Development and implementation of a Type I-C CRISPR-based programmable repression system for Neisseria gonorrhoeae.

Clustered regularly interspaced short palindromic repeats (CRISPR) are prokaryotic adaptive immune systems regularly utilized as DNA-editing tools. While Neisseria gonorrhoeae does not have an endogenous CRISPR, the commensal species Neisseria lactamica encodes a functional Type I-C CRISPR-Cas system. We have established an isopropyl ß-d-1-thiogalactopyranoside added (IPTG)-inducible, CRISPR interference (CRISPRi) platform based on the N. lactamica Type I-C CRISPR missing the Cas3 nuclease to allow locus-specific transcriptional repression. As proof of principle, we targeted a non-phase-variable version of the opaD gene. We show that CRISPRi can downregulate opaD gene and protein expression, resulting in bacterial inability to stimulate neutrophil oxidative responses and to bind to an N-terminal fragment of CEACAM1. Importantly, we used CRISPRi to effectively knockdown all the transcripts of all 11 opa genes using a five-spacer CRISPR array, allowing control of the entire phase-variable opa family in strain FA1090. We also report that repression is reversible following IPTG removal. Finally, we showed that the Type I-C CRISPRi system can conditionally reduce the expression of two essential genes. This CRISPRi system will allow the interrogation of every Gc gene, essential and non-essential, to study physiology and pathogenesis and aid in antimicrobial development.IMPORTANCEClustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have proven instrumental in genetically manipulating many eukaryotic and prokaryotic organisms. Despite its usefulness, a CRISPR system had yet to be developed for use in Neisseria gonorrhoeae (Gc), a bacterium that is the main etiological agent of gonorrhea infection. Here, we developed a programmable and IPTG-inducible Type I-C CRISPR interference (CRISPRi) system derived from the commensal species Neisseria lactamica as a gene repression system in Gc. As opposed to generating genetic knockouts, the Type I-C CRISPRi system allows us to block transcription of specific genes without generating deletions in the DNA. We explored the properties of this system and found that a minimal spacer array is sufficient for gene repression while also facilitating efficient spacer reprogramming. Importantly, we also show that we can use CRISPRi to knockdown genes that are essential to Gc that cannot normally be knocked out under laboratory settings. Gc encodes ~800 essential genes, many of which have no predicted function. We predict that this Type I-C CRISPRi system can be used to help categorize gene functions and perhaps contribute to the development of novel therapeutics for gonorrhea.

The Neisseria gonorrhoeae type IV pilus promotes resistance to hydrogen peroxide- and LL-37-mediated killing by modulating the availability of intracellular, labile iron.

The Neisseria gonorrhoeae Type IV pilus is a multifunctional, dynamic fiber involved in host cell attachment, DNA transformation, and twitching motility. We previously reported that the N. gonorrhoeae pilus is also required for resistance against hydrogen peroxide-, antimicrobial peptide LL-37-, and non-oxidative, neutrophil-mediated killing. We tested whether the hydrogen peroxide, LL-37, and neutrophil hypersensitivity phenotypes in non-piliated N. gonorrhoeae could be due to elevated iron levels. Iron chelation in the growth medium rescued a nonpiliated pilE mutant from both hydrogen peroxide- and antimicrobial peptide LL-37-mediated killing, suggesting these phenotypes are related to iron availability. We used the antibiotic streptonigrin, which depends on free cytoplasmic iron and oxidation to kill bacteria, to determine whether piliation affected intracellular iron levels. Several non-piliated, loss-of-function mutants were more sensitive to streptonigrin killing than the piliated parental strain. Consistent with the idea that higher available iron levels in the under- and non-piliated strains were responsible for the higher streptonigrin sensitivity, iron limitation by desferal chelation restored resistance to streptonigrin in these strains and the addition of iron restored the sensitivity to streptonigrin killing. The antioxidants tiron and dimethylthiourea rescued the pilE mutant from streptonigrin-mediated killing, suggesting that the elevated labile iron pool in non-piliated bacteria leads to streptonigrin-dependent reactive oxygen species production. These antioxidants did not affect LL-37-mediated killing. We confirmed that the pilE mutant is not more sensitive to other antibiotics showing that the streptonigrin phenotypes are not due to general bacterial envelope disruption. The total iron content of the cell was unaltered by piliation when measured using ICP-MS suggesting that only the labile iron pool is affected by piliation. These results support the hypothesis that piliation state affects N. gonorrhoeae iron homeostasis and influences sensitivity to various host-derived antimicrobial agents.

