Protein-Injection Machines in Bacteria.

Many bacteria have evolved specialized nanomachines with the remarkable ability to inject multiple bacterially encoded effector proteins into eukaryotic or prokaryotic cells. Known as type III, type IV, and type VI secretion systems, these machines play a central role in the pathogenic or symbiotic interactions between multiple bacteria and their eukaryotic hosts, or in the establishment of bacterial communities in a diversity of environments. Here we focus on recent progress elucidating the structure and assembly pathways of these machines. As many of the interactions shaped by these machines are of medical importance, they provide an opportunity to develop novel therapeutic approaches to combat important human diseases.

In Situ Molecular Architecture of the Salmonella Type III Secretion Machine.

Type III protein secretion systems have specifically evolved to deliver bacterially encoded proteins into target eukaryotic cells. The core elements of this multi-protein machine are the envelope-associated needle complex, the inner membrane export apparatus, and a large cytoplasmic sorting platform. Here, we report a high-resolution in situ structure of the Salmonella Typhimurium type III secretion machine obtained by high-throughput cryo-electron tomography and sub-tomogram averaging. Through molecular modeling and comparative analysis of machines assembled with protein-tagged components or from different deletion mutants, we determined the molecular architecture of the secretion machine in situ and localized its structural components. We also show that docking of the sorting platform results in significant conformational changes in the needle complex to provide the symmetry adaptation required for the assembly of the entire secretion machine. These studies provide major insight into the structure and assembly of a broadly distributed protein secretion machine.

Host adaptation of a bacterial toxin from the human pathogen Salmonella Typhi.

Salmonella Typhi is an exclusive human pathogen that causes typhoid fever. Typhoid toxin is a S. Typhi virulence factor that can reproduce most of the typhoid fever symptoms in experimental animals. Toxicity depends on toxin binding to terminally sialylated glycans on surface glycoproteins. Human glycans are unusual because of the lack of CMAH, which in other mammals converts N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc). Here, we report that typhoid toxin binds to and is toxic toward cells expressing glycans terminated in Neu5Ac (expressed by humans) over glycans terminated in Neu5Gc (expressed by other mammals). Mice constitutively expressing CMAH thus displaying Neu5Gc in all tissues are resistant to typhoid toxin. The atomic structure of typhoid toxin bound to Neu5Ac reveals the structural bases for its binding specificity. These findings provide insight into the molecular bases for Salmonella Typhi's host specificity and may help the development of therapies for typhoid fever.

An RNA degradation machine sculpted by Ro autoantigen and noncoding RNA.

Many bacteria contain an ortholog of the Ro autoantigen, a ring-shaped protein that binds noncoding RNAs (ncRNAs) called Y RNAs. In the only studied bacterium, Deinococcus radiodurans, the Ro ortholog Rsr functions in heat-stress-induced ribosomal RNA (rRNA) maturation and starvation-induced rRNA decay. However, the mechanism by which this conserved protein and its associated ncRNAs act has been obscure. We report that Rsr and the exoribonuclease polynucleotide phosphorylase (PNPase) form an RNA degradation machine that is scaffolded by Y RNA. Single-particle electron microscopy, followed by docking of atomic models into the reconstruction, suggests that Rsr channels single-stranded RNA into the PNPase cavity. Biochemical assays reveal that Rsr and Y RNA adapt PNPase for effective degradation of structured RNAs. A Ro ortholog and ncRNA also associate with PNPase in Salmonella Typhimurium. Our studies identify another ribonucleoprotein machine and demonstrate that ncRNA, by tethering a protein cofactor, can alter the substrate specificity of an enzyme.

Assembly and architecture of the type III secretion sorting platform.

Type III secretion systems are bacterial nanomachines specialized in protein delivery into target eukaryotic cells. The structural and functional complexity of these machines demands highly coordinated mechanisms for their assembly and operation. The sorting platform is a critical component of type III secretion machines that ensures the timely engagement and secretion of proteins destined to travel this export pathway. However, the mechanisms that lead to the assembly of this multicomponent structure have not been elucidated. Herein, employing an extensive in vivo cross-linking strategy aided by structure modeling, we provide a detailed intersubunit contact survey of the entire sorting platform complex. Using the identified cross-links as signatures for pairwise intersubunit interactions in combination with systematic genetic deletions, we mapped the assembly process of this unique bacterial structure. Insights generated by this study could serve as the bases for the rational development of antivirulence strategies to combat several medically important bacterial pathogens.

