The terminal organelle of M. pneumoniae, also referred to as the attachment organelle or tip structure (Miyata and Hamaguchi, 2016), orchestrates both cytoadherence to host epithelia and gliding motility, which are essential for tissue dissemination (Krause et al., 2018; Widjaja et al., 2020). This specialized polar membrane protrusion has a bipartite architecture: surface-exposed nap-like proteins facilitate host-pathogen interactions, and an intricately organized internal structure enables the generation of mechanical force (Nakane et al., 2015). The adhesion machinery of terminal organelles comprises four evolutionarily conserved surface proteins that mediate gliding and adhesion mechanisms: P1 (MPN141), P90/P40 (encoded by MPN142 as proteolytic cleavage products), and P30 (MPN453). Spatial mapping has demonstrated that the P1 adhesin complex, comprising the P1 and P90/P40 subunits, is strategically localized at the apical tip of the organelle, forming a rigid membrane anchor. Whereas P30 dynamically associates with the complex periphery to regulate force transduction during gliding motility (Nakane et al., 2015; Miyata and Hamaguchi, 2016; Zuo et al., 2024). Internally, the terminal organelle exhibits a sophisticated internal architecture comprising an electron-dense core structure that maintains structural integrity through three specialized components: the terminal button, paired plates, and bowl complex. This core is enveloped by a translucent matrix region. This highly organized core scaffold provides mechanical stability and serves as an assembly platform for the adhesion machinery. The terminal button, situated within the inner peripheral membrane region, is primarily composed of three core proteins: P65 (MPN309), HMW2 (MPN310), and HMW3 (MPN452). Adjacent to this structure, the paired plates system has a stratified organization consisting of four distinct components: HMW1 (MPN447), HMW2 (MPN310), CpsG (MPN066), and HMW3 (MPN452). The paired plates exhibit distinct protein compositions across their morphological subtypes: the thin plates are primarily composed of HMW1 and CpsG, whereas the thick plates are enriched in HMW2. The plate complex occupies a posterior position within the organelle architecture, where the thick and thin plates form an integrated unit through direct adhesion. This composite structure establishes stable connections with bowl-shaped structural elements through precise molecular interactions. Structural analysis of the coiled HMW2 protein revealed critical functional domains: its N-terminal region mediates attachment to the terminal button complex, whereas the C-terminal domain facilitates integration with the bowl structure. This bipolar molecular configuration suggests that HMW2 serves as a key architectural element that bridges distinct organellar components (Kawamoto et al., 2016). The bowl complex itself is composed of seven core components: TopJ (MPN119), P24 (MPN312), Lon protease (MPN332), P200 (MPN567), MPN387, P41 (MPN311), and HMW2 (MPN310) (Nakane et al., 2015; Miyata and Hamaguchi, 2016). MPN387 is specifically localized at the interface between the plates and bowl, suggesting a potential coordinating function. The translucent area occupied by a rigid, electronically transparent substance transmits the force generated by the bowl complex to paired plates (Balish et al., 2003; Kawamoto et al., 2016; Figure 1). As discussed above, the terminal organelle is a highly organized and functionally coordinated structure, and the specific roles of its components are further detailed below.

Sialylated oligosaccharides (SOS) are the terminal glycan modifications that cap the outer branches of glycoconjugates on cell surface-associated glycoproteins and glycolipids. They also serve as essential structural components of mucosal glycoproteins within mucus layers (Miyata, 2008; Kasai et al., 2013; Balish, 2014). Glycoproteins, a class of macromolecules ubiquitously present in biological systems, are strategically localized on the plasma membranes, within the extracellular matrix, and throughout bodily fluids. They exhibit functional diversity across processes such as molecular recognition, immune modulation, and intercellular communication. Among these functions, cell adhesion is primarily mediated by two major families of glycoproteins: integrins and cadherins (Chastney et al., 2025; Jiang et al., 2025). In microbial infection, SOS, which serve as key receptor molecules on the surface of host cells and within the mucus layer, are specifically recognized by a variety of pathogenic microorganisms and exploited to facilitate adhesion and invasion (Table 1; Spahich and St Geme, 2011; Hentrich et al., 2016; Andreae et al., 2018; Marc et al., 2018; Hong et al., 2022; Pronker et al., 2023; Aberg et al., 2024; Ni et al., 2024). Similarly, M. pneumoniae utilizes the terminal organelle to bind to SOS in a “lock-and-key” pattern to initiate its adhesion. Both α-2,6- and α-2,3-sialyllactose support the adherence of M. pneumoniae, of which α-2,3-sialyllactose has a relatively high affinity (Williams et al., 2020; Vizarraga et al., 2021). The interaction between M. pneumoniae and SOS on glycoproteins distributed across multiple organs and tissues may represent a fundamental pathogenic mechanism contributing to the multi-system involvement in M. pneumoniae infections.

