In the present study, a human genome-wide siRNA screen was performed to uncover host proteins required for key events in C. burnetii
infection. The siRNA screen validated the importance of several endocytic Rab proteins in the early stages of CCV biogenesis. Silencing of the genes encoding both Rab5 and Rab7 resulted in strong defects in C. burnetii
replication as indicated by a reduction in the number and size of vacuoles after host cell infection. Our results are consistent with previous data showing that perturbing Rab7 function abrogates formation of a vacuole that supports C. burnetii
). Similarly, silencing of components of the vacuolar ATPase required for acidification of endocytic compartments resulted in fewer and smaller CCVs, which is consistent with data showing that a mature acidified compartment is required for activation of C. burnetii
metabolism and replication (7
) and that preventing acidification of the CCV using bafilomycin A1 to inhibit the the V-ATPase function interferes with C. burnetii
). In addition to components of the V-ATPase, it was found that silencing of the genes encoding the proteins CLN3 and CLCN5 interfered with CCV biogenesis, which is likely due to the role these host proteins have in regulating pH in endolysosomal compartments (38
). These data not only validate the importance of endocytic maturation in the biogenesis of the CCV but also provide confidence that nonbiased genome-wide siRNA screens will identify new host determinants that are involved in the CCV maturation process.
There is evidence that C. burnetii
subverts the autophagy pathway during infection (9
). A large number of host genes are required for a functional autophagy system in mammalian cells, which means that there are a large number of targets that should have been identified in this siRNA screen if the autophagy pathway was critical for intracellular replication of C. burnetii
. Thus, it was somewhat unexpected that the screen did not identify a significant number of genes encoding proteins essential for host autophagy if these host factors are indeed required for C. burnetii
growth (see Table S1
in the supplemental material). This raises the possibility that host autophagy may not be essential for maturation of the CCV to a vacuole that supports replication and indicates that further research is needed to determine whether the association of autophagy proteins with the vacuole containing C. burnetii
has functional significance.
The proteins VPS29 and VPS35 were identified as factors important for C. burnetii
intracellular replication and were subsequently validated using individual siRNA molecules to reduce the levels of these core retromer components in host cells. The involvement of the retromer complex in C. burnetii
replication has not been demonstrated previously, indicating that the genome-wide screen was successful at identifying a new host determinant important for C. burnetii
infection. Because the silencing of genes encoding VPS29 and VPS35 did not affect the uptake of C. burnetii
, the defect observed in replication indicates the CCVs were not progressing through the maturation pathway successfully. The retromer complex is involved primarily in selecting cargo proteins that are sorted by a retrograde pathway from endosomes to the Golgi apparatus. Host receptors sorted by the retromer include proteins such as the cation-independent mannose-6-phosphate receptor, which is involved in delivery of acid hydrolases to lysosomes (40
). Thus, it is possible that perturbing this membrane transport pathway may interfere with the delivery of host factors into the lumen of the CCV that are needed for replication. Additionally, the retromer may be involved in removing proteins and membrane-associated determinants on the CCV that are detrimental to further maturation, which could delay progression of the CCV through the endocytic pathway.
Importantly, it was observed that disrupting VPS29 and VPS35 function interfered with the delivery of effector proteins into host cells by the C. burnetii
Dot/Icm system. Effector protein delivery could be similarly blocked when vacuole biogenesis was perturbed by interfering with Rab7 function or by interfering with vacuole acidification. Given that the Dot/Icm system is essential for C. burnetii
), the defect in C. burnetii
replication observed when retromer function is blocked may in large part be due to the vacuole not providing an environment that enables the Dot/Icm system to efficiently translocate effector proteins.
There is evidence that Rab7 is important for regulating the function of the retromer on endosomal membranes (41
). Thus, Rab7 acquisition may represent a stage that precedes a retromer-dependent step in CCV maturation and suggests that one possible role for Rab7 function in CCV maturation is to promote retromer-mediated membrane-transport processes. Consistent with this hypothesis, the number of intracellular C. burnetii
bacteria that were found in LysoTracker-positive vacuoles in the mock-transfected cells was similar to the number seen in cells where VPS16 or the VPS29 retromer had been silenced, which would be consistent with these factors acting downstream of Rab7 and suggest that the defect in replication observed in these siRNA-silenced cells is not the result of a gross defect in vacuole acidification. It remains possible that defects in retromer function or VPS16 activity delay the kinetics of CCV maturation or result in subtle differences in the pH of these vacuoles and that these effects may delay effector translocation or replication.
