Hanne C. Winther-Larsen

Neisseria gonorrhoeae PilV, a type IV pilus-associated protein essential to human epithelial cell adherence

Type IV pili (Tfp) of Neisseria gonorrhoeae, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Tfp are assumed to play a key role in the initial adherence to human epithelial cells by virtue of the associated adhesin protein PilC. To examine the structural and functional basis for adherence in more detail, we identified potential genes encoding polypeptides sharing structural similarities to PilE (the Tfp subunit) within the N. gonorrhoeae genome sequence database. We show here that a fiber subunit-like protein, termed PilV, is essential to organelle-associated adherence but dispensable for Tfp biogenesis and other pilus-related phenotypes, including autoagglutination, competence for natural transformation, and twitching motility. The adherence defect in pilV mutants cannot be attributed to reduced levels of piliation, defects in fiber anchoring to the bacterial cell surface, or to unstable pilus expression related to organelle retraction. PilV is expressed at low levels relative to PilE and copurifies with Tfp fibers in a PilC-dependent fashion. Purified Tfp from pilV mutants contain PilC adhesin at reduced levels. Taken together, these data support a model in which PilV functions in adherence by promoting the functional display of PilC in the context of the pilus fiber.

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.

Infection of human mucosal tissue by Pseudomonas aeruginosa requires sequential and mutually dependent virulence factors and a novel pilus-associated adhesin

Tissue damage predisposes humans to life‐threatening disseminating infection by the opportunistic pathogen Pseudomonas aeruginosa. Bacterial adherence to host tissue is a critical first step in this infection process. It is well established that P. aeruginosa attachment to host cells involves type IV pili (TFP), which are retractile surface fibres. The molecular details of attachment and the identity of the bacterial adhesin and host receptor remain controversial. Using a mucosal epithelium model system derived from primary human tissue, we show that the pilus‐associated protein PilY1 is required for bacterial adherence. We establish that P. aeruginosa preferentially binds to exposed basolateral host cell surfaces, providing a mechanistic explanation for opportunistic infection of damaged tissue. Further, we demonstrate that invasion and fulminant infection of intact host tissue requires the coordinated and mutually dependent action of multiple bacterial factors, including pilus fibre retraction and the host cell intoxication system, termed type III secretion. Our findings offer new and important insights into the complex interactions between a pathogen and its human host and provide compelling evidence that PilY1 serves as the principal P. aeruginosa adhesin for human tissue and that it specifically recognizes a host receptor localized or enriched on basolateral epithelial cell surfaces.

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.

Genetic, structural, and antigenic analyses of glycan diversity in the O-linked protein glycosylation systems of human Neisseria species.

Bacterial capsular polysaccharides and lipopolysaccharides are well-established ligands of innate and adaptive immune effectors and often exhibit structural and antigenic variability. Although many surface-localized glycoproteins have been identified in bacterial pathogens and symbionts, it not clear if and how selection impacts associated glycoform structure. Here, a systematic approach was devised to correlate gene repertoire with protein-associated glycoform structure in Neisseria species important to human health and disease. By manipulating the protein glycosylation (pgl) gene content and assessing the glycan structure by mass spectrometry and reactivity with monoclonal antibodies, it was established that protein-associated glycans are antigenically variable and that at least nine distinct glycoforms can be expressed in vitro. These studies also revealed that in addition to Neisseria gonorrhoeae strain N400, one other gonococcal strain and isolates of Neisseria meningitidis and Neisseria lactamica exhibit broad-spectrum O-linked protein glycosylation. Although a strong correlation between pgl gene content, glycoform expression, and serological profile was observed, there were significant exceptions, particularly with regard to levels of microheterogeneity. This work provides a technological platform for molecular serotyping of neisserial protein glycans and for elucidating pgl gene evolution.

O-Linked Glycosylation of the PilA Pilin Protein of Francisella tularensis: Identification of the Endogenous Protein-Targeting Oligosaccharyltransferase and Characterization of the Native Oligosaccharide

Findings from a number of studies suggest that the PilA pilin proteins may play an important role in the pathogenesis of disease caused by species within the genus Francisella. As such, a thorough understanding of PilA structure and chemistry is warranted. Here, we definitively identified the PglA protein-targeting oligosaccharyltransferase by virtue of its necessity for PilA glycosylation in Francisella tularensis and its sufficiency for PilA glycosylation in Escherichia coli. In addition, we used mass spectrometry to examine PilA affinity purified from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica and demonstrated that the protein undergoes multisite, O-linked glycosylation with a pentasaccharide of the structure HexNac-Hex-Hex-HexNac-HexNac. Further analyses revealed microheterogeneity related to forms of the pentasaccharide carrying unusual moieties linked to the distal sugar via a phosphate bridge. Type A and type B strains of Francisella subspecies thus express an O-linked protein glycosylation system utilizing core biosynthetic and assembly pathways conserved in other members of the proteobacteria. As PglA appears to be highly conserved in Francisella species, O-linked protein glycosylation may be a feature common to members of this genus.

