فایل ورد کامل بینش هایی نسبت به سیستم CRISPR/Cas در گاردنلاواژینالیس

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تعداد صفحات این فایل: ۲۶ صفحه

بخشی از مقاله انگلیسیعنوان انگلیسی:Insights into the CRISPR/Cas system of Gardnerella vaginalis~~en~~

Abstract

Background: Gardnerella vaginalis is identified as the predominant colonist of the vaginal tracts of women diagnosed with bacterial vaginosis (BV). G. vaginalis can be isolated from healthy women, and an asymptomatic BV state is also recognised. The association of G. vaginalis with different clinical phenotypes could be explained by different cytotoxicity of the strains, presumably based on disparate gene content. The contribution of horizontal gene transfer to shaping the genomes of G. vaginalis is acknowledged. The CRISPR loci of the recently discovered CRISPR/Cas microbial defence system provide a historical view of the exposure of prokaryotes to a variety of foreign genetic elements.

Results: The CRISPR/Cas loci were analysed using available sequence data from three G. vaginalis complete genomes and 18 G. vaginalis draft genomes in the NCBI database, as well as PCR amplicons of the genomic DNA of 17 clinical isolates. The cas genes in the CRISPR/Cas loci of G. vaginalis belong to the E. coli subtype. Approximately 20% of the spacers had matches in the GenBank database. Sequence analysis of the CRISPR arrays revealed that nearly half of the spacers matched G. vaginalis chromosomal sequences. The spacers that matched G. vaginalis chromosomal sequences were determined to not be self-targeting and were presumably neither constituents of mobile-element-associated genes nor derived from plasmids/viruses. The protospacers targeted by these spacers displayed conserved protospacer-adjacent motifs.

Conclusions: The CRISPR/Cas system has been identified in about one half of the analysed G. vaginalis strains. Our analysis of CRISPR sequences did not reveal a potential link between their presence and the virulence of the G. vaginalis strains. Based on the origins of the spacers found in the G. vaginalis CRISPR arrays, we hypothesise that the transfer of genetic material among G. vaginalis strains could be regulated by the CRISPR/Cas mechanism. The present study is the first attempt to determine and analyse the CRISPR loci of bacteria isolated from the human vaginal tract.

۱ Background

Gardnerella vaginalis, a facultatively anaerobic bacterium of the Bifidobacteriaceae family, is strongly associated with bacterial vaginosis (BV): a disease characterised by malodorous vaginal discharge [1-3]. Women with BV are at risk of poor reproductive health outcomes and the acquisition of some sexually transmitted diseases [2,4]. BV is defined as a shift in microbial species from hydrogen peroxide producing Lactobacillus to anaerobic bacteria including G. vaginalis, Atopobium vaginae, Prevotella, Peptostreptococcus, and Bacteroides spp. [5,6]. The gold standard for laboratory diagnosis of BV is the Gram stain, which is used to determine the relative concentrations of lactobacilli and the bacteria characteristic of BV [7]. The state of asymptomatic BV has also been recognised, although Gram stains revealed a decrease in lactobacilli and an increase in the abundance of anaerobes specific to BV [8]. The same G. vaginalis that is recovered as the prevailing inhabitant of the vaginal tracts of women diagnosed with BV is also found in BV-negative women, though at much lower numbers [5,9,10]. The issue of G. vaginalis commensalism is still unclear, as the vaginal bacterial community is dynamic and tends to change during the menstrual cycle to produce transient dominance of G. vaginalis in healthy women [11,12]. Using culture- independent techniques, it was demonstrated that the vaginal microbiota may differ among human populations: Hispanic and non-Hispanic black women have significantly more anaerobes and fewer lactobacilli than Asian and Caucasian women [12]. Thus, low counts of Lactobacillus do not necessarily indicate the BV state [6,13].

The association of G. vaginalis with different clinical phenotypes could be explained by different cytotoxicity of the strains, presumably based on disparities in their gene content. Until recently, surprisingly little has been known about the genetics of G. vaginalis. In 2010, the genomes of several G. vaginalis strains from the vaginas of BV and non-BV patients were sequenced, providing information about the bacterium and enabling comparative genomic analyses [14,15]. Attempts have also been made to expand the knowledge of the genotypic and phenotypic diversity of G. vaginalis strains in terms of virulence factors: particularly vaginolysin, sialidase, and biofilm-forming proteins [16-18]. The development of methods for the genotypic differentiation of G. vaginalis revealed that the genomes exhibit great variability. Therefore, some conventional methods, including pulse field gel electrophoresis, restriction fragment length polymorphism, classical ribotyping with Southern blot, and restriction enzyme analysis, are not applicable for typing this species [19-21]. The amplified ribosomal DNA restriction analysis method, while applicable to the genotypic differentiation of G. vaginalis, has been found to not be discriminatory enough for pathogenetic and epidemiological studies of G. vaginalis [17,18].

Recent data from G. vaginalis comparative genomic analyses have indicated that the bacterium possesses a small core genome, consisting of 746 genes, that accounts for only 27% of the pan-genome of the species [22]. The small number of unique genes (21) in the individual strains of G. vaginalis and the genomic plasticity among the strains suggest that horizontal gene transfer (HGT) is active; but there is frequent homologous recombination among G. vaginalis strains, as well as the intake of foreign DNA from other species [15,22].

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and their associated cas genes constitute a bacterial and archaeal defence mechanism against exogenous nucleic acids [23]. The majority of archaea and approximately half of bacterial genomes contain CRISPR loci [24]. CRISPR loci consist of unique sequences (spacers) that intercalate between short conserved repeat sequences. The spacer sequences often originate from invading viruses and plasmids [25,26]. The CRISPR/Cas defence mechanism relies on RNA interference that prevents bacteriophage infection and plasmid conjugation, thus restricting two routes of HGT [27]. Analyses of CRISPR sequences have been used in a variety of applications including strain genotyping and epidemiological study, detection of evolutionary events and bottlenecks, investigation of the history of virus exposure, and host population dynamics, providing insights into the dominant routes of HGT [28-32]. The current study targeted the detection and analysis of CRISPR loci in the genomes of 17 G. vaginalis strains isolated from the vaginal tracts of women diagnosed with BV [18], and also in the genomes of 21 G. vaginalis strains deposited in the NCBI genome database.

In the current study, we examined the origins of CRISPR spacers representing the immunological memory of G. vaginalis strains, and we hypothesised about the impact of CRISPR/Cas on the emergence of genetic variability of G. vaginalis strains. Also, we demonstrated the restricted distribution of the CRISPR loci among the G. vaginalis strains.

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