Multiple gonococcal opacity proteins are expressed during experimental urethral infection in the male.

The opacity (Opa) proteins of Neisseria gonorrhoeae are a family of outer membrane proteins demonstrating phase and antigenic variation. N. gonorrhoeae strain FA0190 has 11 opa loci that encode at least 8 antigenically distinct Opa proteins. To determine if expression of one Opa protein or a subset of them is favored during gonococcal infection, we inoculated Opa-negative variants of strain FA1090 intraurethrally into male volunteers. The Opa phenotype of gonococci isolated from urine and urethral swab cultures from nine infected subjects was determined. Opa proteins were expressed in a large proportion of the reisolates from the infected subjects. Gonococci cultured from urine or urethral swab samples from six of the subjects were uniformly Opa positive, with the predominant Opa variants differing among subjects. Three different Opa proteins were represented as the predominant type in at least one subject each. In three subjects, there was more heterogeneity in Opa phenotype of the reisolates, including the presence of Opa-negative variants. An increase in the proportion of isolates expressing multiple Opa proteins occurred over time in most subjects. Passage of the inoculum in vitro did not result in similar changes in Opa expression. There was no detectable difference in infectivity of an Opa-negative variant and one expressing an Opa protein (OpaF) that was highly represented in reisolates from the original nine subjects. Reisolates from three infected volunteers inoculated with the OpaF variant showed continued expression of OpaF alone or in conjunction with other Opa proteins. These results demonstrate that there is strong selection for expression of one or more Opa proteins by strain FA1090 in vivo, but that no single protein is preferentially expressed during early infection in the male urethra.

Multiple gonococcal pilin antigenic variants are produced during experimental human infections.

Gonococcal pilin variation is thought to allow immune evasion and change the adherence properties of the pilus. We have examined the process of pilin antigenic variation in human volunteers inoculated with strain FA1090. Our data show that pilin variation occurred throughout the process of infection, that at each time sampled after inoculation multiple pilin variants were present, and that later pilin variants appear to be recombinants between previously expressed genes and the silent storage pilin copies. Thus, during infection a large repertoire of proteins are available to the population to help avoid immune responses, to provide pili with varying functions, and to transmit to a new host.

A homologue of the recombination-dependent growth gene, rdgC, is involved in gonococcal pilin antigenic variation.

Neisseria gonorrhoeae pilin undergoes high-frequency changes in primary amino acid sequence that aid in the avoidance of the host immune response and alter pilus expression. The pilin amino acid changes reflect nucleotide changes in the expressed gene, pilE, which result from nonreciprocal recombination reactions with numerous silent loci, pilS. A series of mini-transposon insertions affecting pilin antigenic variation were localized to three genes in one region of the Gc chromosome. Mutational analysis with complementation showed that a Gc gene with sequence similarity to the Escherichia coli rdgC gene is involved in pilus-dependent colony phase variation and in pilin antigenic variation. Furthermore, we show that the Gc rdgC homologue is transcriptionally linked in an operon with a gene encoding a predicted GTPase. The inability to disrupt expression of this gene suggests it is an essential gene (engA, essential neisserial GTPase). While some of the transposon mutations in rdgC and insertions in the 5'-untranslated portion of engA showed a growth defect, all transposon insertions investigated conferred an aberrant cellular morphology. Complementation analysis showed that the growth deficiencies are due to the interruption of RdgC expression and not that of EngA. The requirement of RdgC for efficient pilin variation suggests a role for this protein in specialized DNA recombination reactions.

Insertionally inactivated and inducible recA alleles for use in Neisseria.

Two classes of recA mutations have been constructed for use in Neisseria gonorrhoeae: three insertionally inactivated ('knockout') mutations and three LacI-regulatable constructs that can be shifted between Rec- and Rec+ by the removal or addition of IPTG. The effects of regulating recA expression on the processes of DNA transformation, DNA repair and pilin-phase variation are described. These regulatable cassettes can also be used to control the expression of any chromosomal gene.

