Microbiology
Type III Secretion System
A 3.5 MDa molecular syringe — Salmonella, Yersinia, Shigella inject ~25 effector proteins into host cells
The Type III secretion system (T3SS), or injectisome, is a ~3.5 MDa transmembrane needle complex assembled from ~20 conserved proteins that pathogenic Gram-negative bacteria use to inject effector proteins directly into the cytoplasm of a eukaryotic host cell. The channel spans the inner membrane, peptidoglycan, outer membrane, an ~80 nm extracellular needle, and the host plasma membrane in a single ~2.5 nm-wide tunnel. About ~10 of the structural proteins are homologous to the bacterial flagellum, indicating shared evolutionary ancestry. Yersinia Yops, Salmonella SPI-1 effectors, Shigella Ipa proteins, enteropathogenic E. coli Tir, and Pseudomonas aeruginosa ExoS/T/U/Y all travel through T3SSs. A typical pathogen carries ~7-25 effectors that rewire host actin, Rho-GTPases, vesicle trafficking, and innate immunity. Discovered in Yersinia in the late 1980s; the canonical structural paper is Kubori, Galán et al. Science 1998.
- Mass~3.5 MDa
- Structural proteins~20 conserved subunits
- Channel width~2.5 nm
- Effectors per pathogen7-25
- Discovered inYersinia, late 1980s-1990s
- Evolutionary cousinBacterial flagellum (~10 homologs)
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Why T3SS matters
- It explains pathogenesis for several billion human infections. Salmonella enterica alone causes ~93 million gastroenteritis cases per year globally; Shigella ~165 million; enteropathogenic and enterohemorrhagic E. coli contribute several hundred thousand deaths annually. T3SS is the central virulence determinant for all of them.
- The injectisome is one of the largest characterized molecular machines. At ~3.5 MDa it is comparable in mass to the bacterial ribosome (~2.5 MDa) and the nuclear pore complex octant. Its needle complex was the first multi-membrane bacterial nanomachine to have a high-resolution cryo-EM structure (Kubori et al. 1998; Hu et al. 2017 sub-nanometer EM).
- Anti-virulence drugs targeting T3SS are in clinical development. Compounds like INP1855 (a thiazolinone) inhibit needle assembly without killing bacteria, exerting weaker selection for resistance than classical antibiotics. Trials against P. aeruginosa in cystic fibrosis are ongoing.
- Effectors are master forgers of eukaryotic enzymes. SopE is a Rho-GEF without any structural homology to host GEFs — convergent evolution of catalytic mechanism. YopJ family proteins are unusual acetyltransferases that block kinase activation loops. Studying T3SS effectors has revealed novel enzyme chemistries that pure cell biology missed.
- It is a paradigm for "shared parts" evolution. The injectisome and flagellum share ~10 core components. Comparing the two has resolved how complex multi-component machines can evolve by gene duplication and specialization without ever passing through a non-functional intermediate — the core argument used by molecular evolutionists against Behe's "irreducible complexity" claims.
- Plant pathogens use the same machinery. Pseudomonas syringae, Xanthomonas campestris, and Erwinia amylovora deploy T3SS to inject effectors into plant cells, suppressing host defenses (PAMP-triggered immunity) and fueling agricultural disease losses estimated at tens of billions of dollars annually.
- The system is a candidate biotechnology delivery platform. Engineered injectisomes have been used to deliver heterologous proteins into mammalian cells in culture — a possible alternative to viral vectors for protein-level interventions where DNA delivery is unwanted.
Common misconceptions
- The needle drills through the host cell. It does not. The translocon (a hetero-oligomer like YopB+YopD or IpaB+IpaC) inserts into the host plasma membrane and forms a passive pore. The needle docks against this translocon; effectors flow through both into the host cytoplasm without mechanical drilling.
- Effectors are folded when they cross the channel. They cannot be — the channel is only ~2.5 nm wide, narrower than a folded globular domain. Substrates are kept partially unfolded by cytoplasmic chaperones (T3SS class IA, IB, II chaperones) and refold after entering the host cytoplasm. This forced unfolding is the energetic cost the ATPase pays.
