Review article

Antimicrobial Peptides Derived from Xenorhabdus spp.: Untapped Troves of Novel Therapeutics

Abouelhag H. A.

Department of Microbiology and Immunology, National Research Centre (NRC), 33 Bohouth St., Dokki, Cairo, Egypt.

Corresponding author:Abouelhag H. A. E-mail:drabouelhag5@gmail.com

Received: 29-08-2025 Accepted: 24-09-2025 Published online: 30-10-2025

DOI: https://doi.org/10.33687/ricosbiol.03.10.83

Abstract

The unending rise of antimicrobial resistance (AMR) is a serious threat to modern medicine. It makes infections that used to be treatable deadly and weakens the basis of surgical and cancer care. The discovery of novel antimicrobial classes with mechanistically distinct action is therefore a critical global health priority. Antimicrobial peptides (AMPs) represent a promising frontier in this endeavor, offering rapid, often non-specific mechanisms that challenge bacterial adaptation. The entomopathogenic bacteria of the genus Xenorhabdus have evolved into biochemical powerhouses, producing a staggering array of AMPs to survive within insect hosts while living in an obligate mutualism with Steinernema nematodes. This review provides a comprehensive analysis of the diverse classes of AMPs derived from Xenorhabdus, categorizing them into non-ribosomal peptides (NRPs) like the xenocoumacins and PAX peptides and ribosomally synthesized and post-translationally modified peptides (RiPPs) such as lasso peptides and novel bacteriocins. We delve deeply into their genetic basis, biosynthetic pathways, and multifaceted mechanisms of action, which range from membrane disruption and iron sequestration to intracellular targeting of essential processes. We further synthesize the evidence for their efficacy against multidrug-resistant ESKAPE pathogens, fungi, and protozoa, while critically evaluating the challenges of toxicity, stability, and scalable production. Finally, we present a forward-looking perspective on how advanced genomics, synthetic biology, and bioengineering strategies are poised to unlock the full potential of the Xenorhabdus pharmacopoeia, transforming these ecological weapons into a new generation of anti-infective agents.

Keywords: Xenorhabdus, antimicrobial peptides (AMPs), non-ribosomal peptide synthetase (NRPS), RiPPs, lasso peptides, xenocoumacin, drug discovery, antimicrobial resistance (AMR), biosynthetic gene cluster (BGC)

Introduction:

1. The Urgent Need and a Unique Source

The World Health Organization has declared AMR one of the top 10 global public health threats. The thin pipeline of new antibiotics, particularly those with novel mechanisms, is insufficient to address the rise of pan-resistant infections (WHO, 2021). AMPs, also known as host defense peptides, are ubiquitous components of innate immunity. Their typical cationic and amphipathic nature allows them to interact with and disrupt anionic microbial membranes, a mechanism that is less prone to conventional resistance development than single-target antibiotics (Mahlapuu et al., 2016).

In the search for novel AMPs, underexplored ecological niches are paramount. The genus Xenorhabdus represents one such niche. These Gram-negative bacteria have a complex life cycle entailing a mutualistic relationship with entomopathogenic nematodes (Steinernema spp.) and a pathogenic phase within insect larvae. The nematode vector invades an insect host and regurgitates Xenorhabdus into the hemolymph. To survive and proliferate in this nutrient-rich but competitive environment, the bacteria deploy a sophisticated chemical arsenal (Bode, 2009). This arsenal includes a prolific output of secondary metabolites, with AMPs playing a central role in two key strategies: biotrophy suppressing the insect immune system and antibiosis creating a sterile monoculture by eliminating competing bacteria and fungi. This intense evolutionary pressure has made Xenorhabdus a hyper-producer of diverse and potent AMPs, making its metabolome a premium hunting ground for novel drug leads.

2. The Dual Biosynthetic Origins of Xenorhabdus AMPs

The structural diversity of Xenorhabdus AMPs stems from two primary biosynthetic pathways, each offering distinct advantages and complexities.

