Intravacuolar Replication Research Articles

A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

Remodeling of host cellular membrane transport pathways is a common pathogenic trait of many intracellular microbes that is essential to their intravacuolar life cycle and proliferation. The bacterium Brucella abortus generates a host endoplasmic reticulum‐derived vacuole (rBCV) that supports its intracellular growth, via VirB Type IV secretion system‐mediated delivery of effector proteins, whose functions and mode of action are mostly unknown. Here, we show that the effector BspF specifically promotes Brucella replication within rBCVs by interfering with vesicular transport between the trans‐Golgi network (TGN) and recycling endocytic compartment. BspF targeted the recycling endosome, inhibited retrograde traffic to the TGN, and interacted with the Arf6 GTPase‐activating Protein (GAP) ACAP1 to dysregulate Arf6‐/Rab8a‐dependent transport within the recycling endosome, which resulted in accretion of TGN‐associated vesicles by rBCVs and enhanced bacterial growth. Altogether, these findings provide mechanistic insight into bacterial modulation of membrane transport used to promote their own proliferation within intracellular vacuoles.

Salmonella exploits the host endolysosomal tethering factor HOPS complex to promote its intravacuolar replication.

Salmonella enterica serovar typhimurium extensively remodels the host late endocytic compartments to establish its vacuolar niche within the host cells conducive for its replication, also known as the Salmonella-containing vacuole (SCV). By maintaining a prolonged interaction with late endosomes and lysosomes of the host cells in the form of interconnected network of tubules (Salmonella-induced filaments or SIFs), Salmonella gains access to both membrane and fluid-phase cargo from these compartments. This is essential for maintaining SCV membrane integrity and for bacterial intravacuolar nutrition. Here, we have identified the multisubunit lysosomal tethering factor—HOPS (HOmotypic fusion and Protein Sorting) complex as a crucial host factor facilitating delivery of late endosomal and lysosomal content to SCVs, providing membrane for SIF formation, and nutrients for intravacuolar bacterial replication. Accordingly, depletion of HOPS subunits significantly reduced the bacterial load in non-phagocytic and phagocytic cells as well as in a mouse model of Salmonella infection. We found that Salmonella effector SifA in complex with its binding partner; SKIP, interacts with HOPS subunit Vps39 and mediates recruitment of this tethering factor to SCV compartments. The lysosomal small GTPase Arl8b that binds to, and promotes membrane localization of Vps41 (and other HOPS subunits) was also required for HOPS recruitment to SCVs and SIFs. Our findings suggest that Salmonella recruits the host late endosomal and lysosomal membrane fusion machinery to its vacuolar niche for access to host membrane and nutrients, ensuring its intracellular survival and replication.

Divergent evolution of Di-lysine ER retention vs. farnesylation motif-mediated anchoring of the AnkB virulence effector to the Legionella-containing vacuolar membrane

Within macrophages and amoeba, the Legionella-containing vacuole (LCV) membrane is derived from the ER. The bona fide F-box AnkB effector protein of L. pneumophila strain AA100/130b is anchored to the cytosolic side of the LCV membrane through host-mediated farnesylation of its C-terminal eukaryotic “CaaX” motif. Here we show that the AnkB homologue of the Paris strain has a frame shift mutation that led to a loss of the CaaX motif and a concurrent generation of a unique C-terminal KNKYAP motif, which resembles the eukaryotic di-lysine ER-retention motif (KxKxx). Our phylogenetic analyses indicate that environmental isolates of L. pneumophila have a potential positive selection for the ER-retention KNKYAP motif. The AnkB-Paris effector is localized to the LCV membrane most likely through the ER-retention motif. Its ectopic expression in HEK293T cells localizes it to the perinuclear ER region and it trans-rescues the ankB mutant of strain AA100/130b in intra-vacuolar replication. The di-lysine ER retention motif of AnkB-Paris is indispensable for function; most likely as an ER retention motif that enables anchoring to the ER-derived LCV membrane. Our findings show divergent evolution of the ankB allele in exploiting either host farnesylation or the ER retention motif to be anchored into the LCV membrane.

Host FIH-Mediated Asparaginyl Hydroxylation of Translocated Legionella pneumophila Effectors.

FIH-mediated post-translational modification through asparaginyl hydroxylation of eukaryotic proteins impacts regulation of protein-protein interaction. We have identified the FIH recognition motif in 11 Legionella pneumophila translocated effectors, YopM of Yersinia, IpaH4.5 of Shigella and an ankyrin protein of Rickettsia. Mass spectrometry analyses of the AnkB and AnkH effectors of L. pneumophila confirm their asparaginyl hydroxylation. Consistent with localization of the AnkB effector to the Legionella-containing vacuole (LCV) membrane and its modification by FIH, our data show that FIH and its two interacting proteins, Mint3 and MT1-MMP are acquired by the LCV in a Dot/Icm type IV secretion-dependent manner. Chemical inhibition or RNAi-mediated knockdown of FIH promotes LCV-lysosomes fusion, diminishes decoration of the LCV with polyubiquitinated proteins, and abolishes intra-vacuolar replication of L. pneumophila. These data show acquisition of the host FIH by a pathogen-containing vacuole and that asparaginyl-hydroxylation of translocated effectors is indispensable for their function.

The transcriptome of Legionella pneumophila-infected human monocyte-derived macrophages.

