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Cyanophycin (multi-l-arginyl-poly-l-aspartic acid; also known as cyanophycin grana peptide [CGP]) is a putative precursor for numerous biodegradable technically used chemicals. Therefore, the biosynthesis and production of the polymer in recombinant organisms is of special interest. The synthesis of cyanophycin derivatives consisting of a wider range of constituents would broaden the applications of this polymer. We applied recombinant Saccharomyces cerevisiae strains defective in arginine metabolism and expressing the cyanophycin synthetase of Synechocystis sp. strain PCC 6308 in order to synthesize CGP with citrulline and ornithine as constituents. Strains defective in arginine degradation (Car1 and Car2) accumulated up to 4% (wt/wt) CGP, whereas strains defective in arginine synthesis (Arg1, Arg3, and Arg4) accumulated up to 15.3% (wt/wt) of CGP, which is more than twofold higher than the previously content reported in yeast and the highest content ever reported in eukaryotes. Characterization of the isolated polymers by different analytical methods indicated that CGP synthesized by strain Arg1 (with argininosuccinate synthetase deleted) consisted of up to 20 mol% of citrulline, whereas CGP from strain Arg3 (with ornithine carbamoyltransferase deleted) consisted of up to 8 mol% of ornithine, and CGP isolated from strain Arg4 (with argininosuccinate lyase deleted) consisted of up to 16 mol% lysine. Cultivation experiments indicated that the incorporation of citrulline or ornithine is enhanced by the addition of low amounts of arginine (2 mM) and also by the addition of ornithine or citrulline (10 to 40 mM), respectively, to the medium.Cyanophycin (multi-l-arginyl-poly-[l-aspartic acid]), also referred to as cyanophycin grana peptide (CGP), represents a polydisperse nonribosomally synthesized polypeptide consisting of poly(aspartic acid) as backbone and arginine residues bound to each aspartate (49) (Fig. (Fig.1).1). One enzyme only, referred to as cyanophycin synthetase (CphA), catalyzes the synthesis of the polymer from amino acids (55). Several CphAs originating from different bacteria exhibit specific features (2, 7, 5, 32, 50, 51). CphAs from the cyanobacteria Synechocystis sp. strain PCC 6308 and Anabaena variabilis ATCC 29413, respectively, exhibit a wide substrate range in vitro (2, 7), whereas CphA from Acinetobacter baylyi or Nostoc ellipsosporum incorporates only aspartate and arginine (23, 24, 32). CphA from Thermosynechococcus elongatus catalyzes the synthesis of CGP primer independently (5); CphA from Synechococcus sp. strain MA19 exhibits high thermostability (22). Furthermore, two types of CGP were observed concerning its solubility behavior: (i) a water-insoluble type that becomes soluble at high or low pH (34, 48) and (ii) a water-soluble type that was only recently observed in recombinant organisms (19, 26, 42, 50, 56). In the past, bacteria were mainly applied for the synthesis of CGP (3, 14, 18, 53), whereas recently there has been greater interest in synthesis in eukaryotes (26, 42, 50). CGP was accumulated to almost 7% (wt/wt) of dry matter in recombinant Nicotiana tabacum and Saccharomyces cerevisiae (26, 50).Open in a separate windowFIG. 1.Chemical structures of dipeptide building blocks of CGP variants detected in vivo. Structure: 1, aspartate-arginine; 2, aspartate-lysine; 3, aspartate-citrulline; 4, aspartate-ornithine. Aspartic acid is presented in black; the second amino acid of the dipeptide building blocks is shown in gray. The nomenclature of the carbon atoms is given.In S. cerevisiae the arginine metabolism is well understood and has been investigated (30) (see Fig. Fig.2).2). Arginine is synthesized from glutamate via ornithine and citrulline in eight successive steps. The enzymes acetylglutamate synthase, acetylglutamate kinase, N-acetyl-γ-glutamylphosphate reductase, and acetylornithine aminotransferase are involved in the formation of N-α-acetylornithine. The latter is converted to ornithine by acetylornithine acetyltransferase. In the next step, ornithine carbamoyltransferase (ARG3) condenses ornithine with carbamoylphosphate, yielding citrulline. Citrulline is then converted to l-argininosuccinate by argininosuccinate synthetase. The latter is in the final step cleaved into fumarate and arginine by argininosuccinate lyase (ARG4). The first five steps occur in the mitochondria, whereas the last three reactions occur in the cytosol (28, 54). Arginine degradation is initiated by arginase (CAR1) and ornithine aminotransferase (CAR2) (10, 11, 38, 39).Open in a separate windowFIG. 