The Yersinia High-Pathogenicity Island Encodes a Siderophore-Dependent Copper Response System in Uropathogenic Escherichia coli.

Siderophores are iron chelators used by microbes to bind and acquire iron, which, once in the cell, inhibits siderophore production through feedback repression mediated by the ferric uptake repressor (Fur). Yersiniabactin (Ybt), a siderophore associated with enhanced pathogenic potential among Enterobacteriaceae, also binds copper ions during human and experimental murine infections. In contrast to iron, we found that extracellular copper ions rapidly and selectively stimulate Ybt production in extraintestinal pathogenic Escherichia coli. The stimulatory pathway requires formation of an extracellular copper-Ybt (Cu(II)-Ybt) complex, internalization of Cu(II)-Ybt entry through the canonical TonB-dependent outer membrane transporter, and Fur-independent transcriptional regulation by the specialized transcription factor YbtA. Dual regulation by iron and copper is consistent with a multifunctional metallophore role for Ybt. Feed-forward regulation is typical of stress responses, implicating Ybt in prevention of, or response to, copper stress during infection pathogenesis. IMPORTANCE Interactions between bacteria and transition metal ions play an important role in encounters between humans and bacteria. Siderophore systems have long been prominent mediators of these interactions. These systems secrete small-molecule chelators that bind oxidized iron(III) and express proteins that specifically recognize and import these complexes as a nutritional iron source. While E. coli and other Enterobacteriaceae secrete enterobactin, clinical isolates often secrete an additional siderophore, yersiniabactin (Ybt), which has been found to also bind copper and other non-iron metal ions. The observation here that an extraintestinal E. coli isolate secretes Ybt in a copper-inducible manner suggests an important gain of function over the enterobactin system. Copper recognition involves using Ybt to bind Cu(II) ions, consistent with a distinctively extracellular mode of copper detection. The resulting Cu(II)-Ybt complex signals upregulation of Ybt biosynthesis genes as a rapid response against potentially toxic extracellular copper ions. The Ybt system is distinguishable from other copper response systems that sense cytosolic and periplasmic copper ions. The Ybt dependence of the copper response presents an implicit feed-forward regulatory scheme that is typical of bacterial stress responses. The distinctive extracellular copper recognition-response functionality of the Ybt system may enhance the pathogenic potential of infection-associated Enterobacteriaceae.

Heightened Virulence of Yersinia Is Associated with Decreased Function of the YopJ Protein.

Despite the maintenance of YopP/J alleles throughout the human-pathogenic Yersinia lineage, the benefit of YopP/J-induced phagocyte death for Yersinia pathogenesis in animals is not obvious. To determine how the sequence divergence of YopP/J has impacted Yersinia virulence, we examined protein polymorphisms in this type III secreted effector protein across 17 Yersinia species and tested the consequences of polymorphism in a murine model of subacute systemic yersiniosis. Our evolutionary analysis revealed that codon 177 has been subjected to positive selection; the Yersinia enterocolitica residue had been altered from a leucine to a phenylalanine in nearly all Yersinia pseudotuberculosis and Yersinia pestis strains examined. Despite this change being minor, as both leucine and phenylalanine have hydrophobic side chains, reversion of YopJF177 to the ancestral YopJL177 variant yielded a Y. pseudotuberculosis strain with enhanced cytotoxicity toward macrophages, consistent with previous findings. Surprisingly, expression of YopJF177L in the mildly attenuated ksgA- background rendered the strain completely avirulent in mice. Consistent with this hypothesis that YopJ activity relates indirectly to Yersinia pathogenesis in vivo, ksgA- strains lacking functional YopJ failed to kill macrophages but actually regained virulence in animals. Also, treatment with the antiapoptosis drug suramin prevented YopJ-mediated macrophage cytotoxicity and enhanced Y. pseudotuberculosis virulence in vivo. Our results demonstrate that Yersinia-induced cell death is detrimental for bacterial pathogenesis in this animal model of illness and indicate that positive selection has driven YopJ/P and Yersinia evolution toward diminished cytotoxicity and increased virulence, respectively.

Genome-wide dissection reveals diverse pathogenic roles of bacterial Tc toxins.

Tc toxins were originally identified in entomopathogenic bacteria, which are important as biological pest control agents. Tc toxins are heteromeric exotoxins composed of three subunit types, TcA, TcB, and TcC. The C-terminal portion of the TcC protein encodes the actual toxic domain, which is translocated into host cells by an injectosome nanomachine comprising the other subunits. Currently the pathogenic roles and distribution of Tc toxins among different bacterial genera remain unclear. Here we have performed a comprehensive genome-wide analysis, and established a database that includes 1,608 identified Tc loci containing 2,528 TcC proteins in 1,421 Gram-negative and positive bacterial genomes. Our findings indicate that TcCs conform to the architecture of typical polymorphic toxins, with C-terminal hypervariable regions (HVR) encoding more than 100 different classes of putative toxic domains, most of which have not been previously recognized. Based on further analysis of Tc loci in the genomes of all Salmonella and Yersinia strains in EnteroBase, a "two-level" evolutionary dynamics scenario is proposed for TcC homologues. This scenario implies that the conserved TcC RHS core domain plays a critical role in the taxonomical specific distribution of TcC HVRs. This study provides an extensive resource for the future development of Tc toxins as valuable agrochemical tools. It furthermore implies that Tc proteins, which are encoded by a wide range of pathogens, represent an important versatile toxin superfamily with diverse pathogenic mechanisms.

Crohn's Disease: Is the Cold Chain Hypothesis Still Hot?

Crohn's disease [CD] is an inflammatory bowel disease of unknown aetiology. During recent decades, significant technological advances led to development of -omic datasets allowing a detailed description of the disease. Unfortunately these have not, to date, resolved the question of the aetiology of CD. Thus, it may be necessary to [re]consider hypothesis-driven approaches to resolve the aetiology of CD. According to the cold chain hypothesis, the development of industrial and domestic refrigeration has led to frequent exposure of human populations to bacteria capable of growing in the cold. These bacteria, at low levels of exposure, particularly those of the genus Yersinia, are believed to be capable of inducing exacerbated inflammation of the intestine in genetically predisposed subjects. We discuss the consistency of this working hypothesis in light of recent data from epidemiological, clinical, pathological, microbiological, and molecular studies.

Two copies of the ail gene found in Yersinia enterocolitica and Yersinia kristensenii.

Yersinia enterocolitica is the most common Yersinia species causing foodborne infections in humans. Pathogenic strains carry the chromosomal ail gene, which is essential for bacterial attachment to and invasion into host cells and for serum resistance. This gene is commonly amplified in several PCR assays detecting pathogenic Y. enterocolitica in food samples and discriminating pathogenic isolates from non-pathogenic ones. We have isolated several non-pathogenic ail-positive Yersinia strains from various sources in Finland. For this study, we selected 16 ail-positive Yersinia strains, which were phenotypically and genotypically characterised. Eleven strains were confirmed to belong to Y. enterocolitica and five strains to Yersinia kristensenii using whole-genome alignment, Parsnp and the SNP phylogenetic tree. All Y. enterocolitica strains belonged to non-pathogenic biotype 1A. We found two copies of the ail gene (ail1 and ail2) in all five Y. kristensenii strains and in one Y. enterocolitica biotype 1A strain. All 16 Yersinia strains carried the ail1 gene consisting of three different sequence patterns (A6-A8), which were highly similar with the ail gene found in high-pathogenic Y. enterocolitica biotype 1B strains (A2). The Ail protein encoded by the ail1 gene was highly conserved compared to the Ail protein encoded by the ail2 gene. Multiple sequence alignment of the ail gene and Ail protein were conducted with MAFF. In total, 10 ail sequence variations have been identified, of which 8 conserved ones belonged to the ail1 gene. According to our results, the detection of ail alone is not sufficient to predict the pathogenicity of Yersinia isolates.

Comparative genomic insights into Yersinia hibernica - a commonly misidentified Yersinia enterocolitica-like organism.

Food-associated outbreaks linked to enteropathogenic Yersinia enterocolitica are of concern to public health. Pigs and their meat are recognized risk factors for transmission of Y. enterocolitica. This study aimed to describe the comparative genomics of Y. enterocolitica along with a number of misclassified Yersinia isolates, now constituting the recently described Yersinia hibernica. The latter was originally cultured from an environmental sample taken at a pig slaughterhouse. Unique features were identified in the genome of Y. hibernica, including a novel integrative conjugative element (ICE), denoted as ICEYh-1 contained within a 255 kbp region of plasticity. In addition, a zebrafish embryo infection model was adapted and applied to assess the virulence potential among Yersinia isolates including Y. hibernica.

The Yersinia Type III Secretion System as a Tool for Studying Cytosolic Innate Immune Surveillance.

Microbial pathogens have evolved complex mechanisms to interface with host cells in order to evade host defenses and replicate. However, mammalian innate immune receptors detect the presence of molecules unique to the microbial world or sense the activity of virulence factors, activating antimicrobial and inflammatory pathways. We focus on how studies of the major virulence factor of one group of microbial pathogens, the type III secretion system (T3SS) of human pathogenic Yersinia, have shed light on these important innate immune responses. Yersinia are largely extracellular pathogens, yet they insert T3SS cargo into target host cells that modulate the activity of cytosolic innate immune receptors. This review covers both the host pathways that detect the Yersinia T3SS and the effector proteins used by Yersinia to manipulate innate immune signaling.

Vaccine Potential of a Recombinant Bivalent Fusion Protein LcrV-HSP70 Against Plague and Yersiniosis.

To counteract the deadly pathogens, i.e., Y. pestis, Y. enetrocolitica, and Y. pseudotuberculosis, we prepared a recombinant DNA construct lcrV-hsp70 encoding the bivalent fusion protein LcrV-HSP70. The lcrV gene of Y. pestis and hsp70 domain II DNA fragment of M. tuberculosis were amplified by PCR. The lcrV amplicon was first ligated in the pET vector using NcoI and BamHI restriction sites. Just downstream to the lcrV gene, the hsp70 domain II was ligated using BamHI and Hind III restriction sites. The in-frame and the orientation of cloned lcrV-hsp70 were checked by restriction analysis and nucleotide sequencing. The recombinant bivalent fusion protein LcrV-HSP70 was expressed in E. coli and purified by affinity chromatography. The vaccine potential of LcrV-HSP70 fusion protein was evaluated in formulation with alum. BALB/c mice were vaccinated, and the humoral and cellular immune responses were studied. The fusion protein LcrV-HSP70 induced a strong and significant humoral immune response in comparison to control animals. We also observed a significant difference in the expression levels of IFN-γ and TNF-α in LcrV-HSP70-immunized mice in comparison to control, HSP70, and LcrV groups. To test the protective efficacy of the LcrV-HSP70 fusion protein against plague and Yersiniosis, the vaccinated mice were challenged with Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis separately. The bivalent fusion protein LcrV-HSP70 imparted 100% protection against the plague. In the case of Yersiniosis, on day 2 post challenge, there was a significant reduction in the number of CFU of Y. enterocolitica and Y. pseudotuberculosis in the blood (CFU/ml) and the spleen (CFU/g) of vaccinated animals in comparison to the LcrV, HSP70, and control group animals.

A Survey of Antimicrobial Resistance Determinants in Category A Select Agents, Exempt Strains, and Near-Neighbor Species.