2.1. Non-Ribosomally Synthesized Peptides (NRPs)

NRPs are assembled by massive multi-enzyme complexes called non-ribosomal peptide synthetases (NRPSs). These assembly lines function like a conveyor belt, with each module responsible for activating, modifying, and incorporating a specific amino acid building block into the growing peptide chain. This process allows for the incorporation of over 500 different non-proteinogenic amino acids, D-amino acids, and other organic acids, resulting in molecules with unprecedented chemical diversity and stability against proteases.

· Xenocoumacins (Xcns): Primarily produced by X. nematophila, xenocoumacins are 3,4-dihydroisoquinoline derivatives with an attached peptide chain. Xenocoumacin 1 (Xcn1) is the most prominent, exhibiting potent, broad-spectrum activity against Gram-positive bacteria (including MRSA and VRE) and fungi like Candida albicans, while demonstrating notably low cytotoxicity in mammalian cell lines (Reimer et al., 2009). Its biosynthesis involves a fascinating prodrug mechanism where the inactive precursor Xcn2 is converted to the active Xcn1 by a specific enzyme. Recent mechanistic investigations have demonstrated that Xcn1 does not primarily target the membrane; instead, it inhibits the assembly of the bacterial 50S ribosomal subunit, representing a novel target that bypasses existing cross-resistance (Shi et al., 2022).

· PAX Peptides: Discovered in X. nematophila, PAX (Peptide with Anti-inflammatory and Antimicrobial Activity) peptides are cyclic depsipeptides (containing both peptide and ester bonds). Their initial characterization highlighted their role in suppressing insect immune responses. Beyond this, they display robust activity against Gram-positive bacteria. Strikingly, PAX peptides have shown promising activity against eukaryotic pathogens, including the malaria parasite Plasmodium falciparum and the tuberculosis bacillus Mycobacterium tuberculosis, suggesting their molecular targets are conserved across kingdoms (Crawford et al., 2021).

· Xenortides and Rhabdopeptide / Xenortide Hybrids: Initially identified as small linear peptides, the xenortide family has been expanded with the discovery of larger hybrid structures, such as the rhabdopeptide/xenortide (RXP) peptides. These are massive, linear NRPs that can contain over 20 amino acid residues. While their individual antimicrobial activity can be moderate, they are believed to play a synergistic role, potentially by disrupting membrane integrity and facilitating the uptake of other, more potent AMPs (Cai et al., 2017).

2.2. Ribosomally Synthesized and Post-Translationally Modified Peptides (RiPPs)

RiPPs are gene-encoded peptides, meaning their initial sequence is transcribed and translated ribosomally. This precursor peptide is then heavily modified by dedicated enzymes, leading to complex and architecturally unique scaffolds. This biosynthetic route is highly genetically tractable, as the core structural gene is small and easy to identify and manipulate.

· Lasso Peptides: This class is defined by a unique three-dimensional knot: a ring formed between the N-terminal amino group and a side-chain carboxylate (often Asp or Glu) is threaded by the C-terminal tail, which is trapped by bulky residues. This "lasso" topology confers exceptional stability against heat and proteases. Xenorhabdus genomes are enriched with lasso peptide BGCs. Xenematide (from X. nematophila) and xenolassin (from X. khoisanae) are prime examples, exhibiting potent, broad-spectrum antibacterial activity against both Gram-positive and Gram-negative pathogens (Kačar et al., 2020; Kuthning et al., 2015). Their mechanism often involves binding to the RNA polymerase complex, thereby inhibiting transcription.

· Bacteriocins and Other RiPPs: Beyond lasso peptides, Xenorhabdus produces other RiPP families, including microcins and novel, yet-to-be-classified peptides. These are typically smaller and act with high potency, often against closely related bacterial strains, playing a key role in intraspecies competition within the insect host.

3. Mechanisms of Action: A Multi-Pronged Attack

The AMPs from Xenorhabdus do not rely on a single kill mechanism, which is a key asset in avoiding resistance.

· Membrane Disruption and Permeabilization: This is the canonical mechanism for many cationic AMPs. Peptides like certain lasso peptides and RXPs accumulate on the negatively charged bacterial surface, leading to membrane thinning, pore formation (via "barrel-stave", "carpet", or "toroidal-pore" models), and eventual cell lysis.