In M. pneumoniae, multiple adhesin proteins localized within the terminal organelle contribute to its attachment to host cells. Among them, P1 functions as the primary adhesin and plays a central role in mediating the bacteria-host interaction. Strains lacking P1 entirely lose their adhesion capability and consequently become non-pathogenic. Through the action of the P1 adhesin, M. pneumoniae attaches to host epithelial cells and localizes within intercellular spaces, thereby evading ciliary clearance and macrophage phagocytosis. This allows the pathogen to release virulence factors and inflict damage on host tissues (He et al., 2016; Widjaja et al., 2020). Although P1 adhesin is an essential component for both adhesion and gliding, it can mediate binding between M. pneumoniae and host receptors only when correctly localized on the terminal organelle (Widjaja et al., 2020). One accessory protein, the DnaJ-like chaperone TopJ, which contains a J-domain, an acidic- and proline-rich region (APR), and a C-terminal domain, efficiently facilitates the delivery of P1 to the surface of the terminal organelle (Cloward and Krause, 2010, 2011). In addition, the P90/P40 adhesins of M. pneumoniae also exhibit specific binding affinity to the terminal sialylated glycans of host oligosaccharides. P90/P40 proteins associate with P1 adhesin to form an adhesin complex that further enhances the adhesive properties of M. pneumoniae (Kenri et al., 2008; Yamazaki and Kenri, 2016; Kenri et al., 2019; Vizarraga et al., 2020; Marseglia et al., 2024). P30, another membrane protein, has been shown to localize at the distal end of the terminal organelle. Research suggested that P30, with a homologous sequence of P1 in a certain domain, implies a potential beneficial association for adhesion (Miyata and Hamaguchi, 2016). Notably, P30 and P65 exhibit a close functional and spatial relationship, wherein P65 interacts with the internal domain of P30 to mediate precise contact between the terminal button and the frontal region of the structural layer (Miyata and Hamaguchi, 2016). Furthermore, the P41 protein governs the polar localization of the terminal organelle and directs the positioning of P24 within the adhesion complex. Together, P41 and P24 ensure proper spatial organization of the adhesin machinery, thereby facilitating effective host adhesion (Hasselbring and Krause, 2007; Krause et al., 2018).

Additionally, the HMW1, HMW2, and HMW3 proteins can also contribute to the adhesion of M. pneumoniae to the airway epithelium (Page and Krause, 2013). The loss of HMW1 results in a deletion at the 3’ end of the p30 gene and disrupts the functional association among HMW2, HMW3, and P65. This disruption consequently impairs the clustering of P1 at the cell’s polar end, thereby weakening the adhesive capacity. Furthermore, dysfunction of HMW3 suppresses P65 expression and causes its diffuse localization, preventing proper positioning of the P1 adhesin at the terminal organelle and further reducing adhesion efficiency (Hahn et al., 1998; Willby and Krause, 2002).

