Inhibition of chlamydiae by primary alcohols correlates with the strain-specific complement of plasticity zone phospholipase D genes

Abstract

Members of the genus Chlamydia are obligate intracellular pathogens that have a unique biphasic developmental cycle and interactions with host cells. Many genes that dictate host infection tropism and, putatively, pathogenic manifestations of disease are clustered in a hypervariable region of the genome termed the plasticity zone (PZ). Comparative genomics studies have determined that an uncharacterized family of PZ genes encoding orthologs of eukaryotic and prokaryotic members of the phospholipase D (PLD) enzyme family varies among chlamydiae. Here, we show that the PZ PLD (pzPLD) of Chlamydia trachomatis are transcribed during both normal and persistent infection and that the corresponding PLD proteins are predominantly localized in reticulate bodies on the inner leaflet of the inclusion membrane. Further, we show that strains of chlamydiae encoding the pzPLD, but not a strain lacking these genes, are inhibited by primary alcohols, potent PLD inhibitors, during growth in HeLa 229 cells. This inhibitory effect is amplified approximately 5,000-fold during recovery from persistent infection. These findings suggest that the chlamydial pzPLD may be important, strain-specific, pathogenesis factors in vivo.

FIG. 1.

Organization of PLD genes in the PZs of chlamydiae. Shown are the arrangements of genes in the PZ, and extra-PZ PLD, in C. trachomatis serovar D, C. muridarum, and C. caviae. The nomenclature is according to designations given by the National Center for Biotechnology Information for accession numbers NC_003361 (29), NC_002620 (28), and NC_000117 (30). PLD genes are shown in red, genes related to the C. muridarum large cytotoxin CT438 are in black, tryptophan biosynthesis genes are colored blue, and nucleotide biosynthesis- or acquisition-related genes are in yellow. PLD orthologs corresponding to pzPLD CT154 to CT158 of C. trachomatis and TC436 to TC432 in C. muridarum are absent in the PZ of C. caviae. C. muridarum encodes two additional pzPLD orthologs (TC440 and TC447) absent from the PZs of both C. trachomatis and C. caviae. CT084 and CT284 of C. trachomatis, TC357 of C. muridarum, and CCA357 and CCA457 of C. caviae encode PLDs that are located outside the PZ (indicated by double curved lines). The genes in white are intervening genes in the PZ which cannot be strictly sorted into any of the four aforementioned categories. The dual-colored C. caviae gene prsA functions in nucleotide and tryptophan biosynthesis. The arrows indicate the direction of gene transcription. Double curved lines indicate nonlinear breaks in the diagram between the extra-pzPLD and depicted portions of the PZ.

FIG. 2.

The pzPLD are expressed in the mid- to late developmental cycle. HeLa 229 cells were infected with C. trachomatis, and total RNA was harvested from EB or infected cells at 1, 3, 8, 16, 24, and 42 h p.i. The copy numbers of different transcripts (indicated in individual graphs) in each sample were determined by qRT-PCR. The experiment was repeated four times in triplicate, and data from a single representative experiment are depicted. The error bars indicate standard deviations of transcript copy numbers. Graph values marked with asterisks indicate transcripts that fell below the limit of reliable detection (approximately 1,000 total transcripts).

FIG. 3.

pzPLD are differentially expressed during normal and persistent infections. HeLa 229 cells were normally or persistently infected (IFN-γ treated) with C. trachomatis at an MOI of 1, and total RNAs were harvested from infected cultures at 24 h p.i. The ratio of specific transcripts (indicated below the x axis) to 16S rRNA measured in normal-infection samples at 24 h was arbitrarily set to 1. Transcript-to-16S rRNA ratios in persistent infections are plotted as a change (n-fold) versus normal infection. The experiment was repeated four times in triplicate, and data from a single representative experiment are shown. The error bars indicate standard deviations of the change.

FIG. 4.

