The mechanistic basis of the membrane-permeabilizing activities of the virulence-associated protein A (VapA) from Rhodococcus equi.

Pathogenic Rhodococcus equi release the virulence-associated protein A (VapA) within macrophage phagosomes. VapA permeabilizes phagosome and lysosome membranes and reduces acidification of both compartments. Using biophysical techniques, we found that VapA interacts with model membranes in four steps: (i) binding, change of mechanical properties, (ii) formation of specific membrane domains, (iii) permeabilization within the domains, and (iv) pH-specific transformation of domains. Biosensor data revealed that VapA binds to membranes in one step at pH 6.5 and in two steps at pH 4.5 and decreases membrane fluidity. The integration of VapA into lipid monolayers was only significant at lateral pressures <20 mN m-1 indicating preferential incorporation into membrane regions with reduced integrity. Atomic force microscopy of lipid mono- and bilayers showed that VapA increased the surface heterogeneity of liquid disordered domains. Furthermore, VapA led to the formation of a new microstructured domain type and, at pH 4.5, to the formation of 5 nm high domains. VapA binding, its integration and lipid domain formation depended on lipid composition, pH, protein concentration and lateral membrane pressure. VapA-mediated permeabilization is clearly distinct from that caused by classical microbial pore formers and is a key contribution to the multiplication of Rhodococcus equi in phagosomes.

The N-terminal domain is required for cell surface localisation of VapA, a member of the Vap family of Rhodococcus equi virulence proteins.

Rhodococcus equi pneumonia is an important cause of mortality in foals worldwide. Virulent equine isolates harbour an 80-85kb virulence plasmid encoding six virulence-associated proteins (Vaps). VapA, the main virulence factor of this intracellular pathogen, is known to be a cell surface protein that creates an intracellular niche for R. equi growth. In contrast, VapC, VapD and VapE are secreted into the intracellular milieu. Although these Vaps share very high degree of sequence identity in the C-terminal domain, the N-terminal domain (N-domain) of VapA is distinct. It has been proposed that this domain plays a role in VapA surface localization but no direct experimental data provides support to such hypothesis. In this work, we employed R. equi 103S harbouring an unmarked deletion of vapA (R. equi ΔvapA) as the genetic background to express C-terminal Strep-tagged Vap-derivatives integrated in the chromosome. The surface localization of these proteins was assessed by flow cytometry using the THE2122;-NWSHPQFEK Tag FITC-antibody. We show that VapA is the only cell surface Vap encoded in the virulence plasmid. We present compelling evidence for the role of the N-terminal domain of VapA on cell surface localization using fusion proteins in which the N-domain of VapD was exchanged with the N-terminus of VapA. Lastly, using an N-terminally Strep-tagged VapA, we found that the N-terminus of VapA is exposed to the extracellular environment. Given the lack of a lipobox in VapA and the exposure of the N-terminal Strep-tag, it is possible that VapA localization on the cell surface is mediated by interactions between the N-domain and components of the cell surface. We discuss the implications of this work on the light of the recent discovery that soluble recombinant VapA added to the extracellular medium functionally complement the loss of VapA.

Biochemically Reconstituted Fusion of Phagosomes with Endosomes and Lysosomes.

Professional phagocytic cells, such as macrophages, ingest large particles into a specialized endocytic compartment, the phagosome, which eventually turns into a phagolysosome and degrades its contents. This phagosome "maturation" is governed by successive fusion of the phagosome with early sorting endosomes, late endosomes, and lysosomes. Further changes occur by fission of vesicles from the maturing phagosome and by on-and-off cycling of cytosolic proteins. We present here a detailed protocol which allows to reconstitute in a cell-free system the fusion events between phagosomes and the different endocytic compartments. This reconstitution can be used to define the identity of, and interplay between, key players of the fusion events.

Rab GTPase regulation of phagosome-lysosome fusion is bypassed in the presence of micromolar Ca2.

