Chlorate: a reversible inhibitor of proteoglycan sulphation in Chlamydia trachomatis-infected cells

Fadel, Sanaa; Eley, A.

doi:10.1099/jmm.0.05497-0

Cited by 22 publications

(20 citation statements)

References 11 publications

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“…The ∼5% of heparan sulfate remaining in the SLC35B2 clone may be due to activity of the second PAPS transporter (PAPST2). Chlorate is a competitive inhibitor of PAPS synthetase and blocks sulfation, which partially protects host cells from Chlamydia infection (31). However, the high concentrations of chlorate required to block Chlamydia infection are toxic to the host cells.…”

Section: Discussionmentioning

confidence: 99%

Attachment ofChlamydia trachomatisL2 to host cells requires sulfation

Rosmarin

Carette

Olive

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Chlamydia trachomatis is a pathogen responsible for a prevalent sexually transmitted disease. It is also the most common cause of infectious blindness in the developing world. We performed a loss-of-function genetic screen in human haploid cells to identify host factors important in C. trachomatis L2 infection. We identified and confirmed B3GAT3 , B4GALT7 , and SLC35B2 , which encode glucuronosyltransferase I, galactosyltransferase I, and the 3′-phosphoadenosine 5′-phosphosulfate transporter 1, respectively, as important in facilitating Chlamydia infection. Knockout of any of these three genes inhibits Chlamydia attachment. In complementation studies, we found that the introduction of functional copies of these three genes into the null clones restored full susceptibility to Chlamydia infection. The degree of attachment of Chlamydia strongly correlates with the level of sulfation of the host cell, not simply with the amount of heparan sulfate. Thus, other, as-yet unidentified sulfated macromolecules must contribute to infection. These results demonstrate the utility of screens in haploid cells to study interactions of human cells with bacteria. Furthermore, the human null clones generated can be used to investigate the role of heparan sulfate and sulfation in other settings not limited to infectious disease.

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Section: Discussionmentioning

confidence: 99%

Attachment ofChlamydia trachomatisL2 to host cells requires sulfation

Rosmarin

Carette

Olive

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…As serovar E binding has been reported to be variably sensitive to heparan sulfate [27], [57], [58], we investigated the role of the FGF2 pathway during epithelial cell infection. We first tested whether FGF2 can enhance serovar E binding to HeLa cells.…”

Section: Resultsmentioning

confidence: 99%

Chlamydia trachomatis Co-opts the FGF2 Signaling Pathway to Enhance Infection

et al. 2011

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The molecular details of Chlamydia trachomatis binding, entry, and spread are incompletely understood, but heparan sulfate proteoglycans (HSPGs) play a role in the initial binding steps. As cell surface HSPGs facilitate the interactions of many growth factors with their receptors, we investigated the role of HSPG-dependent growth factors in C. trachomatis infection. Here, we report a novel finding that Fibroblast Growth Factor 2 (FGF2) is necessary and sufficient to enhance C. trachomatis binding to host cells in an HSPG-dependent manner. FGF2 binds directly to elementary bodies (EBs) where it may function as a bridging molecule to facilitate interactions of EBs with the FGF receptor (FGFR) on the cell surface. Upon EB binding, FGFR is activated locally and contributes to bacterial uptake into non-phagocytic cells. We further show that C. trachomatis infection stimulates fgf2 transcription and enhances production and release of FGF2 through a pathway that requires bacterial protein synthesis and activation of the Erk1/2 signaling pathway but that is independent of FGFR activation. Intracellular replication of the bacteria results in host proteosome-mediated degradation of the high molecular weight (HMW) isoforms of FGF2 and increased amounts of the low molecular weight (LMW) isoforms, which are released upon host cell death. Finally, we demonstrate the in vivo relevance of these findings by showing that conditioned medium from C. trachomatis infected cells is enriched for LMW FGF2, accounting for its ability to enhance C. trachomatis infectivity in additional rounds of infection. Together, these results demonstrate that C. trachomatis utilizes multiple mechanisms to co-opt the host cell FGF2 pathway to enhance bacterial infection and spread.

