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

Abstract

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.

Potential benefits of metformin and pioglitazone combination therapy via gut microbiota and metabolites in high-fat diet-fed mice

“…Therefore, the alterations of these beneficial and harmful microbes indicate significant synergistic effects of combination therapy on gut microbiota modification. However, not all of the potential beneficial microbes that were decreased in HFD can be reversed by combined treatment, such as Lactobacillus, Clostridium_sensu_stricto_1 and Coriobacteriaceae _UCG-002 ( Kong et al, 2019 ; Sultana et al, 2022 ). Notably, Akkermansia, as a beneficial microbiota, showed different trends in the metformin and pioglitazone monotherapy groups and was slightly increased in the combined treatment group, which suggests another form of complementary action between metformin and pioglitazone.…”

Human and mouse angiogenins: Emerging insights and potential opportunities

“…mAng4 demonstrates antibacterial effects against Bacteroides thetaiotaomicron, Enterococcus faecalis, Bifidobacterium longum, Enterococcus gallinarum, Listeria monocytogenes, and Salmonella typhimurium LT2 (Hooper et al, 2003;Walker et al, 2013). Recent 10.3389/fmicb.2022 Frontiers in Microbiology 07 frontiersin.org studies showed that disruption of bacterial membrane is needed to kill bacteria by mAng4 (Sultana et al, 2022). In addition, mAng4 has been attributed to angiogenesis, which is determined by the evaluation of ex vivo angiogenesis through thoracic aorta assay (Crabtree et al, 2007).…”

“…However, Lys59 in mAng4 seems to be crucial for angiogenic activity, since the substitution of Lys59 → Asn59 by mutation abolished angiogenic activity ( Crabtree et al, 2007 ). In addition, structural modification of the cell-binding segment has no appreciable effect on RNase activity ( Crabtree et al, 2007 ; Sultana et al, 2022 ).…”

“…mAng4 demonstrates antibacterial effects against Bacteroides thetaiotaomicron, Enterococcus faecalis, Bifidobacterium longum, Enterococcus gallinarum, Listeria monocytogenes , and Salmonella typhimurium LT2 ( Hooper et al, 2003 ; Walker et al, 2013 ). Recent studies showed that disruption of bacterial membrane is needed to kill bacteria by mAng4 ( Sultana et al, 2022 ).…”

“…Therefore, angiogenesis in gastrointestinal tumor is the focus of research, leading to the development of anticancer drugs. In addition to angiogenesis in gastrointestinal tumors, it has an extensive variety of capabilities, such as neuroprotective, antimicrobial activity, and innate immunity ( Tello-Montoliu et al, 2006 ; Sheng and Xu, 2016 ; Prehn and Jirström, 2020 ; Sultana et al, 2022 ). Further, serum angiogenin levels are associated with different disease conditions, including cancer, cardiovascular, and inflammatory bowel diseases ( Gabriel-Salazar et al, 2018 ; Yu et al, 2018 ; Garcia-Rodriguez et al, 2021 ).…”