Several ATP- and cytosol-dependent fusion processes between membranes of the endocytic and exocytic pathways have been biochemically reconstituted. Here, we present a phagosome-lysosome fusion reaction that is driven by micromolar concentrations of Ca2+ in the absence of ATP and cytosol. Investigating classical fusion and Ca2+-driven fusion (CaFu) side-by-side in vitro, using the same membrane preparations, we show that CaFu is faster than standard fusion (StaFu), leads to larger fusion products and is not blocked by established inhibitors of StaFu. A Ca2+ concentration of ∼120 µM supports maximal membrane attachment, and 15 µM Ca2+ supports maximal membrane fusion, indicating that Ca2+ has both a membrane-binding activity and a fusion-promoting activity. StaFu and CaFu are inhibited by a mutant form of α-SNAP (NAPA) that does not support soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) activation, and both are inhibited by a mixture of the cytosolic domains of three cognate Q-SNARE proteins, demonstrating a role of SNAREs in Ca2+-driven membrane merger. CaFu is independent of the Ca2+-regulated proteins synaptotagmin-7, calmodulin, and annexins A2 and A7. We propose that CaFu corresponds to the last step of phagosome-lysosome fusion, when a raised Ca2+ concentration from the compartment lumen activates SNAREs for fusion.

Differential Effects of Rhodococcus equi Virulence-Associated Proteins on Macrophages and Artificial Lipid Membranes.

Virulence-associated protein A (VapA) of Rhodococcus equi is a pathogenicity factor required for the multiplication of virulent R. equi strains within spacious macrophage vacuoles. The production of VapA is characteristic for R. equi isolates from pneumonic foals. VapB and VapN proteins in R. equi isolates from infected pig (VapB) and cattle (VapN) have amino acid sequences very similar to VapA and consequently have been assumed to be its functional correlates. Using model membrane experiments, phagosome pH acidification analysis, lysosome size measurements, protein partitioning, and degradation assays, we provide support for the view that VapA and VapN promote intracellular multiplication of R. equi by neutralizing the pH of the R. equi-containing vacuole. VapB does not neutralize vacuole pH, is not as membrane active as VapA, and does not support intracellular multiplication. This study also shows that the size of the sometimes enormous R. equi-containing vacuoles or the partitioning of purified Vaps into organic phases are not features that have predictive value for virulence of R. equi, whereas the ability of Vaps to increase phagosome pH is coupled to virulence. IMPORTANCE Rhodococcus equi is a major cause of life-threatening pneumonia in foals and occasionally in immunocompromised persons. Virulence-associated protein A (VapA) promotes R. equi multiplication in lung macrophages, which are the major host cells during foal infection. In this study, we compare cellular, biochemical, and biophysical phenotypes associated with VapA to those of VapB (typically produced by isolates from pigs) or VapN (isolates from cattle). Our data support the hypothesis that only some Vaps support multiplication in macrophages by pH neutralization of the phagosomes that R. equi inhabit.

Consecutive functions of small GTPases guide HOPS-mediated tethering of late endosomes and lysosomes.

The transfer of endocytosed cargoes to lysosomes (LYSs) requires HOPS, a multiprotein complex that tethers late endosomes (LEs) to LYSs before fusion. Many proteins interact with HOPS on LEs/LYSs. However, it is not clear whether these HOPS interactors localize to LEs or LYSs or how they participate in tethering. Here, we biochemically characterized endosomes purified from untreated or experimentally manipulated cells to put HOPS and interacting proteins in order and to establish their functional interdependence. Our results assign Rab2a and Rab7 to LEs and Arl8 and BORC to LYSs and show that HOPS drives LE-LYS fusion by bridging late endosomal Rab2a with lysosomal BORC-anchored Arl8. We further show that Rab7 is absent from sites of HOPS-dependent tethering but promotes fusion by moving LEs toward LYSs via dynein. Thus, our study identifies the topology of the machinery for LE-LYS tethering and elucidates the role of different small GTPases in the process.

Specific preadaptations of Rhodococcus equi cooperate with its Virulence-associated protein A during macrophage infection.

Gram-positive Rhodococcus equi (Prescotella equi) is a lung pathogen of foals and immunocompromised humans. Intra-macrophage multiplication requires production of the bacterial Virulence-associated protein A (VapA) which is released into the phagosome lumen. VapA pH-neutralizes intracellular compartments allowing R. equi to multiply in an atypical macrophage phagolysosome. Here, we show that VapA does not support intra-macrophage growth of several other bacterial species demonstrating that only few bacteria have the specific preadaptations needed to profit from VapA. We show that the closest relative of R. equi, environmental Rhodococcus defluvii (Prescotella defluvii), does not multiply in macrophages at 37°C even when VapA is present because of its thermosensitivity but it does so once the infection temperature is lowered providing rare experimental evidence for 'thermal restriction'. Using growth experiments with isolated macrophage lysosomes and modified infection schemes we provide evidence that R. equi resists the attack by phagolysosome contents at low pH for several hours. During this time, R. equi produces and secretes VapA which enables it to grow at the expense of lysosome constituents. We present arguments that, under natural infection conditions, R. equi is VapA-less during the initial encounter with the host. This has important implications for vaccine development.

