Search

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.

A concurrent outbreak of infection with Yersinia pseudotuberculosis occurred in adult captive African lions ( Panthera leo). Two 17-y-old male lions and one 14-y-old female lion developed respiratory distress, lethargy, ataxia, and hyporexia. Within 3–5 d of the onset of clinical signs, one male and the female lion died and were submitted for postmortem examination. Macroscopically, the liver and spleen had multifocal-to-coalescing, semi-firm, pale-tan nodules throughout the parenchyma. The lungs were non-collapsed and marked by petechiae. Histologic examination identified lytic, necrosuppurative foci in the liver, spleen, lungs, and kidney, with abundant intralesional gram-negative coccobacilli in the male lion. Similar findings were seen in the female lion in the liver, spleen, kidney, and mesenteric lymph node; however, the intralesional bacterial colonies were more pleomorphic, comprising rod and filamentous morphologies. Aerobic bacterial culture of the liver, spleen, and lung revealed Y. pseudotuberculosis growth. The source of infection is unknown, and an epidemiologic study was performed. Sources to be considered are from the predation of rodent and/or bird reservoirs, or contaminated soil or water. Mortality associated with Y. pseudotuberculosis has been described in an African lion cub, however, to our knowledge, Y. pseudotuberculosis has not been reported in adult African lions, and this is only the second report of Y. pseudotuberculosis with aberrant bacterial morphology observed histologically.

Fluorescence dilution approaches can detect bacterial cell division events and can detect if there are differential rates of cell division across individual cells within a population. This approach typically involves inducing expression of a fluorescent protein and then tracking partitioning of fluorescence into daughter cells. However, fluorescence can be diluted very quickly within a rapidly replicating population, such as pathogenic bacterial populations replicating within host tissues. To overcome this limitation, we have generated two revTetR reporter constructs, where either mCherry or yellow fluorescent protein (YFP) is constitutively expressed and repressed by addition of tetracyclines, resulting in fluorescence dilution within defined time frames. We show that fluorescent signals are diluted in replicating populations and that signal accumulates in growth-inhibited populations, including during nitric oxide (NO) exposure. Furthermore, we show that tetracyclines can be delivered to the mouse spleen during Yersinia pseudotuberculosis infection and defined a drug concentration that results in even exposure of cells to tetracyclines. We then used this system to visualize bacterial cell division within defined time frames postinfection. revTetR-mCherry allowed us to detect slow-growing cells in response to NO in culture; however, this strain had a growth defect within mouse tissues, which complicated results. To address this issue, we constructed revTetR-YFP using the less toxic YFP and showed that heightened NO exposure correlated with heightened YFP signal, indicating decreased cell division rates within this subpopulation in vivo. This revTetR reporter will provide a critical tool for future studies to identify and isolate slowly replicating bacterial subpopulations from host tissues.

International audience; Yersinia pseudotuberculosis is an enteric pathogen causing mild enteritis that can lead to mesenteric adenitis in children and septicemia in elderly patients. Most cases are sporadic, but outbreaks have already been described in different countries. We report for the first time a Y. pseudotuberculosis clonal outbreak in France, that occurred in 2020. An epidemiological investigation based on food queries pointed toward the consumption of tomatoes as the suspected source of infection. The Yersinia National Reference Laboratory (YNRL) developed a new cgMLST scheme with 1,921 genes specific to Y. pseudotuberculosis that identified the clustering of isolates associated with the outbreak and allowed to perform molecular typing in real time. In addition, this method allowed to retrospectively identify isolates belonging to this cluster from earlier in 2020. This method, which does not require specific bioinformatic skills, is now used systematically at the YNRL and proves to display an excellent discriminatory power and is available to the scientific community.

Background Yersinia pseudotuberculosis infection can occur in an immunocompromised host. Although rare, bacteremia due to Y. pseudotuberculosis may also occur in immunocompetent hosts. The prognosis and therapeutic strategy, especially for immunocompetent patients with Y. pseudotuberculosis bacteremia, however, remains unknown. Case presentation A 38-year-old Japanese man with a mood disorder presented to our hospital with fever and diarrhea. Chest computed tomography revealed consolidation in the right upper lobe with air bronchograms. He was diagnosed with pneumonia, and treatment with intravenous ceftriaxone and azithromycin was initiated. The ceftriaxone was replaced with doripenem and the azithromycin was discontinued following the detection of Gram-negative rod bacteria in 2 sets of blood culture tests. The isolated Gram-negative rod bacteria were confirmed to be Y. pseudotuberculosis. Thereafter, he developed septic shock. Doripenem was switched to cefmetazole, which was continued for 14 days. He recovered without relapse. Conclusions We herein report a case of septic shock due to Y. pseudotuberculosis infection in an adult immunocompetent patient. The appropriate microorganism tests and antibiotic therapy are necessary to treat patients with Y. pseudotuberculosis bacteremia.

Antibody titers against the superantigen, Yersinia pseudotuberculosis-derived mitogen, suggestive of mediating Kawasaki disease-like manifestation in Y. pseudotuberculosis infections, in immunoglobulin products were evaluated. Trace, but detectable titer was demonstrated in the products. Thus, attention is required when evaluating anti-Y. pseudotuberculosis-derived mitogen IgG titers in patient sera post intravenous immunoglobulin therapy.

