Enteric pathogens induce tissue tolerance and prevent neuronal loss from subsequent infections.

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, dysmotility, and maintains energy balance during subsequent challenge with unrelated pathogens. Bacteria-induced neuroprotection relied on activation of gut-projecting sympathetic neurons and signaling via ß2-adrenergic receptors (ß2AR) on MMs. In contrast, helminth-mediated neuroprotection was dependent on T cells and systemic production of interleukin (IL)-4 and IL-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.

Proinflammatory microenvironments within the intestine regulate the differentiation of tissue-resident CD8⁺ T cells responding to infection.

We report that oral infection with Yersinia pseudotuberculosis results in the development of two distinct populations of pathogen-specific CD8(+) tissue-resident memory T cells (TRM cells) in the lamina propria. CD103(-) T cells did not require transforming growth factor-ß (TGF-ß) signaling but were true resident memory cells. Unlike CD103(+)CD8(+) T cells, which were TGF-ß dependent and were scattered in the tissue, CD103(-)CD8(+) T cells clustered with CD4(+) T cells and CX3CR1(+) macrophages and/or dendritic cells around areas of bacterial infection. CXCR3-dependent recruitment of cells to inflamed areas was critical for development of the CD103(-) population and pathogen clearance. Our studies have identified the 'preferential' development of CD103(-) TRM cells in inflammatory microenvironments within the lamina propria and suggest that this subset has a critical role in controlling infection.

White Adipose Tissue Is a Reservoir for Memory T Cells and Promotes Protective Memory Responses to Infection.

White adipose tissue bridges body organs and plays a fundamental role in host metabolism. To what extent adipose tissue also contributes to immune surveillance and long-term protective defense remains largely unknown. Here, we have shown that at steady state, white adipose tissue contained abundant memory lymphocyte populations. After infection, white adipose tissue accumulated large numbers of pathogen-specific memory T cells, including tissue-resident cells. Memory T cells in white adipose tissue expressed a distinct metabolic profile, and white adipose tissue from previously infected mice was sufficient to protect uninfected mice from lethal pathogen challenge. Induction of recall responses within white adipose tissue was associated with the collapse of lipid metabolism in favor of antimicrobial responses. Our results suggest that white adipose tissue represents a memory T cell reservoir that provides potent and rapid effector memory responses, positioning this compartment as a potential major contributor to immunological memory.

RIPK1 activates distinct gasdermins in macrophages and neutrophils upon pathogen blockade of innate immune signaling.

Injection of effector proteins to block host innate immune signaling is a common strategy used by many pathogenic organisms to establish an infection. For example, pathogenic Yersinia species inject the acetyltransferase YopJ into target cells to inhibit NF-κB and MAPK signaling. To counteract this, detection of YopJ activity in myeloid cells promotes the assembly of a RIPK1-caspase-8 death-inducing platform that confers antibacterial defense. While recent studies revealed that caspase-8 cleaves the pore-forming protein gasdermin D to trigger pyroptosis in macrophages, whether RIPK1 activates additional substrates downstream of caspase-8 to promote host defense is unclear. Here, we report that the related gasdermin family member gasdermin E (GSDME) is activated upon detection of YopJ activity in a RIPK1 kinase-dependent manner. Specifically, GSDME promotes neutrophil pyroptosis and IL-1ß release, which is critical for anti-Yersinia defense. During in vivo infection, IL-1ß neutralization increases bacterial burden in wild-type but not Gsdme-deficient mice. Thus, our study establishes GSDME as an important mediator that counteracts pathogen blockade of innate immune signaling.

Modifying TIMER to generate a slow-folding DsRed derivative for optimal use in quickly-dividing bacteria.

It is now well appreciated that members of pathogenic bacterial populations exhibit heterogeneity in growth rates and metabolic activity, and it is known this can impact the ability to eliminate all members of the bacterial population during antibiotic treatment. It remains unclear which pathways promote slowed bacterial growth within host tissues, primarily because it has been difficult to identify and isolate slow growing bacteria from host tissues for downstream analyses. To overcome this limitation, we have developed a novel variant of TIMER, a slow-folding fluorescent protein, named DsRed42, to identify subsets of slowly dividing bacteria within host tissues. The original TIMER folds too slowly for fluorescence accumulation in quickly replicating bacterial species (Escherichia coli, Yersinia pseudotuberculosis), however DsRed42 accumulates red fluorescence in late stationary phase cultures of E. coli and Y. pseudotuberculosis. We show DsRed42 signal also accumulates during exposure to sources of nitric oxide (NO), suggesting DsRed42 signal detects growth-arrested bacterial cells. In a mouse model of Y. pseudotuberculosis deep tissue infection, DsRed42 signal was detected, and primarily accumulates in bacteria expressing markers of stationary phase growth. There was no significant overlap between DsRed42 signal and NO-exposed subpopulations of bacteria within host tissues, suggesting NO stress was transient, allowing bacteria to recover from this stress and resume replication. This novel DsRed42 variant represents a tool that will enable additional studies of slow-growing subpopulations of bacteria, specifically within bacterial species that quickly divide.