Laboratory Plasticware Induces Expression of a Bacterial Virulence Factor.

Pollution with microplastic has become a prime environmental concern. The various ways in which human-made polymers and microorganisms interact are little understood, and this is particularly true for microplastic and pathogenic microorganisms. Previous reports demonstrated that expression of central virulence-associated protein A (VapA) of the pathogenic bacterium Rhodococcus equi is shut off at 30°C, whereas it is strongly expressed at 37°C, a temperature which may serve as an intrahost cue. Here, we show that cultivation at 30°C in disposable plastic tubes increases mRNA levels of vapA 70-fold compared to growth in conventional glass tubes. Strong expression of vapA in plastic tubes does not seem to be caused by a compound leaching from plastic but rather by tube surface properties. Expression stimulation during growth in plastic is regulated by the R. equi transcription regulators VirR and VirS, indicating that plastic-induced vapA expression is (co)regulated through the canonical vapA expression pathway. Our observations have important implications for the future analysis and assessment of environmental microplastic contaminations in that they show that, in principle, contact of pathogens with environmental plastic can increase their virulence. IMPORTANCE Millions of tons small plastic pieces (microplastic) find their way into the environment every year. They pose digestive and toxicity problems to various life forms in soil, freshwater, and seawater. Additionally, microplastic offers an opportunity for microorganisms to attach and to become an important part of a "plastisphere community." The significance of our study lies in the documentation of a sharp increase in production of a central virulence factor by a bacterial pathogen when the bacterium is in touch with certain makes of plastic. Although this feature may not reflect an increased health risk in case of this particular soilborne pathogen, our data disclose a new facet of how microplastics can endanger life.

Conformational changes of loops highlight a potential binding site in Rhodococcus equi VapB.

Virulence-associated proteins (Vaps) contribute to the virulence of the pathogen Rhodococcus equi, but their mode of action has remained elusive. All Vaps share a conserved core of about 105 amino acids that folds into a compact eight-stranded antiparallel ß-barrel with a unique topology. At the top of the barrel, four loops connect the eight ß-strands. Previous Vap structures did not show concave surfaces that might serve as a ligand-binding site. Here, the structure of VapB in a new crystal form was determined at 1.71 Šresolution. The asymmetric unit contains two molecules. In one of them, the loop regions at the top of the barrel adopt a different conformation from other Vap structures. An outward movement of the loops results in the formation of a hydrophobic cavity that might act as a ligand-binding site. This lends further support to the hypothesis that the structural similarity between Vaps and avidins suggests a potential binding function for Vaps.

Structural Determination of a New Peptidolipid Family from Rhodococcus opacus and the Pathogen Rhodococcus equi by Multiple Stage Mass Spectrometry.

The cell walls of the genus Rhodococcus including the pathogenic bacterium Rhodococcus equi (R. equi) and biotechnologically important bacterium Rhodococcus opacus (R. opacus) contain an abundant peptidolipid (or termed lipopeptide) family whose structures have not been reported previously. Here, we describe a linear ion-trap multiple-stage mass spectrometric (LIT MSn) approach with high resolution mass spectrometry (HRMS), in conjunction with NMR spectroscopy, chemical reactions, and GC/MS analysis to define the structures of these compounds. We employed LIT MSn (n = 2-8) on the [M + Na]+ ion species to establish the peptide sequence, the identity of the fatty acyl substituent, and its location within the molecule, while NMR spectroscopy and GC/MS were used to recognize the Leu and Ile moieties. The major new lipopeptide found in R. opacus is defined as C17H35CH(OH)CH2CO-NHLeu-Ser-Leu-Ile-Thr-Ile-PheCOOH, where a ß-OH fatty acyl (C18-C22) substituent is attached to the N-terminal of the LSLITIF peptide chain via a NH-CO bond. By contrast, the main peptidolipids found in R. equi belong to the cyclopeptidolipid family, which possesses the same peptide sequence and lipid chain, but the ß-OH group of the fatty acyl moiety and the C-terminus of the peptide (i.e., the -COOH) are cyclized by an ester bond formation to a lactone, with a structure similar to iturin-A (Peypoux, F. et al. Biochemistry 1978, 17, 3992-3996). The antibiotic activity test of these new lipids did not reveal an activity against any of seven microorganisms tested.

