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.     

Genome Sequence of the Deep-Rooted Yersinia pestis Strain Angola Reveals New Insights into the Evolution and Pangenome of the Plague Bacterium

To gain insights into the origin and genome evolution of the plague bacterium Yersinia pestis, we have sequenced the deep-rooted strain Angola, a virulent Pestoides isolate. Its ancient nature makes this atypical isolate of particular importance in understanding the evolution of plague pathogenicity. Its chromosome features a unique genetic make-up intermediate between modern Y. pestis isolates and its evolutionary ancestor, Y. pseudotuberculosis. Our genotypic and phenotypic analyses led us to conclude that Angola belongs to one of the most ancient Y. pestis lineages thus far sequenced. The mobilome carries the first reported chimeric plasmid combining the two species-specific virulence plasmids. Genomic findings were validated in virulence assays demonstrating that its pathogenic potential is distinct from modern Y. pestis isolates. Human infection with this particular isolate would not be diagnosed by the standard clinical tests, as Angola lacks the plasmid-borne capsule, and a possible emergence of this genotype raises major public health concerns. To assess the genomic plasticity in Y. pestis, we investigated the global gene reservoir and estimated the pangenome at 4,844 unique protein-coding genes. As shown by the genomic analysis of this evolutionary key isolate, we found that the genomic plasticity within Y. pestis clearly was not as limited as previously thought, which is strengthened by the detection of the largest number of isolate-specific single-nucleotide polymorphisms (SNPs) currently reported in the species. This study identified numerous novel genetic signatures, some of which seem to be intimately associated with plague virulence. These markers are valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies.Yersinia pestis, the causative agent of plague, is a nonmotile Gram-negative bacterial pathogen. The genus Yersinia comprises two other pathogens that cause worldwide infections in humans and animals: Y. pseudotuberculosis and Y. enterocolitica (11, 12, 22, 61, 71). Despite their genetic relationship, these species differ radically in their pathogenicity and transmission. Plague is primarily a disease of wild rodents that is transmitted to other mammals through flea bites. In humans it produces the bubonic form of plague. Y. pestis also can be transmitted from human to human by aerosol, especially during pandemics, causing primarily pneumonic plague. Evolutionarily, it is estimated that Y. pestis diverged from the enteric pathogen Y. pseudotuberculosis within the last 20,000 years, while Y. pseudotuberculosis and Y. enterocolitica lineages separated 0.4 to 1.9 million years ago (2). Y. pestis inhabits a distinct ecological niche, and its transmission is anchored in its unique plasmid inventory: the murine toxin (pMT) and plasminogen activator (pPCP) plasmids. In addition, Y. pestis harbors the low-calcium-response plasmid pCD, which it inherited from its closest relative, Y. pseudotuberculosis (pYV) (12), and it also is found in the more distantly related Y. enterocolitica (71). So-called cryptic plasmids have been described in the literature as part of the Y. pestis mobilome (71), but no sequence data are available to decipher the nature and impact of such plasmids in the epidemiology and pathogenicity of Y. pestis (14). Y. pestis isolates have been historically grouped into the biovars Antiqua (ANT), Medievalis (MED), and Orientalis (ORI), based on metabolic properties such as nitrate reduction and fermentation patterns (72). However, we will use the population-based nomenclature for Y. pestis introduced by Achtman et al. (1), as we believe it better reflects the true evolutionary relationship. Due to its young evolutionary age, only a few genetic polymorphisms have been identified within the Y. pestis genomes sequenced to date (1). Here, we report the comparative analysis of the virulent Y. pestis strain Angola, a representative of one of the most ancient Y. pestis lineages thus far sequenced. We studied adaptive microevolutionary traits Y. pestis has acquired and predicted the global Yersinia pangenome. By comparing the genomes of the three human pathogenic Yersinia species (12, 22), we investigated the global- and species-specific gene reservoir, the genome dynamics, and the degree of genetic diversity that is found within these species. Our genotypic and phenotypic analyses, as well as the refined single-nucleotide polymorphism (SNP)-based phylogeny of Y. pestis, indicate that Angola is a deep-rooted isolate with unique genome characteristics intermediate between modern Y. pestis isolates and Y. pseudotuberculosis.     

