Biology subjects

Laaberki, M.-H.

Publications and source records attributed to Laaberki, M.-H..

8 recordsLinked to original sources

bioRxiv2026

Calcium signals natural transformation in Acinetobacter baumannii

The acquisition of resistance to antibiotics in the opportunistic pathogen Acinetobacter baumannii may be linked to its capacity to undergo natural transformation. This mode of horizontal gene transfer relies on the import of extracellular DNA and its chromosomal integration by homologous recombination. Type IV pilus activity initiates the capture of extracellular DNA, which is then transported to the cytoplasm for recombination in the chromosome. While most Acinetobacter baumannii strains are transformable, the conditions allowing the expression of type IV pilus and other transformation genes remain largely unexplored. By investigating transformation-permissive conditions, we uncovered that calcium is a potent inducer of natural transformation. Type IV pilus genes and other transformation-specific genes (comEA, dprA) are upregulated by submillimolar concentrations of calcium ions, in a growth phase-dependent manner. In contrast, sodium chloride represses expression of pilA, counteracting the calcium-dependent induction, explaining the reported absence of transformation in NaCl-containing medium (such as LB). Independently of transcriptional induction, calcium ions also directly bind the type IV pilus through the calcium-dependent adhesin PilY1. Our data support a model in which calcium strengthens the interaction of PilY1 with the minor pilin complex, increasing pilus dynamics and subsequent pilus-dependent DNA capture. Hence, calcium signals natural transformation through both transcriptional and structural activation of type IV pilus. In addition to providing new insights into the regulation of natural transformation in A. baumannii this work led us to establish a protocol for genetic engineering of A. baumannii by natural transformation. ImportanceAcinetobacter baumannii is a nosocomial pathogen considered a critical research priority due to its resistance to last resort antibiotics. Understanding how A. baumannii evolves and acquires resistance to antibiotics is thus of prime importance. This species is capable of natural transformation, a means to acquire and spread genetic information, including antibiotic resistance genes. However, the conditions under which this process is active in this species remain elusive. We identify calcium ions as potent inducers of natural transformation and propose a model of the signaling of natural transformation by calcium ions. This opens the way for further investigations into the contribution of natural transformation to acquisition of antibiotic resistance. In addition, it provides an efficient way to genetically manipulate most A. baumannii strains.

microbiology↗

bioRxiv2024

Host shapes microevolution of MDR Acinetobacter baumannii during long-term infection

Acinetobacter baumannii is a nosocomial pathogen associated with various infections, including urinary tract infections (UTIs). In the course of an infection, A. baumannii is known to rapidly become resistant to antibiotic therapy, but much less is known about possible adaptation without antibiotic pressure. Through a retrospective study, we investigated within-host genetic diversity during a subclinical five-year UTI in an animal-patient after withdrawal of colistin treatment. We conducted whole-genome sequencing and phenotypic assays on seventeen clonally related isolates from the Sequence Type 25 lineage. Phylogenomic analysis revealed their proximity with animal and human strains from the same country suggesting zoonotic transmission (France). In this case study, the clonally related strains presented variations in genome sizes and nucleotide sequences. Over the course of the infection, A. baumannii underwent genome reduction through insertion sequence (IS) recombination, phage excision, or plasmid curing. Alongside this global genome reduction, we observed an expansion of IS17, initially located on the endogenous large plasmid. Genetic variations were mainly located in biofilm formation and metabolism genes. We observed repeated variation affecting three biofilm genes and two adhesion operons associated with weak biofilm-forming capacity. Conversely, only two metabolic genes were recurrently affected and phenotypic assays indicated a rather stable metabolism profile between the isolates suggesting minor adaptations to its host. Lastly, an overall decreased antibiotic resistance - expected in the absence of antibiotic treatment - contrasted with a conserved colistin resistance due to a pmrB mutation among the isolates. Impact statementThis study brings a new insight on the genome evolution of the opportunistic pathogen Acinetobacter baumannii during a five-year urinary tract infection in the absence of antibiotic treatment. It relies on genomic and phenotypic analyses of multiple isolates from the same animal-patient highlighting the necessity of studying bacterial diversity as opposed to solely on single-isolate approaches. Over the course of the infection, we observed a reduction in genome size, resulting however in a limited loss of metabolism flexibility, but we observed recurrent loss in adhesion capacities and antibiotic resistance. However, this study stands out by the unexpected conserved colistin resistance of the study isolates in the absence of treatment. Data summaryThe newly sequenced genomes are listed in Table S1, these genomes have been submitted to the NCBI and their BioProject number is PRJNA1100485. The publicly available genomes of bacteria from human and animal origin used in this study are listed in Table S2 All the supplementary tables are available in the Supplementary Material File.

microbiology↗

bioRxiv2024

Interplay between the Xer recombination system and the dissemination of antibioresistance in Acinetobacter baumannii

