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

Catalytically active, recombinant fusion proteins of bacteriophage E endosialidase were expressed and purified from Escherichia coli. Constructs with different fusion partners added to the amino terminus of the endosialidase were enzymatically active. A post-translational proteolytic cleavage was shown to occur between serine 706 and aspartate 707 to generate the 76 kDa mature enzyme from the 90 kDa translation product. Endosialidase truncated at the C-terminus from aspartate 707 was observed to have the same 76 kDa molecular weight as wild-type enzyme using denaturing SDS-PAGE but, under native PAGE conditions, was not observed to form the approximately 250 kDa trimeric wild-type enzyme, implying that the C-terminus of the enzyme may be required for correct assembly of active trimer, rather than as part of the active site as has been previously suggested. Mutagenesis of aspartate 138 to alanine greatly reduced enzyme activity whereas conversion of other selected aspartate residues to alanine had less effect, consistent with similarities between the structure and cata-lytic mechanism of bacteriophage E endosialidase and those of exosialidases.     

Sequence-specific DNA uptake in transformation of Neisseria gonorrhoeae

Piliated, competent gonococci are known to preferentially take up homologous transforming DNA into the cell. We examined the mechanism for DNA uptake with pFA10, a hybrid 11.5-kilobase (kb) penicillin-resistant (Pcr) plasmid composed of heterologous DNA from a 7.2-kb Pcr plasmid and homologous DNA from a 4.2-kb gonococcal cryptic plasmid. The presence of the gonococcal cryptic plasmid DNA in the hybrid resulted in markedly increased transformation efficiencies in isogenic crosses as compared with the parent 7.2-kb Pcr plasmid. Uptake of 32P-end-labeled MspI or TaqI restriction fragments of the hybrid was limited to fragments entirely derived from the 4.2-kb gonococcal cryptic plasmid, indicating that DNA uptake was probably dependent on the presence of a specific DNA sequence. Since Haemophilus DNA did not inhibit transformation by the hybrid Pcr plasmid, the gonococcal DNA uptake sequence is different from the known sequence involved in homologous DNA uptake by Haemophilus spp.     

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.     

Opacity determinants of Neisseria gonorrhoeae: gene expression and chromosomal linkage to the gonococcal pilus gene

In N. gonorrhoeae, the expression of pilus and opacity (Op) proteins can be switched on and off and a single cell apparently has a whole repertoire of genes to express many serologically distinguishable protein types. We describe the isolation of several different Op genes and of nonexpressing gene equivalents, all derived from isogenic gonococcal variants. In the E. coli host, Op proteins identical with those made in the respective N. gonorrhoeae strain are produced. The Op genes map near the pilus expression locus. Genomic blotting experiments with an Op gene probe reveal complex hybridization patterns but little heterogeneity among the genes of Op variants. It appears that colonial variation involving the Op protein of N. gonorrhoeae is based on minor sequence alterations, in contrast to the pilus variation system, in which changes in the expression can be evoked by substantial genomic rearrangements.     

Natural transformation of Neisseria gonorrhoeae: from DNA donation to homologous recombination

Gonococci undergo frequent and efficient natural transformation. Transformation occurs so often that the population structure is panmictic, with only one long-lived clone having been identified. This high degree of genetic exchange is likely necessary to generate antigenic diversity and allow the persistence of gonococcal infection within the human population. In addition to spreading different alleles of genes for surface markers and allowing avoidance of the immune response, transformation facilitates the spread of antibiotic resistance markers, a continuing problem for treatment of gonococcal infections. Transforming DNA is donated by neighbouring gonococci by two different mechanisms: autolysis or type IV secretion. All types of DNA are bound non-specifically to the cell surface. However, for DNA uptake, Neisseria gonorrhoeae recognizes only DNA containing a 10-base sequence (GCCGTCTGAA) present frequently in the chromosome of neisserial species. Type IV pilus components and several pilus-associated proteins are necessary for gonococcal DNA uptake. Incoming DNA is subject to restriction, making establishment of replicating plasmids difficult but not greatly affecting chromosomal transformation. Processing and integration of transforming DNA into the chromosome involves enzymes required for homologous recombination. Recent research on DNA donation mechanisms and extensive work on type IV pilus biogenesis and recombination proteins have greatly improved our understanding of natural transformation in N. gonorrhoeae. The completion of the gonococcal genome sequence has facilitated the identification of additional transformation genes and provides insight into previous investigations of gonococcal transformation. Here we review these recent developments and address the implications of natural transformation in the evolution and pathogenesis N. gonorrhoeae.     