Excretory/Secretory Proteome of the Adult Developmental Stage of Human Blood Fluke, Schistosoma japonicum

Schistosomes are the causative agents of schistosomiasis, one of the most prevalent and serious of the parasitic diseases that currently infects ∼200 million people worldwide. Schistosome excretory/secretory (ES) proteins have been shown to play important roles in modulating mammalian host immune systems. In our current study, we performed a global proteomics identification of the ES proteins from adult worms of Schistosoma japonicum, one of the three major schistosome species. Our results unambiguously identified 101 proteins, including 53 putatively secreted proteins. By quantitative analysis, we revealed fatty acid-binding protein as a major constituent of the in vitro ES proteome. Strikingly the heat shock proteins HSP70s, HSP90, and HSP97 constituted the largest protein family in the ES proteome, implying a central role for these proteins in immunomodulation in the host-parasite relationship. Other important S. japonicum ES proteins included actins, 14-3-3, aminopeptidase, enolase, and glyceraldehyde-3-phosphate dehydrogenase, some of which have been considered as viable vaccine candidates and therapeutic targets. A comparison with previous studies suggests that 48.5% of S. japonicum ES proteins are common to other parasite ES products, indicating that the molecular mechanisms involved in evading the host immune response may be conserved across different parasites. Interestingly seven host proteins, including antimicrobial protein CAP18, immunoglobulins, and a complement component, were identified among in vitro S. japonicum ES products likely originating from the schistosome tegument or gut, indicating that host innate and acquired immune systems could defend against schistosome invasion. Our present study represents the first attempt at profiling S. japonicum ES proteins, provides an insight into host-parasite interactions, and establishes a resource for the development of diagnostic agents and vaccines for the control of schistosomiasis.Schistosomes, or blood flukes, are water-borne parasites that are the causative agents of schistosomiasis. An estimated 200 million people worldwide are infected with schistosomes with an additional 650 million people at risk of infection (1). One of the major species of schistosomes, Schistosoma japonicum, is a mammalian parasite endemic in East Asia, especially in China and the Philippines. Schistosomes have complex life cycles. Larval schistosome worms (cercariae) are released by freshwater snails and subsequently invade their definite hosts, human or other mammals, via skin penetration. Once in a host animal, cercariae develop into schistosomula and adult worms, which reside in the portal mesenteric system of the host. When the females lay eggs, some eggs leave the host body and hatch in bodies of water as miracidia. The miracidia seek out and penetrate intermediate host snails, completing the schistosome life cycle.Schistosome strategies for evasion of the host immune system, which permit extended survival in mammalian hosts, are not well understood. These dominant evasion strategies have been described as a system of mimicry capable of producing antigens that are similar to endogenous host components (2–4), antigen disguise through acquisition of host molecules to cover the outer worm surface (5, 6), and immunological modulation through interference with host immune systems (7–10). Among these strategies, schistosome excretory/secretory (ES)¹ products have been shown to elicit host immunological modulation functions (7, 11). Schistosome ES proteins are released or secreted from epithelial surfaces of the gut and/or tegument as well as other specialized ES organs throughout almost all life stages. Schistosoma mansoni primary sporocysts have been reported to synthesize and secrete a wide variety of glycoproteins when cultured in vitro (12, 13). These glycoproteins were shown to have antioxidant activities against potential oxidative killing by mollusk defense systems (14). Similarly ES molecules from schistosome cercariae were also reported to down-regulate host immune responses (7). The anti-inflammatory activity of S. mansoni schistosomula ES products (ESPs) was found to be dominantly associated with Sm16.8 protein (15). In addition, ES proteins from S. mansoni adult worms (16), eggs (17), and miracidia (18) have also been investigated.Identification of all ES complex components is important for understanding how schistosomes regulate host immune systems to establish chronic infections and also other aspects of parasite-host interaction. Importantly this information can be expected to facilitate the discovery of vaccines and new therapeutic drug targets as well as new diagnostic reagents for schistosomiasis control. Proteomics approaches encompass the most efficient and powerful tools for identification of protein complexes and have been widely used to decipher the ES components of the filarial parasite Brugia malayi (19), Leishmania (Trypanosomatidae) (20), nematodes (21–26), and Trematoda (27–37). For the genus Schistosoma, the ES compositions of S. mansoni have been identified in many developmental life stages, including sporocyst (34), cercaria (35, 36), and egg (37) but have not been characterized in the adult worm.Characterization of the S. japonicum ES proteome has not been reported. S. japonicum is significantly different from S. mansoni and Schistosoma hematobium in skin invasion, skin migration, and its developmental patterns of swift migration and maturation (38–40). As such, S. japonicum represents a distinct and valuable model for the study of blood fluke immune evasion strategies. Our research group recently generated and reported a large number of S. japonicum protein-coding genes and expressed sequence tags (ESTs) (41, 42). This preliminary work provides important translated protein sequence data resources for mass spectrum data searching. The present study characterized the in vitro ES proteome of adult worms of S. japonicum (43) using a high throughput LC-MS/MS screening. This life stage spans the longest time frame of parasitic interaction with the host that is distinct from previous reports on proteomics identification of ES compositions in other species. Finally the present study confidently identified 101 S. japonicum ES proteins. This information represents substantial progress toward deciphering the worm ES proteome. These new data provide the basis for further investigations into the molecular basis of schistosome modulation of host immunity, increase the possibility of identifying vaccine candidates and new drug targets, and may aid the development of protein probes for selective and sensitive diagnosis of schistosomiasis.     

