Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy.

Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10-20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.

A clinical Acanthamoeba isolate harboring two distinct bacterial endosymbionts.

Acanthamoebae feed on bacteria but are also frequent hosts of bacterial symbionts. Here, we describe the stable co-occurrence of two symbionts, one affiliated to the genus Parachlamydia and the other to the candidate genus Paracaedibacter (Alphaproteobacteria), within a clinical isolate of Acanthamoeba hatchetti genotype T4. We performed fluorescence in situ hybridization (FISH) and transmission electron microscopy (TEM) to describe this symbiosis. Our study adds to other reports of simultaneous co-occurrence of two symbionts within one Acanthamoeba cell.

The emergence of epitheliocystis in the upper Rhone region: evidence for Chlamydiae in wild and farmed salmonid populations.

We present the first study comparing epitheliocystis in a wild and farmed salmonid in Europe. Sampling three tributaries to the Lake Geneva, including one from headwaters to river mouth, revealed an unequal distribution of epitheliocystis in brown trout (Salmo trutta). When evaluated histologically and comparing sites grouped as wild versus farm, the probability of finding infected trout is higher on farms. In contrast, the infection intensities, as estimated by the number of cysts per gill arch, were higher on average and showed maximum values in the wild trout. Sequence analysis showed the most common epitheliocystis agents were Candidatus Piscichlamydia salmonis, all clustering into a single clade, whereas Candidatus Clavichlamydia salmonicola sequences cluster in two closely related subspecies, of which one was mostly found in farmed fish and the other exclusively in wild brown trout, indicating that farms are unlikely to be the source of infections in wild trout. A detailed morphological analysis of cysts using transmission electron microscopy revealed unique features illustrating the wide divergence existing between Ca. P. salmonis and Ca. C. salmonicola within the phylum Chlamydiae.

Characterization of 'Candidatus Syngnamydia salmonis' (Chlamydiales, Simkaniaceae), a bacterium associated with epitheliocystis in Atlantic salmon (Salmo salar L.).

Two Chlamydiales have previously been found to infect Atlantic salmon (Salmo salar L.), Candidatus Piscichlamydia salmonis and Candidatus Clavichlamydia salmonicola. Both develop intracellularly in cyst-like inclusions in gill cells, generally referred to as epitheliocysts. Here, we present evidence for the association of a novel species of Chlamydiales with epitheliocystis in Atlantic salmon. Based on its partial 16S rRNA gene sequence, it is a new member of the family Simkaniaceae, and a 95.7 % identity to the type species Candidatus Syngnamydia venezia suggests inclusion in the candidate genus Syngnamydia. The presence of the bacterium in epitheliocysts in gills of Atlantic salmon was demonstrated by RNA-RNA hybridization. Ultrastructurally, the novel bacterium produces pleomorphic reticulate bodies and elementary bodies (EBs) with a characteristic morphology. The EBs are short rods with a terminal disc-like cap area, a sub-apical spherical vacuole-like electron-lucent structure and a post-equatorial nucleoid. We propose the name Candidatus Syngnamydia salmonis for this new agent from epitheliocysts in seawater-reared salmon .

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.

Architecture and host interface of environmental chlamydiae revealed by electron cryotomography.

Chlamydiae comprise important pathogenic and symbiotic bacteria that alternate between morphologically and physiologically different life stages during their developmental cycle. Using electron cryotomography, we characterize the ultrastructure of the developmental stages of three environmental chlamydiae: Parachlamydia acanthamoebae, Protochlamydia amoebophila and Simkania negevensis. We show that chemical fixation and dehydration alter the cell shape of Parachlamydia and that the crescent body is not a developmental stage, but an artefact of conventional electron microscopy. We further reveal type III secretion systems of environmental chlamydiae at macromolecular resolution and find support for a chlamydial needle-tip protein. Imaging bacteria inside their host cells by cryotomography for the first time, we observe marked differences in inclusion morphology and development as well as host organelle recruitment between the three chlamydial organisms, with Simkania inclusions being tightly enveloped by the host endoplasmic reticulum. The study demonstrates the power of electron cryotomography to reveal structural details of bacteria-host interactions that are not accessible using traditional methods.

Discovery of chlamydial peptidoglycan reveals bacteria with murein sacculi but without FtsZ.

