Fig 7 and S2). This difference was less apparent with the 500 kDa FITC-labelled dextran.
We hypothesize that Peri-15a may increase the rigidity or decrease the elasticity of the
PM. However, we cannot rule out a differential response to the fixation step in our sample
preparation.

The global chitin-responsive transcription factor ChiS is active in the arthropod
intestine and reduces V. cholerae colonization. V. cholerae can use chitin as a sole
carbon and nitrogen source because its genome encodes multiple chitinases as well as
transporters and enzymes that enable catabolism of chitin degradation products (12, 49-
51). The latter V. cholerae chitin catabolic cascade is under the control of the non-
canonical transcription factor ChiS (12, 50, 52). We previously showed that V. cholerae
is able to digest the Drosophila PM, suggesting that ChiS is active in the Drosophila
intestine (53). To demonstrate this, we measured expression of chiA1 and chiA2, two
chitinase-encoding genes in the ChiS regulon, in the intestines of flies infected either with
WT V. cholerae or a ∆chiS mutant. As shown in Fig 8A, deletion of chiS reduced
expression of both genes, suggesting that ChiS is active in the arthropod intestine.
Chitin and, therefore, ChiS is thought to be critical for growth of V. cholerae in the
presence of arthropods (5, 9, 49, 54). Our results show that the host chitin-binding protein
Peri-15a promotes V. cholerae colonization of the arthropod gut. We, therefore,
questioned how V. cholerae degradation of chitin in the arthropod gut might modulate
colonization and assessed colonization by WT V. cholerae and a V. cholerae ∆chiS
mutant. Mutation of chiS had a small but reproducible positive effect on colonization in a
control fly (Fig 8B). This suggests that metabolism of chitin is not essential for V. cholerae
colonization of the arthropod intestine. We reasoned, instead, that chitin degradation
might damage the PM leading to increased IMD signaling and AMP expression. To test
this directly, we compared colonization by WT V. cholerae and a ∆chiS mutant in the
∆AMP fly. The increased colonization of the ∆chiS mutant was preserved in the ∆AMP
fly background, suggesting that this difference does not rely on a difference in AMP
expression. We then questioned whether improved colonization by the ∆chiS mutant was
dependent on Tk and Peri-15a. As shown in Figs 7C and D, in Tk>TkRNAi and pros>Peri-
15aRNAi flies, the ∆chiS mutant no longer had a colonization advantage over WT V.
cholerae. One possibility is that V. cholerae chitin catabolism removes Peri-15a from the
PM via degradation of chitin attachment sites, thus decreasing colonization of the
arthropod intestine.

Peri-15a is broadly conserved in insects and marine zooplankton. V. cholerae has
been associated with zooplankton in aquatic environments, and there is evidence that
V. cholerae colonizes these organisms using specific adhesins (5, 55-60). Furthermore,
transcription of copepod genes encoding innate immune effectors and chitin binding
proteins, including a homolog of Peri-15a, are increased in response to Vibrio
colonization (33). To understand the relevance of our findings to the natural
environment of V. cholerae, we searched for structural homologs of Peri-15a in the
AlphaFold protein structure database. Highly similar Peri-15a homologs were present in
diverse insects including mosquitos, beetles, butterflies, and sand flies (Fig 8).
Structural homologs of Peri-15a were also identified in the genomes of marine non-
biting midges, rotifers, marine copepods, and fungi, all of which are found in the marine
aquatic environments inhabited by V. cholerae. Taken together, these results suggest
that Peri-15a homologs are present in natural environmental hosts of V. cholerae and
that intestinal colonization with V. cholerae increases expression of these proteins.

Methods
Drosophila husbandry and strains. All fly stocks used in this study were raised on
standard fly food containing 16.5 g/L yeast, 9.5 g/L soy flour, 71 g/L cornmeal, 5.5 g/L
agar, 5.5 g/L malt, 7.5% corn syrup, and 0.4% propionic acid in a humidified 12 hr day-
night cycle incubator at 25°C. Females flies (4-7 day old) were used in all experiments.
The following fly stocks were obtained from the Bloomington Drosophila Stock Center
(BDSC): TkRNAi (BL2500), RelRNAi (BL28943), TkR99DRNAi (BL27513), Cht4RNAi
(BL65001), Peritrophin-15aRNAi (BL55987), and pros-Gal4/Sb (BL84276). Control lines
TRiP (BL36303), y1w1 (BL1495) and Crys MB08319 (BL26106) were also obtained from the
BDSC. The Tk-Gal4 and NP1-Gal4 driver lines were a kind gift from the Veenstra and
Perrimon laboratories (32, 76). The AMP-deficient flies and the corresponding parental
strain were generously provided by the Lemaitre laboratory (37).

