RNase-mediated reprogramming of Yersinia virulence.

RNA degradation is an essential process that allows bacteria to regulate gene expression and has emerged as an important mechanism for controlling virulence. However, the individual contributions of RNases in this process are mostly unknown. Here, we tested the influence of 11 potential RNases in the intestinal pathogen Yersinia pseudotuberculosis on the expression of its type III secretion system (T3SS) and associated effectors (Yops) that are encoded on the Yersinia virulence plasmid. We found that exoribonuclease PNPase and endoribonuclease RNase III inhibit T3SS and yop gene transcription by repressing the synthesis of LcrF, the master activator of Yop-T3SS. Loss of both RNases led to an increase in lcrF mRNA levels. Our work indicates that PNPase exerts its influence via YopD, which accelerates lcrF mRNA degradation. Loss of RNase III, on the other hand, results in the downregulation of the CsrB and CsrC RNAs, thereby increasing the availability of active CsrA, which has been shown previously to enhance lcrF mRNA translation and stability. This CsrA-promoted increase of lcrF mRNA translation could be supported by other factors promoting the protein translation efficiency (e.g. IF-3, RimM, RsmG) that were also found to be repressed by RNase III. Transcriptomic profiling further revealed that Ysc-T3SS-mediated Yop secretion leads to global reprogramming of the Yersinia transcriptome with a massive shift of the expression from chromosomal to virulence plasmid-encoded genes. A similar reprogramming was also observed in the RNase III-deficient mutant under non-secretion conditions. Overall, our work revealed a complex control system where RNases orchestrate the expression of the T3SS/Yop machinery on multiple levels to antagonize phagocytic uptake and elimination by innate immune cells.

Directed Evolution of Protein-Based Sensors for Anaerobic Biological Activation of Methane.

Microbial alkane degradation pathways provide biological routes for converting these hydrocarbons into higher-value products. We recently reported the functional expression of a methyl-alkylsuccinate synthase (Mas) system in Escherichia coli, allowing for the heterologous anaerobic activation of short-chain alkanes. However, the enzymatic activation of methane via natural or engineered alkylsuccinate synthases has yet to be reported. To address this, we employed high-throughput screening to engineer the itaconate (IA)-responsive regulatory protein ItcR (WT-ItcR) from Yersinia pseudotuberculosis to instead respond to methylsuccinate (MS, the product of methane addition to fumarate), resulting in genetically encoded biosensors for MS. Here, we describe ItcR variants that, when regulating fluorescent protein expression in E. coli, show increased sensitivity, improved overall response, and enhanced specificity toward exogenously added MS relative to the wild-type repressor. Structural modeling and analysis of the ItcR ligand binding pocket provide insights into the altered molecular recognition. In addition to serving as biosensors for screening alkylsuccinate synthases capable of methane activation, MS-responsive ItcR variants also establish a framework for the directed evolution of other molecular reporters, targeting longer-chain alkylsuccinate products or other succinate derivatives.

Formation of Amyloid-Like Conformational States of ß-Structured Membrane Proteins on the Example of OMPF Porin from the Yersinia pseudotuberculosis Outer Membrane.

The work presents results of the in vitro and in silico study of formation of amyloid-like structures under harsh denaturing conditions by non-specific OmpF porin of Yersinia pseudotuberculosis (YpOmpF), a membrane protein with ß-barrel conformation. It has been shown that in order to obtain amyloid-like porin aggregates, preliminary destabilization of its structure in a buffer solution with acidic pH at elevated temperature followed by long-term incubation at room temperature is necessary. After heating at 95°C in a solution with pH 4.5, significant conformational rearrangements are observed in the porin molecule at the level of tertiary and secondary structure of the protein, which are accompanied by the increase in the content of total ß-structure and sharp decrease in the value of characteristic viscosity of the protein solution. Subsequent long-term exposure of the resulting unstable intermediate YpOmpF at room temperature leads to formation of porin aggregates of various shapes and sizes that bind thioflavin T, a specific fluorescent dye for the detection of amyloid-like protein structures. Compared to the initial protein, early intermediates of the amyloidogenic porin pathway, oligomers, have been shown to have increased toxicity to the Neuro-2aCCL-131™ mouse neuroblastoma cells. The results of computer modeling and analysis of the changes in intrinsic fluorescence during protein aggregation suggest that during formation of amyloid-like aggregates, changes in the structure of YpOmpF affect not only the areas with an internally disordered structure corresponding to the external loops of the porin, but also main framework of the molecule, which has a rigid spatial structure inherent to ß-barrel.

