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.     

Replacement of the Replication Factors of Porcine Circovirus (PCV) Type 2 with Those of PCV Type 1 Greatly Enhances Viral Replication In Vitro

Porcine circovirus type 1 (PCV1), originally isolated as a contaminant of PK-15 cells, is nonpathogenic, whereas porcine circovirus type 2 (PCV2) causes an economically important disease in pigs. To determine the factors affecting virus replication, we constructed chimeric viruses by swapping open reading frame 1 (ORF1) (rep) or the origin of replication (Ori) between PCV1 and PCV2 and compared the replication efficiencies of the chimeric viruses in PK-15 cells. The results showed that the replication factors of PCV1 and PCV2 are fully exchangeable and, most importantly, that both the Ori and rep of PCV1 enhance the virus replication efficiencies of the chimeric viruses with the PCV2 backbone.Porcine circovirus (PCV) is a single-stranded DNA virus in the family Circoviridae (34). Type 1 PCV (PCV1) was discovered in 1974 as a contaminant of porcine kidney cell line PK-15 and is nonpathogenic in pigs (31-33). Type 2 PCV (PCV2) was discovered in piglets with postweaning multisystemic wasting syndrome (PMWS) in the mid-1990s and causes porcine circovirus-associated disease (PCVAD) (1, 9, 10, 25). PCV1 and PCV2 have similar genomic organizations, with two major ambisense open reading frames (ORFs) (16). ORF1 (rep) encodes two viral replication-associated proteins, Rep and Rep′, by differential splicing (4, 6, 21, 22). The Rep and Rep′ proteins bind to specific sequences within the origin of replication (Ori) located in the intergenic region, and both are responsible for viral replication (5, 7, 8, 21, 23, 28, 29). ORF2 (cap) encodes the immunogenic capsid protein (Cap) (26). PCV1 and PCV2 share approximately 80%, 82%, and 62% nucleotide sequence identity in the Ori, rep, and cap, respectively (19).In vitro studies using a reporter gene-based assay system showed that the replication factors of PCV1 and PCV2 are functionally interchangeable (2-6, 22), although this finding has not yet been validated in a live infectious-virus system. We have previously shown that chimeras of PCV in which cap has been exchanged between PCV1 and PCV2 are infectious both in vitro and in vivo (15), and an inactivated vaccine based on the PCV1-PCV2 cap (PCV1-cap2) chimera is used in the vaccination program against PCVAD (13, 15, 18, 27).PCV1 replicates more efficiently than PCV2 in PK-15 cells (14, 15); thus, we hypothesized that the Ori or rep is directly responsible for the differences in replication efficiencies. The objectives of this study were to demonstrate that the Ori and rep are interchangeable between PCV1 and PCV2 in a live-virus system and to determine the effects of swapped heterologous replication factors on virus replication efficiency in vitro.     

XRCC2 and XRCC3 Regulate the Balance between Short- and Long-Tract Gene Conversions between Sister Chromatids

Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of DNA lesions associated with replication and is thought to be important for suppressing genomic instability. The mechanisms regulating the initiation and termination of SCR in mammalian cells are poorly understood. Previous work has implicated all the Rad51 paralogs in the initiation of gene conversion and the Rad51C/XRCC3 complex in its termination. Here, we show that hamster cells deficient in the Rad51 paralog XRCC2, a component of the Rad51B/Rad51C/Rad51D/XRCC2 complex, reveal a bias in favor of long-tract gene conversion (LTGC) during SCR. This defect is corrected by expression of wild-type XRCC2 and also by XRCC2 mutants defective in ATP binding and hydrolysis. In contrast, XRCC3-mediated homologous recombination and suppression of LTGC are dependent on ATP binding and hydrolysis. These results reveal an unexpectedly general role for Rad51 paralogs in the control of the termination of gene conversion between sister chromatids.DNA double-strand breaks (DSBs) are potentially dangerous lesions, since their misrepair may cause chromosomal translocations, gene amplifications, loss of heterozygosity (LOH), and other types of genomic instability characteristic of human cancers (7, 9, 21, 40, 76, 79). DSBs are repaired predominantly by nonhomologous end joining or homologous recombination (HR), two evolutionarily conserved DSB repair mechanisms (8, 12, 16, 33, 48, 60, 71). DSBs generated during the S or G₂ phase of the cell cycle may be repaired preferentially by HR, using the intact sister chromatid as a template for repair (12, 26, 29, 32, 71). Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of DSBs, which has led to the proposal that SCR protects against genomic instability, cancer, and aging. Indeed, a number of human cancer predisposition genes are implicated in SCR control (10, 24, 45, 57, 75).HR entails an initial processing of the DSB to generate a free 3′ single-stranded DNA (ssDNA) overhang (25, 48, 56). This is coupled to the loading of Rad51, the eukaryotic homolog of Escherichia coli RecA, which polymerizes to form an ssDNA-Rad51 “presynaptic” nucleoprotein filament. Formation of the presynaptic filament is tightly regulated and requires the concerted action of a large number of gene products (55, 66, 68). Rad51-coated ssDNA engages in a homology search by invading homologous duplex DNA. If sufficient homology exists between the invading and invaded strands, a triple-stranded synapse (D-loop) forms, and the 3′ end of the invading (nascent) strand is extended, using the donor as a template for gene conversion. This recombination intermediate is thought to be channeled into one of the following two major subpathways: classical gap repair or synthesis-dependent strand annealing (SDSA) (48). Gap repair entails the formation of a double Holliday junction, which may resolve into either crossover or noncrossover products. Although this is a major pathway in meiotic recombination, crossing-over is highly suppressed in somatic eukaryotic cells (26, 44, 48). Indeed, the donor DNA molecule is seldom rearranged during somatic HR, suggesting that SDSA is the major pathway for the repair of somatic DSBs (26, 44, 49, 69). SDSA terminates when the nascent strand is displaced from the D-loop and pairs with the second end of the DSB to form a noncrossover product. The mechanisms underlying displacement of the nascent strand are not well understood. However, failure to displace the nascent strand might be expected to result in the production of longer gene conversion tracts during