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Lisa Jacobsen Lisa Durso Tyrell Conway Kenneth W. Nickerson 《Applied and environmental microbiology》2009,75(13):4633-4635
Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.Escherichia coli is well known for its ecological versatility (15). A life cycle which includes both gastrointestinal and environmental stages has been stressed by both Savageau (15) and Adamowicz et al. (1). The gastrointestinal stage would be subjected to acid and detergent stress. The environmental stage is implicit in E. coli having transport systems for fungal siderophores (4) as well as pyrroloquinoline quinone-dependent periplasmic glucose utilization (1) because their presence indicates evolution in a location containing fungal siderophores and pyrroloquinoline quinone (1).Since its recognition as a food-borne pathogen, there have been numerous outbreaks of food-borne infection due to E. coli O157:H7, in both ground beef and vegetable crops (6, 13). Cattle are widely considered to be the primary reservoir of E. coli O157:H7 (14), but E. coli O157:H7 does not appear to cause disease in cattle. To what extent is E. coli O157:H7 physiologically unique compared to the other naturally occurring E. coli strains? We feel that the uniqueness of E. coli O157:H7 should be evaluated against a backdrop of other wild-type E. coli strains, and in this regard, we chose the 72-strain ECOR reference collection originally described by Ochman and Selander (10). These strains were chosen from a collection of 2,600 E. coli isolates to provide diversity with regard to host species, geographical distribution, and electromorph profiles at 11 enzyme loci (10).In our study we compared the 72 strains of the ECOR collection against 10 strains of E. coli O157:H7 and six strains of E. coli which had been in laboratory use for many years (Table (Table1).1). The in vitro comparisons were made with regard to factors potentially relevant to the bacteria''s ability to colonize animal guts, i.e., acid tolerance, detergent tolerance, and the presence of the Entner-Doudoroff (ED) pathway (Table (Table2).2). Our longstanding interest in the ED pathway (11) derives in part from work by Paul Cohen''s group (16, 17) showing that the ED pathway is important for E. coli colonization of the mouse large intestine. Growth was assessed by replica plating 88 strains of E. coli under 40 conditions (Table (Table2).2). These included two LB controls (aerobic and anaerobic), 14 for detergent stress (sodium dodecyl sulfate [SDS], hexadecyltrimethylammonium bromide [CTAB], and benzalkonium chloride, both aerobic and anaerobic), 16 for acid stress (pH 6.5, 6.0, 5.0, 4.6, 4.3, 4.2, 4.1, and 4.0), four for the ability to grow in a defined minimal medium (M63 glucose salts with and without thiamine), and four for the presence or absence of a functional ED pathway (M63 with gluconate or glucuronate). All tests were done with duplicate plates in two or three separate trials. The data are available in Tables S1 to S14 in the supplemental material, and they are summarized in Table Table22.
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Open in a separate windowaEight LB controls were run, two for each set of LB experiments: SDS, CTAB, benzalkonium chloride (BAC), and pH stress.bGrowth was measured as either +++, +, or 0 (good, poor, and none, respectively), with +++ being the growth achieved on the LB control plates. “Variable” means that two or three replicates did not agree. All experiments were done at 37°C.c“Anaerobic” refers to use of an Oxoid anaerobic chamber. Aerobic and anaerobic growth data are presented together when the results were identical and separately when the results were not the same or the anaerobic set had not been done. LB plates were measured after 1 (aerobic) or 2 (anaerobic) days, and the M63 plates were measured after 2 or 3 days.dCTAB used at 0.05, 0.2%, and 0.4%.eM63 defined medium (3) was supplemented with glucose, gluconate, or glucuronate, all at 0.2%.fIdentical results were obtained with and without 0.0001% thiamine.gND, not determined. 相似文献
TABLE 1.
E. coli strains used in this studyE. coli strain (n) | Source |
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ECOR strains (72) | Thomas Whittman |
Laboratory adapted (6) | |
K-12 Davis | Paul Blum |
CG5C 4401 | Paul Blum |
K-12 Stanford | Paul Blum |
W3110 | Paul Blum |
B | Tyler Kokjohn |
AB 1157 | Tyler Kokjohn |
O157:H7 (10) | |
FRIK 528 | Andrew Benson |
ATCC 43895 | Andrew Benson |
MC 1061 | Andrew Benson |
C536 | Tim Cebula |
C503 | Tim Cebula |
C535 | Tim Cebula |
ATCC 43889 | William Cray, Jr. |
ATCC 43890 | William Cray, Jr. |
ATCC 43888 | Willaim Cray, Jr. |
ATCC 43894 | William Cray, Jr. |
TABLE 2.
Physiological comparison of 88 strains of Escherichia coliGrowth medium or condition | Oxygenc | No. of strains with type of growthb
| |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
ECOR strains (n = 72)
| Laboratory strains (n = 6)
| O157:H7 strains (n = 10)
| |||||||||||
Good | Poor | None | Variable | Good | Poor | None | Variable | Good | Poor | None | Variable | ||
LB controla | Both | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
1% SDS | Aerobic | 69 | 3 | 0 | 0 | 6 | 0 | 0 | 0 | 8 | 0 | 0 | 2 |
5% SDS | Aerobic | 68 | 4 | 0 | 0 | 6 | 0 | 0 | 0 | 8 | 2 | 0 | 0 |
1% SDS | Anaerobic | 53 | 15 | 4 | 0 | 2 | 3 | 1 | 0 | 1 | 7 | 0 | 2 |
5% SDS | Anaerobic | 0 | 68 | 4 | 0 | 0 | 4 | 2 | 0 | 0 | 7 | 0 | 4 |
CTABd (all) | Both | 0 | 0 | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 |
0.05% BAC | Aerobic | 3 | 11 | 58 | 2 | 0 | 2 | 2 | 2 | 0 | 0 | 9 | 1 |
0.2% BAC | Aerobic | 0 | 1 | 71 | 0 | 1 | 0 | 5 | 0 | 0 | 0 | 10 | 0 |
0.05% BAC | Anaerobic | 2 | 3 | 67 | 0 | 0 | 1 | 5 | 0 | 0 | 0 | 9 | 1 |
0.2% BAC | Anaerobic | 0 | 0 | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 |
pH 6.5 | Both | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
pH 6 | Both | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
pH 5 | Both | 70 | 2 | 0 | 0 | 6 | 0 | 0 | 0 | 9 | 0 | 0 | 1 |
pH 4.6 | Both | 70 | 2 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
pH 4.3 | Aerobic | 14 | 0 | 1 | 57 | 3 | 1 | 2 | 0 | 3 | 2 | 0 | 5 |
pH 4.3 | Anaerobic | 69 | 3 | 0 | 0 | 3 | 1 | 2 | 0 | 1 | 1 | 0 | 0 |
pH 4.1 or 4.2 | Aerobic | 0 | 0 | 72 | 0 | NDg | ND | ||||||
pH 4.0 | Both | 0 | 0 | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 9 | 1 |
M63 with supplemente | |||||||||||||
Glucose | Aerobicf | 69 | 1 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Glucose | Anaerobicf | 70 | 0 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Gluconate | Both | 69 | 1 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Glucuronate | Aerobic | 68 | 2 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Glucuronate | Anaerobic | 69 | 1 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
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Lutz Geue Sabrina Klare Christina Schnick Birgit Mintel Katharina Meyer Franz J. Conraths 《Applied and environmental microbiology》2009,75(21):6947-6953
Twelve cluster groups of Escherichia coli O26 isolates found in three cattle farms were monitored in space and time. Cluster analysis suggests that only some O26:H11 strains had the potential for long-term persistence in hosts and farms. As judged by their virulence markers, bovine enterohemorrhagic O26:H11 isolates may represent a considerable risk for human infection.Shiga toxin (Stx)-producing Escherichia coli (STEC) strains comprise a group of zoonotic enteric pathogens (42). In humans, infections with some STEC serotypes result in hemorrhagic or nonhemorrhagic diarrhea, which can be complicated by hemolytic-uremic syndrome (HUS) (49). These STEC strains are also designated “enterohemorrhagic E. coli” (EHEC). Consequently, EHEC strains represent a subgroup of STEC with a high pathogenic potential for humans. Strains of the E. coli serogroup O26 were originally classified as enteropathogenic E. coli due to their association with outbreaks of infantile diarrhea in the 1940s. In 1977, Konowalchuk et al. (37) recognized that these bacteria produced Stx, and 10 years later, the Stx-producing E. coli O26:H11/H− strains were classified as EHEC. EHEC O26 strains constitute the most common non-O157 EHEC group associated with diarrhea and HUS in Europe (12, 21, 23, 24, 26, 27, 55, 60). Reports on an association between EHEC O26 and HUS or diarrhea from North America including the United States (15, 30, 33), South America (51, 57), Australia (22), and Asia (31, 32) provide further evidence for the worldwide spread of these organisms. Studies in Germany and Austria (26, 27) on sporadic HUS cases between 1996 and 2003 found that EHEC O26 accounted for 14% of all EHEC strains and for ∼40% of non-O157 EHEC strains obtained from these patients. A proportion of 11% EHEC O26 strains was detected in a case-control study in Germany (59) between 2001 and 2003. In the age group <3 years, the number of EHEC O26 cases was nearly equal to that of EHEC O157 cases, although the incidence of EHEC O26-associated disease is probably underestimated because of diagnostic limitations in comparison to the diagnosis of O157:H7/H− (18, 34). Moreover, EHEC O26 has spread globally (35). Beutin (6) described EHEC O26:H11/H−, among O103:H2, O111:H, O145:H28/H−, and O157:H7/H−, as the well-known pathogenic “gang of five,” and Bettelheim (5) warned that we ignore the non-O157 STEC strains at our peril.EHEC O26 strains produce Stx1, Stx2, or both (15, 63). Moreover, these strains contain the intimin-encoding eae gene (11, 63), a characteristic feature of EHEC (44). In addition, EHEC strains possess other markers associated with virulence, such as a large plasmid that carries further potential virulence genes, e.g., genes coding for EHEC hemolysin (EHEC-hlyA), a catalase-peroxidase (katP), and an extracellular serine protease (espP) (17, 52). The efa1 (E. coli factor for adherence 1) gene was identified as an intestinal colonization factor in EHEC (43). EHEC O26 represents a highly dynamic group of organisms that rapidly generate new pathogenic clones (7, 8, 63).Ruminants, especially cattle, are considered the primary reservoir for human infections with EHEC. Therefore, the aim of this study was the molecular characterization of bovine E. coli field isolates of serogroup O26 using a panel of typical virulence markers. The epidemiological situation in the beef herds from which the isolates were obtained and the spatial and temporal behavior of the clonal distribution of E. coli serogroup O26 were analyzed during the observation period. The potential risk of the isolates inducing disease in humans was assessed.In our study, 56 bovine E. coli O26:H11 isolates and one bovine O26:H32 isolate were analyzed for EHEC virulence-associated factors. The isolates had been obtained from three different beef farms during a long-term study. They were detected in eight different cattle in farm A over a period of 15 months (detected on 10 sampling days), in 3 different animals in farm C over a period of 8 months (detected on 3 sampling days), and in one cow on one sampling day in farm D (Table (Table1)1) (28).
