Microbial genome dynamics in CNS pathogenesis.

The balancing act between microbes and their host in commensal and disease states needs to be deciphered in order to fully treat and combat infectious diseases. The elucidation of microbial genome dynamics in each instance is therefore required. In this context, the major bacterial meningitis pathogens are Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae. In prokaryotic CNS pathogenesis both the intact organism as well as its released components can elicit disease, often resulting in neurological sequelae, neurodegeneration or fatal outcome. The study of microbial virulence in CNS disease is expected to generate findings that yield new information on the general mechanisms of brain edema and excitatory neuronal disturbances due to meningitis, with significant potential for discoveries that can directly influence and inspire new strategies for prevention and treatment of this serious disease.

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.

Structural alterations in a type IV pilus subunit protein result in concurrent defects in multicellular behaviour and adherence to host tissue.

The ability of bacteria to establish complex communities on surfaces is believed to require both bacterial-substratum and bacterial-bacterial interactions, and type IV pili appear to play a critical but incompletely defined role in both these processes. Using the human pathogen Neisseria gonorrhoeae, spontaneous mutants defective in bacterial self-aggregative behaviour but quantitatively unaltered in pilus fibre expression were isolated by a unique selective scheme. The mutants, carrying single amino acid substitutions within the conserved amino-terminal domain of the pilus fibre subunit, were reduced in the ability to adhere to a human epithelial cell line. Co-expression of the altered alleles in the context of a wild-type pilE gene confirmed that they were dominant negative with respect to aggregation and human cell adherence. Strains expressing two copies of the altered alleles produced twice as much purifiable pili but retained the aggregative and adherence defects. Finally, the defects in aggregative behaviour and adherence of each of the mutants were suppressed by a loss-of-function mutation in the twitching motility gene pilT. The correlations between self-aggregation and the net capacity of the microbial population to adhere efficiently demonstrates the potential significance of bacterial cell-cell interactions to colonization.

Implications of molecular contacts and signaling initiated by Neisseria gonorrhoeae.

Neisseria gonorrhoeae employs diverse strategies with which to adhere to and invade host cells during the course of infection. These primary encounters provide means by which biologically active molecules can be efficiently targeted to disrupt or exploit normal host cell metabolism and immune response elements, which in turn leads to the pathological responses characteristic of gonococcal disease. Current studies have begun to elucidate in detail the molecular interactions orchestrating these processes and the signaling events that they provoke.

Components and dynamics of fiber formation define a ubiquitous biogenesis pathway for bacterial pili.

Type IV pili (Tfp) are a unique class of multifunctional surface organelles in Gram-negative bacteria, which play important roles in prokaryotic cell biology. Although components of the Tfp biogenesis machinery have been characterized, it is not clear how they function or interact. Using Neisseria gonorrhoeae as a model system, we report here that organelle biogenesis can be resolved into two discrete steps: fiber formation and translocation of the fiber to the cell surface. This conclusion is based on the capturing of an intermediate state in which the organelle is retained within the cell owing to the simultaneous absence of the secretin family member and biogenesis component PilQ and the twitching motility/pilus retraction protein PilT. This finding is the first demonstration of a specific translocation defect associated with loss of secretin function, and additionally confirms the role of PilT as a conditional antagonist of stable pilus fiber formation. These findings have important implications for Tfp structure and function and are pertinent to other membrane translocation systems that utilize a highly related set of components.

Charged tmRNA but not tmRNA-mediated proteolysis is essential for Neisseria gonorrhoeae viability.

tmRNA, through its tRNA and mRNA properties, adds short peptide tags to abnormal proteins, targeting these proteins for proteolytic degradation. Although the conservation of tmRNA throughout the bacterial kingdom suggests that it must provide a strong selective advantage, it has not been shown to be essential for any bacterium. We report that tmRNA is essential in Neisseria gonorrhoeae. Although tagging per se appears to be required for gonococcal viability, tagging for proteolysis does not. This suggests that the essential roles of tmRNA in N.gonorrhoeae may include resolving stalled translation complexes and/or preventing depletion of free ribosomes. Although derivatives of N.gonorrhoeae expressing Escherichia coli tmRNA as their sole tmRNA were isolated, they appear to form colonies only after acquiring an extragenic suppressor(s).