Bacterial strains and media. The wild-type V. cholerae strain and the parental
background for all mutants was quorum sensing-competent V. cholerae 01 El Tor strain
C6706 (C6706 str 2), a kind gift of the late Ronald Taylor. The ∆chiS mutant was
constructed by double homologous recombination as previously described using the
primers listed in Supplementary Data 2 (18). V. cholerae was cultured at 27°C in LB
broth (Miller formulation, Difco) or on LB agar (Difco) supplemented with 100 µg/mL
streptomycin. Where noted, 20-hydroxyecdysone was added to LB broth for Drosophila
colonization studies (10 µM, Sigma).

V. cholerae mutant construction. An in-frame V. cholerae ΔchiS mutant was constructed
by double homologous recombination as previously described (77). ChiS is comprised
of 1177 amino acids. In this mutant, residues 172 to 1053 were deleted including all
conserved domains.

V. cholerae colonization assays. For colonization assays, V. cholerae was cultured
overnight in LB broth and then diluted 10-fold in fresh LB broth. 3 mL of this suspension
was infused into autoclaved cellulose plugs inserted at the bottom of fly vials. 4–7-day
old flies were placed in these prepared vials for 48 hours at 25 oC and then transferred to
fresh vials containing cellulose plugs infused with 3 mL sterile solution of phosphate-
buffered saline for 24-hours to washout non-adherent bacteria. Individual flies treated in
this manner were then homogenized in 200 µL of 1x PBS using a TissueLyser III system
(Qiagen). Serial 10-fold dilutions were prepared, and 20 μl of these dilutions were plated
on LB agar plates supplemented with 100 µg/mL streptomycin. The plates were incubated
at 27°C for 24 hours and colony-forming units (CFU) in each dilution were enumerated.
This was used to calculate V. cholerae CFU/fly.

Drosophila RNA extraction, RNA-seq, and RT-qPCR. Total RNA was isolated from 10-15
fly intestines using TRIzol reagent (Thermo Fisher Scientific 15596026) and the Direct-
zol RNA Miniprep plus kit (Zymo Research NR2070). Three to six biological replicates per
condition/genotype were performed. For RNA sequencing analysis, the RNA was
submitted to the Molecular Biology Core Facilities (MBCF) at the Dana Farber Cancer
Institute (DFCI) for next-generation sequencing (NGS) library preparation, sequencing,
and analysis (https://mbcf.dana-farber.org/totalrnaseq.html). Roche Kapa mRNA
HyperPrep strand specific sample preparation kits were used to prepare libraries on a
Beckman Coulter Biomek i7 from 200ng of purified total RNA. The resulting dsDNA
libraries were quantified by Qubit fluorometer and Agilent TapeStation 4200. Uniquely
dual indexed libraries were pooled in an equimolar ratio and shallowly sequenced on an
Illumina MiSeq to further evaluate library quality and pool balance. The final pool was
sequenced on an Illumina NovaSeq X Plus targeting 40 million 150bp read pairs per
library. Sequenced reads were aligned to the UCSC hg38 reference genome assembly
and gene counts were quantified using STAR (v2.7.3a) and Salmon (78, 79). Differential
gene expression testing was performed by DESeq2 (v1.22.1) (80). RNAseq analysis was
performed using the VIPER snakemake pipeline (81).

For qRT-PCR, the iScript™ cDNA synthesis kit (Bio-Rad 1708891) was used to
synthesize cDNA from 500ng of total RNA. The iTaq™ Universal SYBR® Green Supermix
(Bio-rad 1725121) was used to perform qPCR of target gene transcripts on a
QuantStudio™ Real-Time PCR system 3 (Thermo Fisher Scientific). Quantification cycle
values (Cq) were obtained and used to calculate target gene transcription normalized to
Actin (for D. melanogaster genes) or to rpoA (for V. cholerae genes). Primers used in this
study are listed in Supplementary Data 2.

FITC-labelled dextran feeding. 40 kDa- (Sigma-Aldrich FD40) and 500 kDa- (Sigma-
Aldrich FD500S) labelled dextran were resuspended in a 2.5% sucrose solution and
passed over PD MiniTrap™ G-10 columns (Cytiva) prior to feeding. Adult female flies were
starved for 2 hours and then placed in tubes containing a thin cellulose plug with 200 µL
of the dextran suspension added. The flies were given access to this suspension for 24
hours, after which their guts were dissected and prepared as stated below.