Quantifying Yersinia pseudotuberculosis Type III Secretion System Activity Following Iron Starvation and Anaerobic Growth.

A key virulence mechanism for many Gram-negative pathogens is the type III secretion system (T3SS), a needle-like appendage that translocates cytotoxic or immunomodulatory effector proteins into host cells. The T3SS is a target for antimicrobial discovery campaigns since it is accessible extracellularly and largely absent from non-pathogenic bacteria. Recent studies demonstrated that the T3SS of Yersinia and Salmonella are regulated by factors responsive to iron and oxygen, which are important niche-specific signals encountered during mammalian infection. Described here is a method for iron starvation of Yersinia pseudotuberculosis, with subsequent optional supplementation of inorganic iron. To assess the impact of oxygen availability, this iron starvation process is demonstrated under both aerobic and anaerobic conditions. Finally, incubating the cultures at the mammalian host temperature of 37 °C induces T3SS expression and allows quantification of Yersinia T3SS activity by visualizing effector proteins released into the supernatant. The steps detailed here offer an advantage over the use of iron chelators in the absence of iron starvation, which is insufficient for inducing robust iron starvation, presumably due to efficient Yersinia iron uptake and scavenging systems. Likewise, acid-washing laboratory glassware is detailed to ensure the removal of residual iron, which is essential for inducing robust iron starvation. Additionally, using a chelating agent is described to remove residual iron from media, and culturing the bacteria for several generations in the absence of iron to deplete bacterial iron stores. By incorporating standard protocols of trichloroacetic acid-induced protein precipitation, SDS-PAGE, and silver staining, this procedure demonstrates accessible ways to measure T3SS activity. While this procedure is optimized for Y. pseudotuberculosis, it offers a framework for studies in pathogens with similar robust iron uptake systems. In the age of antibiotic resistance, these methods can be expanded to assess the efficacy of antimicrobial compounds targeting the T3SS under host-relevant conditions.

Yersinia infection induces glucose depletion and AMPK-dependent inhibition of pyroptosis in mice.

Nutritional status and pyroptosis are important for host defence against infections. However, the molecular link that integrates nutrient sensing into pyroptosis during microbial infection is unclear. Here, using metabolic profiling, we found that Yersinia pseudotuberculosis infection results in a significant decrease in intracellular glucose levels in macrophages. This leads to activation of the glucose and energy sensor AMPK, which phosphorylates the essential kinase RIPK1 at S321 during caspase-8-mediated pyroptosis. This phosphorylation inhibits RIPK1 activation and thereby restrains pyroptosis. Boosting the AMPK-RIPK1 cascade by glucose deprivation, AMPK agonists, or RIPK1-S321E knockin suppresses pyroptosis, leading to increased susceptibility to Y. pseudotuberculosis infection in mice. Ablation of AMPK in macrophages or glucose supplementation in mice is protective against infection. Thus, we reveal a molecular link between glucose sensing and pyroptosis, and unveil a mechanism by which Y. pseudotuberculosis reduces glucose levels to impact host AMPK activation and limit host pyroptosis to facilitate infection.

A pyocin-like T6SS effector mediates bacterial competition in Yersinia pseudotuberculosis.

