Substitutions in the N-terminal alpha helical spine of Neisseria gonorrhoeae pilin affect Type IV pilus assembly, dynamics and associated functions.

Type IV pili (Tfp) are multifunctional surface appendages expressed by many Gram negative species of medical, environmental and industrial importance. The N-terminally localized, so called alpha-helical spine is the most conserved structural feature of pilin subunits in these organelles. Prevailing models of pilus assembly and structure invariably implicate its importance to membrane trafficking, organelle structure and related functions. Nonetheless, relatively few studies have examined the effects of missense substitutions within this domain. Using Neisseria gonorrhoeae as a model system, we constructed mutants with single and multiple amino acid substitutions localized to this region of the pilin subunit PilE and characterized them with regard to pilin stability, organelle expression and associated phenotypes. The consequences of simultaneous expression of the mutant and wild-type PilE forms were also examined. The findings document for the first time in a defined genetic background the phenomenon of pilin intermolecular complementation in which assembly defective pilin can be rescued into purifiable Tfp by coexpression of wild-type PilE. The results further demonstrate that pilin subunit composition can impact on organelle dynamics mediated by the PilT retraction protein via a process that appears to monitor the efficacy of subunit-subunit interactions. In addition to confirming and extending the evidence for PilE multimerization as an essential component for competence for natural genetic transformation, this work paves the way for detailed studies of Tfp subunit-subunit interactions including self-recognition within the membrane and packing within the pilus polymer.

Neisseria gonorrhoeae type IV pili undergo multisite, hierarchical modifications with phosphoethanolamine and phosphocholine requiring an enzyme structurally related to lipopolysaccharide phosphoethanolamine transferases.

The zwitterionic phospho-forms phosphoethanolamine and phosphocholine are recognized as influential and important substituents of pathogen cell surfaces. PilE, the major pilin subunit protein of the type IV pilus (Tfp) colonization factor of Neisseria gonorrhoeae undergoes unique, post-translational modifications with these moieties. These phospho-form modifications have been shown to be O-linked alternately to a specific, conserved serine residue of PilE. However, the enzymes and precursors involved in their addition are unknown, and the full spectrum of PilE post-translational modifications has yet to be defined. Here, an intact protein-based mass spectrometric approach was integrated with bioinformatics and reverse genetics to address these matters. Specifically we show that a protein limited in its distribution to pathogenic Neisseria species and structurally related to enzymes implicated in phosphoethanolamine modification of lipopolysaccharide is necessary for PilE covalent modification with phosphoethanolamine and phosphocholine. These findings strongly suggest that protein phospho-form modification is mechanistically similar to processes underlying analogous modifications of prokaryotic saccharolipid glycans. We also show that PilE undergoes multisite and hierarchical phospho-form modifications and that the stoichiometries of site occupancy can be influenced by PilE primary structure and the abundance of the pilin-like protein PilV. Together, these findings have important implications for the structure and antigenicity of PilE.

A conserved set of pilin-like molecules controls type IV pilus dynamics and organelle-associated functions in Neisseria gonorrhoeae.

Type IV pili (Tfp) play central roles in prokaryotic cell biology and disease pathogenesis. As dynamic filamentous polymers, they undergo rounds of extension and retraction modelled as pilin subunit polymerization and depolymerization events. Currently, the molecular mechanisms and components influencing Tfp dynamics remain poorly understood. Using Neisseria gonorrhoeae as a model system, we show that mutants lacking any one of a set of five proteins sharing structural similarity to the pilus subunit are dramatically reduced in Tfp expression and that these defects are suppressed in the absence of the PilT pilus retraction protein. Thus, these molecules are not canonical assembly factors but rather act as effectors of pilus homeostasis by promoting extension/polymerization events in the presence of PilT. Furthermore, localization studies support the conclusion that these molecules form a Tfp-associated complex and influence levels of PilC, the epithelial cell adhesin, in Tfp-enriched shear fractions. This is the first time that the step at which individual pilin-like proteins impact on Tfp expression has been defined. The findings have important implications for understanding Tfp dynamics and fundamental Tfp structure/function relationships.

Unique modifications with phosphocholine and phosphoethanolamine define alternate antigenic forms of Neisseria gonorrhoeae type IV pili.

