Draft Genome Sequences of Yersinia pestis Isolates from Natural Foci of Endemic Plague in China

To gain insights into the evolutionary origin, emergence, and pathogenicity of the etiologic agent of plague, we have sequenced the genomes of four Yersinia pestis strains isolated from the zoonotic rodent reservoir in foci of endemic plague in China. These resources enable in-depth studies of Y. pestis sequence variations and detailed whole-genome comparisons of very closely related genomes from the supposed site of the origin and the emergence of global pandemics of plague.Here we report on the genomes of Yersinia pestis strains B42003004, K1973002, E1979001, and F1991016, which represent a sample of the genetic diversity found in four foci of endemic plague in China (24). Y. pestis bv. orientalis strain F1991016 was isolated in 1991 from Cangyuan County, China, from a rat (Rattus flavipectus), and Y. pestis bv. antiqua strain E1979001 was isolated in 1979 from Jianchuan, China, from a vole (Eothenomys miletus). Both Y. pestis strains K1973002 and B42003004 of biovars medievalis and antiqua, respectively, originate from marmota species (Marmota himalayana Hetian 1973; Marmota baibacina Wenquan 2003) (24). Genome analyses of these key isolates outline the details of microevolution of the plague bacterium, as these isolates represent important evolutionary milestones of the species, which is thought to have originated in Central Asia as a clonal descendant of Yersinia pseudotuberculosis (1). Genomic DNA was subjected to whole-genome shotgun sequencing and closure strategies as previously described (15). Plasmid (pHOS2) and fosmid (pCC1fos) libraries were constructed, with insert sizes of 4 to 6 kb and 30 to 40 kb, respectively. An average of 67,000 high-quality Sanger reads (total, 268,160) was obtained with an 860-bp average read length. The genomes with an average 12-fold read coverage depth were assembled using a Celera Assembler (11) and manually annotated using Manatee (http://manatee.sourceforge.net/). Genomic architectures were compared using Mauve (5, 18), and proteomes were analyzed with the BLAST score ratio tool (17).The young evolutionary history of the species and resulting homogenous population structure is reflected in a high degree of proteome conservation between the sequenced isolates and the modern strain CO92 (16). Y. pestis pathogenicity is anchored in its mobile inventory, and typically, isolates harbor three virulence plasmids, the species-specific plasminogen activator and murine toxin plasmids and the low-calcium-response plasmid pCD (23). Their pCD-borne lcrV antigen shows a genetic makeup identical to that of CO92 (2, 16). The insertion sequence element expansion clearly distinguishes these Central Asian isolates from the progenitor Y. pseudotuberculosis (3, 8). Comprehensive analyses reveal a lack of genome-wide synteny and suggest massive intrachromosomal rearrangements, a characteristic feature of Y. pestis genome evolution (6, 8). Besides insertion sequence element abundance, we observed isolate-specific propagation patterns that not only shaped the reorganization of the genomic architecture but also are known to drive microevolutionary adaptation in Y. pestis (4, 9, 14, 21, 24). Based upon the phenotypic and genotypic features that differentiate these isolates (13, 20, 24), B42003004 belongs to the most ancient Y. pestis lineage known to exist in China; hence, it is phylogenetically thought to be closest to the species progenitor Y. pseudotuberculosis (22). We studied metabolic genes that determine their biovar classification and investigated the underlying genetic determinants (24). Isolate K1973002 is defective in the nitrate reductase napA gene, similar to strain KIM (7), and represents the results of the evolutionary processes implicated in the biovar conversion from antiqua to medievalis. Isolate F1991016 carries an in-frame deletion in the glycerol-3-phosphate dehydrogenase glpD gene (19), similar to strain CO92 (16), and characteristic of the antiqua-to-orientalis conversion. The observed genetic traits strengthen the hypothesis that biovars medievalis and orientalis arose through parallel evolution from a glycerol- and nitrate-positive antiqua progenitor due to the acquisition of independent mutations (1, 10, 14). Variable-number tandem-nucleotide-repeat alleles (12) (allele K, K1973002; allele K, B42003004; allele P, E1979001; allele G, F1991016) are not biovar specific and are not discriminative enough to differentiate these isolates, which clearly supports a population-based phylogeny, as introduced by Achtman et al. (1).The whole-genome draft sequences of these evolutionary key isolates of Y. pestis will facilitate additional bioinformatic and phylogenetic analyses. The availability of high-quality Sanger sequences is crucial to resolve the genetically homogenous population structure and to shed light on Y. pestis speciation. Understanding the plasticity and genome dynamics further aids in forensic and epidemiological analyses by setting up the basis for an accurate and robust typing system for plague surveillance and promotes diagnostics development and control measures.     

The Single Substitution I259T,Conserved in the Plasminogen Activator Pla of Pandemic Yersinia pestis Branches,Enhances Fibrinolytic Activity

