Legionella RavZ Plays a Role in Preventing Ubiquitin Recruitment to Bacteria-Containing Vacuoles.

Bacterial pathogens like Salmonella and Legionella establish intracellular niches in host cells known as bacteria-containing vacuoles. In these vacuoles, bacteria can survive and replicate. Ubiquitin-dependent selective autophagy is a host defense mechanism to counteract infection by invading pathogens. The Legionella effector protein RavZ interferes with autophagy by irreversibly deconjugating LC3, an autophagy-related ubiquitin-like protein, from a phosphoglycolipid phosphatidylethanolamine. Using a co-infection system with Salmonella, we show here that Legionella RavZ interferes with ubiquitin recruitment to the Salmonella-containing vacuoles. The inhibitory activity is dependent on the same catalytic residue of RavZ that is involved in LC3 deconjugation. In semi-permeabilized cells infected with Salmonella, external addition of purified RavZ protein, but not of its catalytic mutant, induced removal of ubiquitin associated with Salmonella-containing vacuoles. The RavZ-mediated restriction of ubiquitin recruitment to Salmonella-containing vacuoles took place in the absence of the host system required for LC3 conjugation. These observations suggest the possibility that the targets of RavZ deconjugation activity include not only LC3, but also ubiquitin.

Bacterial secretion system skews the fate of Legionella-containing vacuoles towards LC3-associated phagocytosis.

The evolutionarily conserved processes of endosome-lysosome maturation and macroautophagy are established mechanisms that limit survival of intracellular bacteria. Similarly, another emerging mechanism is LC3-associated phagocytosis (LAP). Here we report that an intracellular vacuolar pathogen, Legionella dumoffii, is specifically targeted by LAP over classical endocytic maturation and macroautophagy pathways. Upon infection, the majority of L. dumoffii resides in ER-like vacuoles and replicate within this niche, which involves inhibition of classical endosomal maturation. The establishment of the replicative niche requires the bacterial Dot/Icm type IV secretion system (T4SS). Intriguingly, the remaining subset of L. dumoffii transiently acquires LC3 to L. dumoffii-containing vacuoles in a Dot/Icm T4SS-dependent manner. The LC3-decorated vacuoles are bound by an apparently undamaged single membrane, and fail to associate with the molecules implicated in selective autophagy, such as ubiquitin or adaptors. The process requires toll-like receptor 2, Rubicon, diacylglycerol signaling and downstream NADPH oxidases, whereas ULK1 kinase is dispensable. Together, we have discovered an intracellular pathogen, the survival of which in infected cells is limited predominantly by LAP. The results suggest that L. dumoffii is a valuable model organism for examining the mechanistic details of LAP, particularly induced by bacterial infection.

Hijacking the host proteasome for the temporal degradation of bacterial effectors.

To establish infection, intracellular pathogens need to modulate host cellular processes. Modulation of host processes is achieved by the action of various "effector proteins" which are delivered from the bacteria to the host cell cytosol. In order to orchestrate host cell reprogramming, the function of effectors inside host cells is regulated both temporally and spatially. In eukaryotes one of the most prominent processes used to degrade proteins is the ubiquitin-proteasome system. Recently it has emerged that the intracellular pathogen Legionella pneumophila is able to achieve temporal regulation of an effector using the ubiquitin-proteasome system. After establishing its replicative niche, the L. pneumophila effector SidH is degraded by the host proteasome. Most remarkably another effector protein LubX is able to mimic the function of an eukaryotic E3 ubiquitin ligase and polyubiquitinates SidH, targeting it for degradation. In this paper we describe a method to detect the polyubiquitin-modified forms of SidH in vitro and in vivo. Analyzing the temporal profile of polyubiquitination and degradation of bacterial effectors aids towards our understanding of how bacteria hijack host systems.

The machinery at endoplasmic reticulum-plasma membrane contact sites contributes to spatial regulation of multiple Legionella effector proteins.

The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis.

Legionella pneumophila subversion of host vesicular transport by SidC effector proteins.

Tethering proteins play a key role in vesicular transport, ensuring that cargo arrives at a specific destination. The bacterial effector protein SidC and its paralog SdcA have been described as tethering factors encoded by the intracellular pathogen Legionella pneumophila. Here, we demonstrate that SidC proteins are important for early events unique to maturation of vacuoles containing Legionella and discover monoubiquitination of Rab1 as a new SidC-dependent activity. The crystal structure of the SidC N-terminus revealed a novel fold that is important for function and could be involved in Legionella adaptations to evolutionarily divergent host cells it encounters in natural environments.