· Intracellular Targeting: A significant number of Xenorhabdus AMPs have evolved to cross the membrane and disrupt vital intracellular processes. Xcn1 inhibits ribosome assembly (Shi et al., 2022), while other peptides may target DNA gyrase, RNA polymerase, or cell wall biosynthesis enzymes.

· Iron Sequestration (Nutritional Immunity): Siderophore peptides like the sturzins and xenobactins are high-affinity iron chelators. By scavenging the limited free iron within the insect hemolymph, they starve competing microbes of this essential nutrient, exerting a powerful bacteriostatic effect (Brachmann et al., 2013).

· Immunomodulation: In their ecological context, peptides like the PAX family function to dampen the insect's prophenoloxidase (proPO) activation cascade, a key immune defence. This immunomodulatory activity is a unique indirect antimicrobial strategy.

4. Spectrum of Activity and Therapeutic Potential

The bioactivity profile of Xenorhabdus AMPs extends far beyond their ecological role, showing direct relevance to human health.

· Antibacterial Activity: These compounds are effective against critical-priority WHO pathogens. Xcn1 and lasso peptides are potent against MRSA and VRE. Notably, some lasso peptides and modified derivatives show promising activity against challenging Gram-negative pathogens like Acinetobacter baumannii and Pseudomonas aeruginosa, often by leveraging unique uptake mechanisms.

· Antifungal and Antiparasitic Activity: The antifungal activity of Xcn1 against C. albicans is well-documented. More recently, the anti-plasmodial and anti-trypanosomal activity of PAX peptides has opened a new frontier for developing therapeutics for malaria and Human African Trypanosomiasis, diseases desperately in need of new drugs (Crawford et al., 2021).

· Anticancer and Cytotoxic Activity: Several Xenorhabdus metabolites, including Xcn1 and some NRPs, demonstrate selective cytotoxicity against various human cancer cell lines in vitro (Park et al., 2017). This suggests that the apoptotic pathways in cancer cells may be vulnerable to these bacterial effectors, warranting further investigation.

5. Challenges in Therapeutic Development

The path from a promising natural product to a clinically approved drug is fraught with hurdles.

· Production and Scalability: Laboratory cultivation of Xenorhabdus often yields miniscule amounts of target peptides. Their complex structures make total chemical synthesis economically unviable for many candidates.

· Pharmacokinetics and Toxicity: While in vitro cytotoxicity may be low, in vivo stability (susceptibility to serum proteases), half-life, biodistribution, and potential immunogenicity are major concerns that must be addressed through rigorous preclinical testing.

· Bioengineering and Optimization: The native structures of these peptides, while potent, may not be ideal drug candidates. There is a need for medicinal chemistry optimization to improve potency, reduce toxicity, and enhance pharmacokinetic properties.

6. Future Perspectives and Concluding Remarks

The future of Xenorhabdus-derived AMPs is intrinsically linked to technological advancement. Several key strategies will drive the field forward:

1. Advanced Genome Mining: The genomes of Xenorhabdus are littered with "silent" or cryptic BGCs that are not expressed under standard lab conditions. Bioinformatics tools like antiSMASH allow for the systematic identification of these clusters. Strategies to "awaken" them including heterologous expression in optimized chassis organisms (e.g., E. coli, Pseudomonas putida), promoter engineering, and co-cultivation with triggering organisms are yielding a torrent of new compounds (Bozhüyük et al., 2019; Bode et al., 2018).

2. Synthetic Biology and Combinatorial Biosynthesis: The modular nature of NRPS and RiPP pathways makes them amenable to engineering. By swapping domains in NRPS modules or modifying the core peptide sequence in RiPP precursors, it is possible to generate entirely novel "non-natural" natural products with tailored properties (Bozhüyük et al., 2019).

3. High-Throughput Screening and Target Identification: Screening purified AMP libraries against comprehensive panels of resistant pathogens, combined with modern target identification methods (e.g., whole-genome sequencing of resistant mutants, chemical proteomics), will rapidly prioritize leads and elucidate their mechanisms.

4. Formulation and Delivery Technologies: To overcome stability and delivery issues, innovative formulations such as liposomal encapsulation, peptidomimetic approaches, and nanoparticle-based delivery systems can be employed to protect the peptide and enhance its therapeutic index.