While the terminal organelle is essential for primary adherence, M. pneumoniae also expresses adhesion factors that function independently of this structure. Notably, several surface-exposed glycolytic enzymes have recently been identified as non-classical adhesins involved in host-pathogen interactions (Grundel et al., 2015). These enzymes, including lactate dehydrogenase (LDH), phosphoglycerate mutase (PGM), pyruvate kinase (PYK), glyceraldehyde-3-phosphate dehydrogenase (GapA), transketolase (TKT), and pyruvate dehydrogenase subunits A to C (PdhA-C), have been shown to interact with components of the extracellular matrix (ECM), thereby contributing to both adhesion and invasion processes of M. pneumoniae (Thomas et al., 2013; Gründel et al., 2015; Grundel et al., 2016; Grimmer and Dumke, 2019). In addition, elongation factor Tu (MPN665, EF-Tu), a well-known moonlighting protein with multiple non-overlapping functions, has been identified on the surface of M. pneumoniae in several studies (Dallo et al., 2002; Balasubramanian et al., 2008). Besides its canonical role in cytoplasmic biosynthesis and metabolism, EF-Tu has also been shown to bind fibronectin (Fn), thereby mediating the interaction between M. pneumoniae and the ECM (Dallo et al., 2002; Balasubramanian et al., 2008; Grimmer and Dumke, 2019). The ECM consists of two major components: the interstitial connective tissue matrix, which provides structural support, and the basement membrane, a specialized layer that regulates cellular organization and differentiation (Karamanos et al., 2021). Interactions between host cells and the ECM are primarily mediated by laminin (Ln) within the basement membrane, as well as by interstitial matrix proteins such as Fn, vitronectin (Vn), and fibrinogen (Grundel et al., 2016). Integrins, a ubiquitous family of transmembrane receptor proteins expressed on human cells, recognize Arg-Gly-Asp (RGD) motifs within these ECM components and facilitate cellular anchorage through bidirectional mechanotransduction (Sutherland et al., 2023). When surface-associated glycolytic enzymes of M. pneumoniae bind to these ECM proteins, they function as bacterial ligands that interact with host cell receptors, thereby enhancing the adhesion of M. pneumoniae to respiratory epithelial cells (Bao et al., 2023; Pang et al., 2023).

Whether M. pneumoniae utilizes the terminal organelle or surface-associated glycolytic enzymes for adhesion, integrins on the host cell membrane play a central role in mediating this interaction. When M. pneumoniae adhesins engage with integrins, they initiate the assembly of “focal adhesion” (FA) complexes. FA anchors to intracellular actin filaments through adaptor proteins such as talin, vinculin, and paxillin, establishing a stable adhesion interface (Kanchanawong and Calderwood, 2023; Matrullo et al., 2025). This integrin-mediated FA structure has been demonstrated to be critical for static adhesion during host-pathogen interactions (Figure 2; Jones et al., 2019; Kamranvar et al., 2022).

The adhesion of M. pneumoniae to host cells initiates a cascade of events critical for bacterial survival and persistence. A key pathogenic strategy involves the production of hydrogen peroxide (H₂O₂), which induces cytoskeletal reorganization by modulating effector proteins. This process depletes host nutrients and elevates cellular oxygen demand. Importantly, M. pneumoniae undergoes metabolic adaptation by utilizing host-derived glycerol as a carbon and energy source. Enhanced aerobic respiration leads to the accumulation of cytotoxic levels of H₂O₂ (Blotz and Stulke, 2017). Cytoskeletal reorganization of host epithelial cells appears pivotal in producing cytotoxic substances and facilitating bacterial dissemination. Notably, the adhesion of M. pneumoniae not only triggers cytoskeletal rearrangement but also facilitates its translocation across the bronchial mucosal barrier. This process is accompanied by sustained H₂O₂ release, leading to progressive pathological alterations in bronchial epithelial cells, including cellular edema, necrosis, intercellular adhesion, decelerated ciliary motility, and structural deformation (Blotz and Stulke, 2017). Although the precise mechanisms by which M. pneumoniae induces cytoskeletal remodeling remain incompletely defined, current evidence indicates that FA-mediated mechanotransduction plays a pivotal role. Specifically, pathogen adhesion activates focal adhesion kinase (FAK) and Src family kinases (Matrullo et al., 2025), which subsequently initiate Rho GTPase signaling cascades that drive cytoskeletal reorganization (Bement et al., 2024). Recent findings further suggest that force transmission from FAs to the cytoskeleton may represent the proximal mechanical signaling event initiating these structural changes. Notably, such mechanochemical coupling appears transient, potentially explaining the dynamic nature of cytoskeletal rearrangement during infection (Bement et al., 2024; Matrullo et al., 2025).

