Abstract
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.
Similar content being viewed by others
Introduction
Legionnaire’s disease, an atypical pneumonia, is a result of inhalation of the bacteria Legionella pneumophila1,2,3. Within the human host, L. pneumophila primarily reside and replicate within alveolar macrophages4,5,6. Infection of humans is considered to be “accidental”, as the natural hosts for L. pneumophila are protozoa in the aquatic environment7,8. Growth within either host occurs through manipulation of evolutionarily conserved pathways, to avoid fusion to the lysosomes and to remodel the vacuole to become ER-derived, which is designated as the Legionella-containing vacuole (LCV)9,10,11,12,13,14. The Dot/Icm type IVb translocation system, which translocates >320 effector proteins into the host cytosol, is required for biogenesis of the LCV15 and for successful intracellular replication in macrophages and amoebae13,16,17,18,19, and subsequent killing of the host cell20. A plethora of host cell processes are modulated by the translocation of redundant effector proteins21 that allow L. pneumophila to evade innate immunity and acquire nutrients22,23,24,25.
L. pneumophila relies on host amino acids (such as, serine, cysteine, and alanine) to feed into tricarboxylic acid (TCA) cycle as the main source of carbon and energy26,27,28. The bacteria are in such high demand for amino acids that endogenous amounts within the host are below the threshold needed to support robust intracellular replication5,22,29. To raise host cellular levels of amino acids, L. pneumophila translocates the AnkB effector, which is post-translationally modified by the host cell30,31,32, and hijacks the host ubiquitination-proteasome machinery to degrade proteins22,33,34,35, but the host cell also undergo metabolic reprograming in response to infection36.
Early studies pointed to a preference for amino acids as an energy source and in vitro studies identified auxotrophies for seven amino acids in L. pneumophila: threonine, arginine, isoleucine, methionine, leucine, cysteine, and valine26,37. Many of these auxotrophies are shared with the amoeba host which may allow the bacterium to synchronize growth with that of the host, avoiding deleterious growth during times of environmental stress38,39,40. Legionella can enter into a viable but non-culturable (VNBC) state when encountering nutritional stress, which has only been shown to be recovered by co-culturing with amoebae41.
Nutritional virulence studies on L. pneumophila29,42,43 have focused on the generation and utilization of amino acids22,27,42,44,45. Only recently has glucose metabolism been studied for its role during intracellular replication46,47. However, supplementation of glucose in vitro does not enhance growth of L. pneumophila47. Glycolysis plays a minimal role in glucose catabolism, but is predominately metabolized through the ED pathway while the pentose phosphate pathway (PPP) functions only to generate mannose and histidine46,47,48. A gene cluster encoding enzymes for glucose catabolism, through the Entner-Doudoroff (ED) pathway of L. pneumophila has been shown to be required for growth in the A549 epithelial cell line, A/J mouse macrophages, and Acanthamoeba culbertsoni, indicating the importance of the ED pathway in intracellular replication of L. pneumophila47. Initial studies focused on poly-3-hydroxybuyterate (PHB), a 4-carbon storage molecule that is generated by metabolizing glucose, through the ED pathway into pyruvate, which gets converted into acetyl-CoA, then PHB49. PHB is synthesized in late stages of growth and catabolized during stationary growth into acetyl-CoA to feed into the TCA cycle49,50. The primary usage of glucose by L. pneumophila is considered to be conversion into PHB46,50.
How nutrients are imported by L. pneumophila is not well understood51,52. To date, only one amino acid transporter, PhtA, of L. pneumophila has been shown to import threonine and is required for intracellular replication in macrophages53. Given that numerous genes in L. pneumophila have been acquired by inter-kingdom horizontal gene transfer from eukaryotic hosts, we sought to identify nutrient transporters in L. pneumophila based on their similarity to the human solute carrier (SLCs) transporters due to the lack of well annotated amoebal genomes54,55. This superfamily of transporters consists of over 65 families, grouped based on substrate specificity and tissue tropism56. They are considered to be part of a larger, evolutionarily conserved group of transporters known as the Major Facilitator Superfamily (MFS)57,58.
Eleven putative amino acid SLC-like transporters were identified, including a citrate transporter, 7 amino acids transporters and 2 glucose transporters. We focused our studies on the two putative SLC-like glucose transporters, LstA and LstB, to further understand the import of glucose and its role in intracellular replication of L. pneumophila.
