Broad Conservation of Milk Utilization Genes in Bifidobacterium longum subsp. infantis as Revealed by Comparative Genomic Hybridization

Human milk oligosaccharides (HMOs) are the third-largest solid component of milk. Their structural complexity renders them nondigestible to the host but liable to hydrolytic enzymes of the infant colonic microbiota. Bifidobacteria and, frequently, Bifidobacterium longum strains predominate the colonic microbiota of exclusively breast-fed infants. Among the three recognized subspecies of B. longum, B. longum subsp. infantis achieves high levels of cell growth on HMOs and is associated with early colonization of the infant gut. The B. longum subsp. infantis ATCC 15697 genome features five distinct gene clusters with the predicted capacity to bind, cleave, and import milk oligosaccharides. Comparative genomic hybridizations (CGHs) were used to associate genotypic biomarkers among 15 B. longum strains exhibiting various HMO utilization phenotypes and host associations. Multilocus sequence typing provided taxonomic subspecies designations and grouped the strains between B. longum subsp. infantis and B. longum subsp. longum. CGH analysis determined that HMO utilization gene regions are exclusively conserved across all B. longum subsp. infantis strains capable of growth on HMOs and have diverged in B. longum subsp. longum strains that cannot grow on HMOs. These regions contain fucosidases, sialidases, glycosyl hydrolases, ABC transporters, and family 1 solute binding proteins and are likely needed for efficient metabolism of HMOs. Urea metabolism genes and their activity were exclusively conserved in B. longum subsp. infantis. These results imply that the B. longum has at least two distinct subspecies: B. longum subsp. infantis, specialized to utilize milk carbon, and B. longum subsp. longum, specialized for plant-derived carbon metabolism.The newborn infant not only tolerates but requires colonization by commensal microbes for its own development and health (3). The relevance of the gut microbiome in health and disease is reflected by its influence in a number of important physiological processes, from physical maturation of the developing immune system (28) to the altered energy homeostasis associated with obesity (51, 52).Human milk provides all the nutrients needed to satisfy the neonate energy expenditure and a cadre of molecules with nonnutritional but biologically relevant functions (6). Neonatal health is likely dependent on the timely and complex interactions among bioactive components in human milk, the mucosal immune system, and specialized gut microbial communities (30). Human milk contains complex prebiotic oligosaccharides that stimulated the growth of select bifidobacteria (24, 25) and are believed to modulate mucosal immunity and protect the newborn against pathogens (23, 33, 41). These complex oligosaccharides, which are abundantly present in human milk (their structures are reviewed by Ninonuevo et al. [31] and LoCascio et al. [24]), arrive intact in the infant colon (5) and modulate the composition of neonatal gastrointestinal (GI) microbial communities.Bifidobacteria and, frequently, Bifidobacterium longum strains often predominate the colonic microbiota of exclusively breast-fed infants (10, 11). Among the three subspecies of B. longum, only B. longum subsp. infantis grows robustly on human milk oligosaccharides (HMOs) (24, 25). The availability of the complete genome sequences of B. longum subsp. infantis ATCC 15697 (40) and two other B. longum subsp. longum strains (22, 39) made possible the analysis of whole-genome diversity across the B. longum species. Analysis of the B. longum subsp. infantis ATCC 15697 genome has identified regions predicted to enable the metabolism of HMOs (40); however, their distribution across the B. longum spp. remains unknown. We predict that these regions are exclusively conserved in B. longum strains adapted to colonization of the infant gut microbiome and are therefore capable of robust growth on HMOs. In this work, whole-genome microarray comparisons (comparative genomic hybridizations [CGHs]) were used to associate genotypic biomarkers among 15 B. longum strains exhibiting various HMO utilization phenotypes and host associations.     

Distribution of In Vitro Fermentation Ability of Lacto-N-Biose I,a Major Building Block of Human Milk Oligosaccharides,in Bifidobacterial Strains