The outer membrane plasminogen activator Pla of Yersinia pestis is a central virulence factor in plague. The primary structure of the Pla β-barrel is conserved in Y. pestis biovars Antiqua, Medievalis, and Orientalis, which are associated with pandemics of plague. The Pla molecule of the ancestral Y. pestis lineages Microtus and Angola carries the single amino acid change T259I located in surface loop 5 of the β-barrel. Recombinant Y. pestis KIM D34 or Escherichia coli XL1 expressing Pla T259I was impaired in fibrinolysis and in plasminogen activation. Lack of detectable generation of the catalytic light chain of plasmin and inactivation of plasmin enzymatic activity by the Pla T259I construct indicated that Microtus Pla cleaved the plasminogen molecule more unspecifically than did common Pla. The isoform pattern of the Pla T259I molecule was different from that of the common Pla molecule. Microtus Pla was more efficient than wild-type Pla in α₂-antiplasmin inactivation. Pla of Y. pestis and PgtE of Salmonella enterica have evolved from the same omptin ancestor, and their comparison showed that PgtE was poor in plasminogen activation but exhibited efficient antiprotease inactivation. The substitution ²⁵⁹IIDKT/TIDKN in PgtE, constructed to mimic the L5 region in Pla, altered proteolysis in favor of plasmin formation, whereas the reverse substitution ²⁵⁹TIDKN/IIDKT in Pla altered proteolysis in favor of α₂-antiplasmin inactivation. The results suggest that Microtus Pla represents an ancestral form of Pla that has evolved into a more efficient plasminogen activator in the pandemic Y. pestis lineages.Since the year 540, plague has killed some 200 million humans in three pandemics, i.e., the Justinian plague, the Black Death, and the modern plague (36). Genomic studies have estimated that the etiological agent, Yersinia pestis, evolved from the oral-fecal pathogen Yersinia pseudotuberculosis serotype O1b only shortly before the first pandemic, i.e., 5,000 to 20,000 years ago (1, 2, 46), which has made the bacterium a paradigm of the rapid evolution of a severe bacterial pathogen (57). At least four biovars of Y. pestis have been identified through metabolic and genomic studies; of these biovars, Antiqua, Medievalis, and Orientalis may be associated with the three plague pandemics, whereas the fourth biovar, Microtus, is associated with human-attenuated Y. pestis strains from two geographically distant infection foci in China (36, 59-61). A recent molecular analysis indicated that the biovars are not monophyletic and proposed the subdivision of Y. pestis into eight molecular groupings, which represent different evolutionary branches and histories and are only partially compatible with the biovars (1). Y. pestis evolved from Y. pseudotuberculosis along branch 0, which consists of “atypical” Y. pestis strains designated Angola, Microtus, and Pestoides; these are phylogenetically ancestral to the Antiqua, Medievalis, and Orientalis branches (1).As a disease, plague exhibits various pathologies. Bubonic plague is the zoonotic form of the disease, which is usually acquired by humans from the bite of a flea that has been infected through a blood meal on a diseased rodent (36). The bacteria invade at the intradermal flea bite site and migrate to lymphatic vessels and then to regional draining lymph nodes, where they multiply and cause the development of buboes (44). Without early treatment, bubonic plague progresses to life-threatening septicemic plague, and hematogenous spread of the bacterium to lungs leads to pneumonic plague, a rapidly fatal and highly contagious airborne disease. Occasional injection of Y. pestis cells by the flea directly into the circulatory system leads to primary septicemic plague (43).The plasminogen activator Pla is a cell surface protease encoded by the Y. pestis-specific plasmid pPCP1 (10, 48). Pla is essential in the pathogenesis of bubonic (43, 49) and pneumonic plague (28), whereas it has less of a role in primary septicemic plague (43, 49). The pla gene is highly transcribed in buboes of Y. pestis-infected mice (45), and Pla specifically potentiates migration of the bacteria to lymphatic tissue (43). Pla seems to have a different role in pneumonic plague, where it allows Y. pestis to replicate rapidly in the lungs, causing lethal fulminant pneumonia (28). Virulent Y. pestis strains lacking the Pla-encoding plasmid pPCP1 have been isolated in Asia (3), and they can be associated with primary septicemic plague (43).Pla is an aspartic protease (22, 55) that activates human plasminogen (Plg) to the serine protease plasmin (47) and inactivates the plasmin inhibitor α₂-antiplasmin (α₂AP), thus affecting the main control system for plasmin activity (22). Plg is an abundant circulating zymogen, and its activation is central in the pathogenesis of plague (13, 28, 43), and plasmin is a powerful serine protease associated with cell migration and degradation of fibrin clots (29, 32, 37). In accordance with this, Pla-mediated bacterial adherence directs uncontrolled plasmin proteolysis onto basement membranes to enhance bacterial metastasis through tissue barriers (25, 27), and fibrinolysis by Pla-generated plasmin activity plays a role in the pathogenesis of bubonic plague (8).Compared to those of other Y. pestis biovars, Microtus isolates have several unique genomic features that may be involved in their inherent inability to attack the human host, and specific losses of genes or gene functions are thought to be responsible for the human attenuation (59). Interestingly, the attenuation does not apply to the murine host. The predicted amino acid sequence of the Pla polypeptide is remarkably conserved: in the branches Antiqua, Medievalis, and Orientalis, the Pla sequences are completely identical, whereas a single amino acid substitution, T259I, has been detected in atypical Angola and Microtus strains (6, 38, 50). A genetic analysis of 260 isolates of Y. pestis showed that the T259I substitution in Pla is shared by all isolates of biovar Microtus but absent in those of other biovars (59). Many of the Pestoides strains lack the pPCP1 plasmid and hence also the pla gene (12), and pla sequences from Pestoides are not available.Pla is a member of the omptin family of conserved outer membrane proteases/adhesins detected in several gram-negative bacterial pathogens (15, 17, 21). The omptins have the same molecular size, a β-barrel fold of 10 transmembrane β strands, and five surface-exposed loops, L1 to L5 (Fig. ​(Fig.1).1). The catalytic residues and the residues interacting with lipid A in the outer membrane are completely conserved (17, 21-23, 41, 55). The omptins cleave peptide substrates at basic residues (17) but show dramatic heterogeneity in the recognition of biologically important polypeptides, such as Plg, the antiprotease α₂AP, gelatin, and progelatinases. Analyses of hybrid proteins created between Pla and the omptins PgtE of Salmonella enterica and OmpT of Escherichia coli have indicated that the differing polypeptide substrate selectivity of omptins is dictated by sequence variation in the mobile loop structures of the β-barrel (22, 40). Residue T259 in Pla is located at surface loop 5 and oriented inward in the active-site groove of the Pla barrel, close to residue K262, where Pla is autoprocessed (22, 23) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Model of Pla structure (23) and location of residue Thr259. Side (top drawing) and top (bottom drawing) views of the transmembrane β-barrel are shown. L1 to L5 are the surface loops. Catalytic residues Asp84, Asp86, Asp206, and His208 are indicated in green, Thr259 is in red, and the autoprocessing site Lys262 is in yellow. OM is the outer membrane. (C) Amino acid sequence of residues 254 to 273 at L5 and the termini of β-strands 9 and 10 in Pla, Microtus Pla, and PgtE are shown.The omptin β-barrel has spread by horizontal gene transfer in gram-negative bacteria and adapted to the life-styles of host bacteria (15, 17, 21, 22, 40). Overall, the omptins give an example of an evolvable, robust enzyme fold (34) that easily acquires novel or improved functions. The fact that the single substitution T259I associates with ancestral Y. pestis Microtus and Angola populations suggests that Microtus Pla represents a form of the protein that preceded the common Pla protein. The central role of Plg activation in the pathogenesis of plague led us to analyze whether the single substitution T259I affects the fibrinolytic activities of the Pla molecule.     

Complete Genome Sequences of Yersinia pestis from Natural Foci in China

Yersinia pestis, the causative agent of plague, is a deadly bacterium that affects humans. Strain D106004 was isolated from a new plague focus in Yulong County, China, in 2006. To gain insights into the epidemic origin, we have sequenced the genomes of D106004 and strains Z176003 and D182038, isolated from neighboring regions.This article describes genomic comparisons between three respective Yersinia pestis strains isolated from new natural plague foci in China. Y. pestis strain D106004 was isolated from Apodemus chevrieri in Yulong County in 2006, and its genome was compared to those of strain D182038 (isolated from A. chevrieri in 1982 from Jianchuan County) and strain Z176003 (isolated from Marmota himalayana in 1976 in Naqu [Tibet] County).Between 25 October 2005 and 2 November 2005, there was an outbreak of pneumonic plague in Yulong, which was identified as a new natural plague focus (13). The primary Y. pestis reservoirs associated with this outbreak were A. chevrieri, Eothenmys miletus, and Apodemus latronum, and the primary vectors associated with plague transmission were also identified as similar to what was observed in neighboring Jianchuan County (7). However, the Y. pestis strain identified metabolized maltose significantly differently than the previously described strains (6).Whole-genome shotgun and solexa methods were used, as previously described (3), to compare the Y. pestis D106004, D182038, and Z176003 sequences, which consisted of 475, 385, and 413 contigs, respectively, resulting in an average 9-fold coverage across the genomes. All isolates examined possessed a single circular chromosome with the three virulence plasmids (pMT, pCD, and pPCP) associated with classical Y. pestis strains. Automated gene modeling was carried out using the Glimmer3 software program (11) in addition to comparing the respective gene products using the Nt, Nr, KEGG, Swissprot, and COG databases using the basic local alignment search tool for proteins (BLASTP). Open reading frames (ORFs) in the respective 4,626,944-bp, 4,640,720-bp, and 4,553,586-bp genomes of strains D182038, D106004, and Z176003 were predicted to be of 3,642, 3,636, 3,543, and more than 300 bp in length. Strains D182038, D106004, and Z176003 each had six rRNA (16S-23S-5S) genes and 73 (D182038), 70 (D106004), or 68 (Z176003) tRNA genes predicted by the tRNAScan-SE server (9).Comparison of Y. pestis strains 91001 and KIM to Y. pestis strain CO92 identified genetic rearrangements (5, 10, 12) resulting from insertion sequences (2), and pulsed-field gel electrophoresis (PFGE) profile comparisons between D182038 and D106004 suggested that genomic variability of the Y. pestis strains from different foci was caused by genome rearrangement (16). According to our analyses, the Y. pestis strains isolated from the two foci have very different syntenic structures due to rearrangement, but they share high similarity between plates (8). In addition, a unique multiple-locus variable-number tandem repeat analysis (MLVA) type was defined for the strains isolated from Yulong, indicating a new clonal group. These results also suggested that the Yulong strains were closely related to the strains from the Qinghai-Tibet Plateau plague foci (15). Analysis of Y. pestis microevolution has been made possible by comparing single- nucleotide polymorphism (SNP) profiles as previously described (1, 4, 14).The availability of high-quality sequences is crucial in order to resolve the origins of the new strains isolated from natural plague foci.     

