The High-Risk Human Papillomavirus E6 Oncogene Exacerbates the Negative Effect of Tryptophan Starvation on the Development of Chlamydia trachomatis.

Shardulendra P Sherchand,Arnold H Zea,Alison J Quayle,Joyce A Ibana,Ashok Aiyar,Agathe Subtil

doi:10.1371/journal.pone.0163174

Abstract

Chlamydia trachomatis is an obligate intracellular pathogen that requires specific essential nutrients from the host cell, one of which is the amino acid tryptophan. In this context interferon gamma (IFNγ) is the major host protective cytokine against chlamydial infections because it induces the expression of the host enzyme, indoleamine 2,3-dioxygenase 1, that degrades tryptophan, thereby restricting bacterial replication. The mechanism by which IFNγ acts has been dissected in vitro using epithelial cell-lines such as HeLa, HEp-2, or the primary-like endocervical cell-line A2EN. All these cell-lines express the high-risk human papillomavirus oncogenes E6 & E7. While screening cell-lines to identify those suitable for C. trachomatis co-infections with other genital pathogens, we unexpectedly found that tryptophan starvation did not completely block chlamydial development in cell-lines that were HR-HPV negative, such as C33A and 293. Therefore, we tested the hypothesis that HR-HPV oncogenes modulate the effect of tryptophan starvation on chlamydial development by comparing chlamydial development in HeLa and C33A cell-lines that were both derived from cervical carcinomas. Our results indicate that during tryptophan depletion, unlike HeLa, C33A cells generate sufficient intracellular tryptophan via proteasomal activity to permit C. trachomatis replication. By generating stable derivatives of C33A that expressed HPV16 E6, E7 or E6 & E7, we found that E6 expression alone was sufficient to convert C33A cells to behave like HeLa during tryptophan starvation. The reduced tryptophan levels in HeLa cells have a biological consequence; akin to the previously described effect of IFNγ, tryptophan starvation protects C. trachomatis from clearance by doxycycline in HeLa but not C33A cells. Curiously, when compared to the known Homo sapiens proteome, the representation of tryptophan in the HR-HPV E6 & E6AP degradome is substantially lower, possibly providing a mechanism that underlies the lowered intracellular free tryptophan levels in E6-expressing cells during starvation.

Highlights

Chlamydia trachomatis is an obligate intracellular bacterium with a biphasic development in which it alternates between an infectious extracellular elementary body (EB), and an intracellular metabolically active but non-infectious reticulate body (RB) [1, 2]
Because tryptophan is an essential amino acid for both C. trachomatis and its human host cells, we anticipated that the developmental cycle of C. trachomatis would be impeded to a similar extent in a variety of human epithelial cell-lines grown in media lacking tryptophan (Trp-Free Media) prepared as described in experimental procedures
This was tested by comparing the growth of C. trachomatis in Complete Media and Trp-Free Media using the following human epithelial cell-lines, whose origin and characteristics are described in Table 1: 1) HeLa; 2) A2EN; 3) C33A; and 4) 293 [25,26,27,28,29]

Summary

Introduction

Chlamydia trachomatis is an obligate intracellular bacterium with a biphasic development in which it alternates between an infectious extracellular elementary body (EB), and an intracellular metabolically active but non-infectious reticulate body (RB) [1, 2]. IFNγ induces the host enzyme indoleamine 2,3-dioxygenase that catabolizes intracellular tryptophan to kynurenine, thereby limiting the availability of this essential amino acid during chlamydial development [6, 7] Under these conditions, chlamydial development is subject to an alternative program resulting in the formation of inclusions with aberrant RBs (ABs) that do not proceed to yield infectious EBs as long as tryptophan remains limiting [8,9,10,11,12]. Previous studies have shown that exposure to IFNγ reduces the effect of the antibiotic doxycycline on chlamydial development in HeLa cells [18] Our findings indicate this observation to correlate with the amount of free intracellular tryptophan during starvation, such that doxycycline is more effective against chlamydial development when intracellular tryptophan stores are larger. A detailed compositional analysis of proteins targeted for degradation by E6, or its partner ubiquitin ligase E6AP, reveal them to be remarkably, and significantly, poorer in tryptophan when compared to the entire known human proteome (Human genome project release GRCh38)

Experimental Procedures

Results

Discussion