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.     

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.     

Ultrastructural analysis of the effects of erythromycin on the morphology and developmental cycle of Chlamydia trachomatis HAR-13

The effect of erythromycin (10 g/ml) on the morphology and developmental cycle of Chlamydia trachomatis HAR-13 was examined by electron microscopy. When the antibiotic was added later than 24 h post infection, the HAR-13 morphology or developmental cycle was not altered. Addition at 18 or 24 h post infection inhibited glycogen production, blocked the transformation of the reticulate body to elementary body, and produced ghost bodies and reticulate bodies twice the diameter of untreated reticulate bodies. When erythromycin was added within 12 h post infection, the conversion of the elementary body to reticulate body was inhibited. Erythromycin (10 g/ml) was bactericidal to strain HAR-13 throughout the developmental cycle.Abbreviations CXM cycloheximide media - IFU inclusion forming units - MEM minimal essential medium     

Raman microspectroscopy reveals long‐term extracellular activity of chlamydiae

The phylum Chlamydiae consists exclusively of obligate intracellular bacteria. Some of them are formidable pathogens of humans, while others occur as symbionts of amoebae. These genetically intractable bacteria possess a developmental cycle consisting of replicative reticulate bodies and infectious elementary bodies, which are believed to be physiologically inactive. Confocal Raman microspectroscopy was applied to differentiate between reticulate bodies and elementary bodies of Protochlamydia amoebophila and to demonstrate in situ the labelling of this amoeba symbiont after addition of isotope‐labelled phenylalanine. Unexpectedly, uptake of this amino acid was also observed for both developmental stages for up to 3 weeks, if incubated extracellularly with labelled phenylalanine, and P. amoebophila remained infective during this period. Furthermore, P. amoebophila energizes its membrane and performs protein synthesis outside of its host. Importantly, amino acid uptake and protein synthesis after extended extracellular incubation could also be demonstrated for the human pathogen Chlamydia trachomatis, which synthesizes stress‐related proteins under these conditions as shown by 2‐D gel electrophoresis and MALDI‐TOF/TOF mass spectrometry. These findings change our perception of chlamydial biology and reveal that host‐free analyses possess a previously not recognized potential for direct experimental access to these elusive microorganisms.     

Phage infection of the obligate intracellular bacterium, Chlamydia psittaci strain guinea pig inclusion conjunctivitis

The infectious cycle of phiCPG1, a bacteriophage that infects the obligate intracellular pathogen, Chlamydia psittaci strain Guinea Pig Inclusion Conjunctivitis, was observed using transmission electron microscopy of phage-hyperinfected, Chlamydia-infected HeLa cells. Phage attachment to extracellular, metabolically dormant, infectious elementary bodies and cointernalisation are demonstrated. Following entry, phage infection takes place as soon as elementary bodies differentiate into metabolically active reticulate bodies. Phage-infected bacteria follow an altered developmental path whereby cell division is inhibited, producing abnormally large reticulate bodies, termed maxi-reticulate bodies, which do not mature to elementary bodies. These forms eventually lyse late in the chlamydial developmental cycle, releasing abundant phage progeny in the inclusion and, upon lysis of the inclusion membrane, into the cytosol of the host cell. Structural integrity of the hyperinfected HeLa cell is markedly compromised at late stages. Released phage particles attach avidly to the outer leaflet of the outer membranes of lysed and unlysed Chlamydiae at different stages of development, suggesting the presence of specific phage receptors in the outer membrane uniformly during the chlamydial developmental cycle. A mechanism for phage infection is proposed, whereby phage gains access to replicating chlamydiae by attaching to the infectious elementary body, subsequently subverting the chlamydial developmental cycle to its own replicative needs. The implications of phage infection in the context of chlamydial infection and disease are discussed.     