Open in a separate windowastx1/stx2, gene stx1 or stx2.befa1 was detected by two hybridizations (with lifA1-lifA2 and lifA3-lifA4 probes). +/+, complete gene; +/− or −/+, incomplete gene; −/−, efa1 negative.The serotyping of the O26 isolates was confirmed by the results of the fliC PCR-restriction fragment length polymorphism (RFLP) analysis performed according to Fields et al. (25), with slight modifications described by Zhang et al. (62). All O26:H11 isolates showed the H11 pattern described by Zhang et al. (62). In contrast, the O26:H32 isolate demonstrated a different fliC RFLP pattern that was identical to the H32 pattern described by the same authors. It has been demonstrated that EHEC O26:H11 strains belong to at least four different sequence types (STs) in the common clone complex 29 (39). In the multilocus sequence typing analysis for E. coli (61), the tested five EHEC O26:H11 isolates (WH-01/02/003-1, WH-01/20/005-3, WH-01/27/009-9, WH-03/14/004-8, and WH-04/22/001-1) of different farms and clusters were characterized as two sequence types (ST 21 and ST 396). The isolates from farms A and C belong to ST 21, the most frequent ST of EHEC O26:H11 isolates found in humans and animals (39), but the single isolate from farm D was characterized as ST 396.Typing and subtyping of genes (stx1 and/or stx2, eae, tir, espA, espB, EHEC-hlyA, katP, and espP) associated with EHEC were performed with LightCycler fluorescence PCR (48) and different block-cycler PCRs. To identify the subtypes of the stx2 genes and of the locus of enterocyte effacement-encoding genes eae, tir, espA, and espB, the PCR products were digested by different restriction endonucleases (19, 26, 46). The complete pattern of virulence markers was detected in most bovine isolates examined in our study. An stx1 gene was present in all O26 isolates. In addition, an stx2 gene was found in nine O26:H11 isolates in farm A and in three isolates of the same type in farm C, as well as in the O26:H32 isolate. Both Stx1 and Stx2 were closely related to families of Stx1 and Stx2 variants or alleles. EHEC isolates with stx2 genes are significantly more often associated with HUS and other severe disease manifestations than isolates with an stx1 gene, which are more frequently associated with uncomplicated diarrhea and healthy individuals (13). In contrast to STEC strains harboring stx2 gene variants, however, STEC strains of the stx2 genotype were statistically significantly associated with HUS (26). The stx2 genotype was found in all O26 isolates with an stx2 gene, while the GK3/GK4 amplification products after digestion with HaeIII and FokI restriction enzymes showed the typical pattern for this genotype described by Friedrich et al. (26). The nucleotide sequences of the A and B subunits of the stx2 gene of the selected bovine O26:H11 isolate WH-01/27/017-1 (GenBank accession no. ) were identical to the stx2 genes of different sorbitol-fermenting EHEC O157:H− strains associated with human HUS cases and other EHEC infections in Germany ( EU70049110) and 99.3% identical in their DNA sequences to the stx2 gene of the EHEC type strain EDL933, a typical O157:H7 isolate from an HUS patient. A characteristic stx1 genotype was present in all O26 isolates. The nucleotide sequences of the A and B subunits of the stx1 gene of the tested bovine O26:H11 isolate WH-01/27/017-1 (GenBank accession no. ) were nearly identical to those of the stx1 genes of the EHEC O26:H11 reference type strains H19 and DEC10B, which had been associated with human disease outbreaks in Canada and Australia. Nucleotide exchanges typical for stx1c and stx1d subtypes as described by Kuczius et al. ( EU70049038) were not found. All bovine O26:H11 strains produced an Stx1 with high cytotoxicity for Vero cells tested by Stx enzyme-linked immunosorbent assay and Vero cell neutralization assay (53). The Stx2 cytotoxicity for Vero cells was also very high in the O26:H11 isolates.Not only factors influencing the basic and inducible Stx production are important in STEC pathogenesis. It has been suggested that the eae and EHEC-hlyA genes are likely contributors to STEC pathogenicity (2, 3, 13, 50). Ritchie et al. (50) found both genes in all analyzed HUS-associated STEC isolates. In all O26:H11 isolates we obtained, stx genes were present in combination with eae genes. Only the O26:H32 isolate lacked an eae gene. To date, 10 distinct variants of eae have been described (1, 19, 36, 45, 47). Some serotypes were closely associated with a particular intimin variant: the O157 serogroup was linked to γ-eae, the O26 serogroup to β-eae, and the O103 serogroup to ɛ-eae (4, 19, 20, 58). Our study confirms these associations. All bovine O26:H11 isolates were also typed as members of the β-eae subgroup. A translocated intimin receptor gene (tir gene) and the type III secreted proteins encoded by the espA and espB genes were found in all 56 O26:H11 isolates but not in the O26:H32 isolate. These other tested locus of enterocyte effacement-associated genes belonged to the β-subgroups. These results are in accord with the results of China et al. (19), who detected the pathotypes β-eae, β-tir, β-espA, and β-espB in all investigated human O26 strains. Like the eae gene, the EHEC-hlyA gene was found in association with severe clinical disease in humans (52). Aldick et al. (2) showed that EHEC hemolysin is toxic (cytolytic) to human microvascular endothelial cells and may thus contribute to the pathogenesis of HUS. In our study, the EHEC-hlyA gene was detected in 50 of the 56 bovine E. coli O26:H11 isolates which harbored virulence-associated plasmids of different sizes (Table (Table1).1). The presence of virulence-associated plasmids corresponded to the occurrence of additional virulence markers such as the espP and katP genes (17). The katP gene and the espP gene were detected in 49 and 50 of the 56 O26:H11 isolates, respectively. The espP gene was missing in six of the seven bovine O26:H11 isolates in which the katP genes were also absent. Both genes were not found in the O26:H32 isolate (Table (Table1).1). Although we found large plasmids of the same size in O26:H11 isolates, they lacked one or more of the plasmid-associated virulence factors (Table (Table1).1). Two DNA probes were used to detect the efa1 genes by colony hybridization. (DNA probes were labeled with digoxigenin [DIG] with lifA1-lifA2 and lifA3-lifA4 primers [14] using the PCR DIG probe synthesis kit [Roche Diagnostics, Mannheim, Germany]; DIG Easy Hyb solution [Roche] was used for prehybridization and hybridization.) Positive results with both DNA probes were obtained for 52 of 56 E. coli O26:H11 isolates. A positive signal was only found in three isolates with the lifA1-lifA2 DNA probe and in one isolate with the lifA3-lifA4 probe. An efa1 gene was not detected in the O26:H32 isolate (Table (Table11).We also analyzed the spatial and temporal behavior of the O26:H11/H32 isolates in the beef herds by cluster analysis (conducted in PAUP* for Windows version 4.0, 2008 [http://paup.csit.fsu.edu/about.html]). This was performed with distance matrices using the neighbor-joining algorithm, an agglomerative cluster method which generates a phylogenetic tree. The distance matrices were calculated by pairwise comparisons of the fragmentation patterns produced by genomic typing through pulsed-field gel electrophoresis analysis with four restriction endonucleases (XbaI, NotI, BlnI, and SpeI) and the presence or absence of potential virulence markers (Fig. (Fig.11 and Table Table1).1). To this end, the total character difference was used, which counts the pairwise differences between two given patterns. During a monitoring program of 3 years in four cattle farms (29), different O26:H11 cluster groups and one O26:H32 isolate were detected in three different farms. The genetic distance of the O26:H32 isolate was very high relative to the O26:H11 isolates. Therefore, the O26:H32 isolate was outgrouped. The O26:H11 isolates of each farm represented independent cluster groups. The single isolate from farm D fitted better to the isolates from farm C than to those from farm A. This finding is in accord with the geographical distance between the farms. The fact that the farms were located in neighboring villages may suggest that direct or indirect connections between the farms were possible (e.g., by person contacts or animal trade). However, the isolates from farm C and farm D belonged to different sequence types (ST 21 and ST 396), which may argue against a direct connection. Interestingly, O26:H11 isolates with and without stx2 genes were detected in the same clusters. This phenomenon was observed in both farm A and farm C. In farm A, the isolates with additional stx2 genes were found in animal 27 and were grouped in clusters 8 and 9 (day 218). An stx2 gene was repeatedly found (four isolates) in the same animal (animal 27). The isolates grouped in cluster 8 on a later day of sampling (day 478). All other O26:H11 isolates grouped in the same clusters and obtained from the same animals (27 and 29) on different sampling days lacked an stx2 gene. Also, the isolates obtained from animal 27 on previous sampling days, which grouped in clusters 3 and 5, exhibited no stx2 genes. In farm C, the three isolates with additional stx2 genes obtained from animal 7 grouped in clusters 11 and 12. An stx2 gene was absent from all other O26:H11 isolates grouped in the same cluster 12 on later sampling days, and no other isolates of cluster 11 were found later on. However, we detected members of many clusters over relatively long periods (clusters 5, 8, and 9 in farm A and cluster 12 in farm C), but