Bread Feeding Is a Robust and More Physiological Enteropathogen Administration Method Compared to Oral Gavage.

Oral administration is a preferred model for studying infection by bacterial enteropathogens such as Yersinia spp. In the mouse model, the most frequent method for oral infection consists of oral gavage with a feeding needle directly introduced in the animal stomach via the esophagus. In this study, we compared needle gavage to bread feeding as an alternative mode of bacterial administration. Using bioluminescence-expressing strains of Yersinia pseudotuberculosis and Yersinia enterocolitica, we detected very early upon needle gavage a bioluminescent signal in the neck area together with a signal in the abdominal region, highlighting the presence of two independent sites of bacterial colonization and multiplication. Bacteria were often detected in the esophagus and trachea, as well as in the lymph nodes draining the salivary glands, suggesting that lesions made during needle introduction into the animal oral cavity lead to rapid bacterial draining to proximal lymph nodes. We then tested an alternative mode of bacterial administration using pieces of bread containing bacteria. Upon bread feeding infection, mice exhibited a stronger bioluminescent signal in the abdominal region than with needle gavage, and no signal was detected in the neck area. Moreover, Y. pseudotuberculosis incorporated in the bread is less susceptible to the acidic environment of the stomach and is therefore more efficient in causing intestinal infections. Based on our observations, bread feeding constitutes a natural and more efficient administration method which does not require specialized skills, is less traumatic for the animal, and results in diseases that more closely mimic foodborne intestinal infection.

Subcutaneous vaccination with a live attenuated Yersinia pseudotuberculosis plague vaccine.

A single oral inoculation to mice of the live attenuated Yersinia pseudotuberculosis VTnF1 strain producing an F1 pseudocapsule protects against bubonic and pneumonic plague. However oral vaccination can fail in humans exposed to frequent intestinal infections. We evaluated in mice the efficacy of subcutaneous vaccine injection as an alternative way to induce protective immunity, while reducing the dose and avoiding strain release in nature. A single subcutaneous dose of up to 108 CFU induced dose-dependent antibody production. At the dose of 107 CFU, i.e. 10 times less than via the oral route, it caused a modest skin reaction and protected 100% against bubonic and 80% against pneumonic plague, caused by high doses of Yersinia pestis. Bacteria migrating to lymph nodes and spleen, but not feces, were rapidly eliminated. Thus, subcutaneous injection of VTnF1 would represent a good alternative when dissemination in nature and human intestinal responsiveness are limitations.

Real-Time Monitoring of Yersinia pestis Promoter Activity by Bioluminescence Imaging.

Bioluminescence imaging (BLI) has become a major strategy for real-time analysis of dynamic biological processes. In particular, bioluminescent reporter microorganisms have been designed to advance our understanding of infectious diseases. Non-invasive monitoring of light-emitting pathogenic bacteria has revealed novel features of pathogenesis and enabled quantitative and qualitative analysis of antibacterial therapies. Transcriptional gene fusions using the bacterial luciferase operon luxCDABE as a reporter have been successfully used to monitor gene expression in vitro and in vivo, leading to valuable applications and major findings. In this chapter, we describe the construction of Yersinia pestis strains bearing a chromosomal copy of the luxCDABE operon under the control of promoters regulated by temperature and their application to quantify gene expression in real-time in bacteria growing in vitro and in a murine bubonic plague model.

Humoral and cellular immune correlates of protection against bubonic plague by a live Yersinia pseudotuberculosis vaccine.