Antibiotic-resistant infections pose a pressing challenge in clinical settings. Plasmids are widely recognized for hastening the emergence of resistance by facilitating horizontal gene transfer of antibiotic resistance genes among bacteria. We explore this inquiry in Acinetobacter baumannii, a globally emerging nosocomial pathogen responsible for a wide array of infections with worrying accumulation of resistances, notably involving plasmids. In this specie, plasmids of the Rep_3 family harbor adaptive genes within variable regions edged by potential site-specific recombination sites recognized by the XerCD recombinase. We first show that the Xer system of Acinetobacter baumannii functions as described in Escherichia coli, resolving chromosome dimers at the dif site as well as recombining plasmid-borne sites. The multiple Xer recombination sites found in Rep_3 plasmids do not, however, allow excising plasmid fragments. They rather recombine to co-integrate plasmids, which may then further evolve to exchange genes. Co-integrates represent a significative part of the plasmid population and their formation is controlled by the sequence of the recombination sites determining their compatibility between the recombining sites. We conclude that plasmids frequently exchange genes in Acinetobacter baumannii using Xer recombination, allowing a high level yet controlled plasticity involved in the acquisition and combination of resistance genes.

microbiology↗

bioRxiv2023

Manipulation of natural transformation by AbaR-type islands promotes fixation of antibiotic resistance in Acinetobacter baumannii populations

The opportunistic pathogen Acinetobacter baumannii, a major global public health concern, has developed multiple variants of AbaR-type genomic islands that confer multidrug resistance. The mechanisms facilitating the persistence of these potentially costly islands within A. baumannii populations have remained enigmatic. In this study, we employed a combination of investigative methods to shed light on the factors contributing to their selective advantage and long-term persistence. The dissemination of AbaR islands is intricately linked to their horizontal transfer via natural transformation, a process through which bacteria can import and recombine exogenous DNA, facilitating allelic recombination, genetic acquisition, and deletion. In experimental populations, we first quantified the rate at which natural transformation events occur between individuals. Our findings indicate that the rate of AbaR deletion events is marginally higher than the rate of their acquisition. When this result is integrated into a model of population dynamics not exposed to antibiotic selection pressure, it leads to the swift removal of AbaRs from the population, a pattern that stands in contrast to AbaR prevalence in genomes. Yet, genomic analyses show that nearly all AbaRs-carrying A. baumannii have at least one AbaR disrupting comM, a gene encoding a helicase critical for natural transformation. We discovered that such disruption differentially inhibits the rate of genetic acquisition and deletion. Specifically, they significantly impede the removal of AbaRs while still enabling the host cell to acquire and recombine short sequences, such as allelic variants. Through mathematical evolutionary modeling, we demonstrate that AbaRs inserted into comM gain a selective advantage over AbaRs inserted in sites that do not inhibit or completely inhibit transformation, in line with the genomic observations. The persistence of AbaRs within populations can be ascribed to their targeted integration into a gene, substantially diminishing the likelihood of their removal from the bacterial genome. In contrast, this integration enables the host cell to preserve the ability to acquire and eliminate various short heterologous sequences, enabling the host bacterium - and thus its AbaR - to adapt to the unpredictability of the environment and persist over the long term. This work underscores how AbaRs, and potentially other Mobile Genetic Elements (MGEs), can manipulate natural transformation to ensure their persistence in populations, ultimately leading to the high prevalence of multidrug resistance.

microbiology↗

bioRxiv2021

Interbacterial transfer of carbapenem resistance and large antibiotic resistance islands by natural transformation in pathogenic Acinetobacter

Acinetobacter baumannii infection poses a major health threat with recurrent treatment failure due to antibiotic resistance, notably to carbapenems. While genomic analyses of clinical strains indicate that homologous recombination plays a major role in the acquisition of antibiotic resistance genes, the underlying mechanisms of horizontal gene transfer often remain speculative. Our understanding of the acquisition of antibiotic resistance is hampered by the lack of experimental systems able to reproduce genomic observations. We here report the detection of recombination events occurring spontaneously in mixed bacterial populations and which can result in the acquisition of resistance to carbapenems. We show that natural transformation is the main driver of intra-, but also inter-strain recombination events between A. baumannii clinical isolates and pathogenic species of Acinetobacter. We observed that interbacterial natural transformation in mixed populations is more efficient at promoting the acquisition of large resistance islands (AbaR4, AbaR1) than when the same bacteria are supplied with large amounts of purified genomic DNA. Importantly, analysis of the genomes of the recombinant progeny revealed large recombination tracts (from 13 to 123 kb) similar to those observed in the genome of clinical isolates. Moreover, we highlight that transforming DNA availability is a key determinant of the rate of recombinants and results from both spontaneous release and interbacterial predatory behavior. In the light of our results, natural transformation should be considered as a leading mechanism of genome recombination and horizontal gene transfer of antibiotic resistance genes in Acinetobacter baumannii. ImportanceAcinetobacter baumannii is a multidrug resistant pathogen responsible for difficult-to-treat hospital-acquired infections. Understanding the mechanisms leading to the emergence of the multi-drug resistance in this pathogen is today crucial. Horizontal gene transfer is assumed to largely contribute to this multidrug resistance. However, in A. baumannii, the mechanisms leading to genome recombination and the horizontal transfer of resistance genes are poorly understood. We bring experimental evidence that natural transformation, a horizontal gene transfer mechanism recently highlighted in A. baumannii, allows the highly efficient interbacterial transfer of genetic elements carrying resistance to last line antibiotic carbapenems. Importantly, we demonstrated that natural transformation, occurring in mixed populations of Acinetobacter, enables the transfer of large resistance island mobilizing multiple resistance genes.