Challenges and Controversies Concerning Neisseria gonorrhoeae-Neutrophil Interactions in Pathogenesis.

The bacterium Neisseria gonorrhoeae (Ngo) is the main cause of the sexually transmitted infection gonorrhea. The global incidence of 87 million new Ngo infections each year, rising infection rates, and the emergence of Ngo strains that are resistant to all clinically recommended antibiotics have raised the specter of untreatable infections (M. Unemo, H. S. Seifert, E. W. Hook, III, S. Hawkes, et al., Nat Rev Dis Primers 5:79, 2019, https://doi.org/10.1038/s41572-019-0128-6). Given their abundance in symptomatic disease, neutrophils are central to both Ngo infection and consequent damage to host tissues. This article highlights present knowledge and the main open questions about Ngo-neutrophil interactions in immunity versus disease pathogenesis.

Discovery of a New Neisseria gonorrhoeae Type IV Pilus Assembly Factor, TfpC.

Neisseria gonorrhoeae relies on type IV pili (T4p) to promote colonization of their human host and to cause the sexually transmitted infection gonorrhea. This organelle cycles through a process of extension and retraction back into the bacterial cell. Through a genetic screen, we identified the NGO0783 locus of N. gonorrhoeae strain FA1090 as containing a gene encoding a protein required to stabilize the type IV pilus in its extended, nonretracted conformation. We have named the gene tfpC and the protein TfpC. Deletion of tfpC produces a nonpiliated colony morphology, and immuno-transmission electron microscopy confirms that the pili are lost in the ΔtfpC mutant, although there is some pilin detected near the bacterial cell surface. A copy of the tfpC gene expressed from a lac promoter restores pilus expression and related phenotypes. A ΔtfpC mutant shows reduced levels of pilin protein, but complementation with a tfpC gene restored pilin to normal levels. Bioinformatic searches show that there are orthologues in numerous bacterial species, but not all type IV pilin-expressing bacteria contain orthologous genes. Coevolution and nuclear magnetic resonance (NMR) analysis indicates that TfpC contains an N-terminal transmembrane helix, a substantial extended/unstructured region, and a highly charged C-terminal coiled-coil domain.IMPORTANCE Most bacterial species express one or more extracellular organelles called pili/fimbriae that are required for many properties of each bacterial cell. The Neisseria gonorrhoeae type IV pilus is a major virulence and colonization factor for the sexually transmitted infection gonorrhea. We have discovered a new protein of Neisseria gonorrhoeae called TfpC that is required to maintain type IV pili on the bacterial cell surface. There are similar proteins found in other members of the Neisseria genus and many other bacterial species important for human health.

Altering the Neisseria gonorrhoeae pilE Guanine Quadruplex Loop Bases Affects Pilin Antigenic Variation.

Neisseria gonorrhoeae possesses a programmed recombination system that allows the bacteria to alter the major subunit of the type IV pilus, pilin or PilE. An alternate DNA structure known as a guanine quadruplex (G4) is required for pilin antigenic variation (pilin Av). The G-C base pairs within the G4 motif are required for pilin Av, but simple mutation of the loop bases does not affect pilin Av. We show that more substantial changes to the loops, in both size and nucleotide composition, with the core guanines unchanged, decrease or abrogate pilin Av. We investigated why these loop changes might influence the efficiency of pilin Av. RecA is a recombinase required for pilin Av that can bind the pilE G4 in vitro. RecA binds different G4 structures with altered loops with varied affinities. However, changes in RecA binding affinities to the loop mutants do not absolutely correlate with the pilin Av phenotypes. Interestingly, the yeast RecA ortholog, Rad51, also binds the pilE G4 structure with a higher affinity than it binds single-stranded DNA, suggesting that RecA G4 binding is conserved in eukaryotic orthologs. The thermal stability the pilE G4 structure and its loop mutants showed that the parental G4 structure had the highest melting temperature, and the melting temperature of the loop mutants correlated with pilin Av phenotype. These results suggest that the folding kinetics and stability of G4 structures are important for the efficiency of pilin Av.