Within the realm of Gram-negative bacteria, bacteriocins are secreted almost everywhere, and the most representative are colicin and pyocin, which are secreted by Escherichia coli and Pseudomonas aeruginosa, respectively. Signal peptides at the amino terminus of bacteriocins or ABC transporters can secrete bacteriocins, which then enter bacteria through cell membrane receptors and exert toxicity. In general, the bactericidal spectrum is usually narrow, killing only the kin or closely related species. Our previous research indicates that YPK_0952 is an effector of the third Type VI secretion system (T6SS-3) in Yersinia pseudotuberculosis. Next, we sought to determine its identity and characterize its toxicity. We found that YPK_0952 (a pyocin-like effector) can achieve intra-species and inter-species competitive advantages through both contact-dependent and contact-independent mechanisms mediated by the T6SS-3 while enhancing the intestinal colonization capacity of Y. pseudotuberculosis. We further identified YPK_0952 as a DNase dependent on Mg2+, Ni2+, Mn2+, and Co2+ bivalent metal ions, and the homologous immune protein YPK_0953 can inhibit its activity. In summary, YPK_0952 exerts toxicity by degrading nucleic acids from competing cells, and YPK_0953 prevents self-attack in Y. pseudotuberculosis.IMPORTANCEBacteriocins secreted by Gram-negative bacteria generally enter cells through specific interactions on the cell surface, resulting in a narrow bactericidal spectrum. First, we identified a new pyocin-like effector protein, YPK_0952, in the third Type VI secretion system (T6SS-3) of Yersinia pseudotuberculosis. YPK_0952 is secreted by T6SS-3 and can exert DNase activity through contact-dependent and contact-independent entry into nearby cells of the same and other species (e.g., Escherichia coli) to help Y. pseudotuberculosis to exert a competitive advantage and promote intestinal colonization. This discovery lays the foundation for an in-depth study of the different effector protein types within the T6SS and their complexity in competing interactions. At the same time, this study provides a new development for the toolbox of toxin/immune pairs for studying Gram-negative bacteriocin translocation.

Functional versatility of Zur in metal homeostasis, motility, biofilm formation, and stress resistance in Yersinia pseudotuberculosis.

Zur (zinc uptake regulator) is a significant member of the Fur (ferric uptake regulator) superfamily, which is widely distributed in bacteria. Zur plays crucial roles in zinc homeostasis and influences cell development and environmental adaptation in various species. Yersinia pseudotuberculosis is a Gram-negative enteric that pathogen usually serves as a model organism in pathogenicity studies. The regulatory effects of Zur on the zinc transporter ZnuABC and the protein secretion system T6SS have been documented in Y. pseudotuberculosis. In this study, a comparative transcriptomics analysis between a ∆zur mutant and the wild-type (WT) strain of Y. pseudotuberculosis was conducted using RNA-seq. This analysis revealed global regulation by Zur across multiple functional categories, including membrane transport, cell motility, and molecular and energy metabolism. Additionally, Zur mediates the homeostasis not only of zinc but also ferric and magnesium in vivo. There was a notable decrease in 35 flagellar biosynthesis and assembly-related genes, leading to reduced swimming motility in the ∆zur mutant strain. Furthermore, Zur upregulated multiple simple sugar and oligopeptide transport system genes by directly binding to their promoters. The absence of Zur inhibited biofilm formation as well as reduced resistance to chloramphenicol and acidic stress. This study illustrates the comprehensive regulatory functions of Zur, emphasizing its importance in stress resistance and pathogenicity in Y. pseudotuberculosis. IMPORTANCE: Bacteria encounter diverse stresses in the environment and possess essential regulators to modulate the expression of genes in responding to the stresses for better fitness and survival. Zur (zinc uptake regulator) plays a vital role in zinc homeostasis. Studies of Zur from multiple species reviewed that it influences cell development, stress resistance, and virulence of bacteria. Y. pseudotuberculosis is an enteric pathogen that serves a model organism in the study of pathogenicity, virulence factors, and mechanism of environmental adaptation. In this study, transcriptomics analysis of Zur's regulons was conducted in Y. pseudotuberculosis. The functions of Zur as a global regulator in metal homeostasis, motility, nutrient acquisition, glycan metabolism, and nucleotide metabolism, in turn, increasing the biofilm formation, stress resistance, and virulence were reviewed. The importance of Zur in environmental adaptation and pathogenicity of Y. pseudotuberculosis was emphasized.

