The type IV pili component PilO is a virulence determinant of Francisella novicida.

Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. While its ability to replicate within cells has been studied in much detail, the bacterium also encodes a less characterised type 4 pili (T4P) system. T4Ps are dynamic adhesive organelles identified as major virulence determinants in many human pathogens. In F. tularensis, the T4P is required for adherence to the host cell, as well as for protein secretion. Several components, including pilins, a pili peptidase, a secretin pore and two ATPases, are required to assemble a functional T4P, and these are encoded within distinct clusters on the Francisella chromosome. While some of these components have been functionally characterised, the role of PilO, if any, still is unknown. Here, we examined the role of PilO in the pathogenesis of F. novicida. Our results show that the PilO is essential for pilus assembly on the bacterial surface. In addition, PilO is important for adherence of F. novicida to human monocyte-derived macrophages, secretion of effector proteins and intracellular replication. Importantly, the pilO mutant is attenuated for virulence in BALB/c mice regardless of the route of infection. Following intratracheal and intradermal infection, the mutant caused no histopathology changes, and demonstrated impaired phagosomal escape and replication within lung liver as well as spleen. Thus, PilO is an essential virulence determinant of F. novicida.

Idiosyncratic Biogenesis of Intracellular Pathogens-Containing Vacuoles.

While most bacterial species taken up by macrophages are degraded through processing of the bacteria-containing vacuole through the endosomal-lysosomal degradation pathway, intravacuolar pathogens have evolved to evade degradation through the endosomal-lysosomal pathway. All intra-vacuolar pathogens possess specialized secretion systems (T3SS-T7SS) that inject effector proteins into the host cell cytosol to modulate myriad of host cell processes and remodel their vacuoles into proliferative niches. Although intravacuolar pathogens utilize similar secretion systems to interfere with their vacuole biogenesis, each pathogen has evolved a unique toolbox of protein effectors injected into the host cell to interact with, and modulate, distinct host cell targets. Thus, intravacuolar pathogens have evolved clear idiosyncrasies in their interference with their vacuole biogenesis to generate a unique intravacuolar niche suitable for their own proliferation. While there has been a quantum leap in our knowledge of modulation of phagosome biogenesis by intravacuolar pathogens, the detailed biochemical and cellular processes affected remain to be deciphered. Here we discuss how the intravacuolar bacterial pathogens Salmonella, Chlamydia, Mycobacteria, Legionella, Brucella, Coxiella, and Anaplasma utilize their unique set of effectors injected into the host cell to interfere with endocytic, exocytic, and ER-to-Golgi vesicle traffic. However, Coxiella is the main exception for a bacterial pathogen that proliferates within the hydrolytic lysosomal compartment, but its T4SS is essential for adaptation and proliferation within the lysosomal-like vacuole.

An Indispensable Role for the MavE Effector of Legionella pneumophila in Lysosomal Evasion.

