Metabolic remodeling by RNA polymerase gene mutations is associated with reduced β-lactam susceptibility in oxacillin-susceptible MRSA
Authors: Watanabe Shinya , Nsofor Chijioke A. , Thitiananpakorn Kanate , Tan Xin-Ee , Aiba Yoshifumi , Takenouchi Remi , Kiga Kotaro , Sasahara Teppei , Miyanaga Kazuhiko , Veeranarayanan Srivani , Shimamori Yuzuki , Lian Adeline Yeo Syin , Nguyen Thuy Minh , Nguyen Huong Minh , Alessa Ola , Kumwenda Geoffrey Peterkins , Jayathilake Sarangi , Revilleza Jastin Edrian Cocuangco , Baranwal Priyanka , Nishikawa Yutaro , Li Feng-Yu , Kawaguchi Tomofumi , Sankaranarayanan Sowmiya , Arbaah Mahmoud , Zhang Yuancheng , ‌‌ Maniruzzaman , Liu Yi , Sarah Hossain , Li Junjie , Sugano Takashi , Ho Thi My Duyen , Batbold Anujin , Nayanjin Tergel , Cui Longzhu 
Published: May 2024

ABSTRACT

The emergence of oxacillin-susceptible methicillin-resistant
Staphylococcus aureus
(OS-MRSA) has imposed further challenges to the clinical management of MRSA infections. When exposed to β-lactam antibiotics, these strains can easily acquire reduced β-lactam susceptibility through chromosomal mutations, including those in RNA polymerase (RNAP) genes such as
rpoBC
, which may then lead to treatment failure. Despite the increasing prevalence of such strains and the apparent challenges they pose for diagnosis and treatment, there is limited information available on the actual mechanisms underlying such chromosomal mutation-related transitions to reduced β-lactam susceptibility, as it does not directly associate with the expression of
mecA
. This study investigated the cellular physiology and metabolism of six missense mutants with reduced oxacillin susceptibility, each carrying respective mutations on RpoB
H929P
, RpoB
Q645H
, RpoC
G950R
, RpoC
G498D
, RpiA
A64E
, and FruB
A211E
, using capillary electrophoresis-mass spectrometry-based metabolomics analysis. Our results showed that
rpoBC
mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides. These mutations also led to the accumulation of UDP-Glc/Gal and UDP-GlcNAc, which are precursors of UTP-associated peptidoglycan and wall teichoic acid. Excessive amounts of building blocks then contributed to the cell wall thickening of mutant strains, as observed in transmission electron microscopy, and ultimately resulted in decreased susceptibility to β-lactam in OS-MRSA.

IMPORTANCE

The emergence of oxacillin-susceptible methicillin-resistant
Staphylococcus aureus
(OS-MRSA) strains has created new challenges for treating MRSA infections. These strains can become resistant to β-lactam antibiotics through chromosomal mutations, including those in the RNA polymerase (RNAP) genes such as
rpoBC
, leading to treatment failure. This study investigated the mechanisms underlying reduced β-lactam susceptibility in four
rpoBC
mutants of OS-MRSA. The results showed that
rpoBC
mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides and precursors of peptidoglycan as well as wall teichoic acid. This, in turn, caused thickening of the cell wall and ultimately resulted in decreased susceptibility to β-lactam in OS-MRSA. These findings provide insights into the mechanisms of antibiotic resistance in OS-MRSA and highlight the importance of continued research in developing effective treatments to combat antibiotic resistance.