Gonorrhoea.

The bacterium Neisseria gonorrhoeae causes the sexually transmitted infection (STI) gonorrhoea, which has an estimated global annual incidence of 86.9 million adults. Gonorrhoea can present as urethritis in men, cervicitis or urethritis in women, and in extragenital sites (pharynx, rectum, conjunctiva and, rarely, systemically) in both sexes. Confirmation of diagnosis requires microscopy of Gram-stained samples, bacterial culture or nucleic acid amplification tests. As no gonococcal vaccine is available, prevention relies on promoting safe sexual behaviours and reducing STI-associated stigma, which hinders timely diagnosis and treatment thereby increasing transmission. Single-dose systemic therapy (usually injectable ceftriaxone plus oral azithromycin) is the recommended first-line treatment. However, a major public health concern globally is that N. gonorrhoeae is evolving high levels of antimicrobial resistance (AMR), which threatens the effectiveness of the available gonorrhoea treatments. Improved global surveillance of the emergence, evolution, fitness, and geographical and temporal spread of AMR in N. gonorrhoeae, and improved understanding of the pharmacokinetics and pharmacodynamics for current and future antimicrobials in the treatment of urogenital and extragenital gonorrhoea, are essential to inform treatment guidelines. Key priorities for gonorrhoea control include strengthening prevention, early diagnosis, and treatment of patients and their partners; decreasing stigma; expanding surveillance of AMR and treatment failures; and promoting responsible antimicrobial use and stewardship. To achieve these goals, the development of rapid and affordable point-of-care diagnostic tests that can simultaneously detect AMR, novel therapeutic antimicrobials and gonococcal vaccine(s) in particular is crucial.

PacBio Amplicon Sequencing Method To Measure Pilin Antigenic Variation Frequencies of Neisseria gonorrhoeae.

Gene diversification is a common mechanism pathogens use to alter surface structures to aid in immune avoidance. Neisseria gonorrhoeae uses a gene conversion-based diversification system to alter the primary sequence of the gene encoding the major subunit of the pilus, pilE Antigenic variation occurs when one of the nonexpressed 19 silent copies donates part of its DNA sequence to pilE We have developed a method using Pacific Biosciences (PacBio) amplicon sequencing and custom software to determine pilin antigenic variation frequencies. The program analyzes 37 variable regions across the strain FA1090 1-81-S2 pilE gene and can be modified to determine sequence variation from other starting pilE sequences or other diversity generation systems. Using this method, we measured pilin antigenic variation frequencies for various derivatives of strain FA1090 and showed we can also analyze pilin antigenic variation frequencies during macrophage infection.IMPORTANCE Diversity generation systems are used by many unicellular organism to provide subpopulations of cell with different properties that are available when needed. We have developed a method using the PacBio DNA sequencing technology and a custom computer program to analyze the pilin antigenic variation system of the organism that is the sole cause of the sexually transmitted infection, gonorrhea.

Location, Location, Location-Commensalism, Damage and Evolution of the Pathogenic Neisseria.