Results
Identification of human SLC-like transporters in L. pneumophila
We utilized BLAST search of the L. pneumophila Philadelphia strain genome against all human SLC transporters of amino acids and glucose. In addition, since L. pneumophila utilizes pyruvate and citrate to feed its TCA cycle, we search for similarity of the L. pneumophila genome to the mono and tri-carboxylates SLC13 and 25 transporters22,26. Using the primary amino acid sequences from the human amino acid transporter families, SLC1, 3, 7, 17, 36, 38, and 43 against the genome of L. pneumophila strain AA100/130b, eight putative SLC-like amino acid transporters in L. pneumophila were identified by BLAST with similarity of 56–42% and identity of 25–37% (Table 1). In addition, one putative SLC-like transporter of tricarboxylates similar to the SLC13 family, lpg2876 (24%/51%), was identified. In addition, using the primary amino acid sequence from the human SLC2 and SLC5 family, we identified two putative SLC-like glucose transporters, lpg0421 (33%/50%) and lpg1653 (30%/48%) (Table 1). Structural modeling of these proteins was done using the Iterative Threading Assembly Refinement (I-TASSER) server, which is a bioinformatics algorithm for predicting three-dimensional structure based on fold recognition59,60,61. Structural alignment was performed using TM-align, an algorithm that uses known or predicted protein structure to align proteins and determine structural similarity59,60,61. A TM-score is given for each alignment where, 1.0 indicates an exact copy, 0.0 −<0.3 indicates random structural similarity, and 0.5 −<1.0 indicating shared structural topology61. L. pneumophila proteins were compared to the human SLCs to determine structural homology, as measured by TM-scores (Table 1). Structural comparisons of these SLC-like transporters of L. pneumophila with human SLCs showed high structural similarity (TM-scores 0.78–0.977) (Table 1 and see Supplementary Fig. S1). Few homologs of mammalian SLCs have been identified within amoebae, which also can be used to identify L. pneumophila transporters; these are designated as CtrABC in Dictyostelium discodium (see Supplementary Fig. S2). We have designated these putative transporters as Legionella SLC-like transporters, LstA-K (Table 1).
Since the uptake and the role of glucose in intracellular growth and metabolism of L. pneumophila is not well understood, we focused our studies on the two putative SLC-like transporters of L. pneumophila that shared strong structural similarity to SLC2a1/Glut1, LstA and LstB (Fig. 1a,b). LstA of L. pneumophila and Glut1 of humans have a TM-score of 0.903, and LstB and Glut1 a TM-score of 0.922, indicating very strong structural similarity (Fig. 1a,b,d). Members within the Glut family do not share this degree of similarity (Glut1 and Glut3, TM-score of 0.88). When comparing alignment of LstA to LstB, the TM-score is 0.959, indicating potential redundancy (Fig. 1c,d). These two L. pneumophila proteins are smaller in size than their human counterpart proteins but the secondary structural alignment is conserved. LstA and LstB are likely members of the major facilitator superfamily (MFS), which are important transporters that have been maintained, in all domains of life, with little deviation through evolutionary history57. LstA (Lpg0421) has been previously designated as YwtG, but based on its eukaryotic SLC-like structure and function (see below) and as one of a large family of eukaryotic SLC-like proteins in L. pneumophila, we have designated it as LstA47.
Glucose import by LstA and LstB
Predicted substrate binding, by I-TASSER, for LstA and LstB indicate glucose as a putative substrate59,60. Given their high degree of structural similarity to Glut1, we hypothesized that both of these putative transporters were involved in the transport of glucose of L. pneumophila. To test if either LstA or LstB were required for the uptake of glucose, null mutants were generated and uptake of glucose was analyzed by liquid scintillation using 14C-glucose. L. pneumophila strains were grown to post-exponential phase in the presence of 0.1% uniformly labelled 14C-glucose. Broth grown WT L. pneumophila was able to effectively take up 14C-glucose in vitro (Fig. 2). As a control, excess, unlabeled glucose (10 mM) was added, which abolished uptake of 14C-glucose (Student t-test, p < 0.001) (Fig. 2). The lstA and lstB mutants had significantly reduced uptake of 14C-glucose compared to the WT strain (Student t-test, p < 0.001), but glucose uptake was more reduced in the lstA mutant (Fig. 2). Complementation of the single mutants with the respective gene on a plasmid (lstA.C and lstB.C) restored uptake of glucose to that of the WT strain levels (Fig. 2).
To determine whether LstA and LstB were redundant, a lstA/lstB double mutant, was generated. Loss of both transporters abolished uptake of 14C-glucose compared to the WT strain (Student t-test, p < 0.001) (Fig. 2). Upon supplementation of excess, unlabeled glucose (10 mM) uptake of labeled glucose was inhibited in the complemented double mutants (Student t-test, p < 0.001) (Fig. 2). Interestingly, complementation with a single transporter, (lstA.C or lstB.C) restored uptake of 14C-glucose to the double mutant similar to the WT strain levels (Fig. 2). These data show that LstA and LstB are glucose transporters.
The LstA and LstB glucose transporters are required for growth in Acanthamoeba polyphaga and human monocyte-derived macrophages
We determined intracellular replication of glucose transporter single mutants, lstA and lstB, and the double mutant lstA/lstB in human monocyte-derived macrophages (hMDMs) and A. polyphaga. Single transporter null mutants, lstA and lstB, replicated similarly to WT L. pneumophila in A. polyphaga or hMDMs (Figs 3a and 4a), which is consistent with the idea that they are redundant transporters. Given that the lstA/lstB double mutant resulted in a severely diminished uptake of glucose, we determined the ability of the double mutant to replicate intracellularly. In vitro cultures of lstA/lstB grew similarly to the WT strain (see Supplementary Fig. S3).