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“…Although Western blot analysis indicated a reduction in total PARP protein levels with chlorate, production of the cleaved PARP fragment, a definitive indicator of apoptosis, was not observed in every sample (data not shown). A conservative dose of 30 mM chlorate was used in all remaining assays as it achieved significant reductions in cell number without significant increases in cytotoxicity, and was within doses used on other cell types (Fadel and Eley 2004;Yip et al 2002;Zertal-Zidani et al 2007;Zou et al 2004). The effectiveness of this dose was also reflected in its ability to up-regulate ALP activity (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In the context of the current study, TAT-EGFP treatment has indicated chlorate treatment of MG-63 cells as an appropriate method to further analyse the importance of GAGs in osteogenic systems. This method may also be advantageous in the identification of new drugs and treatments capable of altering GAG expression and may be of particular benefit in the development of treatments against pathogens which use cell surface GAGs in their cell invasion process (Carruthers et al 2000;Fadel and Eley 2004;McCormick et al 2000). TAT-PTD transduction could also be helpful in deciphering the paradoxical ability of GAGs to both promote and inhibit tumour progression (Ford et al 2001;Wu 2006).…”

Section: Discussionmentioning

confidence: 97%

A novel use of TAT-EGFP to validate techniques to alter osteosarcoma cell surface glycosaminoglycan expression

Dombrowski¹,

Rider²,

Nurcombe

et al. 2007

J Mol Hist

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Several methods to alter cell surface glycosaminoglycan (GAG) expression have previously been described, including treatments with chlorate to reduce the addition of charged sulfate groups, xyloside compounds to displace GAGs from their core proteins, and GAG lyases, such as heparinase and chondroitinase, to release GAG fragments from the cell layer. While these methods are useful in identifying cellular mechanisms which are dependent on GAGs, they must be stringently validated to assess results in the appropriate context. To determine the most useful technique for the evaluation of GAG function in osteogenesis, MG-63 osteosarcoma cells were systematically treated with these agents and evaluated for changes in cell surface GAGs using a TAT-EGFP fusion protein. TAT, a protein transduction domain from the HIV-1 virus, requires cell surface GAGs to traverse cell membranes. The EGFP component provides a method to assess protein entry into cells in both qualitative and quantitative tests. Here, TAT-EGFP transduction analysis confirmed radiochemical and physiological data that chlorate effectively disrupts GAG expression. TAT-EGFP entry into cells was also inhibited by the exogenous application of commercial heparin and GAGs extracted from MG-63 cells as well as by the pre-treatment of cells with chondroitinase ABC. However, neither heparinase III treatment nor the addition of exogenous chondroitin-6-sulfate affected TAT-EGFP entry into cells. In addition, xyloside-beta-D-naphthol and xyloside-beta-D-cis/trans-decahydro-2-naphthol treatment could not induce significant phenotypic change in these cells, and the unaffected TAT-EGFP transduction confirmed that this was due to an inability to efficiently prime GAG synthesis. The use of TAT-EGFP is thus a useful technique to specifically evaluate cell surface GAG expression in a simple, quantifiable manner, and avoids the complications involved with conventional radiochemical assays or analytical chromatography.

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Chlorate: a reversible inhibitor of proteoglycan sulphation in Chlamydia trachomatis-infected cells

Cited by 22 publications

References 11 publications

Attachment ofChlamydia trachomatisL2 to host cells requires sulfation

Attachment ofChlamydia trachomatisL2 to host cells requires sulfation

Chlamydia trachomatis Co-opts the FGF2 Signaling Pathway to Enhance Infection

A novel use of TAT-EGFP to validate techniques to alter osteosarcoma cell surface glycosaminoglycan expression

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