This study investigated the potential utilization of lacto-N-biose I (LNB) by individual strains of bifidobacteria. LNB is a building block for the human milk oligosaccharides, which have been suggested to be a factor for selective growth of bifidobacteria. A total of 208 strains comprising 10 species and 4 subspecies were analyzed for the presence of the galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) gene (lnpA) and examined for growth when LNB was used as the sole carbohydrate source. While all strains of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, B. breve, and B. bifidum were able to grow on LNB, none of the strains of B. adolescentis, B. catenulatum, B. dentium, B. angulatum, B. animalis subsp. lactis, and B. thermophilum showed any growth. In addition, some strains of B. pseudocatenulatum, B. animalis subsp. animalis, and B. pseudolongum exhibited the ability to utilize LNB. With the exception for B. pseudocatenulatum, the presence of lnpA coincided with LNB utilization in almost all strains. These results indicate that bifidobacterial species, which are the predominant species found in infant intestines, are potential utilizers of LNB. These findings support the hypothesis that GLNBP plays a key role in the colonization of bifidobacteria in the infant intestine.Bifidobacteria are gram-positive anaerobic bacteria that naturally colonize the human intestinal tract and are believed to be beneficial to human health (21, 30). Breastfeeding has been shown to be associated with an infant fecal microbiota dominated by bifidobacteria, whereas the fecal microbiota of infants who are consuming alternative diets has been described as being mixed and adult-like (12, 21). It has been suggested that the selective growth of bifidobacteria observed in breast-fed newborns is related to the oligosaccharides and other factors that are contained in human milk (human milk oligosaccharides [HMOs]) (3, 4, 10, 11, 16, 17, 34). Kitaoka et al. (15) have recently found that bifidobacteria possess a unique metabolic pathway that is specific for lacto-N-biose I (LNB; Galβ1-3GlcNAc) and galacto-N-biose (GNB; Galβ1-3GalNAc). LNB is a building block for the type 1 HMOs [such as lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentaose I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), and lacto-N-difucohexaose I (Fucα1-2Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4Glc)], and GNB is a core structure of the mucin sugar that is present in the human intestine and milk (18, 27). The GNB/LNB pathway, as previously illustrated by Wada et al. (33), involves proteins/enzymes that are required for the uptake and degradation of disaccharides such as the GNB/LNB transporter (29, 32), galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP; LnpA) (15, 24) (renamed from lacto-N-biose phosphorylase after the finding of phosphorylases specific to GNB [23] and LNB [22]), N-acetylhexosamine 1-kinase (NahK) (25), UDP-glucose-hexose 1-phosphate uridylyltransferase (GalT), and UDP-galactose epimerase (GalE). Some bifidobacteria have been demonstrated to be enzymatically equipped to release LNB from HMOs that have a type 1 structure (lacto-N biosidase; LnbB) (33) or GNB from the core 1-type O-glycans in mucin glycoproteins (endo-α-N-acetylgalatosaminidase) (6, 13, 14). It has been suggested that the presence of the LnbB and GNB/LNB pathways in some bifidobacterial strains could provide a nutritional advantage for these organisms, thereby increasing their populations within the ecosystem of these breast-fed newborns (33).The species that predominantly colonize the infant intestine are the bifidobacterial species B. breve, B. longum subsp. infantis, B. longum subsp. longum, and B. bifidum (21, 28). On the other hand, strains of B. adolescentis, B. catenulatum, B. pseudocatenulatum, and B. longum subsp. longum are frequently isolated from the adult intestine (19), and strains of B. animalis subsp. animalis, B. animalis subsp. lactis, B. thermophilum and B. pseudolongum have been shown to naturally colonize the guts of animals (1, 2, 7, 8). However, it is unclear whether there is a relationship between the differential colonization of the bifidobacterial species and the presence of the GNB/LNB pathway. In the present study, we investigated the ability of individual bifidobacterial strains in the in vitro fermentation of LNB and in addition, we also tried to determine whether or not the GLNBP gene (lnpA), which is a key enzyme of the GNB/LNB pathway, was present.     

Coculture Fermentations of Bifidobacterium Species and Bacteroides thetaiotaomicron Reveal a Mechanistic Insight into the Prebiotic Effect of Inulin-Type Fructans