2.Schematic overview of the arginine metabolism in S. cerevisiae. Reactions shown in the shaded area occur in the mitochondria, while the other reactions are catalyzed in the cytosol. Abbreviations: ARG2, acetylglutamate synthase; ARG6, acetylglutamate kinase; ARG5, N-acetyl-γ-glutamyl-phosphate reductase; ARG8, acetylornithine aminotransferase; ECM40, acetylornithine acetyltransferase; ARG1, argininosuccinate synthetase; ARG3, ornithine carbamoyltransferase; ARG4, argininosuccinate lyase; CAR1, arginase; CAR2, ornithine aminotransferase.A multitude of putative applications for CGP derivatives are available (29, 41, 45, 47), thus indicating a need for efficient biotechnological production and for further investigations concerning the synthesis of CGP with alternative properties and different constituents. It is not only the putative application of the polymer as a precursor for poly(aspartic acid), which is used as biodegradable alternative for poly(acrylic acid) or for bulk chemicals, that makes CGP interesting (29, 45-47). In addition, a recently developed process for the production of dipeptides from CGP as a precursor makes the synthesis of CGP variants worthwhile (43). Dipeptides play an important role in medicine and pharmacy, e.g., as additives for malnourished patients, as treatments against liver diseases, or as aids for muscle proliferation (43). Because dipeptides are synthesized chemically (40) or enzymatically (6), novel biotechnological production processes are welcome.  相似文献   

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Complex N-glycans flank the receptor binding sites of the outer domain of HIV-1 gp120, ostensibly forming a protective “fence” against antibodies. Here, we investigated the effects of rebuilding this fence with smaller glycoforms by expressing HIV-1 pseudovirions from a primary isolate in a human cell line lacking N-acetylglucosamine transferase I (GnTI), the enzyme that initiates the conversion of oligomannose N-glycans into complex N-glycans. Thus, complex glycans, including those that surround the receptor binding sites, are replaced by fully trimmed oligomannose stumps. Conversely, the untrimmed oligomannoses of the silent domain of gp120 are likely to remain unchanged. For comparison, we produced a mutant virus lacking a complex N-glycan of the V3 loop (N301Q). Both variants exhibited increased sensitivities to V3 loop-specific monoclonal antibodies (MAbs) and soluble CD4. The N301Q virus was also sensitive to “nonneutralizing” MAbs targeting the primary and secondary receptor binding sites. Endoglycosidase H treatment resulted in the removal of outer domain glycans from the GnTI- but not the parent Env trimers, and this was associated with a rapid and complete loss in infectivity. Nevertheless, the glycan-depleted trimers could still bind to soluble receptor and coreceptor analogs, suggesting a block in post-receptor binding conformational changes necessary for fusion. Collectively, our data show that the antennae of complex N-glycans serve to protect the V3 loop and CD4 binding site, while N-glycan stems regulate native trimer conformation, such that their removal can lead to global changes in neutralization sensitivity and, in extreme cases, an inability to complete the conformational rearrangements necessary for infection.The intriguing results of a recent clinical trial suggest that an effective HIV-1 vaccine may be possible (97). Optimal efficacy may require a component that induces broadly neutralizing antibodies (BNAbs) that can block virus infection by their exclusive ability to recognize the trimeric envelope glycoprotein (Env) spikes on particle surfaces (43, 50, 87, 90). Env is therefore at the center of vaccine design programs aiming to elicit effective humoral immune responses.The amino acid sequence variability of Env presents a significant challenge for researchers seeking to elicit broadly effective NAbs. Early sequence comparisons revealed, however, that the surface gp120 subunit can be divided into discrete variable and conserved domains (Fig. (Fig.1A)1A) (110), the latter providing some hope for broadly effective NAb-based vaccines. Indeed, the constraints on variability in the conserved domains of gp120 responsible for binding the host cell receptor CD4, and coreceptor, generally CCR5, provide potential sites of vulnerability. However, viral defense strategies, such as the conformational masking of conserved epitopes (57), have made the task of eliciting bNAbs extremely difficult.Open in a separate windowFIG. 