members of other clusters were only found on single occasions. This patchy temporal pattern is apparently not a unique property of O26:H11, as we found similar results for cluster groups of other EHEC serotypes of bovine origin (28). The isolates grouped in the dominant cluster 8 were found on 5 of 9 sampling days over a period of 10 months. In contrast, we found the members of clusters 4, 5, 9, and 12 only on two nonconsecutive sampling days. The period during which isolates of these groups were not detected was particularly long for cluster 4 (231 days). We also observed the coexistence of different clusters over long periods in the same farm and in the same cattle (clusters 8 and 9), while one of the clusters dominated. Transmission of clusters between cattle was also observed. These results suggest that some of the EHEC O26:H11 strains had the potential for a longer persistence in the host population, while others had not. The reasons for this difference are not yet clear. Perhaps the incomplete efa1 gene found in isolates of clusters which were only detected once might explain why some strains disappeared rapidly. Efa1 has been discussed as a potential E. coli colonization factor for the bovine intestine used by non-O157 STEC, including O26 (54, 56). The O165:H25 cluster detected during a longer period in farm B may have disappeared after it had lost its efa1 gene (28). The precise biological activity of Efa1 in EHEC O26 is not yet known, but it has been demonstrated that the molecule is a non-Stx virulence determinant which can increase the virulence of EHEC O26 in humans (8).Open in a separate windowFIG. 1.Neighbor-joining tree of bovine E. coli O26:H11/H32 strains based on the restriction pattern obtained after digestion with XbaI, NotI, BlnI, and SpeI.We distinguished 12 different clusters, but complete genetic identity was only found in two isolates. The variations in the O26:H11 clusters may be due to increasing competition between the bacterial populations of the various subtypes in the bovine intestine or to potential interactions between EHEC O26:H11 and the host.The ephemeral occurrence of additional stx2 genes in different clusters and farms may be the result of recombination events due to horizontal gene transfer (16). The loss of stx genes may occur rapidly in the course of an infection, but the reincorporation by induction of an stx-carrying bacteriophage into the O26:H11 strains is possible at any time (9, 40). Nevertheless, an additional stx2 gene may increase the dangerousness of the respective EHEC O26:H11 strains. While all patients involved in an outbreak caused by an EHEC O26:H11 strain harboring the gene encoding Stx2 developed HUS (41), the persons affected by another outbreak caused by an EHEC O26:H11 strain that produced exclusively Stx1 had only uncomplicated diarrhea (60).In conclusion, our results showed that bovine O26:H11 isolates can carry virulence factors of EHEC that are strongly associated with EHEC-related disease in humans, particularly with severe clinical manifestations such as hemorrhagic colitis and HUS. Therefore, strains of bovine origin may represent a considerable risk for human infection. Moreover, some clusters of EHEC O26:H11 persisted in cattle and farms over longer periods, which may increase the risk of transmission to other animals and humans even further. 相似文献
TABLE 1.
Typing of E. coli O26 isolatesSampling day, source, and isolate | Serotype | Virulence profile by: | |||||||||||||||
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fliC PCR-RFLP | stx1 gene | stx2 gene | Stx1 (toxin) | Stx2 (toxin) | Subtype(s) | efa1 geneb | EHEC-hlyA gene | katP gene | espP gene | Plasmid size(s) in kb | Cluster | ||||||
stx1/stx2 | eae | tir | espA | espB | |||||||||||||
Day 15 | |||||||||||||||||
Animal 6 (farm A) | |||||||||||||||||
WH-01/06/002-1 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 7 |
WH-01/06/002-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 7 |
WH-01/06/002-3 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 7 |
Animal 8 (farm A) | |||||||||||||||||
WH-01/08/002-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 7 |
Animal 26 (farm A) | |||||||||||||||||
WH-01/26/001-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12 | 7 |
WH-01/26/001-5 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 7 |
WH-01/26/001-6 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 7 |
WH-01/26/001-7 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/− | + | + | + | 110, 12 | 7 |
Day 29 | |||||||||||||||||
Animal 2 (farm A) | |||||||||||||||||
WH-01/02/003-1 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 6 |
WH-01/02/003-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 6 |
WH-01/02/003-5 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 6 |
WH-01/02/003-6 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | − | + | + | 110, 12 | 6 |
WH-01/02/003-7 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 6 |
WH-01/02/003-8 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | −/+ | + | + | + | 110, 12 | 6 |
WH-01/02/003-9 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110 | 6 |
WH-01/02/003-10 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110 | 6 |
Animal 26 (farm A) | |||||||||||||||||
WH-01/26/002-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12 | 5 |
WH-01/26/002-5 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12 | 5 |
WH-01/26/002-8 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12 | 5 |
WH-01/26/002-9 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | − | − | 110, 12 | 5 |
WH-01/26/002-10 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12 | 5 |
Day 64 | |||||||||||||||||
Animal 20 (farm A) | |||||||||||||||||
WH-01/20/005-3 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | − | − | − | 130, 2.5 | 2 |
Day 78 | |||||||||||||||||
Animal 29 (farm A) | |||||||||||||||||
WH-01/29/002-1 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/− | + | − | − | 130, 12, 2.5 | 4 |
WH-01/29/002-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12, 2.5 | 4 |
WH-01/29/002-3 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12, 2.5 | 4 |
WH-01/29/002-4 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12, 2.5 | 4 |
WH-01/29/002-5 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | − | − | + | 130, 12, 2.5 | 4 |
Day 106 | |||||||||||||||||
Animal 27 (farm A) | |||||||||||||||||
WH-01/27/005-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/− | + | + | + | 145, 110, 12 | 3 |
WH-01/27/005-5 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12, 2.5 | 5 |
WH-01/27/005-6 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | − | − | − | 130, 12, 2.5 | 5 |
Day 113 | |||||||||||||||||
Animal 7 (farm C) | |||||||||||||||||
WH-04/07/001-2 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | − | + | + | 55, 35, 2.5 | 11 |
WH-04/07/001-4 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 55 | 12 |
WH-04/07/001-6 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 55 | 12 |
Day 170 | |||||||||||||||||
Animal 22 (farm C) | |||||||||||||||||
WH-04/22/001-1 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12, 6.3 | 12 |
WH-04/22/001-4 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12, 6.3 | 12 |
WH-04/22/001-5 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12, 6.3 | 12 |
Day 176 | |||||||||||||||||
Animal 14 (farm D) | |||||||||||||||||
WH-03/14/004-8 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | − | + | + | 110 | 10 |
Day 218 | |||||||||||||||||
Animal 27 (farm A) | |||||||||||||||||
WH-01/27/009-1 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110, 12 | 9 |
WH-01/27/009-2 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110, 12 | 9 |
WH-01/27/009-3 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110, 12 | 8 |
WH-01/27/009-8 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | − | − | 110, 12 | 8 |
WH-01/27/009-9 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110, 12 | 9 |
Day 309 | |||||||||||||||||
Animal 29 (farm A) | |||||||||||||||||
WH-01/29/010-1 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 35, 12 | 4 |
WH-01/29/010-2 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | − | − | 130, 55, 35 | 8 |
WH-01/29/010-3 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 130, 35, 12 | 8 |
Day 365 | |||||||||||||||||
Animal 8 (farm C) | |||||||||||||||||
WH-04/08/008-6 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 55 | 12 |
Day 379 | |||||||||||||||||
Animal 9 (farm A) | |||||||||||||||||
WH-01/09/016-2 | O26:H32 | H32 | + | + | − | − | stx1/stx2 | − | − | − | − | −/− | − | − | − | 145, 130, 1.8 | 1 |
Animal 27 (farm A) | |||||||||||||||||
WH-01/27/014-3 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 9 |
WH-01/27/014-4 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 9 |
WH-01/27/014-5 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12 | 8 |
Day 407 | |||||||||||||||||
Animal 29 (farm A) | |||||||||||||||||
WH-01/29/013-4 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12, 2.5 | 8 |
WH-01/29/013-7 | O26:H11 | H11 | + | − | + | − | stx1 | β | β | β | β | +/+ | + | + | + | 110, 12, 2.5 | 8 |
Day 478 | |||||||||||||||||
Animal 27 (farm A) | |||||||||||||||||
WH-01/27/017-1 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110, 12 | 8 |
WH-01/27/017-5 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110, 12 | 8 |
WH-01/27/017-6 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110 | 8 |
WH-01/27/017-7 | O26:H11 | H11 | + | + | + | + | stx1/stx2 | β | β | β | β | +/+ | + | + | + | 110 | 8 |
WH-01/27/017-10 | O26:H11 | H11 | + | − | + | + | stx1 | β | β | β | β | +/+ | + | + | + | 130, 12, 2.5 | 8 |
5.