Several major bacterial pathogens and related commensal species colonizing the human mucosa express phosphocholine (PC) at their cell surfaces. PC appears to impact host-microbe biology by serving as a ligand for both C-reactive protein and the receptor for platelet-activating factor. Type IV pili of Neisseria gonorrhoeae (Ng) and Neisseria meningitidis, filamentous protein structures critical to the colonization of their human hosts, are known to react variably with monoclonal antibodies recognizing a PC epitope. However, the structural basis for this reactivity has remained elusive. To address this matter, we exploited the finding that the PilE pilin subunit in Ng mutants lacking the PilV protein acquired the PC epitope independent of changes in pilin primary structure. Specifically, we show by using mass spectrometry that PilE derived from the pilV background is composed of a mixture of subunits bearing O-linked forms of either phosphoethanolamine (PE) or PC at the same residue, whereas the wild-type background carries only PE at that same site. Therefore, PilV can influence pilin structure and antigenicity by modulating the incorporation of these alternative modifications. The disaccharide covalently linked to Ng pilin was also characterized because it is present on the same peptides bearing the PE and PC modifications and, contrary to previous reports, was found to be linked by means of 2,4-diacetamido-2,4,6-trideoxyhexose. Taken together, these findings provide new insights into Ng type IV pilus structure and antigenicity and resolve long-standing issues regarding the nature of both the PC epitope and the pilin glycan.

An inhibitor of DNA binding and uptake events dictates the proficiency of genetic transformation in Neisseria gonorrhoeae: mechanism of action and links to Type IV pilus expression.

Although natural genetic transformation is a widely disseminated form of genetic exchange in prokaryotic species, the proficiencies with which DNA recognition, uptake and processing occur in nature vary greatly. However, the molecular factors and interactions underlying intra- and interspecies diversity in levels of competence for natural genetic transformation are poorly understood. In Neisseria gonorrhoeae, the Gram-negative aetiologic agent of gonorrhoea, DNA binding and uptake involve components required for Type IV pilus (Tfp) biogenesis as well as those which are structurally related to Tfp biogenesis components but dispensable for organelle expression. We demonstrate here that the gonococcal PilV protein, structurally related to Tfp pilin subunits, is an intrinsic inhibitor of natural genetic transformation which acts ultimately by reducing the levels of sequence-specific DNA uptake into the cell. Specifically, we show that DNA uptake is enhanced in strains bearing pilV mutations and reduced in strains overexpressing PilV. Furthermore, we show that PilV exerts its effect by acting as an antagonist of ComP, a positive effector of sequence-specific DNA binding. As it prevents the accumulation of ComP at a site where it can be purified by shear extraction of intact cells, the data are most consistent with PilV either obstructing ComP trafficking or altering ComP stability. In addition, we report that ComP and PilV play overlapping and partially redundant roles in Tfp biogenesis and document other genetic interactions between comP and pilV together with the pilE and pilT genes required for the expression of retractile Tfp. Together, the results reveal a novel mechanism by which the levels of competence are governed in prokaryotic species and suggest unique ways by which competence might be modulated.

Competence for natural transformation in Neisseria gonorrhoeae: components of DNA binding and uptake linked to type IV pilus expression.

The mechanisms by which DNA is taken up into the bacterial cell during natural genetic transformation are poorly understood. Although related components essential to the uptake of DNA during transformation have been defined in Gram-negative species, it remains unclear whether DNA binding and uptake are dissociable events. Therefore, DNA uptake has been the earliest definable step in any Gram-negative transformation pathway. In the human pathogen Neisseria gonorrhoeae, sequence-specific DNA uptake requires an intact type IV pili (Tfp) biogenesis machinery along with three molecules that are dispensable for Tfp expression: ComP (a pilin subunit-like molecule), PilT (a cytoplasmic protein involved in pilus retraction) and ComE (a periplasmic protein with intrinsic DNA-binding activity). By conditionally altering the levels of ComP and PilT expression, we show here that DNA binding and uptake are resolvable events. Consequently, we are able to demonstrate that PilT is largely dispensable for functional DNA binding and, therefore, contributes specifically to uptake. Furthermore, sequence specificity in this system is imposed at the level of DNA binding, a process that is influenced by both ComP and PilE. However, sequence-specific DNA binding is not attributable to an intrinsic property of the Tfp subunit protein. Finally, we demonstrate the existence of a robust, non-specific DNA-binding activity associated with the expression of both Tfp and PilT, which is unrelated to transformation but obscures the observation of specific binding events.