Genotyping of Candidatus Syngnamydia salmonis (chlamydiales; Simkaniaceae) co-cultured in Paramoeba perurans (amoebozoa; Paramoebidae).

Candidatus Syngnamydia salmonis (Chlamydiales, Simkaniaceae) was described as an epitheliocystis-causing bacterium from the gills of Atlantic salmon (Salmo salar) in Norway. A bacterium showing 99.2% 16S rRNA identity to Cand. S. salmonis is able to multiply in Paramoeba perurans and based on the classification criteria this bacterium could represent the same species as Cand. S. salmonis. Sequencing the genome of the cultured bacterium has made it possible to fulfill the minimal standards for genetic characterization of species within the order Chlamydiales. The complete rRNA genes, the amino acid sequences of SucA, PepF, Adk, HemL, DnaA, FtsK and FabI, are presented in addition to the morphology of the Chlamydia-like morphs in the cytoplasm of P. perurans.

Crossing the border - Solute entry into the chlamydial inclusion.

Chlamydiales comprise important human and animal pathogens as well as endosymbionts of amoebae. Generally, these obligate intracellular living bacteria are characterized by a biphasic developmental cycle, a reduced genome and a restricted metabolic capacity. Because of their metabolic impairment, Chlamydiales essentially rely on the uptake of diverse metabolites from their hosts. Chlamydiales thrive in a special compartment, the inclusion, and hence are surrounded by an additional membrane. Solutes might enter the inclusion through pores and open channels or by redirection of host vesicles, which fuse with the inclusion membrane and release their internal cargo. Recent investigations shed new light on the chlamydia-host interaction and identified an additional way for nutrient uptake into the inclusion. Proteome studies and targeting analyses identified chlamydial and host solute carriers in inclusions of Chlamydia trachomatis infected cells. These transporters are involved in the provision of UDP-glucose and biotin, and probably deliver further metabolites to the inclusion. By the controlled recruitment of specific solute carriers to the inclusion, the chlamydial resident thus can actively manipulate the metabolite availability and composition in the inclusion. This review summarizes recent findings and new ideas on carrier mediated solute uptake into the chlamydial inclusion in the context of the bacterial and host metabolism.

The role of host cell organelles in the development of Simkania negevensis.

Simkania negevensis is an obligate intracellular Chlamydia-like pathogen of the respiratory tract. It infects and multiplies in a wide range of hosts, from unicellular amoeba to a variety of human cells, such as epithelial HeLa and macrophage-like THP1 cells. The Simkania-containing vacuole (SnCV) forms close contacts with the endoplasmic reticulum (ER), and recruits and affects mitochondria of the host cells. Simkania prevent ER stress and require the components of the retrograde transport, as well as several mitochondrial and peroxisomal proteins, for proper development. This review recapitulates our current knowledge about the involvement of various cellular organelles in the life cycle of S. negevensis.

Developmental Cycle and Genome Analysis of Protochlamydia massiliensis sp. nov. a New Species in the Parachlamydiacae Family.

Amoeba-associated microorganisms (AAMs) are frequently isolated from water networks. In this paper, we report the isolation and characterization of Protochlamydia massiliensis, an obligate intracellular Gram-negative bacterium belonging to the Parachlamydiaceae family in the Chlamydiales order, from a cooling water tower. This bacterium was isolated on Vermamoeba vermiformis. It has a multiple range of hosts among amoeba and is characterized by a typical replication cycle of Chlamydiae with a particularity, recently shown in some chlamydia, which is the absence of inclusion vacuoles in the V. vermiformis host, adding by this a new member of Chlamydiae undergoing developmental cycle changes in the newly adapted host V. vermiformis. Draft genome sequencing revealed a chromosome of 2.86 Mb consisting of four contigs and a plasmid of 92 Kb.

Proteomic analysis of the Simkania-containing vacuole: the central role of retrograde transport.