Vertebrate genomic assemblies were analyzed for endogenous sequences related to any known viruses with single-stranded DNA genomes. Numerous high-confidence examples related to the Circoviridae and two genera in the family Parvoviridae, the parvoviruses and dependoviruses, were found and were broadly distributed among 31 of the 49 vertebrate species tested. Our analyses indicate that the ages of both virus families may exceed 40 to 50 million years. Shared features of the replication strategies of these viruses may explain the high incidence of the integrations.It has long been appreciated that retroviruses can contribute significantly to the genetic makeup of host organisms. Genes related to certain other viruses with single-stranded RNA genomes, formerly considered to be most unlikely candidates for such contribution, have recently been detected throughout the vertebrate phylogenetic tree (1, 6, 13). Here, we report that viruses with single-stranded DNA (ssDNA) genomes have also contributed to the genetic makeup of many organisms, stretching back as far as the Paleocene period and possibly the late Cretaceous period of evolution.Determining the evolutionary ages of viruses can be problematic, as their mutation rates may be high and their replication may be rapid but also sporadic. To establish a lower age limit for currently circulating ssDNA viruses, we analyzed 49 published vertebrate genomic assemblies for the presence of sequences derived from the NCBI RefSeq database of 2,382 proteins from known viruses in this category, representing a total of 23 classified genera from 7 virus families. Our survey uncovered numerous high-confidence examples of endogenous sequences related to the Circoviridae and to two genera in the family Parvoviridae: the parvoviruses and dependoviruses (Fig. (Fig.11).Open in a separate windowFIG. 1.Phylogenetic tree of vertebrate organisms and history of ssDNA virus integrations. Times of integration of ancestral dependoviruses (yellow icosahedrons), parvoviruses (blue icosahedrons), and circoviruses (triangles) are approximate.The Dependovirus and Parvovirus genomes are typically 4 to 6 kb in length, include 2 major open reading frames (encoding replicase proteins [Rep and NS1, respectively] and capsid proteins [Cap and VP1, respectively]), and have characteristic hairpin structures at both ends (Fig. (Fig.2).2). For replication, these viruses depend on host enzymes that are recruited by the viral replicase proteins to the hairpin regions, where self-primed viral DNA synthesis is initiated (2). Circovirus genomes are typically ∼2-kb circles. DNA of the type species, porcine circovirus 1 (PCV-1), contains a stem-loop structure within the origin of replication (Fig. (Fig.2),2), and the largest open reading frame includes sequences that are homologous to the Parvovirus replicase open reading frame (9, 11). The circoviruses also depend on host enzymes for replication, and DNA synthesis is self-primed from a 3′-OH end formed by endonucleolytic cleavage of the stem-loop structure (4). The frequency of Dependovirus infection is estimated to be as high as 90% within an individual''s lifetime. None of the dependoviruses have been associated with human disease, but related viruses in the family Parvoviridae (e.g., erythrovirus B19 and possibly human bocavirus) are pathogenic for humans, and members of both the Parvoviridae and the Circoviridae can cause a variety of animal diseases (2, 4).Open in a separate windowFIG. 2.Schematics illustrating the structure and organization of Parvoviridae and Circoviridae genomes and origins of several of the longest-integrated ancestral viral sequences found in vertebrates. Integrations were aligned to the Dependovirus adeno-associated virus 2 (AAV2), the Parvovirus minute virus of mice (MVM), and the Circovirus porcine circovirus 1 (PCV-1). The inverted terminal repeat (ITR) sequences in the Dependovirus and Parvovirus genomes are depicted on an expanded scale. A linear representation of the circular genome of PCV-1 is shown with the 10-bp stem-loop structure on an expanded scale. Horizontal lines beneath the maps indicate the lengths of similar sequences that could be identified by BLAST. The numbers indicate the locations of amino acids in the viral proteins where the sequence similarities in the endogenous insertions start and end. The actual ancestral virus-derived integrated sequences may extend beyond the indicated regions.With some ancestral endogenous sequences that we identified, phylogenetic comparisons can be used to estimate age. For example, as a Dependovirus-like sequence is present at the same location in the genomes of mice and rats, the ancestral virus must have existed before their divergence, more than 20 million years ago. Some Circovirus- and Dependovirus-related integrations also predate the split between dog and panda, about 42 million years ago. However, in most other cases, we rely on an indirect method for estimating age (1). As genomic sequences evolve, they accumulate new stop codons and insertion/deletion-induced frameshifts. The rates of these events can be tied directly to the rates of neutral sequence drift and, therefore, the time of evolution. To apply this method, we first performed a BLAST search of vertebrate genomes for all known ssDNA virus proteins (BLAST options, -p tblastn -M BLOSUM62 -e 1e−4). Candidate sequences were then recorded, along with 5 kb of flanking regions, and then again aligned against the database of ssDNA viruses to find the most complete alignment (BLAST options, -t blastx -F F -w 15 -t 1500 -Z 150 -G 13 -E 1 -e 1e−2). Detected alignments were then compared with a neutral model of genome evolution, as described in the supplemental material, and the numbers of stop codons and frameshifts were converted into the expected genomic drift undergone by the sequences. The age of integration was then estimated from the known phylogeny of vertebrates (7, 10). Using these methods, we discovered that as many as 110 ssDNA virus-related sequences have been integrated into the 49 vertebrate genomes considered, during a time period ranging from the present to over 40 to 60 million years ago (Table (Table1;1; see also Tables S1 to S3 in the supplemental material).
Open in a separate windowaSome ambiguity in choosing the most similar virus is possible. We generally used the alignment with the lowest E value in the BLAST results. However, one or two points in the exponent of an E value were sometimes sacrificed to achieve a longer sequence alignment.baa, amino acids.cThese sequences have long insertions compared to the present-day viruses. In all cases tested, these insertions originated from short interspersed elements (SINEs). These insertions were excluded from the counts of stop codons and frameshifts and the estimation of integration age.dChr, chromosome.It is important to recognize that there is an intrinsic limit on how far back in time we can reach to identify ancient endogenous viral sequences. First, the sequences must be identified with confidence by BLAST or similar programs. This requirement places a lower limit on sequence identity at about 20 to 30% of amino acids, or about 75% of nucleotides (nucleotides evolve nearly 2.5 times slower than the amino acid sequence they encode). Second, the related, present-day virus must have evolved at a rate that is not much higher than that of the endogenous sequences. The viruses for which ancestral endogenous sequences were identified in this study exhibit sequence drift similar to that associated with mammalian genomes. Setting this rate at 0.14% per million years of evolution (8), we arrive at 90 million years as the theoretical limit for the oldest sequences that can be identified using our methods. This limit drops to less than 35 million years for endogenous viral sequences in rodents and even lower for sequences related to viruses that evolve faster than mammalian genomes.The most widespread integrations found in our survey are derived from the dependoviruses. These include nearly complete genomes related to adeno-associated virus (AAV) in microbat, wallaby, dolphin, rabbit, mouse, and baboon (Fig. (Fig.2).2). We did not detect inverted terminal repeats in several integrations tested, even though repeats are common in the present-day dependoviruses. This result could be explained by sequence decay or the absence of such structures in the ancestral viruses. However, we do see sequences that resemble degraded hairpin structures to which Dependovirus Rep proteins bind, with an example from microbat integration mlEDLG-1 shown in Fig. Fig.3.3. The second most widespread endogenous sequences are related to the parvoviruses. They are found in 6 of 49 vertebrate species considered, with nearly complete genomes in rat, opossum, wallaby, and guinea pig (Fig. (Fig.22).Open in a separate windowFIG. 3.Hairpin structure of the inverted terminal repeat of adeno-associated virus 2 (left) and a candidate degraded hairpin structure located close to the 5′ end of the mlEDLG-1 integration in microbats (right). Structures and mountain plots were generated using default parameters of the RNAfold program (5), with nucleotide coloring representing base-pairing probabilities: blue is below average, green is average, and red is above average. Mountain plots represent hairpin structures based on minimum free energy (mfe) calculations and partition function (pf) calculations, as well as the centroid structure (5). Height is expressed in numbers of nucleotides; position represents nucleotide.The Dependovirus AAV2 has strong bias for integration into human chromosome 19 during infection, driven by a host sequence that is recognized by the viral Rep protein(s). Rep mediates the formation of a synapse between viral and cellular sequences, and the cellular sequences are nicked to serve as an origin of viral replication (14). The related integrations in mice and rats, located in the same chromosomal locations, might be explained by such a mechanism. However, the extent of endogenous sequence decay and the frequency of stop codons indicate that these integrations occurred some 30 to 35 million years ago, implying that they are derived from a single event in a rodent ancestor rather than two independent integration events at the same location. Similarly, integrations EDLG-1 in dog and panda lie in chromosomal regions that can be readily aligned (based on University of California—Santa Cruz [UCSC] genome assemblies) and show sequence decay consistent with the age of the common ancestor, about 42 million years. Endogenous sequences related to the family Parvoviridae can thus be traced to over 40 million years back in time, and viral proteins related to this family have remained over 40% conserved.Sequences related to circoviruses were detected in five vertebrate species (Table (Table11 and Table S1 in the supplemental material). At least one of these sequences, the endogenous sequence in opossum, likely represents a recent integration. Several integrations in dog, cat, and panda, on the other hand, appear to date from at least 42 million years ago, which is the last time when pandas and dogs shared a common ancestor. We see evidence for this age in data from sequence degradation (Table (Table1),1), phylogenetic analyses of endogenous Circovirus-like genomes (see Fig. S2 in the supplemental material), and genomic synteny where integration ECLG-3 is surrounded by genes MTA3 and ARID5A in both dog and panda and integration ECLG-2 lies 35 to 43 kb downstream of gene UPF3A. In fact, Circovirus integrations may even precede the split between dogs and cats, about 55 million years ago, although the preliminary assembly and short genomic contigs for cats make synteny analysis impossible.The most common Circovirus-related sequences detected in vertebrate genomes are derived from the rep gene. We speculate that, like those of the Parvoviridae, the ancestral Circoviridae sequences might have been copied using a primer sequence in the host DNA that resembled the viral origin and was therefore recognized by the virus Rep protein. Higher incidence of rep gene identifications may represent higher conservation of this gene with time, or alternatively, possession of these sequences may impart some selective advantage to the host species. The largest Circovirus-related integration detected, in the opossum, comprises a short fragment of what may have been the cap gene immediately adjacent to and in the opposite orientation from the rep gene. This organization is similar to that of the present day Circovirus genome in which these genes share a promoter in the hairpin regions but are translated in opposite directions (Fig. (Fig.22).In summary, our results indicate that sequences derived from ancestral members of the families Parvoviridae and Circoviridae were integrated into their host''s genomes over the past 50 million years of evolution. Features of their replication strategies suggest mechanisms by which such integrations may have occurred. It is possible that some of the endogenous viral sequences could offer a selective advantage to the virus or the host. We note that rep open reading frame-derived proteins from some members of these families kill tumor cells selectively (3, 12). The genomic “fossils” we have discovered provide a unique glimpse into virus evolution but can give us only a lower estimate of the actual ages of these families. However, numerous recent integrations suggest that their germ line transfer has been continuing into present times. 相似文献
TABLE 1.
