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Living third- and fourth-stage larvae (L3 and L4) of Acanthocheilonema viteae were recovered quantitatively from adult Meriones unguiculatus within the first 10 days after subcutaneous inoculation of 60 arthropod-derived larvae (mL3). The average recovery of the inoculated larvae was about one third (28.5%), and the majority (87.7%) were found in muscular tissues. Seventy-two hours after inoculation, larvae could be isolated from all body locations, although the majority still was found near the site of inoculation. Morphological and biometrical data indicated that, at least until molting, the development of the larval population was not synchronous, with molting occurring over a period of 48 hr on days 7 and 8 postinoculation. The stomatal rings of postinvasive L3's and L4's were distinguishable structurally and could be used as stage-specific determinants. Immediately after infection, L3's showed a linear growth in diameter; rapid longitudinal growth started after the molt, leading to a doubling in the length of L4's within 4 days. The time course of shedding was reconstructed in detail using isolated L3/L4 intermediates. A critical event in the life cycle of a parasitic

The gene complex encoding all determinants of the biosynthesis pathway of the capsule of group B meningococci (cps) has been cloned in Escherichia coli. A 24-kilobase large chromosomal fragment is necessary for capsule expression on the E. coli surface. By transposon and deletion mutagenesis, two separate steps in transport of the polysaccharide from the cytoplasm to the periplasm and further to the cell surface became evident. Mutants were also isolated that accumulate soluble poly(sialic acid) in the cytoplasm. The cloned cps complex conferred to E. coli strain GC6 sensitivity for E. coli K1-specific phages; phage sensitivity was enhanced in two distinct classes of cps mutants. Southern blot experiments revealed homology to some or all other Neisseria meningitidis capsular types and other Neisseria species, depending on the fragment of the cps complex used as probe.

An enzyme system with requirements similar to those for replication of phage fd replicative form (RF) DNA in bacteriophage fd-infected cells has been reconstituted with purified fd gene 2 protein, and DNA polymerase III holoenzyme, DNA binding protein I and rep-protein (rep-helicase) of Escherichia coli. The system generates viral circular single strands, which are infective for E. coli spheroplasts. Parental and newly synthesized DNA are covalently connected in early stages of replication, as expected for DNA replication using the rolling circle mechanism. Single-stranded tails of the rolling circle intermediates are cleaved after a full round of replication by gene 2 protein and circularized by the same enzyme molecule.

The gene for Escherichia coli rep helicase (rep protein) was subcloned in a pBR plasmid and the protein overproduced in cells transformed with the hybrid DNA. The effect of purified enzyme on strand unwinding and DNA replication was investigated by electron microscopy. The templates used were partial duplexes of viral DNA from bacteriophage fd::Tn5 and reannealed DNA from bacteriophage Mu. The experiments with the two DNA species show DNA unwinding uncoupled from replication. The single-stranded phage fd::Tn5 DNA with the inverted repeat of transposon Tn5 could be completely replicated in the presence of the E. coli enzymes rep helicase, DNA binding protein I, RNA polymerase and DNA polymerase III holoenzyme. A block in the unwinding step increases secondary initiation events in single-stranded parts of the template, as DNA polymerase III holoenzyme cannot switch across the stem structure of the transposon.

Bacteriophage fd gene 2 protein was specifically labeled with radioactive amino acids and was isolated from membranous cell structures as an apparently homogenous protein. Amino acid sequence analysis revealed that the protein was initiated at two distinct AUG codons close to the ribosome binding site. The two resulting translation products were found to begin with a deformylated methionine residue. Initiation at the first signal was used for 90% of the chains and at the second signal for 10% of the sequenced molecules. The use of one or the other chain start may influence functions of gene 2 protein.

