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1.
Andrew D. Morgan R. Craig MacLean Kristina L. Hillesland Gregory J. Velicer 《Applied and environmental microbiology》2010,76(20):6920-6927
Predator-prey relationships among prokaryotes have received little attention but are likely to be important determinants of the composition, structure, and dynamics of microbial communities. Many species of the soil-dwelling myxobacteria are predators of other microbes, but their predation range is poorly characterized. To better understand the predatory capabilities of myxobacteria in nature, we analyzed the predation performance of numerous Myxococcus isolates across 12 diverse species of bacteria. All predator isolates could utilize most potential prey species to effectively fuel colony expansion, although one species hindered predator swarming relative to a control treatment with no growth substrate. Predator strains varied significantly in their relative performance across prey types, but most variation in predatory performance was determined by prey type, with Gram-negative prey species supporting more Myxococcus growth than Gram-positive species. There was evidence for specialized predator performance in some predator-prey combinations. Such specialization may reduce resource competition among sympatric strains in natural habitats. The broad prey range of the Myxococcus genus coupled with its ubiquity in the soil suggests that myxobacteria are likely to have very important ecological and evolutionary effects on many species of soil prokaryotes.Predation plays a major role in shaping both the ecology and evolution of biological communities. The population and evolutionary dynamics of predators and their prey are often tightly coupled and can greatly influence the dynamics of other organisms as well (1). Predation has been invoked as a major cause of diversity in ecosystems (11, 12). For example, predators may mediate coexistence between superior and inferior competitors (2, 13), and differential trajectories of predator-prey coevolution can lead to divergence between separate populations (70).Predation has been investigated extensively in higher organisms but relatively little among prokaryotes. Predation between prokaryotes is one of the most ancient forms of predation (27), and it has been proposed that this process may have been the origin of eukaryotic cells (16). Prokaryotes are key players in primary biomass production (44) and global nutrient cycling (22), and predation of some prokaryotes by others is likely to significantly affect these processes. Most studies of predatory prokaryotes have focused on Bdellovibrionaceae species (e.g., see references 51, 55, and 67). These small deltaproteobacteria prey on other Gram-negative cells, using flagella to swim rapidly until they collide with a prey cell. After collision, the predator cells then enter the periplasmic space of the prey cell, consume the host cell from within, elongate, and divide into new cells that are released upon host cell lysis (41). Although often described as predatory, the Bdellovibrionaceae may also be considered to be parasitic, as they typically depend (apart from host-independent strains that have been observed [60]) on the infection and death of their host for their reproduction (47).In this study, we examined predation among the myxobacteria, which are also deltaproteobacteria but constitute a monophyletic clade divergent from the Bdellovibrionaceae (17). Myxobacteria are found in most terrestrial soils and in many aquatic environments as well (17, 53, 74). Many myxobacteria, including the model species Myxococcus xanthus, exhibit several complex social traits, including fruiting body formation and spore formation (14, 18, 34, 62, 71), cooperative swarming with two motility systems (64, 87), and group (or “wolf pack”) predation on both bacteria and fungi (4, 5, 8, 9, 15, 50). Using representatives of the genus Myxococcus, we tested for both intra- and interspecific variation in myxobacterial predatory performance across a broad range of prey types. Moreover, we examined whether prey vary substantially in the degree to which they support predatory growth by the myxobacteria and whether patterns of variation in predator performance are constant or variable across prey environments. The latter outcome may reflect adaptive specialization and help to maintain diversity in natural populations (57, 59).Although closely related to the Bdellovibrionaceae (both are deltaproteobacteria), myxobacteria employ a highly divergent mode of predation. Myxobacteria use gliding motility (64) to search the soil matrix for prey and produce a wide range of antibiotics and lytic compounds that kill and decompose prey cells and break down complex polymers, thereby releasing substrates for growth (66). Myxobacterial predation is cooperative both in its “searching” component (6, 31, 82; for details on cooperative swarming, see reference 64) and in its “handling” component (10, 29, 31, 32), in which secreted enzymes turn prey cells into consumable growth substrates (56, 83). There is evidence that M. xanthus employs chemotaxis-like genes in its attack on prey cells (5) and that predation is stimulated by close contact with prey cells (48).Recent studies have revealed great genetic and phenotypic diversity within natural populations of M. xanthus, on both global (79) and local (down to centimeter) scales (78). Phenotypic diversity includes variation in social compatibility (24, 81), the density and nutrient thresholds triggering development (33, 38), developmental timing (38), motility rates and patterns (80), and secondary metabolite production (40). Although natural populations are spatially structured and both genetic diversity and population differentiation decrease with spatial scale (79), substantial genetic diversity is present even among centimeter-scale isolates (78). No study has yet systematically investigated quantitative natural variation in myxobacterial predation phenotypes across a large number of predator genotypes.Given the previous discovery of large variation in all examined phenotypes, even among genetically extremely similar strains, we anticipated extensive predatory variation as well. Using a phylogenetically broad range of prey, we compared and contrasted the predatory performance of 16 natural M. xanthus isolates, sampled from global to local scales, as well as the commonly studied laboratory reference strain DK1622 and representatives of three additional Myxococcus species: M. flavescens (86), M. macrosporus (42), and M. virescens (63) (Table (Table1).1). In particular, we measured myxobacterial swarm expansion rates on prey lawns spread on buffered agar (31, 50) and on control plates with no nutrients or with prehydrolyzed growth substrate.
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TABLE 1.
List of myxobacteria used, with geographical originOrganism abbreviation used in text | Species | Strain | Geographic origin | Reference(s) |
---|---|---|---|---|
A9 | Myxococcus xanthus | A9 | Tübingen, Germany | 78 |
A23 | Myxococcus xanthus | A23 | Tübingen, Germany | 78 |
A30 | Myxococcus xanthus | A30 | Tübingen, Germany | 78 |
A41 | Myxococcus xanthus | A41 | Tübingen, Germany | 78 |
A46 | Myxococcus xanthus | A46 | Tübingen, Germany | 78 |
A47 | Myxococcus xanthus | A47 | Tübingen, Germany | 78 |
A75 | Myxococcus xanthus | A75 | Tübingen, Germany | 78 |
A85 | Myxococcus xanthus | A85 | Tübingen, Germany | 78 |
TV | Myxococcus xanthus | Tvärminne | Tvärminne, Finland | 79 |
PAK | Myxococcus xanthus | Paklenica | Paklenica, Croatia | 79 |
MAD | Myxococcus xanthus | Madeira 1 | Madeira, Portugal | 79 |
WAR | Myxococcus xanthus | Warwick 1 | Warwick, UK | 79 |
TOR | Myxococcus xanthus | Toronto 1 | Toronto, Ontario, Canada | 79 |
SUL2 | Myxococcus xanthus | Sulawesi 2 | Sulawesi, Indonesia | 79 |
KAL | Myxococcus xanthus | Kalalau | Kalalau, HI | 79 |
DAV | Myxococcus xanthus | Davis 1A | Davis, CA | 79 |
GJV1 | Myxococcus xanthus | GJV 1 | Unknown | 35, 72 |
MXFL1 | Myxococcus flavescens | Mx fl1 | Unknown | 65 |
MXV2 | Myxococcus virescens | Mx v2 | Unknown | 65 |
CCM8 | Myxococcus macrosporus | Cc m8 | Unknown | 65 |
2.
Patti J. Miller Claudio L. Afonso Erica Spackman Melissa A. Scott Janice C. Pedersen Dennis A. Senne Justin D. Brown Chad M. Fuller Marcela M. Uhart William B. Karesh Ian H. Brown Dennis J. Alexander David E. Swayne 《Journal of virology》2010,84(21):11496-11504
The biological, serological, and genomic characterization of a paramyxovirus recently isolated from rockhopper penguins (Eudyptes chrysocome) suggested that this virus represented a new avian paramyxovirus (APMV) group, APMV10. This penguin virus resembled other APMVs by electron microscopy; however, its viral hemagglutination (HA) activity was not inhibited by antisera against any of the nine defined APMV serotypes. In addition, antiserum generated against this penguin virus did not inhibit the HA of representative viruses of the other APMV serotypes. Sequence data produced using random priming methods revealed a genomic structure typical of APMV. Phylogenetic evaluation of coding regions revealed that amino acid sequences of all six proteins were most closely related to APMV2 and APMV8. The calculation of evolutionary distances among proteins and distances at the nucleotide level confirmed that APMV2, APMV8, and the penguin virus all were sufficiently divergent from each other to be considered different serotypes. We propose that this isolate, named APMV10/penguin/Falkland Islands/324/2007, be the prototype virus for APMV10. Because of the known problems associated with serology, such as antiserum cross-reactivity and one-way immunogenicity, in addition to the reliance on the immune response to a single protein, the hemagglutinin-neuraminidase, as the sole base for viral classification, we suggest the need for new classification guidelines that incorporate genome sequence comparisons.Viruses from the Paramyxoviridae family have caused disease in humans and animals for centuries. Over the last 40 years, many paramyxoviruses isolated from animals and people have been newly described (16, 17, 22, 29, 31, 32, 36, 42, 44, 46, 49, 58, 59, 62-64). Viruses from this family are pleomorphic, enveloped, single-stranded, nonsegmented, negative-sense RNA viruses that demonstrate serological cross-reactivity with other paramyxoviruses related to them (30, 46). The subfamily Paramyxovirinae is divided into five genera: Respirovirus, Morbillivirus, Rubulavirus, Henipavirus, and Avulavirus (30). The Avulavirus genus contains nine distinct avian paramyxovirus (APMV) serotypes (Table (Table1),1), and information on the discovery of each has been reported elsewhere (4, 6, 7, 9, 12, 34, 41, 50, 51, 60, 68).
Open in a separate windowaRequires the addition of an exogenous protease.bProtease requirement depends on the isolate examined.cPutative.Six of these serotypes were classified in the latter half of the 1970s, when the most reliable assay available to classify paramyxoviruses was the hemagglutination inhibition (HI) assay (61). However, there are multiple problems associated with the use of serology, including the inability to classify some APMVs by comparing them to the sera of the nine defined APMVs alone (2, 8). In addition, one-way antigenicity and cross-reactivity between different serotypes have been documented for many years (4, 5, 14, 25, 29, 33, 34, 41, 51, 52, 60). The ability of APMVs, like other viruses, to show antigenic drift as it evolves over time (37, 43, 54) and the wide use and availability of precise molecular methods, such as PCR and genome sequencing, demonstrate the need for a more practical classification system.The genetic diversity of APMVs is still largely unexplored, as hundreds of avian species have never been surveyed for the presence of viruses that do not cause significant signs of disease or are not economically important. The emergence of H5N1 highly pathogenic avian influenza (HPAI) virus as the cause of the largest outbreak of a virulent virus in poultry in the past 100 years has spurred the development of surveillance programs to better understand the ecology of avian influenza (AI) viruses in aquatic birds around the globe, and in some instances it has provided opportunities for observing other viruses in wild bird populations (15, 53). In 2007, as part of a seabird health surveillance program in the Falkland Islands (Islas Malvinas), oral and cloacal swabs and serum were collected from rockhopper penguins (Eudyptes chrysocome) and environmental/fecal swab pools were collected from other seabirds.While AI virus has not yet been isolated from penguins in the sub-Antarctic and Antarctic areas, there have been two reports of serum antibodies positive to H7 and H10 from the Adélie species (11, 40). Rare isolations of APMV1, both virulent (45) and of low virulence (8), have been reported from Antarctic penguins. Sera positive for APMV1 and AMPV2 have also been reported (21, 24, 38, 40, 53). Since 1981, paramyxoviruses have been isolated from king penguins (Aptenodytes patagonicus), royal penguins (Eudyptes schlegeli), and Adélie penguins (Pygoscelis adeliae) from Antarctica and little blue penguins (Eudyptula minor) from Australia that cannot be identified as belonging to APMV1 to -9 and have not yet been classified (8, 11, 38-40). The morphology, biological and genomic characteristics, and antigenic relatedness of an APMV recently isolated from multiple penguin colonies on the Falkland Islands are reported here. Evidence that the virus belongs to a new serotype (APMV10) and a demonstration of the advantages of a whole genome system of analysis based on random sequencing followed by comparison of genetic distances are presented. Only after all APMVs are reported and classified will epidemiological information be known as to how the viruses are moving and spreading as the birds travel and interact with other avian species. 相似文献
TABLE 1.
Characteristics of prototype viruses APMV1 to APMV9 and the penguin virusStrain | Host | Disease | Distribution | Fusion cleavagec | GI accession no. |
---|---|---|---|---|---|
APMV1/Newcastle disease virus | >250 species | High mortality | Worldwide | GRRQKRF | 45511218 |
Inapparent | Worldwide | GGRQGRLa | 11545722 | ||
APMV2/Chicken/CA/Yucaipa/1956 | Turkey, chickens, psittacines, rails, passerines | Decrease in egg production and respiratory disease | Worldwide | DKPASRF | 169144527 |
APMV3/Turkey/WI/1968 | Turkey | Mild respiratory disease and moderate egg decrease | Worldwide | PRPSGRLa | 209484147 |
APMV3/Parakeet/Netherlands/449/1975 | Psittacines, passerines, flamingos | Neurological, enteric, and respiratory disease | Worldwide | ARPRGRLa | 171472314 |
APMV4/Duck/Hong Kong/D3/1975 | Duck, geese, chickens | None known | Worldwide | VDIQPRF | 210076708 |
APMV5/Budgerigar/Japan/Kunitachi/1974 | Budgerigars, lorikeets | High mortality, enteric disease | Japan, United Kingdom, Australia | GKRKKRFa | 290563909 |
APMV6/Duck/Hong Kong/199/1977 | Ducks, geese, turkeys | Mild respiratory disease and increased mortality in turkeys | Worldwide | PAPEPRLb | 15081567 |
APMV7/Dove/TN/4/1975 | Pigeons, doves, turkeys | Mild respiratory disease in turkeys | United States, England, Japan | TLPSSRF | 224979458 |
APMV8/Goose/DE/1053/1976 | Ducks, geese | None known | United States, Japan | TYPQTRLa | 226343050 |
APMV9/Duck/NY/22/1978 | Ducks | None known | Worldwide | RIREGRIa | 217068693 |
APMV10/Penguin/Falkland Islands/324/2007 | Rockhopper penguins | None Known | Falkland Islands | DKPSQRIa | 300432141 |
3.
4.
Jochen K. Lennerz Jonathan B. Hurov Lynn S. White Katherine T. Lewandowski Julie L. Prior G. James Planer Robert W. Gereau IV David Piwnica-Worms Robert E. Schmidt Helen Piwnica-Worms 《Molecular and cellular biology》2010,30(21):5043-5056
Par-1 is an evolutionarily conserved protein kinase required for polarity in worms, flies, frogs, and mammals. The mammalian Par-1 family consists of four members. Knockout studies of mice implicate Par-1b/MARK2/EMK in regulating fertility, immune homeostasis, learning, and memory as well as adiposity, insulin hypersensitivity, and glucose metabolism. Here, we report phenotypes of mice null for a second family member (Par-1a/MARK3/C-TAK1) that exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling, and normal insulin sensitivity. Knockout mice were protected against high-fat diet-induced obesity and displayed attenuated weight gain, complete resistance to hepatic steatosis, and improved glucose handling with decreased insulin secretion. Overnight starvation led to complete hepatic glycogen depletion, associated hypoketotic hypoglycemia, increased hepatocellular autophagy, and increased glycogen synthase levels in Par-1a−/− but not in control or Par-1b−/− mice. The intercrossing of Par-1a−/− with Par-1b−/− mice revealed that at least one of the four alleles is necessary for embryonic survival. The severity of phenotypes followed a rank order, whereby the loss of one Par-1b allele in Par-1a−/− mice conveyed milder phenotypes than the loss of one Par-1a allele in Par-1b−/− mice. Thus, although Par-1a and Par-1b can compensate for one another during embryogenesis, their individual disruption gives rise to distinct metabolic phenotypes in adult mice.Cellular polarity is a fundamental principle in biology (6, 36, 62). The prototypical protein kinase originally identified as a regulator of polarity was termed partitioning defective (Par-1) due to early embryonic defects in Caenorhabditis elegans (52). Subsequent studies revealed that Par-1 is required for cellular polarity in worms, flies, frogs, and mammals (4, 17, 58, 63, 65, 71, 89). An integral role for Par-1 kinases in multiple signaling pathways has also been established, and although not formally addressed, multifunctionality for individual Par-1 family members is implied in reviews of the list of recognized upstream regulators and downstream substrates (Table (Table1).1). Interestingly, for many Par-1 substrates the phosphorylated residues generate 14-3-3 binding sites (25, 28, 37, 50, 59, 61, 68, 69, 78, 95, 101, 103). 14-3-3 binding in turn modulates both nuclear/cytoplasmic as well as cytoplasmic/membrane shuttling of target proteins, thus allowing Par-1 activity to establish intracellular spatial organization (15, 101). The phosphorylation of Par-1 itself promotes 14-3-3 binding, thereby regulating its subcellular localization (37, 59, 101).
Open in a separate windowaLKB1 also is known as Par-4; MARKK also is known as Ste20-like; (−), inhibitory/negative regulation has been shown; GPCR, G protein-coupled receptors. MARKK is highly homologous to TAO-1 (thousand-and-one amino acid kinase) (46).The mammalian Par-1 family contains four members (Table (Table2).2). Physiological functions of the Par-1b kinase have been studied using targeted gene knockout approaches in mice (9, 44). Two independently derived mouse lines null for Par-1b have implicated this protein kinase in diverse physiological processes, including fertility (9), immune system homeostasis (44), learning and memory (86), the positioning of nuclei in pancreatic beta cells (35, 38), and growth and metabolism (43).
