Delivery Of Effector Proteins Research Articles

Citrobacter rodentium possesses a functional type II secretion system necessary for successful host infection

ABSTRACT Infectious diarrheal diseases are the third leading cause of mortality in young children, many of which are driven by Gram-negative bacterial pathogens. To establish successful host infections these pathogens employ a plethora of virulence factors necessary to compete with the resident microbiota, and evade and subvert the host defenses. The type II secretion system (T2SS) is one such conserved molecular machine that allows for the delivery of effector proteins into the extracellular milieu. To explore the role of the T2SS during natural host infection, we used Citrobacter rodentium, a murine enteric pathogen, as a model of human intestinal disease caused by pathogenic Escherichia coli such as Enteropathogenic and Enterohemorrhagic E. coli (EPEC and EHEC). In this study, we determined that the C. rodentium genome encodes one T2SS and 22 potential T2SS-secreted protein effectors, as predicted via sequence homology. We demonstrated that this system was functional in vitro, identifying a role in intestinal mucin degradation allowing for its utilization as a carbon source, and promoting C. rodentium attachment to a mucus-producing colon cell line. During host infection, loss of the T2SS or associated effectors led to a significant colonization defect and lack of systemic spread. In mice susceptible to lethal infection, T2SS-deficient C. rodentium was strongly attenuated, resulting in reduced morbidity and mortality in infected hosts. Together these data highlight the important role of the T2SS and its effector repertoire during C. rodentium pathogenesis, aiding in successful host mucosal colonization.

NopC/T/L Signal Crosstalk Gene GmPHT1-4.

Symbiotic nodulation between leguminous plants and rhizobia is a critical biological interaction. The type III secretion system (T3SS) employed by rhizobia manipulates the host's nodulation signaling, analogous to mechanisms used by certain bacterial pathogens for effector protein delivery into host cells. This investigation explores the interactive signaling among type III effectors HH103ΩNopC, HH103ΩNopT, and HH103ΩNopL from SinoRhizobium fredii HH103. Experimental results revealed that these effectors positively regulate nodule formation. Transcriptomic analysis pinpointed GmPHT1-4 as the key gene facilitating this effector-mediated signaling. Overexpression of GmPHT1-4 enhances nodulation, indicating a dual function in nodulation and phosphorus homeostasis. This research elucidates the intricate regulatory network governing Rhizobium-soybean (Glycine max (L.) Merr) interactions and the complex interplay between type III effectors.

SPI-1 virulence gene expression modulates motility of Salmonella Typhimurium in a proton motive force- and adhesins-dependent manner.

Both the bacterial flagellum and the evolutionary related injectisome encoded on the Salmonella pathogenicity island 1 (SPI-1) play crucial roles during the infection cycle of Salmonella species. The interplay of both is highlighted by the complex cross-regulation that includes transcriptional control of the flagellar master regulatory operon flhDC by HilD, the master regulator of SPI-1 gene expression. Contrary to the HilD-dependent activation of flagellar gene expression, we report here that activation of HilD resulted in a dramatic loss of motility, which was dependent on the presence of SPI-1. Single cell analyses revealed that HilD-activation triggers a SPI-1-dependent induction of the stringent response and a substantial decrease in proton motive force (PMF), while flagellation remains unaffected. We further found that HilD activation enhances the adhesion of Salmonella to epithelial cells. A transcriptome analysis revealed a simultaneous upregulation of several adhesin systems, which, when overproduced, phenocopied the HilD-induced motility defect. We propose a model where the SPI-1-dependent depletion of the PMF and the upregulation of adhesins upon HilD-activation enable flagellated Salmonella to rapidly modulate their motility during infection, thereby enabling efficient adhesion to host cells and delivery of effector proteins.

Vibrio type III secretion system 2 is not restricted to the Vibrionaceae and encodes differentially distributed repertoires of effector proteins.