Chlamydiae are important pathogens and symbionts with unique cell biological features. They lack the cell-division protein FtsZ, and the existence of peptidoglycan (PG) in their cell wall has been highly controversial. FtsZ and PG together function in orchestrating cell division and maintaining cell shape in almost all other bacteria. Using electron cryotomography, mass spectrometry and fluorescent labelling dyes, here we show that some environmental chlamydiae have cell wall sacculi consisting of a novel PG type. Treatment with fosfomycin (a PG synthesis inhibitor) leads to lower infection rates and aberrant cell shapes, suggesting that PG synthesis is crucial for the chlamydial life cycle. Our findings demonstrate for the first time the presence of PG in a member of the Chlamydiae. They also present a unique example of a bacterium with a PG sacculus but without FtsZ, challenging the current hypothesis that it is the absence of a cell wall that renders FtsZ non-essential.

Crescent and star shapes of members of the Chlamydiales order: impact of fixative methods.

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.

Metabolic features of Protochlamydia amoebophila elementary bodies--a link between activity and infectivity in Chlamydiae.

The Chlamydiae are a highly successful group of obligate intracellular bacteria, whose members are remarkably diverse, ranging from major pathogens of humans and animals to symbionts of ubiquitous protozoa. While their infective developmental stage, the elementary body (EB), has long been accepted to be completely metabolically inert, it has recently been shown to sustain some activities, including uptake of amino acids and protein biosynthesis. In the current study, we performed an in-depth characterization of the metabolic capabilities of EBs of the amoeba symbiont Protochlamydia amoebophila. A combined metabolomics approach, including fluorescence microscopy-based assays, isotope-ratio mass spectrometry (IRMS), ion cyclotron resonance Fourier transform mass spectrometry (ICR/FT-MS), and ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was conducted, with a particular focus on the central carbon metabolism. In addition, the effect of nutrient deprivation on chlamydial infectivity was analyzed. Our investigations revealed that host-free P. amoebophila EBs maintain respiratory activity and metabolize D-glucose, including substrate uptake as well as host-free synthesis of labeled metabolites and release of labeled CO2 from (13)C-labeled D-glucose. The pentose phosphate pathway was identified as major route of D-glucose catabolism and host-independent activity of the tricarboxylic acid (TCA) cycle was observed. Our data strongly suggest anabolic reactions in P. amoebophila EBs and demonstrate that under the applied conditions D-glucose availability is essential to sustain metabolic activity. Replacement of this substrate by L-glucose, a non-metabolizable sugar, led to a rapid decline in the number of infectious particles. Likewise, infectivity of Chlamydia trachomatis, a major human pathogen, also declined more rapidly in the absence of nutrients. Collectively, these findings demonstrate that D-glucose is utilized by P. amoebophila EBs and provide evidence that metabolic activity in the extracellular stage of chlamydiae is of major biological relevance as it is a critical factor affecting maintenance of infectivity.

'Cand. Actinochlamydia clariae' gen. nov., sp. nov., a unique intracellular bacterium causing epitheliocystis in catfish (Clarias gariepinus) in Uganda.

BACKGROUND AND OBJECTIVES: Epitheliocystis, caused by bacteria infecting gill epithelial cells in fish, is common among a large range of fish species in both fresh- and seawater. The aquaculture industry considers epitheliocystis an important problem. It affects the welfare of the fish and the resulting gill disease may lead to mortalities. In a culture facility in Kampala, Uganda, juveniles of the African sharptooth catfish (Clarias gariepinus) was observed swimming in the surface, sometimes belly up, showing signs of respiratory problems. Histological examination of gill tissues from this fish revealed large amounts of epitheliocysts, and also presence of a few Ichthyobodo sp. and Trichodina sp. METHODS AND RESULTS: Sequencing of the epitheliocystis bacterium 16S rRNA gene shows 86.3% similarity with Candidatus Piscichlamydia salmonis causing epitheliocystis in Atlantic salmon (Salmo salar). Transmission electron microscopy showed that the morphology of the developmental stages of the bacterium is similar to that of members of the family Chlamydiaceae. The similarity of the bacterium rRNA gene sequences compared with other chlamydia-like bacteria ranged between 80.5% and 86.3%. Inclusions containing this new bacterium have tubules/channels (termed actinae) that are radiating from the inclusion membrane and opening on the cell surface or in neighbouring cells. CONCLUSIONS: Radiation of tubules/channels (actinae) from the inclusion membrane has never been described in any of the other members of Chlamydiales. It seems to be a completely new character and an apomorphy. We propose the name Candidatus Actinochlamydia clariae gen. nov., sp. nov. (Actinochlamydiaceae fam. nov., order Chlamydiales, phylum Chlamydiae) for this new agent causing epitheliocystis in African sharptooth catfish.

