ActA-mediated PykF acetylation negatively regulates oxidative stress adaptability of
Streptococcus mutans
Authors: Ma Qizhao , Li Jing , Yu Shuxing , Liu Yaqi , Zhou Jing , Wang Xinyue , Wang Lingyun , Zou Jing , Li Yuqing 
Published: September 2024

Dental caries is associated with microbial dysbiosis caused by the excessive proliferation of
Streptococcus mutans
in dental biofilms, where oxidative stress serves as the major stressor to microbial communities. The adaptability of
S. mutans
to oxidative stress is a prerequisite for its proliferation and even for exerting its virulence. Protein acetylation is a reversible and conserved regulatory mechanism enabling bacteria to rapidly respond to external environmental stressors. However, the functions of protein acetylation in regulating oxidative stress adaptability of
S. mutans
are still unknown. Here, we unveil the impact of acetyltransferase ActA-mediated acetylation on regulating the oxidative stress response of
S. mutans. actA
overexpression increased the sensitivity of
S. mutans
to hydrogen peroxide and diminished its competitive ability against
Streptococcus sanguinis
. In contrast,
actA
deletion enhanced oxidative stress tolerance and competitiveness of
S. mutans
. The mass spectrometric analysis identified pyruvate kinase (PykF) as a substrate of ActA, with its acetylation impairing its enzymatic activity and reducing pyruvate production. Supplementation with exogenous pyruvate mitigated oxidative stress sensitivity and restored competitiveness in multi-species biofilms.
In vitro
acetylation analysis further confirmed that ActA directly acetylates PykF, negatively affecting its enzymatic activity. Moreover, 18 potential lysine-acetylated sites on PykF were identified
in vitro
, which account for 75% of lysine-acetylated sites detected
in vivo
. Taken together, our study elucidates a novel regulatory mechanism of ActA-mediated acetylation of PykF in modulating oxidative stress adaptability of
S. mutans
by influencing pyruvate production, providing insights into the importance of protein acetylation in microbial environmental adaptability and interspecies interactions within dental biofilms.

IMPORTANCE

Dental caries poses a significant challenge to global oral health, driven by microbial dysbiosis within dental biofilms. The pathogenicity of
Streptococcus mutans
, a major cariogenic bacterium, is closely linked to its ability to adapt to changing environments and cellular stresses. Our investigation into the protein acetylation mechanisms, particularly through the acetyltransferase ActA, reveals a critical pathway by which
S. mutans
modulates its adaptability to oxidative stress, the dominant stressor within dental biofilms. By elucidating how ActA affects the oxidative stress adaptability and competitiveness of
S. mutans
through the regulatory axis of ActA-PykF-pyruvate, our findings provide insights into the dynamic interplay between cariogenic and commensal bacteria within dental biofilms. This work emphasizes the significance of protein acetylation in bacterial stress response and competitiveness, opening avenues for the development of novel strategies to maintain oral microbial balance within dental biofilms.