Selected endogenous sequences in vertebrate genomes related to single-stranded DNA virusesVirus group and vertebrate species | Initial genomic search using TBLASTN | Best sequence homology identified using BLASTX | Predicted nucleotide drift (%) | Integration label | Age (million yr) or timing of integration based on sequence aging | |||||
---|---|---|---|---|---|---|---|---|---|---|
Chromosomal or scaffold location | Protein | BLAST E value/% sequence identity | Most similar virusa | Protein | Coordinates | No. of stop codons/frameshifts | ||||
Circoviruses | ||||||||||
Cat | Scaffold_62068 | Rep | 6E−05/37 | Canary circovirus | Rep | 4-283 | 3/7 in 268 aab | 14.2 | fcECLG-1 | 82 |
Scaffold_24038 | Rep | 6E−06/51 | Columbid circovirus | Rep | 44-317 | 4/5 in 231 aac | 15.2 | fcECLG-2 | 87 | |
Dog | Chr5d | Rep | 7E−16/46 | Raven circovirus | Rep | 16-263 | 6/5 in 250 aa | 17.6 | cfECLG-1 | 98 |
Chr22 | Rep | 1E−14/43 | Beak and feather disease virus | Rep | 7-264 | 2/1 in 261 aac | 4.5 | cfECLG-2 | 54 | |
Opossum | Chr3 | Rep | 4E−46/44 | Finch circovirus | Rep | 2-291 | 0/2 in 282 aa | 2.3 | mdECLG | 12 |
Cap | 6-36 | 0/0 in 30 aa | ||||||||
Dependoviruses | ||||||||||
Dog | ChrX | Rep | 6E−05/55 | AAV5 | Rep | 239-445 | 3/4 in 200 aa | 14.0 | cfEDLG-1 | 78 |
Dolphin | GeneScaffold1475 | Rep | 8E−39/39 | Avian AAV DA1 | Rep | 79-486 | 3/4 in 379 aac | 6.6 | ttEDLG-2 | 55 |
Cap | 4E−61/47 | Cap | 1-738 | 4/7 in 678 aac | ||||||
Elephant | Scaffold_4 | Rep | 0/55 | AAV5 | Rep | 3-589 | 0/0 in 579 aa | 0.0 | laEDLG | Recent |
Hyrax | GeneScaffold5020 | Cap | 3E−34/53 | AAV3 | Cap | 485-735 | 0/5 in 256 aa | 7.0 | pcEDLG-1 | 29 |
Scaffold_19252 | Rep | 9E−72/47 | Bovine AAV | Rep | 2-348 | 8/4 in 348 aa | 14.3 | pcEDLG-2 | 60 | |
Megabat | Scaffold_5601 | Rep | 2E−13/31 | AAV2 | Rep | 315-479 | 1/5 in 175 aa | 13.1 | pvEDLG-3 | 76 |
Microbat | GeneScaffold2026 | Rep | 1E−117/50 | AAV2 | Rep | 1-617 | 2/5 in 612 aa | 5.8 | mlEDLG-1 | 27 |
Cap | 9E−33/51 | Cap | 1-731 | 2/9 in 509 aac | ||||||
Scaffold_146492 | Cap | 6E−32/42 | AAV2 | Cap | 479-732 | 0/3 in 252 aa | 4.2 | mlEDLG-2 | 19 | |
Mouse | Chr1 | Rep | 2E−06/34 | AAV2 | Rep | 4-206 | 3/5 in 191 aa | 17.1 | mmEDLG-1 | 39 |
Chr3 | Rep | 2E−24/31 | AAV5 | Rep | 71-478 | 12/7 in 389 aa | 16.5 | mmEDLG-2 | 37 | |
Cap | 2E−22/45 | Cap | 22-724 | 12/10 in 649aac | ||||||
Chr8 | Rep | 1E−08/46 | AAV2 | Rep | 314-473 | 3/3 in 147 aa | 13.8 | mmEDLG-3 | 31 | |
Cap | 1-137 | 1/2 in 114 aa | ||||||||
Panda | Scaffold2359 | Rep | 2E−06/37 | Bovine AAV | Rep | 238-426 | 2/3 in 186 aa | 10.4 | amEDLG-1 | 59 |
Pika | Scaffold_9941 | Rep | 4E−14/28 | AAV5 | Rep | 126-415 | 2/2 in 282 aa | 5.4 | opEDLG | 14 |
Platypus | Chr2 | Rep | 9E−10/35 | Bovine AAV | Rep | 297-437 | 4/3 in 138 aa | 17.1 | oaEDLG-1 | 79 |
Cap | 272-419 | 1/2 in 150 aac | ||||||||
Contig12430 | Rep | 2E−09/47 | Bovine AAV | Rep | 353-450 | 3/1 in 123 aa | 12.0 | oaEDLG-2 | 55 | |
Cap | 2E−05/32 | Cap | 253-367 | 2/1 in 116 aa | ||||||
Rabbit | Chr10 | Rep | 3E−97/39 | AAV2 | Rep | 1-619 | 3/9 in 613 aa | 9.3 | ocEDLG | 43 |
Cap | 5E−50/45 | Cap | 1-723 | 10/9 in 675 aa | ||||||
Rat | Chr13 | Rep | 2E−09/33 | AAV2 | Rep | 4-175 | 2/4 in 177 aa | 13.3 | rnEDLG-1 | 28 |
Chr2 | Rep | 4E−18/40 | AAV5 | Rep | 1-461 | 12/12 in 454 aa | 22.7 | rnEDLG-2 | 51 | |
Chr19 | Rep | 2E−07/33 | AAV5 | Rep | 329-464 | 2/4 in 136 aa | 16.1 | rnEDLG-3 | 35 | |
Cap | 31-133 | 2/1 in 93 aa | ||||||||
Tarsier | Scaffold_178326 | Rep | 4E−14/23 | AAV5 | Rep | 96-465 | 2/3 in 356 aa | 5.3 | tsEDLG | 23 |
Parvoviruses | ||||||||||
Guinea pig | Scaffold_188 | Rep | 3E−24/46 | Porcine parvovirus | Rep | 313-567 | 5/3 in 250 aa | 12.3 | cpEPLG-1 | 40 |
Cap | 1E−16/36 | Cap | 10-689 | 11/12 in 672 aa | ||||||
Scaffold_27 | Rep | 1E−50/39 | Canine parvovirus | Rep | 11-640 | 1/4 in 616 aa | 5.3 | cpEPLG-2 | 17 | |
Cap | 1E−38/39 | Porcine parvovirus | Cap | 3-719 | 2/14 in 700 aa | |||||
Tenrec | Scaffold_260946 | Rep | 2E−20/38 | LuIII virus | Rep | 406-598 | 4/4 in 190 aa | 19.0 | etEPLG-2 | 60 |
Cap | 11-639 | 16/15 in 595 aa | ||||||||
Rat | Chr5 | Rep | 6E−10/56 | Canine parvovirus | Rep | 1-282 | 0/0 in 312 aa | 0.6 | rnEPLG | Recent |
Cap | 0/62 | Cap | 637-667 | 0/2 in 760 aa | ||||||
Rep | 0/63 | 1-751 | ||||||||
Opossum | Chr3 | Rep | 2E−39/33 | LuIII virus | Rep | 7-570 | 11/3 in 502 aa | 10.9 | mdEPLG-2 | 56 |
Cap | 7E−8/33 | Cap | 11-729 | 14/7 in 704 aa | ||||||
Chr6 | Rep | 6E−58/44 | Porcine parvovirus | Rep | 16-563 | 3/7 in 534 aac | 4.6 | mdEPLG-3 | 24 | |
Cap | 6E−60/38 | Cap | 10-715 | 2/5 in 707 aac | ||||||
Wallaby | Scaffold_108040 | Rep | 4E−74/62 | Canine parvovirus | Rep | 341-645 | 0/0 in 287 aa | 1.3 | meEPLG-3 | 7 |
Cap | 8E−37/32 | Cap | 35-738 | 0/4 in 687 aa | ||||||
Scaffold_72496 | Rep | 2E−61/42 | Porcine parvovirus | Rep | 23-567 | 4/3 in 531 aa | 5.7 | meEPLG-6 | 30 | |
Cap | 2E−31/38 | Cap | 10-532 | 6/4 in 514 aa | ||||||
Scaffold_88340 | Rep | 7E−37/55 | Mouse parvovirus 1 | Rep | 344-566 | 0/3 in 223 aa | 6.7 | meEPLG-16 | 36 | |
Cap | 7E−22/33 | Cap | 11-713 | 6/9 in 700 aa |
6.