The comP locus of Neisseria gonorrhoeae encodes a type IV prepilin that is dispensable for pilus biogenesis but essential for natural transformation.

The expression of type IV pili (Tfp) by Neisseria gonorrhoeae has been shown to be essential for natural genetic transformation at the level of sequence-specific uptake of DNA. All previously characterized mutants defective in this step of transformation either lack Tfp or are altered in the expression of Tfp-associated properties, such as twitching motility, autoagglutination and the ability to bind to human epithelial cells. To examine the basis for this relationship, we identified potential genes encoding polypeptides sharing structural similarities to PilE, the Tfp subunit, within the N. gonorrhoeae genome sequence database. We found that disruption of one such gene, designated comP (for competence-associated prepilin), leads to a severe defect in the capacity to take up DNA in a sequence-specific manner, but does not alter Tfp biogenesis or expression of the Tfp-associated properties of auto-agglutination, twitching motility and human epithelial cell adherence. Indirect evidence based on immunodetection suggests that ComP is expressed at very low levels relative to that of PilE. The process of DNA uptake in gonococci, therefore, is now known to require the expression of at least three distinct components: Tfp, the recently identified PilT protein and ComP.

Crystallographic structure reveals phosphorylated pilin from Neisseria: phosphoserine sites modify type IV pilus surface chemistry and fibre morphology.

Understanding the structural biology of type IV pili, fibres responsible for the virulent attachment and motility of numerous bacterial pathogens, requires a detailed understanding of the three-dimensional structure and chemistry of the constituent pilin subunit. X-ray crystallographic refinement of Neisseria gonorrhoeae pilin against diffraction data to 2.6 A resolution, coupled with mass spectrometry of peptide fragments, reveals phosphoserine at residue 68. Phosphoserine is exposed on the surface of the modelled type IV pilus at the interface of neighbouring pilin molecules. The site-specific mutation of serine 68 to alanine showed that the loss of the phosphorylation alters the morphology of fibres examined by electron microscopy without a notable effect on adhesion, transformation, piliation or twitching motility. The structural and chemical characterization of protein phosphoserine in type IV pilin subunits is an important indication that this modification, key to numerous regulatory aspects of eukaryotic cell biology, exists in the virulence factor proteins of bacterial pathogens. These O-linked phosphate modifications, unusual in prokaryotes, thus merit study for possible roles in pilus biogenesis and modulation of pilin chemistry for optimal in vivo function.

Competence for natural transformation in Neisseria gonorrhoeae: a model system for studies of horizontal gene transfer.

A combined effort integrating studies of gonococcal Tfp biogenesis, the data made available from the gonococcal genome sequence project and applied molecular genetics have been used to identify the fibrillar filaments themselves, the PilT protein and the ComP protein as essential components for the DNA uptake phase of competence for natural transformation. Our ongoing studies are focused on identifying and understanding the complex interactions which exist between these essential constituents. These studies may be relevant not only to the early steps of genetic transformation but also to the two other venues for horizontal gene transfer based on recent findings. First, the thin pili of IncI1 conjugal plasmids required for liquid mating belong to the type IV family of pili (Yoshida et al., 1998). Secondly, type IV pili are required for lysogenic conversion of Vibrio cholerae by a filamentous phage encoding cholera toxin (Waldor and Mekalanos, 1996). How these highly conserved surface organelles contribute to such diverse forms of DNA translocation across membranes remains to be seen.

Suppression of an absolute defect in type IV pilus biogenesis by loss-of-function mutations in pilT, a twitching motility gene in Neisseria gonorrhoeae.