Four bifidobacteria, each representing a cluster of strains with specific inulin-type-fructan degradation capacities, were grown in coculture fermentations with Bacteroides thetaiotaomicron LMG 11262, a strain able to metabolize both oligofructose and inulin. In a medium for colon bacteria with inulin as the sole added energy source, the ability of the bifidobacteria to compete for this substrate reflected phenotypical variation. Bifidobacterium breve Yakult, a strain that was not able to degrade oligofructose or inulin, was outcompeted by B. thetaiotaomicron LMG 11262. Bifidobacterium adolescentis LMG 10734, a strain that could degrade oligofructose (displaying a preferential breakdown mechanism) but that did not grow on inulin, managed to become competitive when oligofructose and short fractions of inulin started to accumulate in the fermentation medium. Bifidobacterium angulatum LMG 11039^T, a strain that was previously shown to degrade all oligofructose fractions simultaneously and to be able to partially break down inulin, was competitive from the beginning of the fermentation, consuming short fractions of inulin from the moment they appeared. Bifidobacterium longum LMG 11047, representing a cluster of bifidobacteria that shared both high fructose consumption and oligofructose degradation rates and were able to perform partial breakdown of inulin, was the dominating strain in a coculture with B. thetaiotaomicron LMG 11262. These observations indicate that distinct subgroups within the large-intestinal Bifidobacterium population will be stimulated by different groups of prebiotic inulin-type fructans, a variation that could be reflected in differences concerning their health-promoting effects.The vast complexity of the human colon microbiota, the key element of the large-intestinal ecosystem, has inspired researchers to describe it as a postnatally acquired microbial organ located inside a host organ (1, 46). The microbial colon community is estimated to be composed of up to 100 trillion microorganisms, a number exceeding 10 times the total number of somatic and germ cells of a human adult (18, 38). The human microbiome is thought to contain more than 100 times the total number of human genes (1, 18). It not only broadens the digestive abilities of the host (18, 22, 40) but also influences body processes far beyond digestion (7, 33). In spite of its fundamental impact on human health and disease, the human gastrointestinal ecosystem remains largely unexplored (7, 8).Despite the fact that the present knowledge of the composition of the human large-intestinal microbiota is partial, fragmented, and undetailed, the consistency of some observations allows them to be generalized as facts (8, 28, 47). Notwithstanding the huge diversity at the strain level, up to 87% of the human colon inhabitants belong to only two bacterial phyla, the Bacteroidetes and the Firmicutes (1, 8, 14). Within the group of large-intestinal Bacteroidetes, large variations between individuals have been reported (8). However, Bacteroides spp. generally seem to account for up to 20% of the human colon microbiota (26, 32). Moreover, the presence of Bacteroides thetaiotomicron appears to be universal (8, 21). This species, which has been isolated only from human and rodent intestines or feces up to now, has gained importance as a perfect example of a flexible, niche-adapted, human symbiont with a wide carbohydrate consumption range (3, 4, 40).Although B. thetaiotaomicron is considered a human symbiont contributing to the stability of the colon ecosystem, the Bacteroides genus also harbors some notorious pathogens that are linked with severe extraintestinal infections and that have been mentioned as causal agents of acute diarrhea (30, 35). Moreover, besides their enormous saccharolytic potential, Bacteroides spp. are also capable of proteolytic fermentation (22). These considerations make them unsuited as target organisms for stimulation by prebiotics such as inulin-type fructans (23, 31).Most in vivo studies regarding the effect of the addition of inulin or oligofructose to the diet on the composition of the human colon microbiota reveal that Bacteroides spp. are neither stimulated nor repressed through administration of these prebiotics (34). However, at least some Bacteroides spp. are able to degrade inulin-type fructans, including B. thetaiotaomicron (13, 44). Since this species accounts for up to 6% of the colon microbiota (8), it is at least surprising that its numbers are hardly influenced by an increased availability of these prebiotics as substrates for large-intestinal fermentation. A possible explanation for these contradicting observations is to be found in the mechanism of inulin degradation, which in the case of Bacteroides is presumed to be periplasmic or even extracellular (37, 44). Leakage of free fructose toward the extracellular environment appears to be inherent in such breakdown mechanisms (10, 25, 44). Hence, extracellular fructan degraders inevitably provide opportunistic competitors, which are not able to degrade inulin-type fructans themselves, with a valuable source of energy (2, 10, 19). In contrast, a cell-associated or intracellular degradation mechanism is thought to be widespread among Bifidobacterium spp., which are still considered the main target organisms for prebiotic stimulation by inulin-type fructans (15, 16, 39, 44). This mechanism is often reflected in a clearly preferential breakdown of different-chain-length fractions of oligofructose, which approaches degradation of the long fractions only when short ones are depleted (10, 42, 44). The main disadvantage of such a cell-associated or intracellular degradation strategy seems to be the bifidobacterial incapacity to grow on long-chain-length fractions of inulin (36). Reports of the latter are indeed scarce: kinetic pure culture studies report an upper chain length limit for inulin degradation by Bifidobacterium spp., a disadvantage that will presumably not affect extracellular fructan degraders, such as Bacteroides spp. (9). Although the prebiotic effect of inulin-type fructans on the colon Bifidobacterium population is well documented, in vivo stimulation studies usually tend to consider the bifidobacterial community as a whole, ignoring interspecies differences (23). However, since the early days of in vitro prebiotic studies, a large variation in fructan degradation capacities of different Bifidobacterium strains has been reported (17, 36). It is likely that this variety is translated to the in vivo environment, implying that not all bifidobacteria are equally subject to prebiotic stimulation (5, 45). In a recent study, the kinetics of growth, carbohydrate consumption, and metabolite production of 18 Bifidobacterium spp., 17 of which were human intestinal isolates, have been statistically analyzed (9). The existence of four phenotypically distinct clusters among the tested strains, probably reflecting niche-specific adaptation, has been revealed. This rather limited variation was hypothesized to influence the susceptibilities of various bifidobacteria toward prebiotic stimulation by inulin-type fructans and their fitness to compete for these substrates in a complex environment, such as the colon ecosystem (44).The present study aimed at mapping the fructan degradation capacity of B. thetaiotaomicron LMG 11262 growing on oligofructose or inulin. In vitro competitiveness trials with bifidobacterial strains belonging to the different phenotypical clusters mentioned above were designed to investigate the abilities of these strains to compete for inulin in a coculture with an inulin-degrading B. thetaiotaomicron strain.     