Pamela R. Hall Brian Hjelle Hadya Njus Chunyan Ye Virginie Bondu-Hawkins David C. Brown Kathleen A. Kilpatrick Richard S. Larson 《Journal of virology》2009,83(17):8965-8969
Specific therapy is not available for hantavirus cardiopulmonary syndrome caused by Andes virus (ANDV). Peptides capable of blocking ANDV infection in vitro were identified using antibodies against ANDV surface glycoproteins Gn and Gc to competitively elute a cyclic nonapeptide-bearing phage display library from purified ANDV particles. Phage was examined for ANDV infection inhibition in vitro, and nonapeptides were synthesized based on the most-potent phage sequences. Three peptides showed levels of viral inhibition which were significantly increased by combination treatment with anti-Gn- and anti-Gc-targeting peptides. These peptides will be valuable tools for further development of both peptide and nonpeptide therapeutic agents.Andes virus (ANDV), an NIAID category A agent linked to hantavirus cardiopulmonary syndrome (HCPS), belongs to the family Bunyaviridae and the genus Hantavirus and is carried by Oligoryzomys longicaudatus rodents (11). HCPS is characterized by pulmonary edema caused by capillary leak, with death often resulting from cardiogenic shock (9, 16). ANDV HCPS has a case fatality rate approaching 40%, and ANDV is the only hantavirus demonstrated to be capable of direct person-to-person transmission (15, 21). There is currently no specific therapy available for treatment of ANDV infection and HCPS.Peptide ligands that target a specific protein surface can have broad applications as therapeutics by blocking specific protein-protein interactions, such as preventing viral engagement of host cell receptors and thus preventing infection. Phage display libraries provide a powerful and inexpensive tool to identify such peptides. Here, we used selection of a cyclic nonapeptide-bearing phage library to identify peptides capable of binding the transmembrane surface glycoproteins of ANDV, Gn and Gc, and blocking infection in vitro.To identify peptide sequences capable of recognizing ANDV, we panned a cysteine-constrained cyclic nonapeptide-bearing phage display library (New England Biolabs) against density gradient-purified, UV-treated ANDV strain CHI-7913 (a gift from Hector Galeno, Santiago, Chile) (17, 18). To increase the specificity of the peptides identified, we eluted phage by using monoclonal antibodies (Austral Biologicals) prepared against recombinant fragments of ANDV Gn (residues 1 to 353) or Gc (residues 182 to 491) glycoproteins (antibodies 6B9/F5 and 6C5/D12, respectively). Peptide sequences were determined for phage from iterative rounds of panning, and the ability of phage to inhibit ANDV infection of Vero E6 cells was determined by immunofluorescent assay (IFA) (7). Primary IFA detection antibodies were rabbit polyclonal anti-Sin Nombre hantavirus (SNV) nucleoprotein (N) antibodies which exhibit potent cross-reactivity against other hantavirus N antigens (3). ReoPro, a commercially available Fab fragment which partially blocks infection of hantaviruses in vitro by binding the entry receptor integrin β3 (5), was used as a positive control (80 μg/ml) along with the original antibody used for phage elution (5 μg/ml). As the maximum effectiveness of ReoPro in inhibiting hantavirus entry approaches 80%, we set this as a threshold for maximal expected efficacy for normalization. The most-potent phage identified by elution with the anti-Gn antibody 6B9/F5 bore the peptide CPSNVNNIC and inhibited hantavirus entry by greater than 60% (61%) (Table (Table1).1). From phage eluted with the anti-Gc antibody 6C5/D12, those bearing peptides CPMSQNPTC and CPKLHPGGC also inhibited entry by greater than 60% (66% and 72%, respectively).
Open in a separate windowaStandard deviations of four experiments are shown in parentheses. Peptide-bearing phage were added at 109 phage/μl.bP values for the pairwise amino acid alignment score of each peptide versus that of integrin β3 were determined using an unpaired Student''s t test. P values considered statistically significant are shown in bold.To determine whether the peptide sequences of any of the identified inhibitory phage showed homology to integrin β3, a known entry receptor for pathogenic hantaviruses (6, 7), we used the Gap program to perform a pairwise amino acid alignment of each peptide versus the extracellular portion of integrin β3 and determined P values for the alignments. Of 45 phage eluted with the anti-Gn antibody, 6B9/F5, 27 of the peptide sequences showed homology to integrin β3 (P < 0.05), and 9 were highly significant (P ≤ 0.0005) (Fig. (Fig.1A).1A). Of the latter, CKFPLNAAC and CSQFPPRLC map to the hybrid domain (Fig. (Fig.1B),1B), which is proximal to the plexin-semaphorin-integrin domain (PSI) containing residue D39, shown to be critical for viral entry in vitro (19). Five sequences (CPSSPFNH, CPKHVLKVC, CNANKPKMC, CQSQTRNHC, and CDQRTTRLC) map to the I-like (or βA) domain near the binding site of ReoPro (2). Finally, CLPTDPIQC maps to the epidermal growth factor 4 (EGF-4) domain, and CSTRAENQC aligns to a portion of β3 untraceable in the crystal structure, specifically the linker region between the hybrid domain and EGF-1. Although this represents a disordered portion of the protein (22), the location of this loop proximal to the PSI domain is worth noting, due to the role of the PSI domain in facilitating viral entry (19). Therefore, 60% of phage eluted with the anti-Gn antibody showed some homology to integrin β3, and those with highly significant P values predominantly mapped to or proximal to regions of known interest in viral entry.Open in a separate windowFIG. 1.Inhibitory peptides identified through phage panning against ANDV show homology to integrin β3. (A) Alignment of phage peptide sequences with P values for integrin β3 pairwise alignment of less than 0.05. Residues comprising the signal peptide, transmembrane, and cytoplasmic domains, which were not included during pairwise alignment, are underlined. Residues 461 to 548, which are missing in the crystal structure, are italicized. Residues involved in the ReoPro binding site are highlighted in green (2). Residue D39 of the PSI domain is highlighted in yellow (19). Peptides are shown above the sequence of integrin β3, with antibody 6C5/D12-eluted sequences shown in blue text and sequences eluted with antibody 6B9/F5 shown in red. Peptide sequences with alignment P values of ≤0.0005 are highlighted in yellow. Percent inhibition of the peptide-bearing phage is shown in parentheses. (B) View of integrin αvβ3 (PDB ID 1U8C [23]). αv is shown in blue ribbon diagram, and β3 is shown in salmon-colored surface representation, with specific domains circled. Residues corresponding to the ReoPro binding site are shown in green, as in panel A, and D39 is shown in yellow. Regions corresponding to 6C5/D12-eluted peptides with P values of ≤0.0005 for alignment with integrin β3 (highlighted in panel A) are shown in blue, and those corresponding to 6B9/F5-eluted peptides with P values of ≤0.0005 for alignment with integrin β3 are shown in red. Alignment of peptide PLASTRT (P value of 0.0040) adjacent to D39 of the PSI domain is shown in magenta. Graphics were prepared using Pymol (DeLano Scientific LLC, San Carlos, CA).Of the 41 peptide-bearing phage eluted with the anti-Gc antibody 6C5/D12, 14 showed sequence homology to integrin β3 (P < 0.05), 4 of which had P values of ≤0.0005 (Fig. (Fig.1A).1A). Of the latter, sequence CTTMTRMTC mapped to the base of the I-like domain (Fig. (Fig.1B),1B), while CHGVYALHC and CRDTTPWWC mapped to the EGF-3 domain. Finally, sequence CTPTMHNHC mapped to the linker region untraceable in the crystal structure. Therefore, in contrast to peptide sequences identified by competition with the anti-Gn antibody, sequences identified by competition with the anti-Gc antibody 6C5/D12 appear to be mostly unrelated to integrin β3.As a low level of pathogenic hantavirus infection can be seen in cells lacking integrin β3, such as CHO cells (19), we asked if any of the identified peptide sequences could represent a previously unidentified receptor. We used the Basic Local Alignment Search Tool to search a current database of human protein sequences for potential alternate receptors represented by these peptides. However, none of the alignments identified proteins that are expressed at the cell surface, eliminating them as potential candidates for alternate viral entry receptors. This suggests that the majority of the peptides identified here likely represent novel sequences for binding ANDV surface glycoproteins.To determine whether synthetic peptides would also block infection, we synthesized cyclic peptides based on the 10 most-potent peptide-bearing phage. These peptides, in the context of phage presentation, showed levels of inhibition ranging from 44 to 72% (Table (Table2).2). When tested by IFA at 1 mM, four of the synthetic peptides showed inhibition levels significantly lower than those of the same peptide presented in the context of phage. This is not surprising, as steric factors due to the size of the phage and the multivalent presentation of peptide in the context of phage may both contribute to infection inhibition (8). However, there was no significant difference in inhibition by synthetic peptide versus peptide-bearing phage for six of the sequences, implying that inhibition in the context of phage was due solely to the nature of the peptide itself and not to steric factors or valency considerations contributed by the phage, which contrasts with our previous results, determined by using phage directed against αvβ3 integrin (10).
