Elucidating the role of cholesterol and lipid rafts in host-pathogen interactions has been challenging. The majority of studies have relied on cholesterol sequestering agents and biosynthesis inhibitors to remove or deplete host cell cholesterol. Cholesterol sequestering agents such as methyl-ß-cyclodextrin physically remove cholesterol from membranes, in particular the plasma membrane. While this method efficiently depletes cholesterol, several concerns must be recognized. First, standard treatment of cells with methyl-ß-cyclodextrin (5–10 mM) removes up to 90% of the total cellular cholesterol, resulting in dramatic effects on cellular morphology and viability 
. Second, cells tightly regulate total cholesterol levels as well as concentrations across organelles. Removal of plasma membrane cholesterol can therefore alter cholesterol distribution throughout the cell and result in increased cholesterol synthesis and trafficking 
. Finally, cyclodextrins remove cholesterol from both lipid raft and non-lipid raft domains, and there is some evidence that non-cholesterol membrane components such as phospholipids are also extracted 
. As a result, membrane properties such as fluidity and protein distribution are drastically altered. Other agents used to interfere with cholesterol functions, such as filipin and nystatin, have similar negative effects on the host cell 
. Another approach to cholesterol depletion utilizes biosynthesis inhibitors, such as statins and U18668. While inhibitors can efficiently lower cholesterol levels, the approach suffers from two major problems. First, the majority of available inhibitors target enzymes early in the pathway. Consequently, the resulting decrease in all sterols makes it difficult to directly associate results with cholesterol depletion. Second, the majority of the inhibitors have pleotropic and/or off-target effects. For example, U18666A inhibits trafficking of LDL-derived cholesterol 
, de novo
cholesterol synthesis 
, and lipid organization of membranes by directly binding to membranes 
When adapted to serum-free media, DHCR24−/−
MEFs lack cholesterol but contain all of the upstream sterols. In the place of cholesterol these cells accumulate desmosterol, a sterol that differs from cholesterol by a single double bond at the carbon 24 position. Previous work in J774 murine macrophages, which also lack the DHCR24 enzyme, demonstrates that desmosterol can replace cholesterol in the regulation of cellular sterol homeostasis and proliferation 
. However, desmosterol does not functionally replace cholesterol in lipid rafts 
. Indeed, we found lipid raft-mediated uptake and signaling to be dysfunctional in −cholesterol MEFs.
Many studies have examined the role of cholesterol and lipid rafts in pathogen-host cell interactions. However, to our knowledge, these studies have all utilized methyl-ß-cyclodextrin and/or non-specific inhibitors. Our cholesterol-free cell system allowed examination of the importance of cholesterol in host cell colonization of three intracellular bacterial pathogens (C. burnetii, S. Typhimurium, and C. trachomatis) without the pleiotropic effects of these pharmaceutical agents.
Invasion of host cells by the obligate intracellular bacterium C. trachomatis
is promoted by cytosolic translocation of effector proteins via a T3SS 
. However, conflicting reports exist on the role of cholesterol in the invasion process. Jutras and colleagues 
found that C. trachomatis
associates with detergent-resistant plasma membranes, and that bacterial uptake is decreased by 80% in cells treated with methyl-ß-cyclodextrin. In contrast, Gabel et al.
saw no effect on entry when cells were treated with methyl-ß-cyclodextrin, filipin, or nystatin. Our results agree with the latter report, as we saw no difference in C. trachomatis
invasion of MEFs with or without cholesterol.
Once internalized, C. trachomatis
resides in a vacuole (or inclusion) that disconnects from the endocytic pathway and acquires characteristics of a Golgi-derived vesicle 
. Here, infectious EBs differentiate into metabolically active RBs. Several lines of evidence suggest a role for cholesterol in C. trachomatis
inclusion development and replication. Filipin labeling indicates that both the inclusion membrane and bacteria contain sterols 
, while HPLC analysis demonstrates the presence of cholesterol in the bacteria 
. Furthermore, sterol delivery appears to be Golgi-dependent 
. We found that cholesterol was not essential for productive infection by C. trachomatis
, as the number of infectious units at 48 hpi was identical between MEFs with or without cholesterol. However, we did observe a delay in the onset of the logarithmic phase of the organism's growth cycle, suggesting cholesterol is involved in RB to EB transition. This may reflect a defect in trafficking to the inclusion, perhaps resulting in a decrease in nutrients important for C. trachomatis
Our data also demonstrate that cholesterol is not essential for type III secretion and productive infection by S.