Interestingly the lstA/lstB double mutant was severely defective for growth in A. polyphaga and hMDMs (Two-way ANOVA, p < 0.001) (Figs 3b and 4b). Complementation of the double mutant, with individual single transporters, lstA.C or lstB.C, restored intracellular growth of the double mutant in A. polyphaga and hMDMs to almost that of WT L. pneumophila (Figs 3b and 4b). These data show that the two glucose transporters, LstA and LstB, are required for intracellular growth of L. pneumophila within hMDMs and A. polyphaga and these two transporters are most likely to be redundant in their function to import glucose47. Our data show that uptake of glucose is required for intracellular replication of L. pneumophila within evolutionarily distant host cells.
Discussion
L. pneumophila generates copious amounts of host amino acids for carbon and energy but it is also reliant on host glucose46,47,62. Hauslein et al. described L. pneumophila metabolism as being “bipartite”, where amino acids serve as the major energy supply in the exponential phase and carbohydrates at the post-exponential phase are used in anabolic processes48. The role of glucose during intracellular infection can be difficult to study; methods for altering the levels of glucose affect the host cells, which may have detrimental effects on intracellular growth of L. pneumophila independent of the glucose level. The glucose analog, 2-deoxy-D-glucose (2-DG) causes autophagy in macrophages63. Increasing the levels of glucose in macrophages increases the inflammatory response, while starving cells of mimics glucose treatment with 2-DG64,65. Therefore, removing the L. pneumophila’s ability to access glucose by deletion of glucose transporters will best highlight the intracellular need for glucose without altering the host cell response to infection.
Our findings indicate that L. pneumophila utilizes two redundant glucose transporters, LstA and LstB, both of which transport glucose, and are required for growth within hMDMs and A. polyphaga. Surprisingly, the need for intracellular glucose is immediate, despite the fact that glucose is thought to be imported by L. pneumophila at the post-exponential phase and is thought to be primarily used in the late stages of growth for PHB synthesis46,47,49,50. Consistent with this idea, lstA is highly upregulated in the post-exponential growth phase in vitro, when glucose is being utilized47,66. However, lstB expression remains unchanged throughout the growth phases66. This could represent dual usages for glucose; LstB could transport low basal levels of glucose throughout intracellular growth while LstA transports large amounts of glucose when the demand has increased during late stages of growth46,47. Interestingly, LstA has been shown to be induced intracellularly during post-exponential growth of L. pneumophila in THP and Acanthamoeba castellanii, relative to growth in vitro66,67. This supports the idea that glucose is required for intracellular replication but not for in vitro growth of L. pneumophila47. However, loss of either of the two transporters, lstA or lstB, is not sufficient to affect intracellular growth, which is most likely due to functional redundancy.
LstA is situated adjacent to a glucose utilization gene cluster, which is important for the catabolism of glucose via the ED pathway46,47. Conflicting reports have shown that the gene cluster is required for growth in A549, A/J mouse macrophages, and A. culbertsoni, when mutated in L. pneumophila strain AM51147. However, in the Paris strain of L. pneumophila, deletion of one of the genes, zwf, does not result in a growth defect in Acanthamoeba castellanii46. Additionally, the genetic organization of the gene cluster in the AM511 and the Paris strains is different46,47. The lstA gene is 113 bp downstream of the 3′ end of eda of the glucose utilization gene cluster, which is sufficient for lstA to have its own promotor. The genetic organization of lstA is conserved among the Paris and AM511 strains, as well as our strain, AA100/130b.
The glucose transporter LstB/Lpg1653 is part of a myo-inositol catabolism gene cluster in L. pneumophila68. Although, LstB has been shown to transport inositol, inositol transporters are capable of transporting glucose, which is molecularly similar and acts as a competitive inhibitor of myo-inositol transport68,69,70. Therefore, it is possible that LstB has dual, or multi-, substrate specificity. Within L. pneumophila, myo-inositol is also metabolized into acetyl-CoA, which could also support the generation of PHB68.
Supplementation of glucose in vitro does not enhance the growth of L. pneumophila47. This may suggest the intracellular requirement for glucose does not support replication as a source of carbon and energy. Given that PHB is generated from glucose and is essential for survival outside of the host, lack of glucose uptake could be triggering stress response genes that prevent L. pneumophila from replicating49. If this were the case, starving host cells of glucose, would prevent the replication of intracellular L. pneumophila; however, starving host cells of glucose triggers cell death by autophagy71. Glucose is an important requirement for generating a reactive oxygen species (ROS) by amoebae and human macrophages in response to invading pathogens64,65. Uptake of glucose by L. pneumophila could serve dual purposes of sequestering glucose from the host, to dampen the immune response and to provide the precursor for PHB.
In summary, we have identified two redundant glucose transporters, LstA and LstB, which are required for intracellular replication of L. pneumophila in macrophages and amoebae. The requirement for glucose uptake by L. pneumophila is essential for intracellular growth in hMDMs and A. polyphaga, but not during growth in vitro. This presents an interesting question in L. pneumophila biology; why is glucose import required only during intracellular replication? This should be the focus of future studies.