Open in a separate windowaStandard deviations of the results of at least four experiments are shown in parentheses.bMean percent inhibition between phage and synthetic peptide differs significantly (P < 0.05).The three most-potent synthetic peptides were examined for their ability to inhibit ANDV entry in a dose-dependent manner. The concentration of each peptide that produces 50% of its maximum potential inhibitory effect was determined. As shown in Fig. Fig.2A,2A, the 50% inhibitory concentration for each of the peptides was in the range of 10 μM, which from our experience is a reasonable potency for a lead compound to take forward for optimization.Open in a separate windowFIG. 2.Activities of synthetic peptides in inhibition of ANDV infection in vitro. (A) Peptides were examined for their ability to block ANDV infection of Vero E6 cells in a dose-dependent manner by IFA. (B) Peptides were tested in parallel for the ability to block infection of Vero E6 cells by ANDV, SNV, HTNV, and PHV. (C) Peptides were tested, singly or in combination, for the ability to block ANDV infection of Vero E6 cells. For all experiments, controls included media, ReoPro at 80 μg/ml, and monoclonal antibodies 6C5/D12 and 6B9/F5 at 5 μg/ml. All peptides were used at 1 mM. Data points represent n = 2 to 6, with error bars showing the standard errors of the means. Statistical analyses were performed on replicate samples using an unpaired Student''s t test.In order to determine the specificity of the three most-potent synthetic cyclic peptides in blocking ANDV, we examined them for inhibition of ANDV infection versus two other pathogenic hantaviruses, SNV and Hantaan virus (HTNV), or the nonpathogenic hantavirus Prospect Hill virus (PHV). As shown in Fig. Fig.2B,2B, ReoPro, which binds integrin β3, showed inhibition of infection by each of the pathogenic hantavirus strains, known to enter cells via β3, but not the nonpathogenic PHV, which enters via integrin β1 (6, 7). In contrast, peptides selected for the ability to bind ANDV were highly specific inhibitors of ANDV versus SNV, HTNV, or PHV. The specificities of peptides eluted by the anti-Gn monoclonal antibody are not surprising, as they are likely due to global differences in the Gn amino acid sequence. Specifically, sequence homologies between ANDV and SNV, HTNV, and PHV are 61%, 36%, and 51%, respectively, for the region corresponding to the immunogen for antibody 6B9/F5. Although homology between the immunogen for antibody 6C5/D12 and the corresponding Gc region of these viruses is somewhat higher (82% with SNV, 63% with HTNV, and 71% with PHV), the possibility that the monoclonal antibody used here recognizes a three-dimensional epitope lends itself to the high specificity of the peptides.The current model for cellular infection by hantaviruses (14) is as follows. Viral binding of the host cell surface target integrin is followed by receptor-mediated endocytosis and endosome acidification. Lowered pH induces conformational changes in Gn and/or Gc, which facilitate membrane fusion and viral release into the cytosol. As there is currently little information available about whether one glycoprotein is dominant in mediating infection, and as neutralizing epitopes have been found on both Gn and Gc glycoproteins (1, 4, 12, 13, 20), we examined whether combining anti-Gn- and anti-Gc-targeted synthetic peptides would lead to an increased infection blockade compared to those for single treatments. As shown in Fig. Fig.2C,2C, the combination of anti-Gn and anti-Gc peptides CMQSAAAHC and CTVGPTRSC resulted in a significant increase in infection inhibition (P = 0.0207 for CMQSAAAHC, and P = 0.0308 for CTVGPTRSC) compared to that resulting from single treatments. Although the high specificity of the peptides for ANDV makes it unlikely that this combination treatment will lead to more cross-reactivity with other pathogenic hantaviruses, this can be determined only by additional testing. Regardless, these data suggest a unique role for each of these viral proteins in the infection process as well as the benefits of targeting multiple viral epitopes for preventing infection.To our knowledge, the peptides reported here are the first identified that directly target ANDV, and this work further illustrates the power of coupling phage display and selective elution techniques in the identification of novel peptide sequences capable of specific protein-protein interactions from a large, random pool of peptide sequences. These novel peptide inhibitors (R. S. Larson, P. R. Hall, H. Njus, and B. Hjelle, U.S. patent application 61/205,211) provide leads for the development of more-potent peptide or nonpeptide organics for therapeutic use against HCPS. 相似文献
TABLE 1.
Peptide-bearing phage eluted from ANDVPhage | % Inhibition (SD)a | P valueb |
---|---|---|
Phage bearing the following peptides eluted with anti-Gn antibody 6B9/F5 | ||
Group 1 (<30% inhibition) | ||
CDQRTTRLC | 8.45 (15.34) | 0.0002 |
CPHDPNHPC | 9.94 (7.72) | 0.333 |
CQSQTRNHC | 11.76 (13.25) | 0.0001 |
CLQDMRQFC | 13.26 (9.92) | 0.0014 |
CLPTDPIQC | 15.70 (14.05) | 0.0005 |
CPDHPFLRC | 16.65 (15.22) | 0.8523 |
CSTRAENQC | 17.56 (16.50) | 0.0004 |
CPSHLDAFC | 18.98 (20.06) | 0.0017 |
CKTGHMRIC | 20.84 (7.47) | 0.0563 |
CVRTPTHHC | 20.89 (27.07) | 0.1483 |
CSGVINTTC | 21.57 (19.61) | 0.0643 |
CPLASTRTC | 21.65 (5.98) | 0.004 |
CSQFPPRLC | 22.19 (8.26) | 0.0004 |
CLLNKQNAC | 22.34 (7.78) | 0.001 |
CKFPLNAAC | 22.89 (6.15) | 0.0001 |
CSLTPHRSC | 23.63 (16.74) | 0.0563 |
CKPWPMYSC | 23.71 (6.68) | 0.0643 |
CLQHDALNC | 24.01 (7.60) | 1 |
CNANKPKMC | 24.67 (11.67) | 0.0004 |
CPKHVLKVC | 25.30 (28.36) | 0.0003 |
CTPDKKSFC | 26.91 (11.15) | 0.399 |
CHGKAALAC | 27.22 (32.53) | 0.005 |
CNLMGNPHC | 28.08 (21.35) | 0.0011 |
CLKNWFQPC | 28.64 (18.49) | 0.0016 |
CKEYGRQMC | 28.76 (29.33) | 0.0362 |
CQPSDPHLC | 29.44 (31.22) | 0.0183 |
CSHLPPNRC | 29.70 (17.37) | 0.0061 |
Group 2 (30-59% inhibition) | ||
CSPLLRTVC | 33.05 (20.26) | 0.0023 |
CHKGHTWNC | 34.17 (12.50) | 0.0795 |
CINASHAHC | 35.62 (13.03) | 0.3193 |
CWPPSSRTC | 36.75 (26.95) | 0.0006 |
CPSSPFNHC | 37.78 (7.11) | 0.0001 |
CEHLSHAAC | 38.47 (7.60) | 0.0115 |
CQDRKTSQC | 38.74 (9.12) | 0.1802 |
CTDVYRPTC | 38.90 (25.03) | 0.006 |
CGEKSAQLC | 39.11 (27.52) | 0.0013 |
CSAAERLNC | 40.13 (6.33) | 0.0033 |
CFRTLEHLC | 42.07 (5.01) | 0.0608 |
CEKLHTASC | 43.60 (27.92) | 0.1684 |
CSLHSHKGC | 45.11 (49.81) | 0.0864 |
CNSHSPVHC | 45.40 (28.80) | 0.0115 |
CMQSAAAHC | 48.88 (44.40) | 0.5794 |
CPAASHPRC | 51.84 (17.09) | 0.1935 |
CKSLGSSQC | 53.90 (13.34) | 0.0145 |
Group 3 (60-79% inhibition) | ||
CPSNVNNIC | 61.11 (25.41) | 0.1245 |
Negative control | 0 (6.15) | |
6B9/F5 (5 μg/ml) | 26.77 (5.33) | |
ReoPro (80 μg/ml) | 79.86 (4.88) | |
Phage bearing the following peptides eluted with anti-Gc antibody 6C5/D12 | ||
Group 1 (<30% inhibition) | ||
CHPGSSSRC | 1.01 (7.03) | 0.0557 |
CSLSPLGRC | 10.56 (13.62) | 0.7895 |
CTARYTQHC | 12.86 (3.83) | 0.3193 |
CHGVYALHC | 12.91 (7.32) | 0.0003 |
CLQHNEREC | 16.79 (13.72) | 0.0958 |
CHPSTHRYC | 17.23 (14.53) | 0.0011 |
CPGNWWSTC | 19.34(9.91) | 0.1483 |
CGMLNWNRC | 19.48 (19.42) | 0.0777 |
CPHTQFWQC | 20.44 (13.65) | 0.0008 |
CTPTMHNHC | 20.92 (11.68) | 0.0001 |
CDQVAGYSC | 21.79 (23.60) | 0.0063 |
CIPMMTEFC | 24.33 (9.28) | 0.2999 |
CERPYSRLC | 24.38 (9.09) | 0.0041 |
CPSLHTREC | 25.06 (22.78) | 0.1202 |
CSPLQIPYC | 26.30 (34.29) | 0.4673 |
CTTMTRMTC (×2) | 29.27 (8.65) | 0.0001 |
Group 2 (30-59% inhibition) | ||
CNKPFSLPC | 30.09 (5.59) | 0.4384 |
CHNLESGTC | 31.63 (26.67) | 0.751 |
CNSVPPYQC | 31.96 (6.51) | 0.0903 |
CSDSWLPRC | 32.95 (28.54) | 0.259 |
CSAPFTKSC | 33.40 (10.64) | 0.0052 |
CEGLPNIDC | 35.63 (19.90) | 0.0853 |
CTSTHTKTC | 36.28 (13.42) | 0.132 |
CLSIHSSVC | 36.40 (16.44) | 0.8981 |
CPWSTQYAC | 36.81 (32.81) | 0.5725 |
CTGSNLPIC | 36.83 (31.64) | 0.0307 |
CSLAPANTC | 39.73 (4.03) | 0.1664 |
CGLKTNPAC | 39.75 (16.98) | 0.2084 |
CRDTTPWWC | 40.08 (18.52) | 0.0004 |
CHTNASPHC | 40.26 (4.77) | 0.5904 |
CTSMAYHHC | 41.89 (8.61) | 0.259 |
CSLSSPRIC | 42.13 (29.75) | 0.2463 |
CVSLEHQNC | 45.54 (6.55) | 0.5065 |
CRVTQTHTC | 46.55 (8.45) | 0.3676 |
CPTTKSNVC | 49.28 (14.00) | 0.3898 |
CSPGPHRVC | 49.50 (42.60) | 0.0115 |
CKSTSNVYC | 51.20 (4.60) | 0.0611 |
CTVGPTRSC | 57.30 (11.31) | 0.0176 |
Group 3 (60-79% inhibition) | ||
CPMSQNPTC | 65.60 (13.49) | 0.014 |
CPKLHPGGC | 71.88 (27.11) | 0.0059 |
Negative control | 0.26 (4.53) | |
6C5/D12 (5 μg/ml) | 22.62 (8.40) | |
ReoPro (80 μg/ml) | 80.02 (76.64) |
TABLE 2.