CT155 is present in EB and is expressed in the mid- to late developmental cycle. HeLa 229 cells were infected at an MOI of 1 with C. trachomatis serovar D EB, and whole-cell lysates were prepared at various intervals, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted to nitrocellulose membranes. The nitrocellulose membranes were probed with antibodies to chlamydial CT155, MOMP, or the host cell control GAPDH. Both CT155 and MOMP were detected in EB preparations (EB protein equivalent to an MOI of ∼150), but not in mock-infected lysates (labeled M). Strong de novo expression of CT155 and MOMP was detected by 24 h p.i. and continued until the end of the developmental cycle. Equivalent amounts of lysates were loaded in each lane and blot.

FIG. 5.

The pzPLD CT155 is present in RB that associate with the luminal face of the inclusion membrane. HeLa 229 cells were infected at an MOI of 0.2 and prepared for confocal microscopy at 40 h p.i. Paraformaldehyde-fixed, saponin-treated cells were dual labeled with rabbit anti-serovar D MOMP antibodies, mouse anti-serovar D CT155 antibodies, and Alexa-fluor-conjugated secondary antibodies. The micrographs show a single representative cross section from a normal inclusion at 40 h p.i. (A) Anti-CT155 channel in red. (B) Anti-MOMP channel in green. (C) Merged image of panels A and B. The areas of yellow coloration in panel C indicate pixels of CT155 and MOMP colocalization. Scale bars = 10 μm.

FIG. 6.

Alcohol sensitivity of chlamydiae is strain specific and primary alcohol specific and correlates with the complement of pzPLD. HeLa 229 cells were infected at an MOI of 1 with MoPn, C. trachomatis, or GPIC (indicated below the x axis). Following infection, the cultures received normal infection medium (black bars), infection medium supplemented with 1-butanol (open bars), or medium supplemented with 2-butanol (hatched bars). At 48 h p.i., the cultures were harvested, and recoverable IFU (y axis) were enumerated. The experiment was repeated twice in quadruplicate; representative data from a single experiment are depicted. The asterisks above the bars corresponding to 1-butanol treatment of C. trachomatis and MoPn indicate that the recoverable IFU of these cultures were decreased significantly (P < 0.05; 2-tailed unpaired t test) from those of 2-butanol-treated and untreated control cultures. The error bars depict standard deviations of recoverable IFU.

FIG. 7.

C. trachomatis primary-alcohol sensitivity varies during the developmental cycle. HeLa 229 cells were infected with C. trachomatis at an MOI of 1. The infection medium was supplemented with 1-butanol or 2-butanol (indicated below the x axis) during the first 24 h (black bars) or second 24 h (hatched bars) of infection. Control cultures (open bar) received no alcohol during the entire course of infection. At 48 h p.i., the cultures were harvested, and recoverable IFU were enumerated (y axis). The experiment was repeated twice in quadruplicate; representative data from a single experiment are depicted. The asterisk indicates that 1-butanol treatment during the final 24 h of infection significantly decreased recoverable IFU (P < 0.05; two-tailed unpaired t test) from those of 2-butanol-treated and untreated control cultures. The error bars depict standard deviations of recoverable IFU.

FIG. 8.

Primary alcohols block tryptophan rescue of C. trachomatis from IFN-γ-mediated persistence. HeLa 229 cells were seeded in low-tryptophan MDMEM supplemented with 50 U/ml IFN-γ, incubated for 24 h, and infected with C. trachomatis at an MOI of 1. Following infection, the cultures were fed IFN-γ-supplemented MDMEM and incubated for an additional 24 h (IFN-γ treatment phase). The cultures were then washed and refed with MDMEM supplemented with 10× tryptophan and incubated for an additional 24 h (trytophan rescue phase) prior to recoverable-IFU harvest and enumeration (y axis). Experimental cultures were additionally treated with either 1-butanol or 2-butanol (indicated below the x axis) during IFN-γ treatment (black bars) or during tryptophan rescue (hatched bars). Control cultures (open bar) received no alcohol during either IFN-γ treatment or tryptophan rescue. The asterisk indicates that 1-butanol treatment during tryptophan rescue significantly decreased recoverable IFU (P < 0.05; two-tailed unpaired t test) from those of cultures treated with 1-butanol during IFN-γ treatment, cultures treated with 2-butanol during either IFN-γ treatment or tryptophan rescue, and untreated control cultures. The error bars depict standard deviations of recoverable IFU.