Tonje Davidsen

Meningococcal genome dynamics

Neisseria meningitidis (the meningococcus) is an important commensal, pathogen and model organism that faces up to the environment in its exclusive human host with a small but hyperdynamic genome. Compared with Escherichia coli, several DNA-repair genes are absent in N. meningitidis, whereas the gene products of others interact differently. Instead of responding to external stimuli, the meningococcus spontaneously produces a plethora of genetic variants. The frequent genomic alterations and polymorphisms have profound consequences for the interaction of this microorganism with its host, impacting structural and antigenic changes in crucial surface components that are relevant for adherence and invasion as well as antibiotic resistance and vaccine development.

Genome dynamics in major bacterial pathogens.

Pathogenic bacteria continuously encounter multiple forms of stress in their hostile environments, which leads to DNA damage. With the new insight into biology offered by genome sequences, the elucidation of the gene content encoding proteins provides clues toward understanding the microbial lifestyle related to habitat and niche. Campylobacter jejuni, Haemophilus influenzae, Helicobacter pylori, Mycobacterium tuberculosis, the pathogenic Neisseria, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus are major human pathogens causing detrimental morbidity and mortality at a global scale. An algorithm for the clustering of orthologs was established in order to identify whether orthologs of selected genes were present or absent in the genomes of the pathogenic bacteria under study. Based on the known genes for the various functions and their orthologs in selected pathogenic bacteria, an overview of the presence of the different types of genes was created. In this context, we focus on selected processes enabling genome dynamics in these particular pathogens, namely DNA repair, recombination and horizontal gene transfer. An understanding of the precise molecular functions of the enzymes participating in DNA metabolism and their importance in the maintenance of bacterial genome integrity has also, in recent years, indicated a future role for these enzymes as targets for therapeutic intervention.

The outer membrane secretin PilQ from Neisseria meningitidis binds DNA

Neisseria meningitidis is naturally competent for transformation throughout its growth cycle. Transformation in neisserial species is coupled to the expression of type IV pili, which are present on the cell surface as bundled filamentous appendages, and are assembled, extruded and retracted by the pilus biogenesis components. During the initial phase of the transformation process, binding and uptake of DNA takes place with entry through a presumed outer-membrane channel into the periplasm. This study showed that DNA associates only weakly with purified pili, but binds significantly to the PilQ complex isolated directly from meningococcal membranes. By assessing the DNA-binding activity of the native complex PilQ, as well as recombinant truncated PilQ monomers, it was shown that the N-terminal region of PilQ is involved in the interaction with DNA. It was evident that the binding of ssDNA to PilQ had a higher affinity than the binding of dsDNA. The binding of DNA to PilQ did not, however, depend on the presence of the neisserial DNA-uptake sequence. It is suggested that transforming DNA is introduced into the cell through the outer-membrane channel formed by the PilQ complex, and that DNA uptake occurs by non-specific introduction of DNA coupled to pilus retraction, followed by presentation to DNA-binding component(s), including PilQ.

Molecular Diagnostics in Tuberculosis Basis and Implications for Therapy

The processing of clinical specimens in the mycobacterial diagnostic laboratory has undergone remarkable improvements during the last decade. While microscopy and culture are still the major backbone for laboratory diagnosis of tuberculosis on a worldwide basis, new methods including molecular diagnostic tests have evolved over the last two decades. The majority of molecular tests have been focused on (i) detection of nucleic acids, both DNA and RNA, that are specific to Mycobacterium tuberculosis, by amplification techniques such as polymerase chain reaction (PCR); and (ii) detection of mutations in the genes that are associated with resistance to antituberculosis drugs by sequencing or nucleic acid hybridization. Recent developments in direct and rapid detection of mycobacteria, with emphasis on M. tuberculosis species identification by 16S rRNA gene sequence analysis or oligohybridization and strain typing, as well as detection of drug susceptibility patterns, all contribute to these advances. Generally, the balance between genome instability and genome maintenance as the basis for evolutionary development, strain diversification and resistance development is important, because it cradles the resulting M. tuberculosis phenotype. At the same time, semi-automated culture systems have contributed greatly to the increased sensitivity and reduced turnaround time in the mycobacterial analysis of clinical specimens. Collectively, these advances are particularly important for establishing the diagnosis of tuberculosis in children. More basic and operational research to appraise the impact and cost effectiveness of new diagnostic technologies must, however, be carried out. Furthermore, the design and quality of clinical trials evaluating new diagnostics must be improved to allow clinical and laboratory services that would provide rapid response to test results. Thus, important work remains before the new diagnostic tools can be meaningfully integrated into national tuberculosis control programs of high-burden countries.

