The genus Mycobacterium is a highly diverse taxonomic entity, encompassing 170–200 recognized species, whose broad genetic content is reflected in its open pan-genome (16–19). This diversity spans a wide array of ecological lifestyles, beginning with organisms commonly found in environmental reservoirs such as water and soil, from which the M. tuberculosis complex (MTBC) evolved (3, 16, 17). At the end of the pathogenic spectrum are obligate pathogens, primarily the MTBC, which includes Mtb (5, 17) and Mycobacterium leprae (16). The remainder of the genus, referred to as non-tuberculous mycobacteria (NTM), consists mainly of environmental organisms that often exhibit opportunistic pathogenicity and can cause severe diseases such as pulmonary infections, lymphadenitis, or disseminated disease, particularly in immunocompromised individuals or those with pre-existing lung conditions (16, 19). Some of these organisms include the fast-growers Mycobacterium abscessus and Mycobacterium fortuitum; and the slow-growers Mycobacterium ulcerans, Mycobacterium avium, Mycobacterium marinum, Mycobacterium xenopi, Mycobacterium gordonae, and Mycobacterium kansasii (3, 19), among many others.

The ability of mycobacteria to establish persistence in hosts is central to their evolutionary success, although recent evidence indicates that the traditional estimate of MTBC latency in one-third of the global population is a substantial overestimate, with only a small minority of immunoreactive individuals (likely between 1% and 11%) harboring viable latent bacteria (8, 20, 21). Such long-term persistence underscores an intimate host–pathogen coevolution that has unfolded over millennia, driving bacterial adaptations finely tuned to the intracellular environment of macrophages. Genomic and functional analyses highlight key virulence systems that allow mycobacteria to resist immune clearance, modulate host cell biology, and disseminate within tissues (5, 9). In this sense, environmental persistence and host adaptation represent two facets of the same evolutionary strategy: survival under stress.

The remarkable ecological versatility of the Mycobacterium genus underpins its evolutionary transition from environmental saprophytes to opportunistic and obligate pathogens. Many traits that enable persistence in harsh environmental niches—such as slow growth, a hydrophobic and lipid-rich cell envelope, and robust biofilm formation—also confer intrinsic resistance to host immune defenses and antimicrobials (19, 22). Interactions with free-living amoebae (FLA) have likely served as an evolutionary crucible for intracellular adaptation, as NTM can survive within amoebal hosts, gaining protection from environmental stressors while preadapting to life inside macrophages (19, 23). Metabolic flexibility further supports this dual survival strategy: several environmental and opportunistic mycobacteria (including M. smegmatis, M. neoaurum, and M. fortuitum) are capable of degrading sterols and other complex organic compounds, and pathogenic species such as Mtb exploit this same metabolic machinery to utilize host cholesterol as a carbon source during infection (22, 24–28). Together, these ecological, physiological, and metabolic traits illustrate how the genus has been repeatedly shaped by environmental pressures, equipping mycobacteria with the adaptive versatility needed to thrive across the continuum from free-living saprophytes to highly specialized intracellular pathogens.

The transition from a generalist environmental lifestyle to a highly specific, host-adapted existence imposes intense selective pressures that drive diverse evolutionary trajectories within the Mycobacterium genus (29). Host environments exert intense selective forces that drive specific metabolic and genetic adaptations for long-term persistence. For instance, in a study by Vázquez et al. (30), experimental selection of M. bovis BCG in long-term macrophage culture resulted in adapted strains that exhibited improved glucose metabolism (i.e., a switch to glycolytic substrates), increased neutral lipid accumulation, and a lack of peptidoglycan production. Such adaptations correlated directly with increased survival within macrophages and prolonged residence in mice (30).

Obligate intracellular pathogens, such as members of the MTBC, inhabit stable, often sterile host environments, limiting opportunities for horizontal gene exchange and leading to a highly clonal population structure (29). These species evolve predominantly through gene loss and decay, a phenomenon known as reductive evolution, leading to genome downsizing (18, 29). M. leprae and M. ulcerans exemplify extreme cases of this trajectory. M. leprae possesses the highest proportion of pseudogenes known in any prokaryote or eukaryote, approximately 41% (18, 19, 31), resulting in dependence on the host for key nutrients due to the elimination of redundant biosynthetic pathways (31). Similarly, M. ulcerans evolved recently from the environmental species M. marinum through this process, becoming a niche-adapted specialist, evidenced by the proliferation of insertion sequences (IS2404, IS2606) and widespread chromosomal rearrangements (15).

