Electron tomography and cryo-SEM characterization reveals novel ultrastructural features of host-parasite interaction during Chlamydia abortus infection.

Chlamydia (C.) abortus is a widely spread pathogen among ruminants that can be transmitted to women during pregnancy leading to severe systemic infection with consecutive abortion. As a member of the Chlamydiaceae, C. abortus shares the characteristic feature of an obligate intracellular biphasic developmental cycle with two morphological forms including elementary bodies (EBs) and reticulate bodies (RBs). In contrast to other chlamydial species, C. abortus ultrastructure has not been investigated yet. To do so, samples were fixed by high-pressure freezing and processed by different electron microscopic methods. Freeze-substituted samples were analysed by transmission electron microscopy, scanning transmission electron microscopical tomography and immuno-electron microscopy, and freeze-fractured samples were analysed by cryo-scanning electron microscopy. Here, we present three ultrastructural features of C. abortus that have not been reported up to now. Firstly, the morphological evidence that C. abortus is equipped with the type three secretion system. Secondly, the accumulation and even coating of whole inclusion bodies by membrane complexes consisting of multiple closely adjacent membranes which seems to be a C. abortus specific feature. Thirdly, the formation of small vesicles in the periplasmic space of RBs in the second half of the developmental cycle. Concerning the time point of their formation and the fact that they harbour chlamydial components, these vesicles might be morphological correlates of an intermediate step during the process of redifferentiation of RBs into EBs. As this feature has also been shown for C. trachomatis and C. pneumoniae, it might be a common characteristic of the family of Chlamydiaceae.

Identification and evaluation of a combination of chlamydial antigens to support the diagnosis of severe and invasive Chlamydia trachomatis infections.

Chlamydia trachomatis is the most common sexually transmitted organism in industrialized countries. Nucleic acid amplification testing, using non-invasively collected specimens, is considered to be the method of choice for diagnosis of chlamydial infections of the urethra and the lower genital tract. Serological testing has the potential to circumvent the problem of specimen sampling in invasive C. trachomatis infections of the upper genital tract. However, only a few defined chlamydial antigens have been used in a standardized diagnostic assay format. In this study, we used serological two-dimensional proteomic analysis to broaden the spectrum of diagnostically relevant C. trachomatis proteins. The genes encoding an assortment of already known chlamydial antigens, as well as immunogenic proteins that have not been described before, were cloned, and the recombinant proteins were purified in order to compare their diagnostic usefulness in parallel with a newly developed line immunoassay. With 189 sera collected from patients with and without C. trachomatis infection, recombinant major outer membrane protein (MOMP), chlamydial protease-like activity factor (CPAF), outer membrane protein 2 (OMP2), translocated actin-recruiting protein, and polymorphic membrane protein D (PmpD) showed the highest level of diagnostic sensitivity and specificity. In patients suffering from ascending and invasive C. trachomatis infections, such as pelvic inflammatory disease and lymphogranuloma venereum, the sensitivity reached with these proteins ranged between 71% (PmpD) and 94% (OMP2), and the specificity ranged between 82% (PmpD) and 100% (MOMP and OMP2). Recombinant thio-specific antioxidant peroxidase, ribosomal protein S1 (RpsA) and hypothetical protein 17 showed lower sensitivity but comparably high specificity, ranging from 94% to 100%. The novel line immunoassay based on defined recombinant antigens has promise for improved serodiagnosis in severe and invasive C. trachomatis infections.

Screening methods in alveolar echinococcosis: a follow-up study comparing Emc- and Emf-ELISA with Em2plus-ELISA and ultrasonography.

The purpose of the present study was to identify Echinococcus multilocularis infection in follow-up of 95 subjects initially seropostive by Emc-ELISA or Emf-ELISA antibody assays and to compare the utility of these assays with specific Em2plus-ELISA and ultrasound screening for E. multilocularis infection. At follow-up seven subjects were seropositive with both methods, while three were seropositive only with Emc-ELISA and 11 only with Emf-ELISA. All subjects were seronegative with Em2plus-ELISA. There were no manifestations of E. multilocularis infestation by ultrasonographic screening. Seropositivity on Emc-ELISA and Emf-ELISA screening tests does not appear to correlate with manifest alveolar echinococcosis identified by ultrasound. A recommendation for further follow-up of subjects found to be seropositive with Emc-ELISA and Emf-ELISA but with no sonographic evidence of disease is not justified at this time.

Overexpresssion of a Legionella pneumophila homologue of the E. coli regulator csrA affects cell size, flagellation, and pigmentation.

Legionella pneumophila is an inhabitant of the aquatic environment and the causative agent of a bacterial pneumonia. We identified the presence of an L. pneumophila homologue of csrA of E. coli and rsmA of Erwinia carotovora, genes which regulate gene expression by destabilising mRNA and which have been shown to relate to environmental fitness and pathogenicity. The Legionella csrA was able to complement a csrA-negative mutant of E. coli. Overproduction of csrA in L. pneumophila lead to a reduction of flagellation and pigmentation and an increase in bacterial cell size. csrA overproduction was associated with a reduction of fliA and flaA transcripts. This suggests that similar to E. coli and Erwinia, L. pneumophila csrA is a regulator of gene expression and may contribute to the capability of the pathogen to rapidly adapt to changing environments.

A fork head related multigene family is transcribed in Xenopus laevis embryos.

We have isolated and sequenced ten different members of the fork head/HNF-3 multigene family from Xenopus laevis which have been termed Xenopus fork head domain related (XFD) genes 1 to 10. Another four isolated genes (XFD' genes) represent pseudo-allelic variants which arose by an ancient tetraploidization within this species. Whereas all genes of this multigene family exhibit a high degree of sequence homology within the evolutionary conserved fork head domain, sequences outside this module are substantially different. Based upon sequence homologies over the entire coding sequences, XFD-7/7' represent the Xenopus homologs to the rodent hepatocyte nuclear factor HNF-3 alpha, while XFD-3/3' encode the homologs to HNF-3 beta. Here we present an analysis of the temporal transcription pattern of XFD genes 1 to 10 during embryogenesis and in some adult tissues. Eight of these XFD genes are activated during embryonic development, but show different and distinct transcription profiles. The localization of transcripts was determined by whole-mount in situ hybridization. Although transcription of individual XFD genes partially overlaps, each gene is characterized by means of a specific spatial pattern of transcriptional activity.