The gatekeeper of Yersinia type III secretion is under RNA thermometer control.

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.

NO-Stressed Y. pseudotuberculosis Has Decreased Cell Division Rates in the Mouse Spleen.

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.

An RNA thermometer dictates production of a secreted bacterial toxin.

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.

Ultrastructure and Morphological Variability of Non-Culturable Forms of Yersinia pseudotuberculosis Bacteria.

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.

Spatiotemporal Variations in Growth Rate and Virulence Plasmid Copy Number during Yersinia pseudotuberculosis Infection.

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.

Precursor Abundance Influences Divergent Antigen-Specific CD8+ T Cell Responses after Yersinia pseudotuberculosis Foodborne Infection.

Primary infection of C57BL/6 mice with the bacterial pathogen Yersinia pseudotuberculosis elicits an unusually large H-2Kb-restricted CD8+ T cell response to the endogenous and protective bacterial epitope YopE69-77. To better understand the basis for this large response, the model OVA257-264 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 YopE69-77 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-2Kb similar to that of OVA257-264. 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 YopE69-77 over OVA257-264 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 YopE69-77 epitope during Y. pseudotuberculosis infection of C57BL/6 mice.

Septic shock due to Yersinia pseudotuberculosis infection in an adult immunocompetent patient: a case report and literature review.

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.

A Trimeric Autotransporter Enhances Biofilm Cohesiveness in Yersinia pseudotuberculosis but Not in Yersinia pestis.

Cohesion of biofilms made by Yersinia pestis and Yersinia pseudotuberculosis has been attributed solely to an extracellular polysaccharide matrix encoded by the hms genes (Hms-dependent extracellular matrix [Hms-ECM]). However, mutations in the Y. pseudotuberculosis BarA/UvrY/CsrB regulatory cascade enhance biofilm stability without dramatically increasing Hms-ECM production. We found that treatment with proteinase K enzyme effectively destabilized Y. pseudotuberculosiscsrB mutant 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 by csrB, which has been referred to as YadE. Biofilms made by a ΔyadE mutant strain were extremely sensitive to mechanical disruption. Overexpression of yadE in wild-type Y. pseudotuberculosis increased biofilm cohesion, similar to biofilms made by csrB or uvrY mutants. We found that the Rcs signaling cascade, which represses Hms-ECM production, activated expression of yadE The yadE gene appears to be functional in Y. pseudotuberculosis but is a pseudogene in modern Y. pestis strains. Expression of functional yadE in Y. pestis KIM6+ weakened biofilms made by these bacteria. This suggests that although the YadE autotransporter protein increases Y. pseudotuberculosis biofilm stability, it may be incompatible with the Hms-ECM production that is essential for Y. pestis biofilm production in fleas. Inactivation of yadE in Y. pestis may be another instance of selective gene loss in the evolution of flea-borne transmission by this species.IMPORTANCE The evolution of Yersinia pestis from its Y. pseudotuberculosis 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 of Y. pseudotuberculosis but which has been inactivated in Y. pestis, perhaps because it is not compatible with the Hms polysaccharide that is crucial for biofilms inside fleas. We also reveal that the Rcs signaling cascade, which represses Hms expression, activates YadE in Y. pseudotuberculosis The ability of Y. pseudotuberculosis to use polysaccharide or YadE protein for cell-cell adhesion may help it produce biofilms in different environments.

A horse and a zebra: an atypical clinical picture including Guillain-Barré syndrome, recurrent fever and mesenteric lymphadenopathy caused by two concomitant infections.

BACKGROUND: While Campylobacter jejuni represents the most common cause of bacterial gastroenteritis, Yersinia pseudotuberculosis infections are very rarely diagnosed in adults. CASE: We 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 a C. jejuni and Y. pseudotuberculosis coinfection. Antibiotic treatment with doxycycline was effective. CONCLUSION: Broad 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".

Unilateral inguinal lymphadenitis caused by Yersinia pseudotuberculosis. A case report.