Diversion of the Legionella pneumophila-containing vacuole (LCV) from the host endosomal-lysosomal degradation pathway is one of the main virulence features essential for manifestation of Legionnaires' pneumonia. Many of the ∼350 Dot/Icm-injected effectors identified in L. pneumophila have been shown to interfere with various host pathways and processes, but no L. pneumophila effector has ever been identified to be indispensable for lysosomal evasion. While most single effector mutants of L. pneumophila do not exhibit a defective phenotype within macrophages, we show that the MavE effector is essential for intracellular growth of L. pneumophila in human monocyte-derived macrophages (hMDMs) and amoebae and for intrapulmonary proliferation in mice. The mavE null mutant fails to remodel the LCV with endoplasmic reticulum (ER)-derived vesicles and is trafficked to the lysosomes where it is degraded, similar to formalin-killed bacteria. During infection of hMDMs, the MavE effector localizes to the poles of the LCV membrane. The crystal structure of MavE, resolved to 1.8 Å, reveals a C-terminal transmembrane helix, three copies of tyrosine-based sorting motifs, and an NPxY eukaryotic motif, which binds phosphotyrosine-binding domains present on signaling and adaptor eukaryotic proteins. Two point mutations within the NPxY motif result in attenuation of L. pneumophila in both hMDMs and amoeba. The substitution defects of P78 and D64 are associated with failure of vacuoles harboring the mutant to be remodeled by the ER and results in fusion of the vacuole to the lysosomes leading to bacterial degradation. Therefore, the MavE effector of L. pneumophila is indispensable for phagosome biogenesis and lysosomal evasion.IMPORTANCE Intracellular proliferation of Legionella pneumophila within a vacuole in human alveolar macrophages is essential for manifestation of Legionnaires' pneumonia. Intravacuolar growth of the pathogen is totally dependent on remodeling the L. pneumophila-containing vacuole (LCV) by the ER and on its evasion of the endosomal-lysosomal degradation pathway. The pathogen has evolved to inject ∼350 protein effectors into the host cell where they modulate various host processes, but no L. pneumophila effector has ever been identified to be indispensable for lysosomal evasion. We show that the MavE effector localizes to the poles of the LCV membrane and is essential for lysosomal evasion and intracellular growth of L. pneumophila and for intrapulmonary proliferation in mice. The crystal structure of MavE shows an NPxY eukaryotic motif essential for ER-mediated remodeling and lysosomal evasion by the LCV. Therefore, the MavE effector of L. pneumophila is indispensable for phagosome biogenesis and lysosomal evasion.

Evolution and Adaptation of Legionella pneumophila to Manipulate the Ubiquitination Machinery of Its Amoebae and Mammalian Hosts.

The ubiquitin pathway is highly conserved across the eukaryotic domain of life and plays an essential role in a plethora of cellular processes. It is not surprising that many intracellular bacterial pathogens often target the essential host ubiquitin pathway. The intracellular bacterial pathogen Legionella pneumophila injects into the host cell cytosol multiple classes of classical and novel ubiquitin-modifying enzymes that modulate diverse ubiquitin-related processes in the host cell. Most of these pathogen-injected proteins, designated as effectors, mimic known E3-ubiquitin ligases through harboring F-box or U-box domains. The classical F-box effector, AnkB targets host proteins for K48-linked polyubiquitination, which leads to excessive proteasomal degradation that is required to generate adequate supplies of amino acids for metabolism of the pathogen. In contrast, the SidC and SdcA effectors share no structural similarity to known eukaryotic ligases despite having E3-ubiquitin ligase activity, suggesting that the number of E3-ligases in eukaryotes is under-represented. L. pneumophila also injects into the host many novel ubiquitin-modifying enzymes, which are the SidE family of effectors that catalyze phosphoribosyl-ubiquitination of serine residue of target proteins, independently of the canonical E1-2-3 enzymatic cascade. Interestingly, the environmental bacterium, L. pneumophila, has evolved within a diverse range of amoebal species, which serve as the natural hosts, while accidental transmission through contaminated aerosols can cause pneumonia in humans. Therefore, it is likely that the novel ubiquitin-modifying enzymes of L. pneumophila were acquired by the pathogen through interkingdom gene transfer from the diverse natural amoebal hosts. Furthermore, conservation of the ubiquitin pathway across eukaryotes has enabled these novel ubiquitin-modifying enzymes to function similarly in mammalian cells. Studies on the biological functions of these effectors are likely to reveal further novel ubiquitin biology and shed further lights on the evolution of ubiquitin.

Bacterial nucleomodulins: A coevolutionary adaptation to the eukaryotic command center.