Intracellular localisation and innate immune responses following Francisella noatunensis infection of Atlantic cod (Gadus morhua) macrophages

The facultative intracellular bacterium Francisella noatunensis causes francisellosis in Atlantic cod (Gadus morhua), but little is known about its survival strategies or how these bacteria evade the host immune response. In this study we show intracellular localisation of F. noatunensis in cod macrophages using indirect immunofluorescence techniques and green fluorescent labelled bacteria. Transmission electron microscopy revealed that F. noatunensis was enclosed by a phagosomal membrane during the initial phase of infection. Bacteria were at a later stage of the infection found in large electron-lucent zones, apparently surrounded by a partially intact or disintegrated membrane. Immune electron microscopy demonstrated the release of bacterial derived vesicles from intracellular F. noatunensis, an event suspected of promoting phagosomal membrane degradation and allowing escape of the bacteria to cytoplasm. Studies of macrophages infected with F. noatunensis demonstrated a weak activation of the inflammatory response genes as measured by increased expression of the Interleukin (IL)-1β and IL-8. In comparison, a stronger induction of gene expression was found for the anti-inflammatory IL-10 indicating that the bacterium exhibits a role in down-regulating the inflammatory response. Expression of the p40 subunit of IL-12/IL-17 genes was highly induced during infection suggesting that F. noatunensis promotes T cell polarisation. The host macrophage responses studied here showed low ability to distinguish between live and inactivated bacteria, although other types of responses could be of importance for such discriminations. The immunoreactivity of F. noatunensis lipopolysaccharide (LPS) was very modest, in contrast to the strong capacity of Escherichia coli LPS to induce inflammatory responsive genes. These results suggest that F. noatunensis virulence mechanisms cover many strategies for intracellular survival in cod macrophages.

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 α‐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.

Genetic and functional analyses of PptA, a phospho-form transferase targeting type IV pili in Neisseria gonorrhoeae.

The PilE pilin subunit protein of Neisseria gonorrhoeae undergoes unique covalent modifications with phosphoethanolamine (PE) and phosphocholine (PC). The pilin phospho-form transferase A (PptA) protein, required for these modifications, shows sequence relatedness with and architectural similarities to lipopolysaccharide PE transferases. Here, we used regulated expression and mutagenesis as means to better define the relationships between PptA structure and function, as well as to probe the mechanisms by which other factors impact the system. We show here that pptA expression is coupled at the level of transcription to its distal gene, murF, in a division/cell wall gene operon and that PptA can act in a dose-dependent fashion in PilE phospho-form modification. Molecular modeling and site-directed mutagenesis provided the first direct evidence that PptA is a member of the alkaline phosphatase superfamily of metalloenzymes with similar metal-binding sites and conserved structural folds. Through phylogenetic analyses and sequence alignments, these conclusions were extended to include the lipopolysaccharide PE transferases, including members of the disparate Lpt6 subfamily, and the MdoB family of phosphoglycerol transferases. Each of these enzymes thus likely acts as a phospholipid head group transferase whose catalytic mechanism involves a trans-esterification step generating a protein-phospho-form ester intermediate. Coexpression of PptA with PilE in Pseudomonas aeruginosa resulted in high levels of PE modification but was not sufficient for PC modification. This and other findings show that PptA-associated PC modification is governed by as-yet-undefined ancillary factors unique to N. gonorrhoeae.

Pseudomonas aeruginosa Type IV pilus expression in Neisseria gonorrhoeae: effects of pilin subunit composition on function and organelle dynamics.

Type IV pili (TFP) play central roles in the expression of many phenotypes including motility, multicellular behavior, sensitivity to bacteriophages, natural genetic transformation, and adherence. In Neisseria gonorrhoeae, these properties require ancillary proteins that act in conjunction with TFP expression and influence organelle dynamics. Here, the intrinsic contributions of the pilin protein itself to TFP dynamics and associated phenotypes were examined by expressing the Pseudomonas aeruginosa PilA(PAK) pilin subunit in N. gonorrhoeae. We show here that, although PilA(PAK) pilin can be readily assembled into TFP in this background, steady-state levels of purifiable fibers are dramatically reduced relative those of endogenous pili. This defect is due to aberrant TFP dynamics as it is suppressed in the absence of the PilT pilus retraction ATPase. Functionally, PilA(PAK) pilin complements gonococcal adherence for human epithelial cells but only in a pilT background, and this property remains dependent on the coexpression of both the PilC adhesin and the PilV pilin-like protein. Since P. aeruginosa pilin only moderately supports neisserial sequence-specific transformation despite its assembly proficiency, these results together suggest that PilA(PAK) pilin functions suboptimally in this environment. This appears to be due to diminished compatibility with resident proteins essential for TFP function and dynamics. Despite this, PilA(PAK) pili support retractile force generation in this background equivalent to that reported for endogenous pili. Furthermore, PilA(PAK) pili are both necessary and sufficient for bacteriophage PO4 binding, although the strain remains phage resistant. Together, these findings have significant implications for TFP biology in both N. gonorrhoeae and P. aeruginosa.