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.     

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.     

Mutation of the priA gene of Neisseria gonorrhoeae affects DNA transformation and DNA repair

In Escherichia coli, PriA is central to the restart of chromosomal replication when replication fork progression is disrupted and is also involved in homologous recombination and DNA repair. To investigate the role of PriA in recombination and repair in Neisseria gonorrhoeae, we identified, cloned, and insertionally inactivated the gonococcal priA homologue. The priA mutant showed a growth deficiency and decreased DNA repair capability and was completely for deficient in DNA transformation compared to the isogenic parental strain. The priA mutant was also more sensitive to the oxidative damaging agents H2O2 and cumene hydroperoxide compared to the parental strain. These phenotypes were complemented by supplying a functional copy of priA elsewhere in the chromosome. The N. gonorrhoeae priA mutant showed no alteration in the frequency of pilin antigenic variation. We conclude that PriA participates in DNA repair and DNA transformation processes but not in pilin antigenic variation.     

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.     

Control of pilus expression in Neisseria gonorrhoeae as an original system in the family of two-component regulators

We have previously reported the identification of two genes, pilA and pilB, which act in trans to regulate pilus expression in Neisseria gonorrhoeae. Here we show that PilA and PilB have amino acid sequence similarities with members of the two component 'sensor-regulator' family of proteins. PilB has homology with histidine kinase sensors. Alkaline phosphatase fusions to the predicted sensor and transmitter domains are described. Their PhoA activity and cellular location suggest that PilB is inserted in the cytoplasmic membrane and predict periplasmic and cytoplasmic locations for the sensor and the transmitter domains, respectively. PilA has homology with response regulators in its N-terminal part, and with components of the eukaryotic protein secretory apparatus (SRP 54 and SRP receptor) as well as two Escherichia coli gene products in its C-terminal part. In particular, it contains a putative GTP-binding site. Mini-transposon insertions into different regions of pilA were obtained. The phenotypes and genotypes of these mutants and preliminary biochemical studies of the gene products of two of these mutants lend further support to the hypothesis that PilA is a DNA-binding response regulator and confirm that it participates in an essential function in the bacterium.     

Limited variation and maintenance of tight genetic linkage characterize heteroallelic pilE recombination following DNA transformation of Neisseria gonorrhoeae

Genetic recombination impacts on neisserial biology in two ways: (i) specific loci undergo rearrangement at high frequency leading to the formation of many different alleles; and (ii) Neisseria , being naturally competent for DNA transformation, provide a means to disseminate the novel alleles throughout a population. In this study pilE was used as a model system to examine heteroallelic recombination following DNA transformation. When gonococci were transformed with chromosomal donor DNA containing different pilE alleles, the majority of pilE recombinants arose through allelic replacement. Co-conversion analysis across pilE showed that in ∼ 85–90% of recombination events encompassing pilE and an adjacent opa locus, linkage was maintained (i.e. ∼ 10–15% of recombination events terminated within the ∼ 1000 base pair pilE/opaE interval). In addition to those recombinants that arose through allelic replacement, a large pilus-minus subpopulation was also observed (∼ 10% of all recombinants), indicating that many recombination events did not yield recombinant pilE s that could be assembled into functional pili. PilE mosaics increased following transformation with plasmid donor DNAs carrying pilE with limited flanking-sequence homology, suggesting a potential role for flanking-sequence homologies in mosaic formation. Overall, the data support the view that horizontal transmission of chromosomal DNA between gonococci will favour the spread of intact alleles, as opposed to expanding the allelic repertoire through the formation of gene mosaics.     

Structure of the Vibrio cholerae Type IVb Pilus and stability comparison with the Neisseria gonorrhoeae type IVa pilus

Type IV pili are multifunctional filaments displayed on many bacterial pathogens. Members of the Type IVa pilus subclass are found on a diverse group of human pathogens, whereas Type IVb pili are found almost exclusively on enteric bacteria. The Type IVa and IVb subclasses are distinguished by differences in the pilin subunits, including the fold of the globular domain. To understand the implications of the distinct pilin folds, we compared the stabilities of pilin subunits and pilus filaments for the Type IVa GC pilus from Neisseria gonorrhoeae and the Type IVb toxin-coregulated pilus (TCP) from Vibrio cholerae. We show that while recombinant TCP pilin is more stable than GC pilin, the GC pili are more resistant to proteolysis, heat and chemical denaturation than TCP, remaining intact in 8?M urea. To understand these differences, we determined the TCP structure by electron microscopy and three-dimensional image reconstruction. TCP have an architecture similar to that of GC pili, with subunits arranged in a right-handed 1-start helix and related by an 8.4-? axial rise and a 96.8° azimuthal rotation. However, the TCP subunits are not as tightly packed as GC pilins, and the distinct Type IVb pilin fold exposes a segment of the α-helical core of TCP. Hydrophobic interactions dominate for both pilus subtypes, but base stacking by aromatic residues conserved among the Type IVa pilins may contribute to GC pilus stability. The extraordinary stability of GC pili may represent an adaptation of the Type IVa pili to harsh environments and the need to retract against external forces.     

Sequence changes in the pilus subunit lead to tropism variation of Neisseria gonorrhoeae to human tissue

Summary
Pili of Neisseria gonorrhoeae are correlated with Increased bacterial attachment to epithelial cells and undergo both phase and antigenic variation. Phase variation of gonococcal pili can be brought about by recombination events in the pilin structural gene, pilE , or by the on/off switch in expression of PilC, a pilus biogenesis protein for which two loci exist. We have studied the binding to epithelial cell lines and to fixed tissue sections of N. gonorrhoeae MS11 derivatives and mutants carrying structurally defined PilE and PilC proteins, in situ binding studies of N. gonorrhoeae to formalin-fixed tissue sections resulted in a binding pattern similar to that obtained using viable epithelial cell lines of different origin. Piliated gonococcal clones, containing different pilE sequences, varied dramatically from one another in their efficiencies at binding to corneal and conjunctival tissue, but bound equally well to cervical and endometrial tissues. Further, the binding data suggested that PJIC expression by itself, i.e. without pili, cannot confer bacterial binding and that expression of either PilC1 or PiiC2 does not confer different binding properties to the bacterial cells. Possible receptors for piliated gonococci were expressed in human tissues, such as cervix, endometrium, cornea, intestine, stomach, mid-brain and meninges, but not in human kidney. Pretreatment of the target tissues with Proteinase K decreased the gonococcal binding dramatically, whereas pretreatment with neuraminidase and meta-periodate, which cleave carbon-carbon linkages between vicinal hydroxyl groups in carbohydrates, did not affect attachment of gonococci. These data argue that pilus-dependent attachment of N. gonorrhoeae to human tissue may be mediated by a eukaryotic receptor having protein characteristics, and that the pilus subunit sequence may play an important role in the interaction with human cornea.     

Type IV pilus biogenesis in Neisseria meningitidis: PilW is involved in a step occurring after pilus assembly, essential for fibre stability and function

Type IV pili (Tfp) play a critical role in the pathogenic lifestyle of Neisseria meningitidis and N. gonorrhoeae, notably by facilitating bacterial attachment to human cells, but our understanding of their biogenesis, during which the fibres are assembled in the periplasm, then emerge onto the cell surface and are stabilized, remains fragmentary. We therefore sought to identify the genes required for Tfp formation in N. meningitidis by screening a genome-wide collection of mutants for those that were unable to form aggregates, another phenotype mediated by these organelles. Fifteen proteins, of which only seven were previously characterized, were found to be essential for Tfp biogenesis. One novel component, named PilW, was studied in more detail. We found that PilW is an outer-membrane protein necessary for the stabilization of the fibres but not for their assembly or surface localization, because Tfp could be restored on the surface in a pilW mutant by a mutation in the twitching motility gene pilT. However, Tfp-linked properties, including adherence to human cells, were not restored in a pilW/T mutant, which suggests that PilW is also essential for the functionality of the fibres. Together with the finding that PilW is important for the stability of PilQ multimers, our results extend the current model for Tfp biogenesis by suggesting that a multiprotein machinery in the outer-membrane is involved in the terminal stage of Tfp biogenesis during which growing fibres are not only stabilized, but also become perfectly functional.     

