On the use of Legionella/Rickettsia chimeras to investigate the structure and regulation of Rickettsia effector RalF

Folly-Klan, Marcia; Sancerne, B.; Alix, Eric; Roy, Craig R.; Cherfils, Jacqueline; Campanacci, Valérie

doi:10.1016/j.jsb.2014.12.001

Cited by 8 publications

(6 citation statements)

References 19 publications

(43 reference statements)

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“…Several L. pneumophila effectors have been found to have enzymatic activities that are regulated through autoinhibition. The GEF activity of RalF is autoinhibited by a C-terminal domain that functions as a cap that senses host membranes (48)(49)(50), and VipD is a phospholipase that is autoinhibited by an amino-terminal domain that binds to the host protein Rab5 (51)(52)(53). Thus, future studies on the mechanism by which SidJ regulates SidE function could reveal additional strategies by which the activities of L. pneumophila effectors are controlled spatially and temporally during infection.…”

Section: Discussionmentioning

confidence: 99%

Toxicity and SidJ-Mediated Suppression of Toxicity Require Distinct Regions in the SidE Family of Legionella pneumophila Effectors

Havey

Roy

2015

Infect Immun

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Intracellular bacteria use a variety of strategies to evade degradation and create a replicative niche. Legionella pneumophila is an intravacuolar pathogen that establishes a replicative niche through the secretion of more than 300 effector proteins. The function of most effectors remains to be determined. Toxicity in yeast has been used to identify functional domains and elucidate the biochemical function of effectors. A library of L. pneumophila effectors was screened using an expression plasmid that produces low levels of each protein. This screen identified the effector SdeA as a protein that confers a strong toxic phenotype that inhibits yeast replication. The toxicity of SdeA was suppressed in cells producing the effector SidJ. The effector SdeA is a member of the SidE family of L. pneumophila effector proteins. All SidE orthologs encoded by the Philadelphia isolate of Legionella pneumophila were toxic to yeast, and SidJ suppressed the toxicity of each. We identified a conserved central region in the SidE proteins that was sufficient to mediate yeast toxicity. Surprisingly, SidJ did not suppress toxicity when this central region was produced in yeast. We determined that the amino-terminal region of SidE was essential for SidJ-mediated suppression of toxicity. Thus, there is a genetic interaction that links the activity of SidJ and the amino-terminal region of SidE, which is required to modulate the toxic activity displayed by the central region of the SidE protein. This suggests a complex mechanism by which the L. pneumophila effector SidJ modulates the function of the SidE proteins after translocation into host cells.L egionella pneumophila is a Gram-negative, facultative intravacuolar pathogen that is the causative agent of Legionnaires' disease in humans (1-3). L. pneumophila is phagocytosed by host cells and creates a vacuole that supports replication by inhibiting the fusion of host endocytic vesicles and subverting the transport of secretory vesicles (4-7). The bacterially encoded Dot/Icm type IV secretion system (T4SS) is essential for the creation of this Legionella-containing vacuole (LCV) (8, 9). Proteins secreted by the T4SS, known as effectors, have been shown to both decorate the LCV and modify the location and function of host proteins, especially Rab GTPases (8,10,11). Determining the function of these effector proteins has been complicated by the observation that deletion of a single effector or groups of effectors does not typically result in a strong replication defect within host cells (12)(13)(14).The use of the yeast Saccharomyces cerevisiae as a system to study L. pneumophila effector proteins has been successful in previous studies (15)(16)(17)(18)(19). Examples include studies on the effector protein RalF, which was shown to inhibit yeast replication when overproduced, and the effector proteins YlfA and YlfB were both identified in an early yeast toxicity screen (16). Effector toxicity in yeast has also been used to study effector activities. The L. pneumophila effector AnkX is capa...