The Omptins of Yersinia pestis and Salmonella enterica Cleave the Reactive Center Loop of Plasminogen Activator Inhibitor 1

Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule that regulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important human pathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhimurium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavage of PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved, or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteases plasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family of transmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected with the omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptin subfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E. coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novel serpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypass normal control of host proteolysis.Plasminogen activator inhibitor 1 (PAI-1) is a key regulator of the mammalian fibrinolytic/plasminogen system (29, 37). The fibrinolytic system comprises the serine protease zymogen plasminogen, urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), PAI-1, and plasmin inhibitor α₂-antiplasmin (α₂AP) (for a review, see reference 52). Plasminogen is converted to plasmin, which is a broad-spectrum serine protease that dissolves fibrin in blood clots, degrades laminin of basement membranes, and activates matrix metalloproteinases that degrade collagens and gelatins in tissue barriers. Herewith, plasmin controls physiological processes such as fibrinolysis/coagulation, cell migration and invasion, and tumor metastasis (29, 37). PAI-1 maintains normal hemostasis by inhibiting the function of the plasminogen activators tPA and uPA, which are serine proteases and highly specific for cleavage of the plasminogen molecule. tPA binds to fibrin and is associated with plasmin-mediated breakdown of fibrin clots, whereas uPA has low affinity for fibrin and associates with cell surface proteolysis, cellular migration, and damage of tissue barriers (52).The mammalian fibrinolytic and coagulation systems are targeted by invasive bacterial pathogens during infection (reviewed in references 6, 11, 34, and 61). In bacterial sepsis, increased production of fibrin clots at a damaged endothelium results from enhanced thrombin-catalyzed fibrin generation and from an increased serum level of PAI-1. Coagulation can protect the host by activating immune systems or by physically restraining the bacteria (6, 15, 25, 41). On the other hand, several invasive bacterial pathogens enhance fibrinolysis either through direct plasminogen activation or by immobilizing plasminogen/plasmin on the surface (6, 34, 61). Activation of the plasminogen system by bacteria enhances bacterial dissemination and invasiveness through release of bacteria from fibrin deposits and through degradation of tissue barriers. Bacterial plasminogen activators and receptors have been under extensive structural and functional studies, but much less is known about interactions of bacteria with the regulatory proteins of fibrinolysis.PAI-1 is present in a large variety of tissues and is secreted by several human cells (37). In healthy individuals, the level of PAI-1 antigen in human plasma is low (6 to 85 ng/ml), but synthesis and secretion of PAI-1 are strongly elevated in disease states and induced by, e.g., inflammatory cytokines and endotoxin of Gram-negative bacteria (37). PAI-1 is a serine protease inhibitor (serpin), which exists in two forms. In its active form, PAI-1 rapidly inactivates both tPA and uPA by forming a covalent bond between the hydroxyl group of a catalytic serine residue of tPA/uPA and the carboxyl group of the residue R346 at the reactive center loop (RCL) of PAI-1 (52). The RCL of PAI-1 is a 19-amino-acid-long flexible loop which inserts into the catalytic center of tPA/uPA and contains the “bait” residues R346 and M347, which mimic the normal target of tPA/uPA. PAI-1 induces distortion of the active site of tPA/uPA, which prevents completion of the catalytic cycle (70). The active form of PAI-1 is unstable, with a half-life of 2 to 3 h at 37°C, and it changes spontaneously and irreversibly into a latent form, where the RCL is incorporated into a central β-sheet of the PAI-1 molecule and therefore cannot react with tPA or uPA. This conformational change takes place also after proteolytic cleavage of PAI-1 at the R346-M347 bond. The active form of PAI-1 binds with high affinity to vitronectin (Vn), and PAI-1/Vn complex formation increases the half-life of PAI-1 2- to 4-fold (10, 46, 69). Most circulating PAI-1 is thought to be in a complex with Vn, and the complex serves as the reservoir of physiologically active PAI-1 (44).Plague disease caused by Yersinia pestis is associated with imbalance of the fibrinolytic system, and decreased fibrin(ogen) deposition has been observed in both bubonic and pneumonic plague (11, 36). The plasminogen activator Pla, which is encoded by a Y. pestis-specific 9.5-kb virulence plasmid, pPCP1 (59), does not degrade fibrin directly but mimics the action of tPA and uPA in converting plasminogen to plasmin by cleavage at R561-V562. Pla also degrades the serpin α₂AP and thus creates uncontrolled plasmin activity (32, 60). Pla belongs to the omptin superfamily of bacterial β-barrel outer membrane proteases (for reviews of omptins, see references 21 and 23). The omptins share molecular size and transmembrane fold but differ markedly in their substrate selectivities. In their catalytic centers, omptins combine structural features of aspartic and serine proteases (66).Increased fibrinolysis observed in plague led us to investigate whether Y. pestis increases plasminogen activation also indirectly by controlling the activity of PAI-1. We compared Y. pestis to Salmonella enterica serovar Typhimurium and Yersinia pseudotuberculosis, and the study also included omptins of other enterobacterial species.     