A dramatic increase in global antimicrobial resistance (AMR) has been well documented. Of particular concern is the dearth of information regarding the spectrum and prevalence of AMR within Category A Select Agents. Here, we performed a survey of horizontally and vertically transferred AMR determinants among Category A agents and their near neighbors. Microarrays provided broad spectrum screening of 127 Francisella spp., Yersinia spp., and Bacillus spp. strains for the presence/absence of 500+ AMR genes (or families of genes). Detecting a broad variety of AMR genes in each genus, microarray analysis also picked up the presence of an engineered plasmid in a Y. pestis strain. High resolution melt analysis (HRMA) was also used to assess the presence of quinolone resistance-associated mutations in 100 of these strains. Though HRMA was able to detect resistance-causing point mutations in B. anthracis strains, it was not capable of discriminating these point mutations from other nucleotide substitutions (e.g., arising from sequence differences in near neighbors). Though these technologies are well-established, to our knowledge, this is the largest survey of Category A agents and their near-neighbor species for genes covering multiple mechanisms of AMR.

To catch a thief: regulated RIPK1 post-translational modifications as a fail-safe system to detect and overcome pathogen subversion of immune signaling.

Any pathogen worth its salt has mechanisms to evade, subvert, or antagonize host innate immune responses induced by pattern recognition receptors. Resistance against such pathogens therefore requires alternative means to activate protective immune responses. Intriguingly, the receptors that regulate antimicrobial gene expression are coupled to cell death pathways that are activated by blockade of NF-κB and MAPK signaling. In this review, we discuss the regulation of apoptosis in response to pathogen disruption of immune signaling and the role of this cell death response in protection against such pathogens. Stanley often observed that bacterial pathogens are excellent cell biologists and immunologists, and he noted that studying pathogen-host interactions could pave the way to new insights about host biology. Indeed, how Yersinia and other pathogens disrupt innate immune signaling has provided new insight into these pathways and revealed new ways to think about immunogenic properties of apoptosis during bacterial infection.

Unraveling neutrophil- Yersinia interactions during tissue infection.

The human and animal pathogens Yersinia pestis, which causes bubonic and pneumonic plague, and Yersinia pseudotuberculosis and Yersinia enterocolitica, which cause gastroenteritis, share a type 3 secretion system which injects effector proteins, Yops, into host cells. This system is critical for virulence of all three pathogens in tissue infection. Neutrophils are rapidly recruited to infected sites and all three pathogens frequently interact with and inject Yops into these cells during tissue infection. Host receptors, serum factors, and bacterial adhesins appear to collaborate to promote neutrophil- Yersinia interactions in tissues. The ability of neutrophils to control infection is mixed depending on the stage of infection and points to the efficiency of Yops and other bacterial factors to mitigate bactericidal effects of neutrophils. Yersinia in close proximity to neutrophils has higher levels of expression from yop promoters, and neutrophils in close proximity to Yersinia express higher levels of pro-survival genes than migrating neutrophils. In infected tissues, YopM increases neutrophil survival and YopH targets a SKAP2/SLP-76 signal transduction pathway. Yet the full impact of these and other Yops and other Yersinia factors on neutrophils in infected tissues has yet to be understood.

Bacterial adhesion and host cell factors leading to effector protein injection by type III secretion system.

Type III secretion systems (T3SS) play a crucial role for virulence in many Gram-negative bacteria. After tight bacterial contact to host cells, the T3SS injects effector proteins into the host cells, which leads to cell invasion, tissue destruction and/or immune evasion. Over the last decade several attempts were made to characterize the host-cell interactions which precede and determine effector protein injection during infection. The development of the TEM-ß-lactamase reporter was an important breakthrough to achieve this goal. By this means it was demonstrated that during infection with many Gram-negative pathogens such as Salmonella, Pseudomonas or Yersinia the main targets of T3SS are leukocytes of the myeloid lineage such as neutrophils, macrophages or dendritic cells. This is due to the recruitment of these cells to the site of infection, but also due to the specific interplay between bacterial and host cells. Comprehensive studies on Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis effector translocation show that adhesins such as Invasin (Inv), Yersinia adhesin A (YadA) and attachment and invasion locus (Ail) are critical for effector translocation. Here, mainly the complex interaction of YadA and Ail with various host cell receptor repertoires on leukocytes and the modulatory effects of serum factors direct effector translocation predominantly towards myeloid cells. The current understanding suggests that mostly protein based interactions between bacteria and host determine host cell specific effector translocation during Yersinia infection. However, for Shigella dysenteriae infection it was shown that glycan-glycan interactions can also play a critical role for the adhesion preceding effector translocation. In addition, the Shigella infection model revealed that the activation status of cells is a further criterium directing effector translocation into a distinct cell population. In this review the current understanding of the complex and species-specific interaction between bacteria and host cells leading to type III secretion is discussed.

Pairwise visual comparison of small RNA secondary structures with base pair probabilities.

BACKGROUND: Predicted RNA secondary structures are typically visualized using dot-plots for base pair binding probabilities and planar graphs for unique structures, such as the minimum free energy structure. These are however difficult to analyze simultaneously. RESULTS: This work introduces a compact unified view of the most stable conformation of an RNA secondary structure and its base pair probabilities, which is called the Circular Secondary Structure Base Pairs Probabilities Plot (CS2BP2-Plot). Along with our design we provide access to a web server implementation of our solution that facilitates pairwise comparison of short RNA (and DNA) sequences up to 200 base pairs. The web server first calculates the minimum free energy secondary structure and the base pair probabilities for up to 10 RNA or DNA sequences using RNAfold and then provides a two panel comparative view that includes CS2BP2-Plots along with the traditional graph, planar and circular diagrams obtained with VARNA. The CS2BP2-Plots include highlighting of the nucleotide differences between two selected sequences using ClustalW local alignments. We also provide descriptive statistics, dot-bracket secondary structure representations and ClustalW local alignments for compared sequences. CONCLUSIONS: Using circular diagrams and colour and weight-coded arcs, we demonstrate how a single image can replace the state-of-the-art dual representations (dot-plots and minimum free energy structures) for base-pair probabilities of RNA secondary structures while allowing efficient exploration and comparison of different RNA conformations via a web server front end. With that, we provide the community, especially the biologically oriented, with an intuitive tool for ncRNA visualization. Web-server: https://nrcmonsrv01.nrc.ca/cs2bp2plot.

Assessment of Yersinia entomophaga as a control agent of the diamondback moth Plutella xylostella.

The application of the biocontrol bacterium Yersinia entomophaga as a foliar spray was assessed for its efficacy against larvae of the diamondback moth, Plutella xylostella. The bacterium was applied as either a broth suspension, or as a biopolymer-based gel foliar spray and compared with commercial insecticides Dipel (Bacillus thuringiensis) and Spinosad. The performance of Y. entomophaga was comparable with that of Dipel. The gel-based formulation extended leaf persistence over that of the basic broth culture spray, while also providing higher initial foliar deposition rates. The bacterium was found to multiply within the P. xylostella larvae to 5.8 × 105 cells per larva, while the median lethal dose (LD50) was determined to be 2.69 × 103 cells per larva. Importantly, B. thuringiensis Cry1A-resistant, Cry1C-resistant, indoxacarb/pyrethroid-resistant, and Spinosad-resistant P. xylostella larvae were susceptible to Y. entomophaga.

Insecticidal Toxicity of Yersinia frederiksenii Involves the Novel Enterotoxin YacT.