A cloned 5 kb DNA fragment from Neisseria gonorrhoeae strain MS11 promotes expression and excretion of IgA protease in E. coli and other Gram-negative hosts. DNA sequencing reveals a large open reading frame coding for a precursor molecule of 169 kd. The 106 kd mature IgA protease is released from the bacteria in conjunction with a 15 kd soluble precursor segment, the alpha-protein. In contrast, the carboxy terminal portion of the precursor, the beta-protein (45 kd), remains associated with the outer bacterial membrane. The three proteins result form autoproteolytic cleavage at sites in the precursor which are similar to the target site in IgA1. Consensus sequences of the specific cleavage sites are found in a number of relevant human proteins. IgA protease may therefore have other natural substrates besides IgA1. The soluble alpha-protein as well as the membrane bound beta-protein, both associated with IgA protease, may confer additional virulence functions to the gonococcus.

Bacteriophage fd gene 2 was cloned in plasmid pBR325. Cells carrying the hybrid plasmid produce about 200 times more enzymatically active fd gene 2 protein than did cells infected with phage fd wild type, as measured by replication of phage fd replicative form I in vitro. Cloned gene 2 supports replication of an artificial phage fd miniplasmid consisting of the origin of bacteriophage fd replication and a gene coding for kanamycin resistance. This plasmid occurs in high copy numbers and is viable only in cells carrying the cloned fd gene 2 or in cells infected with phage fd. Because the miniplasmid is not propagated in natural hosts, it can be considered a safe cloning vector. Its fusion with the gene 2 hybrid plasmid provides an autonomous replicon independent of the polA function of the host cell. fd gene 2 is the only phage-encoded trans-acting function required for replication of double-stranded fd DNA in vivo.

Gonococcal virulence is thought to rely on multiple characteristics including the production of an extracellular protease specific for human IgA1. Using a sensitive filter assay we have isolated an Escherichia coli clone which harbours the gene of Neisseria gonorrhoeae MS11 IgA protease on a multicopy number plasmid. This clone secrets IgA protease activity to an extent similar to that of the parental MS11 strain. By exonucleolytic digestion of the cloned insert we obtained a fragment of 4.6 kb which could not be shortened further without loss of IgA protease expression. Compared with the cloned IgA protease gene from N. gonorrhoeae F62, this minimal gene segment shows marked differences in the arrangement of restriction sites. We suppose that these differences determine strain-specific variations of N. gonorrhoeae IgA proteases and also affect the secretory properties of the enzyme when produced in E. coli. A novel purification procedure developed for IgA protease of N. gonorrhoeae allowed us to correlate the enzyme activity with a distinct protein band in SDS acrylamide gels. By comparison with the enzyme prepared from the E. coli clone, we identified a 105-kd protein as the extracellular form of gonococcal IgA protease.

Pathogenic Neisseriae vary the expression and properties of two essential surface proteins, thereby abrogating the immune surveillance of their human hosts. A single bacterium contains a wide repertoire of variant genes which are subject to control by intriguing genetic processes; one of these processes depends on homologous recombination while the other does not.

Propionibacterium acnes has been detected in diseased human prostate tissue, and cell culture experiments suggest that the bacterium can establish a low-grade inflammation. Here, we investigated its impact on human primary prostate epithelial cells. Microarray analysis confirmed the inflammation-inducing capability of P. acnes but also showed deregulation of genes involved in the cell cycle. qPCR experiments showed that viable P. acnes downregulates a master regulator of cell cycle progression, FOXM1. Flow cytometry experiments revealed that P. acnes increases the number of cells in S-phase. We tested the hypothesis that a P. acnes-produced berninamycin-like thiopeptide is responsible for this effect, since it is related to the FOXM1 inhibitor siomycin. The thiopeptide biosynthesis gene cluster was strongly expressed; it is present in subtype IB of P. acnes, but absent from type IA, which is most abundant on human skin. A knock-out mutant lacking the gene encoding the berninamycin-like peptide precursor was unable to downregulate FOXM1 and to halt the cell cycle. Our study reveals a novel host cell-interacting activity of P. acnes.