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Section: Discussionmentioning

confidence: 99%

Toxicity and SidJ-Mediated Suppression of Toxicity Require Distinct Regions in the SidE Family of Legionella pneumophila Effectors

Havey

Roy

2015

Infect Immun

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“…RalF proteins from L. pneumophila (LpRalF) and R. prowazekii (RpRalF) and their isolated Sec7 domains were produced and purified as described in refs and . Human Rac1 and the GEF domain of mouse Dock5 were obtained as described in ref .…”

Section: Methodsmentioning

confidence: 99%

Family-wide Analysis of the Inhibition of Arf Guanine Nucleotide Exchange Factors with Small Molecules: Evidence of Unique Inhibitory Profiles

et al. 2017

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Arf GTPases and their guanine nucleotide exchange factors (ArfGEFs) are major regulators of membrane traffic and organelle structure in cells. They are associated with a variety of diseases and are thus attractive therapeutic targets for inhibition by small molecules. Several inhibitors of unrelated chemical structures have been discovered, which have shown their potential in dissecting molecular pathways and blocking disease-related functions. However, their specificity across the ArfGEF family has remained elusive. Importantly, inhibitory responses in the context of membranes, which are critical determinants of Arf and ArfGEF cellular functions, have not been investigated. Here, we compare the efficiency and specificity of four structurally distinct ArfGEF inhibitors, Brefeldin A, SecinH3, M-COPA, and NAV-2729, toward six ArfGEFs (human ARNO, EFA6, BIG1, and BRAG2 and Legionella and Rickettsia RalF). Inhibition was assessed by fluorescence kinetics using pure proteins, and its modulation by membranes was determined with lipidated GTPases in the presence of liposomes. Our analysis shows that despite the intra-ArfGEF family resemblance, each inhibitor has a specific inhibitory profile. Notably, M-COPA is a potent pan-ArfGEF inhibitor, and NAV-2729 inhibits all GEFs, the strongest effects being against BRAG2 and Arf1. Furthermore, the presence of the membrane-binding domain in Legionella RalF reveals a strong inhibitory effect of BFA that is not measured on its GEF domain alone. This study demonstrates the value of family-wide assays with incorporation of membranes, and it should enable accurate dissection of Arf pathways by these inhibitors to best guide their use and development as therapeutic agents.

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“…65 The structure of Legionella RalF showed that the Sec7 domain is strongly autoinhibited by a C-terminal domain that blocks access to the Arf-binding site, 66 and a similar autoinhibited conformation was observed in the Rickettsia RalF homolog. 67 Both bacterial ArfGEFs are strongly activated by membranes, and the membrane-binding site is identical to the elements in the autoinhibitory domain that blocks the Arf-binding site. 68,69 Thus, activation of Arf GTPases by RalF strictly depends on its recruitment to membranes.…”

Section: Regulation Of Bacterial Arfgefs By Membranesmentioning

confidence: 99%

Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

2016

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Arf GTPases assemble protein complexes on membranes to carry out major functions in cellular traffic. An essential step is their activation by guanine nucleotide exchange factors (GEFs), whose Sec7 domain stimulates GDP/GTP exchange. ArfGEFs form 2 major families: ArfGEFs with DCB, HUS and HDS domains (GBF1 and BIG1/BIG2 in humans), which act at the Golgi; and ArfGEFs with a Cterminal PH domain (cytohesin, EFA6 and BRAG), which function at the plasma membrane and endosomes. In addition, pathogenic bacteria encode an ArfGEF with a unique membrane-binding domain. Here we review the allosteric regulation of Arf GTPases and their GEFs at the membrane interface. Membranes contribute several regulatory layers: at the GTPase level, where activation by GTP is coupled to membrane recruitment by a built-in structural device; at the Sec7 domain, which manipulates this device to ensure that Arf-GTP is attached to membranes; and at the level of noncatalytic ArfGEF domains, which form direct or GTPase-mediated interactions with membranes that enable a spectacular diversity of regulatory regimes. Notably, we show here that membranes increase the efficiency of a large ArfGEF (human BIG1) by 32-fold by interacting directly with its Nterminal DCB and HUS domains. The diversity of allosteric regulatory regimes suggests that ArfGEFs can function in cascades and circuits to modulate the shape, amplitude and duration of Arf signals in cells. Because Arf-like GTPases feature autoinhibitory elements similar to those of Arf GTPases, we propose that their activation also requires allosteric interactions of these elements with membranes or other proteins.

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On the use of Legionella/Rickettsia chimeras to investigate the structure and regulation of Rickettsia effector RalF

Cited by 8 publications

References 19 publications

Toxicity and SidJ-Mediated Suppression of Toxicity Require Distinct Regions in the SidE Family of Legionella pneumophila Effectors

Toxicity and SidJ-Mediated Suppression of Toxicity Require Distinct Regions in the SidE Family of Legionella pneumophila Effectors

Family-wide Analysis of the Inhibition of Arf Guanine Nucleotide Exchange Factors with Small Molecules: Evidence of Unique Inhibitory Profiles

Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

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