The genus Yersinia comprises 19 species of which three are known as human and animal pathogens. Some species display toxicity toward invertebrates using the so-called toxin complex (TC) and/or determinants that are not yet known. Recent studies showed a remarkable variability of insecticidal activities when representatives of different Yersinia species (spp.) were subcutaneously injected into the greater wax moth, Galleria mellonella. Here, we demonstrate that Y. intermedia and Y. frederiksenii are highly toxic to this insect. A member of Y. Enterocolitica phylogroup 1B killed G. mellonella larvae with injection doses of approximately 38 cells only, thus resembling the insecticidal activity of Photorhabdus luminescens. The pathogenicity Yersinia spp. displays toward the larvae was higher at 15°C than at 30°C and independent of the TC. However, upon subtraction of all genes of the low-pathogenic Y. enterocolitica strain W22703 from the genomes of Y. intermedia and Y. frederiksenii, we identified a set of genes that may be responsible for the toxicity of these two species. Indeed, a mutant of Y. frederiksenii lacking yacT, a gene that encodes a protein similar to the heat-stable cytotonic enterotoxin (Ast) of Aeromonas hydrophila, exhibited a reduced pathogenicity toward G. mellonella larvae and altered the morphology of hemocytes. The data suggests that the repertoire of virulence determinants present in environmental Yersinia species remains to be elucidated.

Proteomics Goes to Court: A Statistical Foundation for Forensic Toxin/Organism Identification Using Bottom-Up Proteomics.

Bottom-up proteomics is increasingly being used to characterize unknown environmental, clinical, and forensic samples. Proteomics-based bacterial identification typically proceeds by tabulating peptide "hits" (i.e., confidently identified peptides) associated with the organisms in a database; those organisms with enough hits are declared present in the sample. This approach has proven to be successful in laboratory studies; however, important research gaps remain. First, the common-practice reliance on unique peptides for identification is susceptible to a phenomenon known as signal erosion. Second, no general guidelines are available for determining how many hits are needed to make a confident identification. These gaps inhibit the transition of this approach to real-world forensic samples where conditions vary and large databases may be needed. In this work, we propose statistical criteria that overcome the problem of signal erosion and can be applied regardless of the sample quality or data analysis pipeline. These criteria are straightforward, producing a p-value on the result of an organism or toxin identification. We test the proposed criteria on 919 LC-MS/MS data sets originating from 2 toxins and 32 bacterial strains acquired using multiple data collection platforms. Results reveal a > 95% correct species-level identification rate, demonstrating the effectiveness and robustness of proteomics-based organism/toxin identification.

The development of a screening protocol for Salmonella spp. and enteropathogenic Yersinia spp. in samples from wild boar (Sus scrofa) also generating MLVA-data for Y. enterocolitica and Y. pseudotuberculosis.

Salmonellosis and yersiniosis are notifiable human diseases that are commonly associated with contaminated food. Domestic pigs as well as wild boars and other wild-life have been identified as reservoirs of these bacteria. Methods for cultivation and molecular epidemiological investigations of Salmonella spp. are well established, however, cultivation of enteropathogenic Yersinia spp. is time- consuming and the commonly used method for molecular epidemiological investigations, pulsed-field gel electrophoresis, lack in discriminatory power. The aim of this study was to develop and evaluate a screening protocol well suited for wildlife samples and other highly contaminated samples. The method is based on PCR-screening followed by Multiple Loci Variant number tandem repeat Analysis (MLVA) on enrichment broth to obtain molecular epidemiological data for enteropathogenic Yersinia spp. without the need for pure isolates. The performance of the protocol was evaluated using wild boar samples (n=354) including tonsils, faeces and lymph nodes from 90 Swedish wild boars. The new protocol performed as well as or better than the established ISO-standards for detection and cultivation of Y. enterocolitica and Salmonella spp., however for cultivation of Y. pseudotuberculosis, further development is needed. The selection for motility seems beneficial for the enrichment of Salmonella spp. and Y. enterocolitica. Further, the selective enrichment prior to PCR-analysis eliminates inhibitory factors present in the original sample. In total, ten isolates of Y. enterocolitica of various bio-serotypes were obtained, and the MLVA-profile of these isolates were consistent with the profiles from the corresponding enrichment broth. Further, 22 isolates of Salmonella spp. comprising six different serovars were obtained with S. Fulica, S. Hadar and a monophasic S. Typhimurium being the most common. In conclusion, the presented screening protocol offers a rapid and efficient way to obtain prevalence data from a large sample set as well as MLVA-data within a short time frame. These results can hence improve the knowledge on the epidemiology and distribution of these pathogens and their importance to public health.