DOUBLE-STRAND DNA replication of small single-stranded bacteriophages such as ΦX174 and filamentous phage fd is initiated by highly specific endonucleases. This was demonstrated in vitro with the isolation of ΦX cisA protein1 and more recently with that of fd gene II-protein2. Both endonucleases produce one single-strand break in the viral strand of superhelical RFI providing an origin for double-strand (ds) DNA replication3,4. For gene II-protein the strand specificity of nicking was demonstrated by cleavage of asymmetrically labelled fd RFI (viral strand 32P-labelled, complementary strand 3H-labelled) and subsequent alkaline sucrose sedimentation. As a result the viral strand was cleaved into a full length linear form, whereas the complementary strand remained circular (T.F.M. and K.G., submitted). Here we report the position of the nick in the nucleotide sequence of the fd genome.

Filamentous bacteriophages (fd, M13, f1) contain single-stranded circular DNA of about 6400 bases (1). They penetrate the host cell via pili induced by the F-episome of an Escherichia coli cell. During the penetration process their single-stranded genome is converted into double-stranded DNA which is the main viral component in the first minutes after infection. Later in the life cycle, viral single strands are formed and complexed with gene 5 protein. They are assembled in the host membrane into phage particles, which penetrate the cell wall without severe damage of the host. Filamentous phages are quite flexible on the size of the DNA to be packaged. They spontaneously generate miniphages of about 1 kb (2, 3), but they can also comprise artificial DNA sequences up to a length of 15 kb, if the phage packaging signal is provided (4).

Using purified enzymes, double strand replication of phage fd DNA has been dissected into several intermediate steps. (i) Phage fd gene 2 protein cleaves supercoiled phage fd replicative form at a specific site in the viral strand (Meyer, T. F., Geider, K., Kurz, C., and Schaller, H. (1979) Nature 278, 365-367). (ii) Relaxed covalently closed circular replicative form DNA which is also formed by gene 2 protein as a side product in the initiation reaction preceding replication is converted into supercoils by DNA gyrase. (iii) The enzyme forms a noncovalent complex at the generated nick that is necessary for initiation of subsequent unwinding. (iv) The Escherichia coli rep helicase (rep protein) and E. coli DNA binding protein I unwind the double-stranded DNA. (v) Concomitant DNA replication by E. coli DNA polymerase III holoenzyme results in the formation of rolling circle intermediates. The double-stranded core of the rolling circle remains in an open form, thus allowing continued synthesis during several rounds of replication. (vi) Processing of replicated viral DNA can be subdivided into the cleavage and the circularization of viral single strands. Comparative studies of fd and phi X174 replication in vitro have revealed differences in the kinetics of individual steps besides an apparent contrast in the conformation of rolling circle intermediates in the electron microscopy where fd DNA features extended tails rather than looped-back structures observed for phi X174 DNA.

The gram-negative coccus Neisseria gonorrhoeae is a major cause of sexually transmitted disease in humans. This bacterium represents a model for several intriguing virulence mechanisms. Most striking is its tremendous capacity to vary its major surface antigens, assumed to be an effective means for evading the immune surveillance of the infected human host. Antigenic variation of the surface components may further facilitate efficient adaptation of the pathogen to environmental changes in the host during the course of an infection. Of the known surface components pilin, the subunit component of the pili, and opacity protein (Op or PII) of gonococci, as well as the Op-related class 5 proteins of meningococci, have received particular attention (1–3). These proteins seem to play a central role in colonization of the pathogens in the human mucosal tissue (4,5). The expression of pili and Op can be turned on and off (phase variation), and a single cell can give rise to cells that produce structurally and antigenically different proteins (antigenic variation). In addition, more than one variant protein can be produced at a time. The total number of variant proteins expressed appears to be strikingly large. To assess the molecular principles underlying this complex phenomenons, we have isolated and characterized several genes coding for pilin and Op (7,8).