- One T3SS per pathogen. Many bacteria carry two or more, deployed at different infection stages. Salmonella uses SPI-1 (encoded on Salmonella Pathogenicity Island 1) for invasion of intestinal epithelium and SPI-2 for survival inside macrophage phagosomes. Different effectors and partly different structural proteins.
- T3SS = bacterial pathogenesis. Many pathogens rely on T2SS (toxin secretion), T4SS (effector or DNA), T5SS (autotransporters), or T6SS (interbacterial). T3SS is one of six numbered systems. Pathogens like Vibrio cholerae rely heavily on T2SS-secreted cholera toxin and have minimal T3SS contribution.
- The flagellum and injectisome are interchangeable. They share components, but no organism uses one to substitute for the other. T3SS-bearing bacteria still need separate flagella for motility. Component sharing implies common ancestry, not functional equivalence.
- Effectors only target one host pathway. The opposite — most effectors are multi-functional. SipA bundles actin and inhibits actin depolymerizing factor; YopJ acetylates kinase activation loops and is a deubiquitinase. The combinatorial effector cocktail rewires whole networks rather than single pathways.
How the injectisome works
Architecturally the T3SS is divided into three regions. The basal body spans both bacterial membranes and is built from a stack of protein rings — outer membrane secretin (e.g. Salmonella InvG), peptidoglycan ring, and inner membrane MS-ring (PrgH/PrgK). At the cytoplasmic face sits the export apparatus (SpaP/Q/R/SctV homologs), an ATPase (InvC/SctN) hexamer that energizes substrate unfolding, and a sorting platform that selects which substrate to send next. The extracellular needle is a 6-8 nm wide hollow filament (~50 copies of PrgI/SctF in Salmonella) capped by a tip protein (SipD/IpaD/LcrV). Length is regulated by a molecular ruler (InvJ/SctP) that resets at a species-specific length, typically 50-80 nm. The translocon — a pair of hydrophobic proteins (e.g. YopB+YopD, IpaB+IpaC, SipB+SipC) — inserts into the host plasma membrane on contact and forms the channel through which effectors flow.
Substrate selection happens by signal sequence and chaperone gating. The first ~20 amino acids of every T3SS substrate carry a poorly conserved but functionally specific secretion signal that the export apparatus recognizes. Class IA chaperones (e.g. SicP, SycE) bind individual effectors in the cytoplasm, keep them unfolded, and dock them at the sorting platform. The order of secretion is regulated: structural components first (needle subunits, then translocon), then early effectors that prepare the host (e.g. Salmonella SipA, SopE), then late effectors (e.g. SptP, SipA) that reverse host changes once the bacterium is internalized. The switch between hierarchies is mediated by substrate specificity switches (InvE, YscP) that change which class of substrate the export gate accepts.
Once delivered, effectors collectively rewrite host behavior. Salmonella SopE is a Rho-GEF that activates Cdc42 and Rac1 without any structural homology to mammalian GEFs (textbook convergent evolution); the activation drives membrane ruffling that engulfs the bacterium by macropinocytosis. After entry, SptP acts as a GAP to reverse the same GTPases, restoring host morphology so the infection avoids detection. Yersinia Yops do the opposite — six effectors collectively block phagocytosis by macrophages: YopE inactivates Rho-GTPases, YopH dephosphorylates focal adhesion components, YopT proteolyzes Rho, and YopJ/P acetylates MAPK kinases to silence NF-kappaB signaling. The pathogen survives extracellularly while macrophages die or fail to ingest it.
T3SS vs T4SS vs T6SS
| Feature | T3SS (injectisome) | T4SS | T6SS |
|---|---|---|---|
| Substrate | Proteins (effectors) | Proteins and/or DNA | Proteins (toxins) |
| Architecture | Needle complex; flagellum-related | Conjugation-pilus-related | Phage-tail-like contractile sheath |
| Primary target | Eukaryotic host cytoplasm | Eukaryotic or bacterial | Other bacteria (mostly) |
| Energy source | ATPase + PMF | ATPase (VirB11/VirB4) | Sheath contraction (no ATP at firing) |
| Iconic pathogens | Salmonella, Yersinia, Shigella | Agrobacterium, conjugative plasmids, Helicobacter pylori (CagA) | Vibrio cholerae, Pseudomonas aeruginosa |
| Substrate count per pathogen | 7-25 effectors | 1-many proteins; full plasmids | Up to ~10 effectors |
| Trigger | Host contact + low Ca2+ | Recipient-cell contact | Cell-cell contact + signaling |
Famous case studies
- Yersinia pestis and the Yops. The plague bacillus carries a 70 kb pCD1 plasmid encoding the Ysc T3SS and six injected Yops (YopE, YopH, YopT, YopJ, YopM, YopO/YpkA). The Yop cocktail blocks phagocytosis and kills macrophages, allowing the bacterium to multiply extracellularly in lymph nodes (the bubonic phase) before bloodstream dissemination. Yersinia studies in the 1990s defined the field.
- Salmonella enterica SPI-1 and SPI-2. SPI-1 (Salmonella Pathogenicity Island 1) carries a T3SS plus ~13 effectors that invade intestinal epithelium via macropinocytosis. SPI-2 carries a second, distinct T3SS deployed inside the macrophage phagosome (the Salmonella-containing vacuole) where ~20 effectors remodel vesicle trafficking to keep the phagosome hospitable. Galán's lab characterized SPI-1; Wolf-Watz, Holden, and others did SPI-2.
- Shigella flexneri and the Mxi-Spa apparatus. Encodes a T3SS on the 220 kb invasion plasmid; secretes IpaB, IpaC, IpaD as the translocon and IpgB1, IpgD, IpgB2, OspB, OspF, IpaA among ~30 effectors. The combination drives entry into colonic epithelial cells and lateral cell-to-cell spread by actin-based motility (IcsA-mediated, complementing the T3SS).
- Enteropathogenic E. coli (EPEC) and Tir. EPEC injects its own receptor — Tir — into the host membrane, where it binds bacterial intimin and nucleates dramatic actin pedestals beneath the bacterium. The system is the first known example of a pathogen providing the receptor for its own attachment, characterized by Brett Finlay's lab in the 1990s.
- Pseudomonas aeruginosa and ExoU. The acute-infection T3SS effector ExoU is a phospholipase that destroys host membranes within minutes of injection; ExoU+ P. aeruginosa isolates have ~3-fold higher mortality in ventilator-associated pneumonia. The system is the primary anti-virulence drug target in the species.
Frequently asked questions
What is the Type III secretion system in one sentence?
The Type III secretion system, or injectisome, is a ~3.5 MDa transmembrane needle complex built from about 20 conserved proteins that lets pathogenic Gram-negative bacteria inject effector proteins directly across both bacterial membranes and the eukaryotic host plasma membrane in a single secretion event, bypassing the periplasm and the host extracellular space and delivering toxins straight into the host cytoplasm. The needle channel is roughly 2.5 nm wide, forcing effectors through partially unfolded; once in the cytosol they refold and rewire host signaling, cytoskeleton, vesicle trafficking, and immune responses to favor bacterial survival.
How was the T3SS discovered?
The discovery story runs through Yersinia in the late 1980s and early 1990s. Researchers including Hans Wolf-Watz, Gunna Cornelis, and Stanley Falkow's group noticed that pathogenic Yersinia secreted a defined set of proteins called Yops (Yersinia outer proteins) into culture supernatants under low-calcium conditions. The genes lay on a 70 kb virulence plasmid, and when a host cell was present the Yops appeared inside its cytoplasm rather than in the medium. Jorge Galán's lab independently characterized the homologous Inv-Spa secretion machinery in Salmonella in the early 1990s and showed it injected effectors that triggered macropinocytosis. The structural identification of the machine as a needle-like supramolecular complex (Kubori, Macnab, Galán et al. 1998 in Science) cemented the 'molecular syringe' image. The same architecture turned out to underlie virulence in Shigella, EPEC, P. aeruginosa, and plant pathogens, unifying decades of disparate bacteriology under one mechanism.