HR (36, 44, 48, 63).Gene conversion triggered in response to a Saccharomyces cerevisiae or mammalian chromosomal DSB generally results in the copying of a short (50- to 300-bp) stretch of information from the donor (short-tract gene conversion [STGC]) (14, 47, 48, 67, 69). A minority of gene conversions in mammalian cells entail more-extensive copying, generating gene conversion tracts that are up to several kilobases in length (long-tract gene conversion [LTGC]) (26, 44, 51, 54, 64). In yeast, very long gene conversions can result from break-induced replication (BIR), a highly processive form of gene conversion in which a bona fide replication fork is thought to be established at the recombination synapse (11, 36, 37, 39, 61, 63). In contrast, SDSA does not require lagging-strand polymerases and appears to be much less processive than a conventional replication fork (37, 42, 78). BIR in yeast has been proposed to play a role in LOH in aging yeast, telomere maintenance, and palindromic gene amplification (5, 41, 52). It is unclear to what extent a BIR-like mechanism operates in mammalian cells, although BIR has been invoked to explain telomere elongation in tumors lacking telomerase (13). It is currently unknown whether LTGC and STGC in somatic mammalian cells are products of mechanistically distinct pathways or whether they represent alternative outcomes of a common SDSA pathway.Vertebrate cells contain five Rad51 paralogs—polypeptides with limited sequence homology to Rad51—Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3 (74). The Rad51 paralogs form the following two major complexes: Rad51B/Rad51C/Rad51D/XRCC2 (BCDX2) and Rad51C/XRCC3 (CX3) (38, 73). Genetic deletion of any one of the rad51 paralogs in the mouse germ line produces early embryonic lethality, and mouse or chicken cells lacking any of the rad51 paralogs reveal hypersensitivity to DNA-damaging agents, reduced frequencies of HR and of sister chromatid exchanges, increased chromatid-type errors, and defective sister chromatid cohesion (18, 72, 73, 82). Collectively, these data implicate the Rad51 paralogs in SCR regulation. The purified Rad51B/Rad51C complex has been shown to assist Rad51-mediated strand exchange (62). XRCC3 null or Rad51C null hamster cells reveal a bias toward production of longer gene conversion tracts, suggesting a role for the CX3 complex in late stages of SDSA (6, 44). Rad51C copurifies with branch migration and Holliday junction resolution activities in mammalian cell extracts (35), and XRCC3, but not XRCC2, facilitates telomere shortening by reciprocal crossing-over in telomeric T loops (77). These data, taken together with the meiotic defects observed in Rad51C hypomorphic mice, suggest a specialized role for CX3, but not for BCDX2, in resolving Holliday junction structures (31, 58).To further address the roles of Rad51 paralogs in late stages of recombination, we have studied the balance between long-tract (>1-kb) and short-tract (<1-kb) SCR in XRCC2 mutant hamster cells. We found that DSB-induced gene conversion in both XRCC2 and XRCC3 mutant cells is biased in favor of LTGC. These defects were suppressed by expression of wild-type (wt) XRCC2 or XRCC3, respectively, although the dependence upon ATP binding and hydrolysis differed between the two Rad51 paralogs. These results indicate that Rad51 paralogs play a more general role in determining the balance between STGC and LTGC than was previously appreciated and suggest roles for both the BCDX2 and CX3 complexes in influencing the termination of gene conversion in mammals.     

