Identification of Crucial Amino Acid Residues for Antimicrobial Activity of Angiogenin 4 and Its Modulation of Gut Microbiota in Mice

• Published in: Frontiers in microbiology Vol. 13; p. 900948
• Main Authors: Sultana, Mst. Farzana, Suzuki, Maki, Yamasaki, Fumiya, Kubota, Wataru, Takahashi, Kohta, Abo, Hirohito, Kawashima, Hiroto
• Format: Journal Article
• Language: English
• Published: Frontiers Media S.A 06-06-2022
• Subjects: angiogenin 4; antimicrobial peptide; Microbiology; microbiome; ribonuclease
• ISSN: 1664-302X; 1664-302X
• DOI: 10.3389/fmicb.2022.900948

Angiogenin 4 bearing ribonuclease activity is an endogenous antimicrobial protein expressed in small and large intestine. However, the crucial amino acid residues responsible for the antibacterial activity of Ang4 and its impact on gut microbiota remain unknown. Here, we report the contribution of critical amino acid residues in the functional regions of Ang4 to its activity against
Salmonella typhimurium
LT2 and the effect of Ang4 on gut microbiota in mice. We found that Ang4 binds
S. typhimurium
LT2 through two consecutive basic amino acid residues, K58 and K59, in the cell-binding segment and disrupts the bacterial membrane integrity at the N-terminal α-helix containing residues K7 and K30, as evidenced by the specific mutations of cationic residues of Ang4. We also found that the RNase activity of Ang4 was not involved in its bactericidal activity, as shown by the H12 mutant, which lacks RNase activity.
In vivo
administration of Ang4 through the mouse rectum and subsequent bacterial 16S rRNA gene sequencing analyses demonstrated that administration of Ang4 not only increased beneficial bacteria such as
Lactobacillus, Akkermansia
,
Dubosiella
,
Coriobacteriaceae
UCG-002, and
Adlercreutzia
, but also decreased certain pathogenic bacteria, including
Alistipes
and
Enterohabdus
, indicating that Ang4 regulates the shape of gut microbiota composition. We conclude that Ang4 kills bacteria by disrupting bacterial membrane integrity through critical basic amino acid residues with different functionalities rather than overall electrostatic interactions and potentially maintains gut microflora
in vivo
under physiological and pathophysiological conditions.