Distinct Mycobacterium marinum phosphatases determine pathogen vacuole phosphoinositide pattern,phagosome maturation,and escape to the cytosol

The causative agent of tuberculosis, Mycobacterium tuberculosis, and its close relative Mycobacterium marinum manipulate phagocytic host cells, thereby creating a replication‐permissive compartment termed the Mycobacterium‐containing vacuole (MCV). The phosphoinositide (PI) lipid pattern is a crucial determinant of MCV formation and is targeted by mycobacterial PI phosphatases. In this study, we establish an efficient phage transduction protocol to construct defined M. marinum deletion mutants lacking one or three phosphatases, PtpA, PtpB, and/or SapM. These strains were defective for intracellular replication in macrophages and amoebae, and the growth defect was complemented by the corresponding plasmid‐borne genes. Fluorescence microscopy of M. marinum‐infected Dictyostelium discoideum revealed that MCVs harbouring mycobacteria lacking PtpA, SapM, or all three phosphatases accumulate significantly more phosphatidylinositol‐3‐phosphate (PtdIns3P) compared with MCVs containing the parental strain. Moreover, PtpA reduced MCV acidification by blocking the recruitment of the V‐ATPase, and all three phosphatases promoted bacterial escape from the pathogen vacuole to the cytoplasm. In summary, the secreted M. marinum phosphatases PtpA, PtpB, and SapM determine the MCV PI pattern, compartment acidification, and phagosomal escape.     

Mechanism of Rab1b deactivation by the Legionella pneumophila GAP LepB

Legionella pneumophila is an intracellularly surviving pathogen that releases about 270 different proteins into the host cell during infection. A set of secreted proteins takes control of the vesicular trafficking regulator Rab1. Legionella LepB inactivates Rab1 by acting as a GTPase‐activating protein (GAP). We present the crystal structure of the Rab1b:LepB complex together with a thorough biochemical analysis and show that the GAP domain of LepB consists of an unusual fold. LepB acts by an atypical RabGAP mechanism that is reminiscent of classical GAPs and therefore sets the protein apart from mammalian TBC‐like GAPs. Surprisingly, LepB can function as a GAP for Rab3, Rab8, Rab13 and Rab35, too, suggesting that it has a broader cellular role than previously thought.     

Cross‐species functional complementation of cellulose synthase during the development of cellular slime molds

Cellulose is a major and important component of the extracellular matrix during the development of Dictyostelium discoideum. Upon starvation, solitary amoebae of D. discoideum gather and form fruiting bodies in which cells differentiate into stalk cells and spores. The stalk tubes and walls of spores and stalk cells are made of cellulose. In the genus Acytostelium, however, all cells are destined to become spores and the stalks comprise only a cellulose tube, suggesting species‐specific regulation of cellulose synthesis. In this study, we cloned a putative cellulose synthase gene (cesA) of Acytostelium subglobosum and performed comparative analyses with the D. discoideum cellulose synthase gene (dcsA). Although the deduced amino acid sequences were highly conserved between cesA and dcsA, the numbers of transmembrane spans preceding the catalytic domain were dissimilar; 2 and 3, respectively. Since ectopic expression of cesA in dcsA^?null cells failed to restore the developmental defects of the mutant, we constructed a series of chimerical genes for complementation analyses and found that the catalytic domain of cesA was functional in D. discoideum cells if the preceding transmembrane region was swapped with dcsA. The non‐functional products that contained the cesA‐derived transmembrane region were localized to lysosomes. These results indicate that the transmembrane region of cellulose synthase is essential for proper accumulation of cellulose during the development of D. discoideum and that its differential localization in A. subglobosum may be related to the characteristic morphogenesis in this species.     