In Vitro Kinetics of Prebiotic Inulin-Type Fructan Fermentation by Butyrate-Producing Colon Bacteria: Implementation of Online Gas Chromatography for Quantitative Analysis of Carbon Dioxide and Hydrogen Gas Production

Kinetic analyses of bacterial growth, carbohydrate consumption, and metabolite production of five butyrate-producing clostridial cluster XIVa colon bacteria grown on acetate plus fructose, oligofructose, inulin, or lactate were performed. A gas chromatography method was set up to assess H₂ and CO₂ production online and to ensure complete coverage of all metabolites produced. Method accuracy was confirmed through the calculation of electron and carbon recoveries. Fermentations with Anaerostipes caccae DSM 14662^T, Roseburia faecis DSM 16840^T, Roseburia hominis DSM 16839^T, and Roseburia intestinalis DSM 14610^T revealed similar patterns of metabolite production with butyrate, CO₂, and H₂ as the main metabolites. R. faecis DSM 16840^T and R. intestinalis DSM 14610^T were able to degrade oligofructose, displaying a nonpreferential breakdown mechanism. Lactate consumption was only observed with A. caccae DSM 14662^T. Roseburia inulinivorans DSM 16841^T was the only strain included in the present study that was able to grow on fructose, oligofructose, and inulin. The metabolites produced were lactate, butyrate, and CO₂, without H₂ production, indicating an energy metabolism distinct from that of other Roseburia species. Oligofructose degradation was nonpreferential. In a coculture of R. inulinivorans DSM 16841^T with the highly competitive strain Bifidobacterium longum subsp. longum LMG 11047 on inulin, hardly any production of butyrate and CO₂ was detected, indicating a lack of competitiveness of the butyrate producer. Complete recovery of metabolites during fermentations of clostridial cluster XIVa butyrate-producing colon bacteria allowed stoichiometric balancing of the metabolic pathway for butyrate production, including H₂ formation.The implementation of 16S rRNA gene-based analytical techniques in the ongoing exploration of the microbial diversity of the human colon ecosystem has both broadened and sharpened the prevailing image of its population (17, 24, 32). While a rather conservative perception of the composition of the colon microbiota has dominated gut research for several decades (36), recent studies have revealed the importance of previously largely neglected bacterial groups and have reduced historically numerically overestimated subpopulations to their actual (marginal) size (8, 22, 52). The human colon has been shown to be a remarkably selective environment, which is reflected by a rather shallow microbial diversity (32). Species belonging to the bacterial divisions Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria make up more than 98% of the bacterial population of the human colon (2, 17, 24). However, this superficial uniformity only covers an overwhelming diversity at the lower taxonomic levels; the human colon has been estimated to harbor between 500 and 1,000 species, representing over 7,000 strains, with up to 80% of them considered uncultivable using presently available methodologies (14, 28, 53).Assessing identity and abundance of the major microbial groups composing the colon microbiota is a first and indispensable step toward a better understanding of the ecosystem of the large intestine (48). However, defining a complex ecosystem such as the human colon requires more than the construction of a catalog of its members (32). A major challenge of gastrointestinal microbiology lies in linking phylogenetic subgroups with particular ecological habitats and niches (7, 8, 23). The latter requires further development of highly discriminating 16S rRNA gene-targeted probes to monitor spatial bacterial distribution, combined with renewed efforts toward species isolation through the application of innovative cultivation methods and media, and extensive metabolic characterization of representative strains (19, 35, 48).Recently, a global ecological approach, combining efforts in probe development (1, 27), species isolation (3), and metabolic characterization (4, 11, 15, 20), has led to the identification of a functional group of microorganisms, composed of species belonging to the clostridial clusters IV and XIVa, that are responsible for colon butyrate production. As butyrate is regarded as a key metabolite for the maintenance of colon health, this functional subunit of the colon microbiota could have a major influence on human well-being and might be considered as a target for prebiotic dietary interventions (25, 35, 45). Some recently described lactate- and/or acetate-converting colon butyrate producers have been reported to be able to degrade prebiotic inulin-type fructans, although the kinetics of their respective breakdown mechanisms have hardly been investigated (10, 20). The enhancement of colon butyrate production observed after consumption of oligofructose or inulin (6, 31, 40)—the so-called butyrogenic effect—as well as the limited stimulatory effect of these prebiotics on the clostridial cluster IV and XIVa colon populations (16, 30) have been attributed to cross-feeding with bifidobacteria, which are still considered the primary fructan degraders (5, 38). Anaerostipes caccae as well as Roseburia spp. have been shown to be able to (co)metabolize end products of bifidobacterial fructan fermentation (lactate and/or acetate) or to grow on short oligosaccharides and monosaccharides released by Bifidobacterium spp. during fructan degradation (4, 20).Recently, many clostridial cluster IV and XIVa butyrate producers characterized in detail have been shown to produce gases, mainly CO₂ and H₂ (12, 15, 20, 46). Consequently, they might be responsible for an enhancement of gas production as a result of fructan fermentation, through either cross-feeding or direct degradation of inulin-type fructans (15, 16). Indeed, inulin-type fructan consumption has been reported to cause some gastrointestinal discomfort related to gas production—essentially, flatulence and bloating (43)—while bifidobacteria, the main beneficiaries of dietary fructan intake, do not produce gases (19, 49). Although CO₂ and H₂ production by colon butyrate producers could have implications for human intestinal well-being, (in vitro) production has not been satisfactorily monitored up to now, probably due to limited availability of a performant apparatus for (online) gas analysis (15, 20). Moreover, the currently proposed pathway for colon butyrate production does not provide a conclusive quantitative link between bacterial (co)substrate metabolism and H₂ formation (11).This study investigated the kinetics of inulin-type fructan degradation by representatives of the genera Anaerostipes and Roseburia. A method based on online gas chromatography (GC) was developed to assess gas production qualitatively and quantitatively in a continuously sparged fermentation vessel for complete coverage of metabolite production. The competitiveness of inulin-degrading butyrate producers was investigated through coculture fermentations with Bifidobacterium longum subsp. longum LMG 11047, a strain representing a highly competitive cluster of bifidobacteria that share both high fructose consumption and oligofructose degradation rates and are able to perform partial breakdown of inulin (18, 20). A stoichiometrically balanced pathway for butyrate production, including H₂ production, is proposed.     