Through long-term interactions with their hosts, bacterial pathogens have evolved unique arsenals of effector proteins that interact with specific host targets and reprogram the host cell into a permissive niche for pathogen proliferation. The targeting of effector proteins into the host cell nucleus for modulation of nuclear processes is an emerging theme among bacterial pathogens. These unique pathogen effector proteins have been termed in recent years as "nucleomodulins." The first nucleomodulins were discovered in the phytopathogens Agrobacterium and Xanthomonas, where their nucleomodulins functioned as eukaryotic transcription factors or integrated themselves into host cell DNA to promote tumor induction, respectively. Numerous nucleomodulins were recently identified in mammalian pathogens. Bacterial nucleomodulins are an emerging family of pathogen effector proteins that evolved to target specific components of the host cell command center through various mechanisms. These mechanisms include: chromatin dynamics, histone modification, DNA methylation, RNA splicing, DNA replication, cell cycle, and cell signaling pathways. Nucleomodulins may induce short- or long-term epigenetic modifications of the host cell. In this extensive review, we discuss the current knowledge of nucleomodulins from plant and mammalian pathogens. While many nucleomodulins are already identified, continued research is instrumental in understanding their mechanisms of action and the role they play during the progression of pathogenesis. The continued study of nucleomodulins will enhance our knowledge of their effects on nuclear chromatin dynamics, protein homeostasis, transcriptional landscapes, and the overall host cell epigenome.

Paradoxical Pro-inflammatory Responses by Human Macrophages to an Amoebae Host-Adapted Legionella Effector.

Legionella pneumophila has co-evolved with amoebae, their natural hosts. Upon transmission to humans, the bacteria proliferate within alveolar macrophages causing pneumonia. Here, we show L. pneumophila injects the effector LamA, an amylase, into the cytosol of human macrophage (hMDMs) and amoebae to rapidly degrade glycogen to generate cytosolic hyper-glucose. In response, hMDMs shift their metabolism to aerobic glycolysis, which directly triggers an M1-like pro-inflammatory differentiation and nutritional innate immunity through enhanced tryptophan degradation. This leads to a modest restriction of bacterial proliferation in hMDMs. In contrast, LamA-mediated glycogenolysis in amoebae deprives the natural host from the main building blocks for synthesis of the cellulose-rich cyst wall, leading to subversion of amoeba encystation. This is non-permissive for bacterial proliferation. Therefore, LamA of L. pneumophila is an amoebae host-adapted effector that subverts encystation of the amoebae natural host, and the paradoxical hMDMs' pro-inflammatory response is likely an evolutionary accident.

Interaction of the Ankyrin H Core Effector of Legionella with the Host LARP7 Component of the 7SK snRNP Complex.

Species of the Legionella genus encode at least 18,000 effector proteins that are translocated through the Dot/Icm type IVB translocation system into macrophages and protist hosts to enable intracellular growth. Eight effectors, including ankyrin H (AnkH), are common to all Legionella species. The AnkH effector is also present in Coxiella and Rickettsiella To date, no pathogenic effectors have ever been described that directly interfere with host cell transcription. We determined that the host nuclear protein La-related protein 7 (LARP7), which is a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, interacts with AnkH in the host cell nucleus. The AnkH-LARP7 interaction partially impedes interactions of the 7SK snRNP components with LARP7, interfering with transcriptional elongation by polymerase (Pol) II. Consistent with that, our data show AnkH-dependent global reprogramming of transcription of macrophages infected by Legionella pneumophila The crystal structure of AnkH shows that it contains four N-terminal ankyrin repeats, followed by a cysteine protease-like domain and an α-helical C-terminal domain. A substitution within the ß-hairpin loop of the third ankyrin repeat results in diminishment of LARP7-AnkH interactions and phenocopies the ankH null mutant defect in intracellular growth. LARP7 knockdown partially suppresses intracellular proliferation of wild-type (WT) bacteria and increases the severity of the defect of the ΔankH mutant, indicating a role for LARP7 in permissiveness of host cells to intracellular bacterial infection. We conclude that the AnkH-LARP7 interaction impedes interaction of LARP7 with 7SK snRNP, which would block transcriptional elongation by Pol II, leading to host global transcriptional reprogramming and permissiveness to L. pneumophilaIMPORTANCE For intracellular pathogens to thrive in host cells, an environment that supports survival and replication needs to be established. L. pneumophila accomplishes this through the activity of the ∼330 effector proteins that are injected into host cells during infection. Effector functions range from hijacking host trafficking pathways to altering host cell machinery, resulting in altered cell biology and innate immunity. One such pathway is the host protein synthesis pathway. Five L. pneumophila effectors have been identified that alter host cell translation, and 2 effectors have been identified that indirectly affect host cell transcription. No pathogenic effectors have been described that directly interfere with host cell transcription. Here we show a direct interaction of the AnkH effector with a host cell transcription complex involved in transcriptional elongation. We identify a novel process by which AnkH interferes with host transcriptional elongation through interference with formation of a functional complex and show that this interference is required for pathogen proliferation.

