Lateral gene transfer in vitro in the intracellular pathogen Chlamydia trachomatis

Abstract

Genetic recombinants that resulted from lateral gene transfer (LGT) have been detected in sexually transmitted disease isolates of Chlamydia trachomatis, but a mechanism for LGT in C. trachomatis has not been described. We describe here a system that readily detects C. trachomatis LGT in vitro and that may facilitate discovery of its mechanisms. Host cells were simultaneously infected in the absence of antibiotics with an ofloxacin-resistant mutant and a second mutant that was resistant to lincomycin, trimethoprim, or rifampin. Selection for doubly resistant C. trachomatis isolates in the progeny detected apparent recombinant frequencies of 10(-4) to 10(-3), approximately 10(4) times more frequent than doubly resistant spontaneous mutants in progeny from uniparental control infections. Polyclonal doubly resistant populations and clones isolated from them in the absence of antibiotics had the specific resistance-conferring mutations present in the parental mutants; absence of the corresponding normal nucleotides indicated that they had been replaced by homologous recombination. These results eliminate spontaneous mutation, between-strain complementation, and heterotypic resistance as general explanations of multiply resistant C. trachomatis that originated in mixed infections in our experiments and demonstrate genetic stability of the recombinants. The kind of LGT we observed might be useful for creating new strains for functional studies by creating new alleles or combinations of alleles of polymorphic loci and might also disseminate antibiotic resistance genes in vivo. The apparent absence of phages and conjugative plasmids in C. trachomatis suggests that the LGT may have occurred by means of natural DNA transformation. Therefore, the experimental system may have implications for genetically altering C. trachomatis by means of DNA transfer.

FIG. 1.

The 1.04-Mb C. trachomatis chromosome, showing the locations of mutant genes (32) (in bold and italics) that we used to isolate LGT recombinants. Also shown are loci that have relatively numerous between-strain polymorphic differences that could be used to analyze the sizes and possible directionality of chromosome segments that are transferred by LGT. The parC locus is shown because mutations in it can confer resistance to ofloxacin; the potential significance of this is mentioned in the Discussion.

FIG. 2.

Basic format of crosses no. 1 to no. 5, as illustrated with cross no. 1 (OFX^r-1 × LIN^r-1). (P-0) Sets of 10 primary flasks (P-0) containing HeLa cells (pale outlines) were infected with OFX^r-1 (○) (set A), LIN^r-1 (•) (set B), or both (set C). Separately initiated inclusions in a cell fused to form a single inclusion (darkly outlined ovoid objects) that, in the case of set C, allowed both parental types to multiply in the same inclusion in the absence of antibiotics. The thin arrow points to an OFX^r LIN^r recombinant (⊙) that originated in a set C P-0 flask. (P-1) One-tenth of each BH prepared from a P-0 flask was used to infect a first-passage (P-1) HeLa flask containing medium supplemented with OFX and LIN. Excepting rare spontaneous mutants, only OFX^r LIN^r recombinants could multiply in set C P-1 flasks, as shown. (P-2 through P-4) One-half of each bead harvest prepared from a P-1 flask was used to infect a P-2 flask containing OFX plus LIN medium. OFX^r LIN^r IFU present in a P-1 inclusion were liberated by the EB harvesting procedure and initiated separate doubly resistant inclusions in P-2 flasks. P-3 and P-4 flasks were infected with one-half of their antecedent BHs and contained OFX plus LIN medium. (P-4) Phase-contrast microscope images of typical ×200 fields in P-4 flasks. Crosses no. 2 to no. 4 differed from cross no. 1 only with respect to the parental mutant strains and antibiotics that were used (see text describing individual crosses).

FIG. 3.

Phase-contrast microscopic detection of doubly resistant recombinant inclusions at the first passage (P-1) of IFU produced in cells that were simultaneously infected with two kinds of antibiotic-resistant mutants. (A) A normal 48-h p.i. inclusion (thick arrow) formed in the absence of antibiotics used to select for recombinants. Inclusions formed by each strain of C. trachomatis used in this study would have similar appearances in antibiotic-free culture medium or in a medium containing just that antibiotic to which the strain was resistant. (B) A typical 46-h p.i. field in a cross no. 1 (OFX^r-1 × LIN^r-1) set A P-1 flask derived from a primary (P-0) flask in which all cells were infected with only parental strain OFX^r-1. The P-1 flask contained OFX plus LIN. OFX^r-1 is sensitive to LIN and formed tiny, inhibited inclusions in some cells. Similarly, LIN^r-1, the other parental strain in cross no. 1, is sensitive to OFX and would also be unable to form normal inclusions in OFX plus LIN medium (not shown). (C) A 48-h p.i. field in a cross no. 1 set C P-1 flask derived from a P-0 flask in which all cells were simultaneously infected with OFX^r-1 and LIN^r-1. The P-1 flask contained OFX plus LIN medium, in which parental IFU formed inhibited inclusions (small arrows) and OFX^r LIN^r recombinants formed normal inclusions (large arrow). (D) A typical 68-h p.i. field in a cross no. 3 set C P-1 flask derived from a P-0 flask in which all cells had been simultaneously infected with OFX^r-1 and RIF^r-1. The P-1 flask contained OFX plus RIF medium. An OFX^r RIF^r recombinant inclusion is shown (large arrow) along with numerous inhibited inclusions (small arrow) formed by nonrecombinant parental IFU. (E) A typical 46-h p.i. field in a cross no. 4 set C P-1 flask derived from a P-0 flask in which all cells were simultaneously infected with OFX^r-1 and TMP^r-1. The P-1 flask contained medium supplemented with OFX and TMP. An OFX^r TMP^r recombinant inclusion is shown (large arrow). Also shown are examples of relatively large, empty-appearing inclusions (small arrow) that are typically formed by TMP-sensitive strains of C. trachomatis (OFX^r-1 in this experiment) in the presence of TMP. Spontaneous mutants of parental strains would also be able to form doubly resistant inclusions in P-1 flasks containing two antibiotics, but their rarity (<10⁻⁷) would prevent their detection by microscopic scanning in practice.