TABLE 1.
Multifunctionality of Par-1 polarity kinase pathwaysaRegulator or substrate | Function | Reference(s) |
---|---|---|
Regulators (upstream function) | ||
LKB1 | Wnt signaling, Peutz-Jeghers syndrome, insulin signal transduction, pattern formation | 2, 63, 93 |
TAO1 | MEK3/p38 stress-responsive mitogen-activated protein kinase (MAPK) pathway | 46 |
MARKK | Nerve growth factor signaling in neurite development and differentiation | 98 |
aPKC | Ca2+/DAG-independent signal transduction, cell polarity, glucose metabolism | 14, 37, 40, 45, 59, 75, 95 |
nPKC/PKD | DAG-dependent, Ca2+-independent signal transduction (GPCR) | 101 |
PAR-3/PAR-6/aPKC | (−); regulates Par-1, assembly of microtubules, axon-dendrite specification | 19 |
GSK3β | (−); tau phosphorylation, Alzheimer''s dementia, energy metabolism, body patterning | 54, 97 |
Pim-1 oncogene | (−); G2/M checkpoint, effector of cytokine signaling and Jak/STAT(3/5) | 5 |
CaMKI | (−); Ca2+-dependent signal transduction, neuronal differentiation | 99 |
Substrates (downstream function) | ||
Cdc25C | Regulation of mitotic entry by activation of the cdc2-cyclin B complex | 25, 72, 78, 103 |
Class II HDAC | Control of gene expression and master regulator of subcellular trafficking | 28, 50 |
CRTC2/TORC2 | Gluconeogenesis regulator via LKB1/AMPK/TORC2 signaling, PPARγ1a coactivator | 49 |
Dlg/PSD-95 | Synaptogenesis and neuromuscular junction, tumor suppressor (102) | 104 |
Disheveled | Wnt signaling, translocation of Dsh from cytoplasmic vesicles to cortex | 73, 94 |
KSR1 | Regulation of the Ras-MAPK pathway | 68, 69 |
MAP2/4/TAU | Dynamic instability (67, 83) of microtubules, Alzheimer''s dementia (30) | 11, 31-33, 47, 70, 96 |
Mib/Notch | Mind bomb (Mib degradation and repression of Notch signaling results in neurogenesis) | 57, 74, 81 |
Par3/OSKAR/Lgl | Cytoplasmic protein segregation, cell polarity, and asymmetric cell division | 7, 10 |
Pkp2 | Desmosome assembly and organization; nuclear shuttling | 68, 69 |
PTPH1 | Linkage between Ser/Thr and Tyr phosphorylation-dependent signaling | 103 |
Rab11-FIP | Regulation of endocytosis (23), trafficking of E-cadherin (64) | 34 |
TABLE 2.
Terminology and localization of mammalian Par-1 family membersOpen in a separate windowaPar should not to be confused with protease-activated receptor 1 (PAR1 [29]); C-TAK1, Cdc twenty-five C-associated kinase 1; MARK, microtubule affinity regulating kinase; MARKL, MAP/microtubule affinity-regulating kinase-like 1.bBasolateral to a lesser degree than Par-1b (37).cHuman KP78 is asymmetrically localized to the apical surface of epithelial cells (76).dVariant that does not show asymmetric localization in epithelial cells when overexpressed (95).Beyond Par-1b, most information regarding the cell biological functions of the Par-1 kinases comes from studies of Par-1a. Specifically, Par-1a has been implicated in pancreatic (76) and hepatocarcinogenesis (51), as well as colorectal tumors (77), hippocampal function (100), CagA (Helicobacter pylori)-associated epithelial cell polarity disruption (82), and Peutz-Jeghers syndrome (48), although the latter association has been excluded recently (27). As a first step toward determining unique and redundant functions of Par-1 family members, mice disrupted for a second member of the family (Par-1a/MARK3/C-TAK1) were generated. We report that Par-1a−/− mice are viable and develop normally, and adult mice are hypermetabolic, have decreased white and brown adipose tissue mass, and unaltered glucose/insulin handling. However, when challenged by a high-fat diet (HFD), Par-1a−/− mice exhibit resistance to hepatic steatosis, resistance to glucose intolerance, and the delayed onset of obesity relative to that of control littermates. Strikingly, overnight starvation results in a complete depletion of glycogen and lipid stores along with an increase in autophagic vacuoles in the liver of Par-1a−/− but not Par-1b−/− mice. Correspondingly, Par-1a−/− mice develop hypoketotic hypoglycemia. These findings reveal unique metabolic functions of two Par-1 family members. 相似文献5.
6.
Todd J. Ward Thomas Usgaard Peter Evans 《Applied and environmental microbiology》2010,76(19):6680-6684
A 30-probe assay was developed for simultaneous classification of Listeria monocytogenes isolates by lineage (I to IV), major serogroup (4b, 1/2b, 1/2a, and 1/2c), and epidemic clone (EC) type (ECI, ECIa, ECII, and ECIII). The assay was designed to facilitate rapid strain characterization and the integration of subtype data into risk-based inspection programs.Listeria monocytogenes is a facultative intracellular pathogen that can cause serious invasive illness (listeriosis) in humans and other animals. L. monocytogenes is responsible for over 25% of food-borne-disease-related deaths attributable to known pathogens and is a leading cause of food recalls due to microbial adulteration (12, 21). However, not all L. monocytogenes subtypes contribute equally to human illness, and substantial differences in the ecologies and virulence attributes of different L. monocytogenes subtypes have been identified (9, 13, 14, 23, 24, 33, 35, 36). Among the four major evolutionary lineages of L. monocytogenes, only lineages I and II are commonly isolated from contaminated food and human listeriosis patients (19, 27, 29, 33). Lineage I strains are overrepresented among human listeriosis isolates, particularly those associated with epidemic outbreaks, whereas lineage II strains are overrepresented in foods and the environment (13, 14, 24). Lineage III strains account for approximately 1% of human listeriosis cases but are common among animal listeriosis isolates and appear to be a host-adapted group that is poorly adapted to food-processing environments (6, 34-36). The ecological and virulence attributes of lineage IV are poorly understood, as this lineage is rare and was only recently described based on a small number of strains (19, 26, 29, 33).L. monocytogenes is differentiated into 13 serotypes; however, four major serogroups (4b, 1/2b, 1/2a, and 1/2c) from within lineages I and II account for more than 98% of human and food isolates (16, 31). Serogroups refer to evolutionary complexes typified by a predominant serotype but which include very rare serotypes that represent minor evolutionary variants (7, 9, 33). Phylogenetic analyses have indicated that rare serotypes may have evolved recently, or even multiple times, from one of the major serotypes (9), and numerous molecular methods fail to discriminate minor serotypes as independent groups (1, 4, 7, 9, 18, 22, 33, 38, 39). Serotyping is one of the most common methods for L. monocytogenes subtyping, and serogroup classifications are a useful component of strain characterization because ecotype divisions appear largely congruent with serogroup distinctions (16, 34). Serogroup 4b strains are of particular public health concern because contamination with these strains appears to increase the probability that a ready-to-eat (RTE) food will be implicated in listeriosis (16, 28). Serogroup 4b strains account for approximately 40% of sporadic listeriosis and also are responsible for the majority of listeriosis outbreaks despite being relatively rare contaminants of food products (9, 13, 17, 30, 34). In addition, serogroup 4b strains are associated with more severe clinical presentations and higher mortality rates than other serogroups (11, 16, 20, 31, 34). Serogroups 1/2a and 1/2b are overrepresented among food isolates but also contribute significantly to human listeriosis, whereas serogroup 1/2c rarely causes human illness and may pose a lower risk of listeriosis for humans (16). Serogroup-specific differences in association with human listeriosis are consistent with the prevalence of virulence-attenuating mutations in inlA within these serogroups (32, 34); however, a number of additional factors likely contribute to these differences.Four previously described epidemic clones (ECs; ECI, ECIa, ECII, and ECIII) of L. monocytogenes have been implicated in numerous listeriosis outbreaks and have contributed significantly to sporadic illness (15, 34). ECI, ECIa, and ECII are distinct groups within serogroup 4b that were each responsible for repeated outbreaks of listeriosis in the United States and Europe. ECIII is a lineage II clone of serotype 1/2a that persisted in the same processing facility for more than a decade prior to causing a multistate outbreak linked to contaminated turkey (15, 25). While there has been speculation that epidemic clones possess unique adaptations that explain their frequent involvement in listeriosis outbreaks (9, 34, 37), it is not clear that epidemic clones are more virulent than other strains with the same serotype. However, contamination of RTE food with EC strains would be cause for increased concern due to the previous involvement of these clones in major outbreaks of listeriosis (16).As a result of the L. monocytogenes subtype-specific differences in ecology, virulence, and association with human illness, molecular subtyping technologies have the potential to inform assessments of relative risk and to improve risk-based inspection programs. The objective of the present study was to develop a single assay for rapid and accurate classification of L. monocytogenes isolates by lineage, major serogroup, and epidemic clone in order to facilitate strain characterization and the integration of subtype data into inspection programs that are based on assessment of relative risk.A database of more than 5.3 Mb of comparative DNA sequences from 238 L. monocytogenes isolates (9, 33-35) was scanned for single nucleotide polymorphisms that could be used to differentiate lineages, major serogroups, and epidemic clones via a targeted multilocus genotyping (TMLGT) approach. The acronym TMLGT is used to distinguish this approach from previously published multilocus genotyping (MLGT) assays that were lineage specific and designed for haplotype discrimination (9, 33). To provide for simultaneous interrogation of the selected polymorphisms via TMLGT, six genomic regions (Table (Table1)1) were coamplified in a multiplex PCR. While the previous MLGT assays were based on three lineage-specific multiplexes and required prior identification of lineage identity, TMLGT was designed to target variation across all of the lineages simultaneously and is based on a unique set of amplicons. PCR was performed in 50-μl volumes with 1× High Fidelity PCR buffer (Invitrogen Life Technologies), 2 mM MgSO4, 100 μM deoxynucleoside triphosphate (dNTP), 300 nM primer, 1.5 U Platinum Taq high-fidelity DNA polymerase (Invitrogen Life Technologies), and 100 ng of genomic DNA. PCR consisted of an initial denaturation of 90 s at 96°C, followed by 40 cycles of 30 s at 94°C, 30 s at 50°C, and 90 s at 68°C. Amplification products were purified using Montage PCR cleanup filter plates (Millipore) and served as a template for allele-specific primer extension (ASPE) reactions utilizing subtype-specific probes.