The article describes a case of a rare infection caused by Yersinia pseudotuberculosis in a five-year-old boy admitted to the hospital. The infection was manifested by the so-called right lower quadrant syndrome, or terminal ileitis. The Y. pseudotuberculosis strain was isolated from the patient's feces and its biochemical properties are reported. Confirmation was performed by the National Reference Laboratory for E. coli and Shigella. Since pseudotuberculosis is very rare in the Czech Republic, the authors would like to draw attention to this infection. Enlargement of lymph nodes in the right lower quadrant of the abdomen may suggest the infection caused by Y. pseudotuberculosis.

BackgroundWhileCampylobacter jejunirepresents the most common cause of bacterial gastroenteritis,Yersinia pseudotuberculosisinfections are very rarely diagnosed in adults.CaseWe report on a previously healthy patient who presented several times at our hospital with fever, Guillain-Barré syndrome, recurrent abdominal symptoms and distinct mesenteric lymphadenopathy, respectively. This complicated and diagnostically challenging course of disease was caused by aC. jejuniandY. pseudotuberculosiscoinfection. Antibiotic treatment with doxycycline was effective.ConclusionBroad serology testing was crucial to discover that two concomitant infections were causing the symptoms. This case demonstrates that when a clinical picture is not fully explained by one known infection, another infection with the same underlying risk factor has to be considered, hence “a horse and a zebra”.

Yersinia pseudotuberculosis is a Gram-negative bacterium causing infection in humans through contaminated water and/or food. The infection commonly occurs as gastroenteritis and fever, abdominal pain due to mesenteric lymphadenitis and diarrhoea. Bacteraemia is rare and is typically seen in immunocompromised patients and occurs with different clinical presentations like Far East scarlet-like fever, splenic abscess, or mimic appendicitis. This is a case report of Y. pseudotuberculosis bacteraemia and splenic abscess in a Caucasian male.

Significance Our study highlights that hemochromatotic hosts are susceptible to the siderophilic enteropathogenic Yersinia because of dysregulated interleukn (IL)-1β signaling that causes rapid disruption of the intestinal tight junction, leading to acute systemic infection. This study also poses early intervention of anti–IL-1β therapy as a potentially novel strategy for treating hemochromatotic patients with severe siderophilic bacterial infections., Hemachromatosis (iron-overload) increases host susceptibility to siderophilic bacterial infections that cause serious complications, but the underlying mechanisms remain elusive. The present study demonstrates that oral infection with hyperyersiniabactin (Ybt) producing Yersinia pseudotuberculosis Δfur mutant (termed Δfur) results in severe systemic infection and acute mortality to hemochromatotic mice due to rapid disruption of the intestinal barrier. Transcriptome analysis of Δfur-infected intestine revealed up-regulation in cytokine–cytokine receptor interactions, the complement and coagulation cascade, the NF-κB signaling pathway, and chemokine signaling pathways, and down-regulation in cell-adhesion molecules and Toll-like receptor signaling pathways. Further studies indicate that dysregulated interleukin (IL)-1β signaling triggered in hemachromatotic mice infected with Δfur damages the intestinal barrier by activation of myosin light-chain kinases (MLCK) and excessive neutrophilia. Inhibiting MLCK activity or depleting neutrophil infiltration reduces barrier disruption, largely ameliorates immunopathology, and substantially rescues hemochromatotic mice from lethal Δfur infection. Moreover, early intervention of IL-1β overproduction can completely rescue hemochromatotic mice from the lethal infection.

Yersinia pseudotuberculosis is a foodborne pathogen that subverts immune function by translocation of Yersinia outer protein (Yop) effectors into host cells. As adaptive γδ T cells protect the intestinal mucosa from pathogen invasion, we assessed whether Y. pseudotuberculosis subverts these cells in mice and humans. Tracking Yop translocation revealed that the preferential delivery of Yop effectors directly into murine Vγ4 and human Vδ2+ T cells inhibited anti-microbial IFNγ production. Subversion was mediated by the adhesin YadA, injectisome component YopB, and translocated YopJ effector. A broad anti-pathogen gene signature and STAT4 phosphorylation levels were inhibited by translocated YopJ. Thus, Y. pseudotuberculosis attachment and translocation of YopJ directly into adaptive γδ T cells is a major mechanism of immune subversion in mice and humans. This study uncovered a conserved Y. pseudotuberculosis pathway that subverts adaptive γδ T cell function to promote pathogenicity., Author summary Unconventional γδ T cells are a dynamic immune population important for mucosal protection of the intestine against invading pathogens. We determined that the foodborne pathogen Y. pseudotuberculosis preferentially targets an adaptive subset of these cells to subvert immune function. We found that direct injection of Yersinia outer proteins (Yop) into adaptive γδ T cells inhibited their anti-pathogen functions. We screened all Yop effectors and identified YopJ as the sole effector to inhibit adaptive γδ T cell production of IFNγ. We determined that adaptive γδ T cell subversion occurred by limiting activation of the transcription factor STAT4. When we infected mice with Y. pseudotuberculosis expressing an inactive YopJ, this enhanced the adaptive γδ T cell response and led to greater cytokine production from this subset of cells to aid mouse recovery. This mechanism of immune evasion appears conserved in humans as direct injection of Y. pseudotuberculosis YopJ into human γδ T cells inhibited cytokine production. This suggested to us that Y. pseudotuberculosis actively inhibits the adaptive γδ T cell response through YopJ as a mechanism to evade immune surveillance at the site of pathogen invasion.

