PmpA, B, D and H production differs under normal <i>C</i>. <i>psittaci</i> and during penicillin-induced stress culture conditions.

<p><i>C</i>. <i>psittaci</i> infected HeLa cells were fixed at 12, 18, 24, 32 and 48 hpi, and double-stained with a chlamydial LPS-specific antibody and Pmp-specific antibody. For each Pmp subtype, the percentage of positive inclusions was determined by a macro based on co-localization of the two antigens. The results shown here did not take into account smaller inclusions that are formed at 24, 32 and 48 hpi. The data are expressed as box plots: the box represents the 25^th-75^th percentiles, the median is depicted by a bar across the box and the whiskers on each box represent minimum and maximum value. Outliers are depicted by dots. Statistically significant differences (P < 0.05) are indicated with an asterix.</p

The PmpB production profile along development.

<p><i>C</i>. <i>psittaci</i>-infected HeLa cells were fixed at 12, 18, 24, 32 and 48 hpi, and double-stained with chlamydial LPS-specific antibody (FITC-conjugated, green) and PmpB-specific antibody (Alexa Fluor 568-conjugated, red). At 12 and 18 hpi (A), only colored merged images are shown under normal and penicillin-induced persistence culture conditions. At 24 and 48 hpi (B), single channel images are shown in black and white (2 left-most columns), while merged images and insets thereof are shown in color (2 right-most columns). Staining patterns at 32 hpi (not shown) were similar to those at 48 hpi. Bar = 2 μm for the 12 and 18 hpi times, and 24 and 48 hpi insets (i.e. top row and right-most column) and 10 μm for all remaining images.</p

Guinea pig polyclonal antibodies against PmpA, B, D and H are specific for their respective immunizing antigens.

<p>(A) The specificity of polyclonal antibodies raised against rPmpA (anti-PmpA), rPmpB (anti-PmpB), rPmpD (anti-PmpD) and rPmpH (anti-PmpH) was verified by immunoblotting using (A) partially purified recombinant PmpA, B, D, E1, G3 and H as well as (B) density gradient purified EBs of <i>C</i>. <i>psittaci</i> Cal10. The calculated molecular masses of recombinant PmpA, PmpB, PmpD, PmpE1, PmpH and PmpG3 are 92 kDa, 74 kDa, 95 kDa, 74 kDa, 88 kDa and 60 kDa, respectively. The observed molecular masses were 75 kDa, 74 kDa, 95 kDa, 70 kDa, 75 kDa and 60 kDa, respectively. (B) The calculated and observed molecular masses of the protein bands detected in EBs are shown.</p

<i>pmp</i> genes and gene products of <i>C</i>. <i>psittaci</i> Cal10.

<p><i>pmp</i> genes and gene products of <i>C</i>. <i>psittaci</i> Cal10.</p

Transcription of the <i>pmp</i> genes along development.

<p>Transcript levels of <i>pmpA</i> through <i>pmpG8</i> and <i>tufA</i> of <i>C</i>. <i>psittaci</i> Cal10 were measured at 2, 6, 12, 18, 24, 32 and 48 hpi by real-time RT-qPCR. Two biological and two technical replicated were analyzed for each sample. Error bars are based on the standard error of the mean.</p

<i>pmp</i> gene organization in the <i>C</i>. <i>psittaci</i> Cal10 genome.

<p>(A) The <i>pmp</i> CDSs map to 4 distinct loci on the <i>C</i>. <i>psittaci</i> Cal10 genome. Distances (bp) between nearest loci are indicated. (B) Linear representation of the <i>pmp</i> loci. CDSs and inter-CDS regions are drawn to scale. Number of bp between 2 nearest CDSs are indicated. A break (//) is added if the inter-CDS region is bigger than 5000 bp.</p

The PmpA production profile along development.

<p><i>C</i>. <i>psittaci</i>-infected HeLa cells were fixed at 12, 18, 24, 32 and 48 hpi, and double-stained with chlamydial LPS-specific antibody (FITC-conjugated, green) and PmpA-specific antibody (Alexa Fluor 568-conjugated, red). At 12 and 18 hpi (A), only colored merged images are shown under normal and penicillin-induced persistence culture conditions. At 24 and 48 hpi (B), single channel images are shown in black and white (2 left-most columns), while merged images and insets thereof are shown in color (2 right-most columns). Staining patterns at 32 hpi (not shown) were similar to those at 48 hpi. Bar = 2 μm for the 12 and 18 hpi times, and 24 and 48 hpi insets (i.e. top row and right-most column) and 10 μm for all remaining images.</p

The PmpD production profile along development.

<p><i>C</i>. <i>psittaci</i>-infected HeLa cells were fixed at 12, 18, 24, 32 and 48 hpi, and double-stained with chlamydial LPS-specific antibody (FITC-conjugated, green) and PmpD-specific antibody (Alexa Fluor 568-conjugated, red). At 12 and 18 hpi (A), only colored merged images are shown under normal and penicillin-induced persistence culture conditions. At 24 and 48 hpi (B), single channel images are shown in black and white (2 left-most columns), while merged images and insets thereof are shown in color (2 right-most columns). Staining patterns at 32 hpi (not shown) were similar to those at 48 hpi. Bar = 2 μm for the 12 and 18 hpi times, and 24 and 48 hpi insets (i.e. top row and right-most column) and 10 μm for all remaining images.</p

PmpA and PmpH localize to the chlamydial inner and outer membranes, to the inclusion membrane and to small putative outer membrane vesicles.

<p><i>C</i>. <i>psittaci</i> infected HeLa cells were fixed at late times (24 and 48 hpi) and stained with primary PmpA- or PmpH-specific antibody and secondary gold conjugated goat anti-guinea pig antibody. Localization of PmpA and PmpH in the chlamydial inner (IM) and outer (OM) membranes (top row, long arrows), in the inclusion membrane (middle row, short arrows) and in putative OMVs (bottom row, arrowheads) is shown as indicated. Bars = 0.1 μm.</p

The PmpH production profile along development.

<p><i>C</i>. <i>psittaci</i>-infected HeLa cells were fixed at 12, 18, 24, 32 and 48 hpi, and double-stained with chlamydial LPS-specific antibody (FITC-conjugated, green) and PmpH-specific antibody (Alexa Fluor 568-conjugated, red). At 12 and 18 hpi (A), only colored merged images are shown under normal and penicillin-induced persistence culture conditions. At 24 and 48 hpi (B), single channel images are shown in black and white (2 left-most columns), while merged images and insets thereof are shown in color (2 right-most columns). Staining patterns at 32 hpi (not shown) were similar to those at 48 hpi. Bar = 2 μm for the 12 and 18 hpi times, and 24 and 48 hpi insets (i.e. top row and right-most column) and 10 μm for all remaining images.</p