Within-host evolution continues to shape pathogenic populations over shorter timescales, particularly under antimicrobial treatment (32). In the case of Mtb, the evolution and acquisition of drug resistance rely almost exclusively on chromosomal mutation and clonal spread, since this ancient pathogen lacks active horizontal gene transfer (HGT) and does not harbor mobile resistance elements (33–35). The Mtb genome shows no evidence of recombination, meaning that all resistance to antituberculosis drugs arises through spontaneously occurring mutations, typically single nucleotide polymorphisms (SNPs) (33–35). These variants emerge during within-host evolution, where the large bacterial population size [often exceeding 10⁹ colony-forming units (CFUs)] provides a substantial mutational reservoir (34, 35). Under antibiotic pressure, mutants that gain a fitness advantage expand and eventually become the dominant clone within the host before being transmitted onward (34, 35).

Drug resistance evolution in Mtb often follows a pattern of branched evolution, where multiple subpopulations carrying distinct mutations coexist during treatment (32). The spatiotemporal heterogeneity of antimicrobials and bacterial density within lesions, alongside phenotypic drug tolerance, influences this evolutionary path (32). Over time, purifying selection exerted by effective antimicrobials often leads to the dominance of a single, highly fit resistant clone—the “dominant lineage” model (32). Furthermore, recent genomic studies show that Mtb clinical isolates exhibit mutational signatures correlating with host immune environments, highlighting the host's direct role as an evolutionary filter (32).

Genomic comparison studies are essential for elucidating mechanisms of pathogenicity and for revealing the roles of mobile elements, HGT, and accessory gene content in shaping virulence traits. The introduction of genetic material via HGT is a critical mechanism driving the speciation and virulence of some mycobacteria (4, 30, 36). In contrast, the MTBC shows no evidence of ongoing recombination or active HGT, so its pathogenic diversification has occurred primarily through vertical inheritance, gene loss, and spontaneous chromosomal mutations (33–35). In this sense, mobile genetic elements such as insertion sequences (IS) are key drivers of bacterial genome plasticity, virulence, persistence, and drug resistance (18, 37). For instance, M. ulcerans acquired a large plasmid encoding polyketide synthases responsible for producing the necrotic macrolide toxin, mycolactone (15, 30). Moreover, the acquisition of epigenetic modifiers (e.g., a putative DNA methylase, DpnM) in M. abscessus increased pathogenic potential and antibiotic resistance in specific clones (38).

The distribution of specific gene families across the genus reflects adaptation to distinct ecological niches. For instance, virulent mycobacteria show marked expansions of the PE/PPE protein families, duplication, and specialization of ESX (type VII secretion system), and enrichment in complex lipid biosynthetic pathways. These genomic patterns directly link virulence evolution to the pressures of intracellular survival and immune evasion (3, 9).

Parametric genomic analyses have further identified numerous regions of probable foreign origin, often termed genomic or pathogenicity islands (39). These foreign DNA fragments, accounting for roughly 4.5% (199 kb) of the Mtb genome, frequently contain genes with putative or documented virulence functions (39). Examples include the Rv0986–Rv0988 operon, likely acquired from a γ-proteobacterium, which contributed to the emergence of the ancestral Mtb lineage as a successful intracellular pathogen (6). Other acquired regions code for critical adaptation factors, such as components required for anaerobic growth, reflecting adaptation for survival under reduced oxygen conditions, as found in granulomas (6). The espACD locus, which regulates secretion of the ESAT-6 virulence factor, also appears to have been laterally transferred into related species such as Mycobacterium decipiens (5).

In parallel, reductive evolution has been a defining feature of pathogenic specialization. The MTBC genome (4.4 Mb) is markedly smaller than that of its environmental relatives, such as M. marinum (6.6 Mb) and M. kansasii (6.4 Mb), reflecting the streamlining associated with an obligate intracellular lifestyle. This downsizing is thought to be an adaptation mechanism to the specialized niche of the mammalian host (6). Characteristic deletions, including the TbD1 region, which is lost in modern epidemic lineages (L2, L3, L4), have been linked to increased resistance to oxidative stress and hypoxia (9). Similarly, the loss of the crtEIB pigmentation locus in MTBC members signals adaptation away from environmental habitats and toward mammalian hosts (5). These deletions illustrate how genome contraction can serve as an adaptive response to a more specialized niche.

Finally, comparative analyses of virulence determinants underscore the refinement of secretion and surface-exposed systems during pathogenic evolution. The ESX pathways and the PE/PPE protein families have undergone repeated duplication and diversification (6). The ESX-1 system, a major virulence determinant required for macrophage spread and granuloma formation, is present in Mtb and in pathogenic slow-growing mycobacteria such as M. marinum, suggesting its acquisition was driven by selection for an intracellular niche (6, 17). Lineage-specific mutations within these systems continue to shape virulence phenotypes; for instance, variation in esxW has been associated with enhanced transmissibility in modern lineages, while truncations in esxM have been associated with altered macrophage migration and dissemination (9). Other mutations, such as deletions in fadB4 that increase hydrophobicity and promote intracellular replication, further exemplify ongoing adaptive refinement (9).

Viewed through an evolutionary lens, the genus Mycobacterium encapsulates the continuum from environmental opportunism to obligate pathogenicity. This progressive adaptation to intracellular life provides not only a framework for understanding bacterial evolution but also a window into how ancient interactions with phagocytic hosts forged conserved innate immune mechanisms. The study of mycobacteria across diverse host systems thus reveals how microbial pressures have shaped the architecture and complexity of innate immunity.

FLA are ubiquitous, cell-wall-free, unicellular eukaryotes found in environments like soil and water, and they feed on bacteria (40–42). Laboratory investigations commonly use the social soil amoeba Dictyostelium discoideum (Dd) and waterborne amoebae such as Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis (41, 43, 44). These amoebal groups demonstrate marked differences in their life cycles and responses to environmental stress (42, 45, 46). Social amoebae like Dd spend the majority of their existence as unicellular trophozoites, but in response to starvation or nutrient depletion, they initiate a multicellular developmental program (45). This process results in the formation of aggregates, a migrating slug, and ultimately a fruiting body containing individual spores in the sorus (45, 46). In contrast, solitary amoebae do not undergo multicellular development (40, 42). Instead, when exposed to adverse conditions like starvation, desiccation, or temperature shifts, they form highly resistant single-celled cysts. The cyst wall provides robust protection against environmental stress and can shelter internalized bacteria (40, 42, 46). A key distinction among FLA is their temperature tolerance. Acanthamoeba species can thrive at 32–37°C, a range that coincides with that of mammalian hosts (46), whereas most other FLA (including Dd) grow optimally between 15–25 °C and do not tolerate sustained higher temperatures. This thermotolerance makes Acanthamoeba one of the few environmental protozoan hosts capable of exerting selective pressure at mammalian-like temperatures, suggesting a natural training grounds for intracellular bacterial pathogens, including mycobacteria, allowing them to adapt to conserved eukaryotic processes found in macrophage-like environments (40, 46). These ecological constraints highlight the unique position of amoebae as environmental phagocytes, setting the stage for comparison with later-evolving hosts in which similar pressures shaped conserved defense strategies.

Within this framework, Dd has emerged as a powerful model for studying host–pathogen interactions. Often regarded as a “proto-macrophage,” Dd combines the simplicity of a unicellular organism with a conserved endocytic and signaling machinery that closely parallels that of animal phagocytes (10, 13, 47–51). As depicted in Figure 2, its ecological role as a professional phagocyte has driven the evolution of fundamental defense mechanisms conserved with those of animal innate immune cells, including macrophages and neutrophils (10, 48, 50, 52, 53). This conservation supports the hypothesis that amoebae served as an evolutionary “training ground” for intramacrophage pathogens such as mycobacteria (12, 49–51, 54).

