Early systemic bacterial dissemination and a rapid innate immune response characterize genetic resistance to plague of SEG mice.

BACKGROUND: Although laboratory mice are usually highly susceptible to Yersinia pestis, we recently identified a mouse strain (SEG) that exhibited an exceptional capacity to resist bubonic plague and used it to identify immune mechanisms associated with resistance. METHODS: The kinetics of infection, circulating blood cells, granulopoiesis, lesions, and cellular populations in the spleen, and cytokine production in various tissues were compared in SEG and susceptible C57BL/6J mice after subcutaneous infection with the virulent Y. pestis CO92. RESULTS: Bacterial invasion occurred early (day 2) but was transient in SEG/Pas mice, whereas in C57BL/6J mice it was delayed but continuous until death. The bacterial load in all organs significantly correlated with the production of 5 cytokines (granulocyte colony-stimulating factor, keratinocyte-derived chemokine (KC), macrophage cationic peptide-1 (MCP-1), interleukin 1α, and interleukin 6) involved in monocyte and neutrophil recruitment. Indeed, higher proportions of these 2 cell types in blood and massive recruitment of F4/80(+)CD11b(-) macrophages in the spleen were observed in SEG/Pas mice at an early time point (day 2). Later times after infection (day 4) were characterized in C57BL/6J mice by destructive lesions of the spleen and impaired granulopoiesis. CONCLUSION: A fast and efficient Y. pestis dissemination in SEG mice may be critical for the triggering of an early and effective innate immune response necessary for surviving plague.

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.

The complete genome sequence of Yersinia pseudotuberculosis IP31758, the causative agent of Far East scarlet-like fever.

The first reported Far East scarlet-like fever (FESLF) epidemic swept the Pacific coastal region of Russia in the late 1950s. Symptoms of the severe infection included erythematous skin rash and desquamation, exanthema, hyperhemic tongue, and a toxic shock syndrome. The term FESLF was coined for the infection because it shares clinical presentations with scarlet fever caused by group A streptococci. The causative agent was later identified as Yersinia pseudotuberculosis, although the range of morbidities was vastly different from classical pseudotuberculosis symptoms. To understand the origin and emergence of the peculiar clinical features of FESLF, we have sequenced the genome of the FESLF-causing strain Y. pseudotuberculosis IP31758 and compared it with that of another Y. pseudotuberculosis strain, IP32953, which causes classical gastrointestinal symptoms. The unique gene pool of Y pseudotuberculosis IP31758 accounts for more than 260 strain-specific genes and introduces individual physiological capabilities and virulence determinants, with a significant proportion horizontally acquired that likely originated from Enterobacteriaceae and other soil-dwelling bacteria that persist in the same ecological niche. The mobile genome pool includes two novel plasmids phylogenetically unrelated to all currently reported Yersinia plasmids. An icm/dot type IVB secretion system, shared only with the intracellular persisting pathogens of the order Legionellales, was found on the larger plasmid and could contribute to scarlatinoid fever symptoms in patients due to the introduction of immunomodulatory and immunosuppressive capabilities. We determined the common and unique traits resulting from genome evolution and speciation within the genus Yersinia and drew a more accurate species border between Y. pseudotuberculosis and Y. pestis. In contrast to the lack of genetic diversity observed in the evolutionary young descending Y. pestis lineage, the population genetics of Y. pseudotuberculosis is more heterogenous. Both Y. pseudotuberculosis strains IP31758 and the previously sequenced Y. pseudotuberculosis strain IP32953 have evolved by the acquisition of specific plasmids and by the horizontal acquisition and incorporation of different genetic information into the chromosome, which all together or independently seems to potentially impact the phenotypic adaptation of these two strains.

Genetic structure and distribution of the colibactin genomic island among members of the family Enterobacteriaceae.

