TY - JOUR
T1 - Genetic incorporation of non-canonical amino acid photocrosslinkers in Neisseria meningitidis
T2 - New method provides insights into the physiological function of the function-unknown NMB1345 protein
AU - Takahashi, Hideyuki
AU - Dohmae, Naoshi
AU - Kim, Kwang Sik
AU - Shimuta, Ken
AU - Ohnishi, Makoto
AU - Yokoyama, Shigeyuki
AU - Yanagisawa, Tatsuo
N1 - Funding Information:
This work was supported by JSPS KAKENHI Grant Numbers 15K08485 and 19K07550 (to H.T.), AMED under Grant Number 18fk0108071j0101 (to H.T.), by the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from AMED under Grant Number JP16am0101022 (to S.Y.), and under Grant Number JP17am0101081 (to S.Y.).
Publisher Copyright:
© 2020 Takahashi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2020/8
Y1 - 2020/8
N2 - Although whole-genome sequencing has provided novel insights into Neisseria meningitidis, many open reading frames have only been annotated as hypothetical proteins with unknown biological functions. Our previous genetic analyses revealed that the hypothetical protein, NMB1345, plays a crucial role in meningococcal infection in human brain microvascular endothelial cells; however, NMB1345 has no homology to any identified protein in databases and its physiological function could not be elucidated using pre-existing methods. Among the many biological technologies to examine transient protein-protein interaction in vivo, one of the developed methods is genetic code expansion with non-canonical amino acids (ncAAs) utilizing a pyrrolysyl-tRNA synthetase/tRNAPyl pair from Methanosarcina species: However, this method has never been applied to assign function-unknown proteins in pathogenic bacteria. In the present study, we developed a new method to genetically incorporate ncAAs-encoded photocrosslinking probes into N. meningitidis by utilizing a pyrrolysyl-tRNA synthetase/tRNAPyl pair and elucidated the biological function(s) of the NMB1345 protein. The results revealed that the NMB1345 protein directly interacts with PilE, a major component of meningococcal pili, and further physicochemical and genetic analyses showed that the interaction between the NMB1345 protein and PilE was important for both functional pilus formation and meningococcal infectious ability in N. meningitidis. The present study using this new methodology for N. meningitidis provides novel insights into meningococcal pathogenesis by assigning the function of a hypothetical protein.
AB - Although whole-genome sequencing has provided novel insights into Neisseria meningitidis, many open reading frames have only been annotated as hypothetical proteins with unknown biological functions. Our previous genetic analyses revealed that the hypothetical protein, NMB1345, plays a crucial role in meningococcal infection in human brain microvascular endothelial cells; however, NMB1345 has no homology to any identified protein in databases and its physiological function could not be elucidated using pre-existing methods. Among the many biological technologies to examine transient protein-protein interaction in vivo, one of the developed methods is genetic code expansion with non-canonical amino acids (ncAAs) utilizing a pyrrolysyl-tRNA synthetase/tRNAPyl pair from Methanosarcina species: However, this method has never been applied to assign function-unknown proteins in pathogenic bacteria. In the present study, we developed a new method to genetically incorporate ncAAs-encoded photocrosslinking probes into N. meningitidis by utilizing a pyrrolysyl-tRNA synthetase/tRNAPyl pair and elucidated the biological function(s) of the NMB1345 protein. The results revealed that the NMB1345 protein directly interacts with PilE, a major component of meningococcal pili, and further physicochemical and genetic analyses showed that the interaction between the NMB1345 protein and PilE was important for both functional pilus formation and meningococcal infectious ability in N. meningitidis. The present study using this new methodology for N. meningitidis provides novel insights into meningococcal pathogenesis by assigning the function of a hypothetical protein.
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U2 - 10.1371/journal.pone.0237883
DO - 10.1371/journal.pone.0237883
M3 - Article
C2 - 32866169
AN - SCOPUS:85090103807
SN - 1932-6203
VL - 15
JO - PloS one
JF - PloS one
IS - 8 August 2020
M1 - e0237883
ER -