Development of Transposon Mutagenesis for Chlamydia muridarum

Abstract

Functional genetic analysis of Chlamydia has been a challenge due to the historical genetic intractability of Chlamydia, although recent advances in chlamydial genetic manipulation have begun to remove these barriers. Here, we report the development of the Himar C9 transposon system for Chlamydia muridarum, a mouse-adapted Chlamydia species that is widely used in Chlamydia infection models. We demonstrate the generation and characterization of an initial library of 33 chloramphenicol (Cam)-resistant, green fluorescent protein (GFP)-expressing C. muridarum transposon mutants. The majority of the mutants contained single transposon insertions spread throughout the C. muridarum chromosome. In all, the library contained 31 transposon insertions in coding open reading frames (ORFs) and 7 insertions in intergenic regions. Whole-genome sequencing analysis of 17 mutant clones confirmed the chromosomal locations of the insertions. Four mutants with transposon insertions in glgB, pmpI, pmpA, and pmpD were investigated further for in vitro and in vivo phenotypes, including growth, inclusion morphology, and attachment to host cells. The glgB mutant was shown to be incapable of complete glycogen biosynthesis and accumulation in the lumen of mutant inclusions. Of the 3 pmp mutants, pmpI was shown to have the most pronounced growth attenuation defect. This initial library demonstrates the utility and efficacy of stable, isogenic transposon mutants for C. muridarum The generation of a complete library of C. muridarum mutants will ultimately enable comprehensive identification of the functional genetic requirements for Chlamydia infection in vivoIMPORTANCE Historical issues with genetic manipulation of Chlamydia have prevented rigorous functional genetic characterization of the ∼1,000 genes in chlamydial genomes. Here, we report the development of a transposon mutagenesis system for C. muridarum, a mouse-adapted Chlamydia species that is widely used for in vivo investigations of chlamydial pathogenesis. This advance builds on the pioneering development of this system for C. trachomatis We demonstrate the generation of an initial library of 33 mutants containing stable single or double transposon insertions. Using these mutant clones, we characterized in vitro phenotypes associated with genetic disruptions in glycogen biosynthesis and three polymorphic outer membrane proteins.

Keywords: Chlamydia muridarum; glycogen; pmp; transposon mutagenesis.

FIG 1

Generation of C. muridarum transposon mutants. (A) Schematic of the pCMC5M plasmid used for transformation, containing the pUC ori, Himar1 C9 transposase, and a transposon with cat-gfp and flanking inverted repeat (IR) sequences. (B) Overview of procedure for generating C. muridarum transposon mutants in McCoy cells. (C) Genome map of transposon insertions in the C. muridarum chromosome and plasmid pNigg. Insertions in coding sequences (CDS) are in red; insertions in intergenic regions (IGR) are in gray. Nucleotide markers on C. muridarum chromosome are expressed in kilobase pairs.

FIG 2

Transposon insertions in glgB and macP. (A) Chromosomal sequences of insertion sites. (B) PCR analysis of UWCM007 using primers against flanking sequences in genomic DNA. Lanes: 1, UWCM007 (glgB::Tn and macP::Tn); 2, plaque-cloned UWCM007 (glgB::Tn); 3, plaque-cloned UWCM027 (glgB::Tn); 4, plaque-cloned UWCM007 (glgB::Tn and macP::Tn); 5, 2× plaque-cloned UWCM007 (glgB::Tn and macP::Tn); 6, UWCM007 (glgB::Tn and macP::Tn); 7, plaque-cloned UWCM007 (glgB::Tn); 8, plaque-cloned UWCM027 (glgB::Tn); 9, plaque-cloned UWCM007 (glgB::Tn and macP::Tn); 10, 2× plaque-cloned UWCM007 (glgB::Tn and macP::Tn). (C) Schematic showing the deletion of sequences between TC0303 and TC0306 that occurred with the insertion of an intact transposon (red) in mutant clone UWCM022.

FIG 3

Stability of transposon inserts. Two plaque-purified transposon mutants, UWCM012 (pmpA::Tn) and UWCM027 (glgB::Tn), were grown continuously for 10 generations in the presence (+) or absence (−) of 0.5 μg/ml chloramphenicol (cam). Genomic DNA from the passaged strains was extracted and subjected to PCR analysis using primers designed against genomic sequences flanking the transposon inserts. The ∼2-kb bands denote the sizes of transposon inserts.

FIG 4

Deficient glycogen accumulation in glgB::Tn inclusions. McCoy cells were infected with WT C. muridarum, a glgB::Tn mutant, a plasmid-free strain of C. muridarum (Cm P–), or a complemented glgB::Tn strain expressing WT glgB. Iodine staining of the infected cells at 26 hpi showed glycogen accumulation in WT and complemented inclusions (dark orange), no glycogen in Cm P– inclusions, and amylose accumulation in glgB::Tn inclusions (purple with dark deposits). Representative inclusions in each image are marked with asterisks.

FIG 5

In vitro phenotypes of C. muridarum pmp mutants. (A) Chromosomal locations of transposon insertions in pmp genes. The orientations of inserts are indicated by the gene directionality. (B) Predicted protein topologies of Pmp proteins. Transposon insertion sites are marked with black lines; blue, Pmp middle domain; red, autotransporter domain. (C) Inclusion burst sizes of pmpI::Tn, pmpA::Tn, and pmpD::Tn strains versus an IGR::Tn control strain. McCoy cells infected with the indicated strains were harvested at the times postinfection indicated, and the amount of infectious progeny in each sample was determined by infecting fresh McCoy cells, followed by IFU assays. (D) Attachment of the indicated strains to McCoy cells was determined by inoculating the cells under centrifugation-aided and static conditions. The ratios of centrifugation to static conditions were computed. The data represent means, and the error bars indicate standard deviations. Statistical comparisons between transposon mutants and the IGR::Tn control were analyzed using one-way ANOVA with Dunnett’s test for multiple comparisons. *, P < 0.05. (E) Confocal immunofluorescence analysis of inclusions containing WT C. muridarum, the IGR::Tn or pmpI::Tn mutant, and the complemented pmpI::Tn-plus-WT pmpI strain analyzed at 24 hpi. Green, anti-LPS; blue, DAPI; red, Evans blue. Scale bar, 10 μm.

FIG 6

In vitro growth phenotypes of transposon mutants and complemented strains. The growth of mutants and complemented strains was assessed by the IFU propagation ratio. McCoy cells were infected with each strain in 24-well plates. At 20 to 22 hpi, images were taken for inclusion counting by fluorescence microscopy. At 26 hpi, the cells were lysed and the bacteria were inoculated onto fresh McCoy cells for IFU determination. The data are expressed as means, and the error bars indicate standard deviations. Statistical comparisons between the transposon mutants, complemented strains, and IGR::Tn control were analyzed using one-way ANOVA with Dunnett’s test for multiple comparisons. *, P < 0.05; **, P < 0.01.