Abstract
Plasmid-free Chlamydia trachomatis serovar L2 organisms have been transformed with chlamydial plasmid-based shuttle vectors pGFP::SW2 and pBRCT using β-lactamase as a selectable marker. However, the recommendation of amoxicillin, a β-lactam antibiotics, as one of the choices for treating pregnant women with cervicitis due to C. trachomatis infection has made the existing shuttle vectors unsuitable for transforming sexually transmitted infection (STI)-causing serovars of C. trachomatis. Thus, in the current study, we modified the pGFP::SW2 plasmid by fusing a blasticidin S deaminase gene to the GFP gene to establish blasticidin resistance as a selectable marker and replacing the β-lactamase gene with the Sh ble gene to eliminate the penicillin resistance. The new vector termed pGFPBSD/Z::SW2 was used for transforming plasmid-free C. trachomatis serovar D organisms. Using blasticidin for selection, stable transformants were obtained. The GFP-BSD fusion protein was detected in cultures infected with the pGFPBSD/Z::SW2-trasnformed serovar D organisms. The transformation restored the plasmid property to the plasmid-free serovar D organisms. Thus, we have successfully modified the pGFP::SW2 transformation system for studying the biology and pathogenesis of other STI-causing serovars of C. trachomatis.
Highlights
The species Chlamydia trachomatis consist of many serovars with 3 distinct types of tissue tropism
To develop a shuttle vector that can be used for transforming sexually transmitted infection (STI)-causing serovars of C. trachomatis, we modified the pGFP::SW2 plasmid by first using a blasticidin S deaminase gene (BSD) from the vector pLenti6.3 [17] to replace the chloramphenicol acetyltransferase gene (CAT) as a fusion partner of GFP (Figure 1). This genetic replacement was designed for the vector to code for a GFP-BSD fusion protein since the fusion is known to be stable and to maintain BSD’s deaminase function [22]
The current transformation system is based on chlamydial plasmid shuttle vectors with β-lactamase as a selection marker [9,10,11,12,13]
Summary
The species Chlamydia trachomatis consist of many serovars with 3 distinct types of tissue tropism. Serovars D to K target genital tract epithelial cells for infection and are the leading cause of sexually transmitted bacterial infection. Infection with serovars D to K in the female lower genital tract can cause cervicitis frequently accompanying no apparent clinic symptoms. If untreated, the lower genital tract infection can lead to ascending infection termed pelvic inflammatory disease (PID), which in turn can be symptomatic or subclinical. Timely treatment of cervical infection is the key to prevent complications including preterm delivery among pregnant women [4]. Amoxicillin, a β-lactam antibiotics, has been recommended as one of the choices for treating pregnant women with Chlamydia (http://www.cdc.gov/std/treatment/ 2010/chlamydial-infections.htm#chlampreg)
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