One of the mechanisms underlying the appearance of chronic infections is transition of pathogens into a non-culturable state, which is largely associated with the use of antibiotics. We studied ultrastructure of dormant bacteria Yersinia pseudotuberculosis obtained from the vegetative form of strain 512 by inhibition with kanamycin. On the model of the causative agent of pseudotuberculosis we showed that transition of prokaryotes to a dormant state occurs through apoptosis of bacteria. Fragmentation and condensation of chromatin with the formation of electron-dense fibrils, clumps and large conglomerates characteristic of apoptosis were found in the nucleoid zone of the cytoplasm of inhibited bacterial cells. These results are of great importance for understanding the mechanisms of the existence of pathogens in different conditions, as well as for identifying the causative agents of infectious diseases.

Yersinia pseudotuberculosis (Yptb) is a bacterial pathogen that causes foodborne illness. Defense against the host antimicrobial gas, nitric oxide (NO), by the bacterial NO-detoxifying gene, hmp, promotes Yptb replication in mouse models of infection. Here, we detail the use of fluorescent signals as readouts for NO exposure within individual cells and subsequent detection of heterogeneity within a population, using single-cell imaging and analysis. This protocol quantifies NO exposure in culture, without capturing the full complexity of the host environment. For complete details on the use and execution of this protocol, please refer to Patel et al. (2021).

Mom’s IL-6 rewires baby’s gut immunity Most infections that occur during pregnancy are mild and transient. However, whether such pathogen encounters can shape the long-term trajectory of the offspring’s immune system remains unclear. Lim et al . infected pregnant mice with the common food-borne pathogen Yersinia pseudotuberculosis (YopM) (see the Perspective by Amir and Zeng). Although the infection was maternally restricted and short-lived, the offspring harbored greater numbers of intestinal T helper 17 cells into adulthood. Interleukin-6 (IL-6) mediated this tissue-restricted effect by acting on fetal intestinal epithelium during development. Although offspring from mothers infected with YopM or injected with IL-6 showed enhanced resistance to oral infection with Salmonella Typhimurium, they also exhibited higher susceptibility toward enteric inflammatory disease. —STS

Electron microscopy study revealed changes in the ultrastructure of bacteria of Yersinia pseudotuberculosis strains characterized by significantly reduced reproductive ability and virulence potential after long-term storage at low temperature of 4-8°C. Most bacterial cells contained dark cytosol with reduced cellular material or empty cytosol, while the cell wall was preserved. The revealed ultrastructural changes in the bacterial cells of the static culture of Y. pseudotuberculosis suggest that storage of strains under low positive temperatures could induce the transition of the majority of bacterial cell population to a dormant, non-cultivated state with a decrease in their virulence. This fact is of great scientific and applied importance in studies of causative agents of saprozoonoses, including pseudotuberculosis, which has the etiopathogenetic background of persistent infection.

Infection with Yersinia pseudotuberculosis, a known causal pathogen of human bacterial gastroenteritis, causes various symptoms and complications. A previously healthy 7-year-old girl was admitted because of fever and gastrointestinal symptoms. She was initially diagnosed with intussusception by abdominal ultrasonography. Although the patient was successfully treated by air enema, the fever persisted. The patient was then diagnosed with incomplete Kawasaki disease based on the presence of four principal clinical features. Intravenous immunoglobulin and oral aspirin were initiated. The patient defervesced and the other symptoms subsided after the treatment. Cardiac ultrasound results showed normal coronary arteries. Because of the gastrointestinal symptoms, stool samples were cultured repeatedly, only to yield normal flora. However, serum levels of anti-Y. pseudotuberculosis-derived mitogen antibody were elevated between the 7th and 18th days of the disease, thereby confirming Y. pseudotuberculosis infection. Because Y. pseudotuberculosis infection results in various clinical manifestations, we must be aware of each symptom and address them systematically.

Primary infection of C57BL/6 mice with the bacterial pathogen Yersinia pseudotuberculosis elicits an unusually large H-2K(b)-restricted CD8(+) T cell response to the endogenous and protective bacterial epitope YopE(69–77). To better understand the basis for this large response, the model OVA(257–264) epitope was inserted into YopE in Y. pseudotuberculosis and antigen-specific CD8(+) T cells in mice were characterized after foodborne infection with the resulting strain. The epitope YopE(69–77) elicited significantly larger CD8(+) T cell populations in the small intestine, mesenteric lymph nodes (MLNs), spleen, and liver between 7 and 30 days postinfection, despite residing in the same protein and having an affinity for H-2K(b) similar to that of OVA(257–264). YopE-specific CD8(+) T cell precursors were ∼4.6 times as abundant as OVA-specific precursors in the MLNs, spleens, and other lymph nodes of naive mice, explaining the dominance of YopE(69–77) over OVA(257–264) at early infection times. However, other factors contributed to this dominance, as the ratio of YopE-specific to OVA-specific CD8(+) T cells increased between 7 and 30 days postinfection. We also compared the YopE-specific and OVA-specific CD8(+) T cells generated during infection for effector and memory phenotypes. Significantly higher percentages of YopE-specific cells were characterized as short-lived effectors, while higher percentages of OVA-specific cells were memory precursor effectors at day 30 postinfection in spleen and liver. Our results suggest that a large precursor number contributes to the dominance and effector and memory functions of CD8(+) T cells generated in response to the protective YopE(69–77) epitope during Y. pseudotuberculosis infection of C57BL/6 mice.

