Myxococcus xanthus predation: an updated overview

Frontiers in Microbiology 01 frontiersin.org Myxococcus xanthus predation: an updated overview Francisco JavierContreras-Moreno , JuanaPérez , JoséMuñoz-Dorado , AurelioMoraleda-Muñoz * and FranciscoJavierMarcos-Torres * Departamento de Microbiología, Facultad de Ciencias, Universidad de Granada, Granada, Spain Bacterial predators are widely distributed across a variety of natural environments. Understanding predatory interactions is of great importance since they play a defining role in shaping microbial communities in habitats such as soils. Myxococcus xanthus is a soil-dwelling bacterial predator that can prey on Grampositive and Gram-negative bacteria and even on eukaryotic microorganisms. This model organism has been studied for many decades for its unusual lifecycle, characterized by the formation of multicellular fruiting bodies filled with myxospores. However, less is known about its predatory behavior despite being an integral part of its lifecycle. Predation in M. xanthus is a multifactorial process that involves several mechanisms working synergistically, including motility systems to efficiently track and hunt prey, and a combination of shortrange and contact-dependent mechanisms to achieve prey death and feed on them. In the short-range attack, M. xanthus is best known for the collective production of secondary metabolites and hydrolytic enzymes to kill prey and degrade cellular components. On the other hand, contact-dependent killing is a cell-to-cell process that relies on Tad-like and type III secretion systems. Furthermore, recent research has revealed that metals also play an important role during predation, either by inducing oxidative stress in the prey, or by competing for essential metals. In this paper, wereview the current knowledge about M. xanthus predation, focusing on the different mechanisms used to hunt, kill, and feed on its prey, considering the most recent discoveries and the transcriptomic data available. KEYWORDS myxobacteria, bacterial predation, bacterial interaction, predator-prey interactions, motility, secondary metabolites, hydrolytic enzymes, metals Introduction Myxococcus xanthus is a soil-dwelling bacterial predator renowned for its social and multicellular behavior, which is evident along its complex lifecycle. When nutrients are scarce, M. xanthus cells coordinate to form multicellular structures known as fruiting bodies, where some of them differentiate into resistant myxospores. Depending on external cues, such as nutrient levels or the presence of prey microorganisms, M. xanthus must decide whether to initiate this developmental cycle or to activate its predatory mechanisms to feed (MuñozDorado etal., 2016). Coordinating these two facets of its lifecycle has led to an extensive coevolution between both stages. Indeed, mutations in genes involved in early stages of the developmental cycle, where cells are still to commit to this process, have been found to negatively impact predation. OPEN ACCESS EDITED BY Li Zhoukun, Nanjing Agricultural University, China REVIEWED BY Wei Hu, Microbial Technology Institute/Shandong University, China *CORRESPONDENCE Aurelio Moraleda-Muñoz [email protected] Francisco Javier Marcos-Torres [email protected] RECEIVED 16 November 2023 ACCEPTED 15 January 2024 PUBLISHED 24 January 2024 CITATION Contreras-Moreno FJ, Pérez J, Muñoz-Dorado J, Moraleda-Muñoz A and Marcos-Torres FJ (2024) Myxococcus xanthus predation: an updated overview. Front. Microbiol. 15:1339696. doi: 10.3389/fmicb.2024.1339696 COPYRIGHT © 2024 Contreras-Moreno, Pérez, Muñoz-Dorado, Moraleda-Muñoz and Marcos-Torres. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Mini Review PUBLISHED 24 January 2024 DOI 10.3389/fmicb.2024.1339696 Contreras-Moreno et al. 10.3389/fmicb.2024.1339696 Frontiers in Microbiology 02 frontiersin.org By contrast, genes required in later stages of the development, where cells are fully committed to fruiting-body formation, do not seem to play a role in predation (Pham etal., 2005; Berleman etal., 2008; Pérez et al., 2022). Furthermore, some predator–prey interactions can stimulate fruiting bodies formation even during predation, although cells within the fruiting bodies are unable to differentiate into myxospores (Berleman and Kirby, 2007). When conditions are favorable again, myxospores from a fruiting body will germinate into a population of vegetative cells known as swarm, which will actively hunt for prey to feed on them. Cells within the swarm will cooperate in an attempt to prey with different degrees of success on a great diversity of Gram-negative and Gram-positive bacteria (Figure 1), including nitrogen-fixing bacteria and some human and plant pathogens, as well as fungi and nematodes (MendesSoares and Velicer, 2013; Livingstone etal., 2017; Petters etal., 2021; Sydney etal., 2021). M. xanthus predation is a multifactorial task that combines a broad arsenal of resources to ensure prey death. This process starts with tracking of prey in the environment driven by its motility systems and signal-transduction mechanisms. Upon encountering a suitable prey, M. xanthus uses a combination of short-range and contact-dependent mechanisms to kill and lyse prey cells. While short-range killing mainly involves the production of a battery of secondary metabolites (SMs) such as antibiotics, and of hydrolytic enzymes to degrade and feed on the cellular components of the prey (Muñoz-Dorado etal., 2016), contactdependent lysis is mediated by secretion systems (Seef etal., 2021; Thiery etal., 2022). Moreover, recent studies have shown that metals are also involved in M. xanthus predation either by using metals to provoke oxidative stress or by outcompeting prey for possession of essential metals (Contreras-Moreno etal., 2020; Lee etal., 2020; Dong etal., 2022b; Pérez etal., 2022). While the developmental stage of M. xanthus has been thoroughly studied, less attention has been paid to its predatory behavior. This is, however, not an isolated facet of its lifestyle, but is heavily interconnected to the other traits of M. xanthus biology to shape its lifecycle (Volz etal., 2012; Pérez etal., 2022). In fact, there is mounting evidence that predation has played a major role in the selection of M. xanthus biological features via coevolution with its prey (Nair etal., 2019; La Fortezza etal., 2022). In this minireview wewill discuss the state-of-the-art of the toolset used by M. xanthus to prey, including the most recent findings derived from transcriptomic analyses during predation. Motility systems M. xanthus cells must actively search for prey in the soil to obtain nutrients. To approach the prey, they use two types of motility systems: an individual gliding movement, known as adventurous (A) motility, and a collective twitching-type movement, known as social (S) motility. A-motility relies on a Agl-Glt multiprotein outer-membrane complex that attaches the substrate at fixed sites of focal adhesion. These Agl-Glt complexes move directionally across the inner membrane toward the anterior pole of the cell, following a helical trajectory (Islam etal., 2023). Gliding occurs over an exopolysaccharide slime produced by the bacterium, which facilitates cells to follow the trail of previous cells rather than creating a new one, which enables exploration and prey foraging (Rombouts etal., 2023). On the other hand, S-motility is driven by type-IV pili, which pull the cells forward by extending, attaching to surfaces (or other cells), and then retracting (Chang etal., 2016). In M. xanthus, this is a collective movement where the cells must bein contact with each other, allowing them to coordinate the swarm’s movement (Skotnicka and SøgaardAndersen, 2017). A study using mutants impaired in these two motility systems clearly showed that both are required to efficiently prey on FIGURE1 Predation assays of M. xanthus DK1622 (left) against different Gram-negative [(A) Pseudomonas putida; (B) Sinorhizobium meliloti; (C) Escherichia coli] and Gram-positive bacteria [(D) Staphylococcus aureus; (E) Bacillus subtilis; (F) Micrococcus sp.] after 72 h of interaction, showing its different capabilities to kill and consume prey. Contreras-Moreno et al. 10.3389/fmicb.2024.1339696 Frontiers in Microbiology 03 frontiersin.org Sinorhizobium meliloti (Pérez etal., 2014). In fact, transcriptomic analyses of M. xanthus during predation (predatosomes) have shown upregulation