Interactions between Dd and M. marinum, a close genetic relative of Mtb, illuminate several conserved cell-autonomous defense mechanisms (45, 50, 55–57). Amoebae engulf particles into phagocytic vacuoles, or phagosomes, which are normally intended to progress through an endosomal-lysosomal degradation pathway involving fusion with lysosomes and acquisition of enzymes, ultimately resulting in the intracellular digestion and destruction of the particle (45, 46). However, pathogenic mycobacteria have evolved mechanisms to subvert this endosomal-lysosomal maturation process to establish a mycobacteria-containing vacuole (MCV), within which they can proliferate (45, 46). Depending on the species and environmental context, interactions between mycobacteria and amoebae may result in bacterial killing, host cell death, or the establishment of a stable intracellular relationship, as seen with amoeba-resistant organisms (AROs) (42, 45, 46). In solitary amoebae such as Acanthamoeba, this intracellular persistence can be further reinforced during encystment, where surviving bacteria become enclosed within the cyst wall. This highly protective structure enhances resistance to desiccation and antimicrobial agents and acts as a “Trojan horse” that facilitates long-term environmental survival (58, 59).

At the cellular level, Dd employs an extensive repertoire of ancient, cell-autonomous defense mechanisms that mirror those of mammalian phagocytes. A central component is the divalent cation transporter Nramp1 (Slc11a1), a homolog of the mammalian macrophage resistance protein NRAMP1 (48, 60, 61). When delivered to the phagosomal membrane, Nramp1 exports metal ions (mostly Fe²⁺ and Mn²⁺) and limits bacterial access to essential nutrients (62, 63). Thus, amoebae lacking this transporter are markedly more permissive to M. avium infection (60, 61, 64).

In addition to iron deprivation, Dd utilizes zinc intoxication as a form of nutritional immunity. By elevating intravacuolar Zn²⁺ concentrations (via ZIP-family importers and ZnT-family transporters that regulate intracellular zinc), the amoeba creates a toxic environment that restricts mycobacterial survival. Consistent with this, Dd mutants unable to increase intracellular zinc levels—such as those lacking key ZnT transporters—show significantly impaired M. marinum intracellular growth compared to wild-type hosts (65). To counteract host-imposed zinc stress, M. marinum and related species upregulate the efflux transporter CtpC, and deletion of ctpC severely impairs intracellular growth (65, 66). Together, these strategies illustrate that manipulating metal availability—by both deprivation and poisoning—constitutes an evolutionarily ancient antimicrobial defense. Similar principles of metal-based nutritional immunity reappear in more complex hosts, although diversified through additional regulatory layers.

Although it does not possess canonical antimicrobial peptides (AMPs), Dd expresses small secreted proteins with antimicrobial activity, functionally analogous [i.e., saposin-like proteins (SAPLIPs), also called amoebapore-like peptides (Apls)] (67, 68). In addition, Dd exhibits a combination of conserved and lineage-specific elements in nutritional immunity and metal transport: while core pathways for iron and zinc homeostasis are evolutionarily conserved with mammals, other components represent amoeba-specific adaptations (60, 61, 69).

Phagocytic uptake in Dd also depends on conserved membrane receptors, such as the lysosomal membrane proteins LmpA and LmpB, which are homologous to the mammalian LIMP-2 and CD36 scavenger receptors (48). These proteins mediate bacterial internalization and regulate phagosomal acidification and proteolysis; their disruption increases susceptibility to M. marinum infection (48). Once internalized, pathogenic mycobacteria deploy the ESX-1 secretion system to damage the MCV through membranolytic effectors such as EsxA and phthiocerol dimycocerosate (PDIM), enabling partial escape into the cytosol (45, 54, 66, 70, 71). The host responds by coordinating the activation of membrane repair pathways involving the endosomal sorting complex required for transport (ESCRT) and the autophagy machinery (56, 70–72). The ubiquitin ligase TrafE orchestrates recruitment of these systems to damaged membranes, with ESCRT repairing small perforations while autophagy patches larger lesions (56, 70). Disruption of either process accelerates cytosolic escape of M. marinum, underscoring their cooperative importance in containing infection (56).