Yersinia pseudotuberculosis is an important pathogen for both humans and animals. It can infect livestock, as well as pets and wild animals. During recent years, a number of reports have described the isolation of Y. pseudotuberculosis from zoo animals, mainly birds and mammals, for which the infection was mostly lethal. Between 2005 and 2019, there were at least 17 cases of deceased mammals, belonging to five different species, which suffered from a Y. pseudotuberculosis infection at the Zoo Wuppertal, Germany. Since only scarce information exists on the properties of Y. pseudotuberculosis from zoo animals, we characterized eight isolates, covering all infected species, in detail. All isolates were members of biotype 1, but belonged to five serotypes, five sequence types (STs), and seven core-genome multilocus sequence types (cgMLSTs). Using pulsed-field gel electrophoresis (PFGE) analysis and whole-genome sequencing (WGS), the seven isolates could be discriminated from each other. They differed significantly regarding their virulence genes and mobile genetic elements. While the virulence plasmid pYV existed in all serotypes (five isolates), a complete high-pathogenicity island (HPI) was detected only in the serotypes O:1a, O:1b, and O:13 (four isolates), but not in O:2a and O:2b. Similarly, the content of other plasmids and prophages varied greatly between the isolates. The data demonstrate that the deceased mammals were infected by seven individual isolates and not by a single type predominating in the zoo animals.

A Yersinia pseudotuberculosis outbreak was diagnosed in a male turkey flock in Finland. Y. pseudotuberculosis is a quite rare zoonotic bacterium, which typically causes enteritis in humans and sudden death in animals. In this study, osteomyelitis was diagnosed in small, lame, 11-to 12-wk-old male turkeys. Lameness and slower growth among the turkeys was observed on the farm. During pathological examination, multiple lesions were found in the metaphyseal and physeal areas of the femurs, tibiotarsi, and tarsometatarsi, with multifocal to coalescing mixed heterophilic/granulomatous necrotizing osteomyelitis. Y. pseudotuberculosis was isolated from the femoral and tibiotarsal bones or from the joints of six lame turkeys sent for necropsy. The isolation required homogenizing of lesion tissue in phosphate-mannitol-peptone broth, which was cultured directly -and, if needed, after cold enrichment - on selective cefsulodin-irgasan-novobiocin agar. Whole-genome sequencing was used for identification and typing. All isolates belonged to bio/serotype 1/O:1a and sequence type ST42 (Achtman scheme), which is commonly reported in both human and animal Y. pseudotuberculosis infections in Europe. The isolates from all six turkeys showed only one to two allele differences in the core genome comparison, indicating a common source of infection. All asymptomatic turkeys were slaughtered at the age of 17 weeks. Whole and partial carcass condemnation rates at the slaughterhouse were high, but no macroscopic changes in the skeletal system were found, showing that food chain information is essential. This study confirms earlier findings that Y. pseudotuberculosis can cause osteomyelitis in fattening turkeys, leading to lameness. Food chain information is essential for slaughterhouse operations, to protect the workers and emphasize good working hygiene during slaughter.

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.

Adhesins facilitate bacterial colonization and invasion of host tissues and are considered virulence factors, but their impact on immune-mediated damage as a driver of pathogenesis remains unclear. Yersinia pseudotuberculosis encodes for a multivalent adhesion molecule (MAM), a mammalian cell entry (MCE) family protein and adhesin. MAMs are widespread in Gram-negative bacteria and enable enteric bacteria to colonize epithelial tissues. Their role in bacterial interactions with the host innate immune system and contribution to pathogenicity remains unclear. Here, we investigated howY. pseudotuberculosis MAM contributes to pathogenesis during infection of the Galleria mellonella insect model. We show that Y. pseudotuberculosis MAM is required for efficient bacterial binding and uptake by hemocytes, the host phagocytes. Y. pseudotuberculosis interactions with insect and mammalian phagocytes are determined by bacterial and host factors. Loss of MAM, and deficient microbe–phagocyte interaction, increased pathogenesis in G. mellonella. Diminished phagocyte association also led to increased bacterial clearance. Furthermore, Y. pseudotuberculosis that failed to engage phagocytes hyperactivated humoral immune responses, most notably melanin production. Despite clearing the pathogen, excessive melanization also increased phagocyte death and host mortality. Our findings provide a basis for further studies investigating how microbe- and host-factors integrate to drive pathogenesis in a tractable experimental system.

Bacterial type VI secretion systems (T6SSs) inject toxic effectors into adjacent eukaryotic and prokaryotic cells. It is generally thought that this process requires physical contact between the two cells. Here, we provide evidence of contact-independent killing by a T6SS-secreted effector. We show that the pathogen Yersinia pseudotuberculosis uses a T6SS (T6SS-3) to secrete a nuclease effector that kills other bacteria in vitro and facilitates gut colonization in mice. The effector (Tce1) is a small protein that acts as a Ca2+- and Mg2+-dependent DNase, and its toxicity is inhibited by a cognate immunity protein, Tci1. As expected, T6SS-3 mediates canonical, contact-dependent killing by directly injecting Tce1 into adjacent cells. In addition, T6SS-3 also mediates killing of neighboring cells in the absence of cell-to-cell contact, by secreting Tce1 into the extracellular milieu. Efficient contact-independent entry of Tce1 into target cells requires proteins OmpF and BtuB in the outer membrane of target cells. The discovery of a contact-independent, long-range T6SS toxin delivery provides a new perspective for understanding the physiological roles of T6SS in competition. However, the mechanisms mediating contact-independent uptake of Tce1 by target cells remain unclear., Bacteria can use type VI secretion systems (T6SSs) to inject toxic effector proteins into adjacent cells, in a contact-dependent manner. Here, the authors provide evidence of contact-independent killing by a T6SS effector that is secreted into the extracellular milieu and then taken up by other bacterial cells.