Modulation of the ubiquitination machinery by Legionella.

The bacterial pathogen Legionella pneumophila manipulates its intracellular fate by co-opting host processes. Using bacterial proteins translocated into host cells, L. pneumophila targets pathways shared by unicellular protozoa and higher eukaryotes. In eukaryotes, an important mechanism that regulates numerous cellular processes, including those designed to kill invading microorganisms, is ubiquitination. Post-translational modification of proteins with ubiquitin is a highly regulated process that either targets proteins for degradation or modifies their activity. It is emerging that L. pneumophila possesses functional mimics of eukaryotic E3 ubiquitin ligases that function with the host ubiquitination machinery to select and modify substrates for polyubiquitination. L. pneumophila proteins have been identified that ubiquitinate both host and bacterial proteins, and ubiquitination of the bacterial protein SidH results in its degradation by the host proteasome. This pathway allows L. pneumophila to temporally regulate effector function inside host cells, and facilitates optimal L. pneumophila replication by undefined mechanisms. This review will focus on our current knowledge of the proteins used by L. pneumophila to co-opt the host ubiquitination machinery, and current progress toward understanding the ubiquitin-mediated processes manipulated by L. pneumophila to facilitate intracellular survival and propagation.

Conditional requirement for exopolysaccharide in the Mesorhizobium-Lotus symbiosis.

Rhizobial surface polysaccharides are required for nodule formation on the roots of at least some legumes but the mechanisms by which they act are yet to be determined. As a first step to investigate the function of exopolysaccharide (EPS) in the formation of determinate nodules, we isolated Mesorhizobium loti mutants affected in various steps of EPS biosynthesis and characterized their symbiotic phenotypes on two Lotus spp. The wild-type M. loti R7A produced both high molecular weight EPS and lower molecular weight (LMW) polysaccharide fractions whereas most mutant strains produced only LMW fractions. Mutants affected in predicted early biosynthetic steps (e.g., exoB) formed nitrogen-fixing nodules on Lotus corniculatus and L. japonicus 'Gifu', whereas mutants affected in mid or late biosynthetic steps (e.g., exoU) induced uninfected nodule primordia and, occasionally, a few infected nodules following a lengthy delay. These mutants were disrupted at the stage of infection thread (IT) development. Symbiotically defective EPS and Nod factor mutants functionally complemented each other in co-inoculation experiments. The majority of full-length IT observed harbored only the EPS mutant strain and did not show bacterial release, whereas the nitrogen-fixing nodules contained both mutants. Examination of the symbiotic proficiency of the exoU mutant on various L. japonicus ecotypes revealed that both host and environmental factors were linked to the requirement for EPS. These results reveal a complex function for M. loti EPS in determinate nodule formation and suggest that EPS plays a signaling role at the stages of both IT initiation and bacterial release.

Modulation of host cell function by Legionella pneumophila type IV effectors.

Macrophages and protozoa ingest bacteria by phagocytosis and destroy these microbes using a conserved pathway that mediates fusion of the phagosome with lysosomes. To survive within phagocytic host cells, bacterial pathogens have evolved a variety of strategies to avoid fusion with lysosomes. A virulence strategy used by the intracellular pathogen Legionella pneumophila is to manipulate host cellular processes using bacterial proteins that are delivered into the cytosolic compartment of the host cell by a specialized secretion system called Dot/Icm. The proteins delivered by the Dot/Icm system target host factors that play evolutionarily conserved roles in controlling membrane transport in eukaryotic cells, which enables L. pneumophila to create an endoplasmic reticulum-like vacuole that supports intracellular replication in both protozoan and mammalian host cells. This review focuses on intracellular trafficking of L. pneumophila and describes how bacterial proteins contribute to modulation of host processes required for survival within host cells.

The Anaplasma phagocytophilum-occupied vacuole selectively recruits Rab-GTPases that are predominantly associated with recycling endosomes.

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to reside within a host cell-derived vacuole. The A. phagocytophilum-occupied vacuole (ApV) fails to mature along the endocytic pathway and is non-fusogenic with lysosomes. Rab GTPases regulate membrane traffic. To better understand how the bacterium modulates the ApV's selective fusogencity, we examined the intracellular localization of 20 green fluorescent protein (GFP) or red fluorescent protein (RFP)-tagged Rab GTPases in A. phagocytophilum-infected HL-60 cells. GFP-Rab4A, GFP-Rab10, GFP-Rab11A, GFP-Rab14, RFP-Rab22A and GFP-Rab35, which regulate endocytic recycling, and GFP-Rab1, which mediates endoplasmic reticulum to Golgi apparatus trafficking, localize to the ApV. Fluorescently tagged Rabs are recruited to the ApV upon its formation and remain associated throughout infection. Endogenous Rab14 localizes to the ApV. Tetracycline treatment concomitantly promotes loss of recycling endosome-associated GFP-Rabs and acquisition of GFP-Rab5, GFP-Rab7, and the lysosomal marker, LAMP-1. Wild-type and GTPase- deficient versions, but not GDP-restricted versions of GFP-Rab1, GFP-Rab4A and GFP-Rab11A, localize to the ApV. Strikingly, GFP-Rab10 recruitment to the ApV is guanine nucleotide-independent. These data establish that A. phagocytophilum selectively recruits Rab GTPases that are primarily associated with recycling endosomes to facilitate its intracellular survival and implicate bacterial proteins in regulating Rab10 membrane cycling on the ApV.

Symbiosis-induced cascade regulation of the Mesorhizobium loti R7A VirB/D4 type IV secretion system.

The Mesorhizobium loti R7A symbiosis island contains genes encoding a VirB/D4 type IV secretion system (T4SS) similar to that of Agrobacterium tumefaciens. This system has host-dependent effects on symbiosis that probably are due to translocation of two effector proteins, Msi059 and Msi061, into host cells. Here we report that, as in A. tumefaciens, the M. loti vir genes are transcriptionally regulated by a VirA/VirG two-component regulatory system. A virGN54D mutant gene of M. loti caused constitutive expression of lacZ reporter gene fusions to virB1, virD4, msi059, and msi061. Expression of these gene fusions also was activated by a NodD gene product from Rhizobium leguminosarum in the presence of the inducer naringenin, as was a virA::lacZ fusion. This activation was dependent on a nod box present 851 bp upstream of virA, and a mutant with the nod box deleted formed effective nodules on Leucaena leucocephala, the same symbiotic phenotype as other M. loti vir mutants. In contrast, the wild-type strain formed small, empty nodules whereas a nodD1 mutant was completely Nod-. These results indicate that the M. loti vir genes are induced in a symbiosis-specific manner that involves a two-tiered regulatory cascade, and that the vir effectors act after Nod factor during infection thread formation.

Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium loti strain R7A VirB/D4 type IV secretion system.

The symbiosis island of Mesorhizobium loti strain R7A contains genes with strong similarity to the structural vir genes (virB1-11; virD4) of Agrobacterium tumefaciens that encode the type IV secretion system (T4SS) required for T-DNA transfer to plants. In contrast, M. loti strain MAFF303099 lacks these genes but contains genes not present in strain R7A that encode a type III secretion system (T3SS). Here we show by hybridization analysis that most M. loti strains contain the VirB/D4 T4SS and not the T3SS. Strikingly, strain R7A vir gene mutants formed large nodules containing bacteroids on Leucaena leucocephala in contrast to the wild-type strain that formed only uninfected tumour-like structures. A rhcJ T3SS mutant of strain MAFF303099 also nodulated L. leucocephala, unlike the wild type. On Lotus corniculatus, the vir mutants were delayed in nodulation and were less competitive compared with the wild type. Two strain R7A genes, msi059 and msi061, were identified through their mutant phenotypes as possibly encoding translocated effector proteins. Both Msi059 and Msi061 were translocated through the A. tumefaciens VirB/D4 system into Saccharomyces cerevisiae and Arabidopsis thaliana, as shown using the Cre recombinase Reporter Assay for Translocation (CRAfT). Taken together, these results suggest that the VirB/D4 T4SS of M. loti R7A plays an analogous symbiotic role to that of T3SS found in other rhizobia. The heterologous translocation of rhizobial proteins by the Agrobacterium VirB/D4 T4SS is the first demonstration that rhizobial effector proteins are translocated into plant cells and confirms functional conservation between the M. loti and A. tumefaciens T4SS.