Pilin-like proteins in the extremely thermophilic bacterium Thermus thermophilus HB27: implication in competence for natural transformation and links to type IV pilus biogenesis

The extreme thermophile Thermus thermophilus HB27 exhibits high frequencies of natural transformation. Although we recently reported identification of the first competence genes in Thermus, the molecular basis of DNA uptake is unknown. A pilus-like structure is assumed to be involved. Twelve genes encoding prepilin-like proteins were identified in three loci in the genome of T. thermophilus. Mutational analyses, described in this paper, revealed that one locus, which contains four genes that encode prepilin-like proteins (pilA1 to pilA4), is essential for natural transformation. Additionally, comZ, a new competence gene with no similarity to known genes, was identified. Analysis of the piliation phenotype revealed wild-type piliation of a pilA1-pilA3Deltakat mutant and a comZ mutant, whereas a pilA4 mutant was found to be completely devoid of pilus structures. These findings, together with the significant similarity of PilA4 to prepilins, led to the conclusion that the T. thermophilus pilus structures are type IV pili. Furthermore, the loss of the transformation and piliation phenotype in the pilA4 mutant suggests that type IV pili are implicated in natural transformation of T. thermophilus HB27.     

Attachment of Neisseria gonorrhoeae to the cellular pilus receptor CD46: identification of domains important for bacterial adherence

Pili of Neisseria gonorrhoeae mediate binding of the bacteria to human host cells. Membrane cofactor protein (MCP or CD46), a human cell-surface protein involved in regulation of complement activation, acts as a cellular pilus receptor. In this work, we examined which domains of CD46 mediate bacterial adherence. The CD46 expression was quantified and characterized in human epithelial cell lines. N. gonorrhoeae showed the highest adherence to ME180 cells, which have BC1 as the dominant phenotype. The BC isoforms of CD46 were expressed in all cell lines tested. The adherence was not enhanced by high expression of other isoforms, showing that the BC domain of CD46 is important in adherence of N. gonorrhoeae to human cells. To characterize the pilus-binding site within the CD46 molecule, a set of CD46–BC1 deletion constructs were transfected into COS-7 cells. Piliated N. gonorrhoeae attached well to CD46–BC1-expressing COS-7 cells. We show that the complement control protein repeat 3 (CCP-3) and the serine–threonine–proline (STP)-rich domain of CD46 are important for efficient adherence to host cells. Further, partial deletion of the cytoplasmic tail of CD46 results in low bacterial binding, indicating that the cytoplasmic tail takes part in the process of establishing a stable interaction between N. gonorrhoeae and host cells.     

Differential roles of homologous recombination pathways in Neisseria gonorrhoeae pilin antigenic variation, DNA transformation and DNA repair

Neisseria gonorrhoeae (Gc) pili undergo antigenic variation when the amino acid sequence of the pilin protein is changed, aiding in immune avoidance and altering pilus expression. Pilin antigenic variation occurs by RecA-dependent unidirectional transfer of DNA sequences from a silent pilin locus to the expressed pilin gene through high-frequency recombination events that occur at limited regions of homology. We show that the Gc recQ and recO genes are essential for pilin antigenic and phase variation and DNA repair but are not involved in natural DNA transformation. This suggests that a RecF-like pathway of recombination exists in Gc. In addition, mutations in the Gc recB, recC or recD genes revealed that a Gc RecBCD pathway also exists and is involved in DNA transformation and DNA repair but not in pilin antigenic variation.     

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.