How is the T3SS related to the bacterial flagellum?
The injectisome shares its core export apparatus, ATPase, and several basal-body components with the flagellum — about 10 of the ~20 structural proteins are clearly homologous (e.g. SpaP/FliP, SpaQ/FliQ, SpaR/FliR, the ATPases InvC/FliI). Both systems export proteins through a similar narrow channel by similar energetics: a proton-motive-force-driven export gate plus an accessory ATPase. The most parsimonious phylogeny puts the flagellum and the injectisome as sister machines descended from a common ancestor, though which came first is contested — Susan Gophna and others argue the flagellum was first and the injectisome is a derived secretion-only specialization, while a minority view favors the reverse. The two systems are not interchangeable in any extant organism: T3SS-bearing bacteria still need separate flagella for motility.
What do the effector proteins do inside host cells?
They mimic, hijack, or destroy host signaling molecules. Salmonella SipA and SipC bind actin to drive macropinocytotic ruffling and engulf the bacterium. SopE/SopE2 are guanine-nucleotide exchange factors for Cdc42 and Rac1, switching on host actin polymerization without the host's own GEFs. SptP is a GTPase-activating protein that reverses the SopE signal once the bacterium is internalized. Yersinia YopE and YopT inactivate Rho-family GTPases to inhibit phagocytosis; YopH is a tyrosine phosphatase that disrupts focal adhesion; YopJ/YopP is a deubiquitinase/acetyltransferase that suppresses NF-kappaB and MAPK signaling and triggers apoptosis. Shigella IpaA, IpaB, and IpaC drive entry into colonic epithelial cells. Across the family, the typical pathogen has 7-25 effectors targeting actin, Rho-GTPases, kinase cascades, vesicle trafficking, and innate immune signaling — collectively rewriting the host cell's behavior on a multi-pathway scale.
How does the bacterium know when to fire?
Two cues dominate. Calcium concentration: in the bacterial extracellular environment Ca2+ is at ~mM levels and represses Yop secretion (the so-called low-calcium response — secretion is unleashed only when Ca2+ drops, mimicking the cytoplasmic environment of a host cell). Direct contact: insertion of the translocon (YopB+YopD or IpaB+IpaC) into the host plasma membrane creates the channel and triggers the secretion of effectors that follow. Mechanically the needle tip likely senses contact through compression and propagates the signal back to the cytoplasmic export gate. The combination ensures effectors are not wasted into the medium and are released only when the needle is docked into a real target cell. Salmonella adds environmental cues — bile, oxygen, pH — to control SPI-1 expression in the small intestine versus SPI-2 (a second T3SS) inside macrophage phagosomes.
How does T3SS differ from T4SS and T6SS?
Type III, IV, and VI secretion systems all deliver substrates directly into a target cell, but the architectures and substrates differ. T3SS is the flagellum-related needle complex, secretes proteins (effectors) only, and primarily targets eukaryotic host cells. T4SS is descended from bacterial conjugation machinery, also secretes proteins, but additionally translocates DNA — Agrobacterium tumefaciens uses T4SS to deliver T-DNA into plant cells, and many conjugative plasmids spread via T4SS pili. T6SS is structurally a contractile phage-tail-like puncturing device, primarily fires effectors into other bacteria during inter-bacterial competition (although some target eukaryotic hosts), and is built from a Hcp tube wrapped in a VipA/VipB sheath that contracts to drive a VgrG spike through a target. All three depend on inner membrane ATPases and outer membrane channels, but T3SS is the only one with the flagellar lineage and the dedicated host-cytoplasm targeting that defines the injectisome metaphor.