Immunization with the live-attenuated Yersinia pseudotuberculosis VTnF1 strain producing a Yersinia pestis F1 pseudocapsule efficiently protects mice against bubonic and pneumonic plague. In clinical trials, demonstration of a plague vaccine's efficacy in humans will not be feasible, and correlates of protection will be needed to bridge the immune response of protected animals to that of vaccinated humans. Using serum transfer and vaccination of antibody-deficient µMT mice, we established that both humoral and cellular responses elicited by VTnF1 independently conferred protection against bubonic plague. Thus, correlates were searched for in both responses, using blood only. Mice were vaccinated with increasing doses of VTnF1 to provide a range of immune responses and survival outcomes. The cellular response was evaluated using an in vitro IFNγ release assay, and IFNγ levels were significantly associated with protection, although some survivors were negative for IFNγ, so that IFNγ release is not a fully satisfactory correlate. Abundant serum IgG against the F1 capsule, Yop injectable toxins, and also non-F1 Y.pestis antigens were found, but none against the LcrV antigen. All readouts correlated to survival and to each other, confirming that vaccination triggered multiple protective mechanisms developing in parallel. Anti-F1 IgG was the most stringent correlate of protection, in both inbred BALB/c mice and outbred OF1 mice. This indicates that antibodies (Ab) to F1 play a dominant role for protection even in the presence of Ab to many other targets. Easy to measure, the anti-F1 IgG titer will be useful to evaluate the immune response in other animal species and in clinical trials.

Oral vaccination against plague using Yersinia pseudotuberculosis.

Yersinia pestis, the agent of plague, is among the deadliest bacterial pathogens affecting humans, and is a potential biological weapon. Because antibiotic resistant strains of Yersinia pestis have been observed or could be engineered for evil use, vaccination against plague might become the only means to reduce mortality. Although plague is re-emerging in many countries, a vaccine with worldwide license is currently lacking. The vaccine strategy described here is based on an oral vaccination with an attenuated strain of Yersinia pseudotuberculosis. Indeed, this species is genetically almost identical to Y. pestis, but has a much lower pathogenicity and a higher genomic stability. Gradual modifications of the wild-type Yersinia pseudotuberculosis strain IP32953 were performed to generate a safe and immunogenic vaccine. Genes coding for three essential virulence factors were deleted from this strain. To increase cross-species immunogenicity, an F1-encapsulated Y. pseudotuberculosis strain was then generated. For this, the Y. pestis caf operon, which encodes F1, was inserted first on a plasmid, and subsequently into the chromosome. The successive steps achieved to reach maximal vaccine potential are described, and how each step affected bacterial virulence and the development of a protective immune response is discussed. The final version of the vaccine, named VTnF1, provides a highly efficient and long-lasting protection against both bubonic and pneumonic plague after a single oral vaccine dose. Since a Y. pestis strain deprived of F1 exist or could be engineered, we also analyzed the protection conferred by the vaccine against such strain and found that it also confers full protection against the two forms of plague. Thus, the properties of VTnF1 makes it one of the most efficient candidate vaccine for mass vaccination in tropical endemic areas as well as for populations exposed to bioterrorism.

Complete Protection against Pneumonic and Bubonic Plague after a Single Oral Vaccination.

BACKGROUND: No efficient vaccine against plague is currently available. We previously showed that a genetically attenuated Yersinia pseudotuberculosis producing the Yersinia pestis F1 antigen was an efficient live oral vaccine against pneumonic plague. This candidate vaccine however failed to confer full protection against bubonic plague and did not produce F1 stably. METHODOLOGY/PRINCIPAL FINDINGS: The caf operon encoding F1 was inserted into the chromosome of a genetically attenuated Y. pseudotuberculosis, yielding the VTnF1 strain, which stably produced the F1 capsule. Given orally to mice, VTnF1 persisted two weeks in the mouse gut and induced a high humoral response targeting both F1 and other Y. pestis antigens. The strong cellular response elicited was directed mostly against targets other than F1, but also against F1. It involved cells with a Th1-Th17 effector profile, producing IFNγ, IL-17, and IL-10. A single oral dose (108 CFU) of VTnF1 conferred 100% protection against pneumonic plague using a high-dose challenge (3,300 LD50) caused by the fully virulent Y. pestis CO92. Moreover, vaccination protected 100% of mice from bubonic plague caused by a challenge with 100 LD50 Y. pestis and 93% against a high-dose infection (10,000 LD50). Protection involved fast-acting mechanisms controlling Y. pestis spread out of the injection site, and the protection provided was long-lasting, with 93% and 50% of mice surviving bubonic and pneumonic plague respectively, six months after vaccination. Vaccinated mice also survived bubonic and pneumonic plague caused by a high-dose of non-encapsulated (F1-) Y. pestis. SIGNIFICANCE: VTnF1 is an easy-to-produce, genetically stable plague vaccine candidate, providing a highly efficient and long-lasting protection against both bubonic and pneumonic plague caused by wild type or un-encapsulated (F1-negative) Y. pestis. To our knowledge, VTnF1 is the only plague vaccine ever reported that could provide high and durable protection against the two forms of plague after a single oral administration.

