Chlamydia-containing spheres are a novel and predominant form of egress by the pathogen Chlamydia psittaci.

The egress of intracellular bacteria from host cells and cellular tissues is a critical process during the infection cycle. This process is essential for bacteria to spread inside the host and can influence the outcome of an infection. For the obligate intracellular Gram-negative zoonotic bacterium Chlamydia psittaci, little is known about the mechanisms resulting in bacterial egress from the infected epithelium. Here, we describe and characterize Chlamydia-containing spheres (CCSs), a novel and predominant type of non-lytic egress utilized by Chlamydia spp. CCSs are spherical, low-phase contrast structures surrounded by a phosphatidylserine-exposing membrane with specific barrier functions. They contain infectious progeny and morphologically impaired cellular organelles. CCS formation is a sequential process starting with the proteolytic cleavage of a DEVD tetrapeptide-containing substrate that can be detected inside the chlamydial inclusions, followed by an increase in the intracellular calcium concentration of the infected cell. Subsequently, blebbing of the plasma membrane begins, the inclusion membrane destabilizes, and the proteolytic cleavage of a DEVD-containing substrate increases rapidly within the whole infected cell. Finally, infected, blebbing cells detach and leave the monolayer, thereby forming CCS. This sequence of events is unique for chlamydial CCS formation and fundamentally different from previously described Chlamydia egress pathways. Thus, CCS formation represents a major, previously uncharacterized egress pathway for intracellular pathogens that could be linked to Chlamydia biology in general and might influence the infection outcome in vivo.IMPORTANCEHost cell egress is essential for intracellular pathogens to spread within an organism and for host-to-host transmission. Here, we characterize Chlamydia-containing sphere (CCS) formation as a novel and predominant non-lytic egress pathway of the intracellular pathogens Chlamydia psittaci and Chlamydia trachomatis. CCS formation is fundamentally different from extrusion formation, the previously described non-lytic egress pathway of C. trachomatis. CCS formation is a unique sequential process, including proteolytic activity, followed by an increase in intracellular calcium concentration, inclusion membrane destabilization, plasma membrane blebbing, and the final detachment of a whole phosphatidylserine-exposing former host cell. Thus, CCS formation represents an important and previously uncharacterized egress pathway for intracellular pathogens that could possibly be linked to Chlamydia biology, including host tropism, protection from host cell defense mechanisms, or bacterial pathogenicity.

In vitro maturation of Toxoplasma gondii bradyzoites in human myotubes and their metabolomic characterization.

The apicomplexan parasite Toxoplasma gondii forms bradyzoite-containing tissue cysts that cause chronic and drug-tolerant infections. However, current in vitro models do not allow long-term culture of these cysts to maturity. Here, we developed a human myotube-based in vitro culture model of functionally mature tissue cysts that are orally infectious to mice and tolerate exposure to a range of antibiotics and temperature stresses. Metabolomic characterization of purified cysts reveals global changes that comprise increased levels of amino acids and decreased abundance of nucleobase- and tricarboxylic acid cycle-associated metabolites. In contrast to fast replicating tachyzoite forms of T. gondii these tissue cysts tolerate exposure to the aconitase inhibitor sodium fluoroacetate. Direct access to persistent stages of T. gondii under defined cell culture conditions will be essential for the dissection of functionally important host-parasite interactions and drug evasion mechanisms. It will also facilitate the identification of new strategies for therapeutic intervention.

Analysis of the passage of the marine biotoxin okadaic acid through an in vitro human gut barrier.

The marine biotoxin okadaic acid (OA), produced by dinoflagellates, can accumulate in various bivalve molluscs. In humans, oral consumption of shellfish contaminated with OA induces acute toxic effects like diarrhea, nausea, vomiting and abdominal pain. However, tumorigenic and embryotoxic effects of OA have been also described. Current toxicokinetic studies with mice were performed with high cytotoxic oral doses leading presumably to a paracellular passage of OA through the gastrointestinal barrier. There are no studies available analyzing the absorption at low concentrations, which represent a realistic dietary exposure, making a reliable risk assessment difficult. Therefore, we performed a low-dose study using the human intestinal Caco-2 cell model to simulate the intestinal barrier. Low level exposure of 20-200 nM OA to the cell monolayer allows an only limited passage from the "luminal" to the "blood side". Furthermore, we could detect a significant efflux of OA, which led to the suggestion that active transport mechanisms are involved in the elimination process of OA. In conclusion, our results indicate that besides the well known defense mechanisms of humans against this marine biotoxin--vomiting and diarrhea--further detoxification mechanisms are available to limit the absorption of toxic OA.

Sensitive method for the determination of lipophilic marine biotoxins in extracts of mussels and processed shellfish by high-performance liquid chromatography-tandem mass spectrometry based on enrichment by solid-phase extraction.

A solid-phase extraction (SPE) method for the enrichment and clean-up of lipophilic marine biotoxins from extracts of different species of bivalve molluscs and processed shellfish products was developed. Okadaic acid (OA), pectenotoxin2 (PTX2), azaspiracid1 (AZA1) and yessotoxin (YTX) were determined by LC-MS/MS in hydrolyzed and non-hydrolyzed extracts. Applying a concentration factor of 10 the limit of quantification for the four toxins was determined to be 1 microg/kg. An organized in-house ring trial proved transferability of the method protocol and satisfactory results for all four toxins with a relative standard deviation (RSD) of 5-12%. The precision of the whole method including LC-MS detection was determined by processing seven independent extractions analyzed in independent sequences. RSD ranged between 12% and 24%. This SPE method was tested within a concentration range corresponding to the range of the current European Union regulatory limits (up to 160 microg/kg for the OA group), but it would also be applicable to a lower microg/kg range which is important in view of a possible decrease of regulatory limits as proposed by a working group of the European Food Safety Authority. The potential of SPE as a cleaning tool to cope with matrix effects in LC-MS/MS was studied and compared to liquid-liquid portioning.