Type IV pili of Neisseria gonorrhoeae, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Gonococcal PilT, a protein belonging to a large family of molecules sharing a highly conserved nucleotide binding domain motif, has been shown to be dispensable for organelle biogenesis but essential for twitching motility and competence for genetic transformation. Here, we show that the defect in pilus biogenesis resulting from mutations in the pilC gene, encoding a putative pilus-associated adhesin for human tissue, can be suppressed by the absence of functional PilT. These data conclusively demonstrate that PilT influences the Type IV pilus biogenesis pathway and strongly suggest that organelle expression is a dynamic process. In addition, these findings imply that PilT antagonizes the process of organelle biogenesis and provide the basis for a model for how the counteractive roles of PilT and PilC might relate mechanistically to the phenomenon of twitching motility.

Structure and function of repetitive sequence elements associated with a highly polymorphic domain of the Neisseria meningitidis PilQ protein.

Secretins are a large family of proteins associated with membrane translocation of macromolecular complexes, and a subset of this family, termed PilQ proteins, is required for type IV pilus biogenesis. We analysed the status of PlIQ expression in Neisseria meningitidis (Mc) and found that PlIQ mutants were non-piliated and deficient in the expression of pilus-associated phenotypes. Sequence analysis of the 5' portion of the pilQ ORF of the serogroup B Mc strain 44/76 showed the presence of seven copies of a repetitive sequence element, in contrast to the situation in N. gonorrhoeae (Gc) strains, which carry either two or three copies of the repeat. The derived amino acid sequence of the consensus nucleotide repeat was an octapeptide PAKQQAAA, designated as the small basic repeat (SBR). This gene segment was studied in more detail in a collection of 52 Mc strains of diverse origin by screening for variability in the size of the PCR-generated DNA fragments spanning the SBRs. These strains were found to harbour from four to seven copies of the repetitive element. No association between the number of copies and the serogroup, geographic origin or multilocus genotype of the strains was evident. The presence of polymorphic repeat elements in Mc PilQ is unprecedented within the secretin family. To address the potential function of the repeat containing domain, Mc strains were constructed so as to express chimeric PilQ molecules in which the number of SBR repeats was increased or in which the repeat containing domain was replaced in toto by the corresponding region of the Pseudomonas aeruginosa (Pa) PilQ protein. Although the strain expressing PilQ with an increased number of SBRs was identical to the parent strain in pilus phenotypes, a strain expressing PilQ with the equivalent Pa domain had an eightfold reduction in pilus expression level. The findings suggest that the repeat containing domain of PilQ influences Mc pilus expression quantitatively but not qualitatively.

PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the Gram-negative aetiological agent of gonorrhoeae, is one of many mucosal pathogens of man that expresses competence for natural transformation. Expression of this phenotype by gonococci appears to rely on the expression of type IV pili (Tfp), but the mechanistic basis for this relationship remains unknown. During studies of gonococcal pilus biogenesis, a homologue of the PilT family of proteins, required for Tfp-dependent twitching motility in Pseudomonas aeruginosa and social gliding motility in Myxococcus xanthus, was discovered. Like the findings in these other species, we show here that gonococcal PilT mutants constructed in vitro no longer display twitching motility. In addition, we demonstrate that they have concurrently lost the ability to undergo natural transformation, despite the expression of structurally and morphologically normal Tpf. These results were confirmed by the findings that two classes of spontaneous mutants that failed to express twitching motility and transformability carried mutations in PilT. Piliated PilT mutants and a panel of pilus assembly mutants were found to be deficient in sequence-specific DNA uptake into the cell, the earliest demonstrable step in neisserial competence. The PilT-deficient strains represent the first genetically defined mutants that are defective in DNA uptake but retain Tfp expression.

General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae.