Virulence-associated protein A from Rhodococcus equi is an intercompartmental pH-neutralising virulence factor.

Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane-bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram-positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid-encoded and secreted virulence-associated protein A (VapA) participates in exclusion of the proton-pumping vacuolar-ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH-neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid-less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH-neutral and hence growth-promoting intracellular niche. VapA represents a new type of Gram-positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton-pumping ATPase, and consequently disarming host defences.

The Chaperone UNC93B1 Regulates Toll-like Receptor Stability Independently of Endosomal TLR Transport.

Unc-93 homolog B1 (UNC93B1) is a key regulator of nucleic acid (NA)-sensing Toll-like receptors (TLRs). Loss of NA-sensing TLR responses in UNC93B1-deficient patients facilitates Herpes simplex virus type 1 (HSV-1) encephalitis. UNC93B1 is thought to guide NA-sensing TLRs from the endoplasmic reticulum (ER) to their respective endosomal signaling compartments and to guide the flagellin receptor TLR5 to the cell surface, raising the question of how UNC93B1 mediates differential TLR trafficking. Here, we report that UNC93B1 regulates a step upstream of the differential TLR trafficking process. We discovered that UNC93B1 deficiency resulted in near-complete loss of TLR3 and TLR7 proteins in primary splenic mouse dendritic cells and macrophages, showing that UNC93B1 is critical for maintaining TLR expression. Notably, expression of an ER-retained UNC93B1 version was sufficient to stabilize TLRs and largely restore endosomal TLR trafficking and activity. These data are critical for an understanding of how UNC93B1 can regulate the function of a broad subset of TLRs.

Vacuolar ATPase in phago(lyso)some biology.

Many eukaryotic cells ingest extracellular particles in a process termed phagocytosis which entails the generation of a new intracellular compartment, the phagosome. Phagosomes change their composition over time and this maturation process culminates in their fusion with acidic, hydrolase-rich lysosomes. During the maturation process, degradation and, when applicable, killing of the cargo may ensue. Many of the events that are pathologically relevant depend on strong acidification of phagosomes by the 'vacuolar' ATPase (V-ATPase). This protein complex acidifies the lumen of some intracellular compartments at the expense of ATP hydrolysis. We discuss here the roles and importance of V-ATPase in intracellular trafficking, its distribution, inhibition and activities, its role in the defense against microorganisms and the counteractivities of pathogens.

Sequential actions of phosphatidylinositol phosphates regulate phagosome-lysosome fusion.

Phagosomes mature into phagolysosomes by sequential fusion with early endosomes, late endosomes, and lysosomes. Phagosome-with-lysosome fusion (PLF) results in the delivery of lysosomal hydrolases into phagosomes and in digestion of the cargo. The machinery that drives PLF has been little investigated. Using a cell-free system, we recently identified the phosphoinositide lipids (PIPs) phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 4-phosphate (PI(4)P) as regulators of PLF. We now report the identification and the PIP requirements of four distinct subreactions of PLF. Our data show that (i) PI(3)P and PI(4)P are dispensable for the disassembly and activation of (phago)lysosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptors, that (ii) PI(3)P is required only after the tethering step, and that (iii) PI(4)P is required during and after tethering. Moreover, our data indicate that PI(4)P is needed to anchor Arl8 (Arf-like GTPase 8) and its effector homotypic fusion/vacuole protein sorting complex (HOPS) to (phago)lysosome membranes, whereas PI(3)P is required for membrane association of HOPS only. Our study provides a first link between PIPs and established regulators of membrane fusion in late endocytic trafficking.

Trehalose dimycolate interferes with FcγR-mediated phagosome maturation through Mincle, SHP-1 and FcγRIIB signalling.