Background Between February and April 2016, a slight increase in mortality was observed in a colony consisting of 400 captive Seba’s short-tailed bats (Carollia perspicillata). These animals cohabited with other nocturnal animal species in a dome of a private zoo in Switzerland. Results Gross and histological analysis of two (14.3%) out of the 13 animals submitted for necropsy within this period revealed a necrosuppurative pneumonia, hepatitis, splenitis, enterocolitis, and endometritis, with abundant intralesional colonies of Gram-negative rods. Yersinia (Y.) pseudotuberculosis serotype O:1 and biotype 1 belonging to the sequence type ST90 was isolated from the affected organs in both animals. Following this diagnosis, ¼ of the colony (99 animals) was culled and submitted for gross and histopathological analysis, and a bacterial culture selective for Yersinia spp. of lung, liver, and spleen was performed. From these 99 animals, one gravid female was tested and found to be positive for Y. pseudotuberculosis in the absence of clinical symptoms and histopathological lesions. PCR analysis of altogether three bacterial isolates for virulence factors revealed the presence of the ail gene, and one isolate was also positive for the virF and yadA plasmid genes. Conclusions These findings suggest that Carollia perspicillata are susceptible to lethal yersiniosis but do not represent a regular reservoir for Y. pseudotuberculosis. Culling of ¼ of the population was sufficient to limit the spread of this infection among the colony. Moreover, no infections were detected in cohabitant nocturnal animals and caretakers, indicating that the zoonotic risk in this case was low.

Pathogenic Yersinia spp. depend on the activity of a potent virulence plasmid-encoded ysc/yop type 3 secretion system (T3SS) to colonize hosts and cause disease. It was recently shown that Yersinia pseudotuberculosis upregulates the virulence plasmid copy number (PCN) during infection and that the resulting elevated gene dose of plasmid-encoded T3SS genes is essential for virulence., Pathogenic Yersinia spp. depend on the activity of a potent virulence plasmid-encoded ysc/yop type 3 secretion system (T3SS) to colonize hosts and cause disease. It was recently shown that Yersinia pseudotuberculosis upregulates the virulence plasmid copy number (PCN) during infection and that the resulting elevated gene dose of plasmid-encoded T3SS genes is essential for virulence. When and how this novel regulatory mechanism is deployed and regulates the replication of the virulence plasmid during infection is unknown. In the present study, we applied droplet digital PCR (ddPCR) to investigate the dynamics of Y. pseudotuberculosis virulence PCN variations and growth rates in infected mouse organs. We demonstrated that both PCN and growth varied in different tissues and over time throughout the course of infection, indicating that the bacteria adapted to discrete microenvironments during infection. The PCN was highest in Peyer’s patches and cecum during the clonal invasive phase of the infection, while the highest growth rates were found in the draining mesenteric lymph nodes. In deeper, systemic organs, the PCN was lower and more modest growth rates were recorded. Our study indicates that increased gene dosage of the plasmid-encoded T3SS genes is most important early in the infection during invasion of the host. The described ddPCR approach will greatly simplify analyses of PCN, growth dynamics, and bacterial loads in infected tissues and will be readily applicable to other infection models.

Yersinia pseudotuberculosis has been studied for many decades, and research on this microbe has taught us a great deal about host-pathogen interactions, bacterial manipulation of host cells, virulence factors, and the evolution of pathogens. This microbe should not be cultivated at 37°C because this is a trigger that the bacterium uses to sense its presence within a mammalian host and results in expression of genes necessary to colonize a mammalian host. Prolonged growth at this temperature can result in accumulation of mutations that reduce the virulence of the strain, so all protocols need to be modified for growth at room temperature, or 26°C. This article describes protocols for cultivating this microbe and for its long-term storage and its genetic manipulation by transformation and conjugation. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Growth of Y. pseudotuberculosis from a stock Basic Protocol 2: Growth of Y. pseudotuberculosis in liquid medium from a single colony Basic Protocol 3: Freezing Y. pseudotuberculosis in glycerol for long-term storage Basic Protocol 4: Transformation of Y. pseudotuberculosis by electroporation Basic Protocol 5: Tri-parental mating/conjugation.

Upon acquiring two unique plasmids (pMT1 and pPCP1) and genome rearrangement during the evolution from Yersinia pseudotuberculosis, the plague causative agent Y. pestis is closely related to Y. pseudotuberculosis genetically but became highly virulent. We developed a pentaplex real‐time PCR assay that not only detects both Yersinia species but also differentiates Y. pestis strains regarding their plasmid profiles. The five targets used were Y. pestis‐specific ypo2088, caf1, and pst located on the chromosome, plasmids pMT1 and pPCP1, respectively; Y. pseudotuberculosis‐specific chromosomal gene opgG; and 18S ribosomal RNA gene as an internal control for flea DNA. All targets showed 100% specificity and high sensitivity with limits of detection ranging from 1 fg to 100 fg, with Y. pestis‐specific pst as the most sensitive target. Using the assay, Y. pestis strains were differentiated 100% by their known plasmid profiles. Testing Y. pestis and Y. pseudotuberculosis‐spiked flea DNA showed there is no interference from flea DNA on the amplification of targeted genes. Finally, we applied the assay for testing 102 fleas collected from prairie dog burrows where prairie dog die‐off was reported months before flea collection. All flea DNA was amplified by 18S rRNA; no Y. pseudotuberculosis was detected; one flea was positive for all Y. pestis‐specific targets, confirming local Y. pestis transmission. Our results indicated the assay is sensitive and specific for the detection and differentiation of Y. pestis and Y. pseudotuberculosis. The assay can be used in field investigations for the rapid identification of the plague causative agent., We developed a pentaplex real‐time PCR assay including Yersinia pestis‐specific genes ypo2088, caf1, and pst located on the chromosome, as well as on plasmids pMT1 and pPCP1, respectively. Yersinia pseudotuberculosis‐specific chromosomal gene opgG and the 18S ribosomal RNA gene served as an internal control for flea DNA. All targets showed 100% specificity and high sensitivity with limits of detection ranging from 1 to 100 femtograms. Using the assay, Y. pestis was detected in field‐collected fleas and strains were differentiated 100% by their known plasmid profiles.

Bacterial infections are very difficult to treat when bacteria spread into the bloodstream and begin to replicate within deep tissues, such as the spleen. Subsets of bacteria can survive antibiotic treatment, but it remains unclear if this survival is because of limited drug diffusion into tissues, or if there are changes within the bacteria, promoting survival of some bacterial cells. Here, we have developed a fluorescent reporter to detect doxycycline (Dox) diffusion into host tissues, and we show that Dox impacts the bacterial population within hours of administration and inhibits bacterial growth for 48 h. However, bacterial growth resumes when antibiotic concentrations decrease. Subsets of bacteria, stressed by the host response to infection, survive Dox treatment at a higher rate. These results provide critical information about the dynamics that occur within deep tissues following antibiotic administration and suggest that subsets of bacteria are predisposed to survive inhibitory concentrations of antibiotic before exposure., Severe systemic bacterial infections result in colonization of deep tissues, which can be very difficult to eliminate with antibiotics. It remains unclear if this is because antibiotics are not reaching inhibitory concentrations within tissues, if subsets of bacteria are less susceptible to antibiotics, or if both contribute to limited treatment efficacy. To detect exposure to doxycycline (Dox) present in deep tissues following treatment, we generated a fluorescent transcriptional reporter derived from the tet operon to specifically detect intracellular tetracycline exposure at the single bacterial cell level. Dox exposure was detected in the spleen 2 h after intraperitoneal injection, and by 4 h postinjection, this treatment resulted in a significant decrease in viable Yersinia pseudotuberculosis bacteria in the spleen. Nitric oxide-stressed bacteria preferentially survived treatment, suggesting that stress was sufficient to alter Dox susceptibility. Many bacteria (∼10%) survived a single dose of Dox, and the antibiotic accumulated at the periphery of microcolonies to growth inhibitory concentrations until 48 h posttreatment. After this time point, antibiotic concentrations decreased and bacterial growth resumed. Dox-treated mice eventually succumbed to the infection, albeit with significantly prolonged survival relative to that of untreated mice. These results indicate that Dox delivery by intraperitoneal injection results in rapid diffusion of inhibitory concentrations of antibiotic into the spleen, but stressed cells preferentially survive drug treatment, and bacterial growth resumes once drug concentrations decrease. This fluorescent reporter strategy for antibiotic detection could easily be modified to detect the concentration of additional antimicrobial compounds within host tissues following drug administration.

Cohesion of biofilms made byYersinia pestisandYersinia pseudotuberculosis(Yptb) has been attributed solely to an extracellular polysaccharide matrix encoded by thehmsgenes (Hms-ECM). However, mutations in theYptbBarA/UvrY/CsrB regulatory cascade enhance biofilm stability without dramatically increasing Hms-ECM production. We found that treatment with proteinase K enzyme effectively destabilizedYptb csrBmutant biofilms, suggesting that cell-cell interactions might be mediated by protein adhesins or extracellular matrix proteins. We identified an uncharacterized trimeric autotransporter lipoprotein (YPTB2394), repressed bycsrB, which has been referred to as YadE. Biofilms made by a ΔyadEmutant strain were extremely sensitive to mechanical disruption. Overexpression ofyadEin wild-typeYptbincreased biofilm cohesion, similar to biofilms made bycsrBoruvrYmutants. We found that the Rcs signaling cascade, which represses Hms-ECM production, activated expression ofyadE. TheyadEgene appears to be functional inYptbbut is a pseudogene in modernY. pestis strains. Expression of functionalyadEinY. pestisKIM6+ altered the production of Hms-ECM and weakened biofilms made by these bacteria. This suggests that although the YadE autotransporter protein increasesYptbbiofilm stability, it may be incompatible with Hms-ECM production that is essential forY. pestisbiofilm production in fleas. Inactivation ofyadEinY. pestismay be another instance of selective gene loss in the evolution of flea-borne transmission by this species.IMPORTANCEThe evolution ofYersinia pestisfrom itsY. pseudotuberculosis(Yptb) ancestor involved gene acquisition and gene losses, leading to differences in biofilm production. Characterizing the unique biofilm features of both species may provide better understanding of how each adapts to its specific niches. This study identifies a trimeric autotransporter YadE that promotes biofilm stability ofYptbbut which has been inactivated inY. pestis, likely because it is not compatible with Hms polysaccharide that is crucial for biofilms inside fleas. We also reveal that the Rcs signaling cascade, which represses Hms expression inY. pestis, activates YadE inYptb. The ability ofYptbto use polysaccharide or YadE protein for cell-cell adhesion may help it produce biofilms in different environments.

