Microbial pathogens have developed a variety of strategies to infect the human host and cause disease. Many Gram-negative bacteria use type IV secretion systems (T4SSs) to deliver bacterial proteins, called effectors, into host cells. The effectors help to modulate signaling events within the host in order to create conditions supportive of bacterial growth. We are particularly interested in understanding the virulence mechanism of Legionella pneumophila, the causative agent of a life-threatening pneumonia called Legionnaires'disease. L. pneumophila is ubiquitously found in freshwater habitats such as water fountains, air conditioning systems, or shower heads. Consequently, humans are frequently exposed to this organism, with immune-compromised individuals or the elderly being at an elevated risk of contracting an infection. According to the Center for Disease Control and Prevention (CDC), the number of diagnosed Legionnaires'disease cases within the U.S.has doubled over the past decade, making this microorganism is an emerging public health threat. The primary host cell of L. pneumophila are alveolar macrophages, specialized immune cells within our lung. A crucial step in the elimination of invading microbes by macrophages is phagosomal maturation, a process where the bacteria-containing phagosome gradually fuses with endosomes and lysosomes, resulting in the acidification of its lumen and the degradation of its content. L. pneumophila bypasses the endosomal compartment by a yet unknown mechanism and proficiently replicates within the infected macrophage. Endolysosomal evasion relies on the delivery of almost 300 L. pneumophila effector proteins through the Dot/Icm T4SS into the host cytosol. We and others recently demonstrated that several of the effectors are involved in redirecting proteins and membrane material of the infected cell to the surface of the Legionella-containing vacuole, thereby establishing a protective compartment resembling host-cell endoplasmic reticulum (ER) (for review see Neunuebel &Machner (2012) Small GTPases). Our studies revealed a remarkable level of sophistication where L. pneumophila proteins with antagonistic activities are deployed in a precisely coordinated fashion in order to efficiently hijack host transport vesicles. Over the past funding period, we also investigated the molecular mechanisms underlying endolysosomal avoidance by intracellular L. pneumophila. Endosomal fusion is controlled by the host guanine nucleotide binding protein Rab5, which assembles protein complexes that include the tethering protein early endosomal antigen (EEA) 1 and the phosphatidylinositol (PI) 3-kinase hVps34. hVps34 generates PI(3)P, a phospholipid required for the assembly of EEA1 and other fusion factors on the endosome. Our studies revealed that the effector protein VipD from L. pneumophila exhibits phospholipase A1 (PLA1) activity that is activated only upon binding to endosomal Rab5 or Rab22. When produced within mammalian cells, VipD localizes to endosomes and catalyzes the removal of PI(3)P from endosomal membranes. EEA1 and other transport and fusion factors are consequently depleted from endosomes, rendering them fusion-incompetent. Consequently, we showed that during host cell infection, VipD reduces the contact of Legionella-containing vacuoles with the endosomal compartment and protects their surrounding vacuoles from acquiring Rab5. Thus, by catalyzing PI(3)P depletion in a Rab5-dependent manner, VipD specifically alters the protein composition of endosomes while leaving other host organelles unaffected. We also determined the crystal structure of VipD in complex with constitutively active Rab5. This collaborative effort uncovered how the phospholipase A1 (PLA1) activity of VipD is triggered upon binding to the host cell GTPase Rab5. A comparison of the complexed and uncomplexed form of VipD revealed that an active site-obstructing loop which originates from the C-terminal domain of VipD is repositioned upon Rab5 binding, thereby exposing the catalytic pocket within the N-terminal PLA1 domain. Substitution of individual amino acid residues located within the VipD-Rab5 interface prevented Rab5 binding and PLA1 activation, and caused a failure of VipD mutant proteins to target to Rab5-enriched endosomal structures within cells. In summary, these findings disclosed an unexpected mode of long-range allosteric regulation of the PLA1 activity of VipD and provide the basis for the development of novel therapeutic approaches that, rather than directly targeting the enzymes active site, specifically disturb the host factor-mediated activation process of VipD and related microbial phospholipases.
| Yu, Xiaobo; Noll, Rebecca R; Romero Dueñas, Barbara P et al. (2018) Legionella effector AnkX interacts with host nuclear protein PLEKHN1. BMC Microbiol 18:5 |
| Lin, Yi-Han; Lucas, María; Evans, Timothy R et al. (2018) RavN is a member of a previously unrecognized group of Legionella pneumophila E3 ubiquitin ligases. PLoS Pathog 14:e1006897 |
| Romano-Moreno, Miguel; Rojas, Adriana L; Williamson, Chad D et al. (2017) Molecular mechanism for the subversion of the retromer coat by the Legionella effector RidL. Proc Natl Acad Sci U S A 114:E11151-E11160 |
| Lin, Yi-Han; Machner, Matthias P (2017) Exploitation of the host cell ubiquitin machinery by microbial effector proteins. J Cell Sci 130:1985-1996 |
| Tang, Yanyang; Qiu, Ji; Machner, Matthias et al. (2017) Discovering Protein-Protein Interactions Using Nucleic Acid Programmable Protein Arrays. Curr Protoc Cell Biol 74:15.21.1-15.21.14 |
| Machner, Matthias P; Storz, Gisela (2016) Infection biology: Small RNA with a large impact. Nature 529:472-3 |
| Lin, Yi-Han; Doms, Alexandra G; Cheng, Eric et al. (2015) Host Cell-catalyzed S-Palmitoylation Mediates Golgi Targeting of the Legionella Ubiquitin Ligase GobX. J Biol Chem 290:25766-81 |
| Morrissette, Naomi S; Machner, Matthias P (2015) Ingenious strategies of microbial pathogens. Mol Biol Cell 26:1007 |
| Yu, Xiaobo; Decker, Kimberly B; Barker, Kristi et al. (2015) Host-pathogen interaction profiling using self-assembling human protein arrays. J Proteome Res 14:1920-36 |
| Lucas, María; Gaspar, Andrew H; Pallara, Chiara et al. (2014) Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5. Proc Natl Acad Sci U S A 111:E3514-23 |
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