In this context, Glycerol-3-phosphate (G3P) serves as a critical carbon source at the adhesion site of M. pneumoniae. It is derived from free glycerol, which is transported into the cytoplasm via the glycerol facilitator (GlpF) and subsequently phosphorylated by glycerol kinase (GlpK). The lipoproteins MPN133 and MPN284 might also participate in the delivery of glycerol to GlpF. Additionally, glycerophosphodiesterase (GlpQ) converts glycerophosphocholine (GPC) into an alternative carbon source, which is imported via the recently identified transport protein GlpU (MPN421) (Halbedel et al., 2007; Grosshennig et al., 2013). The metabolism of G3P by glycerol-3-phosphate oxidase (GlpD) produces H₂O₂, which benefits bacterial survival but harms the host (Schmidl et al., 2011; Grosshennig et al., 2013).

As a virulence factor, H₂O₂ plays a critical role in eliciting an oxidative stress response during M. pneumoniae infection. Due to the inadequacy of key antioxidant enzymes, such as superoxide dismutases and catalases, M. pneumoniae cannot effectively detoxify H₂O₂. As a major reactive oxygen species (ROS), pathologically accumulated H₂O₂ induces cellular oxidative stress, damages biomacromolecules, disrupts energy metabolism, and activates inflammatory responses (Park, 2013). For M. pneumoniae, recent studies have shown that H₂O₂ can accelerate the shedding of M. pneumoniae-infected cells. H₂O₂-induced oxidative DNA damage, including base oxidation and single- and double-strand breaks, triggers hyperactivation of poly (ADP-ribose) polymerase 1 (PARP1). This excessive DNA repair response depletes cellular nicotinamide adenine dinucleotide (NAD⁺) reserves, subsequently impairing mitochondrial ATP production. The ensuing bioenergetic collapse drives cells into a parthanatos death pathway, a PARP1-dependent programmed necrotic process characterized by irreversible metabolic failure (Yamamoto et al., 2019). ADP-ribosylation is a biochemical process in which the ADP-ribosyl group is transferred from NAD+ to specific amino acid residues on target proteins by ADP-ribosyl transferase (ADPRT). This reaction is catalyzed by several bacterial exotoxins, which disrupt macromolecular function and homeostasis (Krueger and Barbieri, 1995). Such disruptions facilitate the translocation of apoptosis-inducing factor (AIF) to the nucleus, ultimately resulting in apoptosis and cell death. Although the shedding of infected cells can reduce the infectivity of M. pneumoniae (Liesman et al., 2014), another study suggested that M. pneumoniae mitigates exogenous H₂O₂-induced shedding of infected epithelial cells by closely adhering to host cells and depleting cytosolic NAD⁺, which serves as the substrate for PARP1 activity (Figure 4; Yamamoto et al., 2019).

The production and pathogenic activity of M. pneumoniae community-acquired respiratory distress syndrome toxin (CARDS TX) are mechanistically coupled with its adhesion process (Kannan et al., 2010). Substantial evidence reveals the coordinated upregulation of CARDS TX expression and key adhesins, with spatial colocalization observed at the terminal organelle. This functional synergy suggests that toxin deployment is spatially and temporally coordinated with host cell attachment, potentially amplifying cytotoxic effects through membrane-proximal action (Kannan et al., 2010; Techasaensiri et al., 2010). The expression of the CARDS TX gene, regulated by the mpn372 locus, serves dual functions as both a secreted cytotoxin and an adhesin. CARDS TX is a 591-amino-acid protein structurally divided into an N-terminal mART (D1 domain) and a C-terminal β-trefoil structure (D2 + D3 domain) (Figure 5A; Kannan et al., 2014; Becker et al., 2015).

CARDS TX facilitates host cell invasion through a dual-targeting mechanism that engages both protein receptors and membrane lipids. Notably, it can bind to surfactant protein-A (SP-A) and annexin A2 on the host cell surface, thereby initiating clathrin-mediated endocytosis (Kannan and Baseman, 2006). Furthermore, the C-terminal domains selectively interact with phosphatidylcholine (PC) and sphingomyelin (SM), lipid components enriched in membrane microdomains, thereby stabilizing bacterial adhesion and promoting toxin internalization (Becker et al., 2015). This coordinated engagement of receptors and lipids facilitates close bacterial-host interactions, enabling the localized delivery of CARDS TX at membrane interfaces and ultimately inducing cytotoxic effects (Kannan and Baseman, 2006; He et al., 2016).