Background Legionella pneumophila is an intracellular bacterial pathogen that invades and replicates within alveolar macrophages through injection of ∼300 effector proteins by its Dot/Icm type IV translocation apparatus. The bona fide F-box protein, AnkB, is a nutritional virulence effector that triggers macrophages to generate a surplus of amino acids, which is essential for intravacuolar proliferation. Therefore, the ankB mutant represents a novel genetic tool to determine the transcriptional response of human monocyte-derived macrophages (hMDMs) to actively replicating L. pneumophila.Methodology/Principal FindingsHere, we utilized total human gene microarrays to determine the global transcriptional response of hMDMs to infection by wild type or the ankB mutant of L. pneumophila. The transcriptomes of hMDMs infected with either actively proliferating wild type or non-replicative ankB mutant bacteria were remarkably similar. The transcriptome of infected hMDMs was predominated by up-regulation of inflammatory pathways (IL-10 anti-inflammatory, interferon signaling and amphoterin signaling), anti-apoptosis, and down-regulation of protein synthesis pathways. In addition, L. pneumophila modulated diverse metabolic pathways, particularly those associated with bio-active lipid metabolism, and SLC amino acid transporters expression.Conclusion/SignificanceTaken together, the hMDM transcriptional response to L. pneumophila is independent of intra-vacuolar replication of the bacteria and primarily involves modulation of the immune response and metabolic as well as nutritional pathways.

Complete and ubiquitinated proteome of the Legionella-containing vacuole within human macrophages.

Within protozoa or human macrophages Legionella pneumophila evades the endosomal pathway and replicates within an ER-derived vacuole termed the Legionella-containing vacuole (LCV). The LCV membrane-localized AnkB effector of L. pneumophila is an F-box protein that mediates decoration of the LCV with lysine48-linked polyubiquitinated proteins, which is essential for intravacuolar replication. Using high-throughput LC–MS analysis, we have identified the total and ubiquitinated host-derived proteome of LCVs purified from human U937 macrophages. The LCVs harboring the AA100/130b WT strain contain 1193 proteins including 24 ubiquitinated proteins, while the ankB mutant LCVs contain 1546 proteins with 29 ubiquitinated proteins. Pathway analyses reveal the enrichment of proteins involved in signaling, protein transport, phosphatidylinositol, and carbohydrate metabolism on both WT and ankB mutant LCVs. The ankB mutant LCVs are preferentially enriched for proteins involved in transcription/translation and immune responses. Ubiquitinated proteins on the WT strain LCVs are enriched for immune response, signaling, regulation, intracellular trafficking, and amino acid transport pathways, while ubiquitinated proteins on the ankB mutant LCVs are enriched for vesicle trafficking, signaling, and ubiquitination pathways. The complete and ubiquitinated LCV proteome within human macrophages illustrates complex and dynamic biogenesis of the LCV and provides a rich resource for future studies.

Translocon-independent intracellular replication by Pseudomonas aeruginosa requires the ADP-ribosylation domain of ExoS

Pseudomonas aeruginosa, a significant cause of human morbidity and mortality, uses a type 3 secretion system (T3SS) to inject effector toxins into host cells. We previously reported that P. aeruginosa uses ADP-ribosyltransferase (ADPr) activity of the T3SS effector ExoS for intracellular replication. T3SS translocon (ΔpopB)-mutants, which can export, but not translocate effectors across host membranes, retained intracellular replication. We hypothesized that secreted effectors mediate translocon-independent intracellular replication. Translocon mutants of PAO1 lacking one or more of its three known effectors (ExoS, ExoT and ExoY) were used. All translocon mutants, irrespective of effectors expressed, localized to intracellular vacuoles. Translocon-effector null mutants and translocon-exoS mutants showed defective intracellular replication. Mutants in exoT, exoY or both replicated as efficiently as translocon mutants expressing all effectors. Complementation of translocon-effector null mutants with native exoS or a membrane localization domain mutant of exoS, but not the ADPr mutant exoS (pUCPexoSE381D), restored intracellular replication, correlating with increased bacteria per vacuole. Thus, P. aeruginosa is capable of intravacuolar replication that requires ExoS ADPr activity, but not the translocon. These data suggest that T3SS effectors can participate in pathogenesis without translocon-mediated translocation across host membranes, and that intracellular bacteria can contribute to P. aeruginosa pathogenesis within epithelial cells.

Effect of host fatty acid-binding protein and fatty acid uptake on growth of Chlamydia trachomatis L2

Chlamydia trachomatis is an obligate intracellular bacterium and acquires both building blocks and energy from host cells for growth. The fatty acid-binding protein (FABP) plays an important role in uptake of long-chain fatty acids (LCFA) and energy metabolism by eukaryotic cells. The roles of FABP and LCFA in chlamydial infection were evaluated. Infection of liver cells with chlamydial organisms promoted fatty acid uptake by the infected cells, suggesting that LCFA may benefit chlamydial growth. Introduction of FABP into the liver cells not only enhanced fatty acid uptake, but also increased chlamydial intravacuolar replication and maturation. The FABP-enhanced chlamydial intracellular growth was dependent on the host cell uptake of fatty acids. These results have demonstrated that C. trachomatis can productively infect liver cells and utilize FABP-transported LCFA for its own biosynthesis.

Cleavage of host keratin 8 by a Chlamydia-secreted protease.

Chlamydiae have to replicate within a cytoplasmic vacuole in eukaryotic cells. Expansion of the chlamydia-laden vacuole is essential for chlamydial intravacuolar replication, which inevitably causes host cell cytoskeleton rearrangements. A cleavage fragment of keratin 8 corresponding to the central rod region was detected in the soluble fraction of chlamydia-infected cells. Since keratin 8 is a major component of the intermediate filaments in simple epithelial cells, cleavage of keratin 8 may increase the solubility of the host cell cytoskeleton and thus permit vacuole expansion in chlamydia-infected cells. A chlamydia-secreted protease designated CPAF (chlamydial protease/proteasome-like activity factor) was both necessary and sufficient for keratin 8 cleavage in chlamydia-infected cells, suggesting that chlamydiae have evolved specific mechanisms for modifying the host cell cytoskeleton.