Biology subjects

Caroff, M.

Publications and source records attributed to Caroff, M..

3 recordsLinked to original sources

bioRxiv2026

Exploiting HSD17B11-dependent dialkynylcarbinols cytotoxicity for facile CRISPR/Cas9-based gene inactivation

Several approaches have been developed to improve the efficiency of CRISPR/Cas9-based genome editing, including the co-inactivation of a gene whose loss confers resistance to a cytotoxic compound, thereby enabling enrichment of successfully edited cells. Here, we show across multiple cell lines that inactivation of HSD17B11, a non-essential member of the Short-chain Dehydrogenase/Reductase (SDR) superfamily, confers strong resistance (29- to 131-fold) to a Phenyl diAlkynylCarbinol compound (PAC) in both human and mouse cells, without affecting cell viability or proliferation. We demonstrate that co-inactivation of HSD17B11 followed by PAC selection can be used to rapidly identify efficient guide RNAs targeting a gene of interest and to readily isolate clones inactivated for one or multiple genes. Altogether, these results establish a simple and efficient experimental strategy for generating knockout cells by using PAC selection to enrich for successfully edited cells.

molecular biology↗

bioRxiv2020

Leptospiral LPS escapes mouse TLR4 internalization and TRIF?associated antimicrobial responses through O antigen and associated lipoproteins

Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira spp. All vertebrate species can be infected; humans are sensitive hosts whereas other species, such as rodents, may become long-term renal carrier reservoirs. Upon infection, innate immune responses are initiated by recognition of Microbial Associated Molecular Patterns (MAMPs) by Pattern Recognition Receptors (PRRs). Among MAMPs, the lipopolysaccharide (LPS) is recognized by the Toll-Like-Receptor 4 (TLR4) and activates both the MyD88-dependent pathway at the plasma membrane and the TRIF-dependent pathway after TLR4 internalization. We previously showed that leptospiral LPS is not recognized by the human TLR4, whereas it signals through murine TLR4, which mediates mouse resistance to acute leptospirosis. However, leptospiral LPS has low endotoxicity in mouse cells and is an agonist of TLR2, the sensor for bacterial lipoproteins. Here, using confocal microscopy and flow cytometry, we showed that the LPS of L. interrogans did not induce internalization of TLR4 in mouse macrophages, unlike the LPS of Escherichia coli. Consequently, the LPS failed to induce the production of the TRIF-dependent nitric oxide and RANTES, both important antimicrobial responses. Using shorter O antigen LPS and repurified leptospiral LPS with reporter HEK cells, we further found this TLR4-TRIF escape to be dependent on both the co-purifying lipoproteins and the full-length O antigen. Furthermore, our data suggest that the O antigen could alter the binding of the leptospiral LPS to the co-receptor CD14 that is essential for TLR4-TRIF activation. Overall, we describe here a novel immune escape mechanism linked to leptospiral LPS. We hypothesize that the LPS, already known as a virulence factor, plays a major role in the innate immune evasion of the leptospires, thereby contributing to their stealthiness and chronicity in mice. Author summaryLeptospira interrogans is a bacterial pathogen, responsible for leptospirosis, a worldwide neglected reemerging disease. L. interrogans may cause an acute severe disease in humans, whereas rodents and other animals asymptomatically carry the leptospires in their kidneys. They can therefore excrete live bacteria in urine and contaminate the environment. Leptospires are stealth pathogens known to escape the innate immune defenses of their hosts. They are covered in lipopolysaccharide (LPS), a bacterial motif recognized in mammals through the Toll-like receptor 4 (TLR4), which triggers two different signaling pathways. We showed previously that pathogenic leptospires escape TLR4 recognition in humans. Here we show in mice that the leptospiral LPS triggers only one arm of the TLR4 pathway and escapes the other, hence avoiding production of antimicrobial compounds. Removing the lipoproteins that always co-purify with the leptospiral LPS, or using shorter LPS, restores the stimulation of both pathways. This suggests a novel escape mechanism linked to the LPS and involving lipoproteins that could be instrumental for leptospires to escape the mouse defense and allows for their chronic renal colonization.

immunology↗

bioRxiv2019

Escherichia coli O157:H7 responds to phosphate starvation by modifying LPS involved in biofilm formation

In open environments such as water, enterohemorrhagic Escherichia coli O157:H7 responds to inorganic phosphate (Pi) starvation by inducing the Pho regulon controlled by PhoB. The phosphate-specific transport (Pst) system is the high-affinity Pi transporter. In the {Delta}pst mutant, PhoB is constitutively activated and regulates the expression of genes from the Pho regulon. In E. coli O157:H7, the {Delta}pst mutant, biofilm, and autoagglutination were increased. In the double-deletion mutant {Delta}pst {Delta}phoB, biofilm and autoagglutination were similar to the wild-type strain, suggesting that PhoB is involved. We investigated the relationship between PhoB activation and enhanced biofilm formation by screening a transposon mutant library derived from {Delta}pst mutant for decreased autoagglutination and biofilms mutants. Lipopolysaccharide (LPS) genes involved in the synthesis of the LPS core were identified. Transcriptomic studies indicate the influence of Pi-starvation and pst mutation on LPS biosynthetic gene expression. LPS analysis indicated that the O-antigen was deficient in the {Delta}pst mutant. Interestingly, waaH, encoding a glycosyltransferase associated with LPS modifications in E. coli K-12, was highly expressed in the {Delta}pst mutant of E. coli O157:H7. Deletion of waaH from the {Delta}pst mutant and from the wild-type strain grown in Pi-starvation conditions decreased the biofilm formation but without affecting LPS. Our findings suggest that LPS core is involved in the autoagglutination and biofilm phenotypes of the {Delta}pst mutant and that WaaH plays a role in biofilm in response to Pi-starvation. This study highlights the importance of Pi-starvation in biofilm formation of E. coli O157:H7, which may affect its transmission and persistence. IMPORTANCEEnterohemorrhagic Escherichia coli O157:H7 is a human pathogen responsible for bloody diarrhea and renal failures. In the environment, O157:H7 can survive for prolonged periods of time under nutrient-deprived conditions. Biofilms are thought to participate in this environmental lifestyle. Previous reports have shown that the availability of extracellular inorganic phosphate (Pi) affected bacterial biofilm formation; however, nothing was known about O157:H7 biofilm formation. Our results show that O157:H7 membrane undergoes modifications upon PhoB activation leading to increased biofilm formation. A mutation in the Pst system results in reduced amount of the smooth type LPS and that this could influence the biofilm composition. This demonstrates how the E. coli O157:H7 adapts to Pi starvation increasing its ability to occupy different ecological niches.

microbiology↗