Lipid staining and immunofluorescence of Drosophila intestine. Fly intestines were
dissected in 1x PBS and fixed in a 4% paraformaldehyde (PFA), 0.1% PBS-Tween 20
(PBT) solution for 20 minutes. For lipid staining only, the intestines were then washed
three times for 10 minutes with PBT, followed by incubation in staining solution #1 (PBT
+ 1:1000 DAPI (Invitrogen D1306) + 1:500 BODIPY 493/503 (Invitrogen D3922)) for 2
hours. For lipid staining and Tk-immunofluorescence experiments, intestines were
washed three times for 10 minutes in PBT and then left in blocking solution (PBT + 0.1%
Triton X-100 (Sigma-Aldrich 9002-93-1) + 2% BSA (Sigma-Aldrich 9048-46-8)) for 1 hour.
After this blocking step, intestines were left in Rabbit anti-Tk primary antibody solution
(blocking solution + 1:500 anti-Tk) overnight at 4°C. The next day the guts were washed
three times with PBT for 10 minutes, followed by incubation in staining solution #2
(blocking solution + 1:1000 DAPI (Invitrogen D1306) + 1:500 BODIPY 493/503 (Invitrogen
D3922) + 1:500 Alexa 594-conjugated Goat anti-rabbit secondary antibody (Thermo
Fisher Scientific A11012)) for 2 hours. After incubation in the appropriate staining
solution, intestines were washed three times for 10-minutes in PBT and then mounted in
Vectashield® antifade mounting medium (Vector Laboratories H-1000-10). Intestines
were imaged using a Zeiss LSM 980 confocal microscope using a 40X (Fig 2) or 25X (Fig
4) oil objective. Unless otherwise noted, all steps were done at room temperature. Lipid
accumulation was quantified using ImageJ (FIJI) to measure total BODIPY fluorescence.
The corrected total cell fluorescence (CTCF) was calculated by dividing total BODIPY
fluorescence by the total area and subtracting background fluorescence from this
measurement. Tk-expressing cells were quantified manually. A minimum of six fly
intestines per genotype were evaluated for quantification of Tk-expressing cells and
BODIPY fluorescence.

Statistical analysis and reproducibility. All data were graphed and analyzed using Prism
10.0 software (GraphPad). Measurements shown in each graph represent the mean
values of at least three biological replicates for qRT-PCR, at least six flies for colonization
experiments, and at least six intestines for microscopy. The mean is shown for all
experiments, and error bars represent the standard deviation. Where noted, data were
log-transformed to obtain a normal distribution. Appropriate statistical tests were chosen
based on numbers of conditions assessed and tests of normality and variance. The
statistical test used in each graph is specified in the figure legend. With the exception of
the RNA-seq, all experiments were repeated one or more times with similar results.

Data availability statement. Source data are provided with this paper. The RNA-seq
data generated in this study have been deposited in the NCBI repository (accession no.
GSE294931). Remaining raw data generated are available at
doi:10.5281/zenodo.18624207