The 10 human-restricted Neisseria species all colonize mucosal surfaces, but show a spectrum of pathogenicity. The commensal Neisseria do not normally cause pathology, while the two pathogenic species, Neisseria meningitidis and Neisseria gonorrhoeae, straddle the border between commensalism and pathogenicity. Why the pathogenic Neisseria continue to mediate host damage after thousands of years of co-evolution with their human host, and why the commensal species have not acquired the ability to damage the host, if this capability provides a selective advantage, is not understood. One way the pathogenic species are different from the commensal species is by their ability to induce PMN inflammation, which is dependent on the site of colonization. I discuss how the site of colonization dictates whether copious inflammation occurs with both pathogenic species. I put forth a model that posits that an ancestor of both pathogenic species changed colonization site from the oral cavity to the genital tract of a human or humanoid and had to evolve multiple, new traits - to induce PMN inflammation and avoid adaptive immunity - to allow efficient sexual transmission. This model predicts that PMN inflammation produces the serious sequelae of gonorrhea and increases the probability that N. meningitidis might exit the oral cavity to produce systemic disease. In both cases, the pathology produced by these host-adapted species is an unintended by product of the inflammation but host damage does not provide any selective advantage for these organisms.

A Double-Strand Break Does Not Promote Neisseria gonorrhoeae Pilin Antigenic Variation.

The major subunit of the type IV pilus (T4p) of Neisseria gonorrhoeae undergoes antigenic variation (AV) dependent on a guanine quadruplex (G4) DNA structure located upstream of the pilin gene. Since the presence of G4 DNA induces genome instability in both eukaryotic and prokaryotic chromosomes, we tested whether a double-strand break (DSB) at the site of the pilE G4 sequence could substitute for G4-directed pilin AV. The G4 motif was replaced by an I-SceI cut site, and the cut site was also introduced to locations near the origin of replication and the terminus. Expression of the I-SceI endonuclease from an irrelevant chromosomal site confirmed that the endonuclease functions to induce double-strand breaks at all three locations. No antigenic variants were detected when the G4 was replaced with the I-SceI cut site, but there was a growth defect from having a DSB in the chromosome, and suppressor mutations that were mainly deletions of the cut site and/or the entire pilE gene accumulated. Thus, the pilE G4 does not act to promote pilin AV by generating a DSB but requires either a different type of break, a nick, or more complex interactions with other factors to stimulate this programmed recombination system.IMPORTANCENeisseria gonorrhoeae, the causative agent of gonorrhea, possesses a DNA recombination system to change one of its surface-exposed antigens. This recombination system, known as antigenic variation, uses an alternate DNA structure to initiate variation. The guanine quadruplex DNA structure is known to cause nicks or breaks in DNA; however, much remains unknown about how this structure functions in cells. We show that inducing a break by different means does not allow antigenic variation, indicating that the DNA structure may have a more complicated role.

Neisseria gonorrhoeae Exposed to Sublethal Levels of Hydrogen Peroxide Mounts a Complex Transcriptional Response.

Neisseria gonorrhoeae mounts a substantial transcriptional program in response to hydrogen peroxide (HP), a prominent reactive oxygen species (ROS) encountered during infection. We tested which strain FA1090 genes show differential transcript abundance in response to sublethal amounts of HP to differentiate HP-responsive signaling from widespread cellular death and dysregulation. RNA sequencing (RNA-Seq) revealed that 150 genes were significantly upregulated and 143 genes downregulated following HP exposure. We annotated HP-responsive operons and all transcriptional start sites (TSSs) and identified which TSSs responded to HP treatment. We compared the HP responses and other previously reported genes and found only partial overlapping of other regulatory networks, indicating that the response to HP involves multiple biological functions. Using a representative subset of responsive genes, we validated the RNA-Seq results and found that the HP transcriptome was similar to that of sublethal organic peroxide. None of the genes in the representative subset, however, responded to sublethal levels of HOCl or O2 -. These results support the idea that N. gonorrhoeae may use variations in HP levels as a signal for different stages of infection. IMPORTANCE The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted disease gonorrhea. This bacterium encounters hydrogen peroxide produced from host cells during infection, but the organism survives in the presence of this antimicrobial agent. This work shows that the bacterium responds to hydrogen peroxide by regulating the expression of many genes involved in multiple processes.

Analysis of Pilin Antigenic Variation in Neisseria meningitidis by Next-Generation Sequencing.

