Bacterial secretion systems are complex, multi-protein assemblies that transport proteins, known as effectors or toxins, across bacterial membrane barriers. This export is necessary for bacteria to survive, communicate, and interact with their environment, including a host organism. These specialized systems are categorized into seven major types, designated I through VII, based on their structure, mechanism, and evolutionary relationships. The precise export of molecules allows bacteria to acquire nutrients, assemble surface structures, and, for pathogens, manipulate host cell functions.
Foundational Concepts of Protein Export
The complexity of bacterial secretion is determined by the cell envelope structure. Gram-negative bacteria possess an inner membrane (IM) and an outer membrane (OM), separated by the periplasm. Gram-positive bacteria, in contrast, have only a single cytoplasmic membrane surrounded by a thick peptidoglycan layer.
Many secretion pathways rely on a preliminary step to move proteins across the inner membrane, handled primarily by the Sec (general secretion) and Tat (twin-arginine translocation) pathways. The Sec pathway translocates proteins in an unfolded state across the inner membrane, utilizing energy from ATP hydrolysis. The Tat pathway is distinct, moving proteins that are already folded, often incorporating cofactors, using the proton-motive force.
Specific signal peptides dictate routing through the Sec or Tat machinery. The Sec system is the dominant pathway for protein export, while the Tat pathway is specialized for substrates that must be folded before transport. Secretion systems are classified into two groups: two-step processes, which rely on Sec/Tat to reach the periplasm first, and single-step processes, which span all membrane layers at once.
Direct, Single-Step Secretion Pathways (Types I, III, IV, VI)
Type I Secretion System (T1SS)
The Type I Secretion System (T1SS) is an apparatus that directly spans both the inner and outer membranes of Gram-negative bacteria in a single step. This process avoids a periplasmic intermediate. The system is assembled from three main components: an inner membrane ABC (ATP-binding cassette) transporter, a periplasmic adaptor protein, and an outer membrane channel, such as the TolC family protein.
The ABC transporter recognizes a specific C-terminal secretion signal on the substrate protein. The T1SS transports a wide variety of large substrates, including hemolysins, lipases, proteases, and adhesins, with some reaching 1,500 kDa. For example, the hemolysin A (HlyA) system in E. coli is a well-studied example, mediating the export of this exotoxin. The components transiently assemble into a continuous channel that bridges the bacterial cytoplasm directly to the extracellular space.
Type III Secretion System (T3SS)
The Type III Secretion System (T3SS), often called the “injectisome,” is a sophisticated nanomachine used by many Gram-negative pathogens. This complex acts like a molecular syringe, forming a direct conduit that spans the bacterial membranes and the host cell membrane. The T3SS delivers bacterial effector proteins directly from the bacterial cytoplasm into the cytosol of a eukaryotic host cell.
The apparatus is structurally related to the bacterial flagellum. The injectisome consists of a large, multi-protein needle complex (20–150 nm long), crowned by a tip protein that senses host cell contact. Secretion is controlled by an ATPase motor that unfolds the effector proteins and pushes them through the channel. This direct injection mechanism allows pathogens like Salmonella and Yersinia to rapidly subvert host cell functions, promoting infection.
Type IV Secretion System (T4SS)
The Type IV Secretion System (T4SS) is a versatile transport machine found in both Gram-negative and Gram-positive bacteria. It transports proteins, DNA, or protein-DNA complexes across cell membranes in a single step. T4SSs are grouped into two major subfamilies: conjugation systems and effector translocators.
Conjugation systems facilitate the transfer of genetic material, such as plasmids carrying antibiotic resistance genes, between bacterial cells. Effector translocators are used by pathogens like Legionella pneumophila and Bordetella pertussis to deliver virulence proteins into the eukaryotic host cell cytosol. The T4SS often utilizes a pilus-like structure to mediate contact-dependent transfer with the target cell. This system’s ability to move both proteins and DNA makes it a significant factor in bacterial evolution and the spread of virulence and resistance traits.