1.Glycan biosynthesis and distribution on gp120 and gp41. (A) Putative carbohydrate modifications are shown on gp120 and gp41 secondary structures, based on various published works (26, 42, 63, 74, 119, 128). The gp120 outer domain is indicated, as are residues that form the SOS gp120-gp41 disulfide bridge. The outer domain is divided into neutralizing and silent faces. Symbols distinguish complex, oligomannose, and unknown glycans. Generally, the complex glycans of the outer domain line the receptor binding sites of the neutralizing face, while the oligomannose glycans of the outer domain protect the silent domain (105). Asterisks denote sequons that are unlikely to be utilized, including position 139 (42), position 189 (26, 42), position 406 (42, 74), and position 637 (42). Glycans shown in gray indicate when sequon clustering may lead to some remaining unused, e.g., positions 156 and 160 (42, 119), positions 386, 392, and 397 (42), and positions 611 and 616 (42). There is also uncertainty regarding some glycan identities: glycans at positions 188, 355, 397, and 448 are not classified as predominantly complex or oligomannose (26, 42, 63, 128). The number of mannose moieties on oligomannose glycans can vary, as can the number of antennae and sialic acids on complex glycans (77). The glycan at position 301 appears to be predominantly a tetra-antennary complex glycan, as is the glycan at position 88, while most other complex glycans are biantennary (26, 128). (B) Schematic of essential steps of glycan biosynthesis from the Man9GlcNAc2 precursor to a mature multiantennary complex glycan. Mannosidase I progressively removes mannose moieties from the precursor, in a process that can be inhibited by the drug kifunensine. GnTI then transfers a GlcNAc moiety to the D1 arm of the resulting Man5GlcNAc2 intermediate, creating a hybrid glycan. Mannose trimming of the D2 and D3 arms then allows additional GlcNAc moieties to be added by a series of GnT family enzymes to form multiantennary complexes. This process can be inhibited by swainsonine. The antennae are ultimately capped and decorated by galactose and sialic acid. Hybrid and complex glycans are usually fucosylated at the basal GlcNAc, rendering them resistant to endo H digestion. However, NgF is able to remove all types of glycan.Carbohydrates provide a layer of protection against NAb attack (Fig. (Fig.1A).1A). As glycans are considered self, antibody responses against them are thought to be regulated by tolerance mechanisms. Thus, a glycan network forms a nonimmunogenic “cloak,” protecting the underlying protein from antibodies (3, 13, 20, 29, 39, 54, 65, 67, 74, 85, 96, 98, 117, 119, 120). The extent of this protection can be illustrated by considering the ways in which glycans differ from typical amino acid side chains. First, N-linked glycans are much larger, with an average mass more than 20 times that of a typical amino acid R-group. They are also usually more flexible and may therefore affect a greater volume of surrounding space. In the more densely populated parts of gp120, the carbohydrate field may even be stabilized by sugar-sugar hydrogen bonds, providing even greater coverage (18, 75, 125).The process of N-linked glycosylation can result in diverse structures that may be divided into three categories: oligomannose, hybrid, and complex (56). Each category shares a common Man3GlcNAc2 pentasaccharide stem (where Man is mannose and GlcNAc is N-acetylglucosamine), to which up to six mannose residues are attached in oligomannose N-glycans, while complex N-glycans are usually larger and may bear various sizes and numbers of antennae (Fig. (Fig.1B).1B). Glycan synthesis begins in the endoplasmic reticulum, where N-linked oligomannose precursors (Glc3Man9GlcNAc2; Glc is glucose) are transferred cotranslationally to the free amide of the asparagine in a sequon Asn-X-Thr/Ser, where X is not Pro (40). Terminal glucose and mannose moieties are