Proteomic analysis of the outer membrane of Protochlamydia amoebophila elementary bodies.

Chlamydiae are obligate intracellular bacteria, comprising some of the most important bacterial pathogens of animals and humans. During their unique developmental cycle they have to attach to and enter their eukaryotic host cells, a process mediated by proteins in the chlamydial outer membrane. So far the only experimental data for chlamydial outer membrane proteins are available from members of the Chlamydiaceae, a family comprising exclusively human and animal pathogens. To get further insights into the evolution of the protein composition of the chlamydial outer membrane and into host-dependent differences, we performed an extensive experimental analysis of outer membrane fractions of Protochlamydia amoebophila elementary bodies, which constitute the infectious form of this non-pathogenic member of the Chlamydiae that thrives as a symbiont in Acanthamoeba spp. We used 1-D and 2-DE in combination with MALDI-TOF, MALDI-TOF/TOF and nanoLC-ESI-MS/MS, and compared our experimental results with a previously published in silico analysis of chlamydial outer membrane proteins. This resulted in the identification of 38 proteins supported by both studies and therefore very likely to be located in the P. amoebophila outer membrane. The obtained experimental data provide the first comprehensive overview of outer membrane proteins of a chlamydial organism outside the Chlamydiaceae. They reveal both fundamental differences and convergent evolution between pathogenic and symbiotic chlamydiae.

Characterization of a Neochlamydia-like bacterium associated with epitheliocystis in cultured Arctic charr Salvelinus alpinus.

Infections of branchial epithelium by intracellular gram-negative bacteria, termed epitheliocystis, have limited culture of Arctic charr Salvelinus alpinus. To characterize a bacterium associated with epitheliocystis in cultured charr, gills were sampled for histopathologic examination, conventional and immunoelectron microscopy, in situ hybridization, 16S ribosomal DNA (rDNA) amplification, sequence analysis and phylogenetic inference. Sampling was conducted at the Freshwater Institute (Shepherdstown, West Virginia, USA) during outbreaks of epitheliocystis in April and May 2002. Granular, basophilic, cytoplasmic inclusions in charr gill were shown to stain with Macchiavello, Lendrum's phloxine-tartrazine and Gimenez histochemical techniques. Ultrastructurally, inclusions were membrane-bound and contained round to elongate reticulate bodies that were immunoreactive to an antibody against chlamydial lipopolysaccharide, suggesting the presence of similar epitopes. DNA extracted from gills supported amplification of the most polymorphic and phylogenetically relevant region of the 16S rRNA gene, which had 97 to 100% identity with several uncultured clinical Neochlamydia spp. (order Chlamydiales) Clones WB13 (AY225593.1) and WB258 (AY225594.1). Sequence-specific riboprobes localized to inclusions during in situ hybridization experiments. Taxonomic affiliation was inferred by distance- and parsimony-based phylogenetic analyses of the 16S sequence, which branched with Neochlamydia hartmannellae in the order Chlamydiales with high confidence. This is the first molecular characterization of a chlamydia associated with epitheliocystis in Arctic charr and the fourth Neochlamydia spp. sequence to be associated with epitheliocystis. Presence of a clinical neochlamydial sequence, first identified from a cat, in Arctic charr suggests a possible mammalian and piscine host range for some environmental chlamydiae.

Isolation of Waddlia malaysiensis, a novel intracellular bacterium, from fruit bat (Eonycteris spelaea).

