||<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>