Acute inguinal lymphadenitis is usually caused by lower extremity infection and sexually transmitted diseases, such as chancroid, lymphogranuloma venereum, genital herpes, or syphilis. Yersinia pseudotuberculosis is a non-spore forming, pleomorphic, non-lactose fermenting Gram negative bacillus and a member of the family Enterobacteriaceae, which is associated with diarrheal diseases. It also causes mesenteric lymphadenitis at the terminal ileum, which can be clinically indistinguishable from acute appendicitis (pseudoappendicitis). However, lymphadenitis in other regions caused by the organism is rarely reported. Herein, we report a case of a man in his 20s, who presented with unilateral inguinal lymphadenitis caused by Y. pseudotuberculosis, with discussion regarding the pathogenesis of this rare occurrence.

Frequent transitions of bacterial pathogens between their warm-blooded host and external reservoirs are accompanied by abrupt temperature shifts. A temperature of 37°C serves as reliable signal for ingestion by a mammalian host, which induces a major reprogramming of bacterial gene expression and metabolism. Enteric Yersiniae are Gram-negative pathogens accountable for self-limiting gastrointestinal infections. Among the temperature-regulated virulence genes of Yersinia pseudotuberculosis is cnfY coding for the cytotoxic necrotizing factor (CNFY), a multifunctional secreted toxin that modulates the host’s innate immune system and contributes to the decision between acute infection and persistence. We report that the major determinant of temperature-regulated cnfY expression is a thermo-labile RNA structure in the 5’-untranslated region (5’-UTR). Various translational gene fusions demonstrated that this region faithfully regulates translation initiation regardless of the transcription start site, promoter or reporter strain. RNA structure probing revealed a labile stem-loop structure, in which the ribosome binding site is partially occluded at 25°C but liberated at 37°C. Consistent with translational control in bacteria, toeprinting (primer extension inhibition) experiments in vitro showed increased ribosome binding at elevated temperature. Point mutations locking the 5’-UTR in its 25°C structure impaired opening of the stem loop, ribosome access and translation initiation at 37°C. To assess the in vivo relevance of temperature control, we used a mouse infection model. Y. pseudotuberculosis strains carrying stabilized RNA thermometer variants upstream of cnfY were avirulent and attenuated in their ability to disseminate into mesenteric lymph nodes and spleen. We conclude with a model, in which the RNA thermometer acts as translational roadblock in a two-layered regulatory cascade that tightly controls provision of the CNFY toxin during acute infection. Similar RNA structures upstream of various cnfY homologs suggest that RNA thermosensors dictate the production of secreted toxins in a wide range of pathogens., Author summary Bacterial pathogens closely survey the ambient conditions and induce virulence genes only at appropriate conditions. Upon host contact, many pathogens secrete toxins in order to subvert host defense systems. We find that such a secreted toxin in enteropathogenic Yersinia pseudotuberculosis is produced only at host body temperature. This regulation depends on a temperature-responsive RNA structure, an RNA thermometer, in the 5’-untranslated region of the toxin mRNA, which prevents translation at low temperatures when the bacterium is outside the host. Preventing melting of the RNA structure at 37°C by nucleotide substitutions that stabilize base pairing resulted in avirulent Yersinia strains unable to infect mice. Given that similar RNA thermometer-like structures exist upstream of related toxin genes in various bacterial pathogens, we propose that RNA thermometer-mediated toxin production is an evolutionary conserved mechanism. Interfering with opening of such regulatory structures might thus be a promising strategy targeting a broad spectrum of bacterial pathogens.

Yersinia suppress neutrophil responses by using a type 3 secretion system (T3SS) to inject 6–7 Yersinia effector proteins (Yops) effectors into their cytoplasm. YopH is a tyrosine phosphatase that causes dephosphorylation of the adaptor protein SKAP2, among other targets in neutrophils. SKAP2 functions in reactive oxygen species (ROS) production, phagocytosis, and integrin-mediated migration by neutrophils. Here we identify essential neutrophil functions targeted by YopH, and investigate how the interaction between YopH and SKAP2 influence Yersinia pseudotuberculosis (Yptb) survival in tissues. The growth defect of a ΔyopH mutant was restored in mice defective in the NADPH oxidase complex, demonstrating that YopH is critical for protecting Yptb from ROS during infection. The growth of a ΔyopH mutant was partially restored in Skap2-deficient (Skap2KO) mice compared to wild-type (WT) mice, while induction of neutropenia further enhanced the growth of the ΔyopH mutant in both WT and Skap2KO mice. YopH inhibited both ROS production and degranulation triggered via integrin receptor, G-protein coupled receptor (GPCR), and Fcγ receptor (FcγR) stimulation. SKAP2 was required for integrin receptor and GPCR-mediated ROS production, but dispensable for degranulation under all conditions tested. YopH blocked SKAP2-independent FcγR-stimulated phosphorylation of the proximal signaling proteins Syk, SLP-76, and PLCγ2, and the more distal signaling protein ERK1/2, while only ERK1/2 phosphorylation was dependent on SKAP2 following integrin receptor activation. These findings reveal that YopH prevents activation of both SKAP2-dependent and -independent neutrophilic defenses, uncouple integrin- and GPCR-dependent ROS production from FcγR responses based on their SKAP2 dependency, and show that SKAP2 is not required for degranulation., Author summary Pathogenic Yersinia species carry a virulence plasmid encoding a type 3 secretion system that translocates 6–7 effector Yops into host cells. We demonstrate that YopH protects Yersinia pseudotuberculosis from neutrophil-produced reactive oxygen species (ROS) and degranulation by interfering with signaling pathways downstream of three major receptor classes in neutrophils. We show that a previously identified target of YopH, SKAP2, controls some of the pathways essential for YopH to inactivate during infection. SKAP2 is essential in mediating ROS production downstream of two major receptors; however, it is dispensable for degranulation from the three major receptors tested. Our study illustrates that YopH protects Y. pseudotuberculosis by blocking both SKAP2-dependent and independent signaling pathways that regulate several neutrophil functions.