Intriguingly, mycobacteria exploit these same defenses to their advantage. M. marinum induces autophagy gene expression and recruits autophagosomes to the MCV but simultaneously inhibits autophagic flux in an ESX-1– and TORC1-dependent manner, thereby preventing bacterial degradation (72, 73). Cytoskeletal remodeling further shapes the infection outcome. Host proteins such as the small GTPase RacH and flotillin-like vacuolins regulate actin polymerization and vacuolar dynamics; loss of vacuolin isoforms restricts bacterial growth, whereas RacH deficiency enhances susceptibility (74, 75). Moreover, the ESX-1 system, along with EsxA, is also required for the bacteria's eventual exit from the amoeba, which occurs via a nonlytic, actin-dependent mechanism known as ejectosome-mediated ejection (47, 75, 76). Notably, the autophagic machinery actively ensures this nonlytic transmission process (13, 73). This non-destructive egress parallels actin-based cell-to-cell spread in macrophages, highlighting the evolutionary conservation of intracellular infection strategies. Thus, amoebae already display the core logic of intracellular confrontation that is reiterated, refined, and contextually expanded across invertebrate and vertebrate hosts.

Despite lacking canonical Toll-like receptors (TLRs), Dd expresses a large family of transmembrane proteins containing leucine-rich repeats (LRRs) that function analogously as pattern-recognition molecules (69, 77). Other components of its innate machinery, including nucleotide-binding domain–like receptors (NLRs) and autophagy-related proteins, demonstrate evolutionary conservation with animal innate immune signaling pathways (78–82). The highly conserved nature of these host cell defense pathways, which mirror those in mammalian macrophages, validates the concept of Dd as a model for cell-autonomous defenses (10, 13, 45, 48, 50). Moreover, although ancestral, the reactive oxygen species (ROS) machinery of Dd is functionally equivalent to the vertebrate oxidative burst (83, 84). The presence of these conserved effectors in amoebae provides a baseline for interpreting how later organisms diversified but retained the fundamental architecture of these responses.

The similarities extend beyond general cellular processes to include specific counter-strategies, such as the ESX-1-mediated phagosome damage and the host's use of ESCRT/autophagy repair mechanisms -including core components such as ESCRT-III subunits (e.g., Vps32/CHMP4), ESCRT-I (e.g., Tsg101), TrafE, and accessory proteins like ALIX- and nutritional immunity via metal trafficking (56, 60, 65, 71, 73). The shared virulence strategies and host responses suggest that the interaction between mycobacteria and professional phagocytes originated during their ancient co-evolution in environmental niches (13, 49, 51, 71). Furthermore, Dd utilizes a collaborative exclusion mechanism during its multicellular developmental cycle to exclude infected cells, representing an ancient, multicellular form of innate immunity and clearance (10).

However, Dd also reveals evolutionary divergences that highlight the evolution of immunity. Unlike metazoans, Dd lacks a homolog of the p38 mitogen-activated protein kinase (MAPK) pathway (85). However, it possesses p38-like stress-activated protein kinase (SAPKα) and ERK1/2 cascades that regulate stress responses, cytoskeletal dynamics, and chemotaxis via G protein-coupled rather than receptor tyrosine kinase signaling (86–88). Similarly, while the insulin–FOXO signaling axis is functionally conserved, its architecture in Dd is only partially homologous: the organism retains the ancestral PI3K–Akt–TOR nutrient-sensing module. Still, it lacks the dedicated insulin/IGF ligands and receptors that define the metazoan pathway (89). Moreover, Dd has conserved biochemical systems for ROS, homologous to those in mammals, while lacking a canonical Nitric Oxide Synthase (NOS) enzyme homologous to those found in mammals (90–92).

Finally, at the effector level, Dd can produce DNA extracellular traps (ETs), a mechanism previously thought to be restricted to multicellular organisms, demonstrating that this antimicrobial strategy evolved well before the origin of metazoans (14). DNA ET formation in Dd occurs only during the multicellular slug stage, where specialized Sentinel cells function as an ancient innate immune system capable of releasing DNA ETs. In contrast, Dd lacks canonical inflammasome components and pyroptotic cell-death pathways (93). These absences also clarify which immune innovations emerged later in metazoans, allowing direct comparison with the expanded innate repertoires found in C. elegans, D. melanogaster, and zebrafish.

