Estudio molecular y funcional del gen cassette aadA1
Genetic horizontal transfer (GHT) is one of the most efficient evolutionary strategies of bacteria. Acquisition of foreign genetic material allows to increase the genetic variability of a specific genome, almost instantaneously. Integrons are genetic elements, keys in the capture, expression and dis...
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| Formato: | Tesis de maestría acceptedVersion |
| Lenguaje: | Español |
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Facultad de Farmacia y Bioquímica
2018
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_5917 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_5917.dir/5917.PDF |
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| Sumario: | Genetic horizontal transfer (GHT) is one of the most efficient evolutionary strategies of bacteria. Acquisition of foreign genetic material allows to increase the genetic variability of a specific genome, almost instantaneously. Integrons are genetic elements, keys in the capture, expression and dispersion of genes in form of cassette among gram-negative bacteria. Integrons encode a specific site recombinase, integrase (IntI), which recognizes two different recombination sites, the attI site, adjacent to the intI gene, and the attC sites. Cassettes are the mobile elements of this genetic platform, and are designed by an open reading frame (ORF) and an attC site, the architecture of the typical cassettes inserted in the variable region of the integron are atti1-ORF- attC and attC-ORF-attC. Class 1 and 2 integrons are the most successful in hospital environment, while others have been detected sporadically.\nMost of the antibiotic resistance genes that are found in the clinic currently originated from environmental bacteria, including antibiotic-producing species. Two branches of aadA cassette genes have been described that confer resistance to streptomycin/ spectinomycin associated with integrons in clinical strains. These branches are aadA1 and aadA2. Both are study models, since it has been described that at least the aadA1 cassette gene would have been present prior to the antibiotic era and would have been transmitted through HGT events to the current clinical bacteria. This cassette gene is widely distributed in clinical isolates contributing to the problem of antibiotic multiresistance, which is why we conducted a molecular and functional study of it in this work.\nThrough bioinformatic analysis we identified a group of 37 alleles that we entitled aadA-like. We found that only the aadA1 cassette gene is associated with class 1 and 2 integrases, unlike the remaining aadA-like alleles, which are exclusively associated with class 1 integrase; aadA1 allele is the most abundant and also the one with more arrangements in the variable zone of the integron in which it is contained. In second and third place, are the alleles aadA2 and aadA5 respectively. In the matter of associated cassette genes, we found that aadA1 cassette gene is closely related to dfrA1 cassette gene or is exclusive in the variable zone in type 1 integrons. Additionally, this cassette gene have a tendency to be located in the 3 first positions of the variable zone of the integron, which explains its high resistance expression in clinical isolates.\nRegarding the analysis of dispersion of aadA1 cassette gene, through the study of the dispersion of its attC site, we found a total of 86 associated cassette gene arrays, keeping association with the cassette gene dfrA1 or aadA1 cassette gene as exclusive in variable zone. The association of the aadA1 cassette gene to integrases of class 1 integrons was 84%. aadA1 cassette gene is closely related to dfrA1 cassette gene. Their association is maintained both in class 1 and class 2 integrons.\nConcerning to the study of genetic components of the group aadA-like gene cassettes through analysis of alignment and maximum similarity, the formation of some groups was observed, emphasizing the group that includes what we call aadA1-like alleles (7 alleles).\nUsing CLUSTALW and maximum similarity analysis, 15 variants of the attC site were identified, associated with 37 aadA-like alleles with a greater sequence identity between the nucleotide bases involved in the recombination, specifically in 1L and 1R regions (RYYYAAC and GTTRRRY, respectively). Variant 1 contains most of the alleles: 15. As in the case of the analysis of the ORFs, the grouping of most of the alleles corresponds to the so-called aadA1-like alleles, which confirms the high dispersion of this gene cassette in particular.\nIn functional study of aadA1 cassette gene, we analyzed the molecular characteristics of the attC flanking sites of this cassette gene in the bacterial clones in which we carried out recombination tests. The structure of cloned cassette genes in each of these plasmids corresponds to typical structures observed in different clinic integrons arrays: attCaadA1, attCaadA3, attCaacA1 y attCdfrA1. We analyze the regions involved in the recombination process: the core site or 1R region, the 1L region or inverse core site and the presence and spacing of extrahelical bases.\nWe found that the attCaadA1 site is the only one that fit in completely to consensus sequence of the 1R region or core site 5'-GTTRRRY-3 '. The three remaining attC sites (attCaadA3, attCaacA1 and attCdfrA1) are characterized by having a transversion of a Purine to Pirimidine in one of its nucleotides, but not in those nucleotides where the integrase performs the cut in the recombination process 5'-G ' TT-3 '(The cut-off point is between G and T). The sites attCaadA1 and attCaadA3 have 100% sequence identity between their 1L 5'-GTTTAAC-3' regions and coincide exactly with the consensus sequence of the 1L region,\nwhile those corresponding to the attCaacA1 and attCdfrA1 sites present the transversion of one nucleotide each.\nThe extrahelical bases of the attC sites studied: attCaadA1, attCaadA3, attCaacA1 correspond to Guanine and Thymine except for that observed in the attCdfrA1 site which has as extrahelical bases a cytosine and an adenine. The most evident and significant difference with respect to the extrahelical bases within the attC sites analyzed is the spacing between them, and in those attC sites that share the same extrahelicoidal bases and the same nucleotide distance between them: attCaadA1, attCaadA3, attCaacA1, the most significant differences correspond to the length of the attC site, which apparently would have no influence on recombination frequencies, at least for the attC sites that we analyzed in this work.\nNoticing the determinants of structural recognition of importance for recombination, we can say that the attCaadA1 and attCaadA3 sites maintain an identity regarding their extrahelical bases G and T, as well as the distance between them (6 base pairs), which could be contributing to increase the scission frequencies through recombination. Regarding to attCaacA1 site, the extrahelicoidal bases retain the same G and T identity and the same spacing between them (6 base pairs) and present very similar scission values when this attC site is in the attCaacA1-aadA1-attCaadA1 structure.\nTwo attC recombination sites, the attCaadA3 site of 60 bp and the attCaacA1 site of 118 bp, that are different in length and in their spacer region have very similar recombination frequencies, which indicates that importance of identity of the extrahelical bases and the spacing between them is the fundamental characteristic that influences the frequency of recombination, while the attCdfrA1 site whose extrahelical bases are C and A with a distance between them of 7 to 8 bp presents a marked decrease in the excision frequencies.\nAs regards recombination assays carried out in vivo for the excision of cassette genes, mediated by IntI1, using in each case the clone PMI1-1 (AS) 2 (intI1-attI1) containing the gene of integrase type 1 co-transforming E. coli TOP10 with each of the following clones: pLQ423 (attI1-aadA1-attCaadA1), pLQ443 (attCaadA3-aadA1-attCaadA1), pLQ445 (attCaacA1-aadA1-attCaadA1) or pLQ426 (attCdhfr1-aadA1-attCaadA1). It was observed that the first three structures are favorable targets for IntI1-mediated excision, unlike the cassette gene attCdhfr1-aadA1-attCaadA1 where the excision frequency value was the lowest of all the assays. Regarding the insertion frequencies at the attI1 site, analyzing the net insertion, it was observed that recombination was favored between the attI1 site and the attCaadA3 and attCaacA1 sites through the cointegrated pathway.\nAnalyzing bioinformatics and functional results together we observed that the molecular patterns that favor the excision and net insertion of cassettes differ from each other and therefore must continue to be studied in a particular way to achieve a possible consensus of parameters that brings together all the requirements of the target sites of the integron integrase of class 1. |
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