Biology subjects

Cisneros, D. A.

Publications and source records attributed to Cisneros, D. A..

4 recordsLinked to original sources

bioRxiv2025

Bacteroidales T6SS minor Hcp subunits form heteromers recognising effectors

The type VI secretion system (T6SS) is a macromolecular protein complex found in Gram-negative bacteria that mediates intercellular antagonism. T6SSs are gut colonization factors that influence gut biodiversity. Hemolysin-coregulated proteins (Hcp) are major structural proteins in these systems. Hcp forms hexameric rings that stack to create an inner tube structure essential for translocating effector proteins into target cells. In Bacteroidales, T6SS loci encode multiple Hcp proteins with unknown function. The gut commensal Bacteroides fragilis encodes five Hcp subunits (sHcp and Hcp1-4) that have low sequence similarity. In this study, we investigated the roles of these proteins. Interaction studies showed that sHcp forms homohexamers, which is consistent with a major role of forming the bulk of the inner tube. In contrast, the less abundant minor Hcp1-4 were shown to form an interaction network involving heteromeric complexes. Biochemical analyses demonstrated that Hcp1 and Hcp2 assemble into heterohexamers and that this complex recognizes the secreted effector Bte1. Finally, we showed that Hcp modules, which are encoded in highly syntenic regions in T6SS loci of Bacteroidales, cluster with effectors. These results imply that the minor Hcps genetically cosegregate with cognate effectors, contributing to effector cassette variability. Thus, minor Hcp subunits function as recognition particles for effectors to mediate secretion, which appears to be a conserved trait in Bacteroidales T6SSs. Exploiting these features could facilitate the characterization of unknown effectors by copurifying them with their cognate Hcps. This approach may reveal new insights into bacterial interactions and the mechanisms that establish gut biodiversity.

biochemistry↗

bioRxiv2022

Parallel CRISPR-Cas guided mutagenesis of chromosome and virulence plasmid in Shigella flexneri by cytosine base editing

Shigella is a Gram-negative bacterium that invades the human gut epithelium. The resulting infection, shigellosis, is the deadliest bacterial diarrheal disease. Much of the information about the genes dictating the pathophysiology of Shigella, both on the chromosome and the virulence plasmid, was obtained by classical reverse genetics. However, technical limitations of the prevalent mutagenesis techniques restrict the generation of mutants in a single reaction to a small number, preventing large scale targeted mutagenesis of Shigella and the subsequent assessment of phenotype. We adopted a CRISPR-Cas dependent approach, where a nickase Cas9 and cytidine deaminase fusion is guided by sgRNA to introduce targeted C[->]T transitions, resulting in internal STOP codons and premature termination of translation. In proof-of-principle experiments using an mCherry fluorescent reporter, we were able to generate loss-of-function mutants in both E. coli and Shigella with up to 100% efficacy. Using a modified fluctuation assay, we determined that under optimized conditions, the frequency of untargeted mutations introduced by the Cas9-deaminase fusion is in the same range as spontaneous mutations, making our method a safe choice for bacterial mutagenesis. Further, we programmed the method to mutate well-characterized chromosomal and plasmid-borne Shigella genes and found the mutant phenotype to be similar to that of the reported gene deletion mutants, with no apparent polar effects at the phenotype level. This method can be used in a 96-well plate format to increase the throughput and generate an array of targeted loss-of-function mutants in a few days. ImportanceLoss-of-function mutagenesis is critical in understanding the physiological role of genes. Therefore, high-throughput techniques to generate such mutants are important for facilitating the assessment of gene function at a pace that matches system biology approaches. However, to our knowledge, no such method was available for generating an array of single gene mutants in an important enteropathogen - Shigella. This pathogen causes high morbidity and mortality in children and antibiotic resistant strains are quickly emerging. Therefore, determination of the function of unknown Shigella genes is of utmost importance to develop effective strategies to control infections. Our present work will bridge this gap by providing a rapid method for generating loss-of-function mutants. The highly effective and specific method has the potential to be programmed to generated multiple mutants in a single, massively parallel, reaction. By the virtue of plasmid compatibility, this method can be extended to other members of Enterobacteriaceae.

microbiology↗

bioRxiv2020

Polar mutagenesis of bacterial transcriptional units using Cas12a

Bacterial genes are often organized in functionally related transcriptional units or operons. One such example is the fimAICDFGH operon, which codes for type I fimbriae in Escherichia coli. We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in the fim operon. Cas12a-mediated engineering of a terminator sequence inside the fimA gene occurred with efficiencies between 10 and 30%, whilst other types of mutations, such as a 97 bp deletion, occurred with 100% efficiency. Our results showed that some of the obtained mutants, including one with a single base substitution at the fim locus, had decreased mRNA levels of fimA, suggesting that the regulation of the fim operon was disrupted. We corroborated the polar effect of these mutants by phenotypic assays and quantitative PCR, showing up to a 43 fold decrease in expression of genes downstream fimA. We believe this strategy could be useful in engineering the transcriptional shut-down of multiple genes in one single step. For bio-production in E. coli, this opens the possibility of inhibiting competing metabolic routes.

microbiology↗

bioRxiv2017

CTCF, WAPL and PDS5 proteins control the formation of TADs and loops by cohesin

Mammalian genomes are organized into compartments, topologically-associating domains (TADs) and loops to facilitate gene regulation and other chromosomal functions. Compartments are formed by nucleosomal interactions, but how TADs and loops are generated is unknown. It has been proposed that cohesin forms these structures by extruding loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here we show that cohesin suppresses compartments but is essential for TADs and loops, that CTCF defines their boundaries, and that WAPL and its PDS5 binding partners control the length of chromatin loops. In the absence of WAPL and PDS5 proteins, cohesin passes CTCF sites with increased frequency, forms extended chromatin loops, accumulates in axial chromosomal positions (vermicelli) and condenses chromosomes to an extent normally only seen in mitosis. These results show that cohesin has an essential genome-wide function in mediating long-range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.

genomics↗