Materials and Methods
Strains and cell lines
L. pneumophila strain AA100/130b (ATCC BAA-74) and the T4SS-deficient mutant, dotA were grown on Buffered Charcoal Yeast Extract (BCYE) agar, as we previously described72. To generate isogenic mutants in lstA and lstB, ~2 kb of flanking DNA on either side was amplified using primers listed in Table S2 and cloned into the shuttle vector, pBCSK + lstAKO and pBCSK + lstBKO (Table S1). The entire gene of either lstA or lstB was deleted via inverse PCR using the primers listed in Table S2, resulting in pBCSK + lstAKOi and pBCSK + lstBKOi (Table S1). The kanamycin cassette from the Ez-Tn5 transposon was amplified using primers listed in Table S2 and the resulting PCR product was subcloned into in pBCSK + lstAKOi and pBCSK + lstBKOi between the flanking regions of either lstA or lstB, using standard molecular procedures, resulting in pBCSK + lstAKAN and pBCSK + lstBKAN (Table S1). Each resulting plasmid was independently introduced into L. pneumophila AA100/130b via natural transformation, as we previously described73. After three days, natural transformants were recovered by plated on BCYE supplemented with 50 μg/ml kanamycin, to generate L. pneumophila lstA and L. pneumophila lstB (Table S2). To confirm deletion of either lstB or lstB, the forward primer for sequencing and the reverse primer for generation of the knockout, listed in Table S2 were used. To generate double mutants, a gentamycin cassette was amplified using primers listed in Table S2 and the resulting PCR product was subcloned into in pBCSK + lstAKOi between the flanking regions of either lstA, using standard molecular procedures, resulting in in pBCSK + lstAGENT (Table S1). The resulting plasmid was introduced into L. pneumophila lstB via natural transformation, as previously described73. After three days, natural transformants were recovered by plated on BCYE supplemented with 20 μg/ml kanamycin and 5 μg/ml gentamycin, to generate L. pneumophila lstB/lstA (Table S1). Deletions were confirmed using the same primers as described above.
To generate complement mutants of single deletions and double deletions, lstA or lstB with flanking upstream and downstream sequences were amplified by PCR using the primers listed in Table S2, and subcloned into pBCSK+, generating pBCSK + lstA.C and pBCSK-lstB.C (Table S1). The pBCSK + lstA.C plasmid was introduced into the lstA and lstA/lstB mutants and the pBCSK + lstB.C plasmid was introduced into the lstB and lstA/lstB mutants via electroporation as previously described (Table S1)74. All complement mutants were selected on BCYE plates supplemented with 5 μg/ml chloramphenicol, resulting in the following complement strains: lstA.C, lstB.C, lstA/lstB lstA.C, and lstA/lstB lstB.C (Table S1).
Human monocyte-derived macrophages (hMDMs) were isolated from healthy adult donors and cultured in RPMI 1640 (Corning) supplemented with 10% fetal bovine serum, as previously described72. All methods were carried out and approved in accordance to the University of Louisville Institutional Review Board guidelines and blood donors gave informed consent as required by the University of Louisville Institutional Review Board (IRB # 04.0358). U937 cells were cultured in RPMI 1640 (Corning) supplemented with 10% fetal bovine serum and A. polyphaga was cultured in PYG media, experiments were performed in PY media, as we previously described72.
Structural comparison of glucose transporters
Predicted structures were generated via I-TASSER server from the Zhang Lab (https://zhanglab.ccmb.med.umich.edu/I-TASSER/)59,60,75. Structures generated from I-TASSER were aligned using TM-align to determine structural alignment and TM-scores for similarity (https://zhanglab.ccmb.med.umich.edu/TM-align/)61,76.
Glucose uptake assay
Uptake of glucose was assayed by growing WT L. pneumophila, lstA, lstB, and complemented mutant strains individually in the presence of 14C-label glucose (specific activity, 3.3 MBq/μmol) (PerkinElmer) and the presence of glucose followed into the acid-insoluble fraction, as previously described47. One milliliter cultures were grown in Buffered Yeast Extract (BYE) broth supplemented with 0.1% D-[U-14C] glucose (specific activity, 3.3 MBq/μmol) at 37 °C, shaking to post-exponential phase (>OD550 2.0). For control, 10 mM sterile glucose was added to broth cultures with 0.1% D-[U-14C] glucose. Samples normalized to 109 bacteria in 5 ml 1% Triton X-100 for 30 mins, and then incubated for 30 mins with 5 ml of chilled 10% (w/v) trichloroacetic acid, on ice. To capture radioactivity, samples were filtered through nitrocellulose filters (0.45-μm pore size; Milipore) and rinsed three times with chilled 5% trichloroacetic acid. Radioactivity of the whole sample was determined by liquid scintillation (Tri-Carb 2910 TR, PerkinElimer) with BetaBlend scintillation cocktail (MP Biochemical).