Open in a separate windowaCorresponding nucleotide positions in the complete genome sequence of L. monocytogenes strain EGD-e (GenBank accession number ).bSee IUPAC codes for definition of degenerate bases.ASPE was performed in multiplex reactions including 30 probes, with each lineage (I to IV), major serogroup (4b, 1/2b, 1/2a, and 1/2c), and epidemic clone (ECI, ECIa, ECII, and ECIII) targeted by two different probes (Table NC_003210(Table2).2). In addition, positive-control probes were included to confirm the presence of each amplicon in the multiplex PCR. As serogroups and epidemic clones are nested within a particular lineage, probes for these groups were designed to be specific within the appropriate lineage and values for these probes were evaluated only for isolates of the appropriate lineage. For example, serogroup 1/2a probes were evaluated only for isolates that were positive for lineage II probes. ASPE probes were designed with a unique 5′ sequence tag specific to individual sets of xMAP fluorescent polystyrene microspheres (Luminex Corporation) used to sort extension products. Extension and hybridization reactions were performed as described previously (9) except microspheres were twice pelleted by centrifugation (4 min at 2,250 × g) and resuspended in 75 μl 1× TM buffer prior to being pelleted and resuspended in 100 μl 1× TM buffer containing 2 μg/ml streptavidin-R-phycoerythrin (Invitrogen Life Technologies). Samples were incubated for 15 min at 37°C prior to detecting the microsphere complexes with a Luminex 100 flow cytometer (Luminex Corporation). The median fluorescence intensity (MFI) from biotinylated extension products attached to 100 microspheres was measured for each probe. The average MFI from three template-free control samples was also determined and subtracted from the raw MFI of each sample to account for background fluorescence. Probe performance was initially evaluated via the index of discrimination (ID) as described by Ducey et al. (9), and probes with ID values less than 2.0 were redesigned.
Open in a separate windowaNA, not applicable for positive amplicon control probes.bLuminex microsphere sets (Luminex Corporation) used for hybridization reactions are indicated following the hyphen.cn, number of isolates representing the target subtype among the 906 tested isolates.dThe 5′ sequence tag portions of extension probes are capitalized. See IUPAC codes for definitions of degenerate bases.eID, index of discrimination.Validation of the TMLGT assay was performed using 906 L. monocytogenes isolates for which the lineage, major serogroup, and epidemic clone type had been determined independently (see Table S1 in the supplemental material). A subset of 92 isolates, including at least five isolates from each lineage, serogroup, and epidemic clone type, was used to evaluate the discriminatory power of subtype-specific probes and the repeatability of the assay (see Table S1). Two independent runs of the 30-probe TMLGT assay produced identical results for these 92 isolates. In addition, genotypes matched expectations for all isolate/probe combinations, and the fluorescence intensities for positive genotypes (those targeted by a particular probe) were 3.8 to 58.3 (mean, 18.5) times as high as background values for isolates with negative genotypes (those not targeted by a particular probe) (Table (Table2).2). The performances of individual probes also were assessed in terms of sensitivity and specificity, where sensitivity is defined as the percentage of positive samples that produced positive results and specificity indicates the percentage of negative samples that produce negative results (5). Based on results from all 906 isolates analyzed by TMLGT, probe sensitivity was at least 98.6% and 23 of the 24 subtype-specific probes exhibited 100% sensitivity (Table (Table2).2). The specificities for all probes were also greater than 98.6%, and 21 of the 24 subtype-specific probes exhibited 100% specificity (Table (Table22).All but three of the 906 isolates in the validation panel were fully and accurately typed relative to lineage, serogroup, and epidemic clone by using the TMLGT assay (typeability, 99.9%; accuracy of isolate assignment, 99.8%). One of the lineage II isolates, NRRL B-33880, could not be assigned to a serogroup based on the TMLGT results because this isolate was positive for one of the serogroup 1/2a probes (VGCb-40) and one of the serogroup 1/2c probes (LMO-9). This isolate was previously identified as a member of serogroup 1/2c based on mapping lineage-specific MLGT data onto a multilocus phylogeny (34) but produced a serogroup 1/2a-specific banding pattern (data not shown) with the multiplex PCR assay described by Doumith et al. (7). Similar strains, including the common laboratory strain EGD-e, were found to have genomes that are more similar to serogroup 1/2c strains than to strains from the 1/2a serogroup (8, 33) and likely represent intermediates in the evolution of the 1/2c clade from 1/2a ancestors. There is a poor correlation between genomic and antigenic variation for such isolates (34), consistent with the ambiguous results produced by application of the TMLGT assay to NRRL B-33880. The two other problematic isolates, NRRL B-33555 and NRRL B-33559, were accurately identified based on TMLGT data as lineage II isolates from the 1/2a serogroup. However, these two isolates were positive for both ECIII-specific probes in the TMLGT assay but have lineage-specific MLGT haplotypes (Lm2.46), indicating that they are representatives of a sister group closely related to ECIII (33).In 2005, the Food Safety and Inspection Service (FSIS) implemented an approach to inspection that includes consideration of relative risk in order to determine L. monocytogenes sampling frequency among establishments that produce certain RTE products. This approach incorporates information on production volume, outgrowth potential in the product, steps taken to prevent postlethality contamination, and FSIS sampling history. However, L. monocytogenes subtype-specific variation in ecology and virulence indicates that information on the lineage, major serogroup, and epidemic clone identities of isolates could be used to inform assessments of relative risk and to improve inspection programs that are based on consideration of risk. Several PCR-based methods have been described for differentiation of various combinations of these subgroups (1-3, 5, 7, 10, 35, 37); however, these approaches have focused on a single subgroup or a smaller set of subgroups than is differentiated by TMLGT analysis. Although we previously developed a set of three MLGT assays that can be used to differentiate all of the major serogroups and epidemic clones of L. monocytogenes (9, 33, 34), those assays did not include probes for lineage discrimination and require identification of the lineage prior to application of one of three unique sets of probes. In addition, the MLGT assays were designed to maximize strain discrimination, as opposed to subgroup identification, and require the use of at least twice as many probes as is needed for TMLGT analysis. MLGT data analysis is also more complicated than analysis of TMLGT data, and serogroup or epidemic clone type identification via MLGT requires phylogenetic analyses to place novel haplotypes within an established phylogenetic framework.In the present study, we developed the first assay for simultaneous discrimination of the four lineages, the four major serogroups, and the four previously described epidemic clones of L. monocytogenes. The assay includes multiple markers for each of these subtype probes as well as control probes to ensure that negative probe data were not the result of amplification failure, providing a high degree of internal validation required for use in inspection programs that consider risk in making sampling decisions. In addition, the utility of the assay has been validated with a large and diverse panel of 906 isolates, including 567 isolates from FSIS surveillance of RTE products and processing facilities (see Table S1 in the supplemental material). Data produced by the TMLGT assay are amenable to high-throughput analysis, and a simple spreadsheet utility has been developed to semiautomate subtype identifications and to alert investigators to potentially conflicting probe data (available upon request). In addition to having a potential application in inspection programs, the TMLGT assay provides a rapid and accurate means of characterizing L. monocytogenes isolates from different environments, which would facilitate pathogen tracking and improve understanding of L. monocytogenes ecology. 相似文献
TABLE 1.