Nutrition and Bipartite Metabolism of Intracellular Pathogens.

The host is a nutrient-rich niche for microbial pathogens, but one that comes with obstacles and challenges. Many intracellular pathogens like Legionella pneumophila, Coxiella burnetii, Listeria monocytogenes, and Chlamydia trachomatis have developed bipartite metabolism within their hosts. This style of metabolic regulation enables pathogen sensing of specific nutrients to engage them into catabolic and anabolic processes, and contributes to temporal and spatial pathogen phenotypic modulation. Not only have intracellular pathogens adapted their metabolism to the host, they have also acquired idiosyncratic strategies to exploit host nutritional supplies and intercept metabolites. Francisella tularensis and Anaplasma phagocytophilum alter host autophagy, Shigella flexneri intercepts all host pyruvate, while L. pneumophila induces host protein degradation and blocks protein translation. Strategies of pathogen manipulation of host nutrients could serve as therapeutic targets.

Evasion of phagotrophic predation by protist hosts and innate immunity of metazoan hosts by Legionella pneumophila.

Legionella pneumophila is a ubiquitous environmental bacterium that has evolved to infect and proliferate within amoebae and other protists. It is thought that accidental inhalation of contaminated water particles by humans is what has enabled this pathogen to proliferate within alveolar macrophages and cause pneumonia. However, the highly evolved macrophages are equipped with more sophisticated innate defence mechanisms than are protists, such as the evolution of phagotrophic feeding into phagocytosis with more evolved innate defence processes. Not surprisingly, the majority of proteins involved in phagosome biogenesis (~80%) have origins in the phagotrophy stage of evolution. There are a plethora of highly evolved cellular and innate metazoan processes, not represented in protist biology, that are modulated by L. pneumophila, including TLR2 signalling, NF-κB, apoptotic and inflammatory processes, histone modification, caspases, and the NLRC-Naip5 inflammasomes. Importantly, L. pneumophila infects haemocytes of the invertebrate Galleria mellonella, kill G. mellonella larvae, and proliferate in and kill Drosophila adult flies and Caenorhabditis elegans. Although coevolution with protist hosts has provided a substantial blueprint for L. pneumophila to infect macrophages, we discuss the further evolutionary aspects of coevolution of L. pneumophila and its adaptation to modulate various highly evolved innate metazoan processes prior to becoming a human pathogen.

Evolution of the Arsenal of Legionella pneumophila Effectors To Modulate Protist Hosts.