Oligonucleotide probes constructed from the sequences published for Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) genes and antibody against the purified toxins were used to study the SLT (SLT-IIp) produced by porcine E. coli O138 and O139 strains. By DNA hybridization assays no homology was observed between SLT-I and SLT-IIp. By contrast the oligonucleotide probe derived from the slt-II A gene detected porcine strains of E. coli producing SLT-IIp and E. coli strains associated with human disease producing SLT-II. Homology of nucleotide sequences between SLT-IIp and SLT-II is reflected by serological cross-reactivity as demonstrated by a dot blot ELISA and neutralization of SLT-IIp with anti-SLT-II. The toxins were distinguishable in their ability to kill HeLa S-3 cells. The oligonucleotide probe and anti-SLT-II can facilitate identification of SLT-IIp producing E. coli to further clarify their role in diseased pigs.

The expression of Neisseria gonorrhoeae opacity protein shows frequent phase transitions. The genome contains at least twelve copies of the opa gene. Each copy is complete and different from most of the others in certain hypervariable regions. All opa genes are constitutively transcribed. Part of the leader peptide of all Op's is encoded by repetitive CTTCT pentameric units, the so-called coding repeat (CR). The number of repeat units found in the genes and mRNAs is subject to frequent and precise changes. Such changes affect the expression of individual opa genes at the translational level. This control mechanism is common also to the class 5 proteins of N. meningitidis and the Op-related proteins of N. lactamica.

DNA cloning vectors were developed which utilize the replication origin (ori) of bacteriophage fd for their propagation. These vectors depend on the expression of viral gene 2 that was inserted into phage lambda, which in turn was integrated into the host genome. The constitutive expression of gene 2 in the host cells is sufficient for the propagation of at least 100 pfd plasmids per cell. In addition to the fd ori, the pfd vectors carry various antibiotic-resistance genes and unique restriction sites. Some of these vectors have no homologies to commonly used pBR plasmids or to lambda DNA. The nucleotide sequence of the vectors can be deduced from published sequences. Large DNA inserts can be stably propagated in pfd vectors; these are more stable than similar DNA fragments cloned in intact genomes of filamentous bacteriophage. Inclusion of phage sequences required for efficient phage packaging and infection with a helper phage resulted in formation of phage particles containing single-stranded plasmid genomes. Growth at 42 degrees C without selective pressure results in loss of pfd plasmids.

Gene conversion appears to be the frequent mechanism in Neisseria gonorrhoeae that leads to an altered expression of pilin, the subunit component of the pili. In this process segments of variable sequence information, the minicassettes, are transferred from silent storage loci into an expression locus. As a putative consequence of the rearrangement in the pilE gene, gonococci can enter a different phase of pilin production. Although the removal of a 7-amino acid leader peptide results in the production of typical P+ pilin used to form pili, the loss of an additional 39 amino acids yields S-pilin, a soluble form of pilin that is efficiently secreted into the extracellular environment. Both pilin types can coexist in an apparently homogeneous culture. Ps cells usually are piliated, although less extensively with regard to the length and the number of the pili when compared with P+ cells. Ps cells form T3/T4-type colonies also typical of nonpiliated cells (P-). The observations further suggest that the classical nonsecretory P- phenotype is not generated as a rule by precise gene conversion but rather by genetic changes that cause the production of an over-length pilin (L-pilin).

Summary The expression of the Neisseria gonorrhoeae opacity protein (Op, protein II), a major antigenic determinant of the outer membrane, is subject to frequent phase transitions. At least nine expression loci (opaE) are involved in the production of a large number of serologically distinct Op types. Using opa-specific oligonucleotides as probes in genomic blots, we detect Op-related gene sequences (opr) in N. meninglitidis as well as in N. lactamica. DNA sequence analysis of such opr genes derived from N. meninglitidis reveals distinct regions of homology with gonococcal opa E genes. As shown in the immunoblot, the proteins encoded by ops and opr are serologically related. Like the opa E genes, the 5′-coding sequences of the opr genes include a repetitive sequence composed of pentameric CTCTT units. The number of these coding repeat (CR) units is variable. This finding, together with the observation that all opr genes are constitutively transcribed, regardless of the status of protein production, suggests a translational control mechanism identical to that of the opa genes in gonococci. The related structures and control mechanisms of opa and opr genes imply a general significance of their gene products for the pathogenic character of the investigated Neisseria species.

Antigenic variation is a means by which many infectious organisms evade the host immune response. This phenomenon has been observed in animal viruses (e.g., influenza virus), parasites (e.g., Trypanosoma brucei), and bacteria (e.g., Borrelia recurrentis). Recent work has shown that two surface proteins of Neisseria gonorrhoeae undergo antigenic variation as well. These two virulence factors are the pilus and the opacity (PII) proteins, both of which promote gonococcal binding to host (human) epithelial cells (BUCHANAN and PIERCE 1976; SWANSON 1973). In this chapter, we shall emphasize the work done on the pilus system. We present evidence that phase and antigenic variation are closely linked phenomena; that the regulation of pilus expression is reminiscent of yeast mating type interconversion and of immunoglobulin gene rearrangement; and that the gonococcal pilin belongs to a class or family of bacterial pilins with a common subunit structure.

DNA replication of single-stranded phage fd requires E. coli DNA polymerase III holoenzyme and E. coli DNA binding protein I. Complementary strand synthesis is initiated by E. coli RNA polymerase, and viral strand synthesis is initiated by phage fd gene 2 protein. For unwinding of the double strands, viral strand replication also requires EU coli rep protein. The initiation sites have been localized in the intergenic region of the phage. Due to their orientation the replication of the complementary strand is initiated after almost a complete round of viral strand synthesis.

The chromosome of N. gonorrhoeae contains several complete expression genes coding for variant opacity proteins. DNA sequence analysis of two opacity genes derived from the same locus ( opa E1) of two isogenic gonococcal variants reveals common and variable regions in these genes. Genomic blotting experiments using synthetic probes suggest gene conversion as a principle for the assembly of variant sequence information in opacity genes. The 5′ region of opacity genes is composed of identical pentameric pyrimidine units (CTCTT) encoding the hydrophobic portion of the opacity leader peptide. This coding repeat is variable in a given locus with respect of the number of pentameric units. While all expression loci in a single cell are constitutively transcribed, the production of opacity proteins is determined by the coding repeat sequence on the translational level.

The Neisseria gonorrhoeae pilus protein is one of the major antigenic determinants on the cell's surface. It is comprised of identical subunits of approximately 18 kd and plays a role in the infectivity and virulence of the organism. We have cloned the gene encoding a gonococcal pilus protein into Escherichia coli, and, using one of these clones as a probe in hybridization studies, we have shown that conversion of the pilus positive to pilus negative state in N. gonorrhoeae involves chromosomal rearrangement. Although the pilus protein is produced by E. coli, it does not appear to be assembled on the surface of the cell in native form.

Bacteriophage fd replicative form DNA with a nick in the viral strand serves as a template for DNa replication with purified bacteriophage T4 enzymes. As anticipated from previous in vitro studies carried out with this system (Morris, C. F., Sinha, N. K., and Alberts, B. M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4800-4804), DNA is synthesized by a rolling circle mechanism. We show here that the DNA strands synthesized are processed by the phage fd gene 2 protein into unit length products, providing that the gene 2 protein is present at the moment when this DNA is made. The products are mostly unit length linear single strands, indicating that the circularization step normally catalyzed by gene 2 protein subsequent to its site-specific cleavage of an fd DNA strand occurs only inefficiently in this system. The gene 2 protein reduces the level of DNA synthesis by 2-fold at low concentrations, even though it only cleaves the DNA products efficiently at higher levels of the enzyme. This indicates that there are at least two different effects of the fd gene 2 protein in processing of viral fd DNA.

Fusion proteins comprising the amino-terminal 99 amino acids of the bacteriophage MS2 replicase and various portions of OmpV a major outer membrane protein of Vibrio cholerae were expressed in Escherichia coli K12. These fusions were expressed under the control of the PL promoter of bacteriophage lambda, and expression was controlled using a cIts repressor. Fusions occurring within the secretory signal sequence of OmpV gave rise to the production of mature OmpV. The efficiency, however, decreased with progressive deletion of the signal sequence within the fusions. The reactivity of various OmpV fusions with antisera raised against purified OmpV and whole bacteria demonstrated the existence of two antigenic domains: one present in the denatured form and another in the membrane-associated form of OmpV. These domains correspond to markedly hydrophilic regions of the protein as would be predicted for surface-exposed epitopes.

IgA protease is a putative virulence factor that exists in several allelic forms in Neisseria gonorrhoeae. However, extracellular secretion of these variant IgA proteases occurs by the same pathway involving three steps of autoproteolytic maturation from a large precursor. Two principal precursor types (H1 and H2) can be distinguished with respect to the location of autoproteolytic sites and the sizes of the mature products. By partial DNA sequence analysis, additional variations have been detected which are not unique to one particular gene; rather, otherwise unrelated iga genes often share homology, thus revealing a composite organization. In the context of other gonococcal features, this observation implies that recombination has occurred in vivo between iga genes of different strains, probably via the route of species-specific DNA transformation. This process may be of general significance for the modulation and the natural exchange of virulence properties among pathogenic Neisseriae.

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.

The nucleotide sequence of the ompV gene of Vibrio cholerae was determined. The product of the gene is a 28,000 dalton protein which, after the removal of a 19 amino acid signal sequence, produces a mature outer membrane protein of 26,000 daltons. The cleavage site was determined by amino-terminal amino acid sequencing of the purified mature protein. The DNA upstream of the gene shows the presence of a typical promoter region as judged from the Escherichia coli consensus information; however, the Shine-Dalgarno sequence is associated with a region capable of forming a secondary structure in the mRNA. The formation of this structure would inhibit binding of the mRNA to the ribosome and reduce translation. It is proposed that this structure is recognized by a positive activator in V. cholerae and because of its absence in E. coli ompV is poorly expressed. The distribution of rare codons within ompV suggests that they may serve to slow down the translation of particular domains such that the nascent polypeptide has an opportunity to take up its conformation without interference from the later formed regions. Such a mechanism could aid localization of the protein if export were by a contranslational secretion system.

Bacteriophage T7 gner 4 protein and DNA polymerase of the phage were used to study the viral strand synthesis of bacteriophage fd in vitro. Cleavage of supercoiled phage fd replicative form (RF) by fd gene 2 protein produced a nick at a specific site in the viral strand. The cleaved double-stranded DNA was unwound by T7 gene 4 protein and T7 DNA polymerase and the 3′ end of the nicked strand simultaneously extended according to the rolling circle mechanixm. After a complete round of DNA synthesis fd gene 2 protein cleaved the viral strand presumably at the same site, where the endonuclease cuts fd RF I, and subsequently sealed the single-stranded linear DNA into a circle. The reaction products were analyzed by velocity sedimentation, gel electrophoresis and electron microscopy. Most of the single-stranded DNA synthesized was circular. No host proteins were required for the formation of the single-stranded circles, Strand switching of the T7 DNA polymerase indicated by double stranded tails of the rolling circle structures reduced the yield of viral single-stranded circles in this enzymes system.

We have mapped two regions of the Neisseria gonorrheae genome, pilE1 and pilE2, which are involved in pilus expression. When the cells are in the piliated P+ state, these two loci carry sequences necessary for pilin production. A silent locus, pilS1, also maps near pilE1 and pilE2. pilS1 contains structural gene information but lacks pilus promoter sequences. The pilus gene sequences in pilE1 and pilE2 are identical in strain MS11.

To investigate the significance of silent gene loci for pilus antigenic variation in N. gonorrhoeae, we determined the nucleotide sequence of the major silent locus, pilS1. The pilS1 locus contains six tandem pilus gene copies linked by a 39 bp repeat sequence also present in the expression loci. All silent copies lack the common N-terminal coding sequence of pilin, containing instead variant sequence information that constitutes a semivariable (SV) and a hypervariable (HV) domain. The SV and HV domains are interspersed with short, strictly conserved (C) regions flanking small cassettes of variable sequence information. It appears that such minicassettes from silent copies can be duplicated and transferred to other silent or expression genes by means of gene conversion.

The dependence of the induction of choline acetyltransferase (CAT) on the type of tissue culture, monolayer or piece explants, of spinal cord (SC) and muscle (M) was studied. CAT activities of dissociated SC cells were low bu were increased by M monolayers. In piece explants of SC, high CAT activities were achieved independently of muscle cells (chick, mouse). It is therefore likely that in intact SC tissue factors other than the trophic influence of muscle are able to induce cholinergic maturation. M piece explants on SC monolayers did not induce CAT in, and their outgrowth was inhibited by, SC monolayers.

Gonococcal virulence is thought to rely on multiple phenomena to which antigenic variation of surface proteins as well as the production of an extracellular protease specific for human IgA1 can be accounted. The expression of pilin and opacity (Op) protein in Neisseria gonorrhoeae can be switched on and off and a single cell apparently has a whole repertoire of genes to express many serologically distinguishable protein types. In order to investigate the molecular mechanisms associated with this surface variability, we have cloned the genes for pilin and Op expression in Escherichia coli. Using these genes as probes in DNA filter hybridizations, we revealed two complex gene systems which undergo genomic reorganizations and/or gene conversions which occur concomitant to changes in the expression of these surface proteins. To study the role of IgA proteases in bacterial pathogenesis, we cloned the IgA protease gene of N. gonorrhoeae MS11. This clone promotes expression and extracellular secretion of active enzyme in E. coli and other gram-negative bacterial species.

Phase and antigenic variation of pilin expression in Neisseria gonorrhoeae result from recombination events in which variant sequences from one of the silent loci (pilS) are transferred to the expression locus (pilE). Such rearrangements were originally thought to be gene conversions, but findings showing that phase variation is partially inhibited by DNase I, that piliated (P+) cells are highly competent for DNA uptake and that gonococci readily undergo autolysis in culture, led to the suggestion that pilin variation occurs through transformation by exogenous DNA. We have developed a simple method for the selection of non-piliated (P-) cells and have evaluated naturally occurring P+ to P- transitions. Two primary pathways of pilin variation can be distinguished--transformation-mediated recombination, which is influenced by culture conditions and inhibited by DNase I, and intragenomic reciprocal recombination, which is unaffected by DNase I. Furthermore, we demonstrate that both piliated and revertible P- cells are competent for DNA uptake, an essential prerequisite of the first pathway.

N. gonorrhoeae undergoes pilus phase and antigenic variation. During phase variation, the pilin gene is turned on and off at high frequencies. Two loci on the gonococcal chromosome from strain MS11 function as expression sites for the pilin gene (pilE1 and pilE2); many other sites apparently contain silent variant pilin sequences. We reported previously that during pilus phase variation, when cells switch from the pilus expressing state (P+) to the nonexpressing state (P-), genome rearrangement occurs. We have examined phase variation in more detail, and we report that in most P+ to P- switches a deletion of pilin gene information occurs in one or both expression sites. This deletion is due to either a simple or a multiple-step recombination event involving directly repeated sequences in the expression loci. The deletion explains the state of some P- cells, but not all. In the latter cells pilin expression is probably controlled by an undefined regulator.

