Biology subjects

Ipoutcha, T.

Publications and source records attributed to Ipoutcha, T..

3 recordsLinked to original sources

bioRxiv2023

A synthetic biology approach to assemble and reboot clinically-relevant Pseudomonas aeruginosa tailed phages

The rise in frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa, a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically-relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination (TAR) cloning and rebooted these two phage genomes in two different strains of P. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step towards the construction of clinically-relevant, engineered P. aeruginosa phages. ImportancePseudomonas aeruginosa is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically-relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.

synthetic biology↗

bioRxiv2023

Evolution of the CRISPR-Cas9 defence system following a bacterial host shift

CRISPR-Cas systems are bacterial defences that target bacteriophages and mobile genetic elements. How these defences evolve in novel host environments remains, however, unknown. We studied the evolution of the CRISPR-Cas system in Mycoplasma gallisepticum, a bacterial pathogen of poultry that jumped into a passerine host [~]30 years ago. Over the decade following the host shift, all isolates displayed a functional CRISPR-Cas system were found not only to harbour completely new sets of spacers, but the DNA protospacer adjacent motif (PAM) recognised by the main effector MgCas9 was also different. These changes in CRISPR-Cas diversity and specificity are consistent with a change in the community of phages and mobile elements infecting M. gallisepticum as it colonised the novel host. In the years following the host shift, we also detected a gradual rise in isolates displaying non-functional MgCas9. After 12 years, all circulating isolates harboured inactive forms only. This loss of CRISPR-Cas function comes at a time when the passerine host is known to have evolved widespread resistance, which in turn drove the evolution of increasing M. gallisepticum virulence through antagonistic coevolution. Such striking concordance in the rise of inactivated forms of CRISPR-Cas and the evolution of host resistance suggests that inactivation of the CRISPR-Cas system was necessary for enabling adaptive bacterial responses to host-driven selection. We highlight the need to consider both host and pathogen selection pressures on bacteria for understanding the evolution of CRISPR-Cas systems and the key factors driving the emergence of a pathogenic bacterium in a novel host. Data summaryThe authors confirm all supporting data and protocols have been provided within the article or through supplementary data files available in the online version of this article. GenBank accession numbers of all publicly available M. gallisepticum genomes are listed in Table S3. Sequences of the CRISPR locus of other strains are also provided in Table S3. Impact statementMycoplasma are minimal bacteria involved in many diseases affecting humans and a wide diversity of animals. In this paper, we report the evolution of the Type II CRISPR-Cas system of the bird pathogen, Mycoplasma gallisepticum, following an host jump from its original poultry host into its novel house finch host in the early 90s. Instances in which bacterial pathogens have been documented to jump into and subsequently adapt to a new host are rare, and the well documented case of M. gallisepticum is a unique model to evaluate the effect of any dramatic host environmental change on bacterial CRISPR-Cas defence systems. First, we performed in silico analyses on an extended set of 98 M. gallisepticum genomes to better understand the evolution of the CRISPR-Cas9 system in the novel finch host. We documented several evolutionary events leading to the drastic divergence of spacer sets present in poultry and house finch arrays, as well as the progressive inactivation of the CRISPR-Cas system after 12 years in the novel finch host. Second, using in vitro and in vivo assays, we demonstrated that the evolution of the MgCas9 PI domain, involved in the protospacer adjacent motif (PAM) recognition has led to a major change in the defence system, with a modification of the recognized PAM in the novel host. Such radical change in the CRISPR-Cas defence system of M. gallisepticum may have implications for the its rapid adaptation to its novel host. Together, our results highlight the need to consider not only the host-driven selection pressures a bacterium experiences, but also the complex interplay between phages and defence systems for better understanding the key factors driving the emergence of a pathogenic bacterium in a novel host.

microbiology↗

bioRxiv2022

Unblocking genome editing of major animal mycoplasmas using CRISPR/Cas9 base editor systems

Mycoplasmas are minimal bacteria that infect humans, wildlife, and most economically important livestock species. Mycoplasma infections cause a large range of chronic inflammatory diseases, eventually leading to death in some animals. Due to the lack of efficient recombination and genome engineering tools, the production of mutant strains for the identification of virulence factors and the development of improved vaccine strains is still a bottleneck for many mycoplasma species. Here, we demonstrate the efficacy of a CRISPR-derived genetic tool to introduce targeted mutations in three major pathogenic species that span the phylogenetic diversity of these bacteria: the avian pathogen Mycoplasma gallisepticum and the two most important bovine mycoplasmas, Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides. As a proof of concept, we successfully used an inducible dCas9-cytidine deaminase system to disrupt several major virulence factors in these pathogens. Various induction times and inducer concentrations were evaluated to optimize editing efficiency. The optimized system was sufficiently powerful to disrupt 54 of 55 insertion sequence transposases in a single step. Whole genome sequencing showed that off-target mutations were limited and suggest that most variations detected in the edited genomes are Cas9-independent. This effective, rapid, and easy-to-use genetic tool opens a new avenue for the study of these important animal pathogens and, most likely, the entire class Mollicutes. SignificanceMycoplasmas are minimal wall-less pathogenic bacteria that infect a wide range of hosts, including humans, livestock, and wild animals. Major pathogenic species cause acute to chronic infections involving still poorly characterized virulence factors. The lack of precise genome editing tools has hampered functional studies for many species, leaving multiple questions about the molecular basis of their pathogenicity unanswered. We developed a CRISPR-derived base editor for three major pathogenic species, Mycoplasma gallisepticum, Mycoplasma bovis, and Mycoplasma mycoides subsp. mycoides. Several virulence factors were successfully targeted and we were able to edit up to 54 target sites in a single step. The availability of this efficient and easy-to-use genetic tool will greatly facilitate functional studies in these economically important bacteria.

microbiology↗