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CRISPR-cas subtype I-Fb in Acinetobacter baumannii: evolution and utilization for strain subtyping.

ABSTRACT: Clustered regularly interspaced short palindromic repeats (CRISPR) are polymorphic elements found in the genome of some or all strains of particular bacterial species, providing them with a system of acquired immunity against invading bacteriophages and plasmids. Two CRISPR-Cas systems have been identified in Acinetobacter baumannii, an opportunistic pathogen with a remarkable capacity for clonal dissemination. In this study, we investigated the mode of evolution and diversity of spacers of the CRISPR-cas subtype I-Fb locus in a global collection of 76 isolates of A. baumannii obtained from 14 countries and 4 continents. The locus has basically evolved from a common ancestor following two main lineages and several pathways of vertical descent. However, this vertical passage has been interrupted by occasional events of horizontal transfer of the whole locus between distinct isolates. The isolates were assigned into 40 CRISPR-based sequence types (CST). CST1 and CST23-24 comprised 18 and 9 isolates, representing two main sub-clones of international clones CC1 and CC25, respectively. Epidemiological data showed that some of the CST1 isolates were acquired or imported from Iraq, where it has probably been endemic for more than one decade and occasionally been able to spread to USA, Canada, and Europe. CST23-24 has shown a remarkable ability to cause national outbreaks of infections in Sweden, Argentina, UAE, and USA. The three isolates of CST19 were independently imported from Thailand to Sweden and Norway, raising a concern about the prevalence of CST19 in Thailand. Our study highlights the dynamic nature of the CRISPR-cas subtype I-Fb locus in A. baumannii, and demonstrates the possibility of using a CRISPR-based approach for subtyping a significant part of the global population of A. baumannii.

SUBMITTER: Karah N

PROVIDER: S-EPMC4338279 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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CRISPR-cas subtype I-Fb in Acinetobacter baumannii: evolution and utilization for strain subtyping.

Karah Nabil N Samuelsen Ørjan Ø Zarrilli Raffaele R Sahl Jason W JW Wai Sun Nyunt SN Uhlin Bernt Eric BE

PloS one 20150223 2

Clustered regularly interspaced short palindromic repeats (CRISPR) are polymorphic elements found in the genome of some or all strains of particular bacterial species, providing them with a system of acquired immunity against invading bacteriophages and plasmids. Two CRISPR-Cas systems have been identified in Acinetobacter baumannii, an opportunistic pathogen with a remarkable capacity for clonal dissemination. In this study, we investigated the mode of evolution and diversity of spacers of the ...[more]

PMID: 25706932

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Project description:Acinetobacter baumannii is a well-known human opportunistic pathogen in nosocomial infections, and the emergence of multidrug-resistant Acinetobacter baumannii has become a complex problem for clinical anti-infective treatments. The ways this organism obtains multidrug resistance phenotype include horizontal gene transfer and other mechanisms, such as altered targets, decreased permeability, increased enzyme production, overexpression of efflux pumps, metabolic changes, and biofilm formation. A CRISPR-Cas system generally consists of a CRISPR array and one or more operons of cas genes, which can restrict horizontal gene transfer in bacteria. Nevertheless, it is unclear how CRISPR-Cas systems regulate antibiotic resistance in Acinetobacter baumannii. Thus, we sought to assess how CRISPR-Cas affects biofilm formation, membrane permeability, efflux pump, reactive oxygen species, and quorum sensing to clarify further the mechanism of CRISPR-Cas regulation of Acinetobacter baumannii antibiotic resistance. In the clinical isolate AB43, which has a complete I-Fb CRISPR-Cas system, we discovered that the Cas3 nuclease of this type I-F CRISPR-Cas system regulates Acinetobacter baumannii quorum sensing and has a unique function in changing drug resistance. As a result of quorum sensing, synthase abaI is reduced, allowing efflux pumps to decrease, biofilm formation to become weaker, reactive oxygen species to generate, and drug resistance to decrease in response to CRISPR-Cas activity. These observations suggest that the CRISPR-Cas system targeting endogenous abaI may boost bacterial antibiotic sensitivity. IMPORTANCE CRISPR-Cas systems are vital for genome editing, bacterial virulence, and antibiotic resistance. How CRISPR-Cas systems regulate antibiotic resistance in Acinetobacter baumannii is almost wholly unknown. In this study, we reveal that the quorum sensing regulator abaI mRNA was a primary target of the I-Fb CRISPR-Cas system and the cleavage activity of Cas3 was the most critical factor in regulating abaI mRNA degradation. These results advance our understanding of how CRISPR-Cas systems inhibit drug resistance. However, the mechanism of endogenous targeting of abaI by CRISPR-Cas needs to be further explored.