The VPS26-VPS29-VPS35 trimer associates with sorting nexin proteins to function in retrograde transport of vesicular-bound transmembrane cargo from endosomes to the TGN (42
). In mammalian cells, there are at least two different sorting nexin dimers associated with the VPS complex. Both the SNX1-SNX2 and SNX5-SNX6 dimers are composed of sorting nexins containing a BAR domain that senses membrane curvature, which leads to the assembly of a retromer complex that generates membrane tubules that retrieve cargo proteins from endocytic organelles. The SNX3 protein also associates with the VPS complex but does not have a BAR domain and is involved in retrieval of unique cargo proteins such as the Wnt receptor (44
). Replication of C. burnetii
was reduced when the gene encoding SNX2, but not SNX1, was silenced by siRNA, indicating that SNX2 is involved in the important role the retromer has in CCV biogenesis. Although depletion of SNX1
had no detectable effect on C. burnetii
replication, this could reflect an inability to efficiently silence SNX1
or diminish SNX1 protein levels. Additionally, intracellular growth defects were detected after C. burnetii
infection in cells where genes encoding other retromer-associated sorting nexins were silenced. Silencing of these genes resulted in more-robust defects in the translocation of BlaM-77 by C. burnetii
during infection. Because the BlaM-77 translocation assay provides a quantitative measure for host processes that are important for early CCV maturation events, these data suggest that CCV biogenesis is retarded in the absence of SNX2, SNX3, SNX5, or SNX6. Thus, the strong intracellular growth defect observed in cells where the VPS complex is depleted likely results from interfering with multiple membrane transport pathways requiring different sorting nexins.
Lastly, VPS35 has also been shown to localize to mitochondrion-derived vesicles and thereby facilitate retromer-mediated vesicular transport of cargo between mitochondria and peroxisomes (45
). Although the function of VPS35 in this capacity has not been fully characterized, it is plausible that the retromer participates in communication between mitochondria and peroxisomes that may result in regulation of peroxisome biogenesis or function. Several C. burnetii
Dot/Icm effectors localize to mitochondria, including the CBU0077 effector protein (20
). The role of mitochondria in C. burnetii
pathogenesis is unknown; however, several molecules are shared within the mitochondria and endocytic networks (46
), suggesting a similar overlap with C. burnetii
transport. Thus, the retromer may function during C. burnetii
infection both at the level of membrane sorting at the CCV and also through maintaining important membrane transport pathways at other locations that are indirectly important for C. burnetii
In addition to identifying genes encoding host proteins important for early events in CCV maturation that enable intracellular replication, the genome-wide screen identified phenotypes that included larger or a greater number of vacuoles containing C. burnetii in the infected host cells. Silencing of the gene encoding syntaxin-17 resulted in one of the more interesting phenotypes in this category, which was defined as infected cells having a greater number of CCVs. It was determined that this phenotype was not due to a higher rate of infection, although even at a high multiplicity of infection, it is unusual to find infected cells that have multiple CCVs because of the high degree of homotypic fusion observed between CCVs in the same cell, which ultimately stimulates the coalescing of all CCVs to form one large vacuole. These data suggest that syntaxin-17 has a role in regulating the kinetics of homotypic fusion.
The syntaxin-17 protein is a divergent member of the SNARE family of proteins. SNARE proteins are involved in the fusion of intracellular vesicles. Identified in a yeast two-hybrid screen as a potential syntaxin-3 binding partner (34
), the syntaxin-17 protein is found localized to the endoplasmic reticulum (ER) (47
) and on vesicles that cycle between the ER and the ER-Golgi intermediate compartment (48
). Importantly, this defect in homotypic fusion of CCVs was specific for syntaxin-17, as the phenotype was not observed in cells after silencing of the gene encoding syntaxin-18, which is a related ER-localized SNARE protein. Expression of syntaxin-17 in C. burnetii
-infected HeLa cells reveals staining that overlaps with calnexin-positive structures indicative of ER; however, there is no detectable localization of syntaxin-17 to the CCV. This suggests that a syntaxin-17-dependent transport pathway or some other activity regulated by syntaxin-17 is needed for homotypic fusion of the CCV but that syntaxin-17 itself may not be directly involved in the homotypic fusion reaction. Because reducing syntaxin-17 levels in host cells did not affect either C. burnetii
replication or translocation of BlaM-77, which is in contrast to what is observed when Rab7 or components of the retromer were reduced in cells, these data indicate that homotypic fusion occurs only after a mature vacuole is established that contains metabolically active C. burnetii
capable of translocating effectors using the Dot/Icm system.
Finally, the genome-wide siRNA screen identified the class C vacuolar-protein-sorting protein VPS16 as being required for C. burnetii
replication in HeLa cells, similar to the phenotypes seen for Rab7 and VPS35. However, unlike the Rab7 and retromer knockdowns, there was no corresponding defect observed in translocation of BlaM fusions to three different C. burnetii
effectors. Based on homology to the yeast class C Vps protein, mammalian VPS16 is implicated in vesicle transport within the endolysosomal pathway and may mediate membrane docking and fusion events (49
). As with syntaxin-17, the data for VPS16 indicate that this protein functions only after a mature vacuole is established and therefore may have an important role in the C. burnetii
vacuole biogenesis after effector translocation has occurred.
In conclusion, these data reveal that genome-wide siRNA screening can be used to identify host pathways important for distinct stages of C. burnetii
intracellular infection. With the recent developments in genetic manipulation and axenic cultivation of C. burnetii
), it should be possible to design new screening strategies that utilize C. burnetii
producing fluorescent proteins or BlaM-effector fusions and to monitor infection over time after siRNA treatment to identify pathways important for infection, replication, effector translocation, and vacuole expansion.