The specificity of Burkholderia symbionts in the social amoeba farming symbiosis: Prevalence,species, genetic and phenotypic diversity

The establishment of symbioses between eukaryotic hosts and bacterial symbionts in nature is a dynamic process. The formation of such relationships depends on the life history of both partners. Bacterial symbionts of amoebae may have unique evolutionary trajectories to the symbiont lifestyle, because bacteria are typically ingested as prey. To persist after ingestion, bacteria must first survive phagocytosis. In the social amoeba Dictyostelium discoideum, certain strains of Burkholderia bacteria are able to resist amoebal digestion and maintain a persistent relationship that includes carriage throughout the amoeba's social cycle that culminates in spore formation. Some Burkholderia strains allow their host to carry other bacteria, as food. This carried food is released in new environments in a trait called farming. To better understand the diversity and prevalence of Burkholderia symbionts and the traits they impart to their amoebae hosts, we first screened 700 natural isolates of D. discoideum and found 25% infected with Burkholderia. We next used a multilocus phylogenetic analysis and identified two independent transitions by Burkholderia to the symbiotic lifestyle. Finally, we tested the ability of 38 strains of Burkholderia from D. discoideum, as well as strains isolated from other sources, for traits relevant to symbiosis in D. discoideum. Only D. discoideum native isolates belonging to the Burkholderia agricolaris, B. hayleyella, and B. bonniea species were able to form persistent symbiotic associations with D. discoideum. The Burkholderia–Dictyostelium relationship provides a promising arena for further studies of the pathway to symbiosis in a unique system.     

In situ structure of the Legionella Dot/Icm type IV secretion system by electron cryotomography

Type IV secretion systems (T4SSs) are large macromolecular machines that translocate protein and DNA and are involved in the pathogenesis of multiple human diseases. Here, using electron cryotomography (ECT), we report the in situ structure of the Dot/Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella pneumophila. This is the first structure of a type IVB secretion system, and also the first structure of any T4SS in situ. While the Dot/Icm system shares almost no sequence similarity with type IVA secretion systems (T4ASSs), its overall structure is seen here to be remarkably similar to previously reported T4ASS structures (those encoded by the R388 plasmid in Escherichia coli and the cag pathogenicity island in Helicobacter pylori). This structural similarity suggests shared aspects of mechanism. However, compared to the negative‐stain reconstruction of the purified T4ASS from the R388 plasmid, the L. pneumophila Dot/Icm system is approximately twice as long and wide and exhibits several additional large densities, reflecting type‐specific elaborations and potentially better structural preservation in situ.     

Rapid detection of viable Legionella pneumophila in tap water by a qPCR and RT‐PCR‐based method

Aims

A molecular method for a rapid detection of viable Legionella pneumophila of all serogroups in tap water samples was developed as an alternative to the reference method (ISO). Legionellae are responsible for Legionnaires’ disease, a severe pneumonia in humans with high lethality.

Methods and Results

The developed method is based on a nutritional stimulation and detection of an increase in precursor 16S rRNA as an indicator for viability. For quantification, DNA was detected by qPCR. This method was compared to the ISO method using water samples obtained from public sports facilities in Switzerland. The sensitivity and specificity were 91 and 97%, respectively, when testing samples for compliance with a microbiological criterion of 1000 cell equivalents per l.

Conclusion

The new method is sensitive and specific for Leg. pneumophila and allows results to be obtained within 8 h upon arrival, compared to one week or more by the ISO method.

Significance and Impact of the Study

The method represents a useful tool for a rapid detection of viable Leg. pneumophila of all serogroups in water by molecular biology. It can be used as an alternative to the ISO method for official water analysis for legionellae and particularly when a short test time is required.     

Mycobacterium marinum produces distinct mycobactin and carboxymycobactin siderophores to promote growth in broth and phagocytes

Mycobacterium marinum is a model organism for pathogenic Mycobacterium species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. These pathogens enter phagocytes and replicate within the Mycobacterium‐containing vacuole, possibly followed by vacuole exit and growth in the host cell cytosol. Mycobacteria release siderophores called mycobactins to scavenge iron, an essential yet poorly soluble and available micronutrient. To investigate the role of M. marinum mycobactins, we purified by organic solvent extraction and identified by mass spectrometry the lipid‐bound mycobactin (MBT) and the water‐soluble variant carboxymycobactin (cMBT). Moreover, we generated by specialised phage transduction a defined M. marinum ΔmbtB deletion mutant predicted to be defective for mycobactin production. The M. marinum ΔmbtB mutant strain showed a severe growth defect in broth and phagocytes, which was partially complemented by supplying the mbtB gene on a plasmid. Furthermore, purified Fe‐MBT or Fe‐cMBT improved the growth of wild type as well as ΔmbtB mutant bacteria on minimal plates, but only Fe‐cMBT promoted the growth of wild‐type M. marinum during phagocyte infection. Finally, the intracellular growth of M. marinum ΔmbtB in Acanthamoeba castellanii amoebae was restored by coinfection with wild‐type bacteria. Our study identifies and characterises the M. marinum MBT and cMBT siderophores and reveals the requirement of mycobactins for extra‐ and intracellular growth of the pathogen.     

