Reactive Chlorine Species Reversibly Inhibit DnaB Protein Splicing in Mycobacteria

Lennon, Christopher; Wahl, Daniel R.; Goetz, Jean; Weinberger, Joel

doi:10.1128/spectrum.00301-21

Cited by 10 publications

(8 citation statements)

References 41 publications

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“…The first intein is also susceptible to inhibition by zinc and reactive chlorine species. 57,58 These two inteins also can facilitate conditional alternative protein splicing (CAPS) as a function of time when expressed as part of the same precursor. The M. smegmatis inteins differ considerably, as the first intein is a noncanonical class three intein; 37 class three inteins splice by an alternative mechanism utilizing an internal nucleophile in place of the N-terminal Cys or Ser.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Conditional Alternative Protein Splicing Promoted by Inteins from Haloquadratum walsbyi

2022

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Protein splicing is a post-translational process by which an intervening protein, or an intein, catalyzes its own excision from flanking polypeptides, or exteins, coupled to extein ligation. Four inteins interrupt the MCM helicase of the halophile Haloquadratum walsbyi, two of which are mini-inteins that lack a homing endonuclease. Both inteins can be overexpressed in Escherichia coli and purified as unspliced precursors; splicing can be induced in vitro by incubation with salt. However, one intein can splice in 0.5 M NaCl in vitro, whereas the other splices efficiently only in buffer containing over 2 M NaCl; the organism also requires high salt to grow, with the standard growth media containing over 3 M NaCl and about 0.75 M magnesium salts. Consistent with this difference in salt-dependent activity, an intein-containing precursor protein with both inteins promotes conditional alternative protein splicing (CAPS) to yield different spliced products dependent on the salt concentration. Native Trp fluorescence of the inteins suggests that the difference in activity may be due to partial unfolding of the inteins at lower salt concentrations. This differential salt sensitivity of intein activity may provide a useful mechanism for halophiles to respond to environmental changes.

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Section: ■ Results and Discussionmentioning

confidence: 99%

“…smegmatis. The first intein is also susceptible to inhibition by zinc and reactive chlorine species. , These two inteins also can facilitate conditional alternative protein splicing (CAPS) as a function of time when expressed as part of the same precursor. The M.…”

Section: Resultsmentioning

confidence: 99%

Conditional Alternative Protein Splicing Promoted by Inteins from Haloquadratum walsbyi

2022

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“…Here, we will discuss inteins as potential targets to selectively kill bacterial and fungal pathogens, along with the screening assays that could be used to test the ability of small molecules to inhibit protein splicing. There is proof of concept evidence that divalent cations (in particular zinc and copper), reactive oxygen, nitrogen, and chlorine derivatives, as well as small molecules including cisplatin and diethanolamine can act as splicing inhibitors and impact the fitness of pathogenic mycobacteria and fungi (Gangopadhyay et al, 2003;Zhang et al, 2010;Zhang et al, 2011;Dasari et al, 2015;Topilina et al, 2015a;Kelley et al, 2018;Li et al, 2019;Lennon et al, 2021;Panda et al, 2021) The discovery of drug-like molecules that can successfully inhibit the splicing of proteins essential for survival of pathogens could represent new treatment options. Because inteins are absent in humans, specific intein inhibitors could have limited side effects.…”

Section: Introductionmentioning

confidence: 99%

Intein Inhibitors as Novel Antimicrobials: Protein Splicing in Human Pathogens, Screening Methods, and Off-Target Considerations

Wall

Tarrant

Wang

et al. 2021

Front. Mol. Biosci.

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Protein splicing is a post-translational process by which an intervening polypeptide, or intein, catalyzes its own removal from the flanking polypeptides, or exteins, concomitant with extein ligation. Although inteins are highly abundant in the microbial world, including within several human pathogens, they are absent in the genomes of metazoans. As protein splicing is required to permit function of essential proteins within pathogens, inteins represent attractive antimicrobial targets. Here we review key proteins interrupted by inteins in pathogenic mycobacteria and fungi, exciting discoveries that provide proof of concept that intein activity can be inhibited and that this inhibition has an effect on the host organism’s fitness, and bioanalytical methods that have been used to screen for intein activity. We also consider potential off-target inhibition of hedgehog signaling, given the similarity in structure and function of inteins and hedgehog autoprocessing domains.