The oxidative stress response, in particular the katY gene, is temperature-regulated in Yersinia pseudotuberculosis.

Pathogenic bacteria, such as Yersinia pseudotuberculosis encounter reactive oxygen species (ROS) as one of the first lines of defense in the mammalian host. In return, the bacteria react by mounting an oxidative stress response. Previous global RNA structure probing studies provided evidence for temperature-modulated RNA structures in the 5'-untranslated region (5'-UTR) of various oxidative stress response transcripts, suggesting that opening of these RNA thermometer (RNAT) structures at host-body temperature relieves translational repression. Here, we systematically analyzed the transcriptional and translational regulation of ROS defense genes by RNA-sequencing, qRT-PCR, translational reporter gene fusions, enzymatic RNA structure probing and toeprinting assays. Transcription of four ROS defense genes was upregulated at 37°C. The trxA gene is transcribed into two mRNA isoforms, of which the most abundant short one contains a functional RNAT. Biochemical assays validated temperature-responsive RNAT-like structures in the 5'-UTRs of sodB, sodC and katA. However, they barely conferred translational repression in Y. pseudotuberculosis at 25°C suggesting partially open structures available to the ribosome in the living cell. Around the translation initiation region of katY we discovered a novel, highly efficient RNAT that was primarily responsible for massive induction of KatY at 37°C. By phenotypic characterization of catalase mutants and through fluorometric real-time measurements of the redox-sensitive roGFP2-Orp1 reporter in these strains, we revealed KatA as the primary H2O2 scavenger. Consistent with the upregulation of katY, we observed an improved protection of Y. pseudotuberculosis at 37°C. Our findings suggest a multilayered regulation of the oxidative stress response in Yersinia and an important role of RNAT-controlled katY expression at host body temperature.

Effect of Non-Specific Porins from the Outer Membrane of Yersinia pseudotuberculosis on Mice Brain Cortex Tissues.

It was found that a single-dose immunization of mice with Yersinia pseudotuberculosis porins OmpF and OmpC causes development of pathological changes in the deep layers of cerebral cortex characterized by dystrophic changes in the cells against the background of the increasing titer of specific antibodies. At the same time, the increased level of caspase-3 expression is observed in the neurons, which indicates induction of proapoptotic signaling pathways. The obtained results indicate potential ability of nonspecific pore-forming proteins of the outer membrane of Gram-negative bacteria to initiate development of degenerative changes in brain cells.

Calcium-responsive plasmid copy number regulation is dependent on discrete YopD domains in Yersinia pseudotuberculosis.

Yersinia pathogenicity depends mainly on a Type III Secretion System (T3SS) responsible for translocating effector proteins into the eukaryotic target cell cytosol. The T3SS is encoded on a 70 kb, low copy number virulence plasmid, pYV. A key T3SS regulator, YopD, is a multifunctional protein and consists of discrete modular domains that are essential for pore formation and translocation of Yop effectors. In Y. pseudotuberculosis, the temperature-dependent plasmid copy number increase that is essential for elevated T3SS gene dosage and virulence is also affected by YopD. Here, we found that the presence of intracellular YopD results in increased levels of the CopA-RNA and CopB, two inhibitors of plasmid replication. Secretion of YopD leads to decreased expression of copA and copB, resulting in increased plasmid copy number. Moreover, using a systematic mutagenesis of YopD mutants, we demonstrated that the same discrete modular domains important for YopD translocation are also necessary for both the regulation of plasmid copy number as well as copA and copB expression. Hence, Yersinia has evolved a mechanism coupling active secretion of a plasmid-encoded component of the T3SS, YopD, to the regulation of plasmid replication. Our work provides evidence for the cross-talk between plasmid-encoded functions with the IncFII replicon.

Computational Analysis of the Ligand-Binding Sites of the Molecular Chaperone OppA from Yersinia pseudotuberculosis.