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.     

Bile-Inducible Efflux Transporter from Bifidobacterium longum NCC2705, Conferring Bile Resistance

Bifidobacteria are normal inhabitants of the human gut. Some strains of this genus are considered health promoting or probiotic, being included in numerous food products. In order to exert their health benefits, these bacteria must overcome biological barriers, including bile salts, to colonize and survive in specific parts of the intestinal tract. The role of multidrug resistance (MDR) transporters in bile resistance of probiotic bacteria and the effect of bile on probiotic gene expression are not fully understood. In the present study, the effect of subinhibitory concentrations of bile on the expression levels of predicted MDR genes from three different bifidobacterial strains, belonging to Bifidobacterium longum subsp. longum, Bifidobacterium breve, and Bifidobacterium animalis subsp. lactis, was tested. In this way, two putative MDR genes whose expression was induced by bile, BL0920 from B. longum and its homolog, Bbr0838, from B. breve, were identified. The expression of the BL0920 gene in Escherichia coli was shown to confer resistance to bile, likely to be mediated by active efflux from the cells. To the best of our knowledge, this represents the first identified bifidobacterial bile efflux pump whose expression is induced by bile.In the gut, commensal bacteria, including those that have health-promoting or probiotic activity, are challenged by the presence of several toxic compounds of intestinal origin, such as bile salts. Bile is secreted into the duodenum to an estimated concentration of 0.2 to 2% for a given bile salt (14). Bile salts are detergent-like compounds with strong antimicrobial activity (2). Therefore, intestinal microorganisms have developed strategies to tolerate physiological concentrations of bile salts during passage through, or colonization of, the gut.Bifidobacteria are common inhabitants of the human gastrointestinal tract (GIT) (19, 39). Some strains of this genus are known to have probiotic activity and are used as functional ingredients in food products around the world (40). The health-promoting effects attributed to these microorganisms are numerous (20, 42). To exert beneficial actions, these bacteria must overcome biological barriers including acid in the stomach and bile in the intestine in order to, at least temporarily, colonize specific parts of the GIT. Thus, understanding the mechanisms of resistance of a given commensal or probiotic microorganism to toxic substances, such as bile salts, is important in the context of its physiology in the GIT. Although bifidobacterial bile tolerance mechanisms are currently poorly understood, bifidobacteria with increased resistance to bile salts have been obtained by progressive adaptation to gradually increasing concentrations of these compounds (27, 29). Through the analysis of such bile-tolerant derivatives, it was shown that the acquisition of bile resistance coincides with changes in membrane protein profiles (27) and carbohydrate metabolism (35, 38), while also conferring cross-resistance to other environmental stresses (29, 36). This indicates that the bifidobacterial response to bile entails a complex cellular activation/repression process, which impacts on general metabolic pathways (37).Specific bile resistance mechanisms have been described in intestinal bacteria, with bile efflux and bile salt hydrolysis being the most prevalent (31). In this respect, multidrug resistance (MDR) transporters seem to play a crucial role in conferring a bile resistance phenotype. MDR proteins are present in all organisms and frequently confer resistance against several structurally unrelated toxic compounds (31). Research on these transporters has mainly been focused on their role in antibiotic resistance. However, given their ubiquitous presence this does not seem to be their primary function. In fact, recent studies support a role for various MDR transporters in allowing microorganisms to survive, establish, and persist in their (human) host (31).It has been shown elsewhere that MDR proteins confer resistance to bile in different enteric bacteria (32). Bile was found to upregulate the expression of the multidrug efflux system cmeABC in Campylobacter jejuni (22), and inhibition of this pump was found to reduce the colonization ability of the microorganism by reducing bile resistance (21). In vitro and in vivo induction of acrAB expression by bile was observed in Vibrio cholerae (6), and also other MDR proteins appear to be induced in this microorganism (5). In the intestinal bacterium Bacteroides fragilis, the expression of different MDR pumps was also found to be upregulated by bile (34).Several MDR systems have been identified in gram-positive bacteria (including probiotic bacteria) (8, 28), but their role in conferring resistance to intestinal toxic compounds, such as bile salts, and the effect of bile on gene expression have not received much scientific attention. Transporters able to extrude bile salts have been found in gram-positive bacteria such as Lactococcus lactis (44, 45) and Lactobacillus johnsonii (7). In Lactobacillus plantarum a membrane protein whose expression is induced by bile, both in vitro and in vivo, was previously identified (3). Proteins conferring resistance to bile, and whose expression is induced by it, have also been reported in other lactobacilli (30, 43). Just a couple of studies have been published on MDR transporters in bifidobacteria. Margolles and coworkers identified and characterized two MDR transporters from Bifidobacterium breve, BbmAB and BbmR, conferring resistance to antimicrobials (25, 26). In Bifidobacterium longum an MDR transporter, Ctr, was found to export cholate from the cell, conferring resistance to this compound when cloned in a heterologous bacterial host (33). However, there is still a knowledge gap with regard to possible effects of bile on MDR gene expression and the potential role of MDR transporters in bile resistance in bifidobacteria.In the present study, the effect of subinhibitory concentrations of bile on the expression levels of genes encoding bifidobacterial MDR protein homologs was tested. For this purpose, known or putative MDR genes were selected from the genomes of different Bifidobacterium strains belonging to the species B. longum, B. breve, and Bifidobacterium animalis. A putative MDR-encoding gene, present as a homolog in both B. breve and B. longum and whose expression was strongly induced by bile, was identified, and the B. longum gene was then characterized.     