The causative agent of tuberculosis, Mycobacterium tuberculosis (M. tuberculosis), contains an abundant cell wall glycolipid and a crucial virulence factor, trehalose-6,6'-dimycolate (TDM). TDM causes delay of phagosome maturation and thus promotes survival of mycobacteria inside host macrophages by a not fully understood mechanism. TDM signals through the Monocyte-INducible C-type LEctin (Mincle), a recently identified pattern recognition receptor. Here we show that recruitment of Mincle by TDM coupled to immunoglobulin (Ig)G-opsonised beads during Fcγ receptor (FcγR)-mediated phagocytosis interferes with phagosome maturation. In addition, modulation of phagosome maturation by TDM requires SH2-domain-containing inositol polyphosphate 5' phosphatase (SHP-1) and the FcγRIIB, which strongly suggests inhibitory downstream signalling of Mincle during phagosome formation. Overall, our study reveals important mechanisms contributing to the virulence of TDM.

Disruption of the vacuolar-type H+-ATPase complex in liver causes MTORC1-independent accumulation of autophagic vacuoles and lysosomes.

The vacuolar-type H+-translocating ATPase (v-H+-ATPase) has been implicated in the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (MTORC1), an important regulator of macroautophagy. To reveal the mechanistic links between the v-H+-ATPase and MTORC1, we destablilized v-H+-ATPase complexes in mouse liver cells by induced deletion of the essential chaperone ATP6AP2. ATP6AP2-mutants are characterized by massive accumulation of endocytic and autophagic vacuoles in hepatocytes. This cellular phenotype was not caused by a block in endocytic maturation or an impaired acidification. However, the degradation of LC3-II in the knockout hepatocytes appeared to be reduced. When v-H+-ATPase levels were decreased, we observed lysosome association of MTOR and normal signaling of MTORC1 despite an increase in autophagic marker proteins. To better understand why MTORC1 can be active when v-H+-ATPase is depleted, the activation of MTORC1 was analyzed in ATP6AP2-deficient fibroblasts. In these cells, very little amino acid-elicited activation of MTORC1 was observed. In contrast, insulin did induce MTORC1 activation, which still required intracellular amino acid stores. These results suggest that in vivo the regulation of macroautophagy depends not only on v-H+-ATPase-mediated regulation of MTORC1.

Turn up the lysosome.

Lysosomes are digestive organelles of the endocytic and autophagic pathways. Increasing lysosome enzyme activities could help to clear pathological cellular waste. A recent study shows that lysosomal digestive functions can be promoted in isolated cells and mice by pharmacologically stimulating the autophagy- and lysosome-regulating transcription factors TFEB and ZKSCAN3 through previously unrecognized mTORC1-independent pathways acting via PKC.

Deciphering the roles of phosphoinositide lipids in phagolysosome biogenesis.

Professional phagocytes engulf microbial invaders into plasma membrane-derived phagosomes. These mature into microbicidal phagolysosomes, leading to killing of the ingested microbe. Phagosome maturation involves sequential fusion of the phagosome with early endosomes, late endosomes, and the main degradative compartments in cells, lysosomes. Some bacterial pathogens manipulate the phosphoinositide (PIP) composition of phagosome membranes and are not delivered to phagolysosomes, pointing at a role of PIPs in phagosome maturation. This hypothesis is supported by comprehensive microscopic studies. Recently, cell-free reconstitution of fusion between phagosomes and endo(lyso)somes identified phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol 3-phosphate [PI(3)P] as key regulators of phagolysosome biogenesis. Here, we describe the emerging roles of PIPs in phagosome maturation and we present tools to study PIP involvement in phagosome trafficking using intact cells or purified compartments.

Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis.

Professional phagocytic cells ingest microbial intruders by engulfing them into phagosomes, which subsequently mature into microbicidal phagolysosomes. Phagosome maturation requires sequential fusion of the phagosome with early endosomes, late endosomes, and lysosomes. Although various phosphoinositides (PIPs) have been detected on phagosomes, it remained unclear which PIPs actually govern phagosome maturation. Here, we analyzed the involvement of PIPs in fusion of phagosomes with various endocytic compartments and identified phosphatidylinositol 4-phosphate [PI(4)P], phosphatidylinositol 3-phosphate [PI(3)P], and the lipid kinases that generate these PIPs, as mediators of phagosome-lysosome fusion. Phagosome-early endosome fusion required PI(3)P, yet did not depend on PI(4)P. Thus, PI(3)P regulates phagosome maturation at early and late stages, whereas PI(4)P is selectively required late in the pathway.