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen,Legionella pneumophila

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila‐translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.     

Presence of SopE and mode of infection result in increased Salmonella‐containing vacuole damage and cytosolic release during host cell infection by Salmonella enterica

Salmonella enterica serovar Typhimurium (STM) is an invasive, facultative intracellular pathogen that has evolved sophisticated molecular mechanisms to establish an intracellular niche within a specialised vesicular compartment, the Salmonella‐containing vacuole (SCV). The loss of the SCV and release of STM into the cytosol of infected host cells was observed, and a bimodal intracellular lifestyle of STM in the SCV versus life in the cytosol is currently discussed. We set out to investigate the parameters affecting SCV integrity and cytosolic release. A fluorescent protein‐based cytosolic reporter approach was established to quantify, time‐resolved, and on a single cell level, the release of STM into the cytosol of host cells. We observed that the extent of SCV damage and cytosolic release is highly dependent on experimental conditions such as multiplicity of infection, type of host cell line, and STM strain background. Trigger invasion mediated by the Salmonella Pathogenicity Island 1‐encoded type III secretion system (SPI1‐T3SS) and its effector proteins promoted cytosolic release, whereas cytosolic bacteria were rarely observed if entry was mediated by zipper invasion. Presence of SPI1‐T3SS effector SopE was identified as major factor for damage of the SCV in the early phase after STM invasion and sopE‐expressing strains showed higher levels of cytosolic release.     

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires'' disease"¹. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles^2-4.The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania⁵, Listeria⁶, Mycobacterium⁷, Rhodococcus⁸, Salmonella⁹ or Legionella spp.¹⁰. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)^3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation^12,13 (Fig. 1).A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before¹³. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.     

Modulation of phagosome phosphoinositide dynamics by a Legionella phosphoinositide 3‐kinase

The phagosome harboring the bacterial pathogen Legionella pneumophila is known to be enriched with phosphatidylinositol 4‐phosphate (PtdIns4P), which is important for anchoring a subset of its virulence factors and potentially for signaling events implicated in the biogenesis of the Legionella‐containing vacuole (LCV) that supports intracellular bacterial growth. Here we demonstrate that the effector MavQ is a phosphoinositide 3‐kinase that specifically catalyzes the conversion of phosphatidylinositol (PtdIns) into PtdIns3P. The product of MavQ is subsequently phosphorylated by the effector LepB to yield PtdIns(3,4)P2, whose 3‐phosphate is then removed by another effector SidF to generate PtdIns4P. We also show that MavQ is associated with the LCV and the ∆mavQ mutant displays phenotypes in the anchoring of a PtdIns4P‐binding effector similar to those of ∆lepB or ∆sidF mutants. Our results establish a mechanism of de novo PtdIns4P biosynthesis by L. pneumophila via a catalysis axis comprised of MavQ, LepB, and SidF on the surface of its phagosome.     

Dynamic remodeling of the endosomal system during formation of Salmonella-induced filaments by intracellular Salmonella enterica

The infection by Salmonella enterica results in the massive remodeling of the endosomal system of eukaryotic host cells. One unique consequence is the formation of long tubular endosomal compartments, so-called Salmonella-induced filaments (SIF). Formation of SIF requires the function of type III secretion system and is a requirement of efficient intracellular proliferation of Salmonella. Using high-resolution live cell imaging approaches and electron microscopy, we report for the first time the highly dynamic characteristics of SIF and their ultrastructural properties. In the early phase of infection (4-5 h), SIF display highly dynamic properties in various types of host cells. SIF extend, branch and contract rapidly, and a stabilized network of SIF is formed later (>or=8 h after infection). The velocities of SIF extension and contraction in the different phases of infection were quantified. Our observations lead to novel models for the modification of host cell transport processes by virulence factors of intracellular Salmonella.     

