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.
Highlights
Neisseria gonorrhoeae is a Gram-negative, obligate human pathogen and the etiological agent of gonorrhea [1]
The bacterium responsible for the disease gonorrhea, is capable of undergoing natural DNA transformation, a critical mechanism by which bacteria can take up DNA from the environment
Release of the N-terminal domain (Ntd) allows cells to maintain competence during pilin antigenic variation, a process vital for immune escape that frequently produces cells that cannot form Type IV pili. While these cells were previously thought to be incapable of DNA transformation, our data suggest that S-pilin cleavage evolved as a mechanism that allows DNA transformation and horizontal gene transfer to occur in these cells lacking pili
Summary
Neisseria gonorrhoeae is a Gram-negative, obligate human pathogen and the etiological agent of gonorrhea [1]. The process of DNA transformation in N. gonorrhoeae and the closely related pathogen Neisseria meningitidis has been well-studied with the majority of contributing factors having been identified. All N. gonorrhoeae strains are naturally competent for DNA transformation, and competence is not regulated as the organism is able to undergo transformation during all phases of growth, which contributes to the spread of antibiotic resistance [3,4]. The Tfp is a major virulence factor involved in cellular adherence, microcolony formation, resistance to neutrophil mediated killing, twitching motility, and transformation [5,6,7,8,9,10]. Similar to Haemophilus influenzae, N. gonorrhoeae preferentially takes up its own DNA that contains a 10 or 12 base DNA uptake sequence (DUS) (5’ ATGCCGTCTGAA 3’) [11,12]. The DUS occurs frequently within Neisserial genomes and significantly increases efficiency of DNA transformation compared to the same DNA lacking the sequence [13]
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