Synthetic cyclic peptides inhibit ANDV infectionTarget | Sample | % Inhibition bya:
| |
---|---|---|---|
Peptide-bearing phage | Synthetic peptide | ||
Gn | CMQSAAAHC | 48.88 (44.40) | 59.66 (11.17) |
Gc | CTVGPTRSC | 57.30 (11.31) | 46.47 (7.61) |
Gn | CPSNVNNIC | 61.11 (25.41) | 44.14 (10.74) |
Gn | CEKLHTASC | 43.60 (27.92) | 34.87 (9.26) |
Gc | CPKLHPGGC | 71.88 (27.11) | 30.95 (7.73)b |
Gn | CSLHSHKGC | 45.11 (49.81) | 29.79 (9.34) |
Gc | CPMSQNPTC | 65.60 (13.49) | 18.19 (8.55)b |
Gn | CKSLGSSQC | 53.90 (13.34) | 18.10 (7.55)b |
Gn | CNSHSPVHC | 45.40 (28.80) | 15.52 (10.48) |
Gn | CPAASHPRC | 51.84 (17.09) | 0 (10.72)b |
Integrin β3 | ReoPro | 80.10 (7.72) | |
Gn | 6B9/F5 antibody | 42.72 (6.75) | |
Gc | 6C5/D12 antibody | 31.04 (7.81) |
7.
L. Pilloni P. Bianco C. Manieli G. Senes P. Coni L. Atzori N. Aste G. Faa 《European journal of histochemistry : EJH》2009,53(2)
Basal cell carcinoma (BCC) is a very common malignant skin tumor that rarely metastatizes, but is often locally aggressive. Several factors, like large size (more than 3 cm), exposure to ultraviolet rays, histological variants, level of infiltration and perineural or perivascular invasion, are associated with a more aggressive clinical course. These morphological features seem to be more determinant in mideface localized BCC, which frequently show a significantly higher recurrence rate. An immunohistochemical profile, characterized by reactivity of tumor cells for p53, Ki67 and alpha-SMA has been associated with a more aggressive behaviour in large BCCs. The aim of this study was to verify if also little (<3 cm) basal cell carcinomas can express immunohistochemical markers typical for an aggressive behaviour.Basal cell carcinoma (BCC) is a very common malignant skin tumor that rarely metastatizes, even If Is often locally aggressive. Several factors, like large size (more than 3 cm), face localization, exposure to ultraviolet rays, histological variants, infiltration level and perineural or perivascular invasion, are associated with a more aggressive clinical course. In particular, the incidence of metastasis and/or death correlates with tumors greater than 3 cm in diameter in which setting patients are said to have 1–2 % risk of metastases that increases to 20–25% in lesions greater than 5 cm and to 50% in lesions greater than 10 cm in diameter (Snow et al., 1994). Histologically morpheiform, keratotic types and infiltrative growth of BCC are also considered features of the most aggressive course (Crowson, 2006). This can be explained by the fact that both the superficial and nodular variants of BCC are surrounded by a continuous basement membrane zone comprising collagens type IV and V admixed with laminin, while the aggressive growth variants (i.e. morpheiform, metatypical, and infiltrative growth subtypes) manifest the absence of basement membrane (Barsky et al., 1987).The molecular markers which characterize aggressive BCC include: increased expression of stromolysin (MMP-3) and collagenase-1 (MMP-1) (Cribier et al., 2001), decreased expression of syndecan-1 proteoglycan (Bayer-Garner et al., 2000) and of anti-apoptotic protein bcl-2 (Ramdial et al., 2000; Staibano et al., 2001).C-ras , c-fos (Urabe et al., 1994; Van der Schroeff et al., 1990) and p53 tumor supressor gene mutations (Auepemikiate et al., 2002) are indicative of an aggressive course.Focusing upon bcl-2 and p53 expression in BCC, there have been numerous studies documenting the utility of bcl-2 as a marker of favourable clinical behaviour while p53 expression may be a feature of a more aggressive outcome (Ramdial et al., 2000; Staibano et al., 2001; Bozdogan et al., 2002).An increased expression of cytoskeletal microfilaments like α–smooth muscle actin, frequently found in invasive BCC subtypes (Jones JCR et al., 1989), may explain an enhanced tumor mobility and deep tissue invasion through the stroma. (Cristian et al., 2001; Law et al., 2003). The aim of this preliminary study was to verify if also little (<3 cm) basal cell carcinomas may express aggressive immunohistochemical markers like p53, Ki67 and alpha-SMA. We used 31 excisional BCCs with tumor size less than 2 cm (ranging from 2 up to 20 mm) and with different skin localization (19 in the face, 6 in the trunk and 6 in the body extremities). All cases were immunostained for p53, BCL2, Ki67 and alpha-smooth muscle actin (α-SMA) (Age Sex Location Hystotype Max.Dim Depth Ulc Ess Inf p53 Bcl-2 Ki67 AML 1 61 M Extr Keratotic 10×8 1 No +++ URD +++ + + - 2 61 M Face Adenoid 10×9 4 No + URD +++ - - - 3 64 M Extr Sup mult 11×13 0.8 No + DRD + - - - 4 73 M Face Nodular 10×8 2 Yes + DRD +++ + ++ +++ 5 84 M Face Nodular 9×12 2 Yes + DRD - - - - 6 84 M Face Adenoid 5 0.8 No + URD +++ - - - 7 84 M Extr Nodular 13×10 3 No + DRD +++ + + - 8 52 F Face Nodular 4 0.8 No + URD + + + - 9 76 F Face Adenoid 10×4 4 No + DRD +++ - ++ - 10 77 F Face Morph 8×6 1 Yes +++ DRD +++ - - - 11 86 M Face Morph 8 1 Yes + DRD +++ - + + 12 63 F Face Adenoid 4 1 No + URD ++ + + + 13 76 F Face Nodular 7 1.5 No + DRD +++ + ++ - 14 84 M Face Nodular 11 4 Yes +++ DRD + - - + 15 63 F Face Keratotic 10×6 1.8 No ++ DRD - + ++ - 16 68 F Trunk Sup mult 10×6 0.7 No ++ URD + + - - 17 67 M Face Sup mult 12×6 0.4 No + URD + - + - 18 67 M Extr Sup mult 4×3 0.3 No + URD + +++ + - 19 32 F Extr Sup mult 1×3 0.4 No + URD + + + - 20 45 M Trunk Nodular 7×5 2 Yes +++ URD + + + - 21 62 M Trunk Sup mult 11×7 0.9 No ++ URD - ++ - ++ 22 65 M Trunk Adenoid 7×6 1.5 No + URD +++ + + - 23 72 M Trunk Nodular 12×6 1 No + URD +++ - + + 24 86 F Face Keratotic 20×11 3.1 No ++ DRD + + + - 25 85 M Face Nodular 0.5 1.3 No ++ DRD ++ + + - 26 74 F Extr Nodular 4×4 0.9 No + URD - - + - 27 71 M Face Nodular 6×12 1.7 No + DRD - - + - 28 64 F Trunk Sup mult 1.3×1.5 0.4 No ++ URD +++ - - - 29 78 F Face Nodular 4×3 1.5 No ++ DRD ++ + - +++ 30 80 M Face Keratotic 4×4 1.6 Yes + DRD - - + +++