The function of chaperones is to correct or degrade misfolded proteins inside the cell. Classic molecular chaperones such as GroEL and DnaK have not been found in the periplasm of Yersinia pseudotuberculosis. Some periplasmic substrate-binding proteins could be bifunctional, such as OppA. Using bioinformatic tools, we try to elucidate the nature of the interactions between OppA and ligands from four proteins with different oligomeric states. Using the crystal structure of the proteins Mal12 alpha-glucosidase from Saccharomyces cerevisiae S288C, LDH rabbit muscle lactate dehydrogenase, EcoRI endonuclease from Escherichia coli and THG Geotrichum candidum lipase, a hundred models were obtained in total, including five different ligands from each enzyme with five conformations of each ligand. The best values for Mal12 stem from ligands 4 and 5, with conformation 5 for both; for LDH, ligands 1 and 4, with conformations 2 and 4, respectively; for EcoRI, ligands 3 and 5, with conformation 1 for both; and for THG, ligands 2 and 3, with conformation 1 for both. The interactions were analyzed with LigProt, and the length of the hydrogen bridges has an average of 2.8 to 3.0 Å. The interaction within the OppA pocket is energetically favored due to the formation of hydrogen bonds both of OppA and of the selected enzymes. The Asp 419 residue is important in these junctions.

A secreted effector with a dual role as a toxin and as a transcriptional factor.

Bacteria have evolved multiple secretion systems for delivering effector proteins into the cytosol of neighboring cells, but the roles of many of these effectors remain unknown. Here, we show that Yersinia pseudotuberculosis secretes an effector, CccR, that can act both as a toxin and as a transcriptional factor. The effector is secreted by a type VI secretion system (T6SS) and can enter nearby cells of the same species and other species (such as Escherichia coli) via cell-cell contact and in a contact-independent manner. CccR contains an N-terminal FIC domain and a C-terminal DNA-binding domain. In Y. pseudotuberculosis cells, CccR inhibits its own expression by binding through its DNA-binding domain to the cccR promoter, and affects the expression of other genes through unclear mechanisms. In E. coli cells, the FIC domain of CccR AMPylates the cell division protein FtsZ, inducing cell filamentation and growth arrest. Thus, our results indicate that CccR has a dual role, modulating gene expression in neighboring cells of the same species, and inhibiting the growth of competitors.

Subversion of GBP-mediated host defense by E3 ligases acquired during Yersinia pestis evolution.

Plague has caused three worldwide pandemics in history, including the Black Death in medieval ages. Yersinia pestis, the etiological agent of plague, has evolved a powerful arsenal to disrupt host immune defenses during evolution from enteropathogenic Y. pseudotuberculosis. Here, we find that two functionally redundant E3 ligase of Y. pestis, YspE1 and YspE2, can be delivered via type III secretion injectisome into host cytosol where they ubiquitinate multiple guanylate-binding proteins (GBPs) for proteasomal degradation. However, Y. pseudotuberculosis has no such capability due to lacking functional YspE1/2 homologs. YspE1/2-mediated GBP degradations significantly promote the survival of Y. pestis in macrophages and strongly inhibit inflammasome activation. By contrast, Gbpchr3-/-, chr5-/- macrophages exhibit much lowered inflammasome activation independent of YspE1/2, accompanied with an enhanced replication of Y. pestis. Accordingly, Gbpchr3-/-, chr5-/- mice are more susceptible to Y. pestis. We demonstrate that Y. pestis utilizes E3 ligases to subvert GBP-mediated host defense, which appears to be newly acquired by Y. pestis during evolution.

Chronic Leptin Deficiency Improves Tolerance of Physiological Damage and Host-Pathogen Cooperation during Yersinia pseudotuberculosis Infection.

