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Bordetella bronchiseptica and Bordetella pertussis are two closely related respiratory pathogens that employ their T3SS injectisome to deliver the BteA effector into host cells. In this study, we visualized the needle tip filament of their T3SS injectisome, a structure formed by the Bsp22 protein. We demonstrate that during Bordetella cultivation in Stainer-Scholte medium, Bsp22 filaments are abundant and can dynamically extend up to several micrometers in length through the incorporation of new subunits at their distal ends. In contrast, these filaments become shorter and/or less abundant during infection of host cells. This reduction correlates with decreased bsp22 mRNA expression and lower Bsp22 protein levels, while the levels of bscD mRNA, which encodes the inner membrane ring protein of the injectisome, remain stable. These results highlight the adaptability of the Bordetella T3SS injectisome and show how its tip filament structure changes in response to different environments.

Staphylococcus aureus is a leading cause of infections worldwide. S. aureus utilizes a specialized type VIIb secretion system (T7SSb) to persist in the infected host tissues as well as target competitor bacteria to establish its niche. T7SSb assembles into a multiprotein translocation complex and facilitates secretion of a set of small proteins and larger polymorphic toxins across the cytosolic membrane. Beyond the membrane, secreted proteins were thought to diffuse through the thick yet porous cell wall and release into the environment. Here, we demonstrate for the first time that S. aureus T7SSb extends across the cell wall via its EsaA subunit. Furthermore, accommodation of EsaA within the cell wall requires an associated cell wall hydrolase EssH and is essential for protein secretion via T7SSb. Thus, our findings provide a mechanistic insight for a coordinated cell wall processing and T7SSb assembly to support specialized protein secretion in S. aureus .

Salmonella enterica is an increasing global public health threat. As part of its virulence arsenal, Salmonella relies on a type III secretion system (T3SS) or injectisome, a molecular injection device that translocates effector proteins into host cells to promote invasion and inflammation. A central component of this machine is the SpaO protein, which is produced in two forms: a full-length form and a shorter variant. Here, by studying the functional and structural relationship between the two SpaO forms in their native cellular environment, we define how and when they assemble within the injectisome. Employing quantitative injection assays in cultured cells, we define the shorter SpaO variant as an accessory structural piece that boosts effector delivery. These findings refine our understanding of injectisome assembly and function and provide mechanistic insight to inform future efforts to target T3SS-dependent pathogens through antivirulence strategies.

spp. are major zoonotic bacterial pathogens that can cause a range of diseases in both humans and animals, including gastroenteritis, septicemia, and typhoid fever. During intestinal colonization, relies on the coordinated action of the SPI-4 encoded type I secretion system (T1SS) and SPI-1 encoded type III secretion system (T3SS-1) to breach epithelial barriers. Although the T3SS-1 and its effectors have been widely studied, the T1SS and its associated effectors remain poorly characterized. The T1SS-secreted substrate SiiE binds to host cell surface mucins via its bacterial Ig-like (BIg) domain, facilitating the proper positioning of the T3SS-1 and subsequently triggering bacterial internalization. Given the critical role of the T1SS and SiiE in virulence, they may serve as promising targets for anti-virulence drug development. In this review, we provide an overview of the current knowledge on the T1SS, including its regulatory mechanisms, channel formation, and the functional properties of SiiE.

Initially identified in pathogenic Gram-negative bacteria, the two-partner secretion (TPS) pathway, also known as Type Vb secretion, mediates the translocation across the outer membrane of large effector proteins involved in interactions between these pathogens and their hosts. More recently, distinct TPS systems have been shown to secrete toxic effector domains that participate in inter-bacterial competition or cooperation. The effects of these systems are based on kin vs. non-kin molecular recognition mediated by specific immunity proteins. With these new toxin-antitoxin systems, the range of TPS effector functions has thus been extended from cytolysis, adhesion, and iron acquisition, to genome maintenance, inter-bacterial killing and inter-bacterial signaling. Basically, a TPS system is made up of two proteins, the secreted TpsA effector protein and its TpsB partner transporter, with possible additional factors such as immunity proteins for protection against cognate toxic effectors. Structural studies have indicated that TpsA proteins mainly form elongated β helices that may be followed by specific functional domains. TpsB proteins belong to the Omp85 superfamily. Open questions remain on the mechanism of protein secretion in the absence of ATP or an electrochemical gradient across the outer membrane. The remarkable dynamics of the TpsB transporters and the progressive folding of their TpsA partners at the bacterial surface in the course of translocation are thought to be key elements driving the secretion process.