Shuttle mutagenesis: two mini-transposons for gene mapping and for lacZ transcriptional fusions in Neisseria gonorrhoeae.

Shuttle mutagenesis is a system we developed for producing stable transposon insertions in Saccharomyces cerevisiae [Seifert et al., Proc. Natl. Acad. Sci. USA 83 (1986) 735-739; Hoekstra et al., Methods Enzymol. 194 (1991) 329-342] and Neisseria gonorrhoeae (Gc) [Seifert et al., J. Bacteriol. 172 (1990) 40-46] by transposition in Escherichia coli and transformation into yeast or Gc. In developing the system for use in Gc, a series of mini-transposons (mTn) were derived from mTn3 which confer resistance to chloramphenicol in Gc (mTnCm) (Seifert et al., 1990). Herein, we describe the creation of two mTnCm derivatives for use in Gc. One of these transposons, mTnCmNS, contains the infrequently occurring NheI and SpeI restriction sites to localize genes on the gonococcal macro-restriction map which was recently developed using these restriction sites [Bihlmaier et al., Mol. Microbiol. 5 (1991) 2529-2539; Dempsey et al., J. Bacteriol. 173 (1991) 5476-5486]. The mTnCmLac was developed to generate lacZ transcriptional fusions using transposition. It contains at its end a promoterless lacZ gene which is expressed once the element has transposed downstream from a promoter in a cloned gene. In adapting the use of mTnCmLac to the shuttle mutagenesis system, we have identified some factors which affect the transformation of Gc using cloned chromosomal fragments containing the large heterologous insertion, mTnCmLac. Using mTnCmLac, we have created Gc variants containing a pilE::mTnCmLac fusion to determine that pilE transcription in Gc is not auto-regulated.

Shuttle mutagenesis: a mini-transposon for producing PhoA fusions with exported proteins in Neisseria gonorrhoeae.

Shuttle mutagenesis is a method for producing stable mini-transposon (mTn) insertions into the genome of Neisseria gonorrhoeae (gonococcus, Gc) and other microbes. Using an mTn3 derivative, we have produced an mTn (mTnCmPhoA) which contains a phoA' gene lacking its N-terminal signal sequence useful for isolating genes which encode exported proteins. mTnCmPhoA was characterized in Gc and Escherichia coli using a cloned target containing the Gc genes, opaE1, pilA and pilB. PhoA+ Gc containing pilB::mTnCmPhoA insertions confirm that PilB is an exported protein in Gc. This system will be useful for isolating and characterizing extracytoplasmic virulence factors from Gc and other bacterial pathogens.

Transcriptional control of gonococcal pilE expression: involvement of an alternate sigma factor.

The pilE gene encoding Neisseria gonorrhoeae (Gc) pilin contains two putative promoter sequences 5' to the transcription start point (tsp), a Pribnow box and an RpoN-binding consensus sequence. Sequence analysis shows that the nucleotide (nt) sequence of the pilE promoter region is completely conserved among eight different Gc isolates. Using a pilE::lacZ transcriptional fusion, we demonstrate that the RpoN sigma factor can function in Escherichia coli to increase pilE transcription when the NifA activator from Klebsiella is present in trans. In addition, over-production of the native pilin protein using RpoN and NifA is lethal to E. coli. Finally, we show that the RpoN sigma factor decreases the basal expression of pilE when an activator is not present. These results suggest that, in Gc, pilE transcription may be regulated by RpoN in conjunction with an activator and that sigma 70 can also act to direct transcription of pilE.

The pathogenic neisseriae contain an inactive rpoN gene and do not utilize the pilE sigma54 promoter.