Typhimurium, a finding that directly contradicts previous reports 
. These disparate results may reflect different experimental approaches, with previous studies using methyl-ß-cyclodextrin for cholesterol depletion. As discussed earlier, this treatment alters the membrane properties of cells. Indeed, Garner et al.
observed that methyl-ß-cyclodextrin treated cells “exhibited a rounder morphology,” although they did not find a difference in cell viability. Hayward et al.
utilized in vitro
binding assays to demonstrate binding of the S.
Typhimurium T3SS protein SipB to cholesterol complexed with methyl-ß-cyclodextrin. However, other sterols, such as desmosterol, were not tested, nor was the ability of SipB to bind model membranes, a closer cellular mimic. By immunofluorescence, Hayward et al.
also showed defective secretion of the T3SS effector SopB in methyl-ß-cyclodextrin-treated cells. Using a more sensitive CyaA assay, we demonstrate here the cholesterol-independent translocation of the effectors SopB and SlrP.
Based on filipin staining, the S.
Typhimurium vacuole accumulates cholesterol during host cell infection 
. However, inhibitor studies suggest that non-sterol precursors of the cholesterol biosynthetic pathway are required for S.
Typhimurium intracellular growth, and that cholesterol itself is not essential 
. While these studies were not done in a truly cholesterol-free system (i.e.
, normal serum conditions were used), our data support the conclusion that cholesterol is not essential for S.
Typhimurium growth in host cells.
Unlike C. trachomatis
Typhimurium, C. burnetii
passively enters host cells through receptor-ligand interactions, triggering classical actin-dependent phagocytosis 
. C. burnetii
entry into cholesterol-free cells is significantly decreased, suggesting uptake occurs through cholesterol or lipid raft-mediated pathway. Our data using blocking antibodies and vitronectin demonstrate that αV
integrin is involved in entry into MEFs in a cholesterol-dependent manner. Furthermore, FAK, a key component of integrin signaling, is required for efficient C. burnetii
entry. Together, these data suggest that C. burnetii
utilizes lipid raft-mediated αV
integrin signaling to gain entry into host cells. Although significantly decreased, C. burnetii
entry into −cholesterol MEFs still occurs, suggesting the pathogen can also enter by non-lipid raft-associated receptors that function normally and/or by lipid raft-associated receptors that function inefficiently due to raft disruption.
Based on intense staining by the sterol-binding fluorophor filipin, we previously showed that the membrane of the mature C. burnetii
vacuole is sterol-rich 
. In the same study, inhibitors of host cell sterol biosynthesis and uptake inhibited C. burnetii
vacuole formation and growth 
. Here, we demonstrate that C. burnetii
vacuole formation and replication in −cholesterol MEFs is similar to +cholesterol MEFs. We conclude from these data that precursors of cholesterol, but not cholesterol per se
, are required for optimal infection by C. burnetii
. The C. burnetii
-occupied vacuole of −cholesterol MEFs does show a striking absence of CD63-positive membranous material that we speculate represents MVBs. Thus, trafficking to and fusion with the C. burnetii
vacuole of some vesicular compartments appears to depend on cholesterol, although these events are clearly not required for pathogen replication.
The C. burnetii
human DHCR24 homolog CBU1206 has sterol reductase activity when ectopically expressed in yeast 
. Thus, we postulated that CBU1206 activity during C. burnetii
infection of DHCR24−/−
MEFs might rescue the cholesterol-negative phenotype of these cells to result in enhanced pathogen growth. However, synthesis of cholesterol was not detected in infected cells; thus, the precise role of CBU1206 in C. burnetii
colonization of mammalian cells remains unresolved.
To our knowledge, this is the first study to address the role of cholesterol in host-pathogen interactions without the use of pleiotropic inhibitors or compounds that dramatically change membrane dynamics. While our results argue that cholesterol is not absolutely required for in vitro host cell colonization by three different intracellular pathogens, it does not eliminate the possibility that these pathogens target cholesterol and lipid rafts during in vivo infection, or that cholesterol is important under specific cellular conditions.