Structural basis for the dual catalytic activity of the Legionella pneumophila ovarian tumor (OTU) domain deubiquitinase LotA

Legionella pneumophila, a bacterial pathogen that causes a severe pneumonia known as Legionnaires’ disease, extensively exploits the ubiquitin (Ub) pathway in the infected host cells through certain virulence effectors excreted by the Dot/Icm system. To date, several Dot/Icm effectors have been found to act as Ub ligases, and four effectors, including LotA, LotB, LotC, and Ceg7, have been identified as deubiquitinases (DUBs) from the ovarian tumor (OTU) domain family. LotA is unique among other OTU DUBs because it possesses two distinct DUB domains and exclusively exhibits catalytic activity against K6-linked diUb and polyUb chains. However, the structure of LotA and the molecular mechanism for the dual DUB activity remains elusive. In this study, we solved the structure of LotA in complex with proximally bound Ub and distal covalently bound Ub. Both Ub molecules are bound to the DUB1 domain and mimic a K6-linked diUb. Structural analysis reveals that the DUB1 domain utilizes a distinct mechanism for recognition of the K6-linked diUb within a large S1′ binding site that is uncommon to OTU DUBs. Structural fold of the LotA DUB2 domain closely resembles LotB and LotC, similarly containing an extra α-helix lobe that has been demonstrated to play an important role in Ub binding. Collectively, our study uncovers the structural basis for the dual catalytic activity of the unique OTU family DUB LotA.     

Auxin promotes susceptibility to Pseudomonas syringae via a mechanism independent of suppression of salicylic acid‐mediated defenses

Auxin is a key plant growth regulator that also impacts plant–pathogen interactions. Several lines of evidence suggest that the bacterial plant pathogen Pseudomonas syringae manipulates auxin physiology in Arabidopsis thaliana to promote pathogenesis. Pseudomonas syringae strategies to alter host auxin biology include synthesis of the auxin indole‐3‐acetic acid (IAA) and production of virulence factors that alter auxin responses in host cells. The application of exogenous auxin enhances disease caused by P. syringae strain DC3000. This is hypothesized to result from antagonism between auxin and salicylic acid (SA), a major regulator of plant defenses, but this hypothesis has not been tested in the context of infected plants. We further investigated the role of auxin during pathogenesis by examining the interaction of auxin and SA in the context of infection in plants with elevated endogenous levels of auxin. We demonstrated that elevated IAA biosynthesis in transgenic plants overexpressing the YUCCA 1 (YUC1) auxin biosynthesis gene led to enhanced susceptibility to DC3000. Elevated IAA levels did not interfere significantly with host defenses, as effector‐triggered immunity was active in YUC1‐overexpressing plants, and we observed only minor effects on SA levels and SA‐mediated responses. Furthermore, a plant line carrying both the YUC1‐overexpression transgene and the salicylic acid induction deficient 2 (sid2) mutation, which impairs SA synthesis, exhibited additive effects of enhanced susceptibility from both elevated auxin levels and impaired SA‐mediated defenses. Thus, in IAA overproducing plants, the promotion of pathogen growth occurs independently of suppression of SA‐mediated defenses.     

Maize Iranian mosaic virus infection promotes the energy sources of its insect vector,Laodelphax striatellus

Maize Iranian mosaic virus (MIMV, family Rhabdoviridae) causes an important disease in cereal crops in Iran. It is transmitted by the planthopper Laodelphax striatellus in a persistent, propagative manner. In the present study, the effect of MIMV on the energy reserves of L. striatellus was studied by comparing energy contents in viruliferous and non‐viruliferous insects. Results showed that MIMV‐infected male and female adults, and nymphs stored 1.82, 2.24 and 1.7‐fold more total energy reserves than non‐viruliferous individuals. This is consistent with a 2.55‐fold increase of the total energy (sum of energy sources in nymphs and adults) of viruliferous compared to non‐viruliferous specimens. A significant increase in glycogen (2.26‐fold), carbohydrate (2.13‐fold), lipid (1.63‐fold) and protein (1.96‐fold) was documented in viruliferous insects compared to non‐viruliferous insects. Based on these results, we conclude that MIMV enhances the energy reserves of its vector and therefore, may play a vital role in the ecology of L. striatellus.