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).     

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.     

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.     

Enhancement of human immunodeficiency virus (HIV)-specific CD8+ T cells in cerebrospinal fluid compared to those in blood among antiretroviral therapy-naive HIV-positive subjects

During untreated human immunodeficiency virus type 1 (HIV-1) infection, virus-specific CD8⁺ T cells partially control HIV replication in peripheral lymphoid tissues, but host mechanisms of HIV control in the central nervous system (CNS) are incompletely understood. We characterized HIV-specific CD8⁺ T cells in cerebrospinal fluid (CSF) and peripheral blood among seven HIV-positive antiretroviral therapy-naïve subjects. All had grossly normal brain magnetic resonance imaging and spectroscopy and normal neuropsychometric testing. Frequencies of epitope-specific CD8⁺ T cells by direct tetramer staining were on average 2.4-fold higher in CSF than in blood (P = 0.0004), while HIV RNA concentrations were lower. Cells from CSF were readily expanded ex vivo and responded to a broader range of HIV-specific human leukocyte antigen class I restricted optimal peptides than did expanded cells from blood. HIV-specific CD8⁺ T cells, in contrast to total CD8⁺ T cells, in CSF and blood were at comparable maturation states, as assessed by CD45RO and CCR7 staining. The strong relationship between higher T-cell frequencies and lower levels of viral antigen in CSF could be the result of increased migration to and/or preferential expansion of HIV-specific T cells within the CNS. This suggests an important role for HIV-specific CD8⁺ T cells in control of intrathecal viral replication.Human immunodeficiency virus type 1 (HIV-1) invades the central nervous system (CNS) early during primary infection (21, 30, 35), and proviral DNA persists in the brain throughout the course of HIV-1 disease (7, 25, 29, 47, 77, 83). Limited data from human and nonhuman primate studies suggest that little or no viral replication occurs in the brain during chronic, asymptomatic infection, based on the absence of demonstrable viral RNA or proteins (8, 85). In contrast, cognitive impairment affects approximately 40% of patients who progress to advanced AIDS without highly active antiretroviral therapy (21, 30, 35, 65). During HIV-associated dementia, there is active HIV-1 replication in the brain (23, 52, 61, 81), and viral sequence differences between cerebrospinal fluid (CSF) and peripheral tissues suggest distinct anatomic compartments of replication (18, 19, 22, 53, 75, 76, 78). Host mechanisms that control viral replication in the CNS during chronic, asymptomatic HIV-1 infection are incompletely understood.Anti-HIV CD8⁺ T cells are present in blood and peripheral tissues throughout the course of chronic HIV-1 infection (2, 14). Multiple lines of evidence support a critical role for these cells in controlling HIV-1 replication. During acute HIV-1 infection, the appearance of CD8⁺ T-cell responses correlates temporally with a decline in viremia (11, 43), and a greater proliferative capacity of peripheral blood HIV-specific CD8⁺ T cells correlates with better control of viremia (36, 54). In addition, the presence of certain major histocompatibility complex class I human leukocyte antigen (HLA) alleles, notably HLA-B*57, predicts slower progression to AIDS and death during chronic, untreated HIV-1 infection (55, 62). Finally, in the simian immunodeficiency virus (SIV) model, macaques depleted of CD8⁺ T cells experience increased viremia and rapid disease progression (39, 51, 67).Little is known regarding the role of intrathecal anti-HIV CD8⁺ T cells in HIV neuropathogenesis. Nonhuman primate studies have identified SIV-specific CD8⁺ T cells in the CNS early after infection (16, 80). Increased infiltration of SIV antigen-specific CD8⁺ T cells and cytotoxic T lymphocytes has been detected only in CSF of slow progressors without neurological symptoms (72). In chronically infected macaques with little or no SIV replication in the brain, the frequency of HIV-specific T cells was higher in CSF than in peripheral blood but did not correlate with the level of plasma viremia or CD4⁺ T-cell counts (56). Although intrathecal anti-HIV CD8⁺ T cells may help control viral replication, a detrimental role in the neuropathogenesis of HIV-1 has also been postulated (38). Immune responses contribute to neuropathogenesis in models of other infectious diseases, and during other viral infections cytotoxic T lymphocytes can worsen disease through direct cytotoxicity or release of inflammatory cytokines such as gamma interferon (IFN-γ) (3, 17, 31, 37, 42, 44, 71).We tested the hypothesis that quantitative and/or qualitative differences in HIV-specific CD8⁺ T-cell responses are present in CSF compared to blood during chronic, untreated HIV-1 infection. We characterized HIV-specific CD8⁺ T-cell responses in CSF among seven antiretroviral therapy-naïve adults with chronic HIV-1 infection, relatively high peripheral blood CD4⁺ T-cell counts, and low plasma HIV-1 RNA concentrations. We show that among these HIV-positive individuals with no neurological symptoms and with little or no HIV-1 RNA in CSF, frequencies of HIV-specific T cells are significantly higher in CSF than in blood. These CSF cells are at a state of differentiation similar to that of T cells in blood and are functionally competent for expansion and IFN-γ production. The higher frequency of functional HIV-specific CD8⁺ T cells in CSF, in the context of low or undetectable virus in CSF, suggests that these cells play a role in the control of intrathecal viral replication.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Vaccine protection by live, attenuated simian immunodeficiency virus in the absence of high-titer antibody responses and high-frequency cellular immune responses measurable in the periphery

An attenuated derivative of simian immunodeficiency virus strain 239 deleted of V1-V2 sequences in the envelope gene (SIV239ΔV1-V2) was used for vaccine/challenge experiments in rhesus monkeys. Peak levels of viral RNA in plasma of 10⁴ to 10^6.5 copies/ml in the weeks immediately following inoculation of SIV239ΔV1-V2 were 10- to 1,000-fold lower than those observed with parental SIV239 (∼10^7.3 copies/ml). Viral loads consistently remained below 200 copies/ml after 8 weeks of infection by the attenuated SIV239ΔV1-V2 strain. Viral localization experiments revealed large numbers of infected cells within organized lymphoid nodules of the colonic gut-associated lymphoid tissue at 14 days; double-labeling experiments indicated that 93.5% of the virally infected cells at this site were positive for the macrophage marker CD68. Cellular and humoral immune responses measured principally by gamma interferon enzyme-linked immunospot and neutralization assays were variable in the five vaccinated monkeys. One monkey had responses in these assays comparable to or only slightly less than those observed in monkeys infected with parental, wild-type SIV239. Four of the vaccinated monkeys, however, had low, marginal, or undetectable responses in these same assays. These five vaccinated monkeys and three naïve control monkeys were subsequently challenged intravenously with wild-type SIV239. Three of the five vaccinated monkeys, including the one with strong anti-SIV immune responses, were strongly protected against the challenge on the basis of viral load measurements. Surprisingly, two of the vaccinated monkeys were strongly protected against SIV239 challenge despite the presence of cellular anti-SIV responses of low-frequency and low-titer anti-SIV antibody responses. These results indicate that high-titer anti-SIV antibody responses and high-frequency anti-SIV cellular immune responses measurable by standard assays from the peripheral blood are not needed to achieve strong vaccine protection, even against a difficult, neutralization-resistant strain such as SIV239.The characteristics of human immunodeficiency virus type 1 (HIV-1) infection suggest major difficulty for the development of a preventive vaccine (19, 23). Pessimism regarding the prospects for a vaccine is derived at least in part from the ability of HIV-1 to continually replicate in the face of apparently strong host immune responses, resistance to antibody-mediated neutralization, and the extensive sequence diversity in field strains of the virus. Lack of knowledge regarding the key components of a protective immune response also remains a major scientific obstacle. Vaccine/challenge experiments with macaque monkeys have been used to evaluate the properties and relative effectiveness of different vaccine approaches and to gauge the formidable nature of these difficulties.One lesson that has been learned from vaccine/challenge experiments with macaque monkeys is the importance of challenge strain on outcome. Vaccinated monkeys that have been challenged with strains of simian immunodeficiency virus (SIV) with an HIV-1 envelope (SHIV) have almost invariably exhibited strong, long-term protection against disease, irrespective of the nature of the vaccine. Even peptide immunogens have protected against SHIV-induced disease (6, 12, 38). Vaccine approaches that have protected against SHIV challenge include DNA (5, 13), recombinant poxvirus (4), recombinant adenovirus (57), other viral recombinants (18, 55), prime and boost protocols (3, 53, 65), and purified protein (10, 64). Vaccine protection against pathogenic SIV strains such as SIV239, SIV251, and SIV-E660 has been much more difficult to achieve (2, 11, 27, 63). The identical replication-defective gag-recombinant adenovirus that provided strong protection against SHIV challenge (57) provided little or no protection against SIV239 challenge (11). Disappointing levels of protection against SIV have often been observed in the face of apparently robust vaccine-induced immune responses (see, for example, Vogel et al. [63] and Casimiro et al. [11]). Some partial vaccine protections against these SIV strains have been achieved by recombinant poxvirus (7, 50), replication-competent recombinant adenovirus (51), replication-defective adenovirus (66), recombinant poliovirus (15), recombinant Venezuelan equine encephalitis virus (18), and recombinant Sendai virus (44).Differences between the biological properties of the SIV strains and those of the SHIV strains used for the above-mentioned studies provide clues as to what may be responsible for the differences in outcome. These SIV strains are difficult to neutralize (26, 34), use CCR5 as a coreceptor for entry into cells (21, 52), and induce a chronic, progressive disease course (17), and this course is independent of the infectious dose (17). The SHIV strains used for the above-mentioned studies are easier to neutralize, use CXCR4 for entry, and induce an acute decline in CD4 counts, and the disease course is dose dependent (29, 30, 48, 54). These SIV strains, like HIV-1 in humans, exhibit a marked preference for CD4⁺ CCR5⁺ memory cells, in contrast to the acutely pathogenic SHIV strains which principally target naïve cells (48).Live, attenuated strains of SIV have provided the strongest vaccine protection by far against SIV challenge. Although clinical use of a live, attenuated HIV vaccine is not being considered, understanding the basis of the strong protection afforded by live, attenuated SIV strains remains an important research objective for the insights that can be provided. Most of the attenuated SIV strains that have been used lack a functional nef gene (16, 31, 58, 67). Shacklett et al. (56) used an attenuated SIV strain with modifications in the gp41 transmembrane protein for protection. Here, we describe strong vaccine protection by a replication-competent SIV strain lacking 100 amino acids from the essential gp120 envelope protein in the absence of overtly robust immune responses.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Interaction of decay-accelerating factor with echovirus 7