then trimmed to yield Man5GlcNAc2 (Fig. (Fig.1B).1B). Conversion to a hybrid glycan is then initiated by N-acetylglucosamine transferase I (GnTI), which transfers a GlcNAc moiety to the D1 arm of the Man5GlcNAc2 substrate (19) (Fig. (Fig.1B).1B). This hybrid glycoform is then a substrate for modification into complex glycans, in which the D2 and D3 arm mannose residues are replaced by complex antennae (19, 40, 56). Further enzymatic action catalyzes the addition of α-1-6-linked fucose moiety to the lower GlcNAc of complex glycan stems, but usually not to oligomannose glycan stems (Fig. (Fig.1B)1B) (21, 113).Most glycoproteins exhibit only fully mature complex glycans. However, the steric limitations imposed by the high density of glycans on some parts of gp120 lead to incomplete trimming, leaving “immature” oligomannose glycans (22, 26, 128). Spatial competition between neighboring sequons can sometimes lead to one or the other remaining unutilized, further distancing the final Env product from what might be expected based on its primary sequence (42, 48, 74, 119). An attempt to assign JR-FL gp120 and gp41 sequon use and types, based on various studies, is shown in Fig. Fig.1A1A (6, 26, 34, 35, 42, 63, 71, 74, 119, 128). At some positions, the glycan type is conserved. For example, the glycan at residue N301 has consistently been found to be complex (26, 63, 128). At other positions, considerable heterogeneity exists in the glycan populations, in some cases to the point where it is difficult to unequivocally assign them as predominantly complex or oligomannose. The reasons for these uncertainties might include incomplete trimming (42), interstrain sequence variability, the form of Env (e.g., gp120 or gp140), and the producer cell. The glycans of native Env trimers and monomeric gp120 may differ due to the constraints imposed by oligomerization (32, 41, 77). Thus, although all the potential sequons of HXB2 gp120 were found to be occupied in one study (63), some are unutilized or variably utilized on functional trimers, presumably due to steric limitations (42, 48, 75, 96, 119).The distribution of complex and oligomannose glycans on gp120 largely conforms with an antigenic map derived from structural models (59, 60, 102, 120), in which the outer domain is divided into a neutralizing face and an immunologically silent face. Oligomannose glycans cluster tightly on the silent face of gp120 (18, 128), while complex glycans flank the gp120 receptor binding sites of the neutralizing face, ostensibly forming a protective “fence” against NAbs (105). The relatively sparse clustering of complex glycans that form this fence may reflect a trade-off between protecting the underlying functional domains from NAbs by virtue of large antennae while at the same time permitting sufficient flexibility for the refolding events associated with receptor binding and fusion (29, 39, 67, 75, 98, 117). Conversely, the dense clustering of oligomannose glycans on the silent domain may be important for ensuring immune protection and/or in creating binding sites for lectins such as DC-SIGN (9, 44).The few available broadly neutralizing monoclonal antibodies (MAbs) define sites of vulnerability on Env trimers (reviewed in reference 52). They appear to fall into two general categories: those that access conserved sites by overcoming Env''s various evasion strategies and, intriguingly, those that exploit these very defensive mechanisms. Regarding the first category, MAb b12 recognizes an epitope that overlaps the CD4 binding site of gp120 (14), and MAbs 2F5 and 4E10 (84, 129) recognize adjacent epitopes of the membrane-proximal external region (MPER) at the C-terminal ectodomain of gp41. The variable neutralizing potencies of these MAbs against primary isolates that contain their core epitopes illustrate how conformational masking can dramatically regulate their exposure (11, 118). Conformational masking also limits the activities of MAbs directed to the V3 loop and MAbs whose epitopes overlap the coreceptor binding site (11, 62, 121).A second category of MAbs includes MAb 2G12, which recognizes a tight cluster of glycans in the silent domain of gp120 (16, 101, 103, 112). This epitope has recently sparked considerable interest in exploiting glycan clusters as possible carbohydrate-based vaccines (2, 15, 31, 70, 102, 116). Two recently described