Diversification of a Salmonella virulence protein function by ubiquitin-dependent differential localization.

Many bacterial pathogens and symbionts utilize type III secretion systems to deliver bacterial effector proteins into host cells. These effector proteins have the capacity to modulate a large variety of cellular functions in a highly regulated manner. Here, we report that the phosphoinositide phosphatase SopB, a Salmonella Typhimurium type III secreted effector protein, diversifies its function by localizing to different cellular compartments in a ubiquitin-dependent manner. We show that SopB utilizes the same enzymatic activity to modulate actin-mediated bacterial internalization and Akt activation at the plasma membrane and vesicular trafficking and intracellular bacterial replication at the phagosome. Thus, by exploiting the host cellular machinery, Salmonella Typhimurium has evolved the capacity to broaden the functional repertoire of a virulence factor to maximize its ability to modulate cellular functions.

Cryo-EM structure of the needle filament tip complex of the Salmonella type III secretion injectisome.

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.

A polymorphic helix of a Salmonella needle protein relays signals defining distinct steps in type III secretion.

Type III protein-secretion machines are essential for the interactions of many pathogenic or symbiotic bacterial species with their respective eukaryotic hosts. The core component of these machines is the injectisome, a multiprotein complex that mediates the selection of substrates, their passage through the bacterial envelope, and ultimately their delivery into eukaryotic target cells. The injectisome is composed of a large cytoplasmic complex or sorting platform, a multiring base embedded in the bacterial envelope, and a needle-like filament that protrudes several nanometers from the bacterial surface and is capped at its distal end by the tip complex. A characteristic feature of these machines is that their activity is stimulated by contact with target host cells. The sensing of target cells, thought to be mediated by the distal tip of the needle filament, generates an activating signal that must be transduced to the secretion machine by the needle filament. Here, through a multidisciplinary approach, including solid-state NMR (SSNMR) and cryo electron microscopy (cryo-EM) analyses, we have identified critical residues of the needle filament protein of a Salmonella Typhimurium type III secretion system that are involved in the regulation of the activity of the secretion machine. We found that mutations in the needle filament protein result in various specific phenotypes associated with different steps in the type III secretion process. More specifically, these studies reveal an important role for a polymorphic helix of the needle filament protein and the residues that line the lumen of its central channel in the control of type III secretion.

High-resolution view of the type III secretion export apparatus in situ reveals membrane remodeling and a secretion pathway.

Type III protein secretion systems are essential virulence factors for many important pathogenic bacteria. The entire protein secretion machine is composed of several substructures that organize into a holostructure or injectisome. The core component of the injectisome is the needle complex, which houses the export apparatus that serves as a gate for the passage of the secreted proteins through the bacterial inner membrane. Here, we describe a high-resolution structure of the export apparatus of the Salmonella type III secretion system in association with the needle complex and the underlying bacterial membrane, both in isolation and in situ. We show the precise location of the core export apparatus components within the injectisome and bacterial envelope and demonstrate that their deployment results in major membrane remodeling and thinning, which may be central for the protein translocation process. We also show that InvA, a critical export apparatus component, forms a multiring cytoplasmic conduit that provides a pathway for the type III secretion substrates to reach the entrance of the export gate. Combined with structure-guided mutagenesis, our studies provide major insight into potential mechanisms of protein translocation and injectisome assembly.

Unique features in the intracellular transport of typhoid toxin revealed by a genome-wide screen.

Typhoid toxin is a virulence factor for Salmonella Typhi and Paratyphi, the cause of typhoid fever in humans. This toxin has a unique architecture in that its pentameric B subunit, made of PltB, is linked to two enzymatic A subunits, the ADP ribosyl transferase PltA and the deoxyribonuclease CdtB. Typhoid toxin is uniquely adapted to humans, recognizing surface glycoprotein sialoglycans terminated in acetyl neuraminic acid, which are preferentially expressed by human cells. The transport pathway to its cellular targets followed by typhoid toxin after receptor binding is currently unknown. Through a genome-wide CRISPR/Cas9-mediated screen we have characterized the mechanisms by which typhoid toxin is transported within human cells. We found that typhoid toxin hijacks specific elements of the retrograde transport and endoplasmic reticulum-associated degradation machineries to reach its subcellular destination within target cells. Our study reveals unique and common features in the transport mechanisms of bacterial toxins that could serve as the bases for the development of novel anti-toxin therapeutic strategies.

Role of SpaO in the assembly of the sorting platform of a Salmonella type III secretion system.

Many bacterial pathogens and symbionts use type III secretion machines to interact with their hosts by injecting bacterial effector proteins into host target cells. A central component of this complex machine is the cytoplasmic sorting platform, which orchestrates the engagement and preparation of type III secreted proteins for their delivery to the needle complex, the substructure of the type III secretion system that mediates their passage through the bacterial envelope. The sorting platform is thought to be a dynamic structure whose components alternate between assembled and disassembled states. However, how this dynamic behavior is controlled is not understood. In S. Typhimurium a core component of the sorting platform is SpaO, which is synthesized in two tandemly translated products, a full length (SpaOL) and a short form (SpaOS) composed of the C-terminal 101 amino acids. Here we show that in the absence of SpaOS the assembly of the needle substructure of the needle complex, which requires a functional sorting platform, can still occur although with reduced efficiency. Consistent with this observation, in the absence of SpaOS secretion of effectors proteins, which requires a fully assembled injectisome, is only slightly compromised. In the absence of SpaOS we detect a significant number of fully assembled needle complexes that are not associated with fully assembled sorting platforms. We also find that although binding of SpaOL to SpaOS can be detected in the absence of other components of the sorting platform, this interaction is not detected in the context of a fully assembled sorting platform suggesting that SpaOS may not be a core structural component of the sorting platform. Consistent with this observation we find that SpaOS and OrgB, a component of the sorting platform, share the same binding surface on SpaOL. We conclude that SpaOS regulates the assembly of the sorting platform during type III secretion.

Bacterial injection machines: Evolutionary diverse but functionally convergent.

Many human pathogens use Type III, Type IV, and Type VI secretion systems to deliver effectors into their target cells. The contribution of these secretion systems to microbial virulence was the main focus of a workshop organised by the International University of Andalusia in Spain. The meeting addressed structure-function, substrate recruitment, and translocation processes, which differ widely on the different secretion machineries, as well as the nature of the translocated effectors and their roles in subverting the host cell. An excellent panel of worldwide speakers presented the state of the art of the field, highlighting the involvement of bacterial secretion in human disease and discussing mechanistic aspects of bacterial pathogenicity, which can provide the bases for the development of novel antivirulence strategies.

Generation and Characterization of Typhoid Toxin-Neutralizing Human Monoclonal Antibodies.

Typhoid toxin is a virulence factor of Salmonella enterica serovar Typhi, the causative agent of typhoid fever, and is thought to be responsible for the symptoms of severe disease. This toxin has a unique A2B5 architecture with two active subunits, the ADP ribosyl transferase PltA and the DNase CdtB, linked to a pentameric B subunit, which is alternatively made of PltB or PltC. Here, we describe the generation and characterization of typhoid toxin-neutralizing human monoclonal antibodies by immunizing genetically engineered mice that have a full set of human immunoglobulin variable region genes. We identified several monoclonal antibodies with strong in vitro and in vivo toxin-neutralizing activity and different mechanisms of toxin neutralization. These antibodies could serve as the basis for the development of novel therapeutic strategies against typhoid fever.

Metabolic and fitness determinants for in vitro growth and intestinal colonization of the bacterial pathogen Campylobacter jejuni.

Campylobacter jejuni is one of the leading infectious causes of food-borne illness around the world. Its ability to persistently colonize the intestinal tract of a broad range of hosts, including food-producing animals, is central to its epidemiology since most infections are due to the consumption of contaminated food products. Using a highly saturated transposon insertion library combined with next-generation sequencing and a mouse model of infection, we have carried out a comprehensive genome-wide analysis of the fitness determinants for growth in vitro and in vivo of a highly pathogenic strain of C. jejuni. A comparison of the C. jejuni requirements to colonize the mouse intestine with those necessary to grow in different culture media in vitro, combined with isotopologue profiling and metabolic flow analysis, allowed us to identify its metabolic requirements to establish infection, including the ability to acquire certain nutrients, metabolize specific substrates, or maintain intracellular ion homeostasis. This comprehensive analysis has identified metabolic pathways that could provide the basis for the development of novel strategies to prevent C. jejuni colonization of food-producing animals or to treat human infections.