Echovirus 7 (EV7) belongs to the Enterovirus genus within the family Picornaviridae. Many picornaviruses use IgG-like receptors that bind in the viral canyon and are required to initiate viral uncoating during infection. However, in addition, some of the enteroviruses use an alternative or additional receptor that binds outside the canyon. Decay-accelerating factor (DAF) has been identified as a cellular receptor for EV7. The crystal structure of EV7 has been determined to 3.1-Å resolution and used to interpret the 7.2-Å-resolution cryo-electron microscopy reconstruction of EV7 complexed with DAF. Each DAF binding site on EV7 is near a 2-fold icosahedral symmetry axis, which differs from the binding site of DAF on the surface of coxsackievirus B3, indicating that there are independent evolutionary processes by which DAF was selected as a picornavirus accessory receptor. This suggests that there is an advantage for these viruses to recognize DAF during the initial process of infection.Echoviruses (EVs) belong to the family Picornaviridae, which contains some of the most common viral pathogens of vertebrates (43, 50, 51, 55, 58, 63). Picornaviruses are small, icosahedral, nonenveloped animal viruses. Their capsids have 60 copies each of four viral proteins, VP1, VP2, VP3, and VP4, that form an ∼300-Å-diameter icosahedral shell filled with a positive-sense, single-stranded RNA genome. A distinctive feature of the capsid surface is a depression around the 5-fold axes of symmetry, called the “canyon” (47). The results of both genetic and structural studies have shown that the canyon is the site of receptor binding for many of these viruses (4, 11, 23, 25, 36, 47, 68), including echoviruses, which utilize β-integrins (6, 33, 66). Receptor molecules that bind in the canyon have been found to belong to the immunoglobulin superfamily (49). When these receptor molecules bind within the canyon, they dislodge a “pocket factor” within a pocket immediately below the surface of the canyon. The shape and environment of the pocket factor suggest that it might be a lipid (13, 32, 45, 54). When a receptor binds within the canyon, it depresses the floor of the canyon, corresponding to the roof of the pocket. Similarly, when a lipid or antiviral compound binds to the pocket, it expands the roof of the pocket, corresponding to the floor of the canyon (39, 45). Thus, receptors that bind to the canyon and the pocket factor compete with each other for binding to the virus. An absence of the hydrophobic pocket factor destabilizes the virus and initiates transition to altered “A” particles, a likely prelude to uncoating of the virion, possibly during passage through an endosomal vesicle (45).Not all receptors of picornaviruses bind in the canyon. A minor group of human rhinoviruses (HRV) bind to the low-density-lipoprotein receptor family (17, 34, 61, 62), and some other picornaviruses, including certain coxsackie- and echoviruses, utilize decay-accelerating factor (DAF; also called CD55) as a cellular receptor (9, 28, 40, 52).DAF is a member of a family of proteins that regulate complement activation by binding to and