Within the human host, Legionella pneumophila replicates within alveolar macrophages, leading to pneumonia. However, L. pneumophila is an aquatic generalist pathogen that replicates within a wide variety of protist hosts, including amoebozoa, percolozoa, and ciliophora. The intracellular lifestyles of L. pneumophila within the two evolutionarily distant hosts macrophages and protists are remarkably similar. Coevolution with numerous protist hosts has shaped plasticity of the genome of L. pneumophila, which harbors numerous proteins encoded by genes acquired from primitive eukaryotic hosts through interkingdom horizontal gene transfer. The Dot/Icm type IVb translocation system translocates ∼6,000 effectors among Legionella species and >320 effector proteins in L. pneumophila into host cells to modulate a plethora of cellular processes to create proliferative niches. Since many of the effectors have likely evolved to modulate cellular processes of primitive eukaryotic hosts, it is not surprising that most of the effectors do not contribute to intracellular growth within human macrophages. Some of the effectors may modulate highly conserved eukaryotic processes, while others may target protist-specific processes that are absent in mammals. The lack of studies to determine the role of the effectors in adaptation of L. pneumophila to various protists has hampered the progress to determine the function of most of these effectors, which are routinely studied in mouse or human macrophages. Since many protists restrict L. pneumophila, utilization of such hosts can also be instrumental in deciphering the mechanisms of failure of L. pneumophila to overcome restriction of certain protist hosts. Here, we review the interaction of L. pneumophila with its permissive and restrictive protist environmental hosts and outline the accomplishments as well as gaps in our knowledge of L. pneumophila-protist host interaction and L. pneumophila's evolution to become a human pathogen.

Mammalian Solute Carrier (SLC)-like transporters of Legionella pneumophila.

Acquisition of nutrients during intra-vacuolar growth of L. pneumophila within macrophages or amoebae is poorly understood. Since many genes of L. pneumophila are acquired by inter-kingdom horizontal gene transfer from eukaryotic hosts, we examined the presence of human solute carrier (SLC)-like transporters in the L. pneumophila genome using I-TASSER to assess structural alignments. We identified 11 SLC-like putative transporters in L. pneumophila that are structurally similar to SLCs, eight of which are amino acid transporters, and one is a tricarboxylate transporter. The two other transporters, LstA and LstB, are structurally similar to the human glucose transporter, SLC2a1/Glut1. Single mutants of lstA or lstB have decreased ability to import, while the lstA/lstB double mutant is severely defective for uptake of glucose. While lstA or lstB single mutants are not defective in intracellular proliferation within Acanthamoeba polyphaga and human monocyte-derived macrophages, the lstA/lstB double mutant is severely defective in both host cells. The two phenotypic defects of the lstA/lstB double mutant in uptake of glucose and intracellular replication are both restored upon complementation of either lstA or lstB. Our data show that the two glucose transporters, LstA and LstB, are redundant and are required for intracellular replication within human macrophages and amoebae.

A Legionella pneumophila amylase is essential for intracellular replication in human macrophages and amoebae.

Legionella pneumophila invades protozoa with an "accidental" ability to cause pneumonia upon transmission to humans. To support its nutrition during intracellular residence, L. pneumophila relies on host amino acids as the main source of carbon and energy to feed the TCA cycle. Despite the apparent lack of a requirement for glucose for L. pneumophila growth in vitro and intracellularly, the organism contains multiple amylases, which hydrolyze polysaccharides into glucose monomers. Here we describe one predicted putative amylase, LamB, which is uniquely present only in L. pneumophila and L. steigerwaltii among the ~60 species of Legionella. Our data show that LamB has a strong amylase activity, which is abolished upon substitutions of amino acids that are conserved in the catalytic pocket of amylases. Loss of LamB or expression of catalytically-inactive variants of LamB results in a severe growth defect of L. pneumophila in Acanthamoeba polyphaga and human monocytes-derived macrophages. Importantly, the lamB null mutant is severely attenuated in intra-pulmonary proliferation in the mouse model and is defective in dissemination to the liver and spleen. Our data show an essential role for LamB in intracellular replication of L. pneumophila in amoeba and human macrophages and in virulence in vivo.

Isolation of F. novicida-Containing Phagosome from Infected Human Monocyte Derived Macrophages.