of some genes required for both motility systems as well as some regulators involved in motility, such as the sigma factor SigF (Lee etal., 2020; Pérez etal., 2022). Both types of motility occur simultaneously or alternate in different subpopulations of the swarm to adapt to different prey local distribution (Rombouts etal., 2023). While scouting the area in search for prey, M. xanthus seems to detect some prey molecules such as acyl homoserine lactones, that stimulate motility and facilitate their encounter (Lloyd and Whitworth, 2017; Akbar etal., 2022). Once the predator finds its prey, it must stay in close vicinity to activate its attack mechanisms and then feed on their cellular by-products. Therefore, upon detection, M. xanthus cells “stop” by repeatedly reversing their trajectory to optimize prey lysis (Zhang etal., 2020; Thiery and Kaimer, 2022). These repeated reversals are also important to feed on prey, as mutant predator cells defective in this mechanism tend to abandon prey colonies after lysis, without consuming their cellular remains (McBride and Zusman, 1996; Zhang etal., 2020). Secondary metabolites M. xanthus induces prey cell death and lysis by cooperative production of different lytic factors, acting either in isolation or synergistically (Figure2A). Among them, SMs play an important role in prey killing, especially those with antimicrobial activity. M. xanthus genome holds an outstanding biosynthetic capacity for SM production, including at least 18 nonribosomal peptide synthetases (NRPS), 22 polyketide synthases (PKS), and 6 mixed PKS/NRPS, making a total of 14.5% of its genome (Goldman et al., 2006). Antimicrobial compounds so far isolated from M. xanthus have shown to bemore efficient against Gram-positive bacteria (Xiao etal., 2011; Hoffmann etal., 2018), which could bedue to the protective role of the Gram-negative outer membrane and/or a potential facilitation for the intracellular delivery of outer membrane vesicles (OMVs) cargo molecules (Zwarycz etal., 2023). To date, only 2 M. xanthus SMs have been directly implicated in predation: (i) myxovirescin, a macrocyclic SM able to block bacterial growth by inhibiting type a II signal peptidase (Xiao etal., 2011, 2012), and (ii) myxoprincomide, a SM required for effective predation against Bacillus subtilis (Cortina etal., 2012; Müller etal., 2016). However, the most recent predatosome data suggest that SM production is prey specific. Thus, while genes coding for myxoprincomide, myxovirescin, and myxalamide have been reported as being upregulated against Micrococcus luteus and Escherichia coli, only myxalamide is upregulated when preying on S. meliloti. However, against S. meliloti, additional clusters probably involved in the biosynthesis of unidentified bio-products are also upregulated (Pérez etal., 2022; Wang etal., 2023). Hydrolytic enzymes Besides SMs, M. xanthus requires a battery of hydrolytic enzymes to degrade prey cell components and feed on them (Figure2A). To induce lysis of Gram-positive bacteria, this myxobacterium needs to degrade prey cell-walls via peptidoglycan-lysing enzymes (Hart and FIGURE2 Different resources used by M. xanthus (yellow cells) to kill prey (purple cells). (A) Secondary metabolites (SMs) and hydrolytic enzymes are secreted into the extracellular medium or delivered via outer membrane vesicles (OMVs). (B) Contact-dependent killing mechanisms of M. xanthus. (C) Role of metals during bacterial predation. Competition for iron mediated by siderophores and accumulation of copper at the predator–prey interphase. Contreras-Moreno et al. 10.3389/fmicb.2024.1339696 Frontiers in Microbiology 04 frontiersin.org Zahler, 1966; Sudo and Dworkin, 1972; Arend etal., 2021). A protein with lysozyme-like-activity (LlpM) able to induce cell lysis has been identified, although it is not essential for this process, reinforcing the idea that cell-wall lytic activity of the M. xanthus secretome is a multifactorial process with several hydrolytic enzymes involved (Arend et al., 2021). In fact, secretion of hydrolytic enzymes has shown to drive predation in natural isolates with genomes specially enriched in this type of enzymes (Dong etal., 2022a, 2023). Transcriptomic studies against different