How cells determine where to assemble a macromolecular complex is a fundamental question in biology since the localization of these complexes is directly linked to functions. In bacteria, the type VI secretion system (T6SS) relies on effective positioning to target competitor and host cells in contact-dependent interactions. This study identifies a PpkA-TssW-Fha axis that orchestrates T6SS localization and activation through membrane anchoring and liquid-liquid phase separation at the inner membrane interface. These new insights can help us not only better understand how the T6SS functions but also better design T6SS-based solutions for therapeutic targeting of drug-resistant and T6SS-susceptible bacterial and fungal pathogens.

Contractile injection systems mediate bacterial cell-cell interactions by a bacteriophage tail–like structure. In contrast to extracellular systems, the type 6 secretion system (T6SS) is defined by intracellular localization and attachment to the cytoplasmic membrane. Here we used cryo-focused ion beam milling, electron cryotomography, and functional assays to study a T6SS in Amoebophilus asiaticus. The in situ architecture revealed three modules, including a contractile sheath-tube, a baseplate, and an anchor. All modules showed conformational changes upon firing. Lateral baseplate interactions coordinated T6SSs in hexagonal arrays. The system mediated interactions with host membranes and may participate in phagosome escape. Evolutionary sequence analyses predicted that T6SSs are more widespread than previously thought. Our insights form the basis for understanding T6SS key concepts and exploring T6SS diversity.

The type 9 secretion system (T9SS) is the protein export pathway of bacteria of the Gram-negative Fibrobacteres–Chlorobi–Bacteroidetes superphylum and is an essential determinant of pathogenicity in severe periodontal disease. The central element of the T9SS is a so-far uncharacterized protein-conducting translocon located in the bacterial outer membrane. Here, using cryo-electron microscopy, we provide structural evidence that the translocon is the T9SS protein SprA. SprA forms an extremely large (36-strand) single polypeptide transmembrane β-barrel. The barrel pore is capped on the extracellular end, but has a lateral opening to the external membrane surface. Structures of SprA bound to different components of the T9SS show that partner proteins control access to the lateral opening and to the periplasmic end of the pore. Our results identify a protein transporter with a distinctive architecture that uses an alternating access mechanism in which the two ends of the protein-conducting channel are open at different times. Cryo-electron microscopy structures of the protein-conducting translocon of the type 9 secretion system reveal its architecture and mechanism of translocation.

Type III secretion systems are multiprotein molecular machines required for the virulence of several important bacterial pathogens. The central element of these machines is the injectisome, a ∼5-Md multiprotein structure that mediates the delivery of bacterially encoded proteins into eukaryotic target cells. The injectisome is composed of a cytoplasmic sorting platform, and a membrane-embedded needle complex, which is made up of a multiring base and a needle-like filament that extends several nanometers from the bacterial surface. The needle filament is capped at its distal end by another substructure known as the tip complex, which is crucial for the translocation of effector proteins through the eukaryotic cell plasma membrane. Here we report the cryo-EM structure of the Salmonella Typhimurium needle tip complex docked onto the needle filament tip. Combined with a detailed analysis of structurally guided mutants, this study provides major insight into the assembly and function of this essential component of the type III secretion protein injection machine.

Bacterial secretion systems are nanomolecular complexes that release a diverse set of virulence factors/or proteins into its surrounding or translocate to their target host cells. Among these systems, type VI secretion system ‘T6SS’ is a recently discovered molecular secretion system which is widely distributed in Gram-negative (−ve) bacteria, and shares structural similarity with the puncturing device of bacteriophages. The presence of T6SS is an advantage to many bacteria as it delivers toxins to its neighbour pathogens for competitive survival, and also translocates protein effectors to the host cells, leading to disruption of lipid membranes, cell walls, and cytoskeletons etc. Recent studies have characterized both anti-prokaryotic and anti-eukaryotic effectors, where T6SS is involved in diverse cellular functions including favouring colonization, enhancing the survival, adhesive modifications, internalization, and evasion of the immune system. With the evolution of advanced genomics and proteomics tools, there has been an increase in the number of characterized T6SS effector arsenals and also more clear information about the adaptive significance of this complex system. The functions of T6SS are generally regulated at the transcription, post-transcription and post-translational levels through diverse mechanisms. In the present review, we aimed to provide information about the distribution of T6SS in diverse bacteria, any structural similarity/or dissimilarity, effectors proteins, functional significance, and regulatory mechanisms. We also tried to provide information about the diverse roles played by T6SS in its natural environments and hosts, and further any changes in the microbiome.