Bacteriophage fd gene II-protein was characterized as an endonuclease which specifically nicked supercoiled replicative form (RF) of filamentous phages in the viral strand. No other supercoiled DNAs tested were attacked by the enzyme, nor were doubly closed fd RF in the relaxed state nor phage fd single strands. Maximal activity was found at pH 8.5 and 80 mM KCl using fd RFI of physiological superhelicity. Mg2+, but no other cofactor, was required for the cleavage reaction. A sealing activity was found to be associated with the enzyme. At a higher concentration of Mg2+ up to 40% of the reaction products were found as doubly closed relaxed fd RF. The protein was not found to be tightly attached to the cleaved strand.

AlthoughHelicobacter pylorihas generally been considered an extracellular pathogen, a number of in vitro infection experiments and biopsy examinations have shown that it is capable of occasionally entering mammalian host cells. Here, we characterized this entry process by using AGS cells as a host cell model. In gentamicin protection-invasion assays, the number ofH. pyloricolonies recovered was lower than that forSalmonella entericaserovar Typhimurium X22,Escherichia coliexpressing InvA, andYersinia enterocoliticaYO:9 grown at 25°C but higher than that forNeisseria gonorrhoeaeVP1 andY. enterocoliticaYO:9 grown at 37°C. At the ultrastructural level, the entry process was observed to occur via a zipper-like mechanism. InternalizedH. pyloriwas bound in tight LAMP-1-containing vacuoles in close association with condensed filamentous actin and tyrosine phosphorylation signals. Wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, and calphostin C, an inhibitor of protein kinase C, both inhibited the entry ofH. pyloriin a sensitive and dose-dependent manner; however, the level of entry was enhanced by sodium vanadate, an inhibitor of tyrosine phosphatases and ATPases. Furthermore, the cytokine tumor necrosis factor alpha antagonized the entry ofH. pyloriinto AGS cells. Collectively, these results demonstrate that the entry ofH. pyloriinto AGS cells occurs via a zipper-like mechanism which involves various host signal transduction events.

Helicobacter pylori induces motogenic and cytoskeletal responses in gastric epithelial cells. We demonstrate that these responses can be induced via independent signaling pathways that often occur in parallel. The cag pathogenicity island appears to be nonessential for induction of motility, whereas the elongation phenotype depends on translocation and phosphorylation of CagA.

The transcription factor NF-kappaB is a key mediator of the innate immune system. Although tremendous research efforts over the past decades have led to a more and more detailed understanding of NF-kappaB signaling, there are still missing pieces in the puzzle, especially upstream of the IKK complex and in the termination of the signaling. To identify more of the factors important for this signaling pathway we have conducted an RNA-interference based screen. For this purpose, we have developed an assay for high throughput analysis using a human epithelial cell line stably expressing a p65-GFP-fusion construct. The nuclear translocation of p65-GFP can be quantified by automated microscopic analysis. Three different stimuli were compared: the cytokines TNF-alpha and IL1-beta and the gastric pathogen Helicobacter pylori. We chose H. pylori as inducer because permanent infection with this bacterium can lead to chronic inflammation, ulceration and cancerogenesis and NF-kappaB is thought to be crucial in the promotion of this pathology. Furthermore, using different time points of the activation, we screened not only for factors important for activation, but also for termination of the signal. In terms of activation, the screen identified known factors like IKKalpha and IKKbeta as well as factors so far not linked to the NF-kappaB pathway. Interestingly, two factors were identified that are specific for NF-kappaB activation after H. pylori infection and not necessary for NF-kappaB activation by the cytokines TNFalpha or IL-1beta. Regarding termination, the screen identified among other factors an ubiquitin E3-Ligase so far not linked to the pathway. Upon down-regulation of this E3-Ligase, p65-GFP resides longer in the nucleus. This correlates with a strong degradation of IkappaBalpha. The screen was conducted with a library of siRNAs against 646 kinases and associated proteins, and is currently expanded to a genome wide scale.