A genomic island encoding the biosynthesis and secretion pathway of putative hybrid nonribosomal peptide-polyketide colibactin has been recently described in Escherichia coli. Colibactin acts as a cyclomodulin and blocks the eukaryotic cell cycle. The origin and prevalence of the colibactin island among enterobacteria are unknown. We therefore screened 1,565 isolates of different genera and species related to the Enterobacteriaceae by PCR for the presence of this DNA element. The island was detected not only in E. coli but also in Klebsiella pneumoniae, Enterobacter aerogenes, and Citrobacter koseri isolates. It was highly conserved among these species and was always associated with the yersiniabactin determinant. Structural variations between individual strains were only observed in an intergenic region containing variable numbers of tandem repeats. In E. coli, the colibactin island was usually restricted to isolates of phylogenetic group B2 and inserted at the asnW tRNA locus. Interestingly, in K. pneumoniae, E. aerogenes, C. koseri, and three E. coli strains of phylogenetic group B1, the functional colibactin determinant was associated with a genetic element similar to the integrative and conjugative elements ICEEc1 and ICEKp1 and to several enterobacterial plasmids. Different asn tRNA genes served as chromosomal insertion sites of the ICE-associated colibactin determinant: asnU in the three E. coli strains of ECOR group B1, and different asn tRNA loci in K. pneumoniae. The detection of the colibactin genes associated with an ICE-like element in several enterobacteria provides new insights into the spread of this gene cluster and its putative mode of transfer. Our results shed light on the mechanisms of genetic exchange between members of the family Enterobacteriaceae.

Characterization of chromosomal regions conserved in Yersinia pseudotuberculosis and lost by Yersinia pestis.

The transformation of the enteropathogenic bacterium Yersinia pseudotuberculosis into the plague bacillus, Yersinia pestis, has been accompanied by extensive genetic loss. This study focused on chromosomal regions conserved in Y. pseudotuberculosis and lost during its transformation into Y. pestis. An extensive PCR screening of 78 strains of the two species identified five regions (R1 to R5) and four open reading frames (ORFs; orf1 to orf4) that were conserved in Y. pseudotuberculosis and absent from Y. pestis. Their conservation in Y. pseudotuberculosis suggests a positive selective pressure and a role during the life cycle of this species. Attempts to delete two ORFs (orf3 and orf4) from the chromosome of strain IP32953 were unsuccessful, indicating that they are essential for its viability. The seven remaining loci were individually deleted from the IP32953 chromosome, and the ability of each mutant to grow in vitro and to kill mice upon intragastric infection was evaluated. Four loci (orf1, R2, R4, and R5) were not required for optimal growth or virulence of Y. pseudotuberculosis. In contrast, orf2, encoding a putative pseudouridylate synthase involved in RNA stability, was necessary for the optimal growth of IP32953 at 37 degrees C in a chemically defined medium (M63S). Deletion of R1, a region predicted to encode the methionine salvage pathway, altered the mutant pathogenicity, suggesting that the availability of free methionine is severely restricted in vivo. R3, a region composed mostly of genes of unknown functions, was necessary for both optimal growth of Y. pseudotuberculosis at 37 degrees C in M63S and for virulence. Therefore, despite their loss in Y. pestis, five of the nine Y. pseudotuberculosis-specific chromosomal loci studied play a role in the survival, growth, or virulence of this species.

Plague reappearance in Algeria after 50 years, 2003.

An outbreak of plague occurred in the region of Oran, Algeria, from June to July 2003. Algeria had not reported this disease for >50 years. Eighteen bubonic cases were identified, and Yersinia pestis was isolated from 6 patients. Except for the index case-patient, all patients recovered. Targeted chemoprophylaxis, sanitation, and vector control played a crucial role in controlling the outbreak. Epidemiologic and biomolecular findings strongly suggested the existence of a local animal reservoir during this period, but its origin (resurgence or re-importation) could not be determined. This sudden and unexpected reemergence of plague, close to an important commercial seaport, is a textbook illustration of a public health event of international importance. It also demonstrates that the danger of plague reoccurrence is not limited to the currently indexed natural foci.