Project description:IntroductionThe escalating prevalence of bacterial resistance, particularly multidrug-resistant bacteria like Acinetobacter baumannii, has become a significant global public health concern. The CRISPR-Cas system, a crucial defense mechanism in bacteria against foreign genetic elements, provides a competitive advantage. Type I-Fb and Type I-Fa are two subtypes of CRISPR-Cas systems that were found in A. baumannii, and the I-Fb CRISPR-Cas system regulates antibiotic resistance in A. baumannii. However, it is noteworthy that a majority of clinical isolates of A. baumannii lack or have incomplete CRISPR-Cas systems and most of them are multidrug-resistant. In light of this, our study aimed to examine the impact of antibiotic pressure on the fitness cost of the I-Fb CRISPR-Cas system in A. baumannii.Methods and resultsIn the study, we conducted in vitro competition experiments to investigate the influence of sub-minimum inhibitory concentration (sub-MIC) on the CRISPR-Cas systems' fitness cost in A. baumannii. We found that the fitness cost of the CRISPR-Cas system was increased under sub-MIC conditions. The expression of CRISPR-Cas-related genes was decreased, while the conjugation frequency was increased in AB43 under sub-MIC conditions. Through metabolomic analysis, we identified that sub-MIC conditions primarily affected energy metabolism pathways. In particular, we observed increased carbon metabolism, nitrogen metabolism, and intracellular ATP. Notably, the CRISPR-Cas system demonstrated resistance to the efflux pump-mediated resistance. Furthermore, the expression of efflux pump-related genes was increased under sub-MIC conditions.ConclusionOur findings suggest that the I-Fb CRISPR-Cas system confers a significant competitive advantage in A. baumanni. However, under sub-MIC conditions, its function and the ability to inhibit the energy required for efflux pumps are reduced, resulting in an increased fitness cost and loss of competitive advantage.

Project description:BACKGROUND:The presence and activity of CRISPR-Cas defense systems is a hallmark of many prokaryotic microorganisms. Here, the distribution of sequences related to the highly iterated palindrome 1 (HIP1) element and the DNA methylation of CGATCG motifs embedded within HIP1 as a vital part of the CRISPR1 repeat sequence was analyzed in the cyanobacterium Synechocystis sp. PCC 6803. Previously suggested functions of HIP1 include organization of chromosomal structure, DNA recombination or gene regulation, all of which could be relevant in CRISPR-Cas functionality. RESULTS:The CRISPR1 repeat-spacer array contains more than 50 CGATCG elements that are double-methylated (5mCG6mATCG) by the enzymes M.Ssp6803I and M.Ssp6803III. Hence, more than 200 possible methylation events cluster over a stretch of 3600 bp of double-stranded DNA. Bisulfite sequencing showed that these motifs were highly methylated at the m5CGATCG positions whereas specific motifs within the CRISPR1 cas genes were hypomethylated suggesting a lowered accessibility for the DNA methylase to these regions. Assays for conjugation and CRISPR1-mediated DNA interference revealed a 50% drop in conjugation efficiency in the mutant lacking the 5mC methylation of CGATCG motifs, while the highly efficient DNA interference activity was not affected by the lack of m5CGATCG DNA-methylation, nor was the capability to differentiate between self and non-self targets based on the protospacer adjacent motifs (PAMs) GTA and GTC versus the non-PAM AGC. A third DNA methylation mediated by M.Ssp6803II modifies the first cytosine in the motif GGCC yielding GGm4CC. We found a remarkable absence of GGCC motifs and hence the corresponding methylation over an 11 kb stretch encompassing all the cas genes involved in interference and crRNA maturation but not adaptation of the CRISPR1 system. CONCLUSIONS:The lack of GGCC tetranucleotides along the CRISPR1 interference and maturation genes supports the reported hybrid character of subtype I-D CRISPR-Cas systems. We report tight and very high 5mC methylation of the CRISPR1 repeat sequences. Nevertheless, cells lacking the 5mC methylation activity were unaffected in their CRISPR1-mediated interference response but the efficiency of conjugation was reduced by 50%. These results point to an unknown role of m5CGATCG DNA-methylation marks in conjugation and DNA transformation.

Project description:Acinetobacter baumannii is an emergent bacterial pathogen that provokes many types of infections in hospitals around the world. The genome of this organism consists of a chromosome and plasmids. These plasmids vary over a wide size range and many of them have been linked to the acquisition of antibiotic-resistance genes. Our bioinformatic analyses indicate that A. baumannii plasmids belong to a small number of plasmid lineages. The general structure of these lineages seems to be very stable and consists not only of genes involved in plasmid maintenance functions but of gene sets encoding poorly characterized proteins, not obviously linked to survival in the hospital setting, and opening the possibility that they improve the parasitic properties of plasmids. An analysis of genes involved in replication, suggests that members of the same plasmid lineage are part of the same plasmid incompatibility group. The same analysis showed the necessity of classifying the Rep proteins in ten new groups, under the scheme proposed by Bertini et al. (2010). Also, we show that some plasmid lineages have the potential capacity to replicate in many bacterial genera including those embracing human pathogen species, while others seem to replicate only within the limits of the Acinetobacter genus. Moreover, some plasmid lineages are widely distributed along the A. baumannii phylogenetic tree. Despite this, a number of them lack genes involved in conjugation or mobilization functions. Interestingly, only 34.6% of the plasmids analyzed here possess antibiotic resistance genes and most of them belong to fourteen plasmid lineages of the twenty one described here. Gene flux between plasmid lineages appears primarily limited to transposable elements, which sometimes carry antibiotic resistance genes. In most plasmid lineages transposable elements and antibiotic resistance genes are secondary acquisitions. Finally, broad host-range plasmids appear to have played a crucial role.

Dataset Information

CRISPR-cas subtype I-Fb in Acinetobacter baumannii: evolution and utilization for strain subtyping.

Publications

CRISPR-cas subtype I-Fb in Acinetobacter baumannii: evolution and utilization for strain subtyping.

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