Intracellular replication
L. pneumophila; the isogenic single mutants, dotA, lstA and lstB; the double isogenic mutant lstB/lstA; and the complement mutants lstA.C, lstB.C, lstB/lstA lstB.C, and lstB/lstA lstA.C were grown to post-exponential phase on BCYE plates at 37 °C prior to infection and used to infect hMDMs or A. polyphaga, as previously described72,77. A total of 1 × 105 host cells per well were plated into 96 well plates and infected with L. pneumophila at an MOI of 10 for 1 h then treated with gentamycin to kill remaining extracellular bacteria, as previously described72,77. Host cells were lysed with sterile water (hMDMs) or 0.02% Triton X-100 (A. polyphaga) at various timepoints over a 24 h timecourse and L. pneumophila CFUs were determined by plating serial dilutions onto BCYE agar.
Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary Information files).
References
McDade, J. E. et al. Legionnaires’ disease: isolation of a bacterium and demonstration of its role in other respiratory disease. N.Engl.J.Med. 297, 1197–1203 (1977).
Fraser, D. W. et al. Legionnaires’ disease: description of an epidemic of pneumonia. N.Engl.J.Med. 297, 1189–1197 (1977).
Tsai, T. F. & Fraser, D. W. The diagnosis of Legionnaires’ disease. Ann.Intern.Med. 89, 413–414 (1978).
Molmeret, M., Horn, M., Wagner, M., Santic, M. & Abu Kwaik, Y. Amoebae as training grounds for intracellular bacterial pathogens. Appl. Environ. Microbiol. 71, 20–28 (2005).
Richards, A. M., Von Dwingelo, J. E., Price, C. T. & Abu Kwaik, Y. Cellular microbiology and molecular ecology of Legionella-amoeba interaction. Virulence 4, 307–314, https://doi.org/10.4161/viru.24290 (2013).
Nash, T. W., Libby, D. M. & Horwitz, M. A. Interaction between the legionnaires’ disease bacterium (Legionella pneumophila) and human alveolar macrophages. Influence of antibody, lymphokines, and hydrocortisone. J.Clin.Invest. 74, 771–782 (1984).
Shuman, H. A., Purcell, M., Segal, G., Hales, L. & Wiater, L. A. Intracellular multiplication of Legionella pneumophila: Human pathogen or accidental tourist? Curr.Top.Microbiol.Immunol. 225, 99–112 (1998).
Boamah, D. K., Zhou, G., Ensminger, A. W. & O’Connor, T. J. From Many Hosts, One Accidental Pathogen: The Diverse Protozoan Hosts of Legionella. Frontiers in Cellular and Infection Microbiology 7, https://doi.org/10.3389/fcimb.2017.00477 (2017).
Segal, G. & Shuman, H. A. Legionella pneumophila utilizes the same genes to multiply within Acanthamoeba castellanii and human macrophages. Infect.Immun. 67, 2117–2124 (1999).
Horwitz, M. A. The Legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J.Exp.Med. 158, 2108–2126 (1983).
Horwitz, M. A. Formation of a novel phagosome by the Legionnaires’ disease bacterium (Legionella pneumophila) in human monocytes. J.Exp.Med. 158, 1319–1331 (1983).
Bärlocher, K., Welin, A. & Hilbi, H. Formation of the Legionella Replicative Compartment at the Crossroads of Retrograde Trafficking. Frontiers in Cellular and Infection Microbiology 7, https://doi.org/10.3389/fcimb.2017.00482 (2017).
Swart, A. L., Harrison, C. F., Eichinger, L., Steinert, M. & Hilbi, H. Acanthamoeba and Dictyostelium as Cellular Models for Legionella Infection. Frontiers in Cellular and Infection Microbiology 8, https://doi.org/10.3389/fcimb.2018.00061 (2018).
Allgood, S. C. et al. Legionella Effector AnkX Disrupts Host Cell Endocytic Recycling in a Phosphocholination-Dependent Manner. Front Cell Infect Microbiol 7, 397, https://doi.org/10.3389/fcimb.2017.00397 (2017).
Herweg, J. A. et al. Purification and proteomics of pathogen-modified vacuoles and membranes. Front Cell Infect Microbiol 5, 48, https://doi.org/10.3389/fcimb.2015.00048 (2015).
Coers, J. et al. Identification of icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for legionella pneumophila intracellular growth. Molecular microbiology 38, 719–736 (2000).
Zhu, W. et al. Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila. PLoS One 6, e17638, https://doi.org/10.1371/journal.pone.0017638 (2011).
Burstein, D. et al. Genome-scale identification of Legionella pneumophila effectors using a machine learning approach. PLoS pathogens 5, e1000508, https://doi.org/10.1371/journal.ppat.1000508 (2009).
de Felipe, K. S. et al. Legionella eukaryotic-like type IV substrates interfere with organelle trafficking. PLoS pathogens 4, e1000117, https://doi.org/10.1371/journal.ppat.1000117 (2008).
Casson, C. & Shin, S. Inflammasome-mediated cell death in response to bacterial pathogens that access the host cell cytosol: lessons from legionella pneumophila. Frontiers in Cellular and Infection Microbiology 3, https://doi.org/10.3389/fcimb.2013.00111 (2013).
Ghosh, S. & O’Connor, T. J. Beyond Paralogs: The Multiple Layers of Redundancy in Bacterial Pathogenesis. Front Cell Infect Microbiol 7, 467, https://doi.org/10.3389/fcimb.2017.00467 (2017).