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis worldwide. A distinctive feature of the O3:K6 pandemic clone, and its derivatives, is the presence of a second, phylogenetically distinct, type III secretion system (T3SS2) encoded within the genomic island VPaI-7. The T3SS2 allows the delivery of effector proteins directly into the cytosol of infected eukaryotic cells to subvert key host-cell processes, critical for V. parahaemolyticus to colonize and cause disease. Furthermore, the T3SS2 also increases the environmental fitness of V. parahaemolyticus in its interaction with bacterivorous protists; hence, it has been proposed that it contributed to the global oceanic spread of the pandemic clone. Several reports have identified T3SS2-related genes in Vibrio and non-Vibrio species, suggesting that the T3SS2 gene cluster is not restricted to the Vibrionaceae and can mobilize through horizontal gene transfer events. In this work, we performed a large-scale genomic analysis to determine the phylogenetic distribution of the T3SS2 gene cluster and its repertoire of effector proteins. We identified putative T3SS2 gene clusters in 1130 bacterial genomes from 8 bacterial genera, 5 bacterial families and 47 bacterial species. A hierarchical clustering analysis allowed us to define six T3SS2 subgroups (I-VI) with different repertoires of effector proteins, redefining the concepts of T3SS2 core and accessory effector proteins. Finally, we identified a subset of the T3SS2 gene clusters (subgroup VI) that lacks most T3SS2 effector proteins described to date and provided a list of 10 novel effector candidates for this subgroup through bioinformatic analysis. Collectively, our findings indicate that the T3SS2 extends beyond the family Vibrionaceae and suggest that different effector protein repertories could have a differential impact on the pathogenic potential and environmental fitness of each bacterium that has acquired the Vibrio T3SS2 gene cluster.

Label-free relative quantitative proteomics reveals extracellular vesicles as a vehicle for Salmonella effector protein delivery.

Extracellular vesicles are small vesicles with a diameter of 30-150 nm that are actively secreted by eukaryotic cells and play important roles in intercellular communication, immune responses, and tumorigenesis. Previous studies have shown that extracellular vesicles are involved in the process of Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. However, changes in the protein content of extracellular vesicles elicited by S. Typhimurium infection have not been determined. Here, we extracted the extracellular vesicles with high purity from S. Typhimurium-infected Henle-407 cells, a kind of human intestinal epithelial cells, by ultracentrifugation combined with an extracellular vesicles purification kit, and analyzed their protein composition using label-free relative quantitative proteomics. The extracted extracellular vesicles exhibited an oval vesicular structure under electron microscopy, with a mean diameter of 140.4 ± 32.4 nm. The exosomal marker proteins CD9, CD63, and HSP70 were specifically detected. Compared with the uninfected group, nearly 1,234 specifically loaded proteins were uncovered in S. Typhimurium-infected Henle-407 cells. Among them were 409 S. Typhimurium-derived specific proteins, indicating a significant alteration in protein composition of extracellular vesicles by S. Typhimurium infection. Notably, these proteins included 75 secretory proteins and over 300 non-secretory proteins of S. Typhimurium, implicating novel pathways for bacterial protein delivery, although it remains unclear if their loading into extracellular vesicles is active or passive. To investigate the roles of these extracellular proteins, we exemplified the function of SopB, a well-known T3SS effector protein, and showed that the extracellular SopB could be taken up by RAW264.7 macrophages, activating the phosphorylation of Akt. This study provides new insights into the mechanism of Salmonella infection through extracellular vesicles that transport virulence proteins to uninfected neighboring cells to facilitate further infection.

The Type VI Secretion Systems in Plant-Beneficial Bacteria Modulate Prokaryotic and Eukaryotic Interactions in the Rhizosphere.

Rhizosphere colonizing plant growth promoting bacteria (PGPB) increase their competitiveness by producing diffusible toxic secondary metabolites, which inhibit competitors and deter predators. Many PGPB also have one or more Type VI Secretion System (T6SS), for the delivery of weapons directly into prokaryotic and eukaryotic cells. Studied predominantly in human and plant pathogens as a virulence mechanism for the delivery of effector proteins, the function of T6SS for PGPB in the rhizosphere niche is poorly understood. We utilized a collection of Pseudomonas chlororaphis 30–84 mutants deficient in one or both of its two T6SS and/or secondary metabolite production to examine the relative importance of each T6SS in rhizosphere competence, bacterial competition, and protection from bacterivores. A mutant deficient in both T6SS was less persistent than wild type in the rhizosphere. Both T6SS contributed to competitiveness against other PGPB or plant pathogenic strains not affected by secondary metabolite production, but only T6SS-2 was effective against strains lacking their own T6SS. Having at least one T6SS was also essential for protection from predation by several eukaryotic bacterivores. In contrast to diffusible weapons that may not be produced at low cell density, T6SS afford rhizobacteria an additional, more immediate line of defense against competitors and predators.

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.

T4SEfinder: a bioinformatics tool for genome-scale prediction of bacterial type IV secreted effectors using pre-trained protein language model.

Bacterial type IV secretion systems (T4SSs) are versatile and membrane-spanning apparatuses, which mediate both genetic exchange and delivery of effector proteins to target eukaryotic cells. The secreted effectors (T4SEs) can affect gene expression and signal transduction of the host cells. As such, they often function as virulence factors and play an important role in bacterial pathogenesis. Nowadays, T4SE prediction tools have utilized various machine learning algorithms, but the accuracy and speed of these tools remain to be improved. In this study, we apply a sequence embedding strategy from a pre-trained language model of protein sequences (TAPE) to the classification task of T4SEs. The training dataset is mainly derived from our updated type IV secretion system database SecReT4 with newly experimentally verified T4SEs. An online web server termed T4SEfinder is developed using TAPE and a multi-layer perceptron (MLP) for T4SE prediction after a comprehensive performance comparison with several candidate models, which achieves a slightly higher level of accuracy than the existing prediction tools. It only takes about 3minutes to make a classification for 5000 protein sequences by T4SEfinder so that the computational speed is qualified for whole genome-scale T4SEs detection in pathogenic bacteria. T4SEfinder might contribute to meet the increasing demands of re-annotating secretion systems and effector proteins in sequenced bacterial genomes. T4SEfinder is freely accessible at https://tool2-mml.sjtu.edu.cn/T4SEfinder_TAPE/.

Cryo-EM reveals new species-specific proteins and symmetry elements in the Legionella pneumophila Dot/Icm T4SS.

Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires' disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.0 Å resolution maps of the Dot/Icm T4SS core complex by single particle cryo-EM. The high-resolution structural analysis has allowed us to identify proteins encoded outside the Dot/Icm genetic locus that contribute to the core T4SS structure. We can also now define two distinct areas of symmetry mismatch, one that connects the C18 periplasmic ring (PR) and the C13 outer membrane cap (OMC) and one that connects the C13 OMC with a 16-fold symmetric dome. Unexpectedly, the connection between the PR and OMC is DotH, with five copies sandwiched between the OMC and PR to accommodate the symmetry mismatch. Finally, we observe multiple conformations in the reconstructions that indicate flexibility within the structure.

A Brucella effector modulates the Arf6-Rab8a GTPase cascade to promote intravacuolar replication.

Remodeling of host cellular membrane transport pathways is a common pathogenic trait of many intracellular microbes that is essential to their intravacuolar life cycle and proliferation. The bacterium Brucella abortus generates a host endoplasmic reticulum‐derived vacuole (rBCV) that supports its intracellular growth, via VirB Type IV secretion system‐mediated delivery of effector proteins, whose functions and mode of action are mostly unknown. Here, we show that the effector BspF specifically promotes Brucella replication within rBCVs by interfering with vesicular transport between the trans‐Golgi network (TGN) and recycling endocytic compartment. BspF targeted the recycling endosome, inhibited retrograde traffic to the TGN, and interacted with the Arf6 GTPase‐activating Protein (GAP) ACAP1 to dysregulate Arf6‐/Rab8a‐dependent transport within the recycling endosome, which resulted in accretion of TGN‐associated vesicles by rBCVs and enhanced bacterial growth. Altogether, these findings provide mechanistic insight into bacterial modulation of membrane transport used to promote their own proliferation within intracellular vacuoles.

Identification of a Family of Vibrio Type III Secretion System Effectors That Contain a Conserved Serine/Threonine Kinase Domain.

ABSTRACTVibrio parahaemolyticus is a marine Gram-negative bacterium that is a leading cause of seafood-borne gastroenteritis. Pandemic strains of V. parahaemolyticus rely on a specialized protein secretion machinery known as the type III secretion system 2 (T3SS2) to cause disease. The T3SS2 mediates the delivery of effector proteins into the cytosol of infected cells, where they subvert multiple cellular pathways. Here, we identify a new T3SS2 effector protein encoded by VPA1328 (VP_RS21530) in V. parahaemolyticus RIMD2210633. Bioinformatic analysis revealed that VPA1328 is part of a larger family of uncharacterized T3SS effector proteins with homology to the VopG effector protein in Vibrio cholerae AM-19226. These VopG-like proteins are found in many but not all T3SS2 gene clusters and are distributed among diverse Vibrio species, including V. parahaemolyticus, V. cholerae, V. mimicus, and V. diabolicus and also in Shewanella baltica. Structure-based prediction analyses uncovered the presence of a conserved C-terminal kinase domain in VopG orthologs, similar to the serine/threonine kinase domain found in the NleH family of T3SS effector proteins. However, in contrast to NleH effector proteins, in tissue culture-based infections, VopG did not impede host cell death or suppress interleukin 8 (IL-8) secretion, suggesting a yet undefined role for VopG during V. parahaemolyticus infection. Collectively, our work reveals that VopG effector proteins, a new family of likely serine/threonine kinases, is widely distributed in the T3SS2 effector armamentarium among marine bacteria.IMPORTANCEVibrio parahaemolyticus is the leading bacterial cause of seafood-borne gastroenteritis worldwide. The pathogen relies on a type III secretion system to deliver a variety of effector proteins into the cytosol of infected cells to subvert cellular function. In this study, we identified a novel Vibrio parahaemolyticus effector protein that is similar to the VopG effector of Vibrio cholerae. VopG-like effectors were found in diverse Vibrio species and contain a conserved serine/threonine kinase domain that bears similarity to the kinase domain in the enterohemorrhagic Escherichia coli (EHEC) and Shigella NleH effectors that manipulate host cell survival pathways and host immune responses. Together our findings identify a new family of Vibrio effector proteins and highlight the role of horizontal gene transfer events among marine bacteria in shaping T3SS gene clusters.

Tracking bacterial effector protein delivery into host cells.

Bacterial Type IV secretion systems (T4SSs) are a functionally heterogeneous group of nanomachines that can deliver substrates into a wide range of target cells. The Helicobacter pylori Cag T4SS has an important role in the pathogenesis of gastric cancer. CagA, the only effector protein known to be secreted by the H. pylori Cag T4SS, enters human gastric cells and causes alterations in intracellular signaling that are linked to cancer pathogenesis. Understanding the molecular mechanisms by which CagA is delivered into gastric cells has been hindered by the lack of robust methods for monitoring this process. A publication in this issue of Molecular Microbiology describes a split luciferase assay for monitoring T4SS-mediated translocation of CagA into host cells. The use of this translocation reporter allowed the quantification of CagA translocation in real-time assays, thereby facilitating the analysis of the kinetics of CagA delivery. This system also allowed the tracking of several types of CagA fusion proteins and confirmed that protein unfolding is important for secretion by the Cag T4SS. This commentary discusses T4SS-dependent delivery of H. pylori CagA into host cells and the use of the split luciferase system for monitoring bacterial protein secretion and delivery into target cells.

Efficient intracellular delivery of p53 protein by engineered protein crystals restores tumor suppressing function in vivo

Direct delivery of proteins into cells holds significant potential for basic research and drug development. However, the poor endosomal escape of conventional delivery strategies remains a challenge, thus limiting the clinical translation of many protein therapeutics. Herein, we report that engineered Cry3Aa protein (Pos3Aa) crystals formed naturally within Bacillus thuringiensis can serve as a vehicle for efficient cytosolic delivery of bioactive proteins. We showed that Pos3Aa-mediated delivery of tumor suppressor p53 protein, a promising therapeutic candidate found to be inactivated in nearly half of human cancers, resulted in the restoration of p53 function in p53-deficient cancer cells, and thereby sensitized them to 5-fluorouracil chemotherapy as demonstrated in in vitro and in vivo models. Our results validate that Pos3Aa crystals can be a robust and effective platform for the cytosolic delivery of effector proteins, and suggest that efficient uptake and endosomal escape could be critical for efficacious p53 protein-based cancer therapy.

Salmonella-based platform for efficient delivery of functional binding proteins to the cytosol

Protein-based affinity reagents (like antibodies or alternative binding scaffolds) offer wide-ranging applications for basic research and therapeutic approaches. However, whereas small chemical molecules efficiently reach intracellular targets, the delivery of macromolecules into the cytosol of cells remains a major challenge; thus cytosolic applications of protein-based reagents are rather limited. Some pathogenic bacteria have evolved a conserved type III secretion system (T3SS) which allows the delivery of effector proteins into eukaryotic cells. Here, we enhance the T3SS of an avirulent strain of Salmonella typhimurium to reproducibly deliver multiple classes of recombinant proteins into eukaryotic cells. The efficacy of the system is probed with both DARPins and monobodies to functionally inhibit the paradigmatic and largely undruggable RAS signaling pathway. Thus, we develop a bacterial secretion system for potent cytosolic delivery of therapeutic macromolecules.

The Type VI secretion system in commensal Neisseria spp.

A type 6 secretion system (T6SS) was recently identified in the genome sequence data of an isolate sourced from a throat swab of a volunteer that is believed to be N. subflava. The T6SS is one of the most recently discovered bacterial secretion systems and this is the first time it has been reported in Neisseriaceae. Since this discovery, genome sequence analyses for a number of other commensal Neisseria spp. has identified that in fact, two distinct T6SS types exist across Neisseriaceae. These two types are clearly defined and are different to one another in both their core gene sequences and organisation. The two systems also differ in the number of VgrG proteins required for toxic effector protein delivery, as well as type of effector associated with them. The predicted VgrG/effector combinations identified in our isolate are not common to all members of the same species and further analysis has identified a wide range of diversity in these components between different strains of the same species. The data provides possible evidence that T6SS positive commensal Neisseria spp. can acquire new VgrG/effector combinations through competitor killing. A number of putative effectors have so far been identified within the genome of our original isolate, including hydrolases, phospholipases, and nucleases. Whilst these are predicted to be antibacterial effectors, the conditions under which the T6SS system is activated, as well as demonstration of the function of the effectors still needs to be investigated experimentally.

Molecular Dissection of the Campylobacter jejuni CadF and FlpA Virulence Proteins in Binding to Host Cell Fibronectin.

Campylobacter jejuni, a zoonotic pathogen that frequently colonizes poultry, possesses two Microbial Surface Components Recognizing Adhesive Matrix Molecule(s) (MSCRAMMs) termed CadF and FlpA that bind to the glycoprotein fibronectin (FN). Previous to this study, it was not known whether the CadF and FlpA proteins were functionally redundant or if both were required to potentiate host cell binding and signaling processes. We addressed these questions by generating a complete repertoire of cadF and flpA mutants and complemented isolates, and performing multiple phenotypic assays. Both CadF and FlpA were found to be necessary for the maximal binding of C. jejuni to FN and to host cells. In addition, both CadF and FlpA are required for the delivery of the C. jejuni Cia effector proteins into the cytosol of host target cells, which in turn activates the MAPK signaling pathway (Erk 1/2) that is required for the C. jejuni invasion of host cells. These data demonstrate the non-redundant and bi-functional nature of these two C. jejuni FN-binding proteins. Taken together, the C. jejuni CadF and FlpA adhesins facilitate the binding of C. jejuni to the host cells, permit delivery of effector proteins into the cytosol of a host target cell, and aid in the rewiring of host cell signaling pathways to alter host cell behavior.

Eukaryotic Cell Permeabilisation to Identify New Putative Chlamydial Type III Secretion System Effectors Secreted within Host Cell Cytoplasm.

Chlamydia trachomatis and Waddlia chondrophila are strict intracellular bacteria belonging to the Chlamydiales order. C. trachomatis is the most frequent bacterial cause of genital and ocular infections whereas W. chondrophila is an opportunistic pathogen associated with adverse pregnancy outcomes and respiratory infections. Being strictly intracellular, these bacteria are engaged in a complex interplay with their hosts to modulate their environment and create optimal conditions for completing their life cycle. For this purpose, they possess several secretion pathways and, in particular, a Type III Secretion System (T3SS) devoted to the delivery of effector proteins in the host cell cytosol. Identifying these effectors is a crucial step in understanding the molecular basis of bacterial pathogenesis. Following incubation of infected cells with perfringolysin O, a pore-forming toxin that binds cholesterol present in plasma membranes, we analysed by mass spectrometry the protein content of the host cell cytoplasm. We identified 13 putative effectors secreted by C. trachomatis and 19 secreted by W. chondrophila. Using Y. enterocolitica as a heterologous expression and secretion system, we confirmed that four of these identified proteins are secreted by the T3SS. Two W. chondrophila T3SS effectors (hypothetical proteins Wcw_0499 and Wcw_1706) were further characterised and demonstrated to be early/mid-cycle effectors. In addition, Wcw_1706 is associated with a tetratricopeptide domain-containing protein homologous to C. trachomatis class II chaperone. Furthermore, we identified a novel C. trachomatis effector, CT460 that localises in the eukaryotic nucleus when ectopically expressed in 293 T cells.

Enterococcus faecalis Enhances Expression and Activity of the Enterohemorrhagic Escherichia coli Type III Secretion System.

The gut microbiota can significantly impact invading pathogens and the disease they cause; however, many of the mechanisms that dictate commensal-pathogen interactions remain unclear. Enterohemorrhagic Escherichia coli (EHEC) is a potentially lethal human intestinal pathogen that uses microbiota-derived molecules as cues to efficiently regulate virulence factor expression. Here, we investigate the interaction between EHEC and Enterococcus faecalis, a common human gut commensal, and show that E. faecalis affects both expression and activity of the EHEC type III secretion system (T3SS) via two distinct mechanisms. First, in the presence of E. faecalis there is increased transcription of genes encoding the EHEC T3SS. This leads to increased effector translocation and ultimately greater numbers of pedestals formed on host cells. The same effect was observed with several strains of enterococci, suggesting that it is a general characteristic of this group. In a mechanism separate from E. faecalis-induced transcription of the T3SS, we report that an E. faecalis-secreted protease, GelE, cleaves a critical structural component of the EHEC T3SS, EspB. Our data suggest that this cleavage actually increases effector translocation by the T3SS, supporting a model where EspB proteolysis promotes maximum T3SS activity. Finally, we report that treatment of EHEC with E. faecalis-conditioned cell-free medium is insufficient to induce increased T3SS expression, suggesting that this effect relies on cell contact between E. faecalis and EHEC. This work demonstrates a complex interaction between a human commensal and pathogen that impacts both expression and function of a critical virulence factor.IMPORTANCE This work reveals a complex and multifaceted interaction between a human gut commensal, Enterococcus faecalis, and a pathogen, enterohemorrhagic E. coli We demonstrate that E. faecalis enhances expression of the enterohemorrhagic E. coli type III secretion system and that this effect likely depends on cell contact between the commensal and the pathogen. Additionally, the GelE protease secreted by E. faecalis cleaves a critical structural component of the EHEC type III secretion system. In agreement with previous studies, we find that this cleavage actually increases effector protein delivery into host cells by the secretion system. This work demonstrates that commensal bacteria can significantly shape expression and activity of pathogen virulence factors, which may ultimately shape the progression of disease.

Fitting Pieces into the Puzzle of Pseudomonas aeruginosa Type III Secretion System Gene Expression.

Type III secretion systems (T3SS) are widely distributed in Gram-negative microorganisms and critical for host-pathogen and host-symbiont interactions with plants and animals. Central features of the T3SS are a highly conserved set of secretion and translocation genes and contact dependence wherein host-pathogen interactions trigger effector protein delivery and serve as an inducing signal for T3SS gene expression. In addition to these conserved features, there are pathogen-specific properties that include a unique repertoire of effector genes and mechanisms to control T3SS gene expression. The Pseudomonas aeruginosa T3SS serves as a model system to understand transcriptional and posttranscriptional mechanisms involved in the control of T3SS gene expression. The central regulatory feature is a partner-switching system that controls the DNA-binding activity of ExsA, the primary regulator of T3SS gene expression. Superimposed upon the partner-switching mechanism are cyclic AMP and cyclic di-GMP signaling systems, two-component systems, global regulators, and RNA-binding proteins that have positive and negative effects on ExsA transcription and/or synthesis. In the present review, we discuss advances in our understanding of how these regulatory systems orchestrate the activation of T3SS gene expression in the context of acute infections and repression of the T3SS as P. aeruginosa adapts to and colonizes the cystic fibrosis airways.

SsaV Interacts with SsaL to Control the Translocon-to-Effector Switch in the Salmonella SPI-2 Type Three Secretion System.

Nonflagellar type III secretion systems (nf T3SSs) form a cell surface needle-like structure and an associated translocon that deliver bacterial effector proteins into eukaryotic host cells. This involves a tightly regulated hierarchy of protein secretion. A switch involving SctP and SctU stops secretion of the needle protein. The gatekeeper protein SctW is required for secretion of translocon proteins and controls a second switch to start effector secretion. Salmonella enterica serovar Typhimurium encodes two T3SSs in Salmonella pathogenicity island 1 (SPI-1) and SPI-2. The acidic vacuole containing intracellular bacteria stimulates assembly of the SPI-2 T3SS and its translocon. Sensing the nearly neutral host cytosolic pH is required for effector translocation. Here, we investigated the involvement of SPI-2-encoded proteins SsaP (SctP), SsaU (SctU), SsaV (SctV), and SsaL (SctW) in regulation of secretion. We found that SsaP and SsaU are involved in the first but not the second secretion switch. A random-mutagenesis screen identified amino acids of SsaV that regulate translocon and effector secretion. Single substitutions in subdomain 4 of SsaV or InvA (SPI-1-encoded SctV) phenocopied mutations of their corresponding gatekeepers with respect to translocon and effector protein secretion and host cell interactions. SsaL interacted with SsaV in bacteria exposed to low ambient pH but not after the pH was raised to 7.2. We propose that SsaP and SsaU enable the apparatus to become competent for a secretion switch and facilitate the SsaL-SsaV interaction. This mediates secretion of translocon proteins until neutral pH is sensed, which causes their dissociation, resulting in arrest of translocon secretion and derepression of effector translocation.IMPORTANCESalmonella Typhimurium is an intracellular pathogen that uses the SPI-2 type III secretion system to deliver virulence proteins across the vacuole membrane surrounding intracellular bacteria. This involves a tightly regulated hierarchy of protein secretion controlled by two molecular switches. We found that SPI-2-encoded proteins SsaP and SsaU are involved in the first but not the second secretion switch. We identify key amino acids of the inner membrane protein SsaV that are required to interact with the so-called gatekeeper protein SsaL and show that the dissociation of SsaV-SsaL causes the second switch, leading to delivery of effector proteins. Our results provide insights into the molecular events controlling virulence-associated type III secretion and suggest a broader model describing how the process is regulated.