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Infection and Immunity, November 2004, p. 6699-6701, Vol. 72, No. 11
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.11.6699-6701.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cleavage of the N-Linked Oligosaccharide from the Surfaces of Chlamydia Species Affects Attachment and Infectivity of the Organisms in Human Epithelial and Endothelial Cells

Cho-chou Kuo,* Amy Lee, and Lee Ann Campbell

Department of Pathobiology, University of Washington, Seattle, Washington

Received 14 June 2004/ Returned for modification 19 July 2004/ Accepted 1 August 2004


   ABSTRACT
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Previous studies demonstrated that the high-mannose oligosaccharide N linked to the Chlamydia major outer membrane protein inhibited the attachment and infectivity of the organism. The present study showed that cleavage of the glycan with N-glycanase decreased the attachment and infectivity of chlamydial organisms in human epithelial and endothelial cells.


   TEXT
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Chlamydiae are obligate intracellular bacteria. The uptake of chlamydial elementary bodies (EBs) by nonphagocytic cells is thought to be mediated by receptor-mediated endocytosis (16). Several mechanisms by which chlamydiae attach and enter host cells and various ligands that facilitate attachment have been proposed. These ligands include glycosaminoglycans (GAGs) (17), the major outer membrane protein (MOMP) (11), and heat shock protein 70 (8). Our studies have focused on what role high-mannose oligosaccharide glycan plays in attachment and infectivity (6). These studies were based on the findings that Chlamydia trachomatis MOMP is glycosylated (12) and the carbohydrate structure is an N-linked high-mannose oligosaccharide (6). Hapten inhibition assays with structurally defined oligosaccharides showed that attachment and infectivity of C. trachomatis, C. pneumoniae, and C. psittaci in HeLa cells were inhibited by high-mannose oligosaccharides, suggesting that the glycan moiety of chlamydiae mediates attachment and infectivity (6). The present study investigated if removal of the surface-exposed glycan from live chlamydial organisms affects their infectivity.

Chlamydial organisms purified by density gradient centrifugation were incubated with either N-glycanase (PNGase F) or O-glycanase (endo-{alpha}-acetylgalactosaminidase), with no detectable proteolytic activity (Sigma, St. Louis, Mo.), in 5 mM potassium phosphate buffer, pH 7.5, for 3 h at room temperature. N-glycanase cleaves N-linked oligosaccharides and O-glycanase cleaves O-linked oligosaccharides from the protein backbone. Subsequently, organisms were pelleted, washed once, and resuspended in chlamydial transport medium SPG (5). Positive controls were organisms incubated in buffer alone.

Infectivity was assayed in human epithelial HL (2) or endothelial HMEC-1 (1) cell monolayers prepared in 24-well culture plates containing a 12-mm-diameter round coverslip. Glycanase-treated organisms were inoculated into six wells per treatment at a multiplicity of infection of 10 for HL cells and of 20 for HMEC-1 cells. After 2 h of absorption at 37°C on a rocking platform, the inoculum was removed and culture medium was added. Infected cells were incubated at 37°C for 2 days with C. trachomatis and C. psittaci and for 3 days with C. pneumoniae. Three wells were used for the infectivity assay (first passage), and the remaining three wells were harvested and passed to HL cells (second passage) to assay the growth of organisms in the first passage by determining the number of infectious organisms produced (burst size). In the second passage, centrifugation was applied during absorption and cycloheximide was added to the culture medium for incubation of inoculated cells (5). Infectivity was quantified by counting inclusions following fluorescent-antibody staining and expressed as the number of inclusion forming units per milliliter.

Organisms metabolically labeled with [35S]methionine were used for assaying attachment (7). The binding assay was conducted at 4 and 37°C. At 4°C, organisms attach to but do not enter the host cells, while attachment and internalization do occur at 37°C. After 2 h of absorption in triplicate wells, inocula were removed and monolayers were washed three times with phosphate-buffered saline. Cells were scraped off with 0.5 ml of phosphate-buffered saline, disrupted by sonication, and transferred to scintillation vials, after which 5 ml of Dupont LSC scintillation cocktail (Biotechnology System, Boston, Mass.) was added and radioactivity was counted.