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“…62,63 Two reactive chlorine species, hypochlorous acid and N-chlorotaurine, can reversibly block splicing of the DnaB inteins from Mycobacterium leprae and Msm in vitro. 64 Platinum-containing compounds such as cisplatin inhibit splicing of the RecA intein of Mtb with an IC 50 of 2 μM in vitro. 65 The MIC of cisplatin in Mtb was 12 μg/mL, 65 whereas those in Cne and Cga were 4.5 and 8.1 μg/mL, respectively.…”

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confidence: 99%

“…Zn 2+ also inhibits splicing of the DnaB1 intein of Mycobacterium smegmatis ( Msm ) at 10 μM in an in vitro splicing assay, and EDTA could reverse it . Other metal ions, such as Cd 2+ , Ni 2+ , and Co 2+ , have shown weaker inhibition than Zn 2+ on splicing of the RecA intein from Mycobacterium tuberculosis ( Mtb ) and naturally split DnaE intein from Synechocystis sp. , Two reactive chlorine species, hypochlorous acid and N -chlorotaurine, can reversibly block splicing of the DnaB inteins from Mycobacterium leprae and Msm in vitro …”

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confidence: 99%

Calcimycin Inhibits Cryptococcus neoformans In Vitro and In Vivo by Targeting the Prp8 Intein Splicing

Tharappel

Zhu

et al. 2022

ACS Infect. Dis.

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Drug resistance is a significant concern in the treatment of diseases, including cryptococcosis caused by Cryptococcus neoformans (Cne) and Cryptococcus gattii (Cga). Alternative drug targets are necessary to overcome drug resistance before it attains a critical stage. Splicing of inteins from pro-protein precursors is crucial for activities of essential proteins hosting intein elements in many organisms, including human pathogens such as Cne and Cga. Through a high-throughput screening, we identified calcimycin (CMN) as a potent Prp8 intein splicing inhibitor with a minimum inhibitory concentration (MIC) of 1.5 μg/mL against the wild-type Cne-H99 (Cne-WT or Cne). In contrast, CMN inhibited the intein-less mutant strain (Cne-Mut) with a 16-fold higher MIC. Interestingly, Aspergillus fumigatus and a few Candida species were resistant to CMN. Further studies indicated that CMN reduced virulence factors such as urease activity, melanin production, and biofilm formation in Cne. CMN also inhibited Cne intracellular infection in macrophages. In a target-specific split nanoluciferase assay, the IC50 of CMN was 4.6 μg/mL. Binding of CMN to recombinant Prp8 intein was demonstrated by thermal shift assay and microscale thermophoresis. Treating Cne cells with CMN reduced intein splicing. CMN was fungistatic and showed a synergistic effect with the known antifungal drug amphotericin B. Finally, CMN treatment at 20 mg/kg body weight led to 60% reduction in lung fungal load in a cryptococcal pulmonary infection mouse model. Overall, CMN represents a potent antifungal with a novel mechanism of action to treat Cne and possibly Cga infections.

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Reactive Chlorine Species Reversibly Inhibit DnaB Protein Splicing in Mycobacteria

Abstract: Inteins are both widespread and abundant in microbes, including within several bacterial and fungal pathogens. Inteins are domains translated within host proteins and removed at the protein level by splicing.

Cited by 10 publications

References 41 publications

Conditional Alternative Protein Splicing Promoted by Inteins from Haloquadratum walsbyi

Conditional Alternative Protein Splicing Promoted by Inteins from Haloquadratum walsbyi

Intein Inhibitors as Novel Antimicrobials: Protein Splicing in Human Pathogens, Screening Methods, and Off-Target Considerations

Calcimycin Inhibits Cryptococcus neoformans In Vitro and In Vivo by Targeting the Prp8 Intein Splicing

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