As a virulence factor, M. pneumoniae exhibits both ADPRT and vacuolating activities. Key residues in the N-terminal (R10, D12, R14, H36) and mid-region (S49-T50-S51) bind NAD+ are critical for ADP ribosylation (Figure 5B; Krishnan et al., 2013; Kannan et al., 2014; Becker et al., 2015). As described above, this ADP ribosylation disrupts cell function (Kannan et al., 2014; Somarajan et al., 2014), leading to cytopathic effects (CPE) and eventual cell death (Figure 4; Krueger and Barbieri, 1995). The C-terminal region (Y571-F591) of CARDS TX mediates the binding of the toxin to the mammalian cell surface and induces subsequent endocytosis (Figure 5C; Krishnan et al., 2013; Kannan et al., 2014; Becker et al., 2015). Similar to the purified vacuolating cytotoxin (VacA) protein of H. pylori, internalization is essential for its vacuolation activity. CARDS TX-induced vacuoles are enriched with Rab9 GTPase, which mediates vacuole formation via the Golgi membrane (Johnson et al., 2011). While the vacuolation mechanism in M. pneumoniae remains unclear (Kannan and Baseman, 2006; Kannan et al., 2014; Somarajan et al., 2014; Becker et al., 2015), studies have highlighted similarities between CARDS TX and H. pylori VacA toxins (Table 2, Figure 4; Ansari and Yamaoka, 2020).

The adhesion ability of M. pneumoniae appears to be a prerequisite for activating the immune response during infection. Chaudhry et al. (2005) identified immunodominant regions in the C-terminus of P1 protein (residues 1,125–1,131 and 1,382–1,394) that trigger an immune response once the protein attaches to the mucosal surface. Hoek et al. (2005) demonstrated that adherent M. pneumoniae induces IL-4 production in murine basophils, whereas non-adherent strains fail to elicit significant cytokine responses. This disparity is primarily attributed to the higher abundance of P1 protein in adherent M. pneumoniae. Similarly, Shimizu et al. (2011) found that the wild-type strain of M. pneumoniae stimulates the production of inflammatory cytokines, including IL-1β and tumor necrosis factor-α (TNF-α). In contrast, a mutant strain lacking adhesion ability failed to induce cytokine production.

Besides, M. pneumoniae adheres to the host airway through LAMP, whose lipid portion is recognized by Toll-like receptor (TLR) complexes predominantly expressed on respiratory epithelial cells and immune cells (Malik et al., 2023), activating cellular signal transduction pathways (Zuo et al., 2009). Following adhesion, M. pneumoniae interacts with TLR4 to induce macrophage autophagy, enhancing the synthesis and secretion of pro-inflammatory cytokines, including IL-1β, IL-6, and IL-8 (Shimizu et al., 2014). Furthermore, the recognition of LAMP by TLRs triggers an inflammatory response characterized by lymphocyte, neutrophil, and occasionally eosinophil infiltration, promoting IL-1β, IL-6, IL-8, and TNF-α, as well as inflammatory mediators like ROS (Into et al., 2007; Shimizu et al., 2007, 2008; He et al., 2009).

Although adhesion supports the survival of M. pneumoniae in the host, the immune response it triggers acts as a barrier to pathogenic progression. To counter this, M. pneumoniae adhesion proteins employ various immune evasion strategies to mitigate host defenses (Ferreira et al., 2006; Sluijter et al., 2009; Schmidl et al., 2010; Yu et al., 2020). After adhesion to epithelial cells, the adhesins of M. pneumoniae are phosphorylated by protein kinase PrkC, producing multiple phosphorylated forms that aid in immune evasion (Schmidl et al., 2010; Widjaja et al., 2020). Sluijter et al. (2009) demonstrated that the RecA protein homolog encoded by mpn490 promotes gene exchange between homologous DNA sequences (mainly RepMP) in M. pneumoniae, leading to variations in surface adhesins and facilitating immune evasion. Another study revealed that abundant complement factor H is expressed at the site of M. pneumoniae colonization, which strengthens adherence between mycoplasmas and tracheal epithelial cells. Moreover, M. pneumoniae can bind factor H through specific binding proteins, such as EF-Tu, PDH-B, and PDH-A, thereby mimicking host cells to regulate complement activation and evade immune responses (Ferreira et al., 2006; Yu et al., 2020).