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Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

University Hospital Center and University of Lausanne

Human immunodeficiency virus type 2 (HIV-2)/simian immunodeficiency virus SIV(SM) Vpx is incorporated into virion particles and is thus present during the early steps of infection, when it has been reported to influence the nuclear import of viral DNA. We recently reported that Vpx promoted the accumulation of full-length viral DNA following the infection of human monocyte-derived dendritic cells (DCs). This positive effect was exerted following the infection of DCs with cognate viruses and with retroviruses as divergent as HIV-1, feline immunodeficiency virus, and even murine leukemia virus, leading us to suggest that Vpx counteracted an antiviral restriction present in DCs. Here, we show that Vpx is required, albeit to a different extent, for the infection of all myeloid but not of lymphoid cells, including monocytes, macrophages, and monocytoid THP-1 cells that had been induced to differentiate with phorbol esters. The intracellular localization of Vpx was highly heterogeneous and cell type dependent, since Vpx localized differently in HeLa cells and DCs. Despite these differences, no clear correlation between the functionality of Vpx and its intracellular localization could be drawn. As a first insight into its function, we determined that SIV(SM)/HIV-2 and SIV(RCM) Vpx proteins interact with the DCAF1 adaptor of the Cul4-based E3 ubiquitin ligase complex recently described to associate with HIV-1 Vpr and HIV-2 Vpx. However, the functionality of Vpx proteins in the infection of DCs did not strictly correlate with DCAF1 binding, and knockdown experiments failed to reveal a functional role for this association in differentiated THP-1 cells. Lastly, when transferred in the context of a replication-competent viral clone, Vpx was required for replication in DCs.

Characterization of simian immunodeficiency virus SIVSM/human immunodeficiency virus type 2 Vpx function in human myeloid cells.

Infectious viral DNA constitutes only a small fraction of the total viral DNA produced during retroviral infection, and as such its exact behavior is largely unknown. In the present study, we characterized in detail functional viral DNA produced during the early steps of human immunodeficiency virus type 1 infection by analyzing systematically their kinetics of synthesis and integration in different target cells. In addition, we have compared the functional stability of viral nucleoprotein complexes arrested at their pre-reverse transcription state, and we have attempted to measure the kinetics of loss of capsid proteins from viral complexes through the susceptibility of the early phases of infection to cyclosporine, a known inhibitor of the interaction between viral capsid and cyclophilin A. Overall, our data suggest a model in which loss of capsid proteins from viral complexes and reverse transcription occur concomitantly and in which the susceptibility of target cells to infection results from a competition between the ability of the cellular environment to quickly destabilize viral nucleoprotein complexes and the capability of the virus to escape such targeting by engaging the reverse transcription reaction.

Characterization of the behavior of functional viral genomes during the early steps of human immunodeficiency virus type 1 infection.

Originally composed of the single family Chlamydiaceae, the Chlamydiales order has extended considerably over the last several decades. Chlamydia-related bacteria were added and classified into six different families and family-level lineages: the Criblamydiaceae, Parachlamydiaceae, Piscichlamydiaceae, Rhabdochlamydiaceae, Simkaniaceae, and Waddliaceae. While several members of the Chlamydiaceae family are known pathogens, recent studies showed diverse associations of Chlamydia-related bacteria with human and animal infections. Some of these latter bacteria might be of medical importance since, given their ability to replicate in free-living amoebae, they may also replicate efficiently in other phagocytic cells, including cells of the innate immune system. Thus, a new Chlamydiales-specific real-time PCR targeting the conserved 16S rRNA gene was developed. This new molecular tool can detect at least five DNA copies and show very high specificity without cross-amplification from other bacterial clade DNA. The new PCR was validated with 128 clinical samples positive or negative for Chlamydia trachomatis or C. pneumoniae. Of 65 positive samples, 61 (93.8%) were found to be positive with the new PCR. The four discordant samples, retested with the original test, were determined to be negative or below detection limits. Then, the new PCR was applied to 422 nasopharyngeal swabs taken from children with or without pneumonia; a total of 48 (11.4%) samples were determined to be positive, and 45 of these were successfully sequenced. The majority of the sequences corresponded to Chlamydia-related bacteria and especially to members of the Parachlamydiaceae family.

