Genetic incorporation of non-canonical amino acid photocrosslinkers in Neisseria meningitidis: New method provides insights into the physiological function of the function-unknown NMB1345 protein

Abstract

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/tRNA Pyl 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/tRNA Pyl 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.

Genetic Code Expansion in Pseudomonas putida KT2440

“…pBpa was mainly used as a photocross-linker to detect the interaction between a protein and its binding partners. 18,40 An MjTyrRS-derived tRNA synthetase for pBpa (BpaRS) has been applied to expand the genetic code in various bacteria. 40 To enable the incorporation of pBpa in KT2440, the AzFRS in pSEVA621-AzFRS was replaced with BpaRS.…”

“…We further validated the established genetic system for the Mj TyrRS/tRNA pair through the genetic code expansion of p -benzoyl- l -phenylalanine (pBpa) in KT2440. pBpa was mainly used as a photocross-linker to detect the interaction between a protein and its binding partners. , An Mj TyrRS-derived tRNA synthetase for pBpa (BpaRS) has been applied to expand the genetic code in various bacteria . To enable the incorporation of pBpa in KT2440, the AzFRS in pSEVA621-AzFRS was replaced with BpaRS.…”

“…The discovery of natural decoding of the amber codon with pyrrolysine in Methanosarcina barkeri led to the use of the pyrrolysyl-tRNA synthetase and tRNA Pyl CUA ( Mb PylRS/PylT) pair for the incorporation of primarily lysine analogs. − The natural orthogonality of the two systems further allows the simultaneous encoding of two unAAs in response to two blank codons. , The initial method development almost exclusively used Escherichia coli strains for the directed evolution of orthogonal aaRS/tRNA pairs. , Currently, genetic code expansion has been implemented in more than 20 non-E. coli bacterial species with diverse biotechnological and biomedical interests for bioremediation, biomanufacturing, and fundamental research. − …”

Metabolic labeling probes for interrogation of the host–pathogen interaction

“…Takahashi et al also incorporated pBPA into the bacterium and used it to elucidate the role of NMB1345 in the infection pathway. 133…”

Rebooting life: engineering non-natural nucleic acids, proteins and metabolites in microorganisms

“…Non-natural (or non-canonical) amino acids (ncAAs) are incorporated into proteins to enhance or modify their properties [ 48 ]. To date, more than 200 ncAAs have been successfully incorporated in prokaryotic and eukaryotic organisms [ 49 – 52 ]. Incorporation of ncAAs has been explored for multiple applications, including protein labelling [ 53 ]; biomolecular targeting by binding and reacting with fluorescent probes [ 54 – 56 ]; identifying and understanding protein interactions [ 57 – 59 ]; real-time tracking and in vivo imaging [ 60 , 61 ]; establishment of biocontainment systems [ 62 , 63 ]; making novel biomaterials [ 64 ]; developing antimicrobial peptides [ 65 – 67 ]; and for generating novel biocatalysts [ 68 – 70 ].…”