Roland Richard Ryll

Transformation competence and type-4 pilus biogenesis in Neisseria gonorrhoeae - A review

In Neisseria gonorrhoea (Ngo), the processes of type-4 pilus biogenesis and DNA transformation are functionally linked and play a pivotal role in the life style of this strictly human pathogen. The assembly of pili from its main subunit pilin (PilE) is a prerequisite for gonococcal infection since it allows the first contact to epithelial cells in conjunction with the pilus tip-associated PilC protein. While the components of the pilus and its assembly machinery are either directly or indirectly involved in the transport of DNA across the outer membrane, other factors unrelated to pilus biogenesis appear to facilitate further DNA transfer across the murein layer (ComL, Tpc) and the inner membrane (ComA) before the transforming DNA is rescued in the recipient bacterial chromosome in a RecA-dependent manner. Interestingly, PilE is essential for the first step of transformation, i.e., DNA uptake, and is itself also subject to transformation-mediated phase and antigenic variation. This short-term adaptive mechanism allows Ngo to cope with changing micro-environments in the host as well as to escape the immune response during the course of infection. Given the fact that Ngo has no ecological niche other than man, horizontal genetic exchange is essential for a successful co-evolution with the host. Horizontal exchange gives rise to heterogeneous populations harboring clones which better withstand selective forces within the host. Such extended horizontal exchange is reflected by a high genome plasticity, the existence of mosaic genes and a low linkage disequilibrium of genetic loci within the neisserial population. This led to the concept that rather than regarding individual Neisseria species as independent traits, they comprise a collective of species interconnected via horizontal exchange and relying on a common gene pool.

PilC of Neisseria meningitidis is involved in class II pilus formation and restores pilus assembly, natural transformation competence and adherence to epithelial cells in PilC-deficient gonococci

Type 4 pili produced by the pathogenic Neisseria species constitute primary determinants for the adherence to host tissues. In addition to the major pilin subunit (PilE), neisserial pili contain the variable PilC proteins represented by two variant gene copies in most pathogenic Neisseria isolates. Based upon structural differences in the conserved regions of PilE, two pilus classes can be distinguished in Neisseria meningitidis. For class I pili found in both Neisseria gonorrhoeae and N. meningitidis, PilC proteins have been implicated in pilus assembly, natural transformation competence and adherence to epithelial cells. In this study, we used primers specific for the pilC2 gene of N. gonorrhoeae strain MS11 to amplify, by the polymerase chain reaction, and clone a homologous pilC gene from N. meningitidis strain A1493 which produces class II pili. This gene was sequenced and the deduced amino acid sequence showed 75.4% and 73.8% identity with the gonococcal PilC1 and PilC2, respectively. These values match the identity value of 74.1% calculated for the two N. gonorrhoeae MS11 PilC proteins, indicating a horizontal relationship between the N. gonorrhoeae and N. meningitidispilC genes. We provide evidence that PilC functions in meningococcal class II pilus assembly and adherence. Furthermore, expression of the cloned N. meningitidis pilC gene in a gonococcal pilC1,2 mutant restores pilus assembly, adherence to ME‐180 epithelial cells, and transformation competence to the wild‐type level. Thus, PilC proteins exhibit indistinguishable functions in the context of class I and class II pili.

Identification of cDNAs encoding two novel nuclear proteins, IMUP-1 and IMUP-2, upregulated in SV40-immortalized human fibroblasts

Using a model system of young, senescent and SV40-immortalized WI-38 fibroblasts, we identified two mRNAs upregulated in immortalized cells (imup-1, immortalization-upregulated protein 1, and imup-2). Compared to normal tissues, both genes were more frequently expressed in cancer cells. The open reading frame of imup-1 spans 321bp, coding for a 10.9 kDa protein of 106 amino acids, while an insertion of 59bp in the otherwise identical mRNA of imup-2 leads to a frameshift, resulting in an 8.5 kDa protein of 85 amino acids. Database searches identified these genes on chromosome 19, which could account for the cloned imup-1 and imup-2 transcripts by alternative splicing. Southern blot analysis of digested genomic DNA confirmed that both transcripts are derived from a single locus. The expressed proteins IMUP-1 and IMUP-2 share 46 identical N-terminal amino acids, whereas the C-termini are unrelated. Green fluorescent protein-fusions of both IMUP-1 and IMUP-2 accumulated in the nucleus of HeLa cells. The C-terminus of IMUP-1 contains a bipartite nuclear localization signal, the deletion of which impaired nuclear translocation. In-vitro translated proteins bound to poly(rG), but did not interact with single-stranded DNA or double-stranded DNA. The nuclear localization of IMUP-1 and IMUP-2 as well as the upregulation of both underlying mRNAs in immortalized cells suggest a function in immortalization.