Enrichment of macaque intestinal flora for CEACAM-binding bacteria In the present study we aimed to identify novel macaque CEA-binding bacteria from macaque stool samples. To enrich macaque stool samples for CEACAM-binding bacteria, we established a magnetic bead-based enrichment technique.
For this purpose, we coupled soluble GFP-fused amino-terminal domains of
macaque CEA or, CEACAM8 (control) with anti-GFP antibody coupled magnetic beads and incubated these beads with a pool of fresh stool samples from eleven different rhesus monkeys. The magnetic beads were loaded onto a column which was placed in a magnetic field and washed extensively to separate the bead-bound from the nonbound bacteria (Fig. 4.1A and B). To reveal the identity of the enriched bacteria, the bacterial DNA of the macaque CEA sample, CEACAM8 sample or the stool sample input was extracted and the variable regions 1 and 2 (V1-V2) of the 16S rRNA genes were PCR-amplified using the barcoded universal primers 27F and 338R (Fig. 4.1C).
Pyrosequencing of the 16S rRNA amplicons with a Roche 454 FLX Titanum instrument resulted in about 170.000 high quality sequences with an average length of 360 bp.
Fig. 4.1: Enrichment of CEACAM-binding bacteria from a rhesus monkey stool sample pool using magnetic bead-based isolation method. (A) GFP-fused amino-terminal domains of macaque CEA (macCEA) or CEACAM8 (control) were coupled to anti-GFP antibody coupled magnetic beads and incubated with a pool of fresh stool samples from eleven rhesus monkeys.
The magnetic beads were loaded onto a column, placed in a magnetic field, and extensively washed to enrich for CEACAM-binding bacteria. (B) Similar expression of GFP-fused soluble macCEA or CEACAM8 domains was verified by Western blotting using a monoclonal anti-GFP antibody. (C) The variable regions 1 and 2 (V1-V2) of the 16S rRNA genes from CEACAM-enriched bacteria or the stool sample input were amplified and 454 pyrosequenced.
Bioinformatical analyses of 454 pyrosequencing results identified several members of the genus Prevotella as macaque CEA-binding bacteria
To identify the macaque CEA-enriched bacteria we first identified which of the 16S rRNA fragments (V1-V2) showed significant differences in the abundance over the stool sample input and CEACAM8-enriched bacteria vs. macaque CEA-enriched bacteria. Using the bacterial 16S rRNA sequences present in the SILVA database we then attempted to identify which group of bacterial species these fragments originated from. Thus, all SILVA small subunit (SSU)-rRNA sequences of bacteria were clustered using CD-hit at 95% identity. The cluster representatives were combined with the macaque CEA-enriched fragment sequences (48 sequences) and the combined set was then analyzed using CLuster ANalysis of Sequences (CLANS). CLANS performs an all-against-all BLAST comparison of sequences and represents them as dots in 3D-space.
Each sequence is assigned an 'attraction' value to every other sequence based on their reciprocal BLAST hits. By equilibrating the graph in 3D space, sequences similar to each other move into close proximity while sequences of lesser similarity are located more distantly from each other. This leads to a clustered representation of sequence-space, with large clouds of dots representing large groups of sequences with greater than average similarity to each other.
Using this 3D-CLANS map, we were able to identify all major bacterial groupings from the SILVA database and were able to place our macaque CEA-enriched 16S rRNA fragments into a taxonomic context (Fig. 4.2).
Fig. 4.2: Macaque CEA-enriched sequences, depicted in a 3D-CLANS map of 16S rRNA sequences of the major bacterial groups derived from the SILVA database. To identify the macaque CEA-enriched bacteria, SILVA SSU-rRNA sequences were clustered using CD-hit at 95% identity and the cluster representatives were combined with the macaque CEA enriched 16S rRNA fragment sequences. The combined set was then analyzed using CLANS. CLANS performs an all-against-all BLAST comparison of sequences and represents them as dots in a 3D-space. By equilibrating the graph in 3D space, sequences similar to each other move into close proximity while sequences of lesser similarity are located more distantly from each other.
The 16S rRNA sequences of the macaque CEA enriched sequences are indicated by pink stars. 16S rRNA sequences of Prevotella species (P. intermedia, P. nigrescens and P. corporis)
used in further pulldown assays are indicated by pink stars encircled with black.
Accordingly, the macaque CEA-enriched sequences clustered mainly into two groups, belonging to the phyla Bacteroidetes and Clostridia, respectively.
Interestingly, all macaque CEA-enriched bacteria, belonging to the Bacteroidetes were members of the genus Prevotella. In contrast, the macaque CEA-enriched sequences belonging to Clostridia, were represented by a heterogeneous group of different genera. Thus further investigations were concentrated on the interaction of Prevotella species and macaque CEA.
Members of the genus Prevotella specifically interact with macaque CEA and several human CEACAMs
To confirm the interaction of Prevotella and macaque CEA biochemically, we employed a bacterial pulldown assay. Therefore, we incubated P. intermedia, P. nigrescens, P. corporis (indicated as pink stars encircled with black in Fig.
4.2) or as a positive control N. gonorrhoeae expressing the CEACAM-binding Opa52, with similar amounts of soluble GFP-fused CEACAM1, CEA, CEACAM3,
CEACAM6 or CEACAM8 amino-terminal domains. The bacteria were washed extensively, and the bacteria-associated receptor domains were detected by Western blotting. As already known from previous studies, N. gonorrhoeae Opa52 interacted with human CEACAM1, CEA, CEACAM3 and CEACAM6, but not with human CEACAM8. Interestingly, we observed no interaction between the human-restricted pathogen N. gonorrhoeae and macaque CEA (Fig. 4.3.A).
In line with the bioinformatics results, P. intermedia, P. nigrescens and P. corporis bound to macaque CEA. Surprisingly, we also revealed an
interaction between the Prevotella species and human CEACAM3, CEA, and CEACAM8. In contrast, no significant binding was observed between Prevotella and human CEACAM1 or CEACAM6 (Fig. 4.3A).
Fig. 4.3: Species of the genus Prevotella specifically interact with macaque CEA as well as with several human CEACAMs, but not with other mammalian CEACAMs. (A) Similar amounts of the indicated soluble CEACAM-GFP fusion proteins were used in pulldown assays
together with N. gonorrhoeae MS11 strain expressing the CEACAM-binding Opa52, P. intermedia, P. nigrescens and P. corporis, respectively. Precipitates were probed with a
monoclonal anti-GFP antibody to detect CEACAMs co-precipitating with the bacteria.
(B) P. intermedia and P. nigrescens were incubated with similar amounts of murine, bovine or canine CEACAM1 or macaque CEA. Precipitates were probed with a monoclonal anti-GFP antibody to detect CEACAMs co-precipitating with the bacteria.
Since Prevotella interacts with macaque CEA and different human CEACAMs, we wondered if the bacteria might also interact with additional mammalian CEACAMs. To test this, we performed a pulldown experiment with P. intermedia
or P. nigrescens and soluble GFP-fused amino-terminal domains of macaque
CEA or murine, canine and bovine CEACAM1. As observed before, P. intermedia and P. nigrescens bound to macaque CEA. In sharp contrast,
neither P. intermedia nor P. nigrescens interacted with amino-terminal domains of murine, canine or bovine CEACAM1 (Fig. 4.3B). These results demonstrate that Prevotella specifically engage primate CEACAMs, however they do not bind to more distantly related mammalian CEACAMs. In summary, our data provide evidence that CEACAMs could contribute to the species-specific colonization of mammalian epithelial cells.