An obligate intracellular bacterium was isolated from urine samples from 7 (3.5%) of 202 fruit bats (Eonycteris spelaea) in peninsular Malaysia. The bacterium produced large membrane-bound inclusions in human, simian, and rodent cell lines, including epithelial, fibroblastlike, and lymphoid cells. Thin-section electron microscopy showed reticulate bodies dividing by binary fission and elementary bodies in the inclusions; mitochondria surrounded the inclusions. The inclusions were positive for periodic acid-Schiff stain but could not be stained by fluorescein-labeled anti-Chlamydia trachomatis major outer membrane protein monoclonal antibody. The bacterium was resistant to penicillin and streptomycin (MICs > 256 mg/L) but susceptible to tetracycline (MIC = 0.25 mg/L) and chloramphenicol (MIC = 0.5 mg/L). Sequence analysis of the 16SrRNA gene indicated that it was most closely related to 2 isolates of Waddlia chondrophila (94% and 96% identity). The 16S and 23S rRNA gene signatures were only 91% identical. We propose this novel bacterium be called W. malaysiensis.

[The discovery of naked cluster particles of Parachlamydia and its developmental mechanism].

OBJECTIVE: To study the survival and developmental morphology of Parachlamydia (BN9) within Acanthamoeba. METHODS: The morphology of BN9 within Acanthamoeba was studied by inverted phase contrast microscope, electron microscope, Gimenez and AO-staining with amoebal co-culture. RESULTS: The endosomal maturation-blocked were formed after the egress of BN9. Two developmental stages-elementary and reticulate bodies, were both observed within the vacuoles. The reticulate bodies, multiplicated by binary fission, were located mainly within the vacuoles, while the elementary bodies can also be located in the plasma individually. The naked cluster particles were observed after the trophozoites cytolysis with Gimenez-staining. The light infectious trophozoites could encyst, and elementary bodies could survive within the mature cysts. CONCLUSION: The egress of BN9 could form the endosomal maturation-blocked, which was presented in two developmental stages-elementary and reticulate bodies. It exhibited the cytolysin activity that could lyse the infectious trophozoites and were expelled in the vesicles. A few light infected amoeba could encyst with survival elementary bodies in the plasma.

Parachlamydia acanthamoebae enters and multiplies within human macrophages and induces their apoptosis [corrected].

Parachlamydia acanthamoebae is an obligately intracellular bacterium that naturally infects free-living amoebae. It is a potential human pathogen and may survive in human macrophages. We studied P. acanthamoebae entry into, and multiplication within, human monocyte-derived macrophages. After 8 h of incubation, 80% of macrophages were infected with a mean of 3.8 P. acanthamoebae organisms per cell. Electron microscopy demonstrated that parachlamydiae were in an intracellular vacuole. After infection with living organisms, the number of parachlamydiae per macrophage increased 4 times from day 0 to day 4, whereas heat-inactivated parachlamydiae were eliminated during the same period. Quantitative PCR confirmed that P. acanthamoebae replicates within macrophages. Transcriptional activity of P. acanthamoeba was detected by reverse transcription-PCR targeting the gene encoding ADP-ATP translocase (tlc). P. acanthamoebae exerted a cytopathic effect on macrophages. When macrophages were infected with living bacteria, their number decreased significantly from day 0 to day 4 due to apoptosis, as shown by annexin-V binding and electron microscopy. This study shows that P. acanthamoebae enters and multiplies within human macrophages before inducing their apoptosis.

Infections with the chlamydia-like microorganism Simkania negevensis, a possible emerging pathogen.

Although evidence for the existence of numerous chlamydia-like microorganisms has been discovered in both environmental samples and clinical specimens, very few have been grown in vitro, and little is known of their pathogenic potential. Of all such organisms, Simkania negevensis is probably the most extensively studied. This review summarizes current knowledge about this intracellular bacterium, focusing especially on human infections.

Crescent bodies of Parachlamydia acanthamoeba and its life cycle within Acanthamoeba polyphaga: an electron micrograph study.

Parachlamydiaceae are endosymbionts of free-living amoeba first identified in 1997. Two developmental stages, elementary and reticulate bodies, were observed; however, their localization and proportions according to culture condition and duration remain unknown. The life cycle of Parachlamydia acanthamoeba within Acanthamoeba polyphaga was studied by transmission electron microscopy of 8-, 36-, and 144-h coculture. Morphometry and quantification were performed using SAMBA software. The elementary body, the predominant stage within the amoebae, was located mainly within their vacuoles. The multiplication of Parachlamydia bacteria by binary fission of reticulate bodies was independently associated with culture in PYG broth (odds ratio [OR] = 4.4; 95% confidence interval [CI], 1.55 to 12.46) and with the presence of reticulate bodies within the amoebae (OR = 2.10; 95% CI, 1.53 to 2.89). A third developmental stage was observed, the crescent body. Its presence outside and inside the amoebae was associated mainly with prolonged incubation time (OR = 3.98; 95% CI, 1.49 to 10.68, and OR = 5.98; 95% CI, 1.75 to 20.4, respectively). Elementary and crescent bodies were released into the extracellular medium within vesicles or after amoebal lysis. For both, phagocytosis was their mode of entry. This electron micrograph study revealed another infective developmental stage, the crescent body, and provided quantitative analysis of the life cycle of P. acanthamoeba within A. polyphaga.