Long-Term Effects of Continuous Subcutaneous Insulin Infusion on Glucose Control and Microvascular Complications in Patients With Type 1 Diabetes.

BACKGROUND: We investigated the long-term effects of continuous subcutaneous insulin infusion (CSII) on glucose control and microvascular complications in patients with type 1 diabetes (T1D). METHODS: A total of 157 patients (59 M/98 W; age 39.1 ± 14.8 years) with T1D who switched from multiple daily injections to CSII and used CSII for at least one year were included. HbA1c levels and status of microvascular complications before and while under CSII were analyzed, retrospectively. RESULTS: The follow-up period was 4.0 ± 1.5 years. HbA1c significantly decreased from 8.4 ± 1.3 to 7.7 ± 1.3% (68 ± 14 to 61 ± 14 mmol/mol) after 1-year CSII and remained lower than pre-CSII levels during four years. Patients with pre-CSII HbA1c >8.0% (64 mmol/mol) showed significant improvement of HbA1c for four years, while those with pre-CSII HbA1c <8.0% showed no significant change. The prevalence of retinopathy, albuminuria, and chronic kidney disease (CKD) were respectively 39%, 12%, and 9% at CSII initiation. During follow-up, the incidence of retinopathy, albuminuria, and CKD were 3.6, 2.5 and 1.4/100 patient-years. Onset or progression of retinopathy occurred in 16 (27.1%) patients with diabetes duration >15 years, and in three (4.3%) patients with diabetes duration <15 years ( P < .01). CONCLUSION: CSII was effective in improving HbA1c for up to four years, specifically in patients with HbA1c >8% (64 mmol/mol) prior to CSII. Incidence and progression rates of retinopathy and albuminuria were low, particularly in patients with a diabetes duration <15 years at CSII initiation. These results argue for not delaying a proposal of CSII initiation in T1D with sustained HbA1c >8% (64 mmol/mol).

A putative DNA adenine methyltransferase is involved in Yersinia pseudotuberculosis pathogenicity.

Some adenine methyltransferases have been shown not only to protect specific DNA restriction sites from cleavage by a restriction endonuclease, but also to play a role in various bacterial processes and sometimes in bacterial virulence. This study focused on a type I restriction-modification system (designated yrmI) of Y. pseudotuberculosis. This system is composed of three adjacent genes which could potentially encode an N6-adenine DNA methylase (YamA), an enzyme involved in site-specific recognition (YrsA) and a restriction endonuclease (YreA). Screening of 85 isolates of Y. pestis and Y. pseudotuberculosis indicated that the yrmI system has been lost by Y. pestis and that yamA (but not yrsA or yreA) is present in all Y. pseudotuberculosis strains tested, suggesting that it may be important at some stages of the epidemiological cycle of this species. To further investigate the role of yamA in Y. pseudotuberculosis survival, multiplication or virulence, a DeltayamA mutant of Y. pseudotuberculosis IP32953 was constructed by allelic exchange with a kanamycin cassette. The fact that DeltayamA mutants were obtained indicated that this gene is not essential for Y. pseudotuberculosis viability. The IP32953DeltayamA mutant strain grew as well as the wild-type in a rich medium at both 28 degrees C and 37 degrees C. It also grew normally in a chemically defined medium at 28 degrees C, but exhibited a growth defect at 37 degrees C. In contrast to the Dam adenine methyltransferase, a mutation in yamA did not impair the functions of DNA repair or resistance to detergents. However, the DeltayamA mutant exhibited a virulence defect in a mouse model of intragastric infection. The in silico analysis indicated that the chromosomal region carrying the Y. pseudotuberculosis yrmI locus has been replaced in Y. pestis by a horizontally acquired region which potentially encodes another methyltransferase. YamA might thus be dispensable for Y. pestis growth and virulence because this species has acquired another gene fulfilling the same functions.

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.