Microscopic Analysis of the Chlamydia abortus Inclusion and Its Interaction with Those Formed by Other Chlamydial Species

Abstract

The Chlamydiae are obligate intracellular pathogens that develop and multiply within a poorly characterized parasitophorous vacuole (the inclusion) during growth. Chlamydia abortus is a major pathogen of sheep and other ruminants, and its inclusion development is poorly characterized. We used immunofluorescence microscopy, quantitative culture, and qPCR to examine C. abortus inclusion development and to examine the interaction of C. abortus inclusions with those formed by other species. Antibodies used in these studies include sera from ewes from production facilities that were naturally infected with C. abortus. Multiple inclusions are often found in C. abortus-infected cells, even in populations infected at very low multiplicity of infection. Labeling of fixed cells with sera from infected sheep revealed fibrous structures that extend away from the inclusion into the cytoplasm of the host cell. C. abortus inclusions fused with C. caviae and C. psittaci inclusions in coinfected cells. Inclusions formed by C. abortus and C. caviae did not fuse with inclusions formed by C. trachomatis, C. pneumoniae, or C. pecorum. The ability of inclusions to fuse was correlated with the overall genomic relatedness between species, and with sequence similarity in the inclusion membrane protein IncA. Quantitative PCR data demonstrated that C. abortus grows at a decreased rate during coinfections with C. caviae, while C. caviae growth was unaffected. The collected data add depth to our understanding of inclusion development in this significant zoonotic veterinary pathogen.

Keywords: Chlamydia abortus; chlamydial inclusion; intracellular bacteria.

FIG 1

Full genome identity and IncA amino sequence similarity among Chlamydia spp. examined in this work. All alignments are based on the following sequence accession numbers: C. trachomatis D/UW-3/CX (NC_000117); C. psittaci 6BC (NC_017287); C. pneumoniae CWL029 (NC_00092); C. muridarum VR123 (NZ_CP063055.1); C. pecorum E58 (NC_015408.1); C. caviae GPIC (NC_003361.3); C. abortus S26/3 (NC_004552). Panel A: Whole genome alignment for the indicated species. A full alignment was conducted using the Progressive Mauve algorithm and ClustalW (34), with automatic calculations of seed weight and minimum LCB score for colinear genomes. More detailed whole genome trees can be found in references and . Panel B: Similarity analysis of the full length IncA polypeptide sequences. Panel C: Examination of sequence similarity within SNARE motifs in each predicted IncA sequence. An N-terminal and C-terminal SNARE motif is present in each IncA sequence (13), downstream of the predicted transmembrane domain of the protein. As is seen in the analysis of the full genomes and the full-length IncA sequence, similarity within the SNARE domains is highest among those species combinations that form cross-species fused inclusions.

FIG 2

Chlamydia abortus development within murine McCoy cells. Panels A to D: Cells were fixed with methanol and labeled with anti-LOS antibodies (red) and the DNA-specific dye 4′,6-diamidino-2-phenylindole (DAPI; blue). Panel A: McCoy cells infected with C. abortus strain LW203 fixed at 17 hpi. Panel B: Similar infections fixed at 51 hpi. Panel C: A recent clinical isolate (OP5P5) infected at a low MOI (0.2 C. abortus EBs per cell), fixed 50 hpi. Panel D: Similar monolayers as those in (C) but infected at an MOI of 2. Panel E: Quantification of C. abortus-infected cells carrying 2 or more distinct inclusions, measured as a function of MOI. The MOI was varied in 10 folds from 0.002 through 2 ifu per cell. Cells were evaluated for the number of individual inclusions using the 40X objective.

FIG 3

Labeling of C. abortus-infected cells with sera from naturally infected sheep. Immunofluorescence labeling of C. abortus LW203-infected cells fixed with methanol 48 hpi and probed with serum from a naturally infected ewe (panels A, B, D) and anti-MOMP (panel C). Panels A and B represent an identical set of cells examined in different focal planes. Panels C and D show identical cells visualized for the different antigens. The arrows in B and D show Inc-protein-laden fibers that extend away from the inclusion within infected cells. The scale bar in panel C equals 10 microns for all images.

FIG 4

Examination of cells infected with different combinations of Chlamydia species. (A) C. trachomatis and C. caviae. (B) C. caviae and C. pneumoniae. (C) C. pecorum and C. caviae. (D) C. abortus and C. trachomatis. (E) C. abortus and C. pneumoniae. (F) C. pecorum and C. abortus. The scale bar in D shows indicates 10 μm for panels A-F. Panel G. Microscopic quantification of inclusion fusion in coinfected cells. Infected cells were quantified in three or more experiments, with the exception of combinations between C. abortus and C. pneumoniae, which were analyzed in two experiments. The different species included in the experiments were C caviae (C.c.), C. trachomatis (C.t.), C. abortus (C.a.), and C. pneumoniae (C.p.). Panels H-J: McCoy cells were infected with C. abortus and C. caviae (MOI∼1 for each), followed by incubated for 48 h at 37°C, followed by fixation and examination either with the 40X objective (panel H) or at high magnification (panels I and J). These infected cells were visualized using antibodies to C. caviae IncA (green) and C. abortus MOMP (red), and DAPI was used to label DNA. The scale bar in panel J represents 5 μm for panels I and J.

FIG 5

Confocal microscopy of methanol-fixed cells coinfected with C. abortus and C. caviae. McCoy cells were infected and incubated for 48 h at 37°C and then fixed with methanol. Cells were triple labeled with antibodies to C. abortus MOMP (panel A), C. caviae MOMP (panel B), and C. caviae IncA (panel C). Panel D is a merged image of the labeling. The arrows indicate two cells that are infected with C. abortus alone and the asterisks are to the right of doubly infected cells. The scale bar in A shows 10 microns for all images.

FIG 6

Immunofluorescence images of inclusion structure in C. psittaci CP3 and C. abortus LW203-coinfected cells. Cells were infected for 40 h and then fixed with methanol and labeled with (genus-common) anti-LOS and (species-specific) anti-C. abortus MOMP antibodies. Panel A: DAPI labeling. Panel B: anti-LOS. Panel C: anti-C. abortus MOMP. Panel D is a merged image. Cells infected singly with C. abortus (block arrow) or C. psittaci (arrow) are indicated in panel B. The two cells at the top of the images are infected with both pathogens. The scale bar in B represents 10 microns for all images.

FIG 7

Coculture with C. caviae negatively affects the bacterial load of C. abortus within infected cells. qPCR was performed on DNA extracted from coinfections with C. abortus and C. caviae. (A) Bacterial load of C. abortus as effected by changes in starting MOI of C. caviae (B) Bacterial load of C. caviae as effected by changes in starting MOI of C. abortus. The bacterial load was extrapolated from ompA copies per mL for each species. Results are given as mean and standard deviation (± SD) of replicates from a representative experiment.