<p>The <italic>Chlamydiaceae</italic> are Gram-negative, obligate intracellular bacteria that are significant pathogens of humans and animals. Intracellularly, the bacteria reside in a membrane-bound vacuole, called the inclusion, from which they manipulate host processes to create a niche optimal for survival and propagation. Acquisition of host-derived lipids is essential for chlamydial growth, yet the source of lipids and mechanisms of trafficking to the inclusion are not well-established. The inclusion avoids interaction with several classical membrane and lipid transport pathways. In a functional genomic screen to identify host modulating chlamydial proteins, our lab identified cytosolic lipid droplets (LDs) as potential target organelles of <italic>Chlamydia</italic>. LDs are postulated to function in many cellular processes, such as lipid metabolism and transport, membrane trafficking, and cell signaling; therefore, we hypothesized that LDs may be important for <italic>Chlamydia</italic> pathogenesis as a source of lipids or as a platform for regulating other cellular functions. Here, we characterize the interaction between eukaryotic LDs and the chlamydial inclusion.</p><p> We find that LDs are recruited to the <italic>Chlamydia</italic> inclusion, chlamydial infection disrupts neutral lipid homeostasis, and pharmacological prevention of LD formation inhibits chlamydial replication. <italic>Chlamydia</italic> produces proteins (Ldas) that localize with LDs in yeast and mammalian cells when transiently expressed and are exported out of the inclusion to peripheral lipid-rich structures during infection. By electron microscopy and live cell imaging, we observe the translocation of intact LDs into the <italic>Chlamydia</italic> inclusion lumen. Biochemical and microscopic analysis of LDs from infected cells reveals that LD translocation may occur at specialized subregions of the inclusion membrane. The <italic>Chlamydia</italic> Lda3 protein is implicated in LD tethering to the inclusion membrane, and displacement of the protective coat protein, ADRP, from LD surfaces. This phenomenon could provide access for lipases to the LD core for utilization by the replicating bacteria. Additionally, the functional domains of Lda3 involved in binding to LD and inclusion membranes are identified. </p><p> In these studies, we identify eukaryotic lipid droplets (LDs) as a novel target organelle important for <italic>Chlamydia</italic> pathogenesis and describe a unique mechanism of whole organelle sequestration not previously observed for bacterial pathogens. These results represent a fundamental shift in our understanding of host interactions with the chlamydial inclusion, and may represent a new area for research in the field of cellular microbiology.</p> / Dissertation

Confinement of the obligate intracellular bacterium Chlamydia trachomatis to a membrane-bound vacuole, termed an inclusion, within infected epithelial cells neither prevents secretion of chlamydial antigens into the host cytosol nor protects chlamydiae from innate immune detection. However, the details leading to chlamydial antigen presentation are not clear. By immunoelectron microscopy of infected endometrial epithelial cells and in isolated cell secretory compartments, chlamydial major outer membrane protein (MOMP), lipopolysaccharide (LPS) and the inclusion membrane protein A (IncA) were localized to the endoplasmic reticulum (ER) and co-localized with multiple ER markers, but not with markers of the endosomes, lysosomes, Golgi nor mitochondria. Chlamydial LPS was also co-localized with CD1d in the ER. Since the chlamydial antigens, contained in everted inclusion membrane vesicles, were found within the host cell ER, these data raise additional implications for antigen processing by infected uterine epithelial cells for classical and non-classical T cell antigen presentation.

Several chlamydial antigens have been detected in the infected epithelial cell cytosol and on the host cell surface prior to their presumed natural release at the end of the 72-96 h developmental cycle. These extra-inclusion antigens are proposed to influence vital host cell functions, antigen trafficking and presentation and, ultimately, contribute to a prolonged inflammatory response. To begin to dissect the mechanisms for escape of these antigens from the chlamydial inclusion, which are enhanced on exposure to antibiotics, polarized endometrial epithelial cells (HEC-1B) were infected with Chlamydia trachomatis serovar E for 36 h or 48 h. Infected cells were then exposed to chemotactic human polymorphonuclear neutrophils not loaded or pre-loaded in vitro with the antibiotic azithromycin. Viewed by electron microscopy, the azithromycin-mediated killing of chlamydiae involved an increase in chlamydial outer membrane blebbing followed by the appearance of the blebs in larger vesicles (i) everting from but still associated with the inclusion as well as (ii) external to the inclusion. Evidence that the vesicles originated from the chlamydial inclusion membrane was shown by immuno-localization of inclusion membrane proteins A, F, and G on the vesicular membranes. Chlamydial heat shock protein 60 (chsp60) copies 2 and 3, but not copy 1, were released from RB and incorporated into the everted inclusion membrane vesicles and delivered to the infected cell surface. These data represent direct evidence for one mechanism of early antigen delivery, albeit membrane-bound, beyond the confines of the chlamydial inclusion.

Chlamydia trachomatis serovars D-K are the leading cause of bacterially-acquired sexually transmitted infections in the United States. As an obligate intracellular pathogen, C. trachomatis infects columnar epithelial cells of the genital mucosae and can cause deleterious sequelae such as pelvic inflammatory disease, infertility, and ectopic pregnancy. Several chlamydial antigens reach the host cell cytosol prior to the natural release of chlamydiae at the end of the developmental cycle. While some of these extra-inclusion antigens traffic to the host cell surface, others remain intracellular where they are proposed to influence vital host cell functions and antigen trafficking and presentation. The research herein examines the escape and trafficking of the immunodominant chlamydial antigens MOMP, LPS, and cHsp60 within C. trachomatis serovar E-infected polarized human endometrial epithelial cells. Studies using high-resolution transmission electron microscopy (TEM) and immuno-TEM report the novel escape mechanism of chlamydial antigens via vesicles everted/pinched off from the inclusion membrane, an occurrence observed both in the presence and absence of the antibiotic azithromycin. These extra-inclusion vesicles were differentiated from Golgi vesicles and were shown to deliver chlamydial heat shock protein 60 (cHsp60)-homologs 2 and 3, but not homolog 1, to the infected cell surface. Examination of the iron-responsiveness of the three cHsp60 homologs by immuno-TEM revealed a significant increase in cHsp60-2 following iron deprivation. Further investigation of the trafficking of chlamydial MOMP and LPS antigens enveloped within the protective everted inclusion membrane vesicles within host cells involved density gradient centrifugation for the separation of epithelial secretory pathway components followed by SDS-PAGE and Western blot to determine whether the chlamydial antigen-containing vesicles could fuse with and deliver the antigens to host cell organelles. Coupled with immuno-TEM, these data confirmed the presence of major chlamydial antigens within the endoplasmic reticulum of infected host cells. Additionally, chlamydial lipopolysaccharide (LPS) was co-localized with CD1d, a lipid antigen-presenting molecule. Collectively, these studies (i) establish a novel escape mechanism for chlamydial antigens, (ii) identify cHsp60-2 as a marker of iron stress response in C. trachomatis, and (iii) define for the first time the host cell ER as a destination for selected chlamydial antigens during infection.