Francisella is a gram-negative bacterial pathogen, which causes tularemia in humans and animals. A crucial step of Francisella infection is its invasion of macrophage cells. Biogenesis of the Francisella-containing phagosome (FCP) is arrested for ~15 min at the endosomal stage, followed by gradual bacterial escape into the cytosol, where the microbe proliferates. The crucial step in pathogenesis of tularemia is short and transient presence of the bacterium within phagosome. Isolation of FCPs for further studies has been challenging due to the short period of time of bacterial residence in it and the characteristics of the FCP. Here, we will for the first time present the method for isolation of the FCPs from infected human monocytes-derived macrophages (hMDMs). For elimination of lysosomal compartment these organelles were pre-loaded with dextran coated colloidal iron particles prior infection and eliminated by magnetic separation of the post-nuclear supernatant (PNS). We encountered the challenge that mitochondria has similar density to the FCP. To separate the FCP in the PNS from mitochondria, we utilized iodophenylnitrophenyltetrazolium, which is converted by the mitochondrial succinate dehydrogenase into formazan, leading to increased density of the mitochondria and allowing separation by the discontinuous sucrose density gradient ultracentrifugation. The purity of the FCP preparation and its acquisition of early endosomal markers was confirmed by Western blots, confocal and transmission electron microscopy. Our strategy to isolate highly pure FCPs from macrophages should facilitate studies on the FCP and its biogenesis.

Structural Mimicry by a Bacterial F Box Effector Hijacks the Host Ubiquitin-Proteasome System.

Ankyrin B (AnkB/LegAU13) is a translocated F box effector essential for the intracellular replication of the pathogen Legionella pneumophila. AnkB co-opts a host ubiquitin ligase to decorate the pathogen-containing vacuole with K48-linked polyubiquitinated proteins and degrade host proteins as a source of energy. Here, we report that AnkB commandeers the host ubiquitin-proteasome system through mimicry of two eukaryotic protein domains. Using X-ray crystallography, we determined the 3D structure of AnkB in complex with Skp1, a component of the human SCF ubiquitination ligase. The structure confirms that AnkB contains an N-terminal F box similar to Skp2 and a C-terminal substrate-binding domain similar to eukaryotic ankyrin repeats. We identified crucial amino acids in the substrate-binding domain of AnkB and showed them to be essential for the function of AnkB in L. pneumophila intracellular proliferation. The study reveals how Legionella uses molecular mimicry to manipulate the host ubiquitination pathway and proliferate intracellularly.

F. novicida-Infected A. castellanii Does Not Enhance Bacterial Virulence in Mice.

Francisella tularensis is a facultative intracellular bacterium that causes tularemia in humans and animals. Epidemiology of tularemia worldwide is often associated with water-borne transmission, which includes mosquitoes and amoebae as the potential host reservoirs of the bacteria in water environment. In vitro studies showed intracellular replication of F. tularensis within Acanthamoeba castellanii and Hartmanella vermiformis cells. While infection of amoeba by Legionella pneumophila has been shown to enhance infectivity of L. pneumophila the role of F. tularensis-infected protozoa in the pathogenesis of tularemia is not known. We used 6 h coculture of A. castellanii and F. novicida for investigation of the effect of inhaled amoeba on the pathogenesis of tularemia on in vivo model. Balb/c mice were infected intratracheally with F. novicida or with F. novicida-infected A. castellanii. Surprisingly, infection with F. novicida-infected A. castellanii did not lead to bronchopneumonia in Balb/c mice, and Francisella did not disseminate into the liver and spleen. Upon inhalation, F. novicida infects a variety of host cells, though neutrophils are the predominant cells early during infection in the lung infiltrates of pulmonary tularemia. The numbers of neutrophils in the lungs of Balb/c mice were significantly lower in the infection of mice with F. novicida-infected A. castellanii in comparison to group of mice infected only with F. novicida. These results demonstrate that following inoculation of mice with F. novicida-infected A. castellanii, mice did not develop tularemia.

Lysine11-Linked Polyubiquitination of the AnkB F-Box Effector of Legionella pneumophila.