Type VI Secretion System (T6SS)
The Type VI Secretion System (T6SS) operates with a unique, spring-loaded mechanism homologous to the contractile tail of a bacteriophage virus. Found predominantly in Gram-negative bacteria, it functions primarily in inter-bacterial competition, acting as a weapon against neighboring cells. The core is a sheath-like structure that assembles within the cytoplasm and is anchored to the inner membrane by a basal structure.
The apparatus cycles between an extended state and a rapidly contracted state. During contraction, the T6SS rapidly fires a sharp, inner tube structure, tipped with effector proteins, out of the cell and into an adjacent target cell. This forceful contraction provides the energy to propel the toxic cargo across cell membranes. The effectors often kill competing bacteria, demonstrating the T6SS’s role in maintaining microbial community stability.
Two-Step and Specialized Secretion Pathways (Types II, V, VII)
Type II Secretion System (T2SS)
The Type II Secretion System (T2SS) is a two-step pathway widespread among Gram-negative bacteria. It is dependent on the Sec or Tat pathways for its initial step, translocating proteins across the inner membrane into the periplasm. There, the proteins typically fold into their correct three-dimensional structure.
Once in the periplasm, the T2SS transports the folded proteins across the outer membrane into the extracellular environment. The apparatus is a large, multi-protein complex spanning the cell envelope, featuring a unique, piston-like structure known as the pseudopilus. The pseudopilus is thought to extend and retract, pushing the cargo through a pore in the outer membrane called the secretin. The T2SS secretes a variety of toxins and degradative enzymes, such as proteases, lipases, and cholera toxin in Vibrio cholerae.
Type V Secretion System (T5SS)
The Type V Secretion System (T5SS) is known as the Autotransporter pathway because the secreted protein is largely responsible for its own translocation across the outer membrane. It is a two-step process, requiring the Sec machinery to first move the protein into the periplasm. Once there, the C-terminal portion of the protein inserts into the outer membrane, forming a \(\beta\)-barrel structure.
This \(\beta\)-barrel acts as a channel through which the larger, N-terminal functional domain, known as the passenger domain, is transported to the cell surface. The passenger domain often functions as an adhesin or protease, contributing to bacterial virulence. After translocation, the passenger domain is frequently cleaved from the \(\beta\)-barrel domain and released into the extracellular space. The T5SS is the largest family of secreted proteins in Gram-negative bacteria.
Type VII Secretion System (T7SS)
The Type VII Secretion System (T7SS), also known as the ESX system, is a specialized pathway primarily associated with Actinobacteria, such as Mycobacterium, which includes M. tuberculosis. These bacteria possess an unusual, waxy, and relatively impermeable cell envelope. The T7SS complex is large, embedded in the inner membrane, and often forms a six-sided structure.
The T7SS is essential for the virulence of Mycobacterium tuberculosis, secreting key virulence factors like the small proteins EsxA and EsxB. These proteins are often secreted as folded heterodimers and intervene in host cellular signaling pathways, promoting bacterial survival. The M. tuberculosis genome encodes multiple T7SS clusters (ESX-1 to ESX-5), each having a distinct role; for instance, the ESX-1 system is implicated in the lysis of the host cell phagosome.
Significance in Virulence and Therapeutic Targets
Bacterial secretion systems are direct contributors to the virulence of many pathogens. Systems like the T3SS, T4SS, and T6SS inject toxins and effector molecules into host cells, allowing bacteria to manipulate host defenses and cellular processes. A pathogen with a non-functional secretion system is often rendered avirulent, highlighting the dependency of the infection process on these molecular machines.
This dependency makes secretion systems highly attractive targets for anti-virulence therapies. Instead of attempting to kill the bacteria outright, which drives selection pressure for antibiotic resistance, anti-virulence drugs aim to disarm the pathogen by blocking its ability to cause disease. Researchers are identifying small molecules that inhibit the assembly or function of systems like the T3SS and T4SS, effectively neutralizing the bacterium’s weaponry.
Targeting these virulence-associated structures is appealing because the secretion systems are often unique to pathogenic bacteria, meaning beneficial bacteria would not be affected. Understanding the molecular architecture and mechanism of each system is a fundamental step in developing new treatments and effective vaccines. By focusing on these nanomachines, scientists hope to overcome the challenge of antimicrobial resistance by developing compounds that prevent infection without imposing selective pressure.