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In prior work using the Drosophila melanogaster model, it was shown that ingestion of Vibrio cholerae led to biofilm formation in the fly gut and induced the immunodeficiency (Imd) pathway, which increased tachykinin and antimicrobial peptide expression. The present manuscript examines this infection model further exploring the role of peritrophin-15a in V. cholerae colonization.\n\nThe importance of Peri-15a in the reported Vibrio colonization effect is clear and convincing. Multiple approaches were used to demonstrate that Vibrio colonization is improved by increases in Peri-15a, or that reduction of Peri-15a hinders Vibrio colonization. The relevance of Peri-15a in understanding common principles of Vibrio colonization in nature is compelling, as similar proteins were identified in other animals present in natural environments of V. cholerae. This work sets up future investigation into Peri-15a and its homologs as colonization factors in other animal hosts. We note below some comments/feedback on the manuscript.\n\nMajor comments:\nLines 233-235: “To determine the impact of chitin degradation on V. cholerae colonization of the arthropod gut, we assessed colonization by a V. cholerae ΔchiS mutant, which is unable to degrade chitin and utilize its metabolic products.” The construction of this mutant is well-described in the methods section, but there is no evidence that this newly generated mutant is unable to degrade or utilize chitin and its metabolic products. Growth curves of the parental and mutant strains on chitin as the sole carbon source would provide the necessary evidence that the mutation has the intended effect on chitin metabolism.\n\nIt is still unclear why inhibiting microbial chitin degradation (∆chiS) improves colonization while inhibiting host chitinase activity (Cht4-RNAi) decreases colonization. It is suggested that Cht4 increases porosity of the PM and allows for improved Tk activation, while chiS dependent chitinases thin the PM and cause detachment of Peri-15a. Evidence for the effect of Cht4 is provided through measurement of expression of Tk and Peri-15a with and without Cht4-RNAi, but would be strengthened by use of experimental evidence of increased porosity (such as FITC-dextran feeding). However, there is no evidence for the suggested effect of microbial chitinases on Peri-15a attachment. Although the difference in effect of Cht4-RNAi and ∆chiS is addressed extensively in the discussion section, there is no suggested explanation as to why chitinases from arthropods and microbes would have vastly different effects on PM structure, where arthropod chitinases affect porosity while microbial chitinases affect thickness. Further discussion of potential specificity of different chitinases for different components of the PM, or differences in general enzyme function, could support the proposed hypothesis. The authors may also want to consider situations when a microbe would want vs not want to attach to the peritrophic matrix - especially as it is a dynamic structure that is continually synthesized and degraded, built in the proventriculus and moves along with food to be expelled/broken down in the hind gut.\n\nMinor comments:\nFig. 2A: The positions of Peri-15a and b are unclear from the text labels on the volcano plot.\nLines 194-195: “Evidence from other insects suggests that Peri-15a and b do not decrease V. cholerae colonization by disrupting the integrity of the PM.” Could use citations and/or more detail; unclear what this means.", "reviewer_2": "In this manuscript the authors investigate the role of tachykinin in promoting V. cholerae colonization in an insect model. Tk was found to activate expression of chitin-binding proteins and chitinases. Chitin-binding proteins were found to be important for colonization. A variety of methods were used, including RNA-Seq, RNAi knockdowns of various genes, CFU quantification, and fluorescence microscopy, which is a strength. Overall the findings are potentially interesting. However, some of the conclusions are not strongly supported by the date, especially in reference to the chiS mutant. Additionally, the text is very heavy in jargon and abbreviations and is extremely hard to follow. If one is not familiar with Drosophila notation and jargon it becomes nearly impossible to follow. Significant rewriting is necessary to make it palatable to a general audience.\n\nSpecific points:\n1) There is an overly high number of abbreviations used in the text that makes it extremely difficult to follow. Not every longish word needs to be abbreviated. For example, both EC and EEC are used as abbreviations- you can just say enterocytes instead of EC. The reader should not need to keep referring back in the text to remember what all the abbreviations stand for. Similarly, it is not clear why tachykinin needs to be abbreviated when it is not referring to a gene or construct. Others are similar. If the abbreviation shortens a long word or phrase, then it is useful but if the sentence consists largely of a series of abbreviations it becomes unreadable.\n2) Line 123: what is Rel- RNAi? This abbreviation is not described earlier that I can see. Relish?\n3) Line 130: what is NP1?\n4) Fig 3: “Rel” is used to refer to both “relative” and whatever else “Rel” stands for. This is very confusing.\n5) Lines 235-236: while the difference between WT and the chiS mutant was apparently statistically significant, in fact the data almost entirely overlap and it is improbable that this is biologically significant.