Simkania negevensis is an obligate intracellular bacterial pathogen that grows in amoeba or human cells within a membrane-bound vacuole forming endoplasmic reticulum (ER) contact sites. The membrane of this Simkania-containing vacuole (SnCV) is a critical host-pathogen interface whose origin and molecular interactions with cellular organelles remain poorly defined. We performed proteomic analysis of purified ER-SnCV-membranes using label free LC-MS(2) to define the pathogen-containing organelle composition. Of the 1,178 proteins of human and 302 proteins of Simkania origin identified by this strategy, 51 host cell proteins were enriched or depleted by infection and 57 proteins were associated with host endosomal transport pathways. Chemical inhibitors that selectively interfere with trafficking at the early endosome-to-trans-Golgi network (TGN) interface (retrograde transport) affected SnCV formation, morphology and lipid transport. Our data demonstrate that Simkania exploits early endosome-to-TGN transport for nutrient acquisition and growth.

Permissivity of insect cells to Waddlia chondrophila, Estrella lausannensis and Parachlamydia acanthamoebae.

Recent large scale studies questioning the presence of intracellular bacteria of the Chlamydiales order in ticks and fleas revealed that arthropods, similarly to mammals, reptiles, birds or fishes, can be colonized by Chlamydia-related bacteria with a predominant representation of the Rhabdochlamydiaceae and Parachlamydiaceae families. We thus investigated the permissivity of two insect cell lines towards Waddlia chondrophila, Estrella lausannensis and Parachlamydia acanthamoebae, three bacteria representative of three distinct families within the Chlamydiales order, all documented in ticks and/or in other arthropods. We demonstrated that W. chondrophila and E. lausannensis are able to very efficiently multiply in these insect cell lines. E. lausannensis however induced a rapid cytopathic effect, which somehow restricted its replication. P. acanthamoebae was not able to grow in these cell lines even if inclusions containing a few replicating bacteria could occasionally be observed.

In contrast to Chlamydia trachomatis, Waddlia chondrophila grows in human cells without inhibiting apoptosis, fragmenting the Golgi apparatus, or diverting post-Golgi sphingomyelin transport.

The Chlamydiales are an order of obligate intracellular bacteria sharing a developmental cycle inside a cytosolic vacuole, with very diverse natural hosts, from amoebae to mammals. The clinically most important species is Chlamydia trachomatis. Many uncertainties remain as to how Chlamydia organizes its intracellular development and replication. The discovery of new Chlamydiales species from other families permits the comparative analysis of cell-biological events and may indicate events that are common to all or peculiar to some species and more or less tightly linked to "chlamydial" development. We used this approach in the infection of human cells with Waddlia chondrophila, a species from the family Waddliaceae whose natural host is uncertain. Compared to C. trachomatis, W. chondrophila had slightly different growth characteristics, including faster cytotoxicity. The embedding in cytoskeletal structures was not as pronounced as for the C. trachomatis inclusion. C. trachomatis infection generates proteolytic activity by the protease Chlamydia protease-like activity factor (CPAF), which degrades host substrates upon extraction; these substrates were not cleaved in the case of W. chondrophila. Unlike Chlamydia, W. chondrophila did not protect against staurosporine-induced apoptosis. C. trachomatis infection causes Golgi apparatus fragmentation and redirects post-Golgi sphingomyelin transport to the inclusion; both were absent from W. chondrophila-infected cells. When host cells were infected with both species, growth of both species was reduced. This study highlights differences between bacterial species that both depend on obligate intracellular replication inside an inclusion. Some features seem principally dispensable for intracellular development of Chlamydiales in vitro but may be linked to host adaptation of Chlamydia and the higher virulence of C. trachomatis.

Trafficking of Estrella lausannensis in human macrophages.