Together, these features position Dd as a living window into the evolutionary origins of innate immunity. Its genome encodes conserved effectors for phagocytosis, metal trafficking, autophagy, and ROS-mediated killing, yet lacks the complex cytokine networks and multicellular coordination that characterize animal immunity. This balance of conservation and simplicity makes Dd an invaluable comparative model for understanding how ancient, cell-autonomous defenses were progressively co-opted and expanded into the integrated innate immune systems of metazoans.

C. elegans is a ubiquitous nematode that lives in soil and feeds on bacteria and is a widely used model organism for studying genetics, immunology, and host-pathogen interactions (94–97). Several bacterial species—including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis—cause intestinal infection in C. elegans, leading to pathogen proliferation in the gut lumen, epithelial damage, and nematode death (94, 98). As an invertebrate, C. elegans relies exclusively on its innate immune system and lacks both adaptive immunity and professional immune cells such as macrophages (95, 96, 99). Its defense, therefore, depends mainly on epithelial immunity, particularly in the intestine, which is composed of only 20 non-renewable cells that act as both physical and immunological barriers (11, 96). Compared with unicellular amoebae, C. elegans thus illustrates how ancient cell-autonomous defenses are redeployed within a simple metazoan tissue context.

The relationship between C. elegans and mycobacteria provides a crucial model for studying the pathogenesis of TB and other mycobacterial infections (97). For instance, infection with pathogenic M. marinum results in high morbidity and mortality (e.g., >80% mortality within 24 h), whereas the non-pathogenic M. smegmatis causes minimal mortality (< 15%) (97). Pathogenic M. marinum causes irreversible pathological changes in the nematode, including loss of pigmentation and “bagging” (embryo retention leading to the death of the adult worm, which is a stress response) (97). These conserved pathological outcomes and host responses indicate that the underlying pathogenic mechanisms exploited by M. marinum in the simple host are relevant to mammalian infection (Figure 2) (97). Similar virulence hierarchies and pathological signatures for M. marinum vs. non-pathogenic species are also observed in amoebae, flies, and zebrafish, underscoring that core mycobacterial strategies are conserved across very distant hosts.

The fruit fly D. melanogaster has emerged as a crucial invertebrate model for dissecting the fundamental mechanisms of innate immunity and host-pathogen interactions, particularly in the context of mycobacterial infections and TB pathogenesis (124–128). The fly's reliance exclusively on its innate immune system, coupled with its genetic tractability and the high degree of conservation of immunological and disease-related genes with mammals (approximately 75% of human disease-causing genes have homologs in the fly genome), makes it highly valuable for this comparative approach (124–131). Infection studies primarily utilize M. marinum, a close genetic relative of Mtb that causes a TB-like disease in cold-blooded animals, as a model for TB (125, 128, 129, 132). More recently, emerging pathogens such as M. abscessus have also been used to study mechanisms of virulence and host adaptation (126, 128, 133). In this sense, D. melanogaster occupies an intermediate position between nematodes and vertebrates, retaining an innate-only immune system but adding professional phagocytes and complex organ physiology that more closely approximate mammalian infection biology.

The larvae of the greater wax moth, Galleria mellonella, have emerged as a powerful non-mammalian host model for studying mycobacterial pathogenesis and innate immunity (150, 151). This system is cost-effective, ethically favorable, and physiologically relevant, relying exclusively on innate immunity and thus eliminating the confounding influence of adaptive responses (150–152).

G. mellonella can be infected with virulent members of the MTBC, including Mtb H37Rv and M. bovis BCG (153–155), as well as numerous NTM like M. abscessus, M. marinum, and M. fortuitum (154–158). Furthermore, G. mellonella experiments can be performed at 37 °C, the optimal temperature for many human pathogens, allowing for the study of mycobacterial physiology under conditions relevant to human disease (150, 154). This versatility, especially the ability to use virulent MTBC strains, provides an advantage over models like Drosophila and zebrafish, which often rely on surrogates such as M. marinum (151, 154). Together with Drosophila and zebrafish, G. mellonella therefore helps span the continuum from invertebrate to vertebrate hosts, enabling direct comparison of mycobacterial virulence strategies and host defenses across increasing levels of immune and anatomical complexity.