\n6) Lines 331-332: while these are two described adhesins it is possible that others exist."}, "author_response": "Response to reviewers:\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn prior work using the Drosophila melanogaster model, it was shown that\ningestion of Vibrio cholerae led to biofilm formation in the fly gut and induced the\nimmunodeficiency (Imd) pathway, which increased tachykinin and antimicrobial\npeptide expression. The present manuscript examines this infection model\nfurther exploring the role of peritrophin-15a in V. cholerae colonization.\nThe importance of Peri-15a in the reported Vibrio colonization effect is clear and\nconvincing. Multiple approaches were used to demonstrate that Vibrio\ncolonization is improved by increases in Peri-15a, or that reduction of Peri-15a\nhinders Vibrio colonization. The relevance of Peri-15a in understanding common\nprinciples of Vibrio colonization in nature is compelling, as similar proteins were\nidentified in other animals present in natural environments of V. cholerae. This\nwork sets up future investigation into Peri-15a and its homologs as colonization\nfactors in other animal hosts. We note below some comments/feedback on the\nmanuscript.\nMajor comments:\n1) Lines 233-235: “To determine the impact of chitin degradation on V. cholerae\ncolonization of the arthropod gut, we assessed colonization by a V. cholerae\nΔchiS mutant, which is unable to degrade chitin and utilize its metabolic\nproducts.” The construction of this mutant is well-described in the methods\nsection, but there is no evidence that this newly generated mutant is unable to\ndegrade or utilize chitin and its metabolic products. Growth curves of the parental\nand mutant strains on chitin as the sole carbon source would provide the\nnecessary evidence that the mutation has the intended effect on chitin\nmetabolism.\nChiS is a transcription factor whose regulon is well defined in the literature as is its\nability to facilitate growth on chitin1, 2, 3. Rather than repeat these published\ncharacterizations of the action of ChiS, we have added experiments in Fig 8A showing\nthat ChiS activates V. cholerae chitinase expression within the fly intestine. This proves\nthat V. cholerae encounters sufficient chitin within the arthropod intestine to activate\nChiS and its regulon, which includes ChiA1 and ChiA2, is highly relevant to the current\nexperiments, and supports our colonization data.\n2) It is still unclear why inhibiting microbial chitin degradation (∆chiS) improves\ncolonization while inhibiting host chitinase activity (Cht4-RNAi) decreases\ncolonization. It is suggested that Cht4 increases porosity of the PM and allows for\nimproved Tk activation, while chiS dependent chitinases thin the PM and cause\ndetachment of Peri-15a. Evidence for the effect of Cht4 is provided through\nmeasurement of expression of Tk and Peri-15a with and without Cht4-RNAi, but\nwould be strengthened by use of experimental evidence of increased porosity\n(such as FITC-dextran feeding).\nWe very much appreciated the suggestion of this assay by the reviewer and used it to\nour advantage to test the impact of Peri-15a knockdown on PM permeability. These\nresults are now shown in Fig 7. Because we observed no penetration of the peritrophic\nmatrix by either 40 kDa or 500 kDa FITC-dextran in control flies, we did not expect\nCht4RNAi, which we hypothesized would decrease PM porosity further, to show\ndifferential permeability of 40 kDa or 500 kDa FITC-dextran. Instead, we used Cht4RNAi\nas a negative control for our Peri-15A experiments. The Lemaitre lab previously\nshowed that the chitin-binding protein Crystallin is essential for the integrity of the PM4.\nWe used a Crys mutant as a positive control. These experiments show that Peri-15a\nknockdown does not increase the porosity of the PM, but it does seem to alter the\npattern of FITC-dextran accumulation in the PMG suggesting a difference in rigidity or\nelasticity of the PM.\nHowever, there is no evidence for the suggested effect of microbial chitinases on\nPeri-15a attachment.\nWe have not yet gathered experimental evidence for this model. Therefore, it is\npresented as a hypothesis.\nAlthough the difference in effect of Cht4-RNAi and ∆chiS is addressed extensively\nin the discussion section, there is no suggested explanation as to why chitinases\nfrom arthropods and microbes would have vastly different effects on PM\nstructure, where arthropod chitinases affect porosity while microbial chitinases\naffect thickness. Further discussion of potential specificity of different chitinases for\ndifferent components of the PM, or differences in general enzyme function,\ncould support the proposed hypothesis. The authors may also want to consider\nsituations when a microbe would want vs not want to attach to the peritrophic\nmatrix - especially as it is a dynamic structure that is continually synthesized and\ndegraded, built in the proventriculus and moves along with food to be\nexpelled/broken down in the hind gut.\nThe reviewer raises fascinating questions and suggests valid experiments. At present,\nwe do not feel comfortable proposing an explanation for the observed differences\nbetween the actions of the host and pathogen chitinases as there is not enough known\nabout the intestinal distributions and targets of these chitinases. However, we\nappreciate the reviewer’s suggestion as an intriguing direction for future investigation.