Price, C. T., Al-Quadan, T., Santic, M., Rosenshine, I. & Abu Kwaik, Y. Host proteasomal degradation generates amino acids essential for intracellular bacterial growth. Science 334, 1553–1557, https://doi.org/10.1126/science.1212868 (2011).
Rolando, M. et al. Legionella pneumophila effector RomA uniquely modifies host chromatin to repress gene expression and promote intracellular bacterial replication. Cell Host Microbe 13, 395–405, https://doi.org/10.1016/j.chom.2013.03.004 (2013).
Fontana, M. F. et al. Secreted bacterial effectors that inhibit host protein synthesis are critical for induction of the innate immune response to virulent Legionella pneumophila. PLoS pathogens 7, e1001289, https://doi.org/10.1371/journal.ppat.1001289 (2011).
Luo, Z. Q. Legionella secreted effectors and innate immune responses. Cell Microbiol, https://doi.org/10.1111/j.1462-5822.2011.01713.x (2011).
Tesh, M. J., Morse, S. A. & Miller, R. D. Intermediary metabolism in Legionella pneumophila: utilization of amino acids and other compounds as energy sources. J.Bacteriol. 154, 1104–1109 (1983).
Schunder, E. et al. Amino Acid Uptake and Metabolism of Legionella pneumophila Hosted by Acanthamoeba castellanii. The Journal of biological chemistry 289, 21040–21054, https://doi.org/10.1074/jbc.M114.570085 (2014).
Fonseca, M. V. & Swanson, M. S. Nutrient salvaging and metabolism by the intracellular pathogen Legionella pneumophila. Front Cell Infect Microbiol 4, 12, https://doi.org/10.3389/fcimb.2014.00012 (2014).
Manske, C. & Hilbi, H. Metabolism of the vacuolar pathogen Legionella and implications for virulence. Frontiers in Cellular and Infection Microbiology 4, https://doi.org/10.3389/fcimb.2014.00125 (2014).
Price, C. et al. Host FIH-Mediated Asparaginyl Hydroxylation of Translocated Legionella pneumophila Effectors. Front Cell Infect Microbiol 7, 54, https://doi.org/10.3389/fcimb.2017.00054 (2017).
Qiu, J. & Luo, Z.-Q. Hijacking of the Host Ubiquitin Network by Legionella pneumophila. Frontiers in Cellular and Infection Microbiology 7, https://doi.org/10.3389/fcimb.2017.00487 (2017).
Kubori, T., Bui, X. T., Hubber, A. & Nagai, H. Legionella RavZ Plays a Role in Preventing Ubiquitin Recruitment to Bacteria-Containing Vacuoles. Frontiers in Cellular and Infection Microbiology 7, https://doi.org/10.3389/fcimb.2017.00384 (2017).
Al-Quadan, T. & Kwaik, Y. A. Molecular Characterization of Exploitation of the Polyubiquitination and Farnesylation Machineries of Dictyostelium Discoideum by the AnkB F-Box Effector of Legionella Pneumophila. Frontiers in microbiology 2, 23, https://doi.org/10.3389/fmicb.2011.00023 (2011).
Lomma, M. et al. The Legionella pneumophila F-box protein Lpp2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication. Cell Microbiol 12, 1272–1291, https://doi.org/10.1111/j.1462-5822.2010.01467.x (2010).
Price, C. T., Al-Khodor, S., Al-Quadan, T. & Abu Kwaik, Y. Indispensable role for the eukaryotic-like ankyrin domains of the ankyrin B effector of Legionella pneumophila within macrophages and amoebae. Infection and immunity 78, 2079–2088, https://doi.org/10.1128/IAI.01450-09 (2010).
Eisenreich, W., Heesemann, J., Rudel, T. & Goebel, W. Metabolic host responses to infection by intracellular bacterial pathogens. Front Cell Infect Microbiol 3, 24, https://doi.org/10.3389/fcimb.2013.00024 (2013).
Tesh, M. J. & Miller, R. D. Amino acid requirements for Legionella pneumophila growth. J.Clin.Microbiol. 13, 865–869 (1981).
Price, C. & Abu Kwaik, Y. Amoebae and Mammals Deliver Protein-Rich Atkins Diet Meals to Legionella. Microbe 7, 506–513 (2012).
Price, C. T., Richards, A. M., Von Dwingelo, J. E., Samara, H. A. & Abu Kwaik, Y. Amoeba host-Legionella synchronization of amino acid auxotrophy and its role in bacterial adaptation and pathogenic evolution. Environmental microbiology 16, 350–358, https://doi.org/10.1111/1462-2920.12290 (2014).
Abu Khweek, A. et al. Biofilm-derived Legionella pneumophila evades the innate immune response in macrophages. Frontiers in Cellular and Infection Microbiology 3, https://doi.org/10.3389/fcimb.2013.00018 (2013).
Garcia, M. T., Jones, S., Pelaz, C., Millar, R. D. & Abu Kwaik, Y. Acanthamoeba polyphaga resuscitates viable non-culturable Legionella pneumophila after disinfection. Environmental microbiology 9, 1267–1277, https://doi.org/10.1111/j.1462-2920.2007.01245.x (2007).
