Mycobacteriophage SWU1 gp39 can potentiate multiple antibiotics against Mycobacterium via altering the cell wall permeability

Abstract

M. tuberculosis is intrinsically tolerant to many antibiotics largely due to the imperviousness of its unusual mycolic acid-containing cell wall to most antimicrobials. The emergence and increasingly widespread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) revitalized keen interest in phage-inspired therapy. SWU1gp39 is a novel gene from mycobacteriophage SWU1 with unknown function. SWU1gp39 expressed in M. smegmatis conferred the host cell increased susceptibility to multiple antibiotics, including isoniazid, erythromycin, norfloxacin, ampicillin, ciprofloxacin, ofloxacin, rifampicin and vancomycin, and multiple environment stresses such as H2O2, heat shock, low pH and SDS. By using EtBr/Nile red uptake assays, WT-pAL-gp39 strain showed higher cell wall permeability than control strain WT-pAL. Moreover, the WT-pAL-gp39 strain produced more reactive oxygen species and reduced NAD+/NADH ratio. RNA-Seq transcriptomes of the WT-pAL-gp39 and WT-pAL revealed that the transcription of 867 genes was differentially regulated, including genes associated with lipid metabolism. Taken together, our results implicated that SWU1gp39, a novel gene from mycobacteriophage, disrupted the lipid metabolism of host and increased cell wall permeability, ultimately potentiated the efficacy of multiple antibiotics and stresses against mycobacteria.

Insights into the Physiology and Metabolism of a Mycobacterial Cell in an Energy-Compromised State

“…Log-phase cultures (OD 600 , ϳ0.6) of all the strains were harvested and washed with 1ϫ phosphate-buffered saline-Tween 80. Two hundred microliters of the cell suspension was added to a 96-well plate, to which Nile red or ethidium bromide was added to final concentrations of 2 M and 2 g/ml, respectively (34,61). Intracellular accumulation of these dyes was recorded by measuring the fluorescence on a SpectraMax M5 plate reader, with excitation and emission wavelengths of 540 and 630 nm, respectively, being used for Nile Red and 545 and 600 nm, respectively, being used for ethidium bromide.…”

“…SEM and TEM. For scanning electron microscopy (SEM), the procedure was adapted as described previously (34,53), with some modifications, as detailed in the supplemental material. Briefly, the bacterial cell pellet obtained from a log-phase culture was fixed in the required buffer, attached to stubs, sputter coated with gold, and imaged on an UltraPlus scanning electron microscope (Zeiss, Germany) at 10.0 kV.…”

“…Additionally, we performed a Congo red (CR) dye-binding assay. CR dye is known to bind to lipoproteins present on the mycobacterial surface and is frequently used to identify cell wall modifications (34). Our ΔatpD mutant exhibited an altered colony morphology on a CR agar plate.…”

“…Here, we hypothesized that the enhanced sensitivity to drugs in the case of the atpD mutant either was due to an increased uptake because of altered cellular permeability or was because the generally poor fitness of the mutant bacterium makes it more susceptible to antibiotics. In order to examine the former, we determined the intracellular accumulation of Nile red dye and ethidium bromide, which are known to be indicative of the uptake of hydrophobic and hydrophilic compounds, respectively (34). We observed an enhanced uptake of both the Nile red and ethidium bromide dyes by the atpD mutant compared to the wild-type and the complemented strains ( Fig.…”

Looking beyond Typical Treatments for Atypical Mycobacteria

“…As an example, Neith et al [180] successfully delivered the mycobacteriophage TM4 inside giant liposomes into monocytic THP-1 cells. In many studies, the nonvirulent species of mycobacteria M. smegmatis was used as a mycobacteriophage delivery system [181][182][183], since it naturally infects phagocytic cells that host virulent mycobacteria, while allowing, at the same time, the proliferation of the phages inside them [178]. The mycobacteriophage TM4 was effective against M. avium and Mtb in vitro [182] and in vivo [183], significantly reducing the number of bacilli.…”