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.

Ultrastructural Changes of Bacteria in Static Cultures of Yersinia pseudotuberculosis under Long Storage under Conditions of Low Temperature.

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.

The invasin D protein from Yersinia pseudotuberculosis selectively binds the Fab region of host antibodies and affects colonization of the intestine.

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 ß-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.

Grasping the nettle: A bacterial invasin that targets immunoglobulin variable domains.

In a new paper, the protein InvD from Yersinia pseudotuberculosis, a zoonotic pathogen, is shown to assist late-stage invasion of intestinal epithelia. Remarkably, InvD acts by binding the Fab region of IgG or IgA. It straddles adjacent light-chain and heavy-chain variable domains, but its binding is different from that of antigens in that complementarity-determining regions do not participate. Structure determination revealed that its Fab-interacting domain adopts an immunoglobulin-like fold, fused to the preceding immunoglobulin-like domain and carried on a long stalk anchored to the bacterial outer membrane. Possible roles of this unusual host-pathogen interaction include avoidance of clearance from the intestine by secretory IgA.

Iron-Sulfur Cluster Repair Contributes to Yersinia pseudotuberculosis Survival within Deep Tissues.

To successfully colonize host tissues, bacteria must respond to and detoxify many different host-derived antimicrobial compounds, such as nitric oxide (NO). NO has direct antimicrobial activity through attack on iron-sulfur (Fe-S) cluster-containing proteins. NO detoxification plays an important role in promoting bacterial survival, but it remains unclear if repair of Fe-S clusters is also important for bacterial survival within host tissues. Here we show that the Fe-S cluster repair protein YtfE contributes to the survival of Yersinia pseudotuberculosis within the spleen following nitrosative stress. Y. pseudotuberculosis forms clustered centers of replicating bacteria within deep tissues, where peripheral bacteria express the NO-detoxifying gene hmp. ytfE expression also occurred specifically within peripheral cells at the edges of microcolonies. In the absence of ytfE, the area of microcolonies was significantly smaller than that of the wild type (WT), consistent with ytfE contributing to the survival of peripheral cells. The loss of ytfE did not alter the ability of cells to detoxify NO, which occurred within peripheral cells in both WT and ΔytfE microcolonies. In the absence of NO-detoxifying activity by hmp, NO diffused across ΔytfE microcolonies, and there was a significant decrease in the area of microcolonies lacking ytfE, indicating that ytfE also contributes to bacterial survival in the absence of NO detoxification. These results indicate a role for Fe-S cluster repair in the survival of Y. pseudotuberculosis within the spleen and suggest that extracellular bacteria may rely on this pathway for survival within host tissues.

A Recombinant Attenuated Yersinia pseudotuberculosis Vaccine Delivering a Y. pestis YopENt138-LcrV Fusion Elicits Broad Protection against Plague and Yersiniosis in Mice.

In this study, a novel recombinant attenuated Yersinia pseudotuberculosis PB1+ strain (χ10069) engineered with ΔyopK ΔyopJ Δasd triple mutations was used to deliver a Y. pestis fusion protein, YopE amino acid 1 to 138-LcrV (YopENt138-LcrV), to Swiss Webster mice as a protective antigen against infections by yersiniae. χ10069 bacteria harboring the pYA5199 plasmid constitutively synthesized the YopENt138-LcrV fusion protein and secreted it via the type 3 secretion system (T3SS) at 37°C under calcium-deprived conditions. The attenuated strain χ10069(pYA5199) was manifested by the establishment of controlled infection in different tissues without developing conspicuous signs of disease in histopathological analysis of microtome sections. A single-dose oral immunization of χ10069(pYA5199) induced strong serum antibody titers (log10 mean value, 4.2), secretory IgA in bronchoalveolar lavage (BAL) fluid from immunized mice, and Yersinia-specific CD4+ and CD8+ T cells producing high levels of tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and interleukin 2 (IL-2), as well as IL-17, in both lungs and spleens of immunized mice, conferring comprehensive Th1- and Th2-mediated immune responses and protection against bubonic and pneumonic plague challenges, with 80% and 90% survival, respectively. Mice immunized with χ10069(pYA5199) also exhibited complete protection against lethal oral infections by Yersinia enterocolitica WA and Y. pseudotuberculosis PB1+. These findings indicated that χ10069(pYA5199) as an oral vaccine induces protective immunity to prevent bubonic and pneumonic plague, as well as yersiniosis, in mice and would be a promising oral vaccine candidate for protection against plague and yersiniosis for human and veterinary applications.