YpfΦ: a filamentous phage acquired by Yersinia pestis.

Yersinia pestis, the plague bacillus, has an exceptional pathogenicity for humans. The plague bacillus emerged very recently (≈3,000 years ago) from the enteropathogen Y. pseudotuberculosis. Early after its emergence, Y. pestis became infected by a filamentous phage named YpfΦ. During the microevolution of the plague bacillus, the phage remained in the various lineages as an unstable extrachromosomal element. However, in the sub branch that caused the third plague pandemic, YpfΦ integrated itself into the bacterial chromosome to become a stable prophage. The genome of this phage has the same genetic organization as that of other filamentous phages such as the Vibrio cholerae CTXΦ phage, and shares high sequence identity with the CUS-1 filamentous phage of a high-virulence Escherichia coli K1 clone. In addition to genes involved in phage physiology, YpfΦ carries at each extremity of its genome two open reading frames with no predicted functions. This filamentous phage confers some selective properties to Y. pestis during the infectious process, which may explain why it was conserved duringY. pestis microevolution, despite its instability as an extrachromosomal element in most branches.

Inheritance of the lysozyme inhibitor Ivy was an important evolutionary step by Yersinia pestis to avoid the host innate immune response.

BACKGROUND: Yersinia pestis (the plague bacillus) and its ancestor, Yersinia pseudotuberculosis (which causes self-limited bowel disease), encode putative homologues of the periplasmic lysozyme inhibitor Ivy and the membrane-bound lysozyme inhibitor MliC. The involvement of both inhibitors in virulence remains subject to debate. METHODS: Mutants lacking ivy and/or mliC were generated. We evaluated the mutants' ability to counter lysozyme, grow in serum, and/or counter leukocytes; to produce disease in wild-type, neutropenic, or lysozyme-deficient rodents; and to induce host inflammation. RESULTS: MliC was not required for lysozyme resistance and the development of plague. Deletion of ivy decreased Y. pestis' ability to counter lysozyme and polymorphonuclear neutrophils, but it did not affect the bacterium's ability to grow in serum or resist macrophages. Y. pestis lacking Ivy had attenuated virulence, unless animals were neutropenic or lysozyme deficient. The Ivy mutant induced inflammation to a degree similar to that of the parental strain. Last, Y. pseudotuberculosis did not require Ivy to counter lysozyme and for virulence. CONCLUSIONS: Ivy is required to counter lysozyme during infection, but its role as a virulence factor is species dependent. Our study also shows that a gene that is not necessary for the virulence of an ancestral bacterium may become essential in the emergence of a new pathogen.

Imaging of bubonic plague dynamics by in vivo tracking of bioluminescent Yersinia pestis.

Yersinia pestis dissemination in a host is usually studied by enumerating bacteria in the tissues of animals sacrificed at different times. This laborious methodology gives only snapshots of the infection, as the infectious process is not synchronized. In this work we used in vivo bioluminescence imaging (BLI) to follow Y. pestis dissemination during bubonic plague. We first demonstrated that Y. pestis CO92 transformed with pGEN-luxCDABE stably emitted bioluminescence in vitro and in vivo, while retaining full virulence. The light produced from live animals allowed to delineate the infected organs and correlated with bacterial loads, thus validating the BLI tool. We then showed that the first step of the infectious process is a bacterial multiplication at the injection site (linea alba), followed by a colonization of the draining inguinal lymph node(s), and subsequently of the ipsilateral axillary lymph node through a direct connection between the two nodes. A mild bacteremia and an effective filtering of the blood stream by the liver and spleen probably accounted for the early bacterial blood clearance and the simultaneous development of bacterial foci within these organs. The saturation of the filtering capacity of the spleen and liver subsequently led to terminal septicemia. Our results also indicate that secondary lymphoid tissues are the main targets of Y. pestis multiplication and that colonization of other organs occurs essentially at the terminal phase of the disease. Finally, our analysis reveals that the high variability in the kinetics of infection is attributable to the time the bacteria remain confined at the injection site. However, once Y. pestis has reached the draining lymph nodes, the disease progresses extremely rapidly, leading to the invasion of the entire body within two days and to death of the animals. This highlights the extraordinary capacity of Y. pestis to annihilate the host innate immune response.