MAbs, PG9 and PG16 (L. M. Walker and D. R. Burton, unpublished data), also target epitopes regulated by the presence of glycans that involve conserved elements of the second and third variable loops and depend largely on the quaternary trimer structure and its in situ presentation on membranes. Their impressive breadth and potency may come from the fact that they target the very mechanisms (variable loops and glycans) that are generally thought to protect the virus from neutralization. Like 2G12, these epitopes are likely to be constitutively exposed and thus may not be subject to conformational masking (11, 118).The above findings reveal the importance of N-glycans both as a means of protection against neutralization as well as in directly contributing to unique neutralizing epitopes. Clearly, further studies on the nature and function of glycans in native Env trimers are warranted. Possible approaches may be divided into four categories, namely, (i) targeted mutation, (ii) enzymatic removal, (iii) expression in the presence of glycosylation inhibitors, and (iv) expression in mutant cell lines with engineered blocks in the glycosylation pathway. Much of the available information on the functional roles of glycans in HIV-1 and simian immunodeficiency virus (SIV) infection has come from the study of mutants that eliminate glycans either singly or in combination (20, 54, 66, 71, 74, 91, 95, 96). Most mutants of this type remain at least partially functional (74, 95, 96). In some cases these mutants have little effect on neutralization sensitivity, while in others they can lead to increased sensitivity to MAbs specific for the V3 loop and CD4 binding site (CD4bs) (54, 71, 72, 74, 106). In exceptional cases, increased sensitivity to MAbs targeting the coreceptor binding site and/or the gp41 MPER has been observed (54, 66, 72, 74).Of the remaining approaches for studying the roles of glycans, enzymatic removal is constrained by the extreme resistance of native Env trimers to many common glycosidases, contrasting with the relative sensitivity of soluble gp120 (67, 76, 101). Alternatively, drugs can be used to inhibit various stages of mammalian glycan biosynthesis. Notable examples are imino sugars, such as N-butyldeoxynojirimycin (NB-DNJ), that inhibit the early trimming of the glucose moieties from Glc3Man9GlcNAc2 precursors in the endoplasmic reticulum (28, 38, 51). Viruses produced in the presence of these drugs may fail to undergo proper gp160 processing or fusion (37, 51). Other classes of inhibitor include kifunensine and swainsonine, which, respectively, inhibit the trimming of the Man9GlcNAc2 precursor into Man5GlcNAc2 or inhibit the removal of remaining D2 and D3 arm mannoses from the hybrid glycans, thus preventing the construction of complex glycan antennae (Fig. (Fig.1B)1B) (17, 33, 76, 104, 119). Unlike NB-DNJ, viruses produced in the presence of these drugs remain infectious (36, 76, 79, 100).Yet another approach is to express virus in insect cells that can only modify proteins with paucimannose N-glycans (58). However, the inefficient gp120/gp41 processing by furin-like proteases in these cells prevents their utility in functional studies (123). Another option is provided by ricin-selected GnTI-deficient cell lines that cannot transfer GlcNAc onto the mannosidase-trimmed Man5GlcNAc2 substrate, preventing the formation of hybrid and complex carbohydrates (Fig. (Fig.1B)1B) (17, 32, 36, 94). This arrests glycan processing at a well-defined point, leading to the substitution of complex glycans with Man5GlcNAc2 rather than with the larger Man9GlcNAc2 precursors typically obtained with kifunensine treatment (17, 32, 33, 104). With this in mind, here we produced HIV-1 pseudoviruses in GnTI-deficient cells to investigate the role of complex glycan antennae in viral resistance neutralization. By replacing complex glycans with smaller Man5GlcNAc2 we can determine the effect of “lowering the glycan fence” that surrounds the receptor binding sites, compared to the above-mentioned studies of individual glycan deletion mutants, whose effects are analogous to removing a fence post. Furthermore, since oligomannose glycans are sensitive to certain enzymes, such as endoglycosidase H (endo H), we investigated the effect of dismantling the glycan fence on Env function and stability. Our results suggest that the antennae of complex glycans protect against certain specificities but that glycan stems regulate trimer conformation with often more dramatic consequences for neutralization sensitivity and in extreme cases, infectious function.  相似文献   