Oliva, G., Sahr, T. & Buchrieser, C. The Life Cycle of L. pneumophila: Cellular Differentiation Is Linked to Virulence and Metabolism. Frontiers in Cellular and Infection Microbiology 8, https://doi.org/10.3389/fcimb.2018.00003 (2018).
Eisenreich, W., Rudel, T., Heesemann, J. & Goebel, W. To Eat and to Be Eaten: Mutual Metabolic Adaptations of Immune Cells and Intracellular Bacterial Pathogens upon Infection. Frontiers in Cellular and Infection Microbiology 7, https://doi.org/10.3389/fcimb.2017.00316 (2017).
Abu Kwaik, Y. & Bumann, D. Microbial quest for food in vivo: ‘Nutritional virulence’ as an emerging paradigm. Cell Microbiol 15, 882–890, https://doi.org/10.1111/cmi.12138 (2013).
Bruckert, W. M., Price, C. T. & Abu Kwaik, Y. Rapid nutritional remodeling of the host cell upon attachment of Legionella pneumophila. Infection and immunity 82, 72–82, https://doi.org/10.1128/IAI.01079-13 (2014).
Eylert, E. et al. Isotopologue profiling of Legionella pneumophila: role of serine and glucose as carbon substrates. The Journal of biological chemistry 285, 22232–22243, https://doi.org/10.1074/jbc.M110.128678 (2010).
Harada, E., Iida, K., Shiota, S., Nakayama, H. & Yoshida, S. Glucose metabolism in Legionella pneumophila: dependence on the Entner-Doudoroff pathway and connection with intracellular bacterial growth. Journal of bacteriology 192, 2892–2899, https://doi.org/10.1128/JB.01535-09 (2010).
Hauslein, I., Manske, C., Goebel, W., Eisenreich, W. & Hilbi, H. Pathway analysis using (13) C-glycerol and other carbon tracers reveals a bipartite metabolism of Legionella pneumophila. Molecular microbiology 100, 229–246, https://doi.org/10.1111/mmi.13313 (2016).
James, B. W., Mauchline, W. S., Dennis, P. J., Keevil, C. W. & Wait, R. Poly-3-hydorxyburyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments. Appl.Environ.Microbiol. 65, 822–827 (1999).
Gillmaier, N. et al. Growth-Related Metabolism of the Carbon Storage Poly-3-Hydroxybutyrate in Legionella pneumophila. The Journal of biological chemistry, https://doi.org/10.1074/jbc.M115.693481 (2016).
Lama, A., Drennan, S. L., Johnson, R. C., Rubenstein, G. L. & Cambronne, E. D. Identification of Conserved ABC Importers Necessary for Intracellular Survival of Legionella pneumophila in Multiple Hosts. Frontiers in Cellular and Infection Microbiology 7, https://doi.org/10.3389/fcimb.2017.00485 (2017).
Price, C. T., Richards, A. M. & Abu Kwaik, Y. Nutrient generation and retrieval from the host cell cytosol by intra-vacuolar Legionella pneumophila. Front Cell Infect Microbiol 4, 111, https://doi.org/10.3389/fcimb.2014.00111 (2014).
Sauer, J. D., Bachman, M. A. & Swanson, M. S. The phagosomal transporter A couples threonine acquisition to differentiation and replication of Legionella pneumophila in macrophages. Proceedings of the National Academy of Sciences of the United States of America 102, 9924–9929, https://doi.org/10.1073/pnas.0502767102 (2005).
de Felipe, K. S. et al. Evidence for acquisition of Legionella type IV secretion substrates via interdomain horizontal gene transfer. Journal of bacteriology 187, 7716–7726, https://doi.org/10.1128/JB.187.22.7716-7726.2005 (2005).
Gomez-Valero, L. et al. Extensive recombination events and horizontal gene transfer shaped the Legionella pneumophila genomes. BMC Genomics 12, 536, https://doi.org/10.1186/1471-2164-12-536 (2011).
Schlessinger, A., Yee, S. W., Sali, A. & Giacomini, K. M. SLC classification: an update. Clin Pharmacol Ther 94, 19–23, https://doi.org/10.1038/clpt.2013.73 (2013).
Quistgaard, E. M., Löw, C., Guettou, F. & Nordlund, P. Understanding transport by the major facilitator superfamily (MFS): structures pave the way. Nature Reviews Molecular Cell Biology 17, 123, https://doi.org/10.1038/nrm.2015.25.
Yan, N. Structural Biology of the Major Facilitator Superfamily Transporters. Annual Review of Biophysics 44, 257–283, https://doi.org/10.1146/annurev-biophys-060414-033901 (2015).
Yang, J. et al. The I-TASSER Suite: protein structure and function prediction. Nat Methods 12, 7–8, https://doi.org/10.1038/nmeth.3213 (2015).
Yang, J. & Zhang, Y. Protein Structure and Function Prediction Using I-TASSER. Curr Protoc Bioinformatics 52, 5 8 1–5 8 15, https://doi.org/10.1002/0471250953.bi0508s52 (2015).