To combat infections, hosts employ a combination of antagonistic and cooperative defense strategies. The former refers to pathogen killing mediated by resistance mechanisms, while the latter refers to physiological defense mechanisms that promote host health during infection independent of pathogen killing, leading to an apparent cooperation between the host and the pathogen. Previous work has shown that Leptin, a pleiotropic hormone that plays a central role in regulating appetite and energy metabolism, is indispensable for resistance mechanisms, while a role for Leptin signaling in cooperative host-pathogen interactions remains unknown. Using a mouse model of Yersinia pseudotuberculosis (Yptb) infection, an emerging pathogen that causes fever, diarrhea, and mesenteric lymphadenitis in humans, we found that the physiological effects of chronic Leptin-signaling deficiency conferred protection from Yptb infection due to increased host-pathogen cooperation rather than greater resistance defenses. The protection against Yptb infection was independent of differences in food consumption, lipolysis, or fat mass. Instead, we found that the chronic absence of Leptin signaling protects from a shift to lipid utilization during infection that contributes to Yptb lethality. Furthermore, we found that the survival advantage conferred by Leptin deficiency was associated with increased liver and kidney damage. Our work reveals an additional level of complexity for the role of Leptin in infection defense and demonstrates that in some contexts, in addition to tolerating the pathogen, tolerating organ damage is more beneficial for survival than preventing the damage.

Crystal structures of WrbA, a spurious target of the salicylidene acylhydrazide inhibitors of type III secretion in Gram-negative pathogens, and verification of improved specificity of next-generation compounds.

The enterohemorrhagic Escherichia coli pathotype is responsible for severe and dangerous infections in humans. Establishment of the infection requires colonization of the gastro-intestinal tract, which is dependent on the Type III Secretion System. The Type III Secretion System (T3SS) allows attachment of the pathogen to the mammalian host cell and cytoskeletal rearrangements within the host cell. Blocking the functionality of the T3SS is likely to reduce colonization and therefore limit the disease. This route offers an alternative to antibiotics, and problems with the development of antibiotics resistance. Salicylidene acylhydrazides have been shown to have an inhibitory effect on the T3SS in several pathogens. However, the main target of these compounds is still unclear. Past work has identified a number of putative protein targets of these compounds, one of which being WrbA. Whilst WrbA is considered an off-target interaction, this study presents the effect of the salicylidne acylhydrazide compounds on the activity of WrbA, along with crystal structures of WrbA from Yersinia pseudotuberculosis and Salmonella serovar Typhimurium; the latter also containing parts of the compound in the structure. We also present data showing that the original compounds were unstable in acidic conditions, and that later compounds showed improved stability.

Expression of membrane beta-barrel protein in E. coli at low temperatures: Structure of Yersinia pseudotuberculosis OmpF porin inclusion bodies.

The recombinant OmpF porin of Yersinia pseudotuberculosis as a model of transmembrane protein of the ß-barrel structural family was used to study low growth temperature effect on the structure of the produced inclusion bodies (IBs). This porin showed a very low expression level in E. coli at a growth temperature below optimal 37 °C. The introduction of a N-terminal hexahistidine tag into the mature porin molecule significantly increased the biosynthesis of the protein at low cultivation temperatures. The recombinant His-tagged porin (rOmpF-His) was expressed in E. coli at 30 and 18 °C as inclusion bodies (IB-30 and IB-18). The properties and structural organization of IBs, as well as the structure of rOmpF-His solubilized from the IBs with urea and SDS, were studied using turbidimetry, electron microscopy, dynamic light scattering, optical spectroscopy, and amyloid-specific dyes. IB-18, in comparison with IB-30, has a higher solubility in denaturants, suggesting a difference between IBs in the conformation of the associated polypeptide chains. The spectroscopic analysis revealed that rOmpF-His IBs have a high content of secondary structure with a tertiary-structure elements, including a native-like conformation, the proportion of which in IB-18 is higher than in IB-30. Solubilization of the porin from IBs is accompanied by a modification of its secondary structure. The studied IBs also contain amyloid-like structures. The results obtained in this study expand our knowledge of the structural organization of IBs formed by proteins of different structural classes and also have a contribution into the new approaches development of producing functionally active recombinant membrane proteins.

Yersinia pseudotuberculosis doxycycline tolerance strategies include modulating expression of genes involved in cell permeability and tRNA modifications.