prey have revealed upregulation of several M. xanthus genes coding cell-wall lysing proteins. Thus, when the Gram-negative S. meliloti and the Grampositive Streptomyces coelicolor were used as prey, two genes coding N-acetylmuramoyl-L-alanine amidases were upregulated (Lee etal., 2020; Pérez etal., 2022). Moreover, five genes coding for proteins with LysM-domains were overexpressed against S. meliloti and two of them were also upregulated in co-cultures with E. coli (Livingstone etal., 2018; Pérez etal., 2022). However, during predation on Pseudomonas aeruginosa or M. luteus, no gene involved in cell-wall lysis was differentially expressed (Wang etal., 2023). These discrepancies may be due to characteristics of the prey, but also to variances in the methodology used on each assay. In addition to cell-walls, M. xanthus must also hydrolyze prey proteins and lipids to use them as nutrients or as building blocks for metabolic processes. Until now, only the peptidase MepA has been experimentally studied and it seems to contribute to predation by degrading proteins released from lysed cells (Berleman etal., 2014). Indeed, mepA is upregulated in co-culture with S. meliloti and with S. coelicolor, supporting its role in predation (Lee etal., 2020; Pérez et al., 2022). M. xanthus predatosomes against different bacteria indicate that, to achieve full prey lysis, the predator needs to induce a plethora of hydrolytic enzymes. In fact, besides those aforementioned, M. xanthus genes coding for several hydrolytic enzymes are transcriptionally upregulated in co-culture with S. meliloti, E. coli, or M. luteus, including peptidases, metalloproteases, alpha/beta fold hydrolases, lipases, and nucleases (Pérez etal., 2022; Wang etal., 2023). The delivery of this lethal cocktail to the prey is facilitated by OMVs, which include in their cargo many putative hydrolytic proteins and molecules associated with antibiotic activities (Kahnt etal., 2010; Berleman etal., 2014; Remis etal., 2014). In fact, isolated M. xanthus OMVs can kill Gram-positive and Gram-negative bacteria (Evans etal., 2012; Remis etal., 2014; Livingstone etal., 2018; Zwarycz etal., 2023). Moreover, it has been proved that OMV killing activity against different bacteria correlates with the predatory activity of M. xanthus. However, the absence of correlation between OMV killing activity and their ability to fuse with different prey cell-walls reinforces the idea that the composition of the OMVs cargo is more critical than their delivery for the predatory activity (Zwarycz etal., 2023). Contact-dependent killing Although the predatory strategy of M. xanthus is usually described as a cooperative process, single myxobacteria can kill individual prey cells (Zhang etal., 2020; Arend etal., 2021; Seef etal., 2021). This one-to-one interaction requires a contact-dependent mechanism (Figure2B). In this sense, many bacteria have evolved specialized nanomachines to export proteins and/or virulence factors across the cell envelope into the surroundings, or to inject them into eukaryotic or prokaryotic cells (Costa et al., 2015; Galán and Waksman, 2018). Although analyses of the M. xanthus genome have revealed a large potential for secretion (Konovalova etal., 2010), only two secretion systems have been so far related to cell contact-dependent prey killing: a degenerate type III-like system (T3SS*) and a tight adherence (Tad) secretion apparatus, also known as “Kil complex” (Seef etal., 2021; Thiery etal., 2022). T3SSs are multiprotein complexes encoded by a broad range of bacteria with pathogenic or interbacterial antagonism (Galán and Waksman, 2018). While several pathogenic enterobacteria use specialized T3SS to deliver effector proteins into eukaryotic cells (Wagner etal., 2018), other species have adapted flagella-type T3SS for cytotoxin export (Dongre etal., 2018; Halte and Erhardt, 2021). However, M. xanthus T3SS* lacks a dedicated outer membrane secretin and any homologs to needle and translocon components (Figure2B), thus being classified as “non-flagellar, needle-less” T3SS (Konovalova et al., 2010; Abby and Rocha, 2012; Diepold and Armitage, 2015). On the other hand, Tad-like secretion systems are considered members of the type IV