Estrella lausannensis is a new member of the Chlamydiales order. Like other Chlamydia-related bacteria, it is able to replicate in amoebae and in fish cell lines. A preliminary study investigating the pathogenic potential of Chlamydia-related bacteria found a correlation between antibody response to E. lausannensis and pneumonia in children. To further investigate the pathogenic potential of E. lausannensis, we determined its ability to grow in human macrophages and its intracellular trafficking. The replication in macrophages resulted in viable E. lausannensis; however, it caused a significant cytopathic effect. The intracellular trafficking of E. lausannensis was analyzed by determining the interaction of the Estrella-containing inclusions with various endocytic markers as well as host organelles. The E. lausannensis inclusion escaped the endocytic pathway rapidly avoiding maturation into phagolysosomes by preventing both EEA-1 and LAMP-1 accumulation. Compared to Waddlia chondrophila, another Chlamydia-related bacteria, the recruitment of mitochondria and endoplasmic reticulum was minimal for E. lausannensis inclusions. Estrella lausannensis appears to use a distinct source of nutrients and energy compared to other members of the Chlamydiales order. In conclusion, we hypothesize that E. lausannensis has a restricted growth in human macrophages, due to its reduced capacity to control programmed cell death.

Amoebal endosymbiont Parachlamydia acanthamoebae Bn9 can grow in immortal human epithelial HEp-2 cells at low temperature; an in vitro model system to study chlamydial evolution.

Ancient chlamydiae diverged into pathogenic and environmental chlamydiae 0.7-1.4 billion years ago. However, how pathogenic chlamydiae adapted to mammalian cells that provide a stable niche at approximately 37 °C, remains unknown, although environmental chlamydiae have evolved as endosymbionts of lower eukaryotes in harsh niches of relatively low temperatures. Hence, we assessed whether an environmental chlamydia, Parachlamydia Bn9, could grow in human HEp-2 cells at a low culture temperature of 30 °C. The assessment of inclusion formation by quantitative RT-PCR revealed that the numbers of bacterial inclusion bodies and the transcription level of 16SrRNA significantly increased after culture at 30 °C compared to at 37 °C. Confocal microscopy showed that the bacteria were located close to HEp-2 nuclei and were actively replicative. Transmission electron microscopy also revealed replicating bacteria consisting of reticular bodies, but with a few elementary bodies. Cytochalasin D and rifampicin inhibited inclusion formation. Lactacystin slightly inhibited bacterial inclusion formation. KEGG analysis using a draft genome sequence of the bacteria revealed that it possesses metabolic pathways almost identical to those of pathogenic chlamydia. Interestingly, comparative genomic analysis with pathogenic chlamydia revealed that the Parachlamydia similarly possess the genes encoding Type III secretion system, but lacking genes encoding inclusion membrane proteins (IncA to G) required for inclusion maturation. Taken together, we conclude that ancient chlamydiae had the potential to grow in human cells, but overcoming the thermal gap was a critical event for chlamydial adaptation to human cells.

The role of peptidoglycan in chlamydial cell division: towards resolving the chlamydial anomaly.

Chlamydiales are obligate intracellular bacteria including some important pathogens causing trachoma, genital tract infections and pneumonia, among others. They share an atypical division mechanism, which is independent of an FtsZ homologue. However, they divide by binary fission, in a process inhibited by penicillin derivatives, causing the formation of an aberrant form of the bacteria, which is able to survive in the presence of the antibiotic. The paradox of penicillin sensitivity of chlamydial cells in the absence of detectable peptidoglycan (PG) was dubbed the chlamydial anomaly, since no PG modified by enzymes (Pbps) that are the usual target of penicillin could be detected in Chlamydiales. We review here the recent advances in this field with the first direct and indirect evidences of PG-like material in both Chlamydiaceae and Chlamydia-related bacteria. Moreover, PG biosynthesis is required for proper localization of the newly described septal proteins RodZ and NlpD. Taken together, these new results set the stage for a better understanding of the role of PG and septal proteins in the division mechanism of Chlamydiales and illuminate the long-standing chlamydial anomaly. Moreover, understanding the chlamydial division mechanism is critical for the development of new antibiotics for the treatment of chlamydial chronic infections.