Molecular Diagnostics in Tuberculosis

The processing of clinical specimens in the mycobacterial diagnostic laboratory has undergone remarkable improvements during the last decade. While microscopy and culture are still the major backbone for laboratory diagnosis of tuberculosis on a worldwide basis, new methods including molecular diagnostic tests have evolved over the last two decades. The majority of molecular tests have been focused on (i) detection of nucleic acids, both DNA and RNA, that are specific to Mycobacterium tuberculosis, by amplification techniques such as polymerase chain reaction (PCR); and (ii) detection of mutations in the genes that are associated with resistance to antituberculosis drugs by sequencing or nucleic acid hybridization. Recent developments in direct and rapid detection of mycobacteria, with emphasis on M. tuberculosis species identification by 16S rRNA gene sequence analysis or oligohybridization and strain typing, as well as detection of drug susceptibility patterns, all contribute to these advances. Generally, the balance between genome instability and genome maintenance as the basis for evolutionary development, strain diversification and resistance development is important, because it cradles the resulting M. tuberculosis phenotype.At the same time, semi-automated culture systems have contributed greatly to the increased sensitivity and reduced turnaround time in the mycobacterial analysis of clinical specimens. Collectively, these advances are particularly important for establishing the diagnosis of tuberculosis in children.More basic and operational research to appraise the impact and cost effectiveness of new diagnostic technologies must, however, be carried out. Furthermore, the design and quality of clinical trials evaluating new diagnostics must be improved to allow clinical and laboratory services that would provide rapid response to test results. Thus, important work remains before the new diagnostic tools can be meaningfully integrated into national tuberculosis control programs of high-burden countries.

Genetic Interactions of DNA Repair Pathways in the Pathogen Neisseria meningitidis

The current increase in the incidence and severity of infectious diseases mandates improved understanding of the basic biology and DNA repair profiles of virulent microbes. In our studies of the major pathogen and model organism Neisseria meningitidis, we constructed a panel of mutants inactivating genes involved in base excision repair, mismatch repair, nucleotide excision repair (NER), translesion synthesis, and recombinational repair pathways. The highest spontaneous mutation frequency among the N. meningitidis single mutants was found in the MutY-deficient strain as opposed to mutS mutants in Escherichia coli, indicating a role for meningococcal MutY in antibiotic resistance development. Recombinational repair was recognized as a major pathway counteracting methyl methanesulfonate-induced alkylation damage in the N. meningitidis. In contrast to what has been shown in other species, meningococcal NER did not contribute significantly to repair of alkylation-induced DNA damage, and meningococcal recombinational repair may thus be one of the main pathways for removal of abasic (apurinic/apyrimidinic) sites and strand breaks in DNA. Conversely, NER was identified as the main meningococcal defense pathway against UV-induced DNA damage. N. meningitidis RecA single mutants exhibited only a moderate decrease in survival after UV exposure as opposed to E. coli recA strains, which are extremely UV sensitive, possibly reflecting the lack of a meningococcal SOS response. In conclusion, distinct differences between N. meningitidis and established DNA repair characteristics in E. coli and other species were identified.