Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2024.10.05.616838

Fluorescence-based CRISPR interference system for controlled genetic repression and live single-cell imaging in mycobacteria

Laudouze, J.·Point, V.·Achache, W.·Crauste, C.·Canaan, S.·SANTUCCI, P.
View original source ↗Export metadata ↓

Abstract

Mycobacterial genetics has played a pivotal role over the last 35 years in our understanding of mycobacterial physiology, pathogenesis and antibiotic resistance. Numerous approaches are now available worldwide to dissect the contribution of genes of interest in biological processes. However, many of these approaches can be fastidious, difficult to perform and time-consuming, especially when working with slow-growing mycobacteria or in bio-safety level two/three settings. The recent development of CRISPRi-mediated targeted gene repression has revolutionized the way research groups can perform genetics in mycobacteria, providing a fast, robust and efficient alternative to study the function of specific genes including essential genes. In this research letter, we report the development and validation of a new subset of fluorescence-based CRISPRi tools for our scientific community. The pJL series is directly derived from the original integrative pIRL2 and pIRL117 CRISPRi vectors and conserved all the elements required to perform inducible targeted gene repression. In addition, these vectors carry two distinct fluorescent markers for which the expression is driven by the strong and constitutive promotor psmyc to simplify the selection of recombinant clones. We demonstrate the functionality of these vectors by targeting the expression of the non-essential glycopeptidolipid translocase mmpL4b and the essential genes rpoB and mmpL3. Finally, we describe an efficient single-step procedure to co-transform mycobacterial species with this integrative genetic tool alongside replicative vectors. Such tools and approaches should be useful to foster discovery in mycobacterial research. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/616838v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@212460org.highwire.dtl.DTLVardef@1db5529org.highwire.dtl.DTLVardef@b426e2org.highwire.dtl.DTLVardef@164f628_HPS_FORMAT_FIGEXP M_FIG C_FIG Development and validation of a new subset of E. coli-Mycobacteria shuttle vectors that enable simultaneous CRISPRI-mediated gene silencing and fluorescence based single-cell imaging.

Source connections

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Laudouze, J., Point, V., Achache, W., Crauste, C., Canaan, S., SANTUCCI, P.. 2024-10-06. Fluorescence-based CRISPR interference system for controlled genetic repression and live single-cell imaging in mycobacteria. https://doi.org/10.1101/2024.10.05.616838

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A conserved cysteine-histidine-glutamate metal site identifies DUF501 (Rv1025), an essential uncharacterised protein family of Mycobacterium tuberculosis, as a candidate metalloenzyme and drug target

A substantial fraction of the Mycobacterium tuberculosis proteome remains functionally uncharacterised. Rv1025, a 155-residue protein carrying the domain of unknown function DUF501 (Pfam PF04417), is essential by transposon mutagenesis and vulnerable by CRISPR interference, an attractive but neglected drug target, yet has never been functionally described. The family (4,370 proteins, no Gene Ontology term, no solved structure) is uncharacterised across all organisms and essential in three Actinobacterial genera. A Foldseek search of the AlphaFold model against complete structural databases finds no significant homolog, indicating a novel fold. The operon eno-divIC-Rv1025-ppx2 is conserved across the Actinobacteria phylum, yet AlphaFold-Multimer finds no direct complex between Rv1025 and its neighbour DivIC. Instead, conservation across 8,700 homologous sequences reveals a near-invariant Cys113-His115-Glu59 cluster forming a pocket. Holo AlphaFold3 predictions with Zn, Fe and Mn confidently place a divalent metal on this triad at 2.25-2.47 A; mutating the triad relocates the metal, and an independent backbone-geometry predictor recovers the same site, confirming specificity. The triad is universal across the family: present in all 1,472 near-complete bacterial sequences of the Pfam alignment, with no non-conservative substitution among the 2,228 sequences examined, a defining feature of bacterial DUF501 rather than a mycobacterial peculiarity. We propose that DUF501 is a metal-binding protein and candidate metalloenzyme, the first functional hypothesis for this family, whose conserved, essential metal pocket is a promising drug target. As the predictions build on a conservation-defined site within a fully computational study, they are supportive rather than proof of metal occupancy and warrant experimental validation.

microbiology↗
bioRxiv2026

Mycoplasmal endosymbionts of Trichomonas vaginalis are associated with reduced risk for Chlamydia trachomatis endometrial infection in asymptomatic, coinfected, women.

Trichomonas vaginalis is a protozoan parasite that causes trichomoniasis, the most common curable non-viral sexually transmitted infection, and Chlamydia trachomatis is a bacterial pathogen that can ascend to the upper genital tract and cause pelvic inflammatory disease, infertility, and ectopic pregnancy. T. vaginalis harbors bacterial endosymbionts, including Candidatus Malacoplasma girerdii, an obligate symbiont, and Metamycoplasma hominis, which can live freely or symbiotically. In a 16S rRNA sequencing study of the cervicovaginal microbiome of women at high risk for chlamydial infection, Ca. M. girerdii abundance was one of 13 features predicting lack of chlamydial spread to the endometrium, despite no direct association between T. vaginalis infection and reduced chlamydial ascension. Investigating the relationship between these microorganisms further, we found that T. vaginalis vaginal abundance correlated positively with chlamydial burden in women whose infection was confined to the cervix, while a nonsignificant inverse relationship was seen in women with endometrial spread. Among participants with high chlamydial burden, Ca. M. girerdii was detected exclusively in women without endometrial infection. Both endosymbionts trended toward more frequent detection, and higher abundance, in coinfected women without endometrial spread, while M. hominis abundance correlated strongly with T. vaginalis burden in this group. These findings suggest that mycoplasmal endosymbionts of T. vaginalis, rather than T. vaginalis itself, are microbial factors limiting chlamydial ascension, and point to a three-way interaction between parasite, endosymbiont, and bacterial pathogen that shapes upper genital tract C. trachomatis infection risk.

microbiology↗
bioRxiv2026

Understanding the physiological alterations of Vibrio cholerae upon exposure to L-ascorbic acid

The scourge of cholera remains a major global public health threat. It affects up to 4 million people worldwide and causes tens of thousands of deaths each year. The disease is experiencing a concerning resurgence in many parts of Africa, the Middle East, and Asia. To effectively tackle cholera and circumvent rising antimicrobial resistance, targeted biological and preventive approaches, complementing traditional rehydration, are urgently needed. In this regard, our group has demonstrated the efficacy of L-ascorbic acid in controlling the growth and pathogenesis of Vibrio cholerae in vitro. The present work further provides a mechanistic elucidation of the L-ascorbic acid-mediated physiological changes in V. cholerae and also bolsters such a non-antibiotic approach to control cholera.

microbiology↗