A bovine model of a respiratory Parachlamydia acanthamoebae infection.

The aim of this study was to evaluate the pathogenicity of Parachlamydia (P.) acanthamoebae as a potential agent of lower respiratory tract disease in a bovine model of induced lung infection. Intrabronchial inoculation with P. acanthamoebae was performed in healthy calves aged 2-3 months using two challenge doses: 10(8) and 10(10) bacteria per animal. Controls received 10(8) heat-inactivated bacteria. Challenge with 10(8) viable Parachlamydia resulted in a mild degree of general indisposition, whereas 10(10) bacteria induced a more severe respiratory illness becoming apparent 1-2 days post inoculation (dpi), affecting 9/9 (100%) animals and lasting for 6 days. The extent of macroscopic pulmonary lesions was as high as 6.6 (6.0)% [median (range)] of lung tissue at 2-4 dpi and correlated with parachlamydial genomic copy numbers detected by PCR, and with bacterial load estimated by immunohistochemistry in lung tissue. Clinical outcome, acute phase reactants, pathological findings and bacterial load exhibited an initial dose-dependent effect on severity. Animals fully recovered from clinical signs of respiratory disease within 5 days. The bovine lung was shown to be moderately susceptible to P. acanthamoebae, exhibiting a transient pneumonic inflammation after intrabronchial challenge. Further studies are warranted to determine the precise pathophysiologic pathways of host-pathogen interaction.

The chlamydial protease CPAF: important or not, important for what?

The protease CPAF is only found in Chlamydiales and in at least most bacteria that share with Chlamydia the biphasic life-style in a cytosolic inclusion. CPAF is intriguing: it appears to be secreted from the inclusion across the inclusion membrane into the cytosol. A bacterial protease ravaging in the cytosol of a human cell may cause a plethora of effects. Curiously, very few are known. The current discussion is bogged down by a focus on experimental artifact, while proposed functions of CPAF remain speculative. I here make the attempt to summarize what we know about CPAF.

High-temperature adapted primitive Protochlamydia found in Acanthamoeba isolated from a hot spring can grow in immortalized human epithelial HEp-2 cells.

To elucidate how ancient pathogenic chlamydiae could overcome temperature barriers to adapt to human cells, we characterized a primitive chlamydia found in HS-T3 amoebae (Acanthamoeba) isolated from a hot spring. Phylogenetic analysis revealed the primitive species to be Protochlamydia. In situ hybridization staining showed broad distribution into the amoebal cytoplasm, which was supported by transmission electron microscopic analysis showing typical chlamydial features, with inclusion bodies including both elementary and reticular bodies. Interestingly, although most amoebae isolated from natural environments show reduced growth at 37°C, the HS-T3 amoebae harbouring the Protochlamydia grew well at body temperature. Although infection with Protochlamydia did not confer temperature tolerance to the C3 amoebae, the number of infectious progenies rapidly increased at 37°C with amoebal lysis. In immortalized human epithelial HEp-2 cells, fluorescence microscopic study revealed atypical inclusion of the Protochlamydia, and quantitative real-time polymerase chain reaction analyses also showed an increase in 16S ribosomal RNA DNA amounts. Together, these results showed that the Protochlamydia found in HS-T3 amoebae isolated from a hot spring successfully adapted to immortalized human HEp-2 cells at 37°C, providing further information on the evolution of ancient Protochlamydia to the present pathogenic chlamydiae.

Early expression of the type III secretion system of Parachlamydia acanthamoebae during a replicative cycle within its natural host cell Acanthamoeba castellanii.