accelerating the decay of both classical and alternative pathway C3 and C5 convertases (7, 18, 26), the central amplification enzymes of the complement cascade. DAF is expressed on virtually all cell surfaces, protecting self cells from the immune system by rapidly dissociating any convertases that assemble, thereby halting the progression of a complement attack directed at the cell. Recent work (15, 27, 29, 56) has shown that DAF also participates in T-cell antiviral immunity (56) and protects against T-cell autoimmunity (29) by regulating complement that is produced locally by immune cells. The functional region of DAF consists of four short consensus repeats (SCR1, -2, -3, and -4). The structures have been determined for the SCR2-SCR3 fragment, the SCR3-SCR4 fragment, and the full four-domain region (30, 60, 65). Each of the SCR domains contains about 60 residues and is folded into a β structure stabilized by disulfide bridges. The four SCR domains form a relatively rigid extended rod with dimensions of 160 by 50 by 30 Å (30). The four domains rise about 180 Å above the plasma membrane, on a serine- and threonine-rich stalk of 94 amino acids, 11 of which are O-glycosylated, and is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor.Structural and genetic studies have shown that closely related picornaviruses have adapted to bind to DAF at different sites on the receptor surface (9, 31, 38, 42, 52, 64). Although DAF binding is likely to facilitate viral adsorption, the availability of DAF receptor molecules on the host is normally not sufficient for echovirus 7 (EV7) to enter cells. Presumably, viral adaptation to bind DAF offers some advantage to the virus, such as increasing the efficiency of infection.In an earlier publication (14), a 16-Å-resolution cryo-electron microscopy (cryo-EM) density map of the EV7-DAF complex was interpreted with the homologous structures of coxsackievirus B3 (CVB3) for EV7 (74% sequence identity) and virus complement protein for DAF (25% sequence identity). Because of the limited resolution of the earlier cryo-EM reconstruction, it was concluded that DAF bound to EV7 by laying across the icosahedral 2-fold axes. This implied that there were two alternative DAF binding modes occupying the same site, but with DAF oriented in opposite directions, and that only one of these alternative sites could be occupied at a time. Here we describe an improved, 7.2-Å-resolution cryo-EM reconstruction of DAF bound to EV7 and 3.1-Å-resolution X-ray crystal structures of EV7. Together with previously determined structures of DAF (30), we now show that 2-fold axis-related DAF molecules bind close to the icosahedral 2-fold axes on the viral surface but (in contradiction to the earlier results and consistent with predictions made by Pettigrew et al. [38]) do not cross these axes. This is consistent with the results of DAF binding to EV12, which binds DAF similarly to the manner reported here and also predicted for EV7 (38). Thus, the binding modes of DAF to EV12 and EV7 are now shown to be similar, but not the same, and are completely different from the binding mode of DAF to CVB3.