filament superfamily, and are related to bacterial adhesion, biofilm stabilization, and contact-dependent regulation of adhesion (Ellison etal., 2017; Denise etal., 2019; Seef etal., 2021). M. xanthus T3SS* and Tad-like components have been shown to beinterdependently, but coordinately, accumulated at the predator– prey interface for killing E. coli prey cells, exhibiting a functional interplay and different functions during the predatory interaction. Thus, while the Tad-like apparatus is instrumental in prey cell death (but does not cause prey disintegration), the T3SS* is required for cell lysis (Thiery etal., 2022). Nevertheless, both systems are required for utilizing live prey as a nutrient source, although they are not directly involved in the degradation or uptake of dead prey biomass (Thiery etal., 2022). Homologous genes encoding Tad-like complexes have been identified in different genera of bacterial predators (Seef etal., 2021; Wang etal., 2023). However, the co-occurrence of T3SS* and Tad complexes is restricted to the sub-order Cystobacterineae of the Myxococcales, suggesting a specialized function of these secretion systems in myxobacterial predation (Abby and Rocha, 2012; Wang etal., 2023). Gram-negative and Gram-positive prey elicit similar responses by M. xanthus regarding the formation of Tad and T3SS* foci, since both can lead to aggregation of these multiprotein complexes (Wang etal., 2023). Thus, the transcriptomic analysis of the co-culture of M. xanthus with E. coli and M. luteus revealed that part of the genes encoding the T3SS*, as well as part of one of the two clusters encoding the Tad-like apparatus, were significantly upregulated in both cases (Wang etal., 2023). A similar result was reported from the interaction between M. xanthus and S. meliloti, although in this case the upregulation of genes from both clusters coding the Tad-like complex was detected (Pérez etal., 2022). However, during co-culture with S. coelicolor, transcriptomic data indicated that only some genes coding the T3SS*, but not those coding the Tad-like apparatus, were upregulated (Lee etal., 2020). Similarly, P. aeruginosa failed to induce accumulation of Tad complexes, turning out to be resistant to Contreras-Moreno et al. 10.3389/fmicb.2024.1339696 Frontiers in Microbiology 05 frontiersin.org M. xanthus predation (Seef etal., 2021). Interestingly, the coordinated accumulation of the T3SS*/Tad complexes has not been observed during contact with other M. xanthus cells, but only during the interaction with prey cells, which implies that contact-dependent killing mechanisms discriminate between kin and prey cells (Wall, 2016; Seef etal., 2021; Thiery etal., 2022). Role of metals in the predatory interaction The use of metals by bacterial predators has gained relevance in the recent years as a new mechanism of inducing prey death. Two metals have been reported to beinvolved in the predatory behavior of M. xanthus: copper and iron (Figure2C). These two metals exhibit a dual role on living cells since they are cofactors of enzymes that are essential for vital functions, but toxic at high concentrations (Rensing and McDevitt, 2013; Ladomersky and Petris, 2015; Li etal., 2021). In the case of copper, it has been shown that M. xanthus utilizes this metal to poison S. meliloti. This metal accumulates inside the cells at the interface where predator collides with the prey, provoking changes in both partners of the interaction and helping the predator to penetrate in the prey colony (Contreras-Moreno etal., 2020). While M. xanthus upregulates the expression of copper detoxification genes such as those coding for the P1B-ATPase CopA, the multicopper oxidase CuoA, and the copper efflux pumps Cus2 and Cus3 (Sánchez-Sutil etal., 2007; Moraleda-Muñoz etal., 2010a,b; Pérez etal., 2018), S. meliloti produces a brown pigment that has been identified as melanin (Contreras-Moreno etal., 2020). Predatory analyses have revealed that melanin is overproduced by the prey during predation, indicating that copper is being used to generate oxidative stress and that the pigment functions as a defensive shield for the prey (Contreras-Moreno et al., 2020). However, it remains to beelucidated how the predator