Many pathogenic microbes evade host immune surveillance by varying the surface antigens, a process termed antigenic variation. While the process of pilin antigenic variation has been extensively studied in the human pathogen Neisseria gonorrhoeae (gonococcus [Gc]), relatively few studies of pilin antigenic variation have been conducted with Neisseria meningitidis (meningococcus [Mc]). Mc is usually a commensal organism that colonizes the human nasopharynx, but when it translocates to the bloodstream or meninges, it results in the severe and often deadly meningococcal disease. The type IV pili of Mc isolates play a critical role in host surface adherence, and its major pilin component (PilE) can undergo antigenic variation. In this study, Roche 454 pyrosequencing was used to examine the pilin antigenic variation of Mc strain 8013, as well as 8013 recA, recX, recQ, rep, and recJ mutants, Gc orthologues which have been shown to play a role in pilin antigenic variation. This study confirms that the Mc recA, rep, and recJ genes are essential for pilin antigenic variation. While the Mc recQ and recX gene products contribute to normal frequencies of antigenic variation, the loss of these factors does not alter the types of pilin variants produced. Overall, this study shows that the mechanisms of pilin antigenic variation are conserved between Gc and Mc.IMPORTANCE Antigenic variation is a strategy used by many pathogens to escape host immune surveillance and establish persistent infections. This study successfully applies next-generation sequencing to study pilin antigenic variation in the human pathogen Neisseria meningitidis This assay provides an affordable and efficient solution for quantifying antigenic variation frequency in mutant strains and for defining the recombination products of the process. We determined that there is a nonuniformity of silent donor copies used during meningococcus antigenic variation, and by the analysis of selected mutants deficient for specific recombination pathways, we show for the first time that the processes are conserved between N. meningitidis and Neisseria gonorrhoeae.

Above and Beyond Watson and Crick: Guanine Quadruplex Structures and Microbes.

Advances in understanding mechanisms of nucleic acids have revolutionized molecular biology and medicine, but understanding of nontraditional nucleic acid conformations is less developed. The guanine quadruplex (G4) alternative DNA structure was first described in the 1960s, but the existence of G4 structures (G4-S) and their participation in myriads of biological functions are still underappreciated. Despite many tools to study G4s and many examples of roles for G4s in eukaryotic molecular processes and issues with uncontrolled G4-S formation, there is relatively little knowledge about the roles of G4-S in viral or prokaryotic systems. This review summarizes the state of the art with regard to G4-S in eukaryotes and their potential roles in human disease before discussing the evidence that G4-S have equivalent importance in affecting viral and bacterial life.

The low-molecular-mass, penicillin-binding proteins DacB and DacC combine to modify peptidoglycan cross-linking and allow stable Type IV pilus expression in Neisseria gonorrhoeae.

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea and is adapted to survive in humans, its only host. The N. gonorrhoeae cell wall is critical for maintaining envelope integrity, resisting immune cell killing and production of cytotoxic peptidoglycan (PG) fragments. Deletion of the N. gonorrhoeae strain FA1090 genes encoding two predicted low-molecular-mass, penicillin-binding proteins (LMM PBPs), DacB and DacC, substantially altered the PG cross-linking. Loss of the DacB peptidase resulted in global alterations to the PG composition, while loss of the DacC protein affected a much narrower subset of PG peptide components. A double ΔdacB/ΔdacC mutant resembled the ΔdacB single mutant, but had an even greater level of cross-linked PG. While single ΔdacB or ΔdacC mutants did not show any major phenotypes, the ΔdacB/ΔdacC mutant displayed an altered cellular morphology, decreased resistance to antibiotics and increased sensitivity to detergent-mediated death. Loss of the two proteins also drastically reduced the number of Type IV pili (Tfp), a critical virulence factor. The decreased piliation reduced transformation efficiency and correlated with increased growth rate. While these two LMM PBPs differentially alter the PG composition, their overlapping effects are essential to proper envelope function and expression of factors critical for pathogenesis.

Neisseria gonorrhoeae host adaptation and pathogenesis.

The host-adapted human pathogen Neisseria gonorrhoeae is the causative agent of gonorrhoea. Consistent with its proposed evolution from an ancestral commensal bacterium, N. gonorrhoeae has retained features that are common in commensals, but it has also developed unique features that are crucial to its pathogenesis. The continued worldwide incidence of gonorrhoeal infection, coupled with the rising resistance to antimicrobials and the difficulties in controlling the disease in developing countries, highlights the need to better understand the molecular basis of N. gonorrhoeae infection. This knowledge will facilitate disease prevention, surveillance and control, improve diagnostics and may help to facilitate the development of effective vaccines or new therapeutics. In this Review, we discuss sex-related symptomatic gonorrhoeal disease and provide an overview of the bacterial factors that are important for the different stages of pathogenesis, including transmission, colonization and immune evasion, and we discuss the problem of antibiotic resistance.

Analyzing Neisseria gonorrhoeae Pilin Antigenic Variation Using 454 Sequencing Technology.

UNLABELLED: Many pathogens use homologous recombination to vary surface antigens in order to avoid immune surveillance. Neisseria gonorrhoeae, the bacterium responsible for the sexually transmitted infection gonorrhea, achieves this in part by changing the sequence of the major subunit of the type IV pilus in a process termed pilin antigenic variation (Av). The N. gonorrhoeae chromosome contains one expression locus (pilE) and many promoterless, partial-coding silent copies (pilS) that act as reservoirs for variant pilin information. Pilin Av occurs by high-frequency gene conversion reactions, which transfer pilS sequences into the pilE locus. We have developed a 454 sequencing-based assay to analyze the frequency and characteristics of pilin Av that allows a more robust analysis of pilin Av than previous assays. We used this assay to analyze mutations and conditions previously shown to affect pilin Av, confirming many but not all of the previously reported phenotypes. We show that mutations or conditions that cause growth defects can result in Av phenotypes when analyzed by phase variation-based assays. Adapting the 454 sequencing to analyze pilin Av demonstrates the utility of this technology to analyze any diversity generation system that uses recombination to develop biological diversity. IMPORTANCE: Measuring and analyzing complex recombination-based systems constitute a major barrier to understanding the mechanisms used to generate diversity. We have analyzed the contributions of many gonococcal mutations or conditions to the process of pilin antigenic variation.

Seminal Plasma Promotes Neisseria gonorrhoeae Aggregation and Biofilm Formation.

UNLABELLED: Neisseria gonorrhoeae causes the human-specific disease gonorrhea and is transmitted from person to person primarily via sexual contact. During transmission, N. gonorrhoeae is often exposed to seminal fluid and must adapt to this change in environment. Previous work demonstrated that seminal fluid facilitates N. gonorrhoeae motility and alters epithelial cell interactions. In this study, exposure to seminal fluid was found to decrease surface adherence of gonococci in a manner that was independent of Opa adhesin proteins or type IV pilus retraction. Semen was also shown to cause dispersal of bacteria that had previously established surface adherence. Although surface adherence decreased, interbacterial interactions were increased by seminal plasma both in long-term static culture and on a cell-to-cell basis over shorter time periods. The result of increased bacterium-bacterium interactions resulted in the formation of microcolonies, an important step in the N. gonorrhoeae infectious process. Seminal fluid also facilitated increased bacterial aggregation in the form of shear-resistant three-dimensional biofilms. These results emphasize the importance of the gonococcal response to the influx of seminal fluid within the genital niche. Further characterization of the N. gonorrhoeae response to semen will advance our understanding of the mechanisms behind the establishment of infection in naive hosts and the process of transmission. IMPORTANCE: N. gonorrhoeae is the causative agent of the globally prevalent sexually transmitted infection gonorrhea. An understudied aspect of this human-adapted pathogen is the change in bacterial physiology that occurs during sexual transmission. N. gonorrhoeae encounters semen when transmitted from host to host, and it is known that, when N. gonorrhoeae is exposed to seminal fluid, alterations in bacterial motility and type IV pilus arrangement occur. This work extends our previous observations on this modulation of gonococcal physiology by seminal fluid and demonstrates that seminal plasma decreases surface adherence, promotes interbacterial interactions, and enhances biofilm formation.

The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation.

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.