Attachment ofChlamydia trachomatisL2 to host cells requires sulfation

Abstract

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.

Intercellular trafficking of the nuclear oncoprotein DEK

“…His-DEK fluorescence intensity of all samples in a given group are indicated by the horizontal lines in the scatter dot plot. To further confirm that HSPGs are necessary for cellular DEK uptake, we turned to a previously described elegant genetic system (33). We used HAP1 cells and HAP1 cells deficient in either (33), genes that specify part of the biochemical pathway required for the synthesis and translocation of HSPGs.…”

“…To further confirm that HSPGs are necessary for cellular DEK uptake, we turned to a previously described elegant genetic system (33). We used HAP1 cells and HAP1 cells deficient in either (33), genes that specify part of the biochemical pathway required for the synthesis and translocation of HSPGs. The HAP1 cell line is derived from the haploid myeloid KBM7 cell line that was originally isolated from a patient with leukemia (34).…”

“…B4GALT7 and SLC35B2 were disrupted in HAP1 cells by retrovirusmediated insertional mutagenesis. Cells were then reconstituted via lentiviral transduction with either empty vector or functional B4GALT7 or SLC35B2 and selected by puromycin resistance (33). MDMs were isolated from the blood of healthy donors and synovial macrophages derived from patients with JIA as described previously (27,28).…”

Development of retroviral vectors for insertional mutagenesis in medaka haploid cells

“…Gene trapping by retroviral vector in nearly haploid human cancer cell line has generated insertions in N 98% genes expressed in the cell (Carette et al, 2011a). Host factors essential for virus infection (Carette et al, 2009(Carette et al, , 2011bPapatheodorou et al, 2011;Rosmarin et al, 2012;Jae et al, 2013) and genes essential for various cellular processes (Carette et al, 2011a;Reiling et al, 2011;Birsoy et al, 2013;Lee et al, 2013;Timms et al, 2013) have been identified using the mutated cells. IM combined with haploid cell lines has become a powerful system, which will identify more and more novel genes involved in these kinds of cellular processes.…”

Haploid animal cells

“…Both cell systems have distinct strengths and weaknesses. For example, screening in human tumor cells can identify genes in a clinically relevant genome (Birsoy et al, 2013;Carette et al, 2009;Carette et al, 2011;Guimaraes et al, 2011;Reiling et al, 2011;Rosmarin et al, 2012), but tumor-specific genetic aberrations and oncogenic signals could interfere with the particular aim of a genetic screen. The stable haploid karyotype of human tumor cells and ease of culture are additional technical advantages over haploid mouse ESCs.…”