achieves accumulation of copper in the prey to kill it by generating reactive oxygen species. Iron also seems to play an important role during myxobacterial predation. Several transcriptomes of M. xanthus against diverse prey have been published, and in all of them siderophore biosynthesis is upregulated in both predator and prey (Lee etal., 2020; Pérez etal., 2022; Soto etal., 2023; Wang etal., 2023; Whitworth etal., 2023). Moreover, the same result has been observed during predation of another myxobacterium, Cystobacter ferrugineus, against Pseudomonas putida (Akbar and Stevens, 2021), indicating that competition for iron may bea general predatory mechanism among myxobacteria. Experimentally, it has been demonstrated that depletion of iron triggers the biosynthesis of the antibiotic actinorhodin in S. coelicolor to prevent predation from M. xanthus (Lee etal., 2020). Moreover, a mutant of M. xanthus that produces less siderophores (myxochelins) is defective in predation against P. aeruginosa (Dong etal., 2022b). Similarly, mutants in a putative TonB-dependent transporter for ferrimyxochelins and in components of the ABC transporter that introduces ferrimyxochelins into the cytoplasm also exhibit less efficient predation on this prey (Dong etal., 2022b). All these data seem to indicate that competition for iron plays a decisive role in the myxobacterial predatory interaction with the prey. Concluding remarks Bacterial predation is a key factor in shaping ecosystems and establishing microbial diversity in soils. Understanding these interactions will contribute to improve soil conditions in agriculture. Moreover, some bacterial predators are considered “microfactories” of SMs that can beused to overcome the current antibiotic crisis (Pérez etal., 2016, 2020). Predation is an integral part of M. xanthus biology and, therefore, this bacterium has developed a diverse toolset to adapt to its predatory lifestyle. Among the best-understood facets of its predatory activity are the mechanisms used to kill and lyse their prey. The cooperative production of SMs and hydrolytic enzymes along with cell-to-cell contact killing via T3SS* and Tad-like complexes, are well-established predatory mechanisms. Future research in this topic will provide new information about less known aspects of M. xanthus predation and uncover new tools used by this microorganism to kill prey. In fact, recent studies have led to the identification of metals as additional weapons used by myxobacteria to kill their prey. Since fluctuations in metal concentration may occur in the habitats because of several activities, it is expected that they determine which population will predominate in the myxobacterial predatory interaction, which may have a significant impact on the environment and agriculture. Author contributions FC-M: Writing – original draft, Writing – review & editing. JP: Writing – original draft, Writing – review & editing. JM-D: Writing – original draft, Writing – review & editing, Funding acquisition. AM-M: Writing – original draft, Writing – review & editing, Funding acquisition. FM-T: Conceptualization, Writing – original draft, Writing – review & editing, Funding acquisition. Funding The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work has been supported by grant PID2020-112634GBI00 funded by MCIN/ AEI/10.13039/501100011033 (to AM-M and JM-D) and by the Marie Skłodowska-Curie Action (MSCA) grant LysM Ref. 101106411 (to FM-T and JM-D). Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could beconstrued as a potential conflict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Contreras-Moreno et al. 10.3389/fmicb.2024.1339696 Frontiers in Microbiology 06 frontiersin.org References Abby, S. S., and Rocha, E. P. C. (2012). The non-flagellar type III secretion system evolved from the bacterial flagellum and diversified into host-cell adapted systems. PLoS Genet. 8:e1002983. doi: 10.1371/journal.pgen.1002983 Akbar, S., Phillips, K. E., Misra, S. K., Sharp, J. S., and Stevens, D. C. (2022). Differential response to prey quorum signals indicates predatory specialization of myxobacteria and the ability to predate Pseudomonas aeruginosa. Environ. 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