Developmental cycle and host interaction of Rhabdochlamydia porcellionis, an intracellular parasite of terrestrial isopods
Summary Environmental chlamydiae are a diverse group of obligate intracellular bacteria related to well‐known pathogens of humans. To date, only very little is known about chlamydial species infecting arthropods. In this study, we used cocultivation with insect cells for recovery and maintenance of...
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| Published in | Environmental microbiology Vol. 15; no. 11; pp. 2980 - 2993 |
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| Main Authors | , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Oxford
Blackwell Publishing Ltd
01.11.2013
Blackwell Wiley Subscription Services, Inc |
| Subjects | |
| Online Access | Get full text |
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| Summary: | Summary
Environmental chlamydiae are a diverse group of obligate intracellular bacteria related to well‐known pathogens of humans. To date, only very little is known about chlamydial species infecting arthropods. In this study, we used cocultivation with insect cells for recovery and maintenance of Rhabdochlamydia porcellionis, a parasite of the crustacean host Porcellio scaber. In vitro, the infection cycle of R. porcellionis was completed within 7 days, resulting in the release of infectious particles by host cell lysis. Lack of apoptosis induction during the entire course of infection, combined with a reduced sensitivity of infected cultures to experimentally induced programmed cell death, indicates that R. porcellionis like its human pathogenic relatives counteracts this host defence mechanism. Interestingly, the rod‐shaped variant of R. porcellionis, proposed to represent their mature infective stage, was not detected in cell culture, suggesting that its development may require prolonged maturation or may be triggered by specific conditions encountered only in the animal host. This first cell culture‐based system for the cultivation and investigation of an arthropod‐associated chlamydial species will help to better understand the biology of a so far neglected group of chlamydiae and its recently suggested potential to cause disease in humans. |
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| Bibliography: | COST Action - No. FA0701 Austrian Science Fund - No. Y277-B03 ark:/67375/WNG-JF54FJV6-D ArticleID:EMI12252 istex:E667F1A1F9C76B98A4314D9EEEF83C61A8D9EC2C Slovenian Research Agency (ARRS) research program - No. P1-0184 Fig. S1. FISH-based identification of bacteria released from the hepatopancreas of P. scaber and of bacteria recovered by cocultivation. A. Homogenates of glands from putatively infected animals were analysed by FISH using the general bacterial probe mix EUB-338-I-III (green) in combination with the Chlamydiae-specific probe Chls-0523 or the R. porcellionis-specific probe Rhabdo-0922 (red). B. The success of the transfer of R. porcellionis into cultured Sf9 insect cells by cocultivation was confirmed by FISH 6 days after inoculation with gland homogenates. Bacteria were detected with the probe mix EUB-338-I-III (green) combined with the specific probe Rhabdo-0992 (red). In (A) and (B), representative confocal fluorescence microscopic images and phase contrast images are shown. Adjacent images represent the same microscopic field. Bars indicate 20 μm. Note that all detected bacteria in (B) were also targeted by the species-specific probe, demonstrating the absence of additional Chlamydia-like bacteria or other contaminants in insect cell cultures.Fig. S2. Fluorescence microscopic infection analysis accompanying the analysis of R. porcellionis 15C-induced host cell death. In two independent experimental set-ups, Sf9 cells were infected with R. porcellionis 15C at a MOI of 100 ('purification without bead lysis') and were monitored either during the early/mid-stage of infection (daily until 6 days p.i.) or at late stages (daily from 4 d to 13 days p.i.). In parallel to the analysis of host cell death shown in Fig. , the progress of infection was monitored by fluorescence microscopy. Bacteria were detected by immunostaining (yellow). Host cells were stained with HCS cytoplasmic stain (white). Representative confocal images are shown in (A). Bars indicate 20 μm. Results from the quantitative infection analysis are shown in (B). The infection level in individual cells was assessed by counting the number of intracellular bacteria (uninfected: 0; very low: 1-5; low: 6-15; medium: 16-50; high: > 50). Displayed values correspond to means and standard deviations of three replicate infections. Time points between which a statistically significant increase in the proportion of infected cells was first observed are indicated (t-test; ***, P ≤ 0.001). Note that a small proportion of cells classified as heavily infected appeared as early as 2 days p.i., indicating that multiple initial invasions of individual cells, which occurred as a consequence of the more infectious inoculum, led to a faster progression of infection.Fig. S3. Absence of apoptotic nuclear changes in continuous Sf9 cultures infected with R. porcellionis 15C. A representative image taken from a Sf9 culture about 12 months after cocultivation is shown. Bacteria were detected by FISH using a combination of the Chlamydiae-specific probe Chls-0523 (green) and the species-specific probe Rhabdo-0992 (red) (yellow signals, due to the overlap of green and red signals). DNA was stained with DAPI (blue). The bar indicates 20 μm. Note the absence of characteristic apoptotic changes of nuclear morphology, such as condensation or fragmentation, in infected cells.Fig. S4. Failure of R. porcellionis 15C to grow in different amoeba strains. Acanthamoeba sp. UWC1, A. castellanii Neff, A. polyphaga DOME and H. vermiformis A1Hsp were infected with R. porcellionis 15C at a MOI of 50 ('purification without bead lysis'), followed by incubation at 27°C. At 3, 5, 9 and 12 days p.i., the infection was analysed by fluorescence microscopy. Bacteria were detected by immunostaining (yellow) and are additionally highlighted by white arrows. Host cells were stained with HCS cytoplasmic stain (white). The bar indicates 20 μm.Fig. S5. Effect of bead beating and storage on the recovery and preservation of infectious R. porcellionis 15C. R. porcellionis 15C was purified from Sf9 cells by two different procedures, 'purification with bead lysis', including mechanical disruption with glass beads, and 'purification without bead lysis'. Bacteria were either directly applied to Sf9 cells ('freshly purified') or after about 3-6 months storage at −80°C ('frozen stock'). Infections were carried out at MOI 50 and 100, as indicated. The percentage of infected cells was determined at 72 h p.i. after detection of intracellular bacteria by immunostaining. At least 300 cells per replicate were evaluated. Means and standard deviations of three replicate infections are shown. Statistically significant differences are indicated [ANOVA; ***, P ≤ 0.001; ns, not significant (P > 0.05)]. Note that the use of the optimized protocol resulted in a significantly improved infection, while storage had only minor effects on infectivity, especially when higher inoculum sizes were used. R. porcellionis grown in cell culture may thus be more fragile than infectious particles of other chlamydial species and steps including harsh mechanical disruption should thus be avoided from the purification procedure.Table S1. PCR primers and probes used in this study. DOC-fFORTE fellowship European Research Council (ERC StG 'EvoChlamy') ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 1462-2912 1462-2920 1462-2920 |
| DOI: | 10.1111/1462-2920.12252 |