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.In animals, RNA interference (RNAi) was first described for Caenorhabditis elegans (27). The production or introduction of double-stranded RNA (dsRNA) in cells leads to the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of mRNAs. Central to RNAi is the production of 21- to 26-nucleotide small interfering RNAs (siRNAs) from dsRNA and the assembly of an RNA-induced silencing complex (RISC), followed by the degradation of the target mRNA (23, 84). RNAi is a known antiviral strategy of plants (3, 53) and insects (21, 39, 51). Study of Drosophila melanogaster in particular has given important insights into RNAi responses against pathogenic viruses and viral RNAi inhibitors (31, 54, 83, 86, 91). RNAi is well characterized for Drosophila, and orthologs of antiviral RNAi genes have been found in Aedes and Culex spp. (13, 63).Arboviruses, or arthropod-borne viruses, are RNA viruses mainly of the families Bunyaviridae, Flaviviridae, and Togaviridae. The genus Alphavirus within the family Togaviridae contains several mosquito-borne pathogens: arboviruses such as Chikungunya virus (16) and equine encephalitis viruses (88). Replication of the prototype Sindbis virus and Semliki Forest virus (SFV) is well understood (44, 71, 74, 79). Their genome consists of a positive-stranded RNA with a 5′ cap and a 3′ poly(A) tail. The 5′ two-thirds encodes the nonstructural polyprotein P1234, which is cleaved into four replicase proteins, nsP1 to nsP4 (47, 58, 60). The structural polyprotein is encoded in the 3′ one-third of the genome and cleaved into capsid and glycoproteins after translation from a subgenomic mRNA (79). Cytoplasmic replication complexes are associated with cellular membranes (71). Viruses mature by budding at the plasma membrane (35).In nature, arboviruses are spread by arthropod vectors (predominantly mosquitoes, ticks, flies, and midges) to vertebrate hosts (87). Little is known about how arthropod cells react to arbovirus infection. In mosquito cell cultures, an acute phase with efficient virus production is generally followed by the establishment of a persistent infection with low levels of virus production (9). This is fundamentally different from the cytolytic events following arbovirus interactions with mammalian cells and pathogenic insect viruses with insect cells. Alphaviruses encode host response antagonists for mammalian cells (2, 7, 34, 38).RNAi has been described for mosquitoes (56) and, when induced before infection, antagonizes arboviruses and their replicons (1, 4, 14, 15, 29, 30, 32, 42, 64, 65). RNAi is also functional in various mosquito cell lines (1, 8, 43, 49, 52). In the absence of RNAi, alphavirus and flavivirus replication and/or dissemination is enhanced in both mosquitoes and Drosophila (14, 17, 31, 45, 72). RNAi inhibitors weakly enhance SFV replicon replication in tick and mosquito cells (5, 33), posing the questions of how, when, and where RNAi interferes with alphavirus infection in mosquito cells.Here we use an A. albopictus-derived mosquito cell line to study RNAi responses to SFV. Using reporter-based assays, we demonstrate that SFV cannot avoid or efficiently inhibit the establishment of an RNAi response. We also demonstrate that the RNAi signal can spread between mosquito cells. SFV cannot inhibit cell-to-cell spread of the RNAi signal, and spread of the virus-induced RNAi signal (dsRNA/siRNA) can inhibit the replication of incoming SFV in neighboring cells. Furthermore, we show that SFV expression of a siRNA-binding protein increases levels of virus replication mainly by enhancing virus spread between cells rather than replication in initially infected cells. Taken together, these findings suggest a novel mechanism, cell-to-cell spread of antiviral dsRNA/siRNA, by which RNAi limits SFV dissemination in mosquito cells.     

Extensive Antibody Cross-reactivity among Infectious Gram-negative Bacteria Revealed by Proteome Microarray Analysis

Antibodies provide a sensitive indicator of proteins displayed by bacteria during sepsis. Because signals produced by infection are naturally amplified during the antibody response, host immunity can be used to identify biomarkers for proteins that are present at levels currently below detectable limits. We developed a microarray comprising ∼70% of the 4066 proteins contained within the Yersinia pestis proteome to identify antibody biomarkers distinguishing plague from infections caused by other bacterial pathogens that may initially present similar clinical symptoms. We first examined rabbit antibodies produced against proteomes extracted from Y. pestis, Burkholderia mallei, Burkholderia cepecia, Burkholderia pseudomallei, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella flexneri, and Escherichia coli, all pathogenic Gram-negative bacteria. These antibodies enabled detection of shared cross-reactive proteins, fingerprint proteins common for two or more bacteria, and signature proteins specific to each pathogen. Recognition by rabbit and non-human primate antibodies involved less than 100 of the thousands of proteins present within the Y. pestis proteome. Further antigen binding patterns were revealed that could distinguish plague from anthrax, caused by the Gram-positive bacterium Bacillus anthracis, using sera from acutely infected or convalescent primates. Thus, our results demonstrate potential biomarkers that are either specific to one strain or common to several species of pathogenic bacteria.Plague is a disease of historical epidemics that remains an important public health problem in limited areas of the world (1). Disease transmission usually occurs through transfer of the bacillus Yersinia pestis by the bite of a flea. However, less frequent direct transfer of viable bacteria by respiratory droplets may result in primary pneumonic infection. A transient intracellular infection of phagocytic cells (2) occurs during the earliest stage of bubonic plague followed by rapid extracellular expansion of bacteria in lymph nodes. The prototypical lymphatic infection of bubonic plague may also progress to bacteremic or pneumonic infection with a very high rate of fatality if there is not rapid intervention by antibiotic treatment (3). Among the reported cases occurring annually in the United States, 15% were fatal in 2006 (4). Although only small numbers of human cases occur each year in North America, a more substantial incidence of plague is found in wild animal populations (5) with seroprevalence rates of up to 100% among mammalian carnivores in endemic areas (6). The geographic range of infection within feral populations is presently unknown but may contribute significantly to the reservoir of potential disease transmission to humans.Diagnostic tests and prophylactic vaccines or therapies must rapidly distinguish or protect against the many infectious diseases that present similar initial symptoms. Specific diagnostic tests and vaccines for plague are public health priorities primarily because of the threat from potential acts of terrorism. Because human deaths may occur within 48 h of infection (7), delays in proper diagnosis have led to disease complications and fatalities from plague (8). Yet the identification of bacterial sepsis at the earliest stage of clinical presentation is challenging because of the generalized nature of disease symptoms and the difficulty in culturing infectious agents or isolating sufficient material to identify the infectious agent by amplification of genetic markers. Although host antibody responses provide a sensitive indicator of current or past infection, insufficient numbers of validated biomarkers are available, and extensive antibody cross-reactivity among Gram-negative pathogens (9–12) complicates the direct analysis of serum.Identification of plague-specific antibody interactions is a daunting task because of the complexity of the bacterial proteome encountered by the host during infection. The chromosome of Y. pestis CO92 encodes ∼3885 proteins, whereas an additional 181 are episomally expressed by pCD1, pMT1, and pPCP1. For comparison, the proteome of Y. pestis KIM¹ contains 4202 individual proteins (13), 87% in common with CO92 (14), and the closely related enteric pathogen Yersinia pseudotuberculosis (15, 16) contains ∼4038 proteins (chromosome plus plasmids). Recent technical advances have facilitated the development of microarrays comprising full-length, functional proteins that represent nearly complete proteomes. For example, Zhu et al. (17) reported the development of a proteome microarray containing the full-length, purified expression products of over 93% of the 6280 protein-coding genes of the yeast Saccharomyces cerevisiae, and Schmid et al. (18) described the human antibody repertoire for vaccinia virus recognition by using a viral proteome microarray. This approach opens the possibility of examining the entire bacterial proteome to elucidate proteins or protein pathways that are essential to pathogenicity or host immunity. We sought to identify biomarkers that could distinguish plague from diseases caused by other bacterial pathogens by measuring host antibody recognition of individual proteins contained within the Y. pestis proteome. The previously reported genomic sequences of Y. pestis strains KIM (13) and CO92 (14), sharing 95% identity, were used for reference. Approximately 77% of the putative Y. pestis proteome can be classified by known homologies. We successfully expressed and purified the majority (70%) of the 4066 ORFs encoded by the chromosome and plasmids of Y. pestis KIM and arrayed these products onto glass slides coated with nitrocellulose. The Y. pestis ORFs subcloned into expression vectors were fully sequenced to confirm quality and identity before use. Different approaches for studying the antibody repertoire for plague in rabbits and non-human primates were compared. Based on results from experiments using the Y. pestis proteome microarray, we identified new candidates for antibody biomarkers of bacterial infections and patterns of cross-reactivity that may be useful diagnostic tools.     

