Cruz-Lorenzo, E. | bioRxiv Science

bioRxiv2026

Eukaryotic domestication of a bacterial immune protein following horizontal transfer

Many components of eukaryotic innate immunity originated from bacterial immune systems. However, it is unclear how eukaryotes acquire these genes and how the components are domesticated into eukaryotic physiology. Here, we discovered a recent instance of bacteria-eukaryote horizontal transfer and used it to characterize the genetic and biochemical changes that accompanied eukaryotic acquisition. We focus on Toll/interleukin-1 receptor (TIR) domains, which are widespread yet potentially costly immune modules associated with inflammation and/or cell death. By generating an atlas of TIR diversity across the tree of life, we phylogenetically categorized the domains and uncovered highly diverged, eukaryotic TIR families. This analysis revealed a horizontal transfer event that created the TirBCD protein family of Dictyostelium amoebae, which is closely related to the bacterial immune protein TIR-STING. While the TIR domain was transferred into amoebae, the genomic locus did not contain known regulatory domains nor other components of a bacterial operon. Nevertheless, TirC retained biochemical and physiological similarities to TIR-STING. TirC is a highly potent NADase, capable of oligomerizing into large complexes and depleting NAD+ even at very low protein concentrations. When expressed in heterologous systems, TirC was spontaneously active and highly toxic. In contrast, the natural amoeba host tolerated expression of full length TirC, showing that the cells can typically regulate its activity and avoid autoimmunity. However, a truncated form of TirC induced rapid rounding and cell lysis. These results suggest that amoebae have used TirC to retool a form of bacterial cell death for use in eukaryotic cells. Overall, this study uncovers recent eukaryotic TIR evolution that captures features of both bacterial and eukaryotic immunity. We also expect that the TIR domain atlas will be useful to researchers across model systems as they explore the vast diversity of TIR molecular and cellular functions.

microbiology↗

bioRxiv2023

L. pneumophila resists its self-harming metabolite HGA via secreted factors and collective peroxide scavenging

Many pathogenic bacteria, including Legionella pneumophila, infect humans from environmental reservoirs. To survive in these reservoirs, bacteria must withstand microbe-on-microbe competition. We previously discovered that L. pneumophila can compete with neighboring bacteria via an antimicrobial metabolite called homogentisic acid (HGA) (Levin, Goldspiel, and Malik 2019). Curiously, L. pneumophila strains that secrete HGA are not wholly immune to its effects: low-density bacteria are strongly inhibited by HGA whereas high-density cells are tolerant. How do these bacteria tolerate HGA and avoid self-harm during interbacterial competition? Here, we find that HGA toxicity occurs via the production of toxic hydroperoxides and multiple factors facilitate high-density tolerance. First, HGA only becomes fully toxic after >1 hour of oxidation. While this manifests as a delay in killing within well-mixed liquid cultures, in a biofilm environment, this could provide time for HGA to diffuse away before becoming toxic. Second, HGA generates quantities of hydroperoxides that can be collectively scavenged by high-density, but not low-density cells. And third, high-density cells produce one or more secreted factors that are transiently protective from HGA. In combination, we propose that the bacteria are able to deploy HGA to generate a pool of reactive oxygen species surrounding their own biofilms, while maintaining non-toxic conditions within them. Overall, these findings help to explain how broadly toxic molecules can be used as inter-bacterial weapons. They also provide insights about why some of our current decontamination methods to control L. pneumophila are ineffective, leading to recurrent disease outbreaks.

microbiology↗