The general secretion pathway (GSP) of Vibrio cholerae is required for secretion of proteins including chitinase, enterotoxin, and protease through the outer membrane. In this study, we report the cloning and sequencing of a DNA fragment from V. cholerae, containing 12 open reading frames, epsC to -N, which are similar to GSP genes of Aeromonas, Erwinia, Klebsiella, Pseudomonas, and Xanthomonas spp. In addition to the two previously described genes, epsE and epsM (M. Sandkvist, V. Morales, and M. Bagdasarian, Gene 123: 81-86, 1993; L. J. Overbye, M. Sandkvist, and M. Bagdasarian, Gene 132:101-106, 1993), it is shown here that epsC, epsF, epsG, and epsL also encode proteins essential for GSP function. Mutations in the eps genes result in aberrant outer membrane protein profiles, which indicates that the GSP, or at least some of its components, is required not only for secretion of soluble proteins but also for proper outer membrane assembly. Several of the Eps proteins have been identified by use of the T7 polymerase-promoter system in Escherichia coli. One of them, a pilin-like protein, EpsG, was analyzed also in V. cholerae and found to migrate as two bands on polyacrylamide gels, suggesting that in this organism it might be processed or otherwise modified by a prepilin peptidase. We believe that TcpJ prepilin peptidase, which processes the subunit of the toxin-coregulated pilus, TcpA, is not involved in this event. This is supported by the observations that apparent processing of EpsG occurs in a tcpJ mutant of V. cholerae and that, when coexpressed in E. coli, TcpJ cannot process EpsG although the PilD peptidase from Neisseria gonorrhoeae can.

Bacterial pathogenesis: a variation on variation in Lyme disease.

The discovery of antigenic variation in Borrelia burgdorferi, the bacterium that causes Lyme disease, provides a potential explanation for the chronic nature of infection as well as new insights into the genetic structure of highly recombinogenic loci responsible for combinatorial genetic diversification.

The pilus colonization factor of pathogenic neisserial species: organelle biogenesis and structure/function relationships--a review.

Type-IV pilus expression plays a critical role in the interactions between Neisseria gonorrhoeae, Neisseria meningitidis and their human host. We have focused on experiments designed to elucidate the mechanisms of organelle biogenesis as one means of understanding the complexities of pilus biology in these species. Employing a variety of approaches, genes and gene products essential to pilus biogenesis have been identified and characterized. The findings indicate that the neisserial type-IV pilus biogenesis machinery is most closely related to that operating in Pseudomonas aeruginosa and other pseudomonad species. This interrelatedness is documented at the levels of gene organization, DNA homologies and identities between the primary structures of the components. Despite these similarities, the biological correlates of pilus expression in the pathogenic Neisseria are quite unique. The current status of our embryonic understanding of the factors influencing organelle biogenesis is presented. In the context of this workshop, emphasis has been placed on specific contributions made through studies of gonococci and meningococci to the field as a whole..

PilP, a pilus biogenesis lipoprotein in Neisseria gonorrhoeae, affects expression of PilQ as a high-molecular-mass multimer.

Studies of gonococcal pilus biogenesis are fundamental to understanding organelle structure/function relationships and identifying new approaches to controlling disease. This area of research is also relevant to elucidating the basic mechanisms of outer membrane translocation of macromolecules, which requires components highly related to those involved in type IV pilus expression. Previous studies have shown that products of several ancillary pil genes are required for organelle biogenesis but of these only PilQ, a member of the GspD protein family, is a component of the outer membrane. DNA sequencing of the region upstream of pilQ revealed the presence of two open reading frames (ORFs) whose deduced polypeptides shared significant identities with proteins required for pilus expression in Pseudomonas aeruginosa and Pseudomonas syringae, the genes for which are arrayed upstream of a gene encoding a PilQ homologue. Gonococcal mutants bearing transposon insertions in these ORFs were non-piliated and failed to express pilus-associated phenotypes, and the corresponding genes were designated PilO and pilP. The piliation defects in the mutants could not be ascribed to polarity on distal pilQ expression as shown by direct measurement of PilQ antigen in those backgrounds and the use of a novel technique to create tandem duplications in the gonococcus (Gc) genome. As predicted by the presence of a consensus lipoprotein signal sequence, PilP expressed in both Escherichia coli and Gc could be labelled with [3H]-palmitic acid. PilP- as well as PilQ- mutants shed PilC, a protein which facilitates pilus assembly and is implicated in epithelial cell adherence, in a soluble form. Combined with the finding that levels of multimerized PiIQ were greatly reduced in PilP- mutants, the results suggest that PilP is required for PilQ function and that PilQ and PilC may interact during the terminal stages of pilus biogenesis. The findings also support the hypothesis that the Gc PilQ multimer corresponds to a physiologically relevant form of the protein required for pilus biogenesis.

