Spatiotemporal localization of proteins in mycobacteria

Zhu, Junhao; Wolf, Ian D.; Dulberger, Charles L.; Won, Harim I.; Kester, Jemila C.; Judd, Julius; Wirth, Samantha E.; Clark, Ryan R.; Li, Yawei; Luo, Yuan; Gray, Todd A.; Wade, Joseph T.; Derbyshire, Keith M.; Fortune, Sarah M.; Rubín, Eric J.

doi:10.1016/j.celrep.2021.110154

Cited by 23 publications

(22 citation statements)

References 90 publications

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“…To identify biological features that distinguish g2g-L2 strains, we phenotyped 23 g2g and 11 control L2 strains using a high throughput imaging-based phenotyping platform( 29 ) ( Fig. 5A, Fig.…”

Section: Resultsmentioning

confidence: 99%

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A FLOT1 host regulatory allele is associated with a recently expanded Mtb clade in patients with tuberculosis

Luo

Huang

Liu

et al. 2022

Preprint

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The outcome of infectious diseases may depend on the interaction between human and pathogen genomic variations. We explore this relationship in tuberculosis (TB) by conducting a genome-to-genome (g2g) study of paired genomes from humans and the infectious agent Mycobacterium tuberculosis (Mtb) in 1,556 Peruvian TB patients. We identified a significant association between a human variant in the FLOT1 gene and a unique Mtb Lineage 2 (L2) subclade. The host allele affects FLOT1 expression in multiple tissue and cell types including lung, the primary site of TB disease. Phylogenetic analysis shows that the Mtb subclade has expanded rapidly in Peru since its emergence in the 1950s. Unbiased phenotypic profiling demonstrates that strains from the interacting Mtb subclade display different redox metabolism from other L2 strains. This study presents clear evidence that human and bacterial genetic variation interact together to produce different clinical outcomes.

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Section: Resultsmentioning

confidence: 99%

“…Semi-automated imaging was carried out using a customized Nikon JOBS script to locate imaging fields of interest, 24 images were taken for each strain. Cell segmentation and quantification was performed using our previously published pipeline, MOMIA( 29 ).…”

Section: Methodsmentioning

confidence: 99%

A FLOT1 host regulatory allele is associated with a recently expanded Mtb clade in patients with tuberculosis

Luo

Huang

Liu

et al. 2022

Preprint

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“…(B) Cell lengths of uninduced and induced strains. Measurements were obtained by GEMATRIA and MOMIA image analysis pipelines (35). Student’s t test used to calculate the statistical difference in mean cell lengths.…”

Section: Resultsmentioning

confidence: 99%

“…All Mab cultures were fixed with 7H9 + 3% paraformaldehyde (PFA) for 1 hr and resuspended in PBS + 0.05% Tween80 prior to imaging. Cellular features including cell length, width, and fluorescence signal were analyzed using the MOMIA and GEMATRIA image analysis pipelines developed in the lab (35).…”

Section: Methodsmentioning

confidence: 99%

“…The resulting images were analyzed using the recently published MOMIA and GEMATRIA programs, which quantify the fluorescent signal across cells normalized by cell length (35). We first analyzed cells that expressed solely mRFP-PBP-lipo and demonstrated that mid-cell localization of PBP-lipo was only present in the longest cells in the population (Figure 5B).…”

Section: Pbp-lipo Localizes To the Septum After Ftszmentioning

confidence: 99%

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High-density transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding lipo-protein (PBP-lipo) involved in septal peptidoglycan synthesis and antibiotic sensitivity

Akusobi

et al. 2021

Preprint

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Mycobacterium abscessus (Mab) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of infections. Treatment of Mab infections is difficult because the bacterium is intrinsically resistant to many classes of antibiotics. Developing new and effective treatments against Mab requires a better understanding of the unique vulnerabilities that can be targeted for future drug development. To achieve this, we identified essential genes in Mab by conducting transposon-sequencing (TnSeq) on the reference Mab strain ATCC 19977. We generated ~51,000 unique transposon mutants and used this high-density library to identify 362 essential genes for in vitro growth. To investigate species-specific vulnerabilities in Mab, we further characterized MAB_3167c, a predicted penicillin-binding-lipoprotein (PBP-lipo) that is essential in Mab and non-essential in M. tuberculosis (Mtb). We found that PBP-lipo primarily localizes to the subpolar region and later to the septum as cells prepare to divide. Knockdown of Mab PBP-lipo causes cells to elongate, develop ectopic branches, and form multiple septa. Dual knockdown of PBP-lipo with PBPs, PbpB, DacB1, and a carboxypeptidase, MAB_0519 lead to synergistic growth arrest. In contrast, these genetic interactions were absent in the Mtb model organism, M. smegmatis, indicating that the PBP-lipo homologs in the two species exist in distinct genetic networks. Finally, repressing PBP-lipo sensitized the reference strain and 11 Mab clinical isolates to several classes of antibiotics, including the beta-lactams, ampicillin and amoxicillin by greater than 128-fold. Altogether, this study presents PBP-lipo as a key enzyme to study Mab specific processes in cell wall synthesis and importantly positions PBP-lipo as an attractive drug target to treat Mab infections.

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

Laudouze,

Point,

Achache

et al. 2024

FEBS Letters

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In this research letter, we report the development and validation of a new subset of fluorescence‐based CRISPR interference (CRISPRi) tools for our scientific community. The pJL series is directly derived from the original pIRL CRISPRi vectors and conserves all the elements to perform inducible targeted gene repression. These vectors carry two distinct fluorescent markers under the constitutive promoter psmyc to simplify the selection of recombinant clones. We demonstrate the functionality of these vectors by targeting the expression of the 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 episomal vectors. Such tools and approaches should be useful to foster discovery in mycobacterial research.

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Spatiotemporal localization of proteins in mycobacteria

Abstract: Highlights d MOMIA and GEMATRIA efficiently model mycobacterial protein localization d Polar exclusion of mycobacterial ribosomes relies on active translation d GEMATRIA reveals spatial partitioning of mycobacterial membrane proteins

Cited by 23 publications

References 90 publications

A FLOT1 host regulatory allele is associated with a recently expanded Mtb clade in patients with tuberculosis

A FLOT1 host regulatory allele is associated with a recently expanded Mtb clade in patients with tuberculosis

High-density transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding lipo-protein (PBP-lipo) involved in septal peptidoglycan synthesis and antibiotic sensitivity

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

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