The sigma54 promoter (P3) upstream of the pilE gene in Neisseria gonorrhoeae was shown to be non-functional by transcriptional analysis of a PpilE::lacZ fusion containing only P3. A region on the chromosome of N. gonorrhoeae strain MS11-A was identified that potentially encodes a protein with a significant similarity to the Escherichia coli RpoN protein. However, this region (designated RLS for rpoN-like sequence) does not contain a single open reading frame (ORF) capable of encoding a functional RpoN protein. It appears that RLS may have arisen from an ancestral rpoN homologue that underwent a deletion removing the sequence encoding the essential helix-turn-helix (HTH) motif, and changing the subsequent reading frame. An RLS has been identified in several strains of N. gonorrhoeae and N. meningitidis. A 90-kDa gonococcal protein has previously been shown to react with a monoclonal antibody raised against the RpoN from Salmonella typhimurium. However, mutagenesis and Western blot analysis confirmed that the gene encoding this protein is not contained within RLS.

Rapid recombination among transfected plasmids, chimeric episome formation and trans gene expression in Plasmodium falciparum.

Although recombination is known to be important to generating diversity in the human malaria parasite P. falciparum, the low efficiencies of transfection and the fact that integration of transfected DNA into chromosomes is observed only after long periods (typically 12 weeks or more) have made it difficult to genetically manipulate the blood stages of this major human pathogen. Here we show that co-transfection of a P. falciparum line with two plasmids, one expressing a green fluorescent protein (gfp) reporter and the other expressing a drug resistance marker (Tgdhfr-ts M23), allowed selection of a population in which about approximately 30% of the parasites produce GFP. In these GFP-producing parasites, the transfected plasmids had recombined into chimeric episomes as large as 20 kb and could be maintained under drug pressure for at least 16 weeks. Our data suggest that chimera formation occurs early (detected by 7--14 days) and that it involves homologous recombination favored by presence of the same P. falciparum 5'hrp3 UTR promoting transcription from each plasmid. This indicates the presence of high levels of homologous recombination activity in blood stage parasites that can be used to drive rapid recombination of newly introduced DNA, study mechanisms of recombination, and introduce genes for trans expression in P. falciparum.

Direct transformation of Neisseria gonorrhoeae by gel-isolated DNA.

The naturally competent organism, Neisseria gonorrhoeae, can efficiently transform a marker carried on DNA purified in low-melting-temperature agarose without prior purification or dilution. Neither the agarose or buffer components inhibit transformation frequencies, but exposure to UV irradiation completely abrogates transformation.

[Molecular bases of virulence in Neisseria gonorrhoeae]. / Bases moléculaires de la virulence de Neisseria gonorrhoeae.

Gonorrhea remains of clinical concern, due to its frequency, complications, sequelae, increasing prevalence of antibiotic-resistant strains and absence of vaccine. A better understanding of the first stages of infection as well as of mechanisms of escape to immune response appears important. Many pathogenic bacteria express pili on their all surfaces. These structures mediate binding of bacteria to host tissues. Furthermore, gonococcal pili are submitted to a high rate antigenic variation, allowing the escape to host immune response. Pilin antigenic variation occurs by DNA recombination between one of the silent partial variant gene segments and an expressed pilin genes. We have shown that transformation of living bacteria by DNA liberated from lysed cells is a critical strep for antigenic variation. This constitutes the first specific function for a DNA transformation system. Piliation and virulence can change with culture conditions. This observation suggests that pilin expression would be subjected to an adaptative response. We have identified and characterized two genes which act in trans to regulate pilus expression. They determine synthesis of a response regulator and a membrane located sensor. They appear to regulate expression of other genes, possibly also involved in virulence. We present evidence for several environmental factors which may control the degree of piliation.

[Oral long-term administration of probiotic B. cereus--an alternative for the the prevention of enterotoxemia?]. / Orale Dauerapplikation des probiotischen B. cereus--eine Alternative zur Verhütung der Enterotoxämie?

Aetiology and epizootiology of enterotoxaemia, especially the connection between development of the disease and the management of feeding are described and newer knowledge about the physiopathology of the disease is presented. A comparative description of prophylactic schemes which include immuno- and chemoprophylaxis follows. The principles of the application and the effect of probiotic organisms as a new approach to prevent the disease are explained. The probiotic effect of apathogenic non-haemolysing and non-toxic B. cereus strains which have proven to be able to suppress enteropathies and also enterotoxaemia is described.