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.

Yersinia pseudotuberculosis is a Gram-negative bacterium and zoonotic pathogen responsible for a wide range of diseases, ranging from mild diarrhea, enterocolitis, lymphatic adenitis to persistent local inflammation. The Y. pseudotuberculosis invasin D (InvD) molecule belongs to the invasin (InvA)-type autotransporter proteins, but its structure and function remain unknown. In this study, we present the first crystal structure of InvD, analyzed its expression and function in a murine infection model, and identified its target molecule in the host. We found that InvD is induced at 37 °C and expressed in vivo 2–4 days after infection, indicating that InvD is a virulence factor. During infection, InvD was expressed in all parts of the intestinal tract, but not in deeper lymphoid tissues. The crystal structure of the C-terminal adhesion domain of InvD revealed a distinct Ig-related fold that, apart from the canonical β-sheets, comprises various modifications of and insertions into the Ig-core structure. We identified the Fab fragment of host-derived IgG/IgA antibodies as the target of the adhesion domain. Phage display panning and flow cytometry data further revealed that InvD exhibits a preferential binding specificity toward antibodies with VH3/VK1 variable domains and that it is specifically recruited to a subset of B cells. This finding suggests that InvD modulates Ig functions in the intestine and affects direct interactions with a subset of cell surface-exposed B-cell receptors. In summary, our results provide extensive insights into the structure of InvD and its specific interaction with the target molecule in the host.

Many bacterial pathogens use a type III secretion system (T3SS) as molecular syringe to inject effector proteins into the host cell. In the foodborne pathogen Yersinia pseudotuberculosis, delivery of the secreted effector protein cocktail through the T3SS depends on YopN, a molecular gatekeeper that controls access to the secretion channel from the bacterial cytoplasm. Here, we show that several checkpoints adjust yopN expression to virulence conditions. A dominant cue is the host body temperature. A temperature of 37°C is known to induce the RNA thermometer (RNAT)-dependent synthesis of LcrF, a transcription factor that activates expression of the entire T3SS regulon. Here, we uncovered a second layer of temperature control. We show that another RNAT silences translation of the yopN mRNA at low environmental temperatures. The long and short 5’-untranslated region of both cellular yopN isoforms fold into a similar secondary structure that blocks ribosome binding. The hairpin structure with an internal loop melts at 37°C and thereby permits formation of the translation initiation complex as shown by mutational analysis, in vitro structure probing and toeprinting methods. Importantly, we demonstrate the physiological relevance of the RNAT in the faithful control of type III secretion by using a point-mutated thermostable RNAT variant with a trapped SD sequence. Abrogated YopN production in this strain led to unrestricted effector protein secretion into the medium, bacterial growth arrest and delayed translocation into eukaryotic host cells. Cumulatively, our results show that substrate delivery by the Yersinia T3SS is under hierarchical surveillance of two RNATs., Author summary Temperature serves as reliable external cue for pathogenic bacteria to recognize the entry into or exit from a warm-blooded host. At the molecular level, a temperature of 37°C induces various virulence-related processes that manipulate host cell physiology. Here, we demonstrate the temperature-dependent synthesis of the secretion regulator YopN in the foodborne pathogen Yersinia pseudotuberculosis, a close relative of Yersinia pestis. YopN blocks secretion of effector proteins through the type III secretion system unless host cell contact is established. Temperature-specific regulation relies on an RNA structure in the 5’-untranslated region of the yopN mRNA, referred to as RNA thermometer, which allows ribosome binding and thus translation initiation only at an infection-relevant temperature of 37°C. A mutated variant of the thermosensor resulting in a closed conformation prevented synthesis of the molecular gatekeeper YopN and led to permanent secretion and defective translocation of virulence factors into host cells. We suggest that the RNA thermometer plays a critical role in adjusting the optimal cellular concentration of a surveillance factor that maintains the controlled translocation of virulence factors.

The enteric nervous system (ENS) controls several intestinal functions including motility and nutrient handling, which can be disrupted by infection-induced neuropathies or neuronal cell death. We investigated possible tolerance mechanisms preventing neuronal loss and disruption in gut motility after pathogen exposure. We found that following enteric infections, muscularis macrophages (MMs) acquire a tissue-protective phenotype that prevents neuronal loss and dysmotility during subsequent challenge with unrelated pathogens. Bacteria-induced neuroprotection relied on activation of gut-projecting sympathetic neurons and signaling via β2-adrenergic receptors (β2AR) on MMs. In contrast, helminth-mediated neuroprotection was dependent on T cells and systemic production of interleukin (IL)-4 and -13 by eosinophils, which induced arginase-expressing MMs that prevented neuronal loss from an unrelated infection located in a different intestinal region. Collectively, these data suggest that distinct enteric pathogens trigger a state of disease- or tissue tolerance that preserves ENS number and functionality.