Characterization of Two Novel α-Glucosidases from Bifidobacterium breve UCC2003

Two α-glucosidase-encoding genes (agl1 and agl2) from Bifidobacterium breve UCC2003 were identified and characterized. Based on their similarity to characterized carbohydrate hydrolases, the Agl1 and Agl2 enzymes are both assigned to a subgroup of the glycosyl hydrolase family 13, the α-1,6-glucosidases (EC 3.2.1.10). Recombinant Agl1 and Agl2 into which a His₁₂ sequence was incorporated (Agl1_His and Agl2_His, respectively) exhibited hydrolytic activity towards panose, isomaltose, isomaltotriose, and four sucrose isomers—palatinose, trehalulose, turanose, and maltulose—while also degrading trehalose and, to a lesser extent, nigerose. The preferred substrates for both enzymes were panose, isomaltose, and trehalulose. Furthermore, the pH and temperature optima for both enzymes were determined, showing that Agl1_His exhibits higher thermo and pH optima than Agl2_His. The two purified α-1,6-glucosidases were also shown to have transglycosylation activity, synthesizing oligosaccharides from palatinose, trehalulose, trehalose, panose, and isomaltotriose.The gastrointestinal tract is inhabited by a complex community of microorganisms, also referred to as the microbiota, which are believed to play an important role in human health and disease (39). This concept has been driving extensive attempts to positively influence the composition and/or activity of the intestinal microbiota through the use of so-called probiotics and/or prebiotics. A probiotic has been defined as “a preparation or a product containing viable, defined microorganisms in sufficient numbers, which alter the microflora (by implantation or colonization) in a compartment of the host and by that exert beneficial health effect in this host” (48). A prebiotic has recently been (re)defined as “a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host well-being and health” (42). Finally, a synbiotic is the combination of a probiotic and a prebiotic (16).One of the dominant bacteria of the intestinal microbiota of humans and animals (51) is bifidobacteria. These are gram-positive, pleomorphic, and anaerobic bacteria that have received increasing scientific attention in recent years due to their perceived probiotic activity (15, 27). The growth of gut-derived bifidobacteria has been shown to be selectively stimulated by various dietary carbohydrates that can thus be considered as prebiotics (30). In this context, it is interesting to note that more than 8% of the identified genes on bifidobacterial genomes are predicted to be involved in sugar metabolism, thus indicative of extensive carbon source-degrading abilities (55, 56).Carbohydrate degradation has been extensively studied in a variety of different Bifidobacterium species (reviewed in reference 53). For example, various α- and β-galactosidases have been characterized in Bifidobacterium breve 203 (60), Bifidobacterium adolescentis DSM20083 (20), Bifidobacterium bifidum NCIMB41171 (18), and Bifidobacterium longum MB219 (43). A number of studies have also shown that Bifidobacterium spp. produce various α- and β-glucosidase activities (reviewed in reference 53), while Bifidobacterium infantis ATCC 15697 (58), Bifidobacterium lactis DSM10140(T) (13), and B. breve UCC2003 (45) have been reported to produce β-fructofuranosidases during growth on fructooligosaccharides. Additionally, starch-, amylopectin-, and pullulan-degrading activities in bifidobacteria have been investigated (36, 44). Several β-glucosidases have been biochemically characterized from a number of strains of bifidobacteria, e.g., B. adolescentis Int-57 (8), B. breve clb (35,) and Bifidobacterium sp. strain SEN (59). To date, only two α-glucosidases (AglA and AglB) have been described from B. adolescentis DSM20083 (54). AglA was shown to preferentially hydrolyze isomaltotriose, while AglB exhibits a high preference to maltose. Both AglA and AglB were also demonstrated to have transglycosylation activity. Aside from this report, little is known about the biochemical characteristics of α-glucosidase enzymes from bifidobacteria, although it is a common activity observed among these bacteria (41).Carbohydrates other than the commercially exploited prebiotics, e.g., fructooligosaccharides (such as inulin) and trans-galactooligosaccharides (42), have received relatively little attention with regard to their possible prebiotic properties. Such potential prebiotics are, for example, honey oligosaccharides, some of which are also interesting because of their noncariogenic properties (14). One of the predominant fractions of noncariogenic sugars in honey is isomaltulose (5, 46), also called palatinose or 6-O-α-d-glucopyranosyl-d-fructose, which is a reducing disaccharide and a functional isomer of sucrose. Palatinose possesses approximately one-third of the sweetness of sucrose and is very resistant to acid and invertase hydrolysis (29, 32). The hydrolysis and adsorption of palatinose in the small intestine thus occurs at a much slower rate than does those of sucrose (17), which results in a reduction of the postprandial plasma glucose and insulin levels (3), which means that most palatinose passes through the small intestine to present a growth substrate for elements of the colonic microbiota.In this study, we describe the identification of two genes, agl1 and agl2, present in the genome of B. breve UCC2003 and responsible for the hydrolysis of α-glycosidic linkages, such as those present in palatinose.     

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.     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.     

Caenorhabditis elegans as an Alternative Model Host for Legionella pneumophila,and Protective Effects of Bifidobacterium infantis

The survival times of Caenorhabditis elegans worms infected with Legionella pneumophila from day 7.5 or later after hatching were shorter than those of uninfected worms. However, nematodes fed bifidobacteria prior to Legionella infection were resistant to Legionella. These nematodes may act as a unique alternative host for Legionella research.Legionella pneumophila, an environmental bacterium naturally found in fresh water, is the major causative agent of Legionnaires'' disease (7). Fresh water amoebas, a natural host of Legionella, have been used as an infection model to study invasion of Legionella into human macrophages and subsequent intracellular growth (15). However, analyses using these protozoa have inevitably concentrated on the intracellular lifestyle of L. pneumophila. The fate of Legionella organisms in nonmammalian metazoans had not been described (10) until a very recent report by Brassinga et al. (6).Numerous authors have reported Caenorhabditis elegans to be a suitable model to investigate virulence-associated factors of human pathogens (2, 8, 11, 14, 16, 20, 23, 24, 30, 31, 33). In the present study, we examined whether C. elegans can serve as an alternative host for L. pneumophila. Although the nematocidal activity of Legionella has been described recently, the nematodes in the previous study were infected with the pathogen on buffered charcoal yeast extract (BCYE) agar plates, which can support Legionella growth (6). In contrast, our experiments were independently performed on simple agar plates to exclude the possibility that the inoculated pathogen would have proliferated regardless of whether it had successfully infected the nematodes and derived nutrition from the hosts. Garsin et al. showed that nutrition available in agar plates does influence the virulence of pathogens on the medium (9). Furthermore, some pathogens produce toxic metabolites on nutrient medium in situ (3), and thus, we also avoided this possibility. Moreover, we focused on the effects of worm age, since Legionella is prone to infect elderly people.Age at infection is likely one of the most important determinants of disease morbidity and mortality (18). Since Legionella organisms are prone to infect elderly people opportunistically, infections in young and older nematodes were compared. Furthermore, survival curves were compared between worms fed Escherichia coli OP50 (OP), an international standard food for these organisms, and those fed bifidobacteria prior to infection with Legionella organisms, since lactic acid bacteria exert beneficial effects on human and animal health (21).     