The early life stages of zebrafish possess a functional innate immune system, active from approximately 1 day post-fertilization, composed primarily of macrophages and neutrophils (11, 172, 179–183). Zebrafish macrophages are the first immune cells to arrive at the infection site and efficiently engulf the invading mycobacteria (179, 180, 182, 184). However, they possess a dichotomous role: while they restrict extracellular mycobacterial proliferation (172, 182), they also serve as replication niches and vehicles for dissemination, transporting mycobacteria to new tissues and thereby establishing secondary infection foci (11, 172, 174, 180, 182, 185, 186).

Pathogenic mycobacteria actively manipulate macrophage recruitment and polarization. The virulence lipid PGL attracts Ccr²⁺ monocytes that are permissive to bacterial growth (172, 174, 180), whereas PDIM masks TLR ligands, allowing the bacteria to evade detection by microbicidal, iNOS-expressing macrophages (66, 172, 174). These strategies enable the pathogen to skew early host responses toward a phenotype favorable for intracellular persistence.

Neutrophils, by contrast, exhibit distinct roles in the innate defense landscape. They are recruited to infection sites but rarely engulf extracellular M. marinum (180, 182). Instead, they perform efferocytosis—engulfing and clearing dead, infected macrophages—to prevent necrotic tissue damage and bacterial spread (180, 182, 187). A subset of neutrophils is capable of killing phagocytosed mycobacteria through the production of ROS and nitric oxide (NO) via NADPH oxidase activity (172, 174, 180, 182). Enhancing NO production by stabilizing Hypoxia-Inducible Factor 1-alpha (Hif-1) signaling in neutrophils has been shown to modulate susceptibility and reduce bacterial burden, highlighting the conserved role of hypoxia-responsive pathways in antimicrobial defense (172, 174, 186, 188, 189).

Optically transparent zebrafish larvae have been fundamental in revealing that granuloma formation is an active, dynamic process driven by both the host and the pathogen, initiated entirely within the context of innate immunity (172, 174, 180, 190). These structures arise when infected macrophages aggregate to form inflammatory lesions that serve both protective and pathogenic roles (11, 180, 182, 190).

The formation and expansion of early granulomas are actively driven by mycobacterial virulence (172, 174, 180, 186). The ESX-1 is essential for this process (172, 186), mediating programmed cell death (necrosis and pyroptosis) of infected macrophages (172, 174, 182, 186). Mutational studies in zebrafish have shown that early ESX-1 effectors, including those encoded in the RD1 locus and the accessory proteins EspK and EspL, are crucial for macrophage aggregation and early granuloma formation in M. marinum, although EspK appears dispensable for virulence in M. bovis and Mtb (66). This controlled cell death releases both bacteria and intracellular contents, attracting neighboring macrophages that engulf the debris and become secondarily infected. This process can also involve cell fusion between infected and uninfected macrophages (11, 172, 174, 180, 182, 186, 187, 191). The resulting cyclic recruitment and infection amplify bacterial dissemination and promote the development of multicellular granulomas.

Chemokine signaling plays a pivotal role in this process. The mycobacterial effector EsxA induces the expression of host matrix metalloproteinase 9 (MMP-9) in adjacent epithelial cells, which acts as a chemotactic cue for macrophage recruitment, thereby facilitating granuloma maturation and expansion (172, 185, 186, 190). This mechanism highlights how mycobacteria subvert host chemokine pathways to recruit permissive phagocytes and sustain infection.