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.     

Elastase Digests: New Ammunition for Shotgun Membrane Proteomics

Despite many advances in membrane proteomics during the last decade the fundamental problem of accessing the transmembrane regions itself has only been addressed to some extent. The present study establishes a method for the nano-LC-based analysis of complex membrane proteomes on the basis of a methanolic porcine pancreatic elastase digest to increase transmembrane coverage. Halobacterium salinarium purple and Corynebacterium glutamicum membranes were successfully analyzed by using the new protocol. We demonstrated that elastase digests yield a large proportion of transmembrane peptides, facilitating membrane protein identification. The potential for characterization of a membrane protein through full sequence coverage using elastase is there but is restricted to the higher abundance protein components. Compatibility of the work flow with the two most common mass spectrometric ionization techniques, ESI and MALDI, was shown. Currently better results are obtained using ESI mainly because of the low response of MALDI for strictly neutral peptides. New findings concerning elastase specificity in complex protein mixtures reveal a new prospect beyond the application in shotgun experiments. Furthermore peptide mass fingerprinting with less specific enzymes might be done in the near future but requires an adaptation of current search algorithms to the new proteases.Upon the introduction of modern mass spectrometric ionization techniques, such as MALDI (1) and ESI (2), extremely powerful and valuable tools were given to researchers for the identification and characterization of proteins. Nevertheless the intricate analysis of membrane subproteomes still represents one of the major challenges despite the amount of “success” reported in the literature. Until now, there was no general protocol available to address membrane proteomes as a whole, demonstrating their degree of difficulty and complexity.During the past years, two different strategies in proteome analysis have evolved: the more widespread bottom-up and top-down proteomics. The latter approach has been shown to provide access to the transmembranal regions of membrane proteins and has the power to characterize complete protein primary structures including labile covalent modifications (3, 4). Nevertheless there are limitations due to sample complexity, emphasizing the need for a liquid chromatographic separation on the protein level, which is comparable to LC peptide separation. Therefore, the bottom-up variant is the most commonly used method for the proteomics analysis of complex membrane samples. Improvements in the bottom-up work flow were achieved by adapting sample cleanup and prefractionation processes (5–10) and subsequently by the development of modified and optimized separation techniques. The two main work flows, the gel-based approach mainly carried out with 2D¹ SDS-PAGE (11, 12) and the shotgun identification of proteolytically digested protein mixtures and their multidimensional separation via liquid chromatography (13), had to be adapted. The separation of proteins via classical 2D SDS-PAGE is only possible up to a GRAVY score of ∼0.4 (14, 15). Despite lacking the separation power of the IEF-SDS-PAGE system, derived techniques like the doubled SDS- (16) and 16-benzyldimethyl-n-hexadecylammonium chloride/SDS-PAGE (17) represent an improvement for hydrophobic proteins. Advances in the nLC separation of hydrophobic peptides, e.g. the separation at elevated temperatures (18) and the use of LC-compatible detergents (19), yielded significant success. When combining both prominent separation techniques, the one-dimensional SDS-LC analysis proved to be more effective than 2D PAGE as well (20, 21).One of the remaining steps still offering room for improvements is the proteolytic procedure. The original multidimensional protein identification technique using trypsin has been successfully improved by the application of proteinase K under high pH conditions, yielding an increased number of membrane protein identifications (22). Further modification of this method by recleaving the isolated membranous parts with cyanogen bromide increased the accessibility to membrane-spanning peptides (18).Despite the suitability of proteinase K for the shotgun analysis of membrane proteomes, trypsin is the prevalent enzyme choice in most current proteomics approaches because of its very specific cleavage behavior. Calculations have implied that alternative proteases are preferentially suited for the analysis of membrane proteomes (23). Proteases other than trypsin have been utilized only to a small degree and mostly for the targeted analysis of protein complexes. Pepsin, for example, was used for the characterization of an aquaporin (24), and elastase and subtilisin were used as proteases in a triple digest approach of protein complexes and lens tissue (25). Further improvements in the accessibility of the membrane proteome have been shown for tryptic (26, 27) and tryptic/chymotryptic (28) digests by performing the proteolytic treatment in the presence of methanol.During membrane proteomics method development, purple membranes from Halobacterium sp. NRC-1, consisting to a large extent of the H⁺-ATPase bacteriorhodopsin, have widely been used as reference in a variety of cases. Full BR sequence coverage has been reported by several publications using different techniques (27, 29). Such exorbitant sequence coverage amounts are only achievable for the most abundant proteins within a complex mixture. The identification of less expressed proteins, which certainly are more in the researcher''s interest, occurs via lower peptide numbers (27).Generally the basic tryptic cleavage sites are predominantly located in the loops facing the cytoplasm and to a lesser extent in the extracellular or periplasmic loops but are very scarce within the transmembrane helix stretches. This mostly limits the potential tryptic fragments to loop components and larger peptides containing at least one TM helix. The use of another protease, preferentially cleaving after neutral aliphatic residues, should not succumb to this lack of cleavage sites.One of the previously mentioned proteases, porcine pancreatic elastase, was reported to possess potential cleavage specificity at the carboxyl-terminal side of small neutral amino acids (30). It has been used previously for the examination of single protein phosphorylations (31) but was described not to be suited for the cleavage of complex membrane-containing samples because of a supposedly limited activity when applied to them (22). Despite this finding, elastase has been successfully utilized in a mass spectrometric analysis as early as 1974 when it was regarded as an ideal protease for future mass spectrometric studies (32). At the same time, most of the experiments to characterize elastase by analyzing its S₁ pocket (for nomenclature, see Ref. 33) binding capabilities (34) as well as cleavage preferences of ester and protein substrates (35–39) were performed.Based on previous research concerning elastase, we set up an nLC-based membrane proteomics analysis. This large data set was used to characterize the protease behavior of elastase and the physicochemical properties of the detected peptides. The PM model was used to establish a method to analyze more complex Corynebacterium glutamicum membranes, which have been analyzed previously using different 2D techniques (7, 28). We demonstrated that a combination of elastase and methanol is particularly suitable for an nLC-based membrane proteome analysis and results in a significantly increased number of TM peptides. Additionally the promising PMF application of elastase digests is discussed.     

Fundamental Difference in the Content of High-Mannose Carbohydrate in the HIV-1 and HIV-2 Lineages

The virus-encoded envelope proteins of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) typically contain 26 to 30 sites for N-linked carbohydrate attachment. N-linked carbohydrate can be of three major types: high mannose, complex, or hybrid. The lectin proteins from Galanthus nivalis (GNA) and Hippeastrum hybrid (HHA), which specifically bind high-mannose carbohydrate, were found to potently inhibit the replication of a pathogenic cloned SIV from rhesus macaques, SIVmac239. Passage of SIVmac239 in the presence of escalating concentrations of GNA and HHA yielded a lectin-resistant virus population that uniformly eliminated three sites (of 26 total) for N-linked carbohydrate attachment (Asn-X-Ser or Asn-X-Thr) in the envelope protein. Two of these sites were in the gp120 surface subunit of the envelope protein (Asn244 and Asn460), and one site was in the envelope gp41 transmembrane protein (Asn625). Maximal resistance to GNA and HHA in a spreading infection was conferred to cloned variants that lacked all three sites in combination. Variant SIV gp120s exhibited dramatically decreased capacity for binding GNA compared to SIVmac239 gp120 in an enzyme-linked immunosorbent assay (ELISA). Purified gp120s from six independent HIV type 1 (HIV-1) isolates and two SIV isolates from chimpanzees (SIVcpz) consistently bound GNA in ELISA at 3- to 10-fold-higher levels than gp120s from five SIV isolates from rhesus macaques or sooty mangabeys (SIVmac/sm) and four HIV-2 isolates. Thus, our data indicate that characteristic high-mannose carbohydrate contents have been retained in the cross-species transmission lineages for SIVcpz-HIV-1 (high), SIVsm-SIVmac (low), and SIVsm-HIV-2 (low).The envelope proteins of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) are heavily glycosylated. N-linked carbohydrate is attached to the nascent protein at the asparagine of the consensus sequence N-X-S or N-X-T, where X is any amino acid except a proline (31, 52, 53). The number of potential N-linked carbohydrate attachment sites in the surface subunit of Env (gp120) ranges from 18 to 33, with a median of 25 (34, 65). There are typically 3 or 4 potential N-linked sites in the ectodomain of the Env transmembrane protein (gp41) (34).N-linked glycosylation of a protein consists of the en bloc transfer of the carbohydrate core oligosaccharide (two N-acetylglucosamines, nine mannoses, and three glucoses) from dolichol to the asparagine of the N-linked attachment site (8, 60). Initially the attached carbohydrate is processed into the high-mannose type (8). In the Golgi complex, high-mannose carbohydrate may be further processed into complex or hybrid oligosaccharides (58). Incomplete processing of N-linked carbohydrate results in the production of high-mannose carbohydrate chains, which terminate in mannose (58). Fully processed complex carbohydrate chains terminate in galactose, N-acetylglucosamine, sialic acid, or glucose (33, 57). Hybrid carbohydrate chains have two branches from the core, one that terminates in mannose and one that terminates in a sugar of the complex type (63).Glycoproteins exist as a heterogeneous population, exhibiting heterogeneity with respect to the proportion of potential glycosylation sites that are occupied and to the oligosaccharide structure observed at each site. Factors that influence the type of carbohydrate chain that is attached at any one N-linked site are the accessibility of the carbohydrate chain to processing enzymes (49), protein sequences surrounding the site (5, 40), and the type of cell from which the protein is produced (19).The N-linked carbohydrate chains of HIV and SIV Env are critical for the proper folding and cleavage of the fusion-competent envelope spike (20, 59, 61). After Env is assembled, enzymatic removal of N-linked carbohydrate does not dramatically affect the functional conformation (2, 6, 7, 13, 24, 38). It is generally accepted that the carbohydrate attached to Env limits the ability of the underlying protein to be recognized by B cells (11, 48, 62). This carbohydrate also shields protein epitopes that would otherwise be the direct targets of antibodies that neutralize viral infection (41, 48, 62, 64). Furthermore, the high-mannose carbohydrates of HIV and SIV Env bind dynamically to an array of lectin proteins that are part of the host lymphoreticular system. The interaction of viral high-mannose carbohydrate with host lectin proteins has been associated with the enhancement (9, 16, 17, 43-45) or suppression (42, 56) of viral infection of CD4-positive T cells. The high-mannose carbohydrate of Env is also known to activate the release of immune-modulatory proteins from a subset of host antigen-presenting cells (12, 54).The plant lectin proteins from Galanthus nivalis (GNA) and Hippeastrum hybrid (HHA) specifically bind terminal α-1,3- and/or α-1,6-mannose of high-mannose oligosaccharides but not hybrid oligosaccharides (28, 55). GNA and HHA inhibit the replication of HIV-1 and SIVmac251, and uncloned, resistant populations of virus have been selected (3, 14). In this report, we define two N-linked sites in the external surface glycoprotein gp120 and one in the transmembrane glycoprotein gp41 whose mutation imparts high-level resistance to the inhibitory effects of GNA and HHA to cloned SIVmac239. Furthermore, using a GNA-binding enzyme-linked immunosorbent assay (ELISA), we show that assorted HIV-1 and SIVcpz gp120s consistently are considerably higher in mannose content than assorted gp120s from SIVmac, SIVsm, and HIV-2. These results shed new light on the impact of virus-host evolutionary dynamics on viral carbohydrate composition, and they may have important implications for the mechanisms by which long-standing natural hosts such as sooty mangabeys can resist generalized lymphoid activation and disease despite high levels of SIV replication.     

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.