The product of the pilQ gene is essential for the biogenesis of type IV pili in Neisseria gonorrhoeae.

The product of the Neisseria gonorrhoeae omc gene possesses regions homologous to those found in members of a protein superfamily that are associated with the translocation of proteins and DNA-protein complexes across the outer membrane. Amongst its protein homologues, Omc has higher overall homology to PilQ, which is required for type IV pilus expression in Pseudomonas aeruginosa, and OrfE, which is required for sequence-specific DNA uptake by Haemophilus influenzae. The function of Omc, however, is unknown and gonococcal omc mutants have not been described. We constructed gonococcal mutants expressing truncated forms of the protein, and found that these mutants are severely defective for both pilus expression and competence for natural transformation. To be consistent with pre-existing pilus gene nomenclature, we have redesignated the gene pilQ instead of omc, and its product, PilQ instead of Omc. The MS11 gene was sequenced and found to differ from the DNA sequence reported for that of another gonococcal strain; these differences were associated with a repeated DNA element, suggesting a genetic basis for structural variation in PilQ. The results also show that PilQ- mutants are distinct from previously described gonococcal pilus-assembly mutants and P. aeruginosa PilQ- mutants by virtue of their expression of rare pilus filaments. Taking these data into account, PilQ is proposed to function in the terminal steps of organelle biogenesis by acting as a pilus channel or pore.

Single amino acid substitutions in the N-terminus of Vibrio cholerae TcpA affect colonization, autoagglutination, and serum resistance.

The toxin-coregulated pilus (TCP) of Vibrio cholerae O1 is required for successful infection of the host. TcpA, the structural subunit of TCP, belongs to the type IV family of pilins, which includes the PilE pilin of Neisseria gonorrhoeae. Recently, single amino acid changes in the N-terminus of PilE were found to abolish autoagglutination in gonococci. As type IV pilins demonstrate some similarities in function and amino acid sequence, site-directed mutagenesis and allelic exchanges were used to create corresponding mutations in TcpA. All four mutant strains demonstrated autoagglutination defects, and all were highly defective for colonization in the infant mouse model. These results support the previously proposed correlation between autoagglutination and colonization. Finally, all four mutants are serum sensitive, indicating that TcpA plays a role in serum resistance, a phenotype previously attributed to TcpC. As the mutations have similar effects in N. gonorrhoeae and V. cholerae, our results support the idea that type IV pilins have similar functions in a variety of pathogenic bacteria.

Identification and characterization of pilG, a highly conserved pilus-assembly gene in pathogenic Neisseria.

Expression of type IV pili appears to be a requisite determinant of infectivity for the strict human pathogens Neisseria gonorrhoeae and Neisseria meningitidis. The assembly of these colonization factors is a complex process. This report describes a new pilus-assembly gene, pilG, that immediately precedes the gonococcal (Gc) pilD gene encoding the pre-pilin leader peptidase. The nucleotide sequence of this region revealed a single complete open reading frame whose derived polypeptide displayed significant identities to the pilus-assembly protein PilC of Pseudomonas aeruginosa and other polytopic integral cytoplasmic membrane constituents involved in protein export and competence. A unique polypeptide of M(r) 38 kDa corresponding to the gene product was identified. A highly related gene and flanking sequences were cloned from a group B polysaccharide-producing strain of N. meningitidis (Mc). The results indicate that the pilG genes and genetic organization at these loci in Gc and Mc are extremely conserved. Hybridization studies strongly suggest that pilG-related genes exist in commensal Neisseria species and other species known to express type IV pili. Defined genetic lesions were created by using insertional and transposon mutagenesis and moved into the Gc and Mc chromosomes by allelic replacement. Chromosomal pilG insertion mutants were devoid of pili and displayed dramatically reduced competence for transformation. These findings could not be ascribed to pilin-gene alterations or to polarity exerted on pilD expression. The results indicated that PilG exerts its own independent role in neisserial pilus biogenesis.