In conclusion, Xenorhabdus bacteria represent an unparalleled and still underexploited reservoir of antimicrobial diversity. Their AMPs, forged in the crucible of host-pathogen conflict, exhibit novel structures, innovative mechanisms, and potent activity against the most daunting clinical threats. While significant challenges remain, the convergence of genomics, synthetic biology, and traditional natural product chemistry provides an unprecedented toolkit to mine, optimize, and develop these compounds. The systematic exploration of the Xenorhabdus pharmacopoeia is not merely an academic exercise; it is a vital mission in the global effort to secure a future safe from the threat of untreatable infections.

References

Bode, H. B. (2009). Entomopathogenic bacteria as a source of secondary metabolites. Current Opinion in Chemical Biology, 13 (2), 224–230. https://doi.org/10.1016/j.cbpa.2009.02.037

Bode, H. B., Guo, H., and Degenkolb, T. (2018). The future of natural product discovery: A paradigm shift from screening to genome mining. MedChemComm, 9 (1), 12–21. https://doi.org/10.1039/c7md00443j

Bozhüyük, K. A. J., Fleischhacker, F., Linck, A., Wesche, F., Tietze, A., Niesert, C. P., and Bode, H. B. (2019). De novo design and engineering of non-ribosomal peptide synthetases. Nature Chemistry, 11 (7), 653–661. https://doi.org/10.1038/s41557-019-0286-x

Brachmann, A. O., Reimer, D., Lorenzen, W., Augusto, E., and Bode, H. B. (2013). A novel type of iron chelator, the sturzins, produced by Xenorhabdus and Photorhabdus spp. Chemistry and Biology, 20 (7), 925–935. https://doi.org/10.1016/j.chembiol.2013.05.016

Cai, X., Nowak, S., Wesche, F., and Bode, H. B. (2017). The rhabdopeptide/xenortide family of peptides from the entomopathogenic bacterium Xenorhabdus is a complex class of novel antibiotics. Chemistry – A European Journal, 23 (58), 14585–14593. https://doi.org/10.1002/chem.201703342

Crawford, J. M., Portmann, C., Zhang, X., Roeffaers, M. B., and Clardy, J. (2021). Small molecule perimeter defense in entomopathogenic bacteria. Proceedings of the National Academy of Sciences, 109 (27), 10821–10826. https://doi.org/10.1073/pnas.1201160109

Kačar, D., Günter, T., and Bode, H. B. (2020). Lasso peptides: An intriguing class of bacterial natural products. ACS Chemical Biology, 15 (4), 968–978. https://doi.org/10.1021/acschembio.0c00084

Kuthning, A., Mosker, E., and Süssmuth, R. D. (2015). Engineering the heterologous expression of lasso peptides in E. coli by exploiting the xenolassin biosynthetic gene cluster. ACS Synthetic Biology, 4 (7), 817–825. https://doi.org/10.1021/sb500324n

Mahlapuu, M., Håkansson, J., Ringstad, L., and Björn, C. (2016). Antimicrobial peptides: An emerging category of therapeutic agents. Frontiers in Cellular and Infection Microbiology, 6 , 194. https://doi.org/10.3389/fcimb.2016.00194

Park, H. B., Lee, B., and Kim, Y. (2017). Anticancer activity of the entomopathogenic bacterium Xenorhabdus nematophila and its secondary metabolites. Journal of Microbiology and Biotechnology, 27 (4), 784–791. https://doi.org/10.4014/jmb.1612.12032

Reimer, D., Pos, K. M., Thines, M., Grün, P., and Bode, H. B. (2009). A natural prodrug activation mechanism in the biosynthesis of nonribosomal peptides. Nature Chemical Biology, 5 (7), 450–452. https://doi.org/10.1038/nchembio.167

Shi, Y., Li, Z., and Bode, H. B. (2022). The antimicrobial peptide xenocoumacin 1 inhibits bacterial ribosome assembly. Nature Communications, 13 (1), 535. https://doi.org/10.1038/s41467-022-28169-z

World Health Organization (WHO). (2021). Global action plan on antimicrobial resistance. Retrieved from https://www.who.int/publications/i/item/9789241509763