Developmental Cycle and Genome Analysis of "Rubidus massiliensis," a New Vermamoeba vermiformis Pathogen

Abstract

The study of amoeba-associated Chlamydiae is a dynamic field in which new species are increasingly reported. In the present work, we characterized the developmental cycle and analyzed the genome of a new member of this group associated with Vermamoeba vermiformis, we propose to name "Rubidus massiliensis." This bacterium is well-adapted to its amoeba host and do not reside inside of inclusion vacuoles after phagocytosis. It has a developmental cycle typical of this family of bacteria, with a transition from condensed elementary bodies to hypodense replicative reticulate bodies. Multiplication occurs through binary fission of the reticulate bodies. The genome of "R. massiliensis" consists of a 2.8 Mbp chromosome and two plasmids (pRm1, pRm2) consisting of 39,075 bp and 80,897 bp, respectively, a feature that is unique within this group. The Re-analysis of the Chlamydiales genomes including the one of "R. massiliensis" slightly modified the previous phylogeny of the tlc gene encoding the ADP/ATP translocase. Our analysis suggested that the tlc gene could have been transferred to plant and algal plastids before the transfer to Rickettsiales, and that this gene was probably duplicated several times.

Keywords: Rubidus massiliensis; Vermamoeba vermiformis; chlamydiae; co-culture; host specificity.

Figure 1

Ultrastructural features of the R. massiliensis replication cycle in V. vermiformis (A) Adhesion and phagocytosis of a R. massiliensis elementary body, by a trophozoite of V. vermiformis at 0 h p.i. (B) One EB already engulfed and internalized within the cytoplasm, seen near the nucleus. EB do not reside within vacuole (arrowhead). (C) Another typical EB can be seen within the cell host cytoplasm at 6 h p.i (arrowhead). In contrast to Rickettsia and other Chlamydia related to Acanthamoeba, no electron-translucent layers surrounding the intra-cellularly located bacteria could be observed. This is an evidence for the absence of inclusion vacuoles. (D) Arrowheads indicating constrictions of Reticulate bodies (RBs) undergoing their replicative stage where we observe an increase in size and a decrease in density. (E) Full replicative stage at 24 h p.i showing an increased number of R. massiliensis particles, hypodense RBs, at different stages of morphogenesis. (F) Higher magnification of the arrowed area in (E), we can see the bacterium at the typical binary fission stage. (Tomographic reconstruction in Movie S1). (G) Completely infected V. vermiformis at 36 h p.i. After the growth and binary division, RBs reorganize, condensing to form infectious EBs. We note differentiation from hypodense to intermediate condensed particle and numerous bacteria scattered throughout the cytoplasm in various stages of differentiation. Condensed DNA is clearly visible in the two forming daughter cells. (Tomographic reconstruction in Movie S2). (H) Different stages of the R. massiliensis developmental cycle. RBs (black arrows), and EBs (white arrow) can be observed simultaneously within the cytoplasm of the V. vermiformis host cell, and do not reside within vacuoles. At 42 h p.i the co-presence of RBs and EBs signals the asynchronous cycle of R. massiliensis. (I) Ultrathin section of an infected amoeba, harboring the newly synthetized bacterial committee. The newly synthetized bacteria occupy the whole cell cytoplasm area. At 48 h p.i, we can see the different shapes of the newly differentiated, and highly condensed EBs but corresponding to only one rounded shape of the hyper dense EBs. (Tomographic reconstruction in Movie S3).

Figure 2

Histogram of R. massiliensis cycle growth in V. vermiformis measured by real-time PCR. X-axis corresponds to the cycle time points in hours (from 0 to 48 h). No R.M corresponds to the negative control, which is the non-infected amoeba). Y-axis to the right corresponds to the log of bacterial load (the log values are obtained after conversion of the Cycle threshold (Ct.) values based on standard curves realized with serial 1: 10 dilution starting with 10⁷ bacterial particles). Y-axis to the left corresponds to the percentage of amoeba concentrations quantified on kovaslides. This relative quantification by real-time PCR showed the increase in bacterial multiplication coming along with the decrease of the amoeba concentration. No bacterial DNA was detected from H0 till H6. Bacterial titers begin to be detected at H12 p.i, Higher titers are from H18 till H30 with a plateau from H36 until H48 p.i.

Figure 3

Circular representation of the R. massiliensis chromosome. Circles from the center to the outside: GC skew (green/purple), GC content (black), RNA on forward strand (tRNA in blue, rRNA in purple), RNA on reverse strand (tRNA in blue, rRNA in purple), scaffolds in alternative grays, genes on forward strand colored by COGs categories, genes on reverse strand colored by COGs.

Figure 4

Chlamydiales members clustering according to a phylogenetic tree analysis. Maximum-likelihood (PhyML) phylogenetic tree calculated with JTT+G substitution model With the RNA 16S sequences of 26 Chlamydiales members. Bootstrap Proportion values are indicates at the node.

Figure 5

The maximum-likelihood (PhyML) phylogenetic tree inferred from amino acid sequences of the ADP/ATP translocase of Parachlamydia acanthamoebae UV-7, Candidatus, Protochlamydia, Waddlia chondrophila, Simkania negevensis, R. massiliensis, Chlamydiaceae, Rickettsiales, and plant and algal plastids. Bootstrap proportion values are indicated at the node.