Visualization and characterization of individual type III protein secretion machines in live bacteria.

Type III protein secretion machines have evolved to deliver bacterially encoded effector proteins into eukaryotic cells. Although electron microscopy has provided a detailed view of these machines in isolation or fixed samples, little is known about their organization in live bacteria. Here we report the visualization and characterization of the Salmonella type III secretion machine in live bacteria by 2D and 3D single-molecule switching superresolution microscopy. This approach provided access to transient components of this machine, which previously could not be analyzed. We determined the subcellular distribution of individual machines, the stoichiometry of the different components of this machine in situ, and the spatial distribution of the substrates of this machine before secretion. Furthermore, by visualizing this machine in Salmonella mutants we obtained major insights into the machine's assembly. This study bridges a major resolution gap in the visualization of this nanomachine and may serve as a paradigm for the examination of other bacterially encoded molecular machines.

In Situ Structures of Polar and Lateral Flagella Revealed by Cryo-Electron Tomography.

The bacterial flagellum is a sophisticated self-assembling nanomachine responsible for motility in many bacterial pathogens, including Pseudomonas aeruginosa, Vibrio spp., and Salmonella enterica The bacterial flagellum has been studied extensively in the model systems Escherichia coli and Salmonella enterica serovar Typhimurium, yet the range of variation in flagellar structure and assembly remains incompletely understood. Here, we used cryo-electron tomography and subtomogram averaging to determine in situ structures of polar flagella in P. aeruginosa and peritrichous flagella in S Typhimurium, revealing notable differences between these two flagellar systems. Furthermore, we observed flagellar outer membrane complexes as well as many incomplete flagellar subassemblies, which provide additional insight into mechanisms underlying flagellar assembly and loss in both P. aeruginosa and S Typhimurium.IMPORTANCE The bacterial flagellum has evolved as one of the most sophisticated self-assembled molecular machines, which confers locomotion and is often associated with virulence of bacterial pathogens. Variation in species-specific features of the flagellum, as well as in flagellar number and placement, results in structurally distinct flagella that appear to be adapted to the specific environments that bacteria encounter. Here, we used cutting-edge imaging techniques to determine high-resolution in situ structures of polar flagella in Pseudomonas aeruginosa and peritrichous flagella in Salmonella enterica serovar Typhimurium, demonstrating substantial variation between flagella in these organisms. Importantly, we observed novel flagellar subassemblies and provided additional insight into the structural basis of flagellar assembly and loss in both P. aeruginosa and S Typhimurium.

Salmonella enterica serovar-specific transcriptional reprogramming of infected cells.

Despite their high degree of genomic similarity, different Salmonella enterica serovars are often associated with very different clinical presentations. In humans, for example, the typhoidal S. enterica serovar Typhi causes typhoid fever, a life-threatening systemic disease. In contrast, the non-typhoidal S. enterica serovar Typhimurium causes self-limiting gastroenteritis. The molecular bases for these different clinical presentations are incompletely understood. The ability to re-program gene expression in host cells is an essential virulence factor for typhoidal and non-typhoidal S. enterica serovars. Here, we have compared the transcriptional profile of cultured epithelial cells infected with S. Typhimurium or S. Typhi. We found that both serovars stimulated distinct transcriptional responses in infected cells that are associated with the stimulation of specific signal transduction pathways. These specific responses were associated with the presence of a distinct repertoire of type III secretion effector proteins. These observations provide major insight into the molecular bases for potential differences in the pathogenic mechanisms of typhoidal and non-typhoidal S. enterica serovars.

Bacterial type III secretion systems: specialized nanomachines for protein delivery into target cells.

One of the most exciting developments in the field of bacterial pathogenesis in recent years is the discovery that many pathogens utilize complex nanomachines to deliver bacterially encoded effector proteins into target eukaryotic cells. These effector proteins modulate a variety of cellular functions for the pathogen's benefit. One of these protein-delivery machines is the type III secretion system (T3SS). T3SSs are widespread in nature and are encoded not only by bacteria pathogenic to vertebrates or plants but also by bacteria that are symbiotic to plants or insects. A central component of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins across the bacterial envelope. Working in conjunction with several cytoplasmic components, the needle complex engages specific substrates in sequential order, moves them across the bacterial envelope, and ultimately delivers them into eukaryotic cells. The central role of T3SSs in pathogenesis makes them great targets for novel antimicrobial strategies.