The Role of Amino Acid Substitution in HepT Toward Menaquinone Isoprenoid Chain Length Definition and Lysocin E Sensitivity in Staphylococcus aureus.

Suresh Panthee,Atmika Paudel,Anne-Catrin Uhlemann,Kazuhisa Sekimizu,Hiroshi Hamamoto

doi:10.3389/fmicb.2020.02076

Suresh Panthee, Atmika Paudel + Show 3 more

Open Access

https://doi.org/10.3389/fmicb.2020.02076

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Abstract

ObjectivesStaphylococcus aureus Smith strain is a historical strain widely used for research purposes in animal infection models for testing the therapeutic activity of antimicrobial agents. We found that it displayed higher sensitivity toward lysocin E, a menaquinone (MK) targeting antibiotic, compared to other S. aureus strains. Therefore, we further explored the mechanism of this hypersensitivity.MethodsMK production was analyzed by high-performance liquid chromatography and mass spectrometric analysis. S. aureus Smith genome sequence was completed using a hybrid assembly approach, and the MK biosynthetic genes were compared with other S. aureus strains. The hepT gene was cloned and introduced into S. aureus RN4220 strain using phage mediated recombination, and lysocin E sensitivity was analyzed by the measurement of colony-forming units.ResultsWe found that Smith strain produced MKs with the length of the side chain ranging between 8 and 10, as opposed to other S. aureus strains that produce MKs 7–9. We revealed that Smith strain possessed the classical pathway for MK biosynthesis like the other S. aureus. HepT, a polyprenyl diphosphate synthase involved in chain elongation of isoprenoid, in Smith strain harbored a Q25P substitution. Introduction of hepT from Smith to RN4220 led to the production of MK-10 and an increased sensitivity toward lysocin E.ConclusionWe found that HepT was responsible for the definition of isoprenoid chain length of MKs and antibiotic sensitivity.

Highlights

Menaquinone (MK), found in the cytoplasmic membrane, is an essential component of the electron transport chain in Gram-positive bacteria
We found that S. aureus Smith strain was consistently more sensitive toward lysocin E with a 2-fold lower MIC value compared with other S. aureus strains
As lysocin E targets MK (Hamamoto et al, 2015), and S. aureus has MK as the sole quinone known to be utilized for respiration (Bentley and Meganathan, 1982), we speculated that the MKs in Smith strain could be different from other S. aureus strains

Summary

Introduction

Menaquinone (MK), found in the cytoplasmic membrane, is an essential component of the electron transport chain in Gram-positive bacteria. Apart from respiration, it plays vital roles in oxidative phosphorylation and the formation of transmembrane potential. It has been shown that MK analogs inhibit the bacterial growth (Schlievert et al, 2013) and several enzymes involved in MK biosynthesis such-as isoprenoid precursor (Jomaa et al, 1999); naphthoquinone (Fang et al, 2010); and incorporation of the isoprenoid side chain to naphthoquinone moiety (Dhiman et al, 2009) can independently be targeted for antimicrobial agent discovery against Grampositive and acid-fast microbes. RH2180-5, directly targets MK in the bacterial membrane exerting rapid and potent bactericidal activity (Hamamoto et al, 2015)

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