Biology subjects

Moreno, A. B.

Publications and source records attributed to Moreno, A. B..

4 recordsLinked to original sources

bioRxiv2024

Host-specific adaptation of Legionella pneumophila to single and multiple hosts

Legionella pneumophila is an endosymbiotic bacterial species able to infect and reproduce in various protist and human hosts. Upon entry in human lungs, they may infect lung macrophages, causing Legionnaires disease (LD), an atypical pneumonia, using similar mechanisms as in their protozoan hosts, despite the two hosts being separated by a billion years of evolution. In this study, we used experimental evolution to identify genes conferring host-specificity to L. pneumophila. To this end, we passaged L. pneumophila in two different hosts - Acanthamoeba castellanii and the human macrophage-like cells U937 - separately and by switching between the hosts twice a week for a year. In total, we identified 1518 mutations present in at least 5% of the population at the time of sampling. Half of these were localized in five groups of repeated sequences, likely to be recombination hotspots. Forty-nine mutations were fixed in the 18 populations at the end of the experiment, representing four different groups. The first two groups involve adaptation to the specific selection conditions, including (i) two specific mutations in the 30S ribosomal protein S12 (RpsL) that conferred resistance to streptomycin, to which the bacteria were unexpectedly exposed to during serial passage and (ii) two mutations, one in the 30S ribosomal protein S4 (RpsD), and one in the chaperonin GroES, both of which are likely to be fitness-restoring compensatory mutations to the original RpsL mutations. Two more interesting groups of mutations included (iii) mutations in 4 different strain-specific genes involved in LPS synthesis, found only in the lineages passaged with A. castellanii and (iv) mutations in the gene coding for LerC, a key regulator of protein effector expression, which was independently mutated in 6 lineages grown in presence of the U937 cells. We propose that the mutations degrading the function of the regulator LerC improve the fitness of L. pneumophila in human-derived cells, and that modifications in the LPS are beneficial for growth in A. castellanii. This study is a first step in further investigating determinants of host specificity in L. pneumophila.

evolutionary biology↗

bioRxiv2022

High-efficiency transfection of Acanthamoeba castellanii using a cationic polymer

The free-living amoeba Acanthamoeba castellanii is an ecologically, clinically, and evolutionarily important microorganism. A. castellanii amoebae are directly pathogenic to humans and serve as reservoirs for bacterial pathogens (e.g., Legionella pneumophila) but also regulate the proliferation of other microorganisms in the soil. Despite their importance, no reliable genetic system has been developed, hampering the use of A. castellanii and related species as model organisms. Transfecting A. castellanii with plasmids is possible with commercial kits, but it is expensive, inefficient, and vulnerable to product discontinuation. In this contribution, we present a method for efficient transfection of A. castellanii with readily available and inexpensive polyethylenimines. We systematically explore the methods parameters, obtaining up to 100-fold higher efficiency than currently used protocols. The method presented here provides a robust step towards a complete genetic toolbox for A. castellanii, hence expanding its use as a model organism.

microbiology↗

bioRxiv2022

Rapid, in-patient adaptations of Legionella pneumophila to the human host

Legionella pneumophila are host-adapted bacteria that infect and reproduce primarily in amoeboid protists. Using similar infection mechanisms, they infect human macrophages, and cause Legionnaires disease, an atypical pneumonia, and the milder Pontiac fever. We hypothesized that, despite these similarities, the hosts are different enough so that there exist high-selective value mutations that would dramatically increase the fitness of Legionella inside the human host. By comparing a large number of isolates from independent infections, we identified two genes, mutated in three unrelated patients, despite the short duration of the incubation period (2-14 days). One is a gene coding for an outer membrane protein (OMP) belonging to the OmpP1/FadL family. The clinical strain, carrying the mutated OMP homolog, grows faster in macrophages than the wild type strain, and thus appears to be better adapted to the human host. The other is a gene coding for a protein involved in cyclic-di-GMP regulation, which in turn modulates flagellar activity. As human-to-human transmission is very rare, fixation of these mutations into the population and spread into the environment is unlikely. Therefore, convergent evolution - here mutations in the same genes observed in independent human infections - could point to adaptations to the accidental human host. These results suggest that despite its ability to infect, replicate, and disperse from amoebae, L. pneumophila is not well adapted to the human host. Impact statementLegionella pneumophila is primarily infecting amoeboid protists, but occasionally infects human lung macrophages, causing Legionnaires disease, an atypical pneumonia. By comparing 171 isolates from patients to their probable environmental source, we identified 119 mutations that presumably occurred in-patient. Among these, several mutations occurred in the gene. In particular, two genes were mutated thrice, significantly more often than expected by chance alone, and are likely to represent adaptations to the human host. We experimentally show that, for one mutation at least, the mutated strain grows faster in human macrophages than in amoebae. By specifically investigating in-patient mutations, we were able to identify two genes that might be involved in human host-specific adaptations of L. pneumophila. This result suggests that L. pneumophila is not particularly well adapted to the human host, as mutations get fixed in-patient, during the short course of an infection (2-14 days), indicating a very high selective value. Data SummaryThe sequencing data generated in this study are available in the NCBI database under the BioProject accession number: PRJEB52976.

microbiology↗

bioRxiv2020

Longitudinal whole-exome sequencing of cell-free DNA unravels the metastatic evolutionary dynamics of BRCA2 mutated breast cancer

Little is known about the metastatic evolutionary dynamics of BRCA2-mutated cancers. Here, we applied whole-exome sequencing (WES) of primary tumor (PT), local relapse (LR) and eight serial plasma cfDNA samples collected from disease progression to depict the 12 years evolutionary trajectory of a metastatic BRCA2-mutated breast cancer. While longitudinal WES-cfDNA recapitulated clonal and subclonal mutations and copy number profiles detected in LR, emergence of plasma-exclusive mutations in TSC2 and HDAC9 cancer-related genes and loss of HLA loci as an immune escape mechanism were also detected. Lastly, mutation signature 3, associated with homologous recombination deficiency and response to platinum-based therapy raised profoundly from 19% in PT to 60% in late stage disease. In conclusion, we show for the first time that longitudinal WES-cfDNA enables the evolutionary trajectory of advanced cancer to be uncovered and that increment of MS3 and loss of HLA are key players in this BRCA2-mutated breast metastasis.

cancer biology↗