Pedro Escoll

Targeting of host organelles by pathogenic bacteria: a sophisticated subversion strategy

Many bacterial pathogens have evolved the ability to subvert and exploit host functions in order to enter and replicate in eukaryotic cells. For example, bacteria have developed specific mechanisms to target eukaryotic organelles such as the nucleus, the mitochondria, the endoplasmic reticulum and the Golgi apparatus. In this Review, we highlight the most recent advances in our understanding of the mechanisms that bacterial pathogens use to target these organelles. We also discuss how these strategies allow bacteria to manipulate host functions and to ultimately enable bacterial infection.

Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy.

Significance Legionella pneumophila is the causative agent of Legionnaires’ disease. It translocates a large repertoire of effectors into the host cell through a specialized secretion system to subvert cellular defenses. A key characteristic of this pathogen is that the majority of its effectors are encoded by eukaryotic-like genes acquired through horizontal gene transfer. We determined the crystal structure of one of these effectors, sphingosine-1 phosphate lyase (LpSpl), and show that it has high similarity with its eukaryotic homologue. We demonstrate that LpSpl possesses lyase activity and that it disrupts sphingolipid metabolism in the host cells. LpSpl plays a critical and previously unknown role in decreasing autophagy and is a unique virulence factor facilitating intracellular replication of L. pneumophila. Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen’s Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.

Ordering human CD34+CD10−CD19+ pre/pro-B-cell and CD19− common lymphoid progenitor stages in two pro-B-cell development pathways

Studies here respond to two long-standing questions: Are human “pre/pro-B” CD34+CD10−CD19+ and “common lymphoid progenitor (CLP)/early-B” CD34+CD10+CD19− alternate precursors to “pro-B” CD34+CD19+CD10+ cells, and do the pro-B cells that arise from these progenitors belong to the same or distinct B-cell development pathways? Using flow cytometry, gene expression profiling, and Ig VH-D-JH sequencing, we monitor the initial 10 generations of development of sorted cord blood CD34highLineage− pluripotential progenitors growing in bone marrow S17 stroma cocultures. We show that (i) multipotent progenitors (CD34+CD45RA+CD10−CD19−) directly generate an initial wave of Pax5+TdT− “unilineage” pre/pro-B cells and a later wave of “multilineage” CLP/early-B cells and (ii) the cells generated in these successive stages act as precursors for distinct pro-B cells through two independent layered pathways. