YopD Self-assembly and Binding to LcrV Facilitate Type III Secretion Activity by Yersinia pseudotuberculosis

YopD-like translocator proteins encoded by several Gram-negative bacteria are important for type III secretion-dependent delivery of anti-host effectors into eukaryotic cells. This probably depends on their ability to form pores in the infected cell plasma membrane, through which effectors may gain access to the cell interior. In addition, Yersinia YopD is a negative regulator essential for the control of effector synthesis and secretion. As a prerequisite for this functional duality, YopD may need to establish molecular interactions with other key T3S components. A putative coiled-coil domain and an α-helical amphipathic domain, both situated in the YopD C terminus, may represent key protein-protein interaction domains. Therefore, residues within the YopD C terminus were systematically mutagenized. All 68 mutant bacteria were first screened in a variety of assays designed to identify individual residues essential for YopD function, possibly by providing the interaction interface for the docking of other T3S proteins. Mirroring the effect of a full-length yopD gene deletion, five mutant bacteria were defective for both yop regulatory control and effector delivery. Interestingly, all mutations clustered to hydrophobic amino acids of the amphipathic domain. Also situated within this domain, two additional mutants rendered YopD primarily defective in the control of Yop synthesis and secretion. Significantly, protein-protein interaction studies revealed that functionally compromised YopD variants were also defective in self-oligomerization and in the ability to engage another translocator protein, LcrV. Thus, the YopD amphipathic domain facilitates the formation of YopD/YopD and YopD/LcrV interactions, two critical events in the type III secretion process.     

Genetically Engineered Frameshifted YopN-TyeA Chimeras Influence Type III Secretion System Function in Yersinia pseudotuberculosis

Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis, we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia-host cell contact.     

A Potent Anti-SpuE Antibody Allosterically Inhibits Type III Secretion System and Attenuates Virulence of Pseudomonas Aeruginosa

Multidrug-resistant gram-negative bacteria infection is particularly severe within the designated ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), which underscores the urgent need to explore alternative therapeutic strategies. The type III secretion system (T3SS) is considered to be a key virulence factor in many gram-negative bacteria, and T3SS is in turn regulated by SpuE in P. aeruginosa, which is a spermidine binding protein from an ATP-binding cassette transporter family and highly conserved within ESKAPE pathogens. Here, we identified a potent anti-SpuE antagonistic antibody that allosterically inhibits the expression of T3SS and attenuates virulence of P. aeruginosa. X-ray crystallography and molecular dynamics simulations revealed that binding of antibody to SpuE induces a change in the dynamics of SpuE, which in turn may reduce spermidine uptake by P. aeruginosa. The antibody could serve as a template for developing novel biologics to target a broad spectrum of gram-negative bacteria.     

Cross-talk between Type Three Secretion System and Metabolism in Yersinia

Pathogenic yersiniae utilize a type three secretion system (T3SS) to inject Yop proteins into host cells in order to undermine their immune response. YscM1 and YscM2 proteins have been reported to be functionally equivalent regulators of the T3SS in Yersinia enterocolitica. Here, we show by affinity purification, native gel electrophoresis and small angle x-ray scattering that both YscM1 and YscM2 bind to phosphoenolpyruvate carboxylase (PEPC) of Y. enterocolitica. Under in vitro conditions, YscM1, but not YscM2, was found to inhibit PEPC with an apparent IC₅₀ of 4 μm (K_i = 1 μm). To analyze the functional roles of PEPC, YscM1, and YscM2 in Yop-producing bacteria, cultures of Y. enterocolitica wild type and mutants defective in the formation of PEPC, YscM1, or YscM2, respectively, were grown under low calcium conditions in the presence of [U-¹³C₆]glucose. The isotope compositions of secreted Yop proteins and nine amino acids from cellular proteins were analyzed by mass spectrometry. The data indicate that a considerable fraction of oxaloacetate used as precursor for amino acids was derived from [¹³C₃]phosphoenolpyruvate by the catalytic action of PEPC in the wild-type strain but not in the PEPC^- mutant. The data imply that PEPC is critically involved in replenishing the oxaloacetate pool in the citrate cycle under virulence conditions. In the YscM1^- and YscM2^- mutants, increased rates of pyruvate formation via glycolysis or the Entner-Doudoroff pathway, of oxaloacetate formation via the citrate cycle, and of amino acid biosynthesis suggest that both regulators trigger the central metabolism of Y. enterocolitica. We propose a “load-and-shoot cycle” model to account for the cross-talk between T3SS and metabolism in pathogenic yersiniae.Type three secretion systems (T3SSs)³ are used by several Gram-negative bacteria as microinjection devices to deliver effector proteins into host cells (1). The translocated effector proteins reprogram the host cell in favor of the microbial invader or symbiont. Pathogenic yersiniae (the enteropathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis and the plague bacillus Yersinia pestis) utilize a plasmid-encoded T3SS to undermine the host primary immune response (2). This is mediated by the injection of a set of effector proteins called Yops (Yersinia outer proteins) into host cells, in particular into cells with innate immune functions, such as macrophages, dendritic cells, and neutrophils (3). The concerted action of Yops, targeting multiple signaling pathways, results in actin cytoskeleton disruption, suppression of proinflammatory signaling, and induction of apoptosis. This strategy enables yersiniae to multiply extracellularly in host tissue.Expression of the Yersinia T3SS is up-regulated at 37 °C, and translocation of Yops across the host cell membrane is triggered by cell contact (4, 5). Pathogenic yersiniae cultivated under low calcium conditions at 37 °C express a phenotype referred to as “low calcium response” (LCR). The LCR is characterized by growth restriction as well as massive expression and secretion of Yops into the culture medium (6–9). The allocation of energy and metabolites for the massive synthesis and transport of Yops is demanding, and this burden is believed to be responsible for the observed growth inhibition (10). To give an idea of the metabolic requirements, Yops are secreted to the culture supernatant in 10-mg amounts per liter of culture within 2 h after calcium depletion of the medium. Furthermore, post-translationally secreted substrates need to be unfolded by a T3SS-specific ATPase prior to secretion (11–14). In addition, T3SS-dependent transport of Yops requires the proton motive force (15). However, there is evidence that growth cessation and Yop expression can be uncoupled (16, 17), suggesting a coordinated regulation of metabolism and protein transport rather than the LCR reflecting an inevitable physiological consequence.What are the candidate proteins that could be involved in such a coordination? YscM1 and YscM2 (57% identical to YscM1) are key candidates, since they act at a major nodal point of the T3SS regulatory network in Y. enterocolitica. In Y. pestis and Y. pseudotuberculosis, only the YscM1 homologue LcrQ exists (99% identical to YscM1). YscM1/LcrQ and YscM2 are secretion substrates of the T3SS that are involved in up-regulation of Yop expression after host cell contact. Upon cell contact, the decrease of intracellular levels of YscM1/LcrQ and YscM2 due to their translocation into host cells results in a derepression of Yop synthesis (18–21). The two yscM copies of Y. enterocolitica were presumed to be functionally equivalent, since deletion of either gene was found to be phenotypically silent (19, 22). Only deletion of both yscM genes could establish the lcrQ phenotype (19, 22), distinguished by temperature sensitivity for growth, derepressed Yop expression, and secretion of LcrV and YopD in the presence of calcium ions.YscM1/LcrQ as well as YscM2 exhibit homology to the N terminus of the effector YopH (19, 23, 24), a fact that may explain their shared assistance by SycH (specific Yop chaperone) (20, 25). It was shown that YscM1/LcrQ and YscM2 exert their influence on Yop expression in concert with the T3SS components SycH, SycD (LcrH in Y. pestis and Y. pseudotuberculosis), and YopD (21, 26–28). It is further described that YscM1 and/or YscM2 interact with several of the T3SS-specific chaperones, in particular with SycH, SycE, SycD, and SycO (20, 29–31). This has led to the model that YscM/LcrQ proteins might function as an interface that senses whether chaperones are loaded with Yops and transduces these signals into control of Yop expression (14).These features of YscM1/LcrQ and YscM2 prompted us to speculate about a key role of these proteins in coordination of metabolism and expression of T3SS components. Using recombinant GST-YscM1 and GST-YscM2 as bait for Y. enterocolitica cytosolic proteins, we identified phosphoenolpyruvate carboxylase (PEPC) as interaction partner of both YscM1 and YscM2. Under in vitro conditions, YscM1 down-regulated PEPC activity and bacterial growth/replication. Isotopologue profiling of Yop proteins and derived amino acids from Y. enterocolitica grown in the presence of [U-¹³C₆]glucose showed the functionality of the PEPC reaction under virulence conditions (isotopologues are molecular entities that differ only in isotopic composition (number of isotopic substitutions); e.g. CH₄, CH₃D, and CH₂D₂). Moreover, biosynthetic rates of amino acids were increased in mutants defective in YscM1 or YscM2, suggesting a general role of these regulators in the metabolism of Y. enterocolitica. Recently, evidence has been accumulating that the metabolic state contributes to the regulation of T3SSs of diverse pathogens, also including the flagellar T3SS in Pseudomonas and Salmonella (32–36).     

The Lon Protease Is Essential for Full Virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa PAO1 lon mutants are supersusceptible to ciprofloxacin, and exhibit a defect in cell division and in virulence-related properties, such as swarming, twitching and biofilm formation, despite the fact that the Lon protease is not a traditional regulator. Here we set out to investigate the influence of a lon mutation in a series of infection models. It was demonstrated that the lon mutant had a defect in cytotoxicity towards epithelial cells, was less virulent in an amoeba model as well as a mouse acute lung infection model, and impacted on in vivo survival in a rat model of chronic infection. Using qRT-PCR it was demonstrated that the lon mutation led to a down-regulation of Type III secretion genes. The Lon protease also influenced motility and biofilm formation in a mucin-rich environment. Thus alterations in several virulence-related processes in vitro in a lon mutant were reflected by defective virulence in vivo.     

Structural and Functional Characterisation of TesA - A Novel Lysophospholipase A from Pseudomonas aeruginosa

TesA from Pseudomonas aeruginosa belongs to the GDSL hydrolase family of serine esterases and lipases that possess a broad substrate- and regiospecificity. It shows high sequence homology to TAP, a multifunctional enzyme from Escherichia coli exhibiting thioesterase, lysophospholipase A, protease and arylesterase activities. Recently, we demonstrated high arylesterase activity for TesA, but only minor thioesterase and no protease activity. Here, we present a comparative analysis of TesA and TAP at the structural, biochemical and physiological levels. The crystal structure of TesA was determined at 1.9 Å and structural differences were identified, providing a possible explanation for the differences in substrate specificities. The comparison of TesA with other GDSL-hydrolase structures revealed that the flexibility of active-site loops significantly affects their substrate specificity. This assumption was tested using a rational approach: we have engineered the putative coenzyme A thioester binding site of E. coli TAP into TesA of P. aeruginosa by introducing mutations D17S and L162R. This TesA variant showed increased thioesterase activity comparable to that of TAP. TesA is the first lysophospholipase A described for the opportunistic human pathogen P. aeruginosa. The enzyme is localized in the periplasm and may exert important functions in the homeostasis of phospholipids or detoxification of lysophospholipids.     

The Periplasmic Protein TolB as a Potential Drug Target in Pseudomonas aeruginosa

The Gram-negative bacterium Pseudomonas aeruginosa is one of the most dreaded pathogens in the hospital setting, and represents a prototype of multi-drug resistant “superbug” for which effective therapeutic options are very limited. The identification and characterization of new cellular functions that are essential for P. aeruginosa viability and/or virulence could drive the development of anti-Pseudomonas compounds with novel mechanisms of action. In this study we investigated whether TolB, the periplasmic component of the Tol-Pal trans-envelope protein complex of Gram-negative bacteria, represents a potential drug target in P. aeruginosa. By combining conditional mutagenesis with the analysis of specific pathogenicity-related phenotypes, we demonstrated that TolB is essential for P. aeruginosa growth, both in laboratory and clinical strains, and that TolB-depleted P. aeruginosa cells are strongly defective in cell-envelope integrity, resistance to human serum and several antibiotics, as well as in the ability to cause infection and persist in an insect model of P. aeruginosa infection. The essentiality of TolB for P. aeruginosa growth, resistance and pathogenicity highlights the potential of TolB as a novel molecular target for anti-P. aeruginosa drug discovery.     

Deciphering Protein Dynamics of the Siderophore Pyoverdine Pathway in Pseudomonas aeruginosa

Pseudomonas aeruginosa produces the siderophore, pyoverdine (PVD), to obtain iron. Siderophore pathways involve complex mechanisms, and the machineries responsible for biosynthesis, secretion and uptake of the ferri-siderophore span both membranes of Gram-negative bacteria. Most proteins involved in the PVD pathway have been identified and characterized but the way the system functions as a whole remains unknown. By generating strains expressing fluorescent fusion proteins, we show that most of the proteins are homogeneously distributed throughout the bacterial cell. We also studied the dynamics of these proteins using fluorescence recovery after photobleaching (FRAP). This led to the first diffusion coefficients ever determined in P. aeruginosa. Cytoplasmic and periplamic diffusion appeared to be slower than in Escherichia coli but membrane proteins seemed to behave similarly in the two species. The diffusion of cytoplasmic and periplasmic tagged proteins involved in the PVD pathway was dependent on the interaction network to which they belong. Importantly, the TonB protein, motor of the PVD-Fe uptake process, was mostly immobile but its mobility increased substantially in the presence of PVD-Fe.     

Exploration of Chlamydial Type III Secretion System Reconstitution in Escherichia coli

Background

Type III secretion system is a virulent factor for many pathogens, and is thought to play multiple roles in the development cycle and pathogenesis of chlamydia, an important human pathogen. However, due to the obligate intracellular parasitical nature of chlamydiae and a lack of convenient genetic methodology for the organisms, very limited approaches are available to study the chlamydial type III secretion system. In this study, we explored the reconstitution of a chlamydial type III secretion in Escherichia coli.

Results

We successfully cloned all 6 genomic DNA clusters of the chlamydial type III secretion system into three bacterial plasmids. 5 of the 6 clusters were found to direct mRNA synthesis from their own promoters in Escherichia coli transformed with the three plasmids. Cluster 5 failed to express mRNA using its own promoters. However, fusion of cluster 5 to cluster 6 resulted in the expression of cluster 5 mRNA. Although only two of the type III secretion system proteins were detected transformed E. coli due to limited antibody availability, type III secretion system-like structures were detected in ultrathin sections in a small proportion of transformed E. coli.

Conclusions

We have successfully generated E. coli expressing all genes of the chlamydial type III secretion system. This serves as a foundation for optimal expression and assembly of the recombinant chlamydial type III secretion system, which may be extremely useful for the characterization of the chlamydial type III secretion system and for studying its role in chlamydial pathogenicity.     

Structure and Interactions of the Cytoplasmic Domain of the Yersinia Type III Secretion Protein YscD

The virulence of a large number of Gram-negative bacterial pathogens depends on the type III secretion (T3S) system, which transports select bacterial proteins into host cells. An essential component of the Yersinia T3S system is YscD, a single-pass inner membrane protein. We report here the 2.52-Å resolution structure of the cytoplasmic domain of YscD, called YscDc. The structure confirms that YscDc consists of a forkhead-associated (FHA) fold, which in many but not all cases specifies binding to phosphothreonine. YscDc, however, lacks the structural properties associated with phosphothreonine binding and thus most likely interacts with partners in a phosphorylation-independent manner. Structural comparison highlighted two loop regions, L3 and L4, as potential sites of interactions. Alanine substitutions at L3 and L4 had no deleterious effects on protein structure or stability but abrogated T3S in a dominant negative manner. To gain insight into the function of L3 and L4, we identified proteins associated with YscD by affinity purification coupled to mass spectrometry. The lipoprotein YscJ was found associated with wild-type YscD, as was the effector YopH. Notably, the L3 and L4 substitution mutants interacted with more YopH than did wild-type YscD. These substitution mutants also interacted with SycH (the specific chaperone for YopH), the putative C-ring component YscQ, and the ruler component YscP, whereas wild-type YscD did not. These results suggest that substitutions in the L3 and L4 loops of YscD disrupted the dissociation of SycH from YopH, leading to the accumulation of a large protein complex that stalled the T3S apparatus.     

Genes as Early Responders Regulate Quorum-Sensing and Control Bacterial Cooperation in Pseudomonas aeruginosa

Quorum-sensing (QS) allows bacterial communication to coordinate the production of extracellular products essential for population fitness at higher cell densities. It has been generally accepted that a significant time duration is required to reach appropriate cell density to activate the relevant quiescent genes encoding these costly but beneficial public goods. Which regulatory genes are involved and how these genes control bacterial communication at the early phases are largely un-explored. By determining time-dependent expression of QS-related genes of the opportunistic pathogen Pseudomonas aerugionsa, we show that the induction of social cooperation could be critically influenced by environmental factors to optimize the density of population. In particular, small regulatory RNAs (RsmY and RsmZ) serving as early responders, can promote the expression of dependent genes (e.g. lasR) to boost the synthesis of intracellular enzymes and coordinate instant cooperative behavior in bacterial cells. These early responders, acting as a rheostat to finely modulate bacterial cooperation, which may be quickly activated under environment threats, but peter off when critical QS dependent genes are fully functional for cooperation. Our findings suggest that RsmY and RsmZ critically control the timing and levels of public goods production, which may have implications in sociomicrobiology and infection control.