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The fermentative metabolism of glucose was redirected to succinate as the primary product without mutating any genes encoding the native mixed-acid fermentation pathway or redox reactions. Two changes in peripheral pathways were together found to increase succinate yield fivefold: (i) increased expression of phosphoenolpyruvate carboxykinase and (ii) inactivation of the glucose phosphoenolpyruvate-dependent phosphotransferase system. These two changes increased net ATP production, increased the pool of phosphoenolpyruvate available for carboxylation, and increased succinate production. Modest further improvements in succinate yield were made by inactivating the pflB gene, encoding pyruvate formate lyase, resulting in an Escherichia coli pathway that is functionally similar to the native pathway in Actinobacillus succinogenes and other succinate-producing rumen bacteria.Succinic acid is used as a specialty chemical in the agricultural, food, and pharmaceutical industries (17, 32). It has also been identified by the U.S. Department of Energy as one of the top 12 building block chemicals (30), because it can be converted into a variety of products, including green solvents, pharmaceutical products, and biodegradable plastics (17, 32). Although succinic acid is currently produced from petroleum-derived maleic anhydride, considerable interest in the fermentative production of succinate from sugars has emerged during the past decade (9, 10, 17).Several natural succinate-producing rumen bacteria that have high rates of succinate production and high succinate yields, such as Anaerobiospirillum succiniciproducens (22), Actinobacillus succinogenes (13, 28), and “Mannheimia succiniciproducens” (15, 16), have been isolated. However, these strains require complex organic nutrients that increase the costs associated with production, purification, and waste disposal (15, 22, 28). Low levels of succinate are produced by native strains of Escherichia coli in complex and mineral salts media (1, 4). Most mutant strains of E. coli that have been described previously as succinate producers also require complex organic nutrients (18, 23-26, 29, 31). Many involve two-step aerobic and anaerobic processes (3, 23-25, 29) and the addition of foreign genes (5, 6, 23-26, 29, 31).Novel E. coli biocatalysts (KJ060, KJ071, and KJ073) for the anaerobic production of succinate in mineral salts medium have been developed recently without the use of foreign genes or resident plasmids (9, 10). These biocatalysts were developed by combining constructed mutations to eliminate alternative routes of NADH oxidation in the mixed-acid pathway with growth-based selection (metabolic evolution). In subsequent studies (33), these strains were found to have recruited the glucose-repressed (7), gluconeogenic pck gene (11, 12, 19, 21, 27), encoding phosphoenolpyruvate carboxykinase (PCK) (derepressed via a point mutation in the promoter region), to replace the native phosphoenolpyruvate carboxylase (ppc) and serve as the primary route for CO2 fixation (Fig. (Fig.1).1). A second acquired mutation was also identified as a frameshift mutation in the carboxy terminus of ptsI, inactivating the phosphoenolpyruvate-dependent phosphotransferase system (33). Glucose uptake by the phosphotransferase system was functionally replaced by galactose permease (galP) and glucokinase (glk).Open in a separate windowFIG. 1.Anaerobic metabolism of E. coli using the mixed-acid fermentation pathway (data from reference 1). The native phosphotransferase system pathway for glucose uptake and the mixed-acid pathway for fermentation are shown with black arrows. Peripheral reactions for glucose uptake, carboxylation, and acetyl-CoA synthesis are shown as dotted green arrows and represent new metabolic functions that have been recruited for succinate production from glucose. Reactions that have been blocked by gene deletions or point mutations are marked with an X. pck* indicates a novel mutation that derepressed pck, allowing the enzyme to serve as the primary route for oxaloacetate production. Pyruvate (boxed) appears at two sites but is presumed to exist as a single intracellular pool.Based on these previous studies, we have now determined the core mutations needed to direct carbon flow from glucose to succinate in E. coli and have constructed new succinate-producing strains with a minimum of genetic change.  相似文献   

