Growth medium consisted of Minimal Essential Medium (MEM) with Earles salts, 25 mM HEPES, without L-Glutamine (GIBCO, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS, BioConcept, Allschwil, Switzerland), 4 mM GlutaMAX-I (200 mM, GIBCO), 1% MEM Non-Essential Amino Acids (100x, GIBCO) and 0.2 mg/ml gentamycin (50 mg/ml, GIBCO). AB are shown for (A) and (B).(TIFF) pone.0134943.s002.tiff (393K) GUID:?1D0DD661-3F89-4522-B2B0-A9D032B227A5 S3 Fig: cAMP, ATP and EHNA/ADO do not cause host cell loss. HeLa cells were infected with or serovar E and exposed to cAMP (1 mM), 8BrcAMP (1 mM), ATP (1 mM), Apyrase (2.5 U), Apyrase (2.5 U) followed by ATP (1 mM), ADO (50 M), EHNA (25 M), or ADO (50 M) plus EHNA (25 M) in incubation medium immediately after infection (T0; A-G) or 14 hours post infection (T14; H,I). Cells were incubated for 35 hours (test; n = 3 A-D and H-I, n = 8 fields per coverslip from a single experiment E-G).(TIFF) pone.0134943.s003.tiff (759K) GUID:?3AAE89DB-FE03-4129-8258-9615A19FB9E4 S4 Fig: DAMP-dependent modulation of infectious EB production depends on host Ziyuglycoside I cell protein synthesis. HeLa cells were infected with (A-B) or serovar E (C-D) and exposed to the DAMPs cAMP (1 mM), ATP (1 mM), or ADO (50 M, plus 25 M EHNA) in incubation medium, in the presence (A, C) or absence (B, D) of 1 1 g/ml cycloheximide, immediately after infection. Cells were incubated for 35 hours (test; values are derived from duplicate determinations within a single experiment).(TIFF) pone.0134943.s004.tiff (348K) GUID:?5BD032D4-ABC2-4118-872F-787A90BE8409 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Persistence, more recently termed the chlamydial stress response, is a viable but noninfectious state constituting a divergence from the characteristic chlamydial biphasic developmental cycle. Damage/danger associated molecular patterns (DAMPs) are normal intracellular components or metabolites Ziyuglycoside I that, when released from cells, signal cellular damage/lysis. Purine metabolite Rabbit Polyclonal to SMC1 (phospho-Ser957) DAMPs, including extracellular ATP and adenosine, inhibit chlamydial development in a species-specific manner. Viral co-infection has been shown to reversibly abrogate inclusion development, suggesting persistence/chlamydial stress. Because viral infection can cause host cell DAMP release, we hypothesized DAMPs may influence chlamydial development. Therefore, we examined the effect of extracellular ATP, adenosine, and cyclic AMP exposure, at 0 and 14 hours post infection, on and serovar E development. In the absence of host protein synthesis, exposure to DAMPs immediately post or at 14 hours post infection reduced inclusion size; however, the effect was less robust upon 14 hours post infection exposure. Additionally, upon exposure to DAMPs immediately post infection, bacteria per inclusion and subsequent infectivity were reduced in both species. These effects were reversible, and exhibited more pronounced recovery from DAMP exposure. Aberrant bodies, typical in virus-induced chlamydial persistence, were absent upon DAMP exposure. In the presence of de protein synthesis, exposure to DAMPs immediately post infection reduced inclusion size, but only variably modulated Ziyuglycoside I chlamydial infectivity. Because chlamydial infection and other infections may increase local DAMP concentrations, DAMPs may influence infection are a genus of Gram-negative, obligate intracellular bacterial pathogens that cause a spectrum of diseases in both humans and agriculturally important animals. infection, for example, causes clinical manifestations in swine ranging from conjunctivitis to abortion [1]. infection in humans can cause medically important conditions, such as trachoma, pelvic inflammatory disease, and infertility. Though chlamydial infections can cause acute diseases, they are most associated with chronic inflammation resulting in significant host tissue damage [2]. The chlamydiae also share a complex developmental cycle. The infectious form (the elementary body or EB), binds to and enters the host cell. After host cell entry, the EB transitions into the more metabolically active, replicative developmental form (the reticulate body or RB). The RB then grow and divide within a cytoplasmic, membrane-bound inclusion. After several rounds of division, RB convert back into infectious EB, which are released from the Ziyuglycoside I host cell [3]. A third stage, historically termed persistence or more recently the chlamydial stress response, is defined as a developmental stage in which the organisms are viable but noninfectious. Persistent/stressed RB are enlarged,.