Date Published: May 23, 2016
Publisher: Public Library of Science
Author(s): Kyle P. Obergfell, H. Steven Seifert, Lotte Søgaard-Andersen
Abstract: The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3’ third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants and suggest there are alternate forms of the Tfp assembly apparatus that mediate various functions including transformation.
Partial Text: Neisseria gonorrhoeae is a Gram-negative, obligate human pathogen and the etiological agent of gonorrhea . Each year, the bacterium causes an estimated 106 million new cases worldwide.The rise of resistance to all available antibiotics coupled with the lack of any viable vaccine candidates have led the Centers for Disease Control to classify N. gonorrhoeae as an urgent threat, underscoring the need for understanding the fundamental virulence mechanisms of this organism.
We have shown in a directed mutational analysis of the 3’ pilE coding region that a variety of pilE mutations that either alter or completely abolish pilus elaboration on the bacterial cell surface do not prevent transformation. A series of site-directed pilE mutants demonstrated that this transformation competence is not due to translational read-through, but that the maintenance of competence requires the pilin Ntd, a putative cleavage product of S-pilin proteolysis. Moreover, naturally occurring pilin variants that have pilin molecules incapable of forming an extended pilus fiber also retain competence. In these variants, S-pilin cleavage is required for transformation to mediate release of the Ntd from the mutated C-terminal head. We propose that the process of S-pilin cleavage releases the Ntd to maintain competence in cells undergoing antigenic variation, a diversity-generation system critical for immune escape during infection.