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Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has traditionally circulated in Africa and Asia, causing human febrile illness accompanied by severe, chronic joint pain. In Africa, epidemic emergence of CHIKV involves the transition from an enzootic, sylvatic cycle involving arboreal mosquito vectors and nonhuman primates, into an urban cycle where peridomestic mosquitoes transmit among humans. In Asia, however, CHIKV appears to circulate only in the endemic, urban cycle. Recently, CHIKV emerged into the Indian Ocean and the Indian subcontinent to cause major epidemics. To examine patterns of CHIKV evolution and the origins of these outbreaks, as well as to examine whether evolutionary rates that vary between enzootic and epidemic transmission, we sequenced the genomes of 40 CHIKV strains and performed a phylogenetic analysis representing the most comprehensive study of its kind to date. We inferred that extant CHIKV strains evolved from an ancestor that existed within the last 500 years and that some geographic overlap exists between two main enzootic lineages previously thought to be geographically separated within Africa. We estimated that CHIKV was introduced from Africa into Asia 70 to 90 years ago. The recent Indian Ocean and Indian subcontinent epidemics appear to have emerged independently from the mainland of East Africa. This finding underscores the importance of surveillance to rapidly detect and control African outbreaks before exportation can occur. Significantly higher rates of nucleotide substitution appear to occur during urban than during enzootic transmission. These results suggest fundamental differences in transmission modes and/or dynamics in these two transmission cycles.Chikungunya virus (CHIKV; Togaviridae: Alphavirus) is an arbovirus (arthropod-borne virus) vectored by Aedes mosquitoes to humans in tropical and subtropical regions of Africa and Asia (Fig. (Fig.1;1; reviewed in references 26 and 46). CHIKV has a single-stranded, positive-sense RNA genome of ∼12 kb and causes chikungunya fever (CHIK), a febrile illness associated with severe arthralgia and rash (2, 15, 31, 35); the name is derived from a Bantu language word describing the severe arthritic signs (32), which can persist for years. Thus, CHIK has enormous economic costs in addition to its public health impact (9). Because the signs and symptoms of CHIK overlap with those of dengue and because CHIKV is transmitted sympatrically in urban areas by the same mosquito vectors, it is grossly underreported in the absence of laboratory diagnostics (10, 37).Open in a separate windowFIG. 1.Distribution of the CHIKV strains used in this study. The map, based on a world map template from http://www.presentationmagazine.com, was edited with permission.CHIKV was first isolated during a 1953 outbreak in present-day Tanzania by Ross (48, 49). Since then, outbreaks have been documented in Africa and Asia, including the Indian subcontinent (Fig. (Fig.1)1) (1, 4). In 2005, CHIKV emerged from East Africa to cause an explosive urban epidemic in popular tourist island destinations in the Indian Ocean (Fig. (Fig.1;1; reviewed in reference 31). In late 2005, CHIKV spread into the Indian subcontinent, where millions of people have been affected (5). However, the geographic source of spread into India, from the mainland of Africa or from the Indian Ocean Islands, has not been delineated. India had seen large epidemics of CHIK in the past (reviewed in reference 30), but CHIKV apparently disappeared during the 1970s (5). Since 2006, CHIKV has been imported into Europe and the western hemisphere (including the United States) via many viremic travelers, and an epidemic was initiated in Italy by a traveler from India (4, 11, 47). The dramatic spread since 1980 of dengue viruses (DENV) throughout tropical America, via the same vectors, portends the severity of the public health problem if CHIKV becomes established in the western hemisphere.The first phylogenetic analysis of CHIKV (45) identified three geographically associated genotypes: the West African (WAf), East/Central/South African (ECSA), and Asian genotypes. More recent analyses indicate that the recent Indian Ocean and Indian strains form a monophyletic group within the ECSA lineage (5, 12, 14, 27, 40, 51, 52). However, most CHIKV phylogenetic studies (1, 14, 28, 29, 38, 40, 41, 47, 52) have utilized only partial sequences from the envelope glycoprotein E1 gene, preventing a robust assessment of some of the relationships among strains and of their evolutionary dynamics.The CHIKV strains represented in different geographic lineages apparently circulate in different ecological cycles. In Asia, CHIKV appears to circulate primarily in an urban transmission cycle involving the peridomestic mosquitoes Aedes aegypti and A. albopictus, as well as humans (25, 45). Asian epidemics typically infect thousands-to-millions of people over the course of several years (46). In contrast, African CHIKV circulates primarily in a sylvatic/enzootic cycle, transmitted by arboreal primatophilic Aedes mosquitoes (e.g., A. furcifer and A. africanus) and probably relies on nonhuman primates as reservoir hosts (reviewed in reference 16). Epidemics in rural Africa usually occur on a much smaller scale than in Asia, likely a result of the lower human population densities, and possibly more stable herd immunity. Although the assignments of “urban” and “sylvatic/enzootic” are based on the most common mode of transmission, CHIKV strains of African origin are capable of urban transmission by A. aegypti and A. albopictus, as evidenced by outbreaks in the Democratic Republic of the Congo (41), Nigeria (36), Kenya (27), and Gabon (42). The ecological differences between the sylvatic/enzootic (henceforth called enzootic) and urban/endemic/epidemic transmission cycles (henceforth called epidemic) such as seasonality of vector larval habitats, vertebrate host abundance and herd immunity, and vector host preferences, prompted us to hypothesize that the evolutionary dynamics of CHIKV may differ between the two transmission cycles. To test this hypothesis, to provide more robust estimates of the evolutionary relationships among the CHIKV strains including the sources of the recent epidemics, and to elucidate the temporal and spatial history of CHIKV evolution, we performed an extensive, genome-scale phylogenetic analysis, utilizing complete open reading frame (ORF) sequences of a large collection of 80 isolates with broad temporal, spatial, and host coverage.  相似文献   

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l-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of human infections and is considered the main pathogen responsible for chronic pneumonia in cystic fibrosis patients (7, 23). P. aeruginosa also infects other organisms, such as insects (4), nematodes (6), plants (18), and amoebae (20). Its ability to thrive as a pathogen and to compete in aquatic and soil environments can be partly attributed to the production and interplay of secreted virulence factors and secondary metabolites. While the importance of many of these exoproducts has been studied, the antimetabolite l-2-amino-4-methoxy-trans-3-butenoic acid (AMB; methoxyvinylglycine) (Fig. (Fig.1)1) has received only limited attention. Identified during a search for new antibiotics, AMB was found to reversibly inhibit the growth of Bacillus spp. (26) and Escherichia coli (25) and was later shown to inhibit the growth and metabolism of cultured Walker carcinosarcoma cells (28). AMB is a γ-substituted vinylglycine, a naturally occurring amino acid with a β,γ-C=C double bond. Other members of this family are aminoethoxyvinylglycine from Streptomyces spp. (19) and rhizobitoxine, made by Bradyrhizobium japonicum (16) and Pseudomonas andropogonis (15) (Fig. (Fig.1).1). As inhibitors of pyridoxal phosphate-dependent enzymes (13, 17, 21, 22), γ-substituted vinylglycines have multiple targets in bacteria, animals, and plants (3, 5, 10, 21, 22, 29). However, the importance of AMB as a toxin in biological interactions with P. aeruginosa has not been addressed, as AMB biosynthesis and the genes involved have not been elucidated.Open in a separate windowFIG. 1.Chemical structures of the γ-substituted vinylglycines AMB, aminoethoxyvinylglycine, and rhizobitoxine.  相似文献   

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