Iron and innate antimicrobial immunity-Depriving the pathogen, defending the host.

The acute-phase response is triggered by the presence of infectious agents and danger signals which indicate hazards for the integrity of the mammalian body. One central feature of this response is the sequestration of iron into storage compartments including macrophages. This limits the availability of this essential nutrient for circulating pathogens, a host defence strategy known as 'nutritional immunity'. Iron metabolism and the immune response are intimately linked. In infections, the availability of iron affects both the efficacy of antimicrobial immune pathways and pathogen proliferation. However, host strategies to withhold iron from microbes vary according to the localization of pathogens: Infections with extracellular bacteria such as Staphylococcus aureus, Streptococcus, Klebsiella or Yersinia stimulate the expression of the iron-regulatory hormone hepcidin which targets the cellular iron-exporter ferroportin-1 causing its internalization and blockade of iron egress from absorptive enterocytes in the duodenum and iron-recycling macrophages. This mechanism disrupts both routes of iron delivery to the circulation, contributes to iron sequestration in the mononuclear phagocyte system and mediates the hypoferraemia of the acute phase response subsequently resulting in the development of anaemia of inflammation. When intracellular microbes are present, other strategies of microbial iron withdrawal are needed. For instance, in macrophages harbouring intracellular pathogens such as Chlamydia, Mycobacterium tuberculosis, Listeria monocytogenes or Salmonella Typhimurium, ferroportin-1-mediated iron export is turned on for the removal of iron from infected cells. This also leads to reduced iron availability for intra-macrophage pathogens which inhibits their growth and in parallel strengthens anti-microbial effector pathways of macrophages including the formation of inducible nitric oxide synthase and tumour necrosis factor. Iron plays a key role in infectious diseases both as modulator of the innate immune response and as nutrient for microbes. We need to gain a more comprehensive understanding of how the body can differentially respond to infection by extra- or intracellular pathogens. This knowledge may allow us to modulate mammalian iron homeostasis pharmaceutically and to target iron-acquisition systems of pathogens, thus enabling us to treat infections with novel strategies that act independent of established antimicrobials.

Structure and mechanism of the two-component α-helical pore-forming toxin YaxAB.

Pore-forming toxins (PFT) are virulence factors that transform from soluble to membrane-bound states. The Yersinia YaxAB system represents a family of binary α-PFTs with orthologues in human, insect, and plant pathogens, with unknown structures. YaxAB was shown to be cytotoxic and likely involved in pathogenesis, though the molecular basis for its two-component lytic mechanism remains elusive. Here, we present crystal structures of YaxA and YaxB, together with a cryo-electron microscopy map of the YaxAB complex. Our structures reveal a pore predominantly composed of decamers of YaxA-YaxB heterodimers. Both subunits bear membrane-active moieties, but only YaxA is capable of binding to membranes by itself. YaxB can subsequently be recruited to membrane-associated YaxA and induced to present its lytic transmembrane helices. Pore formation can progress by further oligomerization of YaxA-YaxB dimers. Our results allow for a comparison between pore assemblies belonging to the wider ClyA-like family of α-PFTs, highlighting diverse pore architectures.

Development of a flow cytometry based assay to determine the invasion of enteropathogenic Yersiniae into C2BBe1 cells.

A rapid method was developed to determine the invasion frequency of enteropathogenic Yersinia into intestinal C2BBe1 cells by means of flow cytometry. Bacteria are labelled with a thiol-cleavable amine-reactive biotin and subsequently incubated with the fluorochrome-labelled biotin-ligand neutravidin. After infection of the intestinal cells with the labelled bacteria, the neutravidin-coupled fluorochrome is detached by breaking up the linker through reduction of the disulphide. Despite reduced adhesion and invasion frequencies of the labelled bacteria into C2BBe1 cells this procedure offers the basis for the development of a fast single-step staining protocol for the recovery of invading bacteria in in a host-pathogen system for further transcriptome or proteome analysis.