Genetic incorporation ofnon-canonical amino acidphotocrosslinkers inNeisseria meningitidis: New method provides insights into the physiological function of the function-unknown NMB1345 protein.

Hideyuki Takahashi,Naoshi Dohmae,Tatsuo Yanagisawa,Kwang Sik Kim,Ken Shimuta,Shigeyuki Yokoyama,Makoto Ohnishi,Nediljko Budisa

doi:10.1371/journal.pone.0237883

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/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.

Highlights

Neisseria meningitidis is a fastidious Gram-negative microorganism that generally exists in the non-invasive so-called “carrier state” at a rate of 0.4–25% in human populations [1, 2]
During the course of our research on the infectious abilities of the sequence type (ST)-2032 N. meningitidis strain mutagenized by signature-tagged mutagenesis (STM) to human brain microvascular endothelial cells (HBMEC), we found that a transposon mutant, in which the gene annotated as NMB1345 in the N. meningitidis MC58 genome [39] was disrupted and resulted in defective infectious ability in HBMEC (Fig 1A)
While progressive genome sequencing has recently provided novel insights into N. meningitidis, more than 50% of the annotated ORFs remain as “hypothetical proteins”, and their biological functions are currently unclear. This may be due to an incomplete understanding of meningococcal pathogenesis, which is largely attributable to the lack of analytical methods for N. meningitidis [34]

Summary

Introduction

Neisseria meningitidis is a fastidious Gram-negative microorganism that generally exists in the non-invasive so-called “carrier state” at a rate of 0.4–25% in human populations [1, 2]. N. meningitidis exhibits the abilities to cross the epithelial layer of the upper respiratory tract, infiltrate the bloodstream, evade the defenses of the human immune system, adhere to the endothelial layers of peripheral and brain vessels, cross the brain-blood barrier, and replicate in cerebrospinal fluid [3]. Epidemiological analyses previously suggested that a human genetic polymorphism [4] and climate conditions [5] are important for predicting the outcomes of infection, the reasons why invasive meningococcal diseases (IMD) occur in some individuals, but not in others remain unclear [6]. Among the factors described above, meningococcal pili, which are mainly composed of the major pilin, PilE, and three minor pilins, PilV, PilX, and ComP, play the most important roles in the meningococcal infection processes involved in interactions with human epithelial and endothelial cells (reviewed in [17, 18]). The mechanisms by which N. meningitidis causes septicemia and meningitis in humans cannot be completely explained by these characterized factors [32]

Methods

Results

Conclusion