Primers used in multiplex amplification for the TMLGT assayAmplicon | Positiona | Gene(s) | Primer | Sequence (5′-3′)b |
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INLa | 455381-456505 | inlA | inl2-a1 | GTCCTTGATAGTCTACTG |
inl2-a2 | ACCAAATTAGTAATCTAGCAC | |||
INLb | 457726-458752 | inlB | inl-f1d | GAATTRTTTAGYCAAGAATGT |
inlb-r | CTACCGGRACTTTATAGTAYG | |||
LMO | 325116-326096 | lmo0298-lmo0300 | lmo-a1 | AAGGCTTACAAGATGGCT |
lmo1a-1r | AAATAATAYGTGATACCGAC | |||
VGCa | 205366-206622 | plcA, hly | plca-f | CTCATCGTATCRTGTGTACC |
hly-r | TCTGGAAGGTCKTGTAGGTTC | |||
VGCb | 208447-209465 | mpl | ra_mpl-f | GTGGAYAGAACTCATAAAGG |
ra_mpl-r | ACTCCCTCCTYGTGATASGCT | |||
VGCc | 209728-211239 | actA | vgc1a-2f | TTCMATRCCAGCAGAACG |
vgc1a-2r | GCAGACCTAATAGCAATGTTG |
TABLE 2.
TMLGT probes and probe performance dataProbeb | Target (n)c | Probe sequenced | IDe | Sensitivity (%) | Specificity (%) |
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VGCb-21 | Lineage I (506) | AATCCTTTCTTTAATCTCAAATCAgcggaagcttgggaagcggtc | 7.3 | 100 | 100 |
VGCa-94 | Lineage I | CTTTCTATCTTTCTACTCAATAATcaacccgatgttcttcctgtc | 51.7 | 100 | 100 |
VGCc-8 | Lineage II (340) | AATCCTTTTACATTCATTACTTACattagctgattcgctttcct | 14.1 | 100 | 100 |
INLb-51 | Lineage II | TCATTTCAATCAATCATCAACAATagcgccaataaagctggc | 21.9 | 100 | 100 |
VGCb-19 | Lineage III (50) | TCAATCAATTACTTACTCAAATACccgctattaaaatgtactcca | 31.0 | 100 | 100 |
VGCb-29 | Lineage III | AATCTTACTACAAATCCTTTCTTTggtataccgctattaaaatgt | 45.1 | 100 | 100 |
LMO-17 | Lineage IV (10) | CTTTAATCCTTTATCACTTTATCAgaaccaaacaatgttattggt | 11.8 | 100 | 100 |
VGCa-27 | Lineage IV | CTTTTCAAATCAATACTCAACTTTttaacgacggtaacgtgccac | 58.3 | 100 | 100 |
INLb-84 | Serogroup 4b (213) | TCAACTAACTAATCATCTATCAATggtaaaaatatgcgaatattg | 9.7 | 100 | 100 |
INLb-85 | Serogroup 4b | ATACTACATCATAATCAAACATCActcgtgaacaagctttcc | 5.5 | 100 | 100 |
INLb-16 | Serogroup 1/2b (293) | AATCAATCTTCATTCAAATCATCAggtaaaaatatgcgtatctta | 11.7 | 100 | 100 |
INLb-100 | Serogroup 1/2b | CTATCTTTAAACTACAAATCTAACgtgaataagctatcggtctat | 13.0 | 100 | 100 |
LMO-42 | Serogroup 1/2a (268) | CTATCTTCATATTTCACTATAAACtggcgttgctgrctaagtttg | 6.6 | 100 | 100 |
VGCb-40 | Serogroup 1/2a | CTTTCTACATTATTCACAACATTAaatcaagcsgctcatatgaag | 10.4 | 100 | 98.6 |
LMO-9 | Serogroup 1/2c (72) | TAATCTTCTATATCAACATCTTACtttactggtgaaatggcg | 13.5 | 100 | 100 |
VGCb-5 | Serogroup 1/2c | CAATTCAAATCACAATAATCAATCaagattacgaatcgcttccac | 20.8 | 98.6 | 100 |
LMO-10 | ECI (111) | ATCATACATACATACAAATCTACAatgattaaaagtcagggaaag | 19.0 | 100 | 100 |
LMO-28 | ECI | CTACAAACAAACAAACATTATCAAaatcgaggcttacgaacgt | 23.7 | 100 | 100 |
VGCc-80 | ECIa (44) | CTAACTAACAATAATCTAACTAACactacaacgaaaacagcgc | 10.7 | 100 | 100 |
VGCa-35 | ECIa | CAATTTCATCATTCATTCATTTCAgttacttttatgtcgagt | 9.2 | 100 | 100 |
LMO-12 | ECII (35) | TACACTTTCTTTCTTTCTTTCTTTataccgattatttggacggtt | 3.8 | 100 | 100 |
LMO-30 | ECII | TTACCTTTATACCTTTCTTTTTACgacttgtagcagttgatttcaa | 7.5 | 100 | 100 |
VGCc-45 | ECIII (10) | TCATTTCACAATTCAATTACTCAActcttatttgcttttgttggtc | 21.1 | 100 | 99.4 |
INLa-3 | ECIII | TACACTTTATCAAATCTTACAATCgagcttaatgaaaatcagcta | 17.0 | 100 | 99.4 |
INLa-1 | INLa control | CTTTAATCTCAATCAATACAAATCagaagtggaagctgggaa | NAa | NA | NA |
INLb-13 | INLb control | CAATAAACTATACTTCTTCACTAAtgcacctaaacctccgac | NA | NA | NA |
LMO-88 | LMO control | TTACTTCACTTTCTATTTACAATCccgtttccttatgccaca | NA | NA | NA |
VGCa-23 | VGCa control | TTCAATCATTCAAATCTCAACTTTcaagycctaagacgccaatcg | NA | NA | NA |
VGCb-25 | VGCb control | CTTTTCAATTACTTCAAATCTTCAgcatgcgttagttcatgrcca | NA | NA | NA |
VGCc-82 | VGCc control | TACATACACTAATAACATACTCATgactgcatgctagaatctaag | NA | NA | NA |
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Haruyuki Nakayama-Imaohji Hideki Hirakawa Minoru Ichimura Shin Wakimoto Satoru Kuhara Tetsuya Hayashi Tomomi Kuwahara 《Journal of bacteriology》2009,191(19):6003-6011
The human gut microbe Bacteroides fragilis can alter the expression of its surface molecules, such as capsular polysaccharides and SusC/SusD family outer membrane proteins, through reversible DNA inversions. We demonstrate here that DNA inversions at 12 invertible regions, including three gene clusters for SusC/SusD family proteins, were controlled by a single tyrosine site-specific recombinase (Tsr0667) encoded by BF0667 in B. fragilis strain YCH46. Genetic disruption of BF0667 diminished or attenuated shufflon-type DNA inversions at all three susC/susD genes clusters, as well as simple DNA inversions at nine other loci, most of which colocalized with susC/susD family genes. The inverted repeat sequences found within the Tsr0667-regulated invertible regions shared the consensus motif sequence AGTYYYN4GDACT. Tsr0667 specifically mediated the DNA inversions of 10 of the 12 regions, even under an Escherichia coli background when the invertible regions were exposed to BF0667 in E. coli cells. Thus, Tsr0667 is an additional globally acting DNA invertase in B. fragilis, which probably involves the selective expression of SusC/SusD family outer membrane proteins.The human gut harbors an abundant and diverse microbiota. Bacteroides is one of the most abundant genera of human gut microflora (10, 17, 20), and the biological activities of Bacteroides species are deeply integrated into human physiology through nutrient degradation, the production of short-chain fatty acids, or immunomodulatory molecules (11-14, 24). Recent genomic analyses of Bacteroides have revealed that the bacteria possess redundant abilities not only to bind and degrade otherwise indigestible dietary polysaccharides but also to produce vast arrays of capsular polysaccharide (5, 19, 38, 39). These functional redundancies have been established by the extensive duplication of various genes that encode molecules such as glycosylhydrolases, glycosyltransferases, and outer membrane proteins of the SusC/SusD family (starch utilization system) known to be involved in polysaccharide recognition and transport (7, 27, 28, 30). It has been assumed that these functional redundancies of Bacteroides contribute to the stability of the gut ecosystem (3, 21, 23, 32, 39).Another characteristic feature common in Bacteroides species is that the expression of some of the genes is altered in an on-off manner by reversible DNA inversions at gene promoters or within the protein-coding regions (5, 9, 19, 38, 39). These phase-variable phenotypes are associated with surface architectures such as capsular polysaccharides and SusC/SusD family proteins (5, 6, 16, 19). Our previous genomic analyses of Bacteroides fragilis strain YCH46 revealed that it contained as many as 31 invertible regions in its chromosome (19). These invertible regions can be grouped into six classes according to the internal motif sequences within inverted repeat sequences (IRs) (Table (Table1).1). The DNA inversions within these regions are thought to be controlled by site-specific DNA invertases specific to each class. B. fragilis strain YCH46 contains 33 tyrosine site-specific recombinases (Tsr) genes and four serine site-specific recombinases (Ssr) genes. Generally, DNA invertases mediate DNA inversions at adjacent regions, such as FimB and FimE, that flip their immediate downstream promoters to generate a phase-variable phenotype of type I pili in Escherichia coli (15). B. fragilis is unique in that this anaerobe possesses not only locally acting DNA invertases but also globally acting DNA invertases that mediate DNA inversions at distant loci (8, 29). It has been reported that B. fragilis possesses at least two types of master DNA invertase that regulate DNA inversions at multiple loci simultaneously (8, 29). One is Mpi, an Ssr that mediates the on-off switching of 13 promoter regions (corresponding to class I regions in B. fragilis strain YCH46), including seven promoter regions for capsular polysaccharide biosynthesis in B. fragilis strain NCTC9343 (8). The other master DNA invertase is Tsr19, a Tsr that regulates DNA inversions at two distantly located promoter regions (corresponding to class IV regions in B. fragilis strain YCH46) associated with the large encapsulation phenotype (6, 26, 29). The invertible regions contain specific consensus motifs within the IRs corresponding to each DNA invertase and constitute a regulatory unit. We designated this type of regulatory unit as an “inverlon,” which consists of at least two invertible regions controlled by a single master DNA invertase.
Open in a separate windowaOur previous classification of the invertible regions identified in B. fragilis strain YCH46 genome (19).bConsensus motif sequences found within IRs are shown. R = A or G, W = A or T, and N = A, G, C, or T.cThe gene identifications in B. fragilis strain YCH46 genome are shown.Our previous studies indicated that an additional inverlon other than the Mpi- and Tsr19-regulated inverlons is present in B. fragilis, based on the finding that at least 10 invertible regions (corresponding to class II regions in B. fragilis strain YCH46) contain a particular consensus motif sequence (AAGTTCN5GAACTT) within their IRs (19) but do not appear to colocalize with a DNA invertase gene. The majority of the class II regions were associated with the selective switching of a particular set of susC/susD family genes. Since the SusC/SusD family of outer membrane proteins play an important role in polysaccharide utilization by Bacteroides (3, 23, 32), the inverlon associated with the phase variation of SusC/SusD family proteins would likely be involved in the survival of this anaerobe in the distal gut.In the present study, we sought to identify the DNA invertase regulating the additional inverlon in B. fragilis. Our results indicated that the Tsr encoded by BF0667 is a master DNA invertase for this inverlon (designated the Tsr0667-inverlon) in B. fragilis. 相似文献
TABLE 1.
Classification of the invertible regions in B. fragilis strain YCH46 based on internal motif sequences within IRsClassa | No. | Consensus motif sequencesb | Master DNA invertase genec | Regulated genes | Source or reference(s) |
---|---|---|---|---|---|
I | 14 | ARACGTWCGT | BF2765 (mpi) | Capsular polysaccharide biosynthesis genes | 8 |
II | 10 | AGTTC{N5}GAACT | BF0667 | susC/susD paralogs | This study |
III | 3 | GTTAC{N7}GTAAC | BF3038, BF4033, BF4283 | Putative outer membrane protein genes | 36 |
IV | 2 | TACTTANTAGGTAANAGAA | BF2766 | Extracellular polysacharide biosynthesis genes | 6, 26, 29 |
V | 1 | TCTGCAAAGNCTTTGCAGA | BF0667 | susC/susD paralogs | This study |
VI | 1 | ACTAAGTTCTATCGG | BF0667 | susC/susD paralogs | This study |
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Robert Ng Lakshmanan Govindasamy Brittney L. Gurda Robert McKenna Olga G. Kozyreva R. Jude Samulski Kristin N. Parent Timothy S. Baker Mavis Agbandje-McKenna 《Journal of virology》2010,84(24):12945-12957
The three-dimensional structure of adeno-associated virus (AAV) serotype 6 (AAV6) was determined using cryo-electron microscopy and image reconstruction and using X-ray crystallography to 9.7- and 3.0-Å resolution, respectively. The AAV6 capsid contains a highly conserved, eight-stranded (βB to βI) β-barrel core and large loop regions between the strands which form the capsid surface, as observed in other AAV structures. The loops show conformational variation compared to other AAVs, consistent with previous reports that amino acids in these loop regions are involved in differentiating AAV receptor binding, transduction efficiency, and antigenicity properties. Toward structure-function annotation of AAV6 with respect to its unique dual glycan receptor (heparan sulfate and sialic acid) utilization for cellular recognition, and its enhanced lung epithelial transduction compared to other AAVs, the capsid structure was compared to that of AAV1, which binds sialic acid and differs from AAV6 in only 6 out of 736 amino acids. Five of these residues are located at or close to the icosahedral 3-fold axis of the capsid, thereby identifying this region as imparting important functions, such as receptor attachment and transduction phenotype. Two of the five observed amino acids are located in the capsid interior, suggesting that differential AAV infection properties are also controlled by postentry intracellular events. Density ordered inside the capsid, under the 3-fold axis in a previously reported, conserved AAV DNA binding pocket, was modeled as a nucleotide and a base, further implicating this capsid region in AAV genome recognition and/or stabilization.Adeno-associated viruses (AAVs) are nonpathogenic single-stranded DNA (ssDNA) parvoviruses that belong to the Dependovirus genus and require helper viruses, such as Adenovirus or Herpesvirus, for lytic infection (4, 8, 22, 67). These viruses package a genome of ∼4.7 kb inside an icosahedral capsid (∼260 Å in diameter) with a triangulation number equal to 1 assembled from a total of 60 copies of their overlapping capsid viral protein (VP) 1 (VP1), VP2, and VP3 in a predicted ratio of 1:1:8/10 (10). The VPs are encoded from a cap open reading frame (ORF). VP3 is 61 kDa and constitutes 90% of the capsid''s protein composition. The less abundant VPs, VP1 (87 kDa) and VP2 (73 kDa), share the same C-terminal amino acid sequence with VP3 but have additional N-terminal sequences. A rep ORF codes for four overlapping proteins required for replication and DNA packaging.To date, more than 100 AAV isolates have been identified (21). Among the human and nonhuman primate AAVs isolated, 12 serotypes (AAV serotype 1 [AAV1] to AAV12) have been described and are classified into six phylogenetic clades on the basis of their VP sequences and antigenic reactivities, with AAV4 and AAV5 considered to be clonal isolates (21). AAV1 and AAV6, which represent clade A, differ by only 6 out of 736 VP1 amino acids (5 amino acids within VP3) and are antigenically cross-reactive. Other clade representatives include AAV2 (clade B), AAV2-AAV3 hybrid (clade C), AAV7 (clade D), AAV8 (clade E), and AAV9 (clade F) (21).The AAVs are under development as clinical gene delivery vectors (e.g., see references 5, 9, 12, 13, 24, 25, 53, and 61), with AAV2, the prototype member of the genus, being the most extensively studied serotype for this application. AAV2 has been successfully used to treat several disorders, but its broad tissue tropism makes it less effective for tissue-specific applications and the prevalence of preexisting neutralizing antibodies in the human population (11, 43) limits its utilization, especially when readministration is required to achieve a therapeutic outcome. Efforts have thus focused on characterizing the capsid-associated tissue tropism and transduction properties conferred by the capsid of representative serotypes of other clades (21). Outcomes of these studies include the observation that AAV1 and AAV6, for example, transduce liver, muscle, and airway epithelial cells more efficiently (e.g., up to 200-fold) than AAV2 (27, 28, 30). In addition, the six residues (Table (Table1)1) that differ between the VPs of AAV1 and AAV6 (a natural recombinant of AAV1 and AAV2 [56]) confer functional disparity between these two viruses. For example, AAV6 shows ∼3-fold higher lung cell epithelium transduction than AAV1 (27), and AAV1 and AAV6 bind terminally sialylated proteoglycans as their primary receptor, whereas AAV6 additionally binds to heparan sulfate (HS) proteoglycans with moderate affinity (70, 71). Therefore, a comparison of the AAV1 and AAV6 serotypes and, in particular, their capsid structures can help pinpoint the capsid regions that confer differences in cellular recognition and tissue transduction.
