High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Chiaraviglio, Lucius; Kang, Yoon‐Suk

doi:10.3791/54841

Cited by 4 publications

(4 citation statements)

References 25 publications

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“…Despite compression of scale, relative light unit (RLU) or relative fluorescence unit (RFU) measurements and CFU for the B. neotomae wild type were highly correlated (R 2 values of 0.98 and 0.96, respectively). Therefore, the reporterlabeled organisms provide a facile and predictive tool for real-time assessment of intracellular bacterial growth (19,20).…”

Section: Resultsmentioning

confidence: 99%

Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila

Kang

2017

Infect Immun

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We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus, B. melitensis, and B. suis, B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila, we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila. Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection.KEYWORDS Brucella, Legionella pneumophila, coinfection, fluorescent image analysis, intracellular pathogens, pathogenesis, phagosomes, reporter genes, type IV secretion system B rucella species are Gram-negative Alphaproteobacteria that cause chronic, systemic infections in mammals and zoonotic infections in humans (1). These pathogens are known to infect the reticuloendothelial system and proliferate significantly in macrophage-rich organs such as liver, spleen, and bone marrow. Chronic, often debilitating bloodstream infection is typical. In humans, a chronic course punctuated by spikes in body temperature is underscored by the descriptive name, undulant fever, given to disease caused by these organisms. Endovascular infection and osteomyelitis are concerning sequelae. Humans may acquire Brucella from airborne exposure related to large quantities of organisms shed from birthing livestock or from ingesting unpasteurized dairy products.B. abortus, B. melitensis, and B. suis, the species responsible for human zoonotic infection, are facultative intracellular pathogens (1). Intracellular growth is thought critical to successful infection of the host. More particularly, each of these pathogens deploys a type IV secretion system (T4SS), a molecular syringe, to inject virulence

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Section: Resultsmentioning

confidence: 99%

Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila

Kang

2017

Infect Immun

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“…Eukaryotic cell cytotoxicity was determined against J774A.1 macrophage and LLC-PK1 proximal tubule kidney cells line during a 5-day incubation. Cytotoxicity was measured using a previously established SYTOX Green exclusion, real-time fluorescence assay [ 20 , 21 ]. Notably CC 50 for these cell lines was >10× higher for S-F than S-D ( Fig 4 , S4 Data ).…”

Section: Resultsmentioning

confidence: 99%

Streptothricin F is a bactericidal antibiotic effective against highly drug-resistant gram-negative bacteria that interacts with the 30S subunit of the 70S ribosome

et al. 2023

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The streptothricin natural product mixture (also known as nourseothricin) was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F (S-F, 1 lysine) and streptothricin D (S-D, 3 lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii. For CRE, the MIC50 and MIC90 for S-F and S-D were 2 and 4 μM, and 0.25 and 0.5 μM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain with minimal or no toxicity. Cryo-EM characterization of S-F bound to the A. baumannii 70S ribosome defines extensive hydrogen bonding of the S-F steptolidine moiety, as a guanine mimetic, to the 16S rRNA C1054 nucleobase (Escherichia coli numbering) in helix 34, and the carbamoylated gulosamine moiety of S-F with A1196, explaining the high-level resistance conferred by corresponding mutations at the residues identified in single rrn operon E. coli. Structural analysis suggests that S-F probes the A-decoding site, which potentially may account for its miscoding activity. Based on unique and promising activity, we suggest that the streptothricin scaffold deserves further preclinical exploration as a potential therapeutic for drug-resistant, gram-negative pathogens.

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“…Eukaryotic cell cytotoxicity was determined against J774A.1 macrophage and LLC-PK1 proximal tubule kidney cells line during a five-day incubation. Cytotoxicity was measured using a previously established SYTOX Green exclusion, real-time fluorescence assay (24,25). Notably CC50 for these cell lines was > 10X higher for S-F than S-D (Fig.…”

Section: Resultsmentioning

confidence: 99%

Profiling thein vitroandin vivoactivity of streptothricin-F against carbapenem-resistant Enterobacterales: a historic scaffold with a novel mechanism of action

Kang

Ab³

et al. 2021

Preprint

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Streptothricins are components of the natural product, nourseothricin; each containing identical streptolidine and gulosamine aminosugar moieties attached to varying numbers of linked b-lysines. Nourseothricin was discovered by Waksman and colleagues in the early 1940's, generating intense interest because of excellent Gram-negative activity. However, the natural product mixture was associated with toxicity, and subsequent exploration was limited. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin-F (S-F, one lysine) and streptothricin D (S-D, three lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE). The MIC50 and MIC90 for S-F and S-D were 2 and 4 µM, and 0.25 and 0.5 µM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. There was >10-fold in vitro selectivity of S-F compared with S-D on LLC-PK1 and J774 cell lines. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain at dosing levels without observable or minimal toxicity. Resistance mutations obtained in single ribosomal operon E. coli identify novel interactions with 16S rRNA helix 34, i.e., C1504A and A1196G/C conferred high level resistance to nourseothricin. Based on promising, unique activity, we suggest that the streptothricin scaffold deserves further pre-clinical exploration as a potential therapeutic for the treatment of CRE and potentially other multidrug-resistant, Gram-negative pathogens

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High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Cited by 4 publications

References 25 publications

Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila

Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila

Streptothricin F is a bactericidal antibiotic effective against highly drug-resistant gram-negative bacteria that interacts with the 30S subunit of the 70S ribosome

Profiling thein vitroandin vivoactivity of streptothricin-F against carbapenem-resistant Enterobacterales: a historic scaffold with a novel mechanism of action

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