Population Structure of the Lyme Borreliosis Spirochete Borrelia burgdorferi in the Western Black-Legged Tick (Ixodes pacificus) in Northern California

Factors potentially contributing to the lower incidence of Lyme borreliosis (LB) in the far-western than in the northeastern United States include tick host-seeking behavior resulting in fewer human tick encounters, lower densities of Borrelia burgdorferi-infected vector ticks in peridomestic environments, and genetic variation among B. burgdorferi spirochetes to which humans are exposed. We determined the population structure of B. burgdorferi in over 200 infected nymphs of the primary bridging vector to humans, Ixodes pacificus, collected in Mendocino County, CA. This was accomplished by sequence typing the spirochete lipoprotein ospC and the 16S-23S rRNA intergenic spacer (IGS). Thirteen ospC alleles belonging to 12 genotypes were found in California, and the two most abundant, ospC genotypes H3 and E3, have not been detected in ticks in the Northeast. The most prevalent ospC and IGS biallelic profile in the population, found in about 22% of ticks, was a new B. burgdorferi strain defined by ospC genotype H3. Eight of the most common ospC genotypes in the northeastern United States, including genotypes I and K that are associated with disseminated human infections, were absent in Mendocino County nymphs. ospC H3 was associated with hardwood-dominated habitats where western gray squirrels, the reservoir host, are commonly infected with LB spirochetes. The differences in B. burgdorferi population structure in California ticks compared to the Northeast emphasize the need for a greater understanding of the genetic diversity of spirochetes infecting California LB patients.In the United States, Lyme borreliosis (LB) is the most commonly reported vector-borne illness and is caused by infection with the spirochete Borrelia burgdorferi (3, 9, 52). The signs and symptoms of LB can include a rash, erythema migrans, fever, fatigue, arthritis, carditis, and neurological manifestations (50, 51). The black-legged tick, Ixodes scapularis, and the western black-legged tick, Ixodes pacificus, are the primary vectors of B. burgdorferi to humans in the United States, with the former in the northeastern and north-central parts of the country and the latter in the Far West (9, 10). These ticks perpetuate enzootic transmission cycles together with a vertebrate reservoir host such as the white-footed mouse, Peromyscus leucopus, in the Northeast and Midwest (24, 35), or the western gray squirrel, Sciurus griseus, in California (31, 46).B. burgdorferi is a spirochete species with a largely clonal population structure (14, 16) comprising several different strains or lineages (8). The polymorphic ospC gene of B. burgdorferi encodes a surface lipoprotein that increases expression within the tick during blood feeding (47) and is required for initial infection of mammalian hosts (25, 55). To date, approximately 20 North American ospC genotypes have been described (40, 45, 49, 56). At least four, and possibly up to nine, of these genotypes are associated with B. burgdorferi invasiveness in humans (1, 15, 17, 49, 57). Restriction fragment length polymorphism (RFLP) and, subsequently, sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) are used as molecular typing tools to investigate genotypic variation in B. burgdorferi (2, 36, 38, 44, 44, 57). The locus maintains a high level of variation between related species, and this variation reflects the heterogeneity found at the genomic level of the organism (37). The IGS and ospC loci appear to be linked (2, 8, 26, 45, 57), but the studies to date have not been representative of the full range of diversity of B. burgdorferi in North America.Previous studies in the northeastern and midwestern United States have utilized IGS and ospC genotyping to elucidate B. burgdorferi evolution, host strain specificity, vector-reservoir associations, and disease risk to humans. In California, only six ospC and five IGS genotypes have been described heretofore in samples from LB patients or I. pacificus ticks (40, 49, 56) compared to approximately 20 ospC and IGS genotypes identified in ticks, vertebrate hosts, or humans from the Northeast and Midwest (8, 40, 45, 49, 56). Here, we employ sequence analysis of both the ospC gene and IGS region to describe the population structure of B. burgdorferi in more than 200 infected I. pacificus nymphs from Mendocino County, CA, where the incidence of LB is among the highest in the state (11). Further, we compare the Mendocino County spirochete population to populations found in the Northeast.     

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.