Analysis of polymorphic membrane protein expression in cultured cells Identifies PmpA and PmpH of Chlamydia psittaci as candidate factors in pathogenesis and immunity to infection

The polymorphic membrane protein (Pmp) paralogous families of Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia abortus are putative targets for Chlamydia vaccine development. To determine whether this is also the case for Pmp family members of C. psittaci, we analyzed transcription levels, protein production and localization of several Pmps of C. psittaci. Pmp expression profiles were characterized using quantitative real-time PCR (RT-qPCR), immunofluorescence (IF) and immuno-electron microscopy (IEM) under normal and stress conditions. We found that PmpA was highly produced in all inclusions as early as 12 hpi in all biological replicates. In addition, PmpA and PmpH appeared to be unusually accessible to antibody as determined by both immunofluorescence and immuno-electron microscopy. Our results suggest an important role for these Pmps in the pathogenesis of C. psittaci, and make them promising candidates in vaccine development

Castor bean organelle genome sequencing and worldwide genetic diversity analysis

Castor bean is an important oil-producing plant in the Euphorbiaceae family. Its high-quality oil contains up to 90% of the unusual fatty acid ricinoleate, which has many industrial and medical applications. Castor bean seeds also contain ricin, a highly toxic Type 2 ribosome-inactivating protein, which has gained relevance in recent years due to biosafety concerns. In order to gain knowledge on global genetic diversity in castor bean and to ultimately help the development of breeding and forensic tools, we carried out an extensive chloroplast sequence diversity analysis. Taking advantage of the recently published genome sequence of castor bean, we assembled the chloroplast and mitochondrion genomes extracting selected reads from the available whole genome shotgun reads. Using the chloroplast reference genome we used the methylation filtration technique to readily obtain draft genome sequences of 7 geographically and genetically diverse castor bean accessions. These sequence data were used to identify single nucleotide polymorphism markers and phylogenetic analysis resulted in the identification of two major clades that were not apparent in previous population genetic studies using genetic markers derived from nuclear DNA. Two distinct sub-clades could be defined within each major clade and large-scale genotyping of castor bean populations worldwide confirmed previously observed low levels of genetic diversity and showed a broad geographic distribution of each sub-clade

Whole-Genome Sequence of Chlamydia gallinacea Type Strain 08-1274/3

The recently introduced bacterial species Chlamydia gallinacea is known to occur in domestic poultry and other birds. Its potential as an avian pathogen and zoonotic agent is under investigation. The whole-genome sequence of its type strain, 08-1274/3, consists of a 1,059,583-bp chromosome with 914 protein-coding sequences (CDSs) and a plasmid (p1274) comprising 7,619 bp with 9 CDSs

Isolation of a new Chlamydia species from the feral Sacred Ibis (Threskiornis aethiopicus): Chlamydia ibidis

Investigations conducted on feral African Sacred Ibises (Threskiornis aethiopicus) in western France led to the isolation of a strain with chlamydial genetic determinants. Ultrastructural analysis, comparative sequence analysis of the 16S rRNA gene, ompA, and of a concatenate of 31 highly conserved genes, as well as determination of the whole genome sequence confirmed the relatedness of the new isolate to members of the Chlamydiaceae, while, at the same time demonstrating a unique position outside the currently recognized species of this family. We propose to name this new chlamydial species Chlamydia ibidis

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

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 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

<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