Antibiotic tolerance is typically associated with a phenotypic change within a bacterial population, resulting in a transient decrease in antibiotic susceptibility that can contribute to treatment failure and recurrent infections. Although tolerant cells may emerge prior to treatment, the stress of prolonged antibiotic exposure can also promote tolerance. Here, we sought to determine how Yersinia pseudotuberculosis responds to doxycycline exposure, to then verify if these gene expression changes could promote doxycycline tolerance in culture and in our mouse model of infection. Only four genes were differentially regulated in response to a physiologically-relevant dose of doxycycline: osmB and ompF were upregulated, tusB and cnfy were downregulated; differential expression also occurred during doxycycline treatment in the mouse. ompF, tusB and cnfy were also differentially regulated in response to chloramphenicol, indicating these could be general responses to ribosomal inhibition. cnfy has previously been associated with persistence and was not a major focus here. We found deletion of the OmpF porin resulted in increased antibiotic accumulation, suggesting expression may promote diffusion of doxycycline out of the cell, while OsmB lipoprotein had a minor impact on antibiotic permeability. Overexpression of tusB significantly impaired bacterial survival in culture and in the mouse, suggesting that tRNA modification by tusB, and the resulting impacts on translational machinery, promotes survival during treatment with an antibiotic classically viewed as bacteriostatic. We believe this may be the first observation of bactericidal activity of doxycycline under physiological conditions, which was revealed by reversing tusB downregulation.

Live imaging of Yersinia translocon formation and immune recognition in host cells.

Yersinia enterocolitica employs a type three secretion system (T3SS) to translocate immunosuppressive effector proteins into host cells. To this end, the T3SS assembles a translocon/pore complex composed of the translocator proteins YopB and YopD in host cell membranes serving as an entry port for the effectors. The translocon is formed in a Yersinia-containing pre-phagosomal compartment that is connected to the extracellular space. As the phagosome matures, the translocon and the membrane damage it causes are recognized by the cell-autonomous immune system. We infected cells in the presence of fluorophore-labeled ALFA-tag-binding nanobodies with a Y. enterocolitica strain expressing YopD labeled with an ALFA-tag. Thereby we could record the integration of YopD into translocons and its intracellular fate in living host cells. YopD was integrated into translocons around 2 min after uptake of the bacteria into a phosphatidylinositol-4,5-bisphosphate enriched pre-phagosomal compartment and remained there for 27 min on average. Damaging of the phagosomal membrane as visualized with recruitment of GFP-tagged galectin-3 occurred in the mean around 14 min after translocon formation. Shortly after recruitment of galectin-3, guanylate-binding protein 1 (GBP-1) was recruited to phagosomes, which was accompanied by a decrease in the signal intensity of translocons, suggesting their degradation or disassembly. In sum, we were able for the first time to film the spatiotemporal dynamics of Yersinia T3SS translocon formation and degradation and its sensing by components of the cell-autonomous immune system.

Force spectroscopy of interactions between Yersinia pseudotuberculosis and Yersinia pestis cells and monoclonal antibodies using optical tweezers.

The interactions of a microbial cell with host cells and humoral factors play an important role in the development of infectious diseases. The study of these mechanisms contributes to the development of effective methods for the treatment of bacterial infections. One of the possible approaches to studying bacterial adhesion to host cells is based on the use of the optical trap method. The aim of this work was to assess the significance of lipopolysaccharide O-antigen on the adhesiveness of Yersinia pseudotuberculosis using a model system including a bacterial cell captured by a laser beam and monoclonal antibodies (mAbs) bound covalently to a glass substrate. Registered interaction forces between Y. pseudotuberculosis cells and complementary antibodies to the O-antigen of lipopolysaccharide (LPS) or the B antigen outer membrane protein were 5.9 ± 3.3 and 2.0 ± 1.8 pN, respectively. Interaction forces between O-antigen deficient Y. pestis cells and the mentioned mAbs were 4.2 ± 2.9 and 9.6 ± 4.9 pN. The results are qualitatively consistent with earlier data obtained by using a model system based on polymer beads sensitized with LPS from Y. pseudotuberculosis and Y. pestis and surfaces coated by the aforementioned antibodies. This indicates that the immunochemical activity of Y. pseudotuberculosis cells is mediated mainly by the lipopolysaccharide. The model described can be used in similar studies of physicochemical and immunochemical mechanisms of bacterial adhesiveness.