Bacterial Toxin Monalysin Blocks Defecation to Keep Infection in Female Flies

When a fruit fly’s gut comes under attack, one of its first lines of defence is deceptively simple: flush the invaders out. Frequent defecation physically expels pathogens from the intestine before they can establish a foothold, and many microbes, in a curious twist, actually seem to accelerate this process, prompting the host to increase intestinal peristalsis and clear them away. But a new study published in PLOS Pathogens reveals that at least one bacterium has evolved a way to slam the door shut. Researchers led by Igor Iatsenko and colleagues, including Marko Rubinić, Yi Yu, Kathirvel Alagesan and Dagmar Frahm, have identified a secreted bacterial protein called Monalysin that suppresses defecation in infected female fruit flies, effectively trapping the pathogen inside the gut and prolonging infection.

The bacterium in question, Pseudomonas entomophila, is a well-known killer of insects and a workhorse of Drosophila infection research. When flies ingest it, the microbe unleashes an arsenal of virulence factors that damage the intestinal lining and eventually kill the host. Yet the new work uncovered a striking and previously underappreciated phenotype: rather than stimulating gut motility the way many other infections do, P. entomophila infection causes a pronounced blockage of defecation, and this effect is particularly strong in female flies. In practical terms, infected females stop expelling waste and, with it, the bacteria dwelling in their intestinal contents, giving the pathogen a chance to persist and multiply in a niche it would otherwise be flushed out of.

To dissect the mechanism behind this defecation blockage, the team turned to genetic mutants of P. entomophila and compared how each one affected the flies’ bowel habits. A mutant lacking GacA, the response regulator of the bacterial GacS/GacA two-component system, only partially inhibited defecation, indicating that this global regulatory pathway controls factors needed for the full phenotype. In contrast, a mutant lacking Hfq, an RNA chaperone that supports the stability and translation of many small-RNA-regulated transcripts, had lost its ability to suppress defecation altogether while remaining avirulent. This pattern told the researchers that the defecation-blocking activity depends on a secreted, thermosensitive protein under GacS/GacA control, but not on Hfq-dependent regulation.

Narrowing the field from a global regulator to a specific toxin required a systematic comparison of the proteins these mutants secrete. The researchers performed a proteomic comparison of the ΔgacA and Δhfq strains, reasoning that factors abundant in the partially active mutant but absent or reduced in the inactive one would be prime candidates for the defecation-suppressing activity. Among the candidates that emerged from this screen was Monalysin, a pore-forming toxin that P. entomophila is already known to deploy against insect hosts. Monalysin assembles into complexes that insert into host cell membranes, punching holes that disrupt epithelial integrity and contribute to the intestinal damage characteristic of P. entomophila infection. What the new study adds is a role for this toxin that goes far beyond simple tissue destruction.

To test whether Monalysin truly drives the defecation blockage, the researchers constructed a mutant strain lacking the toxin and examined its behaviour in infected flies. The result was unambiguous: the Monalysin-deficient mutant failed to fully suppress defecation, allowing infected females to maintain a degree of gut clearance that the wild-type bacterium abolishes. This loss of function came with measurable costs to the pathogen. The toxin-deficient strain showed attenuated virulence, meaning it killed flies less effectively, and it exhibited reduced persistence in the gut, being cleared more readily from the intestinal tract than its toxin-producing counterpart. Together, these findings establish Monalysin as a dual-purpose weapon: it damages the gut epithelium and, at the same time, sabotages the host’s mechanical defence of pathogen expulsion.

The timing of the effect is particularly revealing. The study found that the bacterial factor rapidly reduces defecation frequency, a pattern consistent with a transient suppression of intestinal transit rather than a slow, cumulative paralysis of a dying gut. In other words, Monalysin does not merely stop defecation as a by-product of catastrophic tissue damage late in infection; it acts early enough to matter, shutting down the flow of intestinal contents while the host is still alive and the pathogen is still trying to establish itself. This rapid action suggests that the toxin interferes with the physiological control of gut motility, possibly through its effects on the epithelial cells and the signalling pathways that coordinate peristalsis, although the precise cellular mechanism remains an open question for future work.

Why the effect is so much stronger in female flies is another puzzle the study highlights. Sex differences in gut physiology, defecation behaviour and immune responses are well documented in Drosophila, and the pronounced female bias in infection-induced defecation blockage suggests that hormonal status, reproductive state or sex-specific differences in intestinal architecture may modulate how Monalysin acts or how the gut responds to it. Understanding this dimorphism could have implications beyond flies, since sex differences in gastrointestinal transit and infection outcomes are observed across many animal species, including humans. The Drosophila model, with its powerful genetics, offers a tractable system for unpicking these differences at the level of molecules and neural circuits.

The broader significance of the work lies in what it says about the evolutionary arms race inside the gut. Defecation is an ancient and effective defence: by physically evacuating intestinal contents, the host denies pathogens the time and substrate they need to reach high population densities. Many enteric microbes, from commensals to pathogens, modulate host motility in various ways, and the conventional wisdom has been that bacteria tend to promote, not suppress, gut transit. P. entomophila and its toxin Monalysin now provide a clear counterexample, demonstrating that a pathogen can actively subvert this defence by secreting a factor that slows or halts the conveyor belt of the intestine. This strategy presumably buys the bacterium time to replicate, adhere and inflict the damage that ultimately kills the host.

The study also illustrates the power of comparative proteomics guided by clever genetics. Rather than screening every gene in the P. entomophila genome one by one, the researchers exploited the contrasting phenotypes of two regulatory mutants, one partially active and one inactive, to triangulate the culprit. The GacS/GacA two-component system, which orchestrates the expression of numerous secreted virulence factors, and Hfq, the RNA chaperone that fine-tunes post-transcriptional regulation, served as molecular filters that narrowed a large secretome down to a shortlist of candidates. Monalysin’s emergence from that shortlist, and its confirmation through deletion analysis, virulence assays and gut persistence measurements, exemplifies how phenotype-driven approaches can cut through the complexity of bacterial virulence repertoires.

Looking ahead, the findings raise a series of compelling questions. How does Monalysin, a pore-forming toxin best known for punching holes in epithelial cells, translate membrane damage into a change in defecation behaviour? Which host cells and signalling pathways relay the toxin’s effects to the motor programs of the gut? And do other pore-forming toxins in other pathogens play similar roles in manipulating host transit, a possibility that would suggest this is a widespread and under-recognised virulence strategy rather than a quirk of one insect pathogen? For now, the study by Rubinić, Yu, Alagesan, Frahm and Iatsenko stands as a vivid demonstration that the battle between host and pathogen extends to the most basic of bodily functions, and that a single bacterial protein can turn the gut’s own waste-disposal system against the animal it inhabits.

Subject of Research: How the bacterial toxin Monalysin suppresses infection-induced defecation in female Drosophila

Article Title: A pore-forming toxin monalysin contributes to infection-induced suppression of defecation in female Drosophila

Article References: Rubinić, M., Yu, Y., Alagesan, K., Frahm, D., & Iatsenko, I. (2026). A pore-forming toxin monalysin contributes to infection-induced suppression of defecation in female Drosophila. PLOS Pathogens, 22(10), e1014696. https://doi.org/10.1371/journal.ppat.1014696

Image Credits: AI Generated

DOI: 10.1371/journal.ppat.1014696

Keywords: Monalysin, Pseudomonas entomophila, Drosophila melanogaster, pore-forming toxin, defecation, gut immunity, pathogen expulsion, GacS/GacA, Hfq, virulence, intestinal transit, host-pathogen interaction