Zebrafish models have also elucidated the molecular and cellular pathways underlying innate immune recognition and effector responses. TLR signaling, mediated by the adaptor molecule MyD88, is essential for the induction of proinflammatory cytokines such as IL-1β and TNFα and provides protection during early pathogenesis (181, 183, 192). MyD88 deficiency leads to increased susceptibility and accelerated granuloma formation (174, 179, 181), underscoring the protective role of canonical pattern-recognition receptor pathways in early infection (181). Although TNF is necessary for macrophage microbicidal activity and granuloma stability, zebrafish studies reveal that early granuloma formation can proceed through TNF-independent mechanisms, with metalloproteinase 9 (MMP9) and Type 2 STAT6-driven pathways instead guiding epithelioid transformation and maturation, paralleling findings in mammalian models (66).

The JAK/STAT pathway in zebrafish is conserved and plays a pivotal role in the innate immune defense against mycobacteria, not only through classical cytokine signaling and immune cell recruitment but also by modulating host lipid metabolism within infected phagocytes (82, 193, 194). This dual function exemplifies how zebrafish integrate metabolic and immune responses to limit bacterial growth and inflammation.

Inflammasome signaling also plays a critical role in host defense. Activation of the Asc-dependent inflammasome promotes IL-1β secretion and pyroptotic cell death, thereby restricting intracellular bacterial growth and limiting granuloma expansion (180, 195). Conversely, regulatory NOD-like receptors such as Nlrc3-like act as negative modulators of inflammation. Loss of NLRC3-like enhances resistance by boosting inflammasome activation and proinflammatory cytokine production in infected macrophages (195). These findings illustrate the delicate equilibrium between inflammatory activation and resolution required to maintain effective yet non-pathogenic immunity.

Moreover, the autophagy machinery is a crucial host-protective defense mechanism against M. marinum (11, 172, 174, 180, 191, 196). Selective autophagy receptors (SLRs), such as Optn and p62, promote host resistance against M. marinum infection (196, 197). The DNA-damage-regulated autophagy modulator 1 (Dram1) links mycobacterial recognition via the TLR-MyD88 pathway to the autophagic defense mechanism, promoting the fusion of autophagosomes and lysosomes and enhancing host resistance (172, 174, 180, 196, 197). Nonetheless, pathogenic mycobacteria retain partial resistance to these defenses (197). M. marinum can inhibit phagosome-lysosome fusion and even survive within acidified compartments by expressing MarP, a virulence determinant required for acid tolerance and persistence (66, 198). These findings echo those from Dd and Drosophila, indicating that mycobacteria repeatedly target autophagy and membrane damage–repair pathways across host phyla, while hosts reuse a conserved set of stress- and danger-sensing modules to contain intracellular infection.

In addition, the zebrafish genome encodes orthologs of all key ESCRT components involved in endosomal sorting, membrane remodeling, and repair (199, 200). This conservation suggests that ESCRT-mediated membrane repair and vesicular trafficking play essential, though still underexplored, roles in the cellular response to mycobacterial infection, potentially analogous to their functions in mammalian macrophages.

A balanced inflammatory response is crucial for disease outcome. Both insufficient (hypo-inflammatory) and excessive (hyper-inflammatory) responses promote susceptibility (172, 174, 195). Overproduction of tumor necrosis factor (TNF) or loss of negative regulators such as Ptpn6 or Nlrc3-like precipitates necrotic macrophage death, fueling bacterial expansion (172, 174, 195). High TNF levels are particularly detrimental, as they trigger programmed necrotic cell death (necroptosis) in macrophages, leading to bacterial growth and spread (172, 174). Similarly, the CXCR3–CXCL11 axis, which orchestrates macrophage chemotaxis, is often exploited by mycobacteria to enhance dissemination; its disruption limits granuloma growth and bacterial spread (172, 174, 185).

In summary, the zebrafish larval model, relying entirely on innate immunity, has demonstrated that mycobacteria function as evolutionary drivers by subverting basic host innate mechanisms, exploiting macrophages for dissemination, hijacking chemokine signals for recruitment of permissive cells, actively driving pro-necrotic cell death to expand infection, and tolerating microbicidal strategies like phagolysosome acidification (172, 174, 180, 198). These host–pathogen interactions recapitulate the core immunopathological processes observed in vertebrate TB and underscore the zebrafish as a unique bridge between invertebrate models and mammalian immunity (177, 179, 192) (conceptual overview in Figure 3).