Inclusion Membrane Proteins ofProtochlamydia amoebophilaUWE25 Reveal a Conserved Mechanism for Host Cell Interaction among theChlamydiae

Abstract

Chlamydiae are a group of obligate intracellular bacteria comprising several important human pathogens. Inside the eukaryotic cell, chlamydiae remain within a host-derived vesicular compartment, termed the inclusion. They modify the inclusion membrane through insertion of unique proteins, which are involved in interaction with and manipulation of the host cell. Among chlamydiae, inclusion membrane proteins have been exclusively found in members of the family Chlamydiaceae, which predominantly infect mammalian and avian hosts. Here, the presence of inclusion membrane proteins in Protochlamydia amoebophila UWE25, a chlamydial endosymbiont of free-living amoebae, is reported. A genome-wide screening for secondary structure motifs resulted in the identification of 23 putative inclusion membrane proteins for this organism. Immunofluorescence analysis demonstrated that five of these proteins were expressed, and four of them could be localized to a halo surrounding the intracellular bacteria. Colocalization studies showed an almost complete overlap of the signals obtained for the four putative inclusion membrane proteins, and immuno-transmission electron microscopy unambiguously demonstrated their location in the inclusion membrane. The presence of inclusion membrane proteins (designated IncA, IncQ, IncR, and IncS) in P. amoebophila shows that this strategy for host cell interaction is conserved among the chlamydiae and is used by chlamydial symbionts and pathogens alike.Chlamydiae were once considered a small group of very closely related bacteria which form an evolutionarily well-separated lineage in the tree of life. These obligate intracellular bacteria infect a broad spectrum of at least 60 eukaryotic host organisms including both invertebrates and vertebrates. Until recently, the family Chlamydiaceae was the only described chlamydial family, which is exclusively comprised of human and animal pathogens. It includes Chlamydia trachomatis, the most prevalent sexually transmitted bacteria worldwide, with over 90 million new cases of infection per year (56), and the world's leading cause of preventable blindness (57). The other major human pathogen among the chlamydiae, Chlamydophila pneumoniae (basonym, Chlamydia pneumoniae) (15), causes community-acquired pneumonia and is also implicated in several chronic diseases such as arteriosclerosis and multiple sclerosis (33, 38). The discovery of several chlamydia-like endosymbionts in amoebae in the late 1990s represented the first evidence for a much larger diversity of chlamydiae in the environment (3,7,17,27). Since then, numerous new chlamydial organisms were discovered, which led to the description of several new families (9,25,30).A characteristic feature of all chlamydiae is the developmental cycle, which includes morphologically and physiologically distinct developmental forms (1, 37). Infection of host cells starts with the attachment of the infectious form of the chlamydiae, the elementary body (EB), to a host cell and uptake by a phagocytosis-like mechanism. Onc...

Metabolic Features of Protochlamydia amoebophila Elementary Bodies – A Link between Activity and Infectivity in Chlamydiae

“…Pellets were then resuspended in a small volume PBS and transferred to microscope slides. Bacteria and cell debris were dried (46°C), stained with DAPI (0.5 µg/ml in PBS, 10 min), washed once with PBS, and embedded in mowiol [84]. Images were taken with a CCD camera (AxioCam HRc; Carl Zeiss) connected to an epifluorescence microscope (Axioplan 2 imaging; Carl Zeiss).…”

Identification and Characterization of a Novel Porin Family Highlights a Major Difference in the Outer Membrane of Chlamydial Symbionts and Pathogens

“…Highly enriched fractions of EBs were obtained using two additional centrifugation steps to further remove host cell debris [32]. Highly purified EBs were thawed and centrifuged at 10,621×g for 15 min at 4°C.…”

“…Single colonies were inoculated in LB, and protein expression was induced by addition of 1 mM IPTG at an OD 600 of 0.6. As controls, the vector pet16b without insert and pet16b containing a gene fragment coding for the inclusion membrane protein IncA (pc0399) of P. amoebophila were used [32]. Samples were taken at 0, 10 and 60 min after induction, they were diluted in phosphate buffered saline (PBS, 130 mM NaCl, 10 mM Na x PO 4 ; pH 7.2–7.4) and plated on LB plates.…”

“…Pre-immune serum and antibodies targeting Pc1489 were affinity purified on a polyvinylidene fluoride (PVDF) membrane as previously described to further reduce unspecific background signals [42]. Immunofluorescence analysis of methanol and paraformaldehyde fixed cells and immunogold labelling of ultrathin cryosections was performed as described previously [32].…”

“…Several key mechanisms for host cell interaction, such as a type three secretion system and its effector proteins, are conserved among all chlamydiae [32] [33], [34]. Yet, the genome of the amoeba symbiont Protochlamydia amoebophila encodes no homologue of MOMP [33], and antibodies targeting Chlamydiaceae MOMP did not bind to the outer membrane of these bacteria [35].…”