The type three secretion system (T3SS) operons of Chlamydiales bacteria are distributed in different clusters along their chromosomes and are conserved at both the level of sequence and genetic organization. A complete characterization of the temporal expression of multiple T3SS components at the transcriptional and protein levels has been performed in Parachlamydia acanthamoebae, replicating in its natural host cell Acanthamoeba castellanii. The T3SS components were classified in four different temporal clusters depending on their pattern of expression during the early, mid- and late phases of the infectious cycle. The putative T3SS transcription units predicted in Parachlamydia are similar to those described in Chlamydia trachomatis, suggesting that T3SS units of transcriptional expression are highly conserved among Chlamydiales bacteria. The maximal expression and activation of the T3SS of Parachlamydia occurred during the early to mid-phase of the infectious cycle corresponding to a critical phase during which the intracellular bacterium has (1) to evade and/or block the lytic pathway of the amoeba, (2) to differentiate from elementary bodies (EBs) to reticulate bodies (RBs), and (3) to modulate the maturation of its vacuole to create a replicative niche able to sustain efficient bacterial growth.

Lack of effective anti-apoptotic activities restricts growth of Parachlamydiaceae in insect cells.

The fundamental role of programmed cell death in host defense is highlighted by the multitude of anti-apoptotic strategies evolved by various microbes, including the well-known obligate intracellular bacterial pathogens Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae. As inhibition of apoptosis is assumed to be essential for a successful infection of humans by these chlamydiae, we analyzed the anti-apoptotic capacity of close relatives that occur as symbionts of amoebae and might represent emerging pathogens. While Simkania negevensis was able to efficiently replicate within insect cells, which served as model for metazoan-derived host cells, the Parachlamydiaceae (Parachlamydia acanthamoebae and Protochlamydia amoebophila) displayed limited intracellular growth, yet these bacteria induced typical features of apoptotic cell death, including formation of apoptotic bodies, nuclear condensation, internucleosomal DNA fragmentation, and effector caspase activity. Induction of apoptosis was dependent on bacterial activity, but not bacterial de novo protein synthesis, and was detectable already at very early stages of infection. Experimental inhibition of host cell death greatly enhanced parachlamydial replication, suggesting that lack of potent anti-apoptotic activities in Parachlamydiaceae may represent an important factor compromising their ability to successfully infect non-protozoan hosts. These findings highlight the importance of the evolution of anti-apoptotic traits for the success of chlamydiae as pathogens of humans and animals.

Amoebal host range, host-free survival and disinfection susceptibility of environmental Chlamydiae as compared to Chlamydia trachomatis.

The term 'Chlamydia-like organisms' encompasses obligate intracellular bacterial species phylogenetically close to Chlamydiaceae. Most are associated with free-living amoebae, and several could be responsible for respiratory tract infections and abortion in human and animals. Despite increasing concern about their pathogenic role, the prevalence, biodiversity and ecology of Chlamydia-related bacteria still remain largely unknown. In this study, six members of the Chlamydiales were tested, including Parachlamydia acanthamoebae (two different strains), Protochlamydia naegleriophila, Waddlia chondrophila, Criblamydia sequanensis and Chlamydia trachomatis as a reference. Intracellular growth was tested in 11 different Acanthamoeba strains, demonstrating significant differences in host susceptibilities to infection depending on strains investigated. Survival of host-free bacteria in suspension or dried onto surfaces was also explored, demonstrating that Chlamydia-like organisms present better survival capacity than C. trachomatis. Longer survival times were observed for bacteria suspended in rich culture medium, with survivors being detected after 10 weeks incubation. We also tested susceptibility of host-free Chlamydia-like organisms to several disinfection treatments. Each chemical biocide tested reduced viability of host-free Chlamydia by more than 4 logs. Conversely, all Chlamydia-like organisms tested resisted exposure at 55 °C for 10 min, while C. trachomatis was completely inactivated.

Permissivity of fish cell lines to three Chlamydia-related bacteria: Waddlia chondrophila, Estrella lausannensis and Parachlamydia acanthamoebae.

Epitheliocystis is an infectious disease affecting gills and skin of various freshwater and marine fishes, associated with high mortality and reduced growth of survivors. Candidatus Piscichlamydia salmonis and Clavochlamydia salmonicola have recently been identified as aetiological agents of epitheliocystis in Atlantic Salmon. In addition, several other members of the Chlamydiales order have been identified in other fish species. To clarify the pathogenicity of Chlamydia-like organisms towards fishes, we investigated the permissivity of two fish cell lines, EPC-175 (Fathead Minnow) and RTG-2 (rainbow trout) to three Chlamydia-related bacteria: Waddlia chondrophila, Parachlamydia acanthamoebae and Estrella lausannensis. Quantitative PCR and immunofluorescence demonstrated that W. chondrophila and, to a lesser extent, E. lausannensis were able to replicate in the two cell lines tested. Waddlia chondrophila multiplied rapidly in its host cell and a strong cytopathic effect was observed. During E. lausannensis infection, we observed a limited replication of the bacteria not followed by host cell lysis. Very limited replication of P. acanthamoebae was observed in both cell lines tested. Given its high infectivity and cytopathic effect towards fish cell lines, W. chondrophila represents the most interesting Chlamydia-related bacteria to be used to develop an in vivo model of epitheliocystis disease in fishes.

Estrella lausannensis, a new star in the Chlamydiales order.

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.

Permissivity of Vero cells, human pneumocytes and human endometrial cells to Waddlia chondrophila.

Growing evidence suggests that the bacterium Waddlia chondrophila, a novel member of the Chlamydiales order, is an agent of miscarriage in humans and abortion in ruminants. We thus investigated the permissivity of three epithelial cell lines, primate Vero kidney cells, human A549 pneumocytes and human Ishikawa endometrial cells to this strict intracellular bacteria. Bacterial growth kinetics in these cell lines was assessed by quantitative PCR and immunofluorescence and our results demonstrated that W. chondrophila enters and efficiently multiplies in these epithelial cell lines. Additionally, confocal and electron microscopy indicated that the bacteria co-localize with host cell mitochondria. Within Vero and A549 cells, intracellular growth of W. chondrophila was associated with a significant decrease in host cell viability while no such cytophatic effect was detected in Ishikawa cells. Bacterial cell growth in this endometrial cell line stopped 48 h after infection. This stop in the replication of W. chondrophila coincided with the appearance of large aberrant bodies, a form of the bacteria also observed in Chlamydiaceae and associated with persistence. This persistent state of W. chondrophila may explain recurrent episodes of miscarriage in vivo, since the bacteria might reactivate within endometrial cells following hormonal changes that occur during pregnancy.

Looking inside the box: using Raman microspectroscopy to deconstruct microbial biomass stoichiometry one cell at a time.

Stoichiometry of microbial biomass is a key determinant of nutrient recycling in a wide variety of ecosystems. However, little is known about the underlying causes of variance in microbial biomass stoichiometry. This is primarily because of technological constraints limiting the analysis of macromolecular composition to large quantities of microbial biomass. Here, we use Raman microspectroscopy (MS), to analyze the macromolecular composition of single cells of two species of bacteria grown on minimal media over a wide range of resource stoichiometry. We show that macromolecular composition, determined from a subset of identified peaks within the Raman spectra, was consistent with macromolecular composition determined using traditional analytical methods. In addition, macromolecular composition determined by Raman MS correlated with total biomass stoichiometry, indicating that analysis with Raman MS included a large proportion of a cell's total macromolecular composition. Growth phase (logarithmic or stationary), resource stoichiometry and species identity each influenced each organism's macromolecular composition and thus biomass stoichiometry. Interestingly, the least variable peaks in the Raman spectra were those responsible for differentiation between species, suggesting a phylogenetically specific cellular architecture. As Raman MS has been previously shown to be applicable to cells sampled directly from complex environments, our results suggest Raman MS is an extremely useful application for evaluating the biomass stoichiometry of environmental microorganisms. This includes the ability to partition microbial biomass into its constituent macromolecules and increase our understanding of how microorganisms in the environment respond to resource heterogeneity.