Open in a separate windowaMutant residues in boldface have an AAV6 parental original; those underlined have an AAV1 parental origin.bS, sialic acid; HS, heparan sulfate; HS+, HS positive.cThe sialic acid binding phenotypes of these mutants were not discussed in the respective publications but are assumed to be still present.dThe glycan targets for these mutants were not discussed in this publication; thus, the phenotypes indicated are assumed.eThis mutant is sialic acid independent (S-ind) for cellular transduction.The structures of AAV1 to AAV5 and AAV8 have been determined by X-ray crystallography and/or cryo-electron microscopy and image reconstruction (cryo-EM) (23, 36, 47, 52, 66, 73; unpublished data), and preliminary characterization of crystals has also been reported for AAV1, AAV5, AAV7, and AAV9 (15, 45, 46, 55). The capsid VP structures contain a conserved eight-stranded (βB to βI) β-barrel core and large loop regions between the strands that form the capsid surface. The capsid surface is characterized by depressions at the icosahedral 2-fold axes of symmetry, finger-like projections surrounding the 3-fold axes, and canyon-like depressions surrounding the 5-fold axes. A total of nine variable regions (VRs; VRI to VRIX) were defined when the two most disparate structures, AAV2 and AAV4, were compared (23). The VRs contain amino acids that contribute to slight differences in surface topologies and distinct functional phenotypes, such as in receptor binding, transduction efficiency, and antigenic reactivity (10, 23, 37, 47).The structure of virus-like particles (VLPs) of AAV6, produced in a baculovirus/Sf9 insect cell expression system, has been determined by two highly complementary approaches, cryo-EM and X-ray crystallography. The AAV6 VP structure contains the general features already described for the AAVs and has conformational differences in the VRs compared to the VRs of other AAVs. The 9.7-Å-resolution cryoreconstructed structure enabled the localization of the C-α positions of five of the six amino acids that differ between highly homologous AAV6 and AAV1 but did not provide information on the positions of the side chains or their orientations. The X-ray crystal structure determined to 3.0-Å resolution enabled us to precisely map the atomic positions of these five residues at or close to the icosahedral 3-fold axes of the capsid. Reported mutagenesis and biochemical studies had functionally annotated the six residues differing between AAV1 and AAV6 with respect to their roles in receptor attachment and differential cellular transduction. Their disposition identifies the 3-fold capsid region as playing essential roles in AAV infection. 相似文献
TABLE 1.
Amino acid differences between AAV1 and AAV6 and their reported mutantsAAV | Amino acid at positiona: | Glycan targetb | Reference | ||||||
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129 | 418 | 531 | 532 | 584 | 598 | 642 | |||
AAV1 | L | E | E | D | F | A | N | S | 70 |
AAV1-E/K | L | E | K | D | F | A | N | HS+ (and S)c | 70 |
AAV6 | F | D | K | D | L | V | H | HS and S | 70 |
AAV6.1 | F | D | E | D | L | V | H | HS− (and S)c | 40, 70 |
AAV6.2 | L | D | K | D | L | V | H | HS (and S)c | 40, 70 |
AAV6R2 | L | D | E | D | L | V | H | HS− (and S)c | 40 |
HAE1 | L | E | E | D | L | V | N | (HS− and S)d | 39 |
HAE2 | L | D | K | D | L | V | N | (HS and S)d | 39 |
shH10 | F | D | K | N | L | V | N | HS (and S-inde) | 33 |
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Ashraf A. Khan Elizabeth Ponce M. S. Nawaz Chorng-Ming Cheng Junaid A. Khan Christine S. West 《Applied and environmental microbiology》2009,75(4):1192-1196
A total of 210 Salmonella isolates, representing 64 different serovars, were isolated from imported seafood samples, and 55/210 isolates were found to be resistant to at least one antibiotic. Class 1 integrons from three multidrug-resistant Salmonella enterica strains (Salmonella enterica serovars Newport [strain 62], Typhimurium var. Copenhagen [strain 629], and Lansing [strain 803], originating from Hong Kong, the Philippines, and Taiwan, respectively) were characterized. Southern hybridization of plasmids isolated from these strains, using a class 1 integron probe, showed that trimethoprim-sulfamethoxazole and streptomycin resistance genes were located on a megaplasmid in strain 629. Our study indicates that imported seafood could be a reservoir for Salmonella isolates resistant to multiple antibiotics.Salmonella spp. are recognized as major food-borne pathogens of humans worldwide. In the United States, there are an estimated 800,000 to 4 million Salmonella infections annually, and approximately 500 of the cases are fatal (8, 26). A variety of foods have been implicated as vehicles transmitting salmonellosis to humans, including poultry, beef, pork, eggs, milk, cheese, fish, shellfish, fruits, juice, and vegetables (1, 4, 9, 12, 23). Previous studies by field laboratories of the U.S. Food and Drug Administration have shown the prevalences of Salmonella isolates in imported and domestic seafood as 7.2% and 1.3%, respectively (6, 11, 27).Mobile genetic elements, such as plasmids, transposons, and integrons, which disseminate antibiotic resistance genes by horizontal or vertical transfer, as part of either resistance plasmids or conjugative transposons, play an important role in the evolution and dissemination of multidrug resistance (2, 3, 10, 17). Salmonella genomic island 1 (SGI1), the first genomic island reported to contain an antibiotic resistance gene cluster, was identified in the multidrug-resistant Salmonella enterica serovar Typhimurium strain DT 104 (21).Most studies of the prevalence and characterization of antimicrobial resistance genes and integrons in Salmonella spp. have focused on strains from clinical and veterinary sources. However, little is known about the occurrence of SGI1 and its variants in Salmonella spp. isolated from seafood. We have screened a set of drug-resistant S. enterica strains from seafood belonging to 64 different serovars for SGI1 and class 1 integron conserved sequences (CS). We report the presence of a class I variant integron carrying the dfrXII and aadA2 genes on a megaplasmid in serovar Typhimurium var. Copenhagen and on the chromosome in Salmonella enterica serovar Lansing. We also found the variant class 1 integron carrying the dfrA1 and orfC genes in Salmonella enterica serovar Newport strains from seafood.A total of 210 Salmonella enterica strains isolated from seafood imported into the United States between 2000 and 2005 were identified and serotyped by the Pacific Regional Laboratory-Southwest of the FDA, Irvine, CA. The Salmonella strains represented 20 serogroups (Table (Table1)1) from various imported seafood items. The Salmonella strains were tested with 16 antibiotics (14) commonly used in either human or veterinary medicine on Mueller-Hinton agar (Difco Laboratories, Detroit, MI), using a disk diffusion method. The sensitivity and resistance were determined by the criteria of the Clinical and Laboratory Standards Institute (1999).
Open in a separate windowAll Salmonella strains that were resistant to three or four antibiotics and trimethoprim were screened by PCR for the presence of class 1 integrons, using the CSL1 and CSR1 primers (Table (Table2)2) (14). To confirm other antibiotic resistance genes, we used primers and PCR methods described previously (13, 14, 16). To identify SGI1 in multidrug-resistant strains, PCR was performed by using primers U7-L12/LJ-R1 and 104-RJ/104-D (Table (Table2),2), corresponding to the left and right junctions of SGI1 in the Salmonella chromosome, respectively (16). For a positive control, serovar Typhimurium DT104 strain DT7 (13) was used. As a negative control, Escherichia coli cells or DNA was used. A reagent blank included in each PCR contained distilled water instead of template DNA. For sequencing, the PCR-amplified integrons were purified and cloned into plasmid vector pCR2.1 (Invitrogen Corp., Carlsbad, CA). The clones were investigated for the presence of inserts by isolating the recombinant plasmid, which was confirmed by digestion with the restriction enzyme EcoRI. Sequencing of both strands was performed. DNA sequences were analyzed with Lasergene (DNASTAR, Inc., Madison, WI) software. Oligonucleotide primers and probes were purchased from MWG Biotech (High Point, NC).
Open in a separate windowPlasmid DNA was isolated using an alkaline lysis method with modifications described previously (19). Plasmids were separated by electrophoresis in 1× Tris-acetate-EDTA buffer at 64 V for 2 h on 1.0% agarose gels, stained with 40 μl of ethidium bromide (0.625 mg/ml) for visualization, and then transferred and cross-linked to positively charged nylon membranes (Roche, Indianapolis, IN). The resulting blots were hybridized at 65°C for 18 h with digoxigenin-labeled DNA probes (1.2-kb and 1.9-kb PCR-amplified products), using CSL1 and CSR1 primers specific for class 1 integrons (22). 相似文献
TABLE 1.
Salmonella serotypes isolated from imported foodsNo. of strains | S. enterica serovar(s) or Salmonella group(s) |
---|---|
39 | Weltevreden |
16 | Newport |
13 | Saintpaul |
10 | Senftenberg |
8 | Lexington |
7 | Virchow |
6 | Enteritidis, Bareily |
5 | Bovismorbificans, Brunei, Java, Hvittingfoss |
4 | Paratyphi B var. Java, Thompson |
3 | Aberdeen, Cubana, Stanley, Derby, Lansing |
2 | Montevideo, Hadar, Agona, San Diego, Braenderup, Lanka, Salmonella enterica subsp. diarizonae, Oslo, Bareily variant, Salmonella monophasic group C2 |
1 | Ouakam, Cannstatt, Albany, Newport/Bardo, Adelaide, S. enterica subsp. diarizonae, Houten, Giza, Miami, Onderstepoof, Infantis, Salmonella monophasic group D1, Mbandaka, Salmonella monophasic group G2, Ohio, Rutgers, Salmonella monophasic group D2, Amsterdam, Salmonella enterica subsp. IV serotype 43:z4z23, Paratyphi B var. Java, Wentworth, Potsdam, Muenster var. 15+, 34+, Lexington var. 15+, Weltevreden var. 15+, S. enterica subsp. I, Madella, Alachua, London, Singapore, Uphill, Thielallee, Typhimurium var. Copenhagen |
TABLE 2.