N-glycanase treatment inhibited the infectivity of C. pneumoniae AR-39 for HL and HMEC-1 cells in a dose-dependent manner (Fig. 1). The growth (burst size) of C. pneumoniae in these cells was also inhibited but at higher concentrations (greater than 90% inhibition at ≥10 U in HL cells and at 30 U in HMEC-1 cells). Both infectivity and growth of C. trachomatis E/UW-5/Cx and L2/434/Bu and C. psittaci 6BC were also inhibited by N-glycanase treatment (Table 1). Inhibition of all species following treatment with 2.5 U was partial in HL (≤34%) and HMEC-1 (≤40%) cells. C. pneumoniae and C. trachomatis L2 were the most susceptible to N-glycanase treatment, followed by C. trachomatis E and C. psittaci (Table 1). As expected (12), O-glycanase (0.02 U) had no effect on any of the above strains (data not shown).



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  		FIG. 1. Dose-dependent response of the infectivity of C. pneumoniae AR-39 to treatment with N-glycanase in HL and HMEC-1 cells. Error bars indicate 1 standard deviation from the means.

 

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  		TABLE 1. Inhibition of infectivity of chlamydial species by 30 U of N-glycanase

 
Attachment of radiolabeled organisms to both HL and HMEC-1 cells was significantly inhibited at 4°C (Table 2). C. pneumoniae was inhibited the most, followed by C. trachomatis L2 and C. trachomatis E, in HL and HMEC-1 cells, respectively. With the exception of C. pneumoniae and HMEC-1 cells, greater inhibition was observed at 37°C than at 4°C (Table 2).


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  		TABLE 2. Attachment of chlamydiae is inhibited by treatment with N-glycanase

 
This study confirmed our previous studies showing that chlamydiae use the surface-exposed carbohydrate structure for infection of mammalian host cells and that the oligosaccharide is N linked to the protein backbone (6). The strain differences observed in susceptibility to N-glycanase treatment had also been observed with the binding of EBs to GAGs (9, 10). For example, the attachment of C. trachomatis E was not affected by GAG, while that of C. trachomatis L2 was (3). This finding is similar to the present study showing the greater effect of N-glycanase treatment on C. trachomatis L2 than on C. trachomatis E. It has been shown that the attachment of C. psittaci GPIC is GAG dependent; however, GAG does not prevent productive infections (4). This observation is compatible with the present finding that C. psittaci can also use the mannose oligosaccharide for infection.

In this study, EBs were incubated with N-glycanase at pH 7.5 for 3 h at room temperature, which are not optimal conditions for glycan removal. In previous studies, the glycan was released from gel-purified, glycanated MOMP by treatment with 0.3 U at pH 8.6 for 48 h at 37°C, which resulted in maximum recovery of glycan for use in the infectivity inhibition assay (6, 13). This treatment is not applicable to live chlamydial EBs, as it results in inactivation of the organism. To compensate for the shorter duration of treatment and the use of neutral pH, the concentration of N-glycanase was increased to 30 U. Nevertheless, a significant reduction of infectivity was obtained.

Any effect of proteinase contamination of N-glycanase could be ruled out, as inhibitory effects were identical regardless of whether the proteinase inhibitor phenylmethylsulfonyl fluoride (Sigma) was present or not during treatment and no proteolytic activity was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis following the treatment of organisms.

Metabolic labeling studies demonstrated that the glycan plays a role in both attachment to the host cell and internalization, as an increased inhibitory effect was observed at 37°C in comparison to that seen at 4°C (Table 2). An additional effect of N-glycanase treatment on growth is suggested by the greater inhibition of infectivity observed on the basis of inclusion counts (Table 1 versus Table 2). The greater inhibition of infectivity and the lower percent inhibition of attachment by N-glycanase treatment are analogous to those observed previously with high-mannose oligosaccharides in hapten inhibition studies (6). These results are compatible with the current concept (17) that EBs initially bind to the GAG receptor, which leads to the specific ligand-receptor-mediated interaction for entry (9, 10, 18).

We have previously reported that C. trachomatis contains three glycoproteins with molecular masses of 40, 32, and 18 kDa (12, 14, 15). Therefore, the present study cannot differentiate which glycoprotein(s) is involved in infectivity. However, it is believed that MOMP plays a major role in adhesion and infectivity of chlamydial organisms (9, 10).

In summary, N-glycanase treatment inhibited both attachment and infectivity and susceptibility varied among strains and between cell types.


   ACKNOWLEDGMENTS
 
This work was supported by Public Health Service grant AI-43060 from the National Institute of Allergy and Infectious Diseases.

We thank Sen-itirho Hakomori for advice on carbohydrate chemistry.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pathobiology, Box 357238, University of Washington, Seattle, WA 98195. Phone: (206) 543-8689. Fax: (206) 543-3873. E-mail: cckuo{at}u.washington.edu. Back

Editor: J. T. Barbieri


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Infection and Immunity, November 2004, p. 6699-6701, Vol. 72, No. 11
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.11.6699-6701.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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