Electron Microscopic Observations on the Structure of the Envelopes of Mature Elementary Bodies and Developmental Reticulate Forms of Chlamydia psittaci

Purified suspensions of Chlamydia psittaci were prepared from L cells. Thin sections of intact elementary bodies and intact developmental reticulate bodies and of their purified envelopes were observed by electron microscopy. In both intact organisms and partially purified envelopes, two membranous structures, each appearing in electron micrographs as two darkly stained layers, were observed. In the elementary body sections, the outer membrane was round, apparently rigid, and was not soluble in 0.5% sodium dodecyl sulfate. The inner layer was irregular in shape and was completely removed by detergent treatment. We interpret these results to indicate that the outer rigid layer of the envelope is the cell wall and the inner layer is the cytoplasmic membrane. When the fragile reticulate body envelopes were similarly studied, the outer cell wall was clearly visible, and some evidence of an inner membrane was seen. After treatment with nucleases and detergent, all evidence of inner or cytoplasmic membrane was removed, but the outer cell wall remained. Thus, it appears that the cell wall of this organism is continuous throughout the growth cycle and that the fragility and lack of rigidity of the reticulate body cell is due to changes in chemical composition or structure of the cell wall.     

Description and partial characterization of a new chlamydia-like microorganism

Abstract A new obligate intracellular bacterium which we called 'Z' was isolated as a cell culture contaminant of unknown origin. The organism grew in a variety of cultured cells with a 5–7-day developmental cycle, within cytoplasmic phagosomes, similarly to Chlamydia and some Rickettsia spp. Two alternating developmental forms (elementary bodies and reticulate bodies) were observed by electron microscopy. SDS-PAGE electrophoresis, immunoblotting with chlamydia-specific antibodies, and polymerase chain reaction using chlamydial genus specific primers provided evidence that our bacterium differs significantly from chlamydiae. Further characterization of 'Z' including determination of 16S ribosomal RNA sequences will allow its taxonomic position to be established.     

Type III Secretion, Contact-dependent Model for the Intracellular Development of Chlamydia

The medically significant genus Chlamydia is a class of obligate intracellular bacterial pathogens that replicate within vacuoles in host eukaryotic cells termed inclusions. Chlamydia's developmental cycle involves two forms; an infectious extracellular form, known as an elementary body (EB), and a non-infectious form, known as the reticulate body (RB), that replicates inside the vacuoles of the host cells. The RB surface is covered in projections that are in intimate contact with the inclusion membrane. Late in the developmental cycle, these reticulate bodies differentiate into the elementary body form. In this paper, we present a hypothesis for the modulation of these developmental events involving the contact-dependent type III secretion (TTS) system. TTS surface projections mediate intimate contact between the RB and the inclusion membrane. Below a certain number of projections, detachment of the RB provides a signal for late differentiation of RB into EB. We use data and develop a mathematical model investigating this hypothesis. If the hypothesis proves to be accurate, then we have shown that increasing the number of inclusions per host cell will increase the number of infectious progeny EB until some optimal number of inclusions. For more inclusions than this optimum, the infectious yield is reduced because of spatial restrictions. We also predict that a reduction in the number of projections on the surface of the RB (and as early as possible during development) will significantly reduce the burst size of infectious EB particles. Many of the results predicted by the model can be tested experimentally and may lead to the identification of potential targets for drug design.     

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.     

Miniature Cell Formation in Chlamydia psittaci

In an electron microscope study on the developmental cycle of the goat pneumonitis strain of Chlamydia psittaci in L cells, it was observed that miniature reticulate bodies, measuring approximately 0.2 mum in diameter and surrounded by double unit membranes, were produced infrequently from normal-sized reticulate bodies through a "budding"-like process. As little as 0.1 mug of penicillin per ml greatly increased the frequency of the miniature reticulate body formation.     

Cyclic AMP inhibits developmental regulation of Chlamydia trachomatis.

The effect of cyclic AMP (cAMP) on the chlamydial growth cycle was studied with Chlamydia trachomatis-infected HeLa cells. At concentrations of 1 mM, cAMP had a profound effect on the chlamydial developmental cycle, resulting in small, immature inclusions. Immunoblot analysis revealed the absence of elementary body (EB)-specific antigens in the cAMP-treated cells. This effect was observed only if cAMP was added within the first 12 h of incubation and continued thereafter. Its withdrawal at any time from the medium led to the reappearance of fully mature, infectious organisms. Analogs or breakdown products of cAMP exerted no inhibitory effect on chlamydial development. Intracellular inclusions from the cAMP-treated cells were unable to infect fresh HeLa monolayers, in contrast to the completely infectious nontreated inclusions. Protein profiles of the cAMP-treated organisms (at any time point) resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis very closely resembled reticulate bodies (RB) and did not possess characteristic EB-binding proteins. Collectively, these observations suggest an inhibitory role for cAMP at the RB stage of intracellular development. We also identified a cAMP receptor protein which is associated with RB and not with EB, further supporting a role for this system in the developmental regulation of chlamydiae.     

Criblamydia sequanensis, a new intracellular Chlamydiales isolated from Seine river water using amoebal co-culture

Accumulating evidence supports a role for Chlamydia-related organisms as emerging pathogens for human and animals. Assessment of their pathogenicity requires strain availability, at least for animal models and serological studies. As these obligate intracellular species are able to grow inside amoebae, we used co-culture with Acanthamoeba castellanii in an attempt to recover new Chlamydia-related species from river water. We isolated two strains from eight water samples. The first strain is a new Parachlamydia acanthamoebae strain that differs from previously described isolates by only two bases in the complete 16S rRNA gene sequence. The second isolate is the first representative of a new Chlamydiales family, as demonstrated by genetic and phylogenetic analyses of the 16S rRNA, 23S rRNA, ADP/ATP translocase and RnpB encoding genes. Using fluorescent in situ hybridization and electron microscopy, we demonstrated that it grows in high numbers in amoebae, where it exhibits a Chlamydia-like developmental cycle with reticulate bodies and star-like elementary bodies. Based on these results, we propose to name this new species 'Criblamydia sequanensis'. This work confirmed that amoebal co-culture is a relevant method to isolate new chlamydiae, and that it can be successfully applied to ecosystems colonized with a complex microbial community.     

Reactivity of elementary and reticulate bodies of Chlamydia trachomatis LGV2 with monoclonal antibodies specific for the major outer membrane protein

The reactivity of the major outer membrane protein (MOMP) of Chlamydia trachomatis (LGV2 serotype) with 15 monoclonal antibodies was studied during the course of developmental cycle by immunoblotting and immunofluorescence. The monoclonal antibodies reacted in immunoblots with the MOMP of both elementary bodies (EBs) and reticulate bodies (RBs). Using an immunofluorescence test with LGV2-infected cell cultures, the 15 monoclonal antibodies could be divided into 5 groups, according to the time of appearance of their reactivity with the cell culture.     

Structural and polypeptide differences between envelopes of infective and reproductive life cycle forms of Chlamydia spp

Significant differences in cysteine-containing proteins and detergent-related solubility properties were observed between outer membrane protein complexes of reproductive (reticulate body) and infective (elementary body) forms of Chlamydia psittaci (6BC). Elementary bodies harvested at 48 h postinfection possessed a 40-kilodalton major outer membrane protein and three extraordinarily cysteine-rich outer membrane proteins of 62, 59, and 12 kilodaltons, all of which were not solubilized by sodium dodecyl sulfate in the absence of thiol reagents. Intracellularly dividing reticulate bodies harvested at 21 h postinfection were severely deficient in the cysteine-rich proteins but possessed almost as much major outer membrane protein as did the elementary bodies. Most of the major outer membrane protein of reticulate bodies was solubilized by sodium dodecyl sulfate and was present in envelopes as monomers, although a proportion formed disulfide-cross-linked oligomers. By 21 to 24 h postinfection, reticulate bodies commenced synthesis of the cysteine-rich proteins which were found in outer membranes as disulfide-cross-linked complexes. The outer membranes of reticulate bodies of Chlamydia trachomatis (LGV434) also were found to be deficient in cysteine-rich proteins and to be more susceptible to dissociation in sodium dodecyl sulfate than were outer membranes of elementary bodies.     

Interaction of L Cells and Chlamydia psittaci: Entry of the Parasite and Host Responses to Its Development

The entry and development of Chlamydia psittaci in the L cell was studied by using purified, infectious parasites at high multiplicity. Entry of the parasite was accomplished by an act of phagocytosis by the host which was independent of an adsorption stage but was temperature-dependent. Kinetic studies of phagocytosis performed with (14)C-amino acid-labeled, purified parasites indicated that the rate of phagocytosis was directly proportional to the multiplicity of inoculation. Electron microscopy of cells infected at high multiplicity with purified infectious C. psittaci showed that phagocytosed chlamydiae were segregated in a host phagocytic vacuole throughout their developmental cycle which consisted of the transition of infecting elementary bodies to reticulate bodies dividing by binary fission, followed by the reemergence of a population of elementary bodies. The process of the transition was examined and a proposed sequence of intermediate bodies is presented. In isopycnic gradients of fractionated, infected L cells, chlamydial phagocytic vacuoles were apparent as a dense band distinct from lysosome and mitochondrion peaks, as indicated by acid phosphatase and cytochrome oxidase activities. Chlamydiae inactivated by heat or neutralized by antiserum were phagocytosed and appeared in lysosomes within 12 hr after infection according to electron microscopy; however, chlamydiae which were continuously inhibited in their development by chloramphenicol were retained intact in the cell for 24 hr without lysosomal response. The possibility of a lysosomal inhibitor on the native parasite is discussed.     

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.     

Spatial constraints within the chlamydial host cell inclusion predict interrupted development and persistence

Background  

The chlamydial developmental cycle involves the alternation between the metabolically inert elementary body (EB) and the replicating reticulate body (RB). The triggers that mediate the interchange between these particle types are unknown and yet this is crucial for understanding basic Chlamydia biology.     

The role of pretreatment prognostic nutritional index in esophageal cancer: A meta-analysis

Clinicopathological characteristics and prognosis of esophageal cancer (EC) patients with decreased prognostic nutritional index (PNI) have not been well investigated. So, we conducted this meta-analysis. We performed comprehensive research in PubMed, Embase, and Cochrane databases. The effect size was hazard ratio (HR) with 95% confidence interval (CI) for overall survival (OS) and cancer-specific survival (CSS). The pooled odds ratio (OR) with 95% CI were used to assess the association between PNI and clinicopathological features. A total of 3,425 EC patients were included in the present meta-analysis. Male patients, advanced age, higher tumor stage, and lymph node metastases were associated with reduced PNI level (OR = 1.40, 95% CI: 1.10-1.79; OR = 1.35, 95% CI: 1.10-1.66; OR = 2.37, 95% CI: 1.91-2.94; OR = 1.63, 95% CI: 1.04-2.56). And, the EC patients with decreased PNI held a worse OS and CSS compared with those who carried a higher PNI (HR = 1.29, 95% CI: 1.10-1.50; HR = 2.53, 95% CI: 1.15-5.57). This meta-analysis demonstrated PNI level was associated with tumor stage and lymph nodes metastases and was an independent prognostic factor in EC.     

Identification and characterization of a novel Chlamydia trachomatis reticulate body protein

The genome of the obligate intracellular bacterium Chlamydia trachomatis comprises 894 genes predicted by computer-based analysis. As part of a large-scale proteome analysis of C. trachomatis, a small abundant protein encoded by a previously unrecognized novel 204-bp open reading frame was identified by tandem mass spectrometry. No homology of this protein was observed to proteins from other organisms. The protein was conserved in C. trachomatis but not found in Chlamydia pneumoniae. Using proteomics, we show that the expression of the protein is initiated at the middle of the developmental cycle. The protein is rapidly degraded and is only present in reticulate or intermediate bodies, suggesting a possible function in the intracellular stage of C. trachomatis development. We have termed the protein '7-kDa reticulate body protein'.     

Developmental regulation of the cysteine-rich outer-membrane proteins of murine Chlamydia trachomatis

The developmental cycle of the obligate intracellular prokaryote Chlamydia trachomatis involves the serial alternation of two distinct morphological forms of the organism. To examine the basis of chlamydial differentiation we have searched for developmentally regulated gene products in this species. Chlamydia-infected cells were pulse-labelled with [35S] cysteine at various stages of development and the products of synthesis examined by SDS-PAGE. Our results indicate that the synthesis of the cysteine-rich outer-membrane proteins is developmentally regulated, occurring only late in the cycle during the conversion of reticulate bodies to elementary bodies. Both hydroxyurea and ampicillin block this conversion; as a result of this blockade the cysteine-rich outer-membrane proteins are not produced in the presence of either drug.