Primer pairs for integron PCR and sequencingPrimer | Sequence (5′-3′) | Location | PCR product size (bp) |
---|---|---|---|
CSL1 | GGC ATC CAA GCA GCA AGC | 5′ CS | |
CSR1 | AAG CAG ACT TGA CCT GAT | 3′ CS | |
U7-L12 | ACA CCT TGA GCA GGG CAA AG | thdF | 500 |
LJ-R1 | AGT TCT AAA GGT TCG TAG TCG | ||
104-RJ | TGA CGA GCT GAA GCG AAT TG | S044 | |
104D | ACC AGG GCA AAA CTA CAC AG | yidY | |
aadA2F | TGT TGG TTA CTG TGG CCG TA | aadA2 | 380 |
aadA2R | GCT GCG AGT TCC ATA GCT TC |
19.
Yvonne Qvarnstrom Ana Cristina Aramburu da Silva John L. Teem Robert Hollingsworth Henry Bishop Carlos Graeff-Teixeira Alexandre J. da Silva 《Applied and environmental microbiology》2010,76(15):5287-5289
Angiostrongylus cantonensis is the most common cause of human eosinophilic meningitis. Humans become infected by ingesting food items contaminated with third-stage larvae that develop in mollusks. We report the development of a real-time PCR assay for the species-specific identification of A. cantonensis in mollusk tissue.Angiostrongylus cantonensis is the most common agent associated with eosinophilic meningitis in humans. Young adult worms develop in the brains of rodents and are carried to pulmonary arteries to reach sexual maturity. Eggs are laid in lung tissues, and first-stage (L1) larvae break into air spaces, migrate to the trachea, are swallowed, and are passed with rodent feces. The L1 larvae must infect mollusks to develop into third-stage (L3) larvae; L3 is the infective stage for rodents and other mammals. Humans become infected by ingesting raw produce contaminated with L3 larvae or infected raw or undercooked mollusks or paratenic hosts. The immature worms remain in the human brain, creating tissue damage and inflammation (2, 19, 21).A. cantonensis is endemic in Southeast Asia, parts of the Caribbean, and the Pacific Islands, including Hawaii (7, 12, 15-17). The worm has been detected in host animals in Louisiana (5, 14) and in one human patient from New Orleans (18), but it is currently unclear to what extent the nematode has spread into other U.S. states (8, 9). Ascertaining the geographic presence of the parasite is important to manage and prevent new cases of eosinophilic meningitis associated with ingestion of infective larvae (12, 18).Detection of A. cantonensis in mollusks can be performed by releasing the larvae from the tissue with pepsin digestion (11). However, that procedure requires access to living mollusks, which complicates analysis of large numbers of samples. After a recent outbreak of angiostrongyliasis in Hawaii (12), we developed a conventional PCR assay and applied it to survey the Hawaiian mollusk population using frozen tissue (20). That PCR assay, as well as morphological identification using pepsin digestion, can only identify the larvae on the superfamily level, so additional molecular work is required for species-specific classification. Here we describe a new real-time PCR assay that allows for a direct detection of A. cantonensis at the species level.The 18S rRNA gene is too conserved among nematode species to allow species-specific detection. The first and second internal transcribed spacers (ITS1 and ITS2) are comparatively more variable than the rRNA coding regions and have thus been used for differentiation of closely related species (1, 4, 6, 10, 22, 23). We PCR amplified and sequenced ITS1 from A. costaricensis (two laboratory strains from Costa Rica and Brazil), A. vasorum (from naturally infected hosts in United Kingdom), and A. cantonensis from three geographical regions (one laboratory strain from Japan plus nine environmental isolates from Hawaii and New Orleans, LA) to assess the variability of this potential PCR target. The oligonucleotide primers used were AngioF1674 (5′-GTCGTAACAAGGTATCTGTAGGTG-3′) and 58SR4 (5′-TAGCTGCGTTTTTCATCGATA-3′). The reaction mixtures contained 0.4 μM each primer and AmpliTaq Gold PCR master mix (Applied Biosystems, Foster City, CA) and were cycled 45 times at 94°C for 30 s, 65°C for 30 s, and 72°C for 1 min. PCR products were cloned into pCR2.1 vectors using the TOPO cloning technique (Invitrogen, Carlsbad, CA) and sequenced on both strands as described elsewhere (20).The sequence analysis revealed high interspecific and low intraspecific variability. A TaqMan assay targeting ITS1 was then designed using Primer Express version 2.3 (Applied Biosystems, Foster City, CA). The real-time PCR assay was performed in a 20-μl total volume containing Platinum qPCR Supermix (Invitrogen, Carlsbad, CA), 0.2 μM (each) primers AcanITS1F1 (5′-TTCATGGATGGCGAACTGATAG-3′) and AcanITS1R1 (5′-GCGCCCATTGAAACATTATACTT-3′), and 0.05 μM the TaqMan probe AcanITS1P1 (5′-6-carboxyfluorescein-ATCGCATATCTACTATACGCATGTGACACCTG-BHQ-3′). The standard cycling conditions for TaqMan assays were used (i.e., 40 cycles of 95°C for 15 s and 60°C for 1 min).We evaluated the real-time PCR assay with a set of 26 Parmarion martensi slugs from Hawaii. Seventeen slugs were positive for L3 larvae as determined by pepsin digestion, and nine slugs were negative. DNA was extracted from approximately 25 mg of tissue of each slug using the DNeasy tissue and blood DNA extraction kit (Qiagen, Inc., Valencia, CA). The real-time PCR performed on this set of samples returned an identical result to the morphological analysis. The real-time PCR amplified only DNA from A. cantonensis and did not react with DNA from other nematode species (Table (Table1).1). The detection limit of the assay was determined by serially diluting a recombinant plasmid containing the ITS1 sequence to less than 1 copy per μl of sample. The real-time PCR reliably detected down to 10 plasmid copies in the reaction.
Open in a separate windowaThis number includes three samples positive by PCR but later identified as non-Angiostrongylus nematodes by DNA sequencing analysis of the amplicons (20). These three samples were negative in the real-time PCR assay.bThe conventional PCR detects other Angiostrongylus species besides A. cantonensis.cTwo stool samples containing Strongyloides worms, eight environmental samples containing unclassified free-living nematodes and one of each of the following parasitic nematodes: Dipetalonema sp., Toxocara cati, Dracunculus medinensis, and Ascaris lumbricoides.The real-time PCR assay was then used to analyze a larger set of naturally infected host animals from Hawaii, partly described elsewhere (13, 20), and Island Apple snails (Pomacea insularum) from New Orleans, LA. All samples had previously been characterized by the conventional PCR followed by DNA sequencing analysis (20).Table Table11 summarizes the PCR findings and highlights the enhanced performance of the real-time PCR in comparison to the conventional PCR. In addition, the real-time PCR assay was more practical to use since it did not require DNA sequence confirmation to rule out false positives.The findings from Island Apple snails from New Orleans infected with A. cantonensis concur with previous reports about the potential for angiostrongyliasis transmission in this area (5, 14). Another interesting finding was the positive PCR results in two samples of flatworms from Hawaii. Predatory flatworms that ingest infected mollusks are known to be paratenic hosts of A. cantonensis and have been suspected to be an important source of infection for humans in Japan because they hide in leafy vegetables (3).In conclusion, this real-time PCR assay can be a useful tool for environmental surveys of local wildlife to determine the geographic distribution of this reemerging human parasite. 相似文献
TABLE 1.
Comparison of conventional and real-time PCR for detection of Angiostrongylus cantonensis in mollusks and nematode samplesBiological origin of DNA sample | Geographic origin | No. of samples tested | No. of samples positive by: | |
---|---|---|---|---|
18S rRNA-based conventional PCR | ITS1-based TaqMan PCR | |||
Parmarion martensi | Hawaii | 112 | 75 | 83 |
Veronicella cubensis | Hawaii | 50 | 23a | 22 |
Laevicaulis alte | Hawaii | 5 | 3 | 4 |
Achatina fulica | Hawaii | 6 | 4 | 5 |
Other/unidentified mollusks | Hawaii | 16 | 4 | 5 |
Flatworms | Hawaii | 2 | 2 | 2 |
Slime from infected slugs | Hawaii | 13 | 1 | 1 |
Pomacea insularum | Louisiana | 31 | 5 | 5 |
A. costaricensis | Brazil, Costa Rica | 2 | 2b | 0 |
A. vasorum | United Kingdom | 2 | 2b | 0 |
Other nematodesc | CDC collection | 14 | 0 | 0 |
Total | 253 | 121 | 127 |