From Differential Stains to Next Generation Physiology: Chemical Probes to Visualize Bacterial Cell Structure and Physiology

Hira, Jonathan; Uddin, Jalal; Haugland, Marius M.; Lentz, Christian S.

doi:10.3390/molecules25214949

Cited by 15 publications

(13 citation statements)

References 177 publications

(271 reference statements)

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“…We hypothesized that because E. coli MG1655 is non-hypersusceptible and has a more complete cell envelope than DC2, Bocillin-FL had a reduced ability to penetrate the outer membrane. The literature supports this theory as poor penetration of fluorophore-conjugated molecules has previously been reported in Gram-negative bacteria. − The inability to utilize a fluorescent readout probe for PBP activity in non-hypersusceptible strains poses a significant barrier to the assessment of inhibitor-PBP selectivity profiles.…”

Section: Resultsmentioning

confidence: 85%

Live-Cell Profiling of Penicillin-Binding Protein Inhibitors in Escherichia coli MG1655

2022

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Penicillin-binding proteins (PBPs) make up an essential class of bacterial enzymes that carry out the final steps of peptidoglycan synthesis and regulate the recycling of this polymeric structure. PBPs are an excellent drug target and have been the most clinically relevant antibacterial target since the 1940s with the introduction of β-lactams. Despite this, a large gap in knowledge remains regarding the individual function and regulation of each PBP homologue in most bacteria. This can be attributed to a lack of chemical tools and methods that enable the study of individual PBPs in an activity-dependent manner and in their native environment. The development of such methods in Gram-negative bacteria has been particularly challenging due to the presence of an outer membrane and numerous resistance mechanisms. To address this, we have developed an optimized live-cell assay for screening inhibitors of the PBPs in Escherichia coli MG1655. We utilized EDTA to permeabilize Gram-negative cells, enabling increased penetration of our readout probe, Bocillin-FL, and subsequent analysis of PBP-inhibition profiles. To identify scaffolds for future development of PBP-selective activity-based probes, we screened ten β-lactams, one diazabicyclooctane, and one monobactam for their PBP-selectivity profiles in E. coli MG1655. These results demonstrate the utility of our assay for the screening of inhibitors in live, non-hypersusceptible Gram-negative organisms.

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Section: Resultsmentioning

confidence: 85%

Live-Cell Profiling of Penicillin-Binding Protein Inhibitors in Escherichia coli MG1655

2022

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“…4a, Fig. 5c) and length of 0.2-2 μm, which is shorter than common gram-negative bacteria with a length of 2-5 μm 39,40 . For live SIM imaging, TM7i cells are labeled with Alexa Fluor 555 probes, a nonspecific fluorescence dye that labels all the protein upon and inside the cell.…”

Section: The Tm7i Cell Uses T4p For Twitching Motilitymentioning

confidence: 83%

EpicPCR-Directed Cultivation of a Candidatus Saccharibacteria Symbiont Reveals a Type IV Pili-dependent Epibiotic Lifestyle

Xie

Wang

Nie

et al. 2021

Preprint

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Candidate phyla radiations (CPR), accounting for a major microbial supergroup with remarkably small genomes and reduced sizes, are widely distributed yet mostly uncultured. Limited culture and its obligate reliance upon other bacteria hindered investigation of their lifestyles. In this work we isolated a CPR bacterium, TM7i, with its host Leucobacter aridocollis J1, by combination of Emulsion, Paired Isolation and Concatenation PCR (epicPCR) detection and filtrate co-culture. Genomic profiling of TM7 genomes and microscopic investigation of TM7i-J1 symbiosis suggest the conservation of type IV pili and a pili-dependent lifestyle of TM7. Further, we observed twitching motility of TM7i mediated by pili and its role played in the interaction with its host. Our results shed a light on the lifestyle about this enigmatic bacterial radiation, which may also be adopted by other CPR organisms. The epicPCR-directed isolation method underlines high efficiency of CPR bacteria isolation and thus may be used in other symbiotic or epibiotic microorganisms.

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“…Multiple options are available for chemical probes, including fluorescence, chemiluminescence, light scattering, and radioactivity. Fluorescence is the most popular approach, since it is easily captured by fluorescence scanners/plate readers, time-lapse fluorescence microscopes, ultra-high-resolution microscopes, and flow cytometers ( Hira et al, 2020 ).…”

Section: New Promising Diagnostic Techniquesmentioning

confidence: 99%

Improved Conventional and New Approaches in the Diagnosis of Tuberculosis

Dong

et al. 2022

Front. Microbiol.

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Tuberculosis (TB) is a life-threatening infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). Timely diagnosis and effective treatment are essential in the control of TB. Conventional smear microscopy still has low sensitivity and is unable to reveal the drug resistance of this bacterium. The traditional culture-based diagnosis is time-consuming, since usually the results are available after 3–4 weeks. Molecular biology methods fail to differentiate live from dead M. tuberculosis, while diagnostic immunology methods fail to distinguish active from latent TB. In view of these limitations of the existing detection techniques, in addition to the continuous emergence of multidrug-resistant and extensively drug-resistant TB, in recent years there has been an increase in the demand for simple, rapid, accurate and economical point-of-care approaches. This review describes the development, evaluation, and implementation of conventional diagnostic methods for TB and the rapid new approaches for the detection of M. tuberculosis.

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From Differential Stains to Next Generation Physiology: Chemical Probes to Visualize Bacterial Cell Structure and Physiology

Cited by 15 publications

References 177 publications

Live-Cell Profiling of Penicillin-Binding Protein Inhibitors in Escherichia coli MG1655

Live-Cell Profiling of Penicillin-Binding Protein Inhibitors in Escherichia coli MG1655

EpicPCR-Directed Cultivation of a Candidatus Saccharibacteria Symbiont Reveals a Type IV Pili-dependent Epibiotic Lifestyle

Improved Conventional and New Approaches in the Diagnosis of Tuberculosis

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