Zhang, Y. & Skolnick, J. TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic acids research 33, 2302–2309, https://doi.org/10.1093/nar/gki524 (2005).
Herrmann, V. et al. GamA is a eukaryotic-like glucoamylase responsible for glycogen- and starch-degrading activity of Legionella pneumophila. International journal of medical microbiology: IJMM 301, 133–139, https://doi.org/10.1016/j.ijmm.2010.08.016 (2011).
Matsuda, F., Fujii, J. & Yoshida, S. Autophagy induced by 2-deoxy-D-glucose suppresses intracellular multiplication of Legionella pneumophila in A/J mouse macrophages. Autophagy 5, 484–493 (2009).
Freemerman, A. J. et al. Metabolic Reprogramming of Macrophages: Glucose Transporter 1 (GLUT1)-Mediated Glucose Metabolism Drives a Proinflammatory Phenotype. Journal of Biological Chemistry 289, 7884–7896, https://doi.org/10.1074/jbc.M113.522037 (2014).
Ham, M. et al. Macrophage glucose-6-phosphate dehydrogenase stimulates proinflammatory responses with oxidative stress. Mol Cell Biol 33, 2425–2435, https://doi.org/10.1128/MCB.01260-12 (2013).
Bruggemann, H. et al. Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila. Cell Microbiol 8, 1228–1240, https://doi.org/10.1111/j.1462-5822.2006.00703.x (2006).
Faucher, S. P., Mueller, C. A. & Shuman, H. A. Legionella Pneumophila Transcriptome during Intracellular Multiplication in Human Macrophages. Frontiers in microbiology 2, 60, https://doi.org/10.3389/fmicb.2011.00060 (2011).
Manske, C., Schell, U. & Hilbi, H. Metabolism of myo-Inositol by Legionella pneumophila Promotes Infection of Amoebae and Macrophages. Applied and environmental microbiology 82, 5000–5014, https://doi.org/10.1128/aem.01018-16 (2016).
Fenili, D., Weng, Y.-Q., Aubert, I., Nitz, M. & McLaurin, J. Sodium/myo-Inositol Transporters: Substrate Transport Requirements and Regional Brain Expression in the TgCRND8 Mouse Model of Amyloid Pathology. Plos One 6, e24032, https://doi.org/10.1371/journal.pone.0024032 (2011).
Schneider, S. Inositol transport proteins. FEBS letters 589, 1049–1058, https://doi.org/10.1016/j.febslet.2015.03.012 (2015).
Edinger, A. L. & Thompson, C. B. Death by design: apoptosis, necrosis and autophagy. Current opinion in cell biology 16, 663–669, https://doi.org/10.1016/j.ceb.2004.09.011 (2004).
Al-Khodor, S., Price, C. T., Habyarimana, F., Kalia, A. & Abu Kwaik, Y. A Dot/Icm-translocated ankyrin protein of Legionella pneumophila is required for intracellular proliferation within human macrophages and protozoa. Molecular microbiology 70, 908–923, https://doi.org/10.1111/j.1365-2958.2008.06453.x (2008).
Stone, B. J. & Abu Kwaik, Y. Natural competency for DNA uptake by Legionella pneumophila and its association with expression of type IV pili. J.Bacteriol. 181, 1395–1402 (1999).
Chen, D. Q., Huang, S. S. & Lu, Y. J. Efficient transformation of Legionella pneumophila by high-voltage electroporation. Microbiol Res 161, 246–251, https://doi.org/10.1016/j.micres.2005.09.001 (2006).
Roy, A., Kucukural, A. & Zhang, Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5, 725–738, https://doi.org/10.1038/nprot.2010.5 (2010).
Zhang, Y. & Skolnick, J. Scoring function for automated assessment of protein structure template quality. Proteins 57, 702–710, https://doi.org/10.1002/prot.20264 (2004).
Price, C. T. et al. Molecular mimicry by an F-box effector of Legionella pneumophila hijacks a conserved polyubiquitination machinery within macrophages and protozoa. PLoS pathogens 5, e1000704, https://doi.org/10.1371/journal.ppat.1000704 (2009).
Acknowledgements
The Y.A.K. lab is supported by Public Health Service Awards R01AI120244 from the NIAID and by the Commonwealth of Kentucky Research Challenge Trust Fund. A.B. was supported by a National Science Foundation Graduate Research Fellowship (NSF GRFP) under Grant No. DGE- 1144204.
Author information
Authors and Affiliations
Contributions
Y.A.K. and A.B. conceived the ideas and designed the experiments. A.B. and S.J. performed the experiments. A.B. analysed all the data. A.B. and Y.A.K. wrote the main manuscript text and A.B. prepared all figures. All authors have read and approved of the final manuscript.
Corresponding author
Ethics declarations
Competing Interests
The authors declare no competing interests.
Additional information
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Best, A., Jones, S. & Abu Kwaik, Y. Mammalian Solute Carrier (SLC)-like transporters of Legionella pneumophila. Sci Rep 8, 8352 (2018). https://doi.org/10.1038/s41598-018-26782-x
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-018-26782-x
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.