\nBecause the transcript abundance of gene encoding peritrophins and mucins is highest\nin the cardia/R1, it is logical to assume that the protein products of these genes are\nincorporated into the peritrophic matrix in the cardia and then move distally along the\ngut to be expelled/broken down in the hind gut. However, I am not aware of direct\nexperimental evidence for this in the literature. This is an avenue of future investigation.\nThe question of whether bacterial chitinases promote detachment from the PM under\nparticular conditions is also fascinating. There may be aspects of ChiS regulation that\nhave not been elucidated or additional regulators of the bacterial chitinases that could\nplay a role in detachment from the arthropod gut and environmental dispersal. This is\nan important area to explore. We have added this possibility to the discussion in line\n337 and thank the reviewer for the suggestion.\nMinor comments:\nFig. 2A: The positions of Peri-15a and b are unclear from the text labels on the\nvolcano plot.\nWe have now made the circles representing Peri-15a and b larger and a bright green\ncolor. We believe this increases their visibility\nLines 194-195: “Evidence from other insects suggests that Peri-15a and b do not\ndecrease V. cholerae colonization by disrupting the integrity of the PM.” Could\nuse citations and/or more detail; unclear what this means.\nWe have now added our own experimental evidence in Fig 7 that knockdown of Peri-\n15a does not affect PM permeability.\nReviewer #2 (Remarks to the Author):\nIn this manuscript the authors investigate the role of tachykinin in promoting V.\ncholerae colonization in an insect model. Tk was found to activate expression of\nchitin-binding proteins and chitinases. Chitin-binding proteins were found to be\nimportant for colonization. A variety of methods were used, including RNA-Seq,\nRNAi knockdowns of various genes, CFU quantification, and fluorescence\nmicroscopy, which is a strength. Overall the findings are potentially interesting.\nHowever, some of the conclusions are not strongly supported by the date,\nespecially in reference to the chiS mutant. Additionally, the text is very heavy in\njargon and abbreviations and is extremely hard to follow. If one is not familiar\nwith Drosophila notation and jargon it becomes nearly impossible to follow.\nSignificant rewriting is necessary to make it palatable to a general audience.\nWe have rewritten the manuscript for clarity. In particular, we have explained the\ntechnique used to knockdown gene expression in Drosophila and we have eliminated\nmany of the abbreviations.\nSpecific points:\n1) There is an overly high number of abbreviations used in the text that makes it\nextremely difficult to follow. Not every longish word needs to be abbreviated. For\nexample, both EC and EEC are used as abbreviations- you can just say\nenterocytes instead of EC. The reader should not need to keep referring back in\nthe text to remember what all the abbreviations stand for. Similarly, it is not clear\nwhy tachykinin needs to be abbreviated when it is not referring to a gene or\nconstruct. Others are similar. If the abbreviation shortens a long word or phrase,\nthen it is useful but if the sentence consists largely of a series of abbreviations it\nbecomes unreadable.\nWe have eliminated EC and EEC abbreviations. Tk is an established abbreviation for\nTachykinin, so we have retained this one.\n2) Line 123: what is Rel- RNAi? This abbreviation is not described earlier that I\ncan see. Relish?\nWe have now defined this abbreviation in the main text.\n3) Line 130: what is NP1?\nNp1 refers to the Myo31DF-Gal4 driver, which is commonly used driver for gut\nenterocyte expression. We now described this in line 128-131.\n4) Fig 3: “Rel” is used to refer to both “relative” and whatever else “Rel” stands\nfor. This is very confusing.\nThank you for the comment, we now defined this abbreviation in line 87.\n5) Lines 235-236: while the difference between WT and the chiS mutant was\napparently statistically significant, in fact the data almost entirely overlap and it is\nimprobable that this is biologically significant.\nThis is an important point. The differences are small but significant and reproducible.\nThis is acknowledged in lines 257-8. However, even this small difference is remarkable\nas the opposite phenotype was predicted based on the previous literature.\n6) Lines 331-332: while these are two described adhesins it is possible that others\nexist.\nWe have now added the possibility that it could also be a novel protein in line 349.\n1. Meibom, K.L. et al. The Vibrio cholerae chitin utilization program. Proc Natl Acad\nSci U S A 101, 2524–2529 (2004).\n2. Chourashi, R. et al. Chitin-induced T6SS in Vibrio cholerae is dependent on ChiS\nactivation. Microbiology (Reading) 164, 751–763 (2018).\n3. Li, X. & Roseman, S. The chitinolytic cascade in Vibrios is regulated by chitin\noligosaccharides and a two-component chitin catabolic sensor/kinase. Proc Natl\nAcad Sci U S A 101, 627–631 (2004).\n4. Kuraishi, T., Binggeli, O., Opota, O., Buchon, N. & Lemaitre, B. Genetic evidence\nfor a protective role of the peritrophic matrix against intestinal bacterial infection\nin Drosophila melanogaster. Proc Natl Acad Sci U S A 108, 15966–15971 (2011)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed major criticisms from both reviewers. The edits and new data have strengthened the manuscript."}, "author_response": ""}]

Decision letter