The fate of the polyubiquitinated protein is determined by the lysine linkages involved in the polymerization of the ubiquitin monomers, which has seven lysine residues (K(6), K(11), K(27), K(29), K(33), K(48), and K(63)). The translocated AnkB effector of the intravacuolar pathogen Legionella pneumophila is a bona fide F-box protein, which is localized to the cytosolic side of the Legionella-containing vacuole (LCV) and is essential for intravacuolar proliferation within macrophages and amoebae. The F-box domain of AnkB interacts with the host SCF1 E3 ubiquitin ligase that triggers the decoration of the LCV with K(48)-linked polyubiquitinated proteins that are targeted for proteasomal degradation. Here we report that AnkB becomes rapidly polyubiquitinated within the host cell, and this modification is independent of the F-box domain of AnkB, indicating host-mediated polyubiquitination. We show that the AnkB effector interacts specifically with the host E3 ubiquitin ligase Trim21. Mass spectrometry analyses have shown that AnkB is modified by K(11)-linked polyubiquitination, which has no effect on its stability. This work shows the first example of K(11)-linked polyubiquitination of a bacterial effector and its interaction with the host Trim21 ubiquitin ligase.

Yersinia pestis Requires Host Rab1b for Survival in Macrophages.

Yersinia pestis is a facultative intracellular pathogen that causes the disease known as plague. During infection of macrophages Y. pestis actively evades the normal phagosomal maturation pathway to establish a replicative niche within the cell. However, the mechanisms used by Y. pestis to subvert killing by the macrophage are unknown. Host Rab GTPases are central mediators of vesicular trafficking and are commonly targeted by bacterial pathogens to alter phagosome maturation and killing by macrophages. Here we demonstrate for the first time that host Rab1b is required for Y. pestis to effectively evade killing by macrophages. We also show that Rab1b is specifically recruited to the Yersinia containing vacuole (YCV) and that Y. pestis is unable to subvert YCV acidification when Rab1b expression is knocked down in macrophages. Furthermore, Rab1b knockdown also altered the frequency of association between the YCV with the lysosomal marker Lamp1, suggesting that Rab1b recruitment to the YCV directly inhibits phagosome maturation. Finally, we show that Rab1b knockdown also impacts the pH of the Legionella pneumophila containing vacuole, another pathogen that recruits Rab1b to its vacuole. Together these data identify a novel role for Rab1b in the subversion of phagosome maturation by intracellular pathogens and suggest that recruitment of Rab1b to the pathogen containing vacuole may be a conserved mechanism to control vacuole pH.

Selective requirement of the shikimate pathway of Legionella pneumophila for intravacuolar growth within human macrophages but not within Acanthamoeba.

Legionella pneumophila utilizes the Dot/Icm type IV translocation system to proliferate within a vacuole in a wide variety of natural amoebal hosts and in alveolar macrophages of the human accidental host. Although L. pneumophila utilizes host amino acids as the main sources of carbon and energy, it is not known whether de novo synthesis of amino acids by intravacuolar L. pneumophila contributes to its nutrition. The aroB and aroE genes encode enzymes for the shikimate pathway that generates the aromatic amino acids Phe, Trp, and Tyr. Here we show the aroB and aroE mutants of L. pneumophila to be defective in growth in human monocyte-derived macrophages (hMDMs) but not in Acanthamoeba spp. The aroB and aroE mutants are severely attenuated in intrapulmonary proliferation in the A/J mouse model of Legionnaires' disease, and the defect is fully complemented by the respective wild-type alleles. The two mutants grow normally in rich media but do not grow in defined media lacking aromatic amino acids, and the growth defect is rescued by inclusion of the aromatic amino acids, which are essential for production of the pyomelanin pigment. Interestingly, supplementation of infected hMDMs with the three aromatic amino acids or with Trp alone rescues the intramacrophage defect of the aroE but not the aroB mutant. Therefore, the shikimate pathway of L. pneumophila is differentially required for optimal growth within human macrophages, which are auxotrophic for Trp and Phe, but is dispensable for growth within the Acanthamoeba spp. that synthesize the aromatic amino acids.