An encapsulated Yersinia pseudotuberculosis is a highly efficient vaccine against pneumonic plague.

BACKGROUND: Plague is still a public health problem in the world and is re-emerging, but no efficient vaccine is available. We previously reported that oral inoculation of a live attenuated Yersinia pseudotuberculosis, the recent ancestor of Yersinia pestis, provided protection against bubonic plague. However, the strain poorly protected against pneumonic plague, the most deadly and contagious form of the disease, and was not genetically defined. METHODOLOGY AND PRINCIPAL FINDINGS: The sequenced Y. pseudotuberculosis IP32953 has been irreversibly attenuated by deletion of genes encoding three essential virulence factors. An encapsulated Y. pseudotuberculosis was generated by cloning the Y. pestis F1-encoding caf operon and expressing it in the attenuated strain. The new V674pF1 strain produced the F1 capsule in vitro and in vivo. Oral inoculation of V674pF1 allowed the colonization of the gut without lesions to Peyer's patches and the spleen. Vaccination induced both humoral and cellular components of immunity, at the systemic (IgG and Th1 cells) and the mucosal levels (IgA and Th17 cells). A single oral dose conferred 100% protection against a lethal pneumonic plague challenge (33×LD(50) of the fully virulent Y. pestis CO92 strain) and 94% against a high challenge dose (3,300×LD(50)). Both F1 and other Yersinia antigens were recognized and V674pF1 efficiently protected against a F1-negative Y. pestis. CONCLUSIONS AND SIGNIFICANCE: The encapsulated Y. pseudotuberculosis V674pF1 is an efficient live oral vaccine against pneumonic plague, and could be developed for mass vaccination in tropical endemic areas to control pneumonic plague transmission and mortality.

Insights into the infective properties of YpfΦ, the Yersinia pestis filamentous phage.

YpfΦ is a filamentous phage that infected Yersinia pestis, the plague bacillus, during its emergence. Using an experimental transduction approach, we show here that this phage has the capacity to infect with variable efficiencies, all three pathogenic Yersinia species as well as Escherichia coli. Like other Inovirus phages, its genetic organization comprises three functional modules necessary for the production of infectious virions. Upon infection, YpfΦ integrates into the chromosomal dif site, but extrachromosomal forms are also frequently observed. Several pieces of evidence suggest that the absence of chromosomal YpfΦ in natural non-Orientalis Y. pestis isolates results from a higher chromosomal excision rate rather than from a defective integration machinery. A resident YpfΦ confers some protection against a superinfection. In contrast to other filamentous phages, the incoming YpfΦ genome inserts itself between two copies of the resident prophage. This analysis thus unravels infective properties specific to YpfΦ.

Delineation and analysis of chromosomal regions specifying Yersinia pestis.

Yersinia pestis, the causative agent of plague, has recently diverged from the less virulent enteropathogen Yersinia pseudotuberculosis. Its emergence has been characterized by massive genetic loss and inactivation and limited gene acquisition. The acquired genes include two plasmids, a filamentous phage, and a few chromosomal loci. The aim of this study was to characterize the chromosomal regions acquired by Y. pestis. Following in silico comparative analysis and PCR screening of 98 strains of Y. pseudotuberculosis and Y. pestis, we found that eight chromosomal loci (six regions [R1pe to R6pe] and two coding sequences [CDS1pe and CDS2pe]) specified Y. pestis. Signatures of integration by site specific or homologous recombination were identified for most of them. These acquisitions and the loss of ancestral DNA sequences were concentrated in a chromosomal region opposite to the origin of replication. The specific regions were acquired very early during Y. pestis evolution and were retained during its microevolution, suggesting that they might bring some selective advantages. Only one region (R3pe), predicted to carry a lambdoid prophage, is most likely no longer functional because of mutations. With the exception of R1pe and R2pe, which have the potential to encode a restriction/modification and a sugar transport system, respectively, no functions could be predicted for the other Y. pestis-specific loci. To determine the role of the eight chromosomal loci in the physiology and pathogenicity of the plague bacillus, each of them was individually deleted from the bacterial chromosome. None of the deletants exhibited defects during growth in vitro. Using the Xenopsylla cheopis flea model, all deletants retained the capacity to produce a stable and persistent infection and to block fleas. Similarly, none of the deletants caused any acute flea toxicity. In the mouse model of infection, all deletants were fully virulent upon subcutaneous or aerosol infections. Therefore, our results suggest that acquisition of new chromosomal materials has not been of major importance in the dramatic change of life cycle that has accompanied the emergence of Y. pestis.

A horizontally acquired filamentous phage contributes to the pathogenicity of the plague bacillus.

Yersinia pestis, the plague bacillus, has an exceptional pathogenicity but the factors responsible for its extreme virulence are still unknown. A genome comparison with its less virulent ancestor Yersinia pseudotuberculosis identified a few Y. pestis-specific regions acquired after their divergence. One of them potentially encodes a prophage (YpfPhi), similar to filamentous phages associated with virulence in other pathogens. We show here that YpfPhi forms filamentous phage particles infectious for other Y. pestis isolates. Although it was previously suggested that YpfPhi is restricted to the Orientalis branch, our results indicate that it was acquired by the Y. pestis ancestor. In Antiqua and Medievalis strains, YpfPhi genome forms an unstable episome whereas in Orientalis isolates it is stably integrated as tandem repeats. Deletion of the YpfPhi genome does not affect Y. pestis ability to colonize and block the flea proventriculus, but results in an alteration of Y. pestis pathogenicity in mice. Our results show that transformation of Y. pestis from a classical enteropathogen to the highly virulent plague bacillus was accompanied by the acquisition of an unstable filamentous phage. Continued maintenance of YpfPhi despite its high in vitro instability suggests that it confers selective advantages to Y. pestis under natural conditions.

Evaluation of O-antigen inactivation on Pla activity and virulence of Yersinia pseudotuberculosis harbouring the pPla plasmid.

Yersinia pestis is a species that emerged recently from Yersinia pseudotuberculosis and gained an exceptional pathogenicity potential. Among the major genetic differences between the plague bacillus and its ancestor is the acquisition of the pPla plasmid, which has been associated with the increased virulence of Y. pestis. In a previous study, introduction of pPla into Y. pseudotuberculosis did not lead to any modification of the virulence of the host bacterium. However, it was subsequently demonstrated that the presence of smooth lipopolysaccharide (LPS) inhibits the activity of Pla. In this study, pPla was introduced into a Y. pseudotuberculosis strain expressing smooth LPS, and into a variant in which a mutation that abrogates the formation of O-antigen (O-Ag) repeats (as in natural isolates of Y. pestis) was generated. It was found that in both strains, Pla was synthesized, exported to the bacterial membrane and processed as in Y. pestis. However, the ability of Pla to activate plasminogen was weak and observed only at 37 degrees C in the smooth strain, while this activity was similar to that of Y. pestis and expressed at both 28 and 37 degrees C in the O-Ag mutant strain. Similarly, Pla-mediated inactivation of the antiprotease alpha2-antiplasmin was not detected in the smooth Y. pseudotuberculosis strain grown at 28 degrees C, but was expressed at both temperatures in the O-Ag mutant strain. Despite the more efficient activity of Pla, the Y. pseudotuberculosis O-Ag mutant strain exhibited a lower pathogenicity upon subcutaneous infection of mice. The results thus indicate that, although abrogation of O side chain synthesis in a Y. pseudotuberculosis strain harbouring pPla potentiates the two proteolytic activities of Pla, this is not sufficient to confer to Y. pseudotuberculosis a higher pathogenicity potential. These results also suggest that acquisition of pPla may not have been sufficient to confer an immediate higher pathogenic potential to the ancestor Y. pestis strain.

Identification of multiple genes encoding membrane proteins with undecaprenyl pyrophosphate phosphatase (UppP) activity in Escherichia coli.

The bacA gene product of Escherichia coli was recently purified to near homogeneity and identified as an undecaprenyl pyrophosphate phosphatase activity (El Ghachi, M., Bouhss, A., Blanot, D., and Mengin-Lecreulx, D. (2004) J. Biol. Chem. 279, 30106-30113). The enzyme function is to synthesize the carrier lipid undecaprenyl phosphate that is essential for the biosynthesis of peptidoglycan and other cell wall components. The inactivation of the chromosomal bacA gene was not lethal but led to a significant, but not total, depletion of undecaprenyl pyrophosphate phosphatase activity in E. coli membranes, suggesting that other(s) protein(s) should exist and account for the residual activity and viability of the mutant strain. Here we report that inactivation of two additional genes, ybjG and pgpB, is required to abolish growth of the bacA mutant strain. Overexpression of either of these genes, or of a fourth identified one, yeiU, is shown to result in bacitracin resistance and increased levels of undecaprenyl pyrophosphate phosphatase activity, as previously observed for bacA. A thermosensitive conditional triple mutant delta bacA,delta ybjG,delta pgpB in which the expression of bacA is impaired at 42 degrees C was constructed. This strain was shown to accumulate soluble peptidoglycan nucleotide precursors and to lyse when grown at the restrictive temperature, due to the depletion of the pool of undecaprenyl phosphate and consequent arrest of cell wall synthesis. This work provides evidence that two different classes of proteins exhibit undecaprenyl pyrophosphate phosphatase activity in E. coli and probably other bacterial species; they are the BacA enzyme and several members from a superfamily of phosphatases that, different from BacA, share in common a characteristic phosphatase sequence motif.

Role of the C-terminal lysine residues of streptococcal surface enolase in Glu- and Lys-plasminogen-binding activities of group A streptococci.

Streptococcal surface enolase (SEN) is a major plasminogen-binding protein of group A streptococci. Our earlier biochemical studies have suggested that the region responsible for this property is likely located at the C-terminal end of the SEN molecule. In the present study, the gene encoding SEN was cloned from group A streptococci M6 isolate D471. A series of mutations in the sen gene corresponding to the C-terminal region (428KSFYNLKK435) of the SEN molecule were created by either deleting one or more terminal lysine residues or replacing them with leucine. All purified recombinant SEN proteins with altered C-terminal ends were found to be enzymatically active and were analyzed for their Glu- and Lys-plasminogen-binding activities. Wild-type SEN bound to Lys-plasminogen with almost three times more affinity than to Glu-plasminogen. However, the recombinant mutant SEN proteins with a deletion of Lys434-435 or with K435L and K434-435L replacements showed a significant decrease in Glu- and Lys-plasminogen-binding activities. Accordingly, a streptococcal mutant expressing SEN-K434-435L showed a significant decrease in Glu- and Lys-plasminogen-binding activities. Biochemical and functional analyses of the isogenic mutant strain revealed a significant decrease in its abilities to cleave a chromogenic tripeptide substrate, acquire plasminogen from human plasma, and penetrate the extracellular matrix. Together, these data indicate that the last two C-terminal lysine residues of surface-exposed SEN contribute significantly to the plasminogen-binding activity of intact group A streptococci and hence to their ability to exploit host properties to their own advantage in tissue invasion.

A rapid and simple method for inactivating chromosomal genes in Yersinia.

A polymerase chain reaction (PCR)-based procedure without any cloning step was developed for a rapid mutagenesis/deletion of chromosomal target genes in Yersinia. For this purpose, a PCR fragment carrying an antibiotic resistance gene flanked by regions homologous to the target locus is electroporated into a recipient strain expressing the highly proficient homologous recombination system encoded by plasmid pKOBEG-sacB. Two PCR procedures were tested to generate an amplification product formed of an antibiotic resistance gene flanked by short (55 bp) or long (500 bp) homology extensions. Using this method, three chromosomal loci were successfully disrupted in Yersinia pseudotuberculosis. The use of this technique allows rapid and efficient large-scale mutagenesis of Yersinia target chromosomal genes.