Parachlamydiaceae: potential emerging pathogens.

Parachlamydiaceae, which naturally infect amoebae, form a sister taxon to the Chlamydiaceae on the basis of the Chlamydia-like cycle of replication and 80% to 90% homology of ribosomal RNA genes. Because intra-amoebal growth could increase the virulence of some intracellular bacteria, Parachlamydiaceae may be pathogenic. Arguments supporting a pathogenic role are that Chlamydia pneumoniae, a well-recognized agent of pneumonia, was shown to infect free-living amoebae and that another member of the Chlamydiales, Simkania negevensis, which has 88% homology with Parachlamydia acanthamoebae, has caused pneumonia in adults and acute bronchiolitis in infants. The recent identification of a 16S rRNA gene sequence of a Parachlamydiaceae from bronchoalveolar lavage is additional evidence supporting potential for pathogenicity.

The growth cycle of Simkania negevensis.

Simkania negevensis, a bacterium formerly referred to as 'the micro-organism Z' or 'Simkania Z', belongs to the order Chlamydiales, assigned to the family Simkaniaceae: The purpose of this study was to investigate the production of Simkania negevensis progeny in infected cells in comparison with the well-documented Chlamydiaceae developmental cycle. It was found that replicating Simkania negevensis in Vero cells resembled the reticulate bodies of all known chlamydial species: in electron micrographs they were reticulated, homogeneously staining, and often caught in the process of binary division. These replicative forms were found in low abundance shortly after infection, but by 3 days post-infection they were the most prevalent particles in host cells. Electron-dense forms of Simkania negevensis began to appear on the third day post-infection, but quantitatively did not account for the high titre of infectivity in extracts from these host cells. These had both electron-dense and electron-lucent areas, a characteristic seen only in a few chlamydial species. Simkania negevensis infectivity did not appreciably change during the ensuing 12 days required for host cell lysis, despite an eightfold increase in the proportion of electron-dense bacteria over this time. The emergence of electron-dense bodies, increase in infectivity and host-cell lysis were not synchronized developmental events. This is a novel finding in Chlamydiales spp. and suggests that Simkania negevensis will provide new perspectives in the mechanisms of chlamydial intracellular growth.

Infection of Acanthamoeba polyphaga with Simkania negevensis and S. negevensis survival within amoebal cysts.

Simkania negevensis, a novel microorganism belonging to the family Simkaniaceae in the order Chlamydiales, has an intracellular developmental cycle during which two morphological entities, elementary bodies (EB) and reticulate bodies (RB), are seen by electron microscopy. Rates of seropositivity to the organism are high in certain population groups, and S. negevensis has been associated with respiratory illness in humans. This study reports for the first time the ability of S. negevensis to survive and grow inside Acanthamoeba polyphaga in addition to its known ability to grow in cell cultures of human or simian origin. Infectivity of S. negevensis and growth in amoebae were monitored by immunoperoxidase assays. Long-term persistence and exponential growth of S. negevensis in amoebal trophozoites were demonstrated by infectivity assays and by electron microscopy. EB and dividing RB of S. negevensis were observed within inclusion bodies inside A. polyphaga. When S. negevensis-infected A. polyphaga amoebae were exposed to adverse conditions resulting in encystation of the amoebae, several possible outcomes were observed: cysts containing both normal amoebic cytoplasm and S. negevensis; cysts in which S. negevensis cells were relegated to the space between cyst walls; and cysts containing S. negevensis, but apparently lacking amoebal cytoplasm. S. negevensis within dried amoebal cysts was capable of long-term survival. The possibility that amoebae may have a role in natural transmission of S. negevensis needs to be investigated.