Paradigms Regained : the Uses of Illuminative, Semiotic and Post-Modern Criticism as Modes of Inquiry in Educational Technology /

Glossaire: p. 513-519Comprend des bibliogr.Index: p. 521-54

Correction: Polarized Cell Division of Chlamydia trachomatis.

[This corrects the article DOI: 10.1371/journal.ppat.1005822.]

Polarized Cell Division of <i>Chlamydia trachomatis</i>

<div><p>Bacterial cell division predominantly occurs by a highly conserved process, termed binary fission, that requires the bacterial homologue of tubulin, FtsZ. Other mechanisms of bacterial cell division that are independent of FtsZ are rare. Although the obligate intracellular human pathogen <i>Chlamydia trachomatis</i>, the leading bacterial cause of sexually transmitted infections and trachoma, lacks FtsZ, it has been assumed to divide by binary fission. We show here that <i>Chlamydia</i> divides by a polarized cell division process similar to the budding process of a subset of the Planctomycetes that also lack FtsZ. Prior to cell division, the major outer-membrane protein of <i>Chlamydia</i> is restricted to one pole of the cell, and the nascent daughter cell emerges from this pole by an asymmetric expansion of the membrane. Components of the chlamydial cell division machinery accumulate at the site of polar growth prior to the initiation of asymmetric membrane expansion and inhibitors that disrupt the polarity of <i>C</i>. <i>trachomatis</i> prevent cell division. The polarized cell division of <i>C</i>. <i>trachomatis</i> is the result of the unipolar growth and FtsZ-independent fission of this coccoid organism. This mechanism of cell division has not been documented in other human bacterial pathogens suggesting the potential for developing <i>Chlamydia</i>-specific therapeutic treatments.</p></div

Inhibitors of membrane biosynthesis, peptidoglycan biosynthesis, and MreB prevent the polarized cell division of <i>Chlamydia</i>.

<p>HeLa cells were infected with <i>C</i>. <i>trachomatis</i> serovar L2. At 11 hours post-infection, AFN1252 (A), glyburide (B), or A22 (C) was added to the cells and the cells were subsequently fixed at 16 hours post-infection. (D) Penicillin or carbenicillin was added to infected HeLa cells at 10 hours post-infection and the cells were fixed at 12 hours post-infection, or (F) carbenicillin was added to infected HeLa cells at 18 hours post-infection and the cells were fixed 40 minutes later. In each instance, the cells were permeabilized and incubated with rabbit antibodies against Hsp60 (blue) and goat antibodies against MOMP (green) followed by donkey anti rabbit IgG conjugated to Alexa Fluor 633 and donkey anti-goat IgG conjugated to Alexa Fluor 488. In some instances, the cells were stained with Hoechst 33342 (red) prior to microscopic analysis (A—C and F). Two cells illustrating the effect of AFN1252 (A), glyburide (B) and A22 (C) on the polarized cell division process are shown. The cells in A—C are representative of 100 cells that were analyzed from two independent experiments. Panel D illustrates the various intermediates in polarized cell division observed in penicillin-treated or carbenicillin-treated cells. Asterisks indicate polar (A and B) and diffuse (C) Hsp60. Arrows in F point to polarized cell division intermediates observed following carbenicillin treatment of a more mature inclusion containing multiple cells. The asterisk in F indicates a cell that has almost completed division. Images in A-C and F were acquired by confocal microscopy; images in D were acquired by epifluorescent microscopy. White bars are 0.5μ. (E) Penicillin or carbenicillin were added to infected HeLa cells at 10 hours post-infection and the cells were fixed at 12 hours post-infection. The percentage of <i>C</i>. <i>trachomatis</i> that were undergoing polarized cell division (black bars) or had completed the first division (white bars) were quantified. All of the MOMP and Hsp60 double-positive cells in randomly selected fields were included in the analysis. The data shown represents the analysis of more than 200 cells from two independent experiments.</p

Characterization of the polarized division process of <i>Chlamydia</i> in more mature inclusions.

<p>HeLa cells were infected with C. trachomatis and fixed at 14 hours post-infection. The cells were then permeabilized and incubated with anti-MOMP goat polyclonal antibodies (green) and anti-IncG rabbit polyclonal antibodies (red) followed by donkey anti-goat IgG conjugated to Alexa Fluor 488 and donkey anti-rabbit IgG conjugated to Alexa Fluor 568 secondary antibodies (A and B). The cells were then washed and stained with Hoechst 33342 (blue) prior to confocal analysis. The images in B are consecutive confocal slices from a z-stack. Arrowheads in A and B point to sites of asymmetric membrane expansion. Asterisk in B indicates a cell simultaneously undergoing polar growth from two sites. The white bars are 0.5μ. The numbers in the merged images in B correspond to the position in the Z-stack in microns.</p

Outer membrane and cytosolic markers are polarized in <i>C</i>. <i>trachomatis</i> serovar L2.

<p>HeLa cells infected with <i>C</i>. <i>trachomatis</i> were fixed at 2, 4, 8 (A and B), or 10 (C) hours post-infection (hpi). Following fixation, the cells were permeabilized and LPS localization was determined by incubating cells with anti-LPS mouse monoclonal antibodies followed by donkey anti-mouse IgG conjugated to Alexa Fluor 568 (A; red), Hsp60 localization was determined using anti-Hsp60 rabbit polyclonal antibodies followed by donkey anti-rabbit IgG conjugated to Alexa Fluor 633 (A, B, and C; blue), and MOMP localization was determined using anti-MOMP goat polyclonal antibodies followed by donkey anti-goat IgG conjugated to Alexa Fluor 488 (A, B, and C; green). Following the antibody incubations, the cells were imaged by confocal microscopy. (B and C) DNA was visualized by staining with Hoechst 33342 (red). White bars are 0.5μ.</p