microbiology↗

bioRxiv2020

Curing of large resistance island AbaR results in antibiotic susceptibility and increased natural transformability in Acinetobacter baumannii.

With a great diversity in gene composition including multiple putative antibiotic-resistance genes, AbaR islands are potential contributors to multi-drug resistance in Acinetobacter baumannii. However, the effective contribution of AbaR to antibiotic resistance and bacterial physiology remains elusive. To address this, we sought to accurately remove AbaR islands and restore the integrity of their insertion site. To this end, we devised a versatile scarless genome editing strategy. We performed this genetic modification in two recent A. baumannii clinical strains: the strain AB5075 and the nosocomial strain AYE which carry AbaR11 and AbaR1 islands of 19.7 kbp and 86.2 kbp, respectively. Antibiotic susceptibilities were then compared between the parental strains and their AbaR-cured derivatives. As anticipated by the predicted function of the ORF of this island, the antibiotic resistance profiles were identical between the wild type and the AbaR11-cured AB5075 strains. In contrast, AbaR1 carries 25 ORFs with a predicted resistance to several classes of antibiotics and the AYE AbaR1-cured derivative showed restored susceptibility to multiple classes of antibiotics. Moreover, curing of AbaRs restored high levels of natural transformability. Indeed, most AbaR islands are inserted into the comM gene involved in natural transformation. Our data indicate that AbaR insertion effectively inactivates comM and that the restored comM is functional. Curing of AbaR consistently resulted in highly transformable, and therefore, easily genetically tractable strains. Emendation of AbaR provides insight into the functional consequences of AbaR acquisition.

microbiology↗

bioRxiv2019

Bacterial transformation buffers environmental fluctuations through the reversible integration of mobile genetic elements

Horizontal gene transfer (HGT) is known to promote the spread of genes in bacterial communities, which is of primary importance to human health when these genes provide resistance to antibiotics. Among the main HGT mechanisms, natural transformation stands out as being widespread and encoded by the bacterial core genome. From an evolutionary perspective, transformation is often viewed as a mean to generate genetic diversity and mixing within bacterial populations. However, another recent paradigm proposes that its main evolutionary function would be to cure bacterial genomes from their parasitic mobile genetic elements (MGEs). Here, we propose to combine these two seemingly opposing points of view because MGEs, although costly for bacterial cells, can carry functions that are point-in-time beneficial to bacteria under stressful conditions (e.g. antibiotic resistance genes under antibiotic exposure). Using computational modeling, we show that, in stochastic environments (unpredictable stress exposure), an intermediate transformation rate maximizes bacterial fitness by allowing the reversible integration of MGEs carrying resistance genes but costly for the replication of host cells. By ensuring such reversible genetic diversification (acquisition then removal of MGEs), transformation would be a key mechanism for stabilizing the bacterial genome in the long term, which would explain its striking conservation.

microbiology↗

bioRxiv2018

Fluorescence-based detection of natural transformation in drug resistant Acinetobacter baumannii

Acinetobacter baumannii is a nosocomial agent with a high propensity for developing resistance to antibiotics. This ability relies on horizontal gene transfer mechanisms occurring in the Acinetobacter genus, including natural transformation. To study natural transformation in bacteria, the most prevalent method uses selection for the acquisition of an antibiotic resistance marker in a target chromosomal locus by the recipient cell. Most clinical isolates of A. baumannii are resistant to multiple antibiotics limiting the use of such selection-based method. Here we report the development of a phenotypic and selection-free method based on flow cytometry to detect transformation events in multidrug resistant (MDR) clinical A. baumannii isolates. To this end, we engineered a translational fusion between the abundant and conserved A. baumannii nucleoprotein (HU) and the superfolder green fluorescent protein (sfGFP). The new method was benchmarked against the conventional antibiotic selection-based method. Using this new method, we investigated several parameters affecting transformation efficiencies and identified conditions of transformability one hundred times higher than those previously reported. Using optimized transformation conditions, we probed natural transformation in a set of MDR clinical and non-clinical animal A. baumannii isolates. Regardless of their origin, the majority of the isolates displayed natural transformability, indicative of a conserved trait in the species. Overall, this new method and optimized protocol will greatly facilitate the study of natural transformation in the opportunistic pathogen A. baumannii.

microbiology↗