Development of a new chlamydiales-specific real-time PCR and its application to respiratory clinical samples.

Originally, the Chlamydiales order was represented by a single family, the Chlamydiaceae, composed of several pathogens, such as Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci and Chlamydia abortus. Recently, 6 new families of Chlamydia-related bacteria have been added to the Chlamydiales order. Most of these obligate intracellular bacteria are able to replicate in free-living amoebae. Amoebal co-culture may be used to selectively isolate amoeba-resisting bacteria. This method allowed in a previous work to discover strain CRIB 30, from an environmental water sample. Based on its 16S rRNA gene sequence similarity with Criblamydia sequanensis, strain CRIB 30 was considered as a new member of the Criblamydiaceae family. In the present work, phylogenetic analyses of the genes gyrA, gyrB, rpoA, rpoB, secY, topA and 23S rRNA as well as MALDI-TOF MS confirmed the taxonomic classification of strain CRIB 30. Morphological examination revealed peculiar star-shaped elementary bodies (EBs) similar to those of C. sequanensis. Therefore, this new strain was called "Estrella lausannensis". Finally, E. lausannensis showed a large amoebal host range and a very efficient replication rate in Acanthamoeba species. Furthermore, E. lausannensis is the first member of the Chlamydiales order to grow successfully in the genetically tractable Dictyostelium discoideum, which opens new perspectives in the study of chlamydial biology.

Estrella lausannensis, a new star in the Chlamydiales order.

Evidence is growing for a role of Waddlia chondrophila as an agent of adverse pregnancy outcomes in both humans and ruminants. This emerging pathogen, member of the order Chlamydiales, is also implicated in bronchiolitis and lower respiratory tract infections. Until now, the serological diagnosis of W. chondrophila infection has mainly relied on manually intensive tests including micro-immunofluorescence and Western blotting. Thus, there is an urgent need to establish reliable high throughput serological assays. Using a combined genomic and proteomic approach, we detected 57 immunogenic proteins of W. chondrophila, of which 17 were analysed by mass spectrometry. Two novel hypothetical proteins, Wim3 and Wim4, were expressed as recombinant proteins in Escherichia coli, purified and used as antigens in an ELISA test. Both proteins were recognized by sera of rabbits immunized with W. chondrophila as well as by human W. chondrophila positive sera but not by rabbit pre-immune sera nor human W. chondrophila negative sera. These results demonstrated that the approach chosen is suitable to identify immunogenic proteins that can be used to develop a serological test. This latter will be a valuable tool to further clarify the pathogenic potential of W. chondrophila.

Identification of immunogenic proteins of Waddlia chondrophila.

Members of the Chlamydiales order all share a biphasic lifecycle alternating between small infectious particles, the elementary bodies (EBs) and larger intracellular forms able to replicate, the reticulate bodies. Whereas the classical Chlamydia usually harbours round-shaped EBs, some members of the Chlamydia-related families display crescent and star-shaped morphologies by electron microscopy. To determine the impact of fixative methods on the shape of the bacterial cells, different buffer and fixative combinations were tested on purified EBs of Criblamydia sequanensis, Estrella lausannensis, Parachlamydia acanthamoebae, and Waddlia chondrophila. A linear discriminant analysis was performed on particle metrics extracted from electron microscopy images to recognize crescent, round, star and intermediary forms. Depending on the buffer and fixatives used, a mixture of alternative shapes were observed in varying proportions with stars and crescents being more frequent in C. sequanensis and P. acanthamoebae, respectively. No tested buffer and chemical fixative preserved ideally the round shape of a majority of bacteria and other methods such as deep-freezing and cryofixation should be applied. Although crescent and star shapes could represent a fixation artifact, they certainly point towards a diverse composition and organization of membrane proteins or intracellular structures rather than being a distinct developmental stage.

Julia Lienard

Institute of Microbiology

University Hospital Center and University of Lausanne

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches