Chlamydia trachomatis cytotoxicity associated with complete and partial cytotoxin genes

Abstract

Chlamydia trachomatis is an obligate intracellular human bacterial pathogen that infects epithelial cells of the eye and genital tract. Infection can result in trachoma, the leading cause of preventable blindness worldwide, and sexually transmitted diseases. A common feature of infection is a chronic damaging inflammatory response for which the molecular pathogenesis is not understood. It has been proposed that chlamydiae have a cytotoxic activity that contributes to this pathology, but a toxin has not been identified. The C. trachomatis genome contains genes that encode proteins with significant homology to large clostridial cytotoxins. Here we show that C. trachomatis makes a replication-independent cytotoxic activity that produces morphological and cytoskeletal changes in epithelial cells that are indistinguishable from those mediated by clostridial toxin B. A mouse chlamydial strain that encodes a full-length cytotoxin caused pronounced cytotoxicity, as did a human strain that has a shorter ORF with homology to only the enzymatically active site of clostridial toxin B. Cytotoxin gene transcripts were detected in chlamydiae-infected cells, and a protein with the expected molecular mass was present in lysates of infected epithelial cells. The protein was present transiently in infected cells during the period of cytotoxicity. Together, these data provide compelling evidence for a chlamydial cytotoxin for epithelial cells and imply that the cytotoxin is present in the elementary body and delivered to host cells very early during infection. We hypothesize that the cytotoxin is a virulence factor that contributes to the pathogenesis of C. trachomatis diseases.

Figure 1

Cytotoxin-like genes present in C. trachomatis serovars MoPn, D, and L2 and amino acid sequence alignments with large clostridial cytotoxins. (A) C. trachomatis serovars MoPn (7), D (6), and L2 (ref. and data not shown) ORFs within the PZ region. Serovar MoPn has three large ORFs (TC0437, TC0438, and TC0439) arranged in tandem (TC0437 and TC0439 were not shown to allow detailed comparison of flanking regions). Serovar D has four ORFs (CT165, CT166, CT167, and CT168) with homology at the amino acid level to portions of the large TC0438 ORF of MoPn at the N and C termini (as indicated by the shaded regions). Serovar L2 has a single ORF (CT 168) with homology to the C-terminal region of the large MoPn ORF. (B) Alignment of the conserved “extended DxD motif” (20), present in the superfamily of glycosyltransferases and chlamydial toxin-like ORFs. Arrows indicate conserved amino acid residues that are required for (black arrows) or involved in (gray arrows) glucosyltransferase activity (8). The open circle indicates the “DxD” motif in A and B. Identical (+) and conserved (: and .) amino acid residues are indicated below the sequences. CtD CT166, GenBank AE001273; CtMoPn TC0437, GenBank AE002311_3; CtMoPn TC0438, GenBank AE002311_4; CtMoPn TC0439, GenBank AE002312_1; CpGPIC, C. psittaci toxin ORF, http://www.tigr.org; Cd Toxin A, C. difficile strain VPI 10463 toxin A, GenBank M30307; Cd Toxin B, C. difficile strain VPI 10463 toxin B, GenBank X53138. (C) Alignment of a conserved region of LCTs (8) showing residues required for (black arrow) or involved in (gray arrows) binding the UDP-glucose cosubstrate in LCTs (8). The asterisk indicates the UDP-glucose binding domain in A and C.

Figure 2

C. trachomatis infection-associated cytotoxicity. HeLa cell monolayers were examined at 4 h PI by phase and fluorescent microscopy. (A–D) Phase microscopic images of infected cell monolayers. (A) Uninfected. (B) L2 infected. (C) MoPn infected. (D) D infected. (Magnification, ×400.) A pronounced cytopathic effect characterized by cell rounding is evident in monolayers infected with MoPn and D (see Insets). (E–H) Fluorescent images of cells stained with Oregon Green phalloidin. (E) Uninfected. (F) L2 infected. (G) MoPn infected. (H) D infected. (Magnification, ×1,000.) Cytoskeletal collapse associated with the depolymerization of actin can be seen in MoPn and D infected cells.

Figure 3

Comparative cytotoxicity of C. trachomatis MoPn and D. HeLa cell monolayers were infected with C. trachomatis MoPn, D, and L2 at the MOI indicated and examined by phase microscopy at 4 h PI. (×400.) Cytotoxicity was assessed by cell rounding as described in Table 1. C. trachomatis MoPn had higher levels of cytotoxicity at lower MOIs than serovar D. C. trachomatis L2 had no cytotoxicity at any of the MOIs tested.

Figure 4

RT-PCR analysis of toxin gene expression throughout the C. trachomatis developmental cycle. C. trachomatis infections of HeLa 229 cells were monitored for toxin gene expression throughout the developmental cycle [for serovars MoPn (A) and D (B)]. RT-PCR analyses were done for each time point with oligonucleotide primers specific for each ORF. (A) RT-PCR analysis of TC0438 expression compared with expression of the groEL control gene. TC0438 mRNA was first detected at 16 h PI and at all time points thereafter, whereas groEL mRNA was detected at all time points. (B) Expression of CT166 of serovar D compared with expression of groEL. Similar to the profile of the cytotoxin gene in MoPn, expression was first detected at 16 h PI and continued throughout the developmental cycle.

Figure 5

Toxin protein expression analyzed by immunoblotting. C. trachomatis serovars MoPn, D, and L2 were analyzed for toxin protein expression in purified EBs and infected host cells with antiserum raised to purified CT166. The lanes correspond to EB (purified EBs); t10–t240, time in minutes PI that lysates of infected cells were made for each serovar (MOI 500). (C) Control cells (uninfected t10 only). Strong reactivity to a protein of ≈73 kDa (predicted M_r of CT166 is 74.8 kDa) was observed in serovar D EB preparations and infected cells (indicated by an asterisk). Reactivity in infected cells was transient and was not detected after 120 min PI. A lower M_r product of ≈20 kDa was observed in MoPn EB preparations and infected cells (indicated by an asterisk). This product also appeared to be degraded during the infection and was not present in infected cells at 60 min PI.