Contribution of Lipoproteins and Lipoprotein Processing to Endocarditis Virulence in Streptococcus sanguinis

Streptococcus sanguinis is an important cause of infective endocarditis. Previous studies have identified lipoproteins as virulence determinants in other streptococcal species. Using a bioinformatic approach, we identified 52 putative lipoprotein genes in S. sanguinis strain SK36 as well as genes encoding the lipoprotein-processing enzymes prolipoprotein diacylglyceryl transferase (lgt) and signal peptidase II (lspA). We employed a directed signature-tagged mutagenesis approach to systematically disrupt these genes and screen each mutant for the loss of virulence in an animal model of endocarditis. All mutants were viable. In competitive index assays, mutation of a putative phosphate transporter reduced in vivo competitiveness by 14-fold but also reduced in vitro viability by more than 20-fold. Mutations in lgt, lspA, or an uncharacterized lipoprotein gene reduced competitiveness by two- to threefold in the animal model and in broth culture. Mutation of ssaB, encoding a putative metal transporter, produced a similar effect in culture but reduced in vivo competiveness by >1,000-fold. [³H]palmitate labeling and Western blot analysis confirmed that the lgt mutant failed to acylate lipoproteins, that the lspA mutant had a general defect in lipoprotein cleavage, and that SsaB was processed differently in both mutants. These results indicate that the loss of a single lipoprotein, SsaB, dramatically reduces endocarditis virulence, whereas the loss of most other lipoproteins or of normal lipoprotein processing has no more than a minor effect on virulence.Streptococcus sanguinis is a member of the viridans group of streptococci and is a primary colonizer of teeth (8). The viridans species and, in particular, S. sanguinis (15, 18) are a leading cause of infective endocarditis, a serious infection of the valves or lining of the heart (48). Damage to the heart resulting from rheumatic fever or certain congenital heart defects dramatically increases the risk of developing endocarditis (48, 71). The damage is thought to result in the formation of sterile cardiac “vegetations” composed of platelets and fibrin (48) that can be colonized by certain bacteria during periods of bacteremia. This view is supported by animal studies in which formation of sterile vegetation by cardiac catheterization is required for the efficient establishment of streptococcal endocarditis (17). Prevention of infective endocarditis currently relies upon prophylactic administration of antibiotics prior to dental or other surgical procedures that are likely to produce bacteremia. The growing realization that oral bacteria such as S. sanguinis can enter the bloodstream through routine daily activities such as eating has led the American Heart Association (71) and others (57) to question the value of using antibiotic prophylaxis for dental procedures. Clearly, a better understanding of the bacterial virulence factors that contribute to endocarditis could lead to better preventive measures, such as a vaccine that could potentially afford continuous protection to high-risk patients (71).In a previous study, we used the signature-tagged mutagenesis (STM) technique to search for endocarditis virulence factors of S. sanguinis in a rabbit model (53). This study identified a number of housekeeping enzymes that contribute to endocarditis. Because these proteins are not likely to be surface localized, they hold little promise as vaccine candidates. One class of streptococcal surface proteins that is rich in both virulence factors (4, 7, 25, 33, 38, 60) and promising vaccine candidates (6, 39, 42, 51, 70) is the lipoproteins. Lipoprotein activities that have been suggested to contribute to streptococcal virulence include adhesion (4, 7, 63), posttranslational modification (25, 29, 51), and ATP-binding cassette (ABC)-mediated transport (33, 52, 60). In the last instance, lipoproteins anchored to the cell membrane by their lipid tails appear to serve the same transport function as the periplasmic substrate-binding proteins of gram-negative bacteria (66). STM studies performed with Streptococcus pneumoniae (26, 41, 55) and Streptococcus agalactiae (34) have identified multiple lipoprotein mutants among collections of reduced virulence mutants. In an attempt to determine the cumulative contribution of streptococcal lipoproteins to virulence, some investigators have created mutations in the lgt or lspA genes, encoding lipoprotein-processing enzymes (12, 25, 27, 36). The lgt gene encodes prolipoprotein diacylglyceryl transferase, which catalyzes the transfer of a diacylglycerol lipid unit to a cysteine in the conserved N-terminal “lipobox” of lipoproteins, while lspA encodes the signal peptidase II enzyme that cleaves the signal peptide of the prolipoprotein just prior to the conserved cysteine (59, 65). While mutation of these genes has been shown to be lethal in gram-negative bacteria (21, 73), many gram-positive bacterial species have been shown to tolerate such mutations, often with only minor effects on growth (3, 12, 13, 25, 27, 36, 54). Some of these studies indicated a deleterious effect on the virulence of the lgt (25, 54) or lspA (36) mutation, but others found no effect (12) or an enhancement of virulence (27). It is clear from these and other studies (3, 13) that neither the loss of acylation due to lgt inactivation nor the loss of signal peptidase II-mediated cleavage completely eliminates lipoprotein function, necessitating alternative approaches for assessing the global contribution of lipoproteins to virulence.We have used bioinformatic approaches to identify every putative lipoprotein encoded by S. sanguinis strain SK36. To determine the contribution of these lipoproteins to the endocarditis virulence of S. sanguinis, we have systematically mutagenized each of these genes, as well as the lgt and lspA genes, and evaluated these mutants for virulence by using STM in an animal model. Selected mutants were further examined for virulence in competitive index (CI) assays. A strain with a disrupted ssaB gene, which encodes a putative metal transport protein, was found to exhibit a profound defect in virulence that was far greater than that of any other strain tested, including the lgt or lspA mutant.     

Identification of Yersinia enterocolitica at the Species and Subspecies Levels by Fourier Transform Infrared Spectroscopy

Yersinia enterocolitica and other Yersinia species, such as Y. pseudotuberculosis, Y. bercovieri, and Y. intermedia, were differentiated using Fourier transform infrared spectroscopy (FT-IR) combined with artificial neural network analysis. A set of well defined Yersinia strains from Switzerland and Germany was used to create a method for FT-IR-based differentiation of Yersinia isolates at the species level. The isolates of Y. enterocolitica were also differentiated by FT-IR into the main biotypes (biotypes 1A, 2, and 4) and serotypes (serotypes O:3, O:5, O:9, and “non-O:3, O:5, and O:9”). For external validation of the constructed methods, independently obtained isolates of different Yersinia species were used. A total of 79.9% of Y. enterocolitica sensu stricto isolates were identified correctly at the species level. The FT-IR analysis allowed the separation of all Y. bercovieri, Y. intermedia, and Y. rohdei strains from Y. enterocolitica, which could not be differentiated by the API 20E test system. The probability for correct biotype identification of Y. enterocolitica isolates was 98.3% (41 externally validated strains). For correct serotype identification, the probability was 92.5% (42 externally validated strains). In addition, the presence or absence of the ail gene, one of the main pathogenicity markers, was demonstrated using FT-IR. The probability for correct identification of isolates concerning the ail gene was 98.5% (51 externally validated strains). This indicates that it is possible to obtain information about genus, species, and in the case of Y. enterocolitica also subspecies type with a single measurement. Furthermore, this is the first example of the identification of specific pathogenicity using FT-IR.The genus Yersinia belongs to the bacterial family Enterobacteriaceae and encompasses three well-known human pathogens: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. Pathogenic strains of Y. enterocolitica cause yersiniosis, an acute enteric disease. In Germany and Switzerland, strains of Y. enterocolitica belong to the most frequently isolated pathogens connected with bacterial gastroenteritis (27, 31). Y. enterocolitica also causes other clinical syndromes, such as enterocolitis, acute mesenteric lymphadenitis, mimicking appendicitis, postinfectious arthritis, and systemic infections (7, 21). It is assumed that the main contamination source is food of animal origin, especially pork meat or raw milk (8, 21, 27). Therefore, the focus of diagnosis for these bacteria as food-borne pathogens includes the examination of food samples in food inspection and veterinary controls of livestock.The species Y. enterocolitica sensu lato as described by Frederiksen (9) was recently subdivided into several species: Y. enterocolitica sensu stricto, Y. intermedia, Y. frederiksenii, Y. kristensenii, Y. aldovae, Y. mollaretii, Y. rohdei, and Y. bercovieri (20). The identification of Y. enterocolitica sensu stricto by traditional agar plate techniques (ISO standard 10273:2003) is complicated by the fact that on the commonly used selective agar plates, especially the cefsulodin-irgasan-novobiocin (CIN) agar, several unrelated bacteria also grow (1, 20). In addition, some Yersinia strains are inhibited by CIN agar (10). The differentiation of putative Yersinia strains isolated from the CIN agar is additionally impeded because the commonly used commercial identification systems (for example, API 20E or API Rapid 32IDE) do not include all Yersinia strains in their databases and usually misidentify them as Y. enterocolitica (12). Nevertheless, the biochemical test system API 20E is still used as an affordable tool for the identification of Y. enterocolitica. This probably results in a constant misidentification of certain Yersinia species, particularly Y. bercovieri, Y. rohdei, and Y. intermedia, as Y. enterocolitica (1, 12, 15).Y. enterocolitica sensu stricto comprises pathogenic and nonpathogenic members. The species can be grouped into various biotypes by biochemical tests and independently into different serotypes by immunological tests. Both types are connected with different pathogenic potential. The most common biotype-serotype combinations associated with human diseases were biotype 1B/serotype O:8, 2/O:5,27, 2/O:9, 3/O:3, and 4/O:3 (7). Biotype 1A is deemed to be non- or less pathogenic for humans. Biotype 1B is widespread in the United States and only rarely detected in Europe and Japan (11, 14, 26, 28). Based on different DNA-DNA hybridization values and 16S rRNA gene sequences, it was proposed to name the “American” strains Y. enterocolitica subsp. enterocolitica (19). Biotypes 2 and 4 are often isolated from yersiniosis patients, and biotype 3 seems to be pathogenic but rare (6, 21).Pathogenic strains of Y. enterocolitica harbor certain virulence factors, such as the plasmid-encoded yadA gene and the chromosomally encoded ail gene (17, 32). In contrast, apathogenic strains of Y. enterocolitica do not contain these two genes. However, the plasmid harboring the yadA gene can be lost under certain cultivation conditions in the laboratory (4). This may lead to false-negative results in any test system based on the presence of this plasmid. Therefore, the ail gene appears to be the best-suited marker for the detection of pathogenic Y. enterocolitica strains. The product of the ail gene is an adhesion and invasion factor (17). Therefore, the detection of the ail gene by PCR is used as an indication of the presence of pathogenic strains of Y. enterocolitica in selective enrichments or isolated pure cultures (33).Recently, Fourier transform infrared spectroscopy (FT-IR) has been established as a new method for identification of bacteria, yeasts, and other microorganisms (3, 16, 22, 24, 38). This method analyzes the total composition of all components of the cell using infrared spectroscopy (13, 18). The FT-IR method is rapid and reliable and therefore can be easily adapted to routine analysis. Furthermore, there accrue almost no costs for consumables during sample preparation and measurements. The technique offers a wide range of applications for differentiation at the species and subspecies levels. It has already been used for the differentiation of several food-borne pathogens, like Listeria monocytogenes (25), Escherichia coli (13), and Bacillus cereus (23, 29). Recently, promising results were obtained by combination of FT-IR and multivariate methods for data processing, in particular artificial neural networks (ANN) (25, 35).In the present work, FT-IR combined with ANN analysis was applied for classification of Yersinia strains at the species level and of Y. enterocolitica at the subspecies level. Furthermore, differentiation between pathogenic and apathogenic strains of Y. enterocolitica by FT-IR was attempted.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Clonal Relationship among Atypical Enteropathogenic Escherichia coli Strains Isolated from Different Animal Species and Humans

Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.Enteropathogenic Escherichia coli (EPEC) strains are among the major causes of infantile diarrhea in developing countries (71) and can be classified as typical and atypical, depending on the presence or absence of the E. coli adherence factor plasmid (pEAF), respectively (39).The pathogenesis of EPEC resides in the ability to cause the attaching and effacing (A/E) lesion in the gut mucosa of human or animal hosts, leading to diarrheal illness (40). The genes responsible for the A/E lesion formation are located in a chromosomal pathogenicity island of ∼35 kb, known as the locus of enterocyte effacement (LEE) (23, 47). LEE encodes an adhesin called intimin (38), its translocated receptor (Tir) (42), components of a type III secretion system (36), and effector molecules, named E. coli-secreted proteins (Esp proteins) (41). These virulence factors have a crucial role in A/E lesion formation, and their detection in EPEC strains is an indicator of their potential to produce these lesions (19, 56).Atypical EPEC strains have been associated with diarrhea outbreaks in developed countries (31, 73, 77) and with sporadic cases of diarrhea in developing and developed countries (1, 12, 26, 52, 55). At present, the prevalence of atypical EPEC is higher than that of typical EPEC in several countries (1, 12, 26, 52, 55, 65).Different from the situation in developed countries, where atypical EPEC outbreaks and sporadic infections are associated with children and adults, atypical EPEC infection in Brazil is mainly associated with children''s illnesses (32, 71).Typical EPEC strains are rarely isolated from animals, and humans are the major natural reservoir for these pathogens (14, 32, 53, 71). In contrast, atypical EPEC strains are present in both healthy and diseased animals (dog, monkey, cats, and bovines) and humans (4, 6, 18, 28, 71). Some studies have associated pets and farm and wild animals as reservoirs and infection sources of atypical EPEC strains for humans (32). However, these studies did not compare atypical EPEC strains isolated from humans and animals by gold-standard molecular methods like multilocus sequence typing (MLST) or pulsed-field gel electrophoresis (PFGE) (15, 35, 43, 53). For this reason, there are some doubts about whether atypical EPEC strains isolated from animals represent risks for human health and whether animals really play the role of reservoirs of atypical EPEC.The aim of this study was to compare atypical EPEC strains isolated from humans and different animals, including pets (cats and dogs), farm animals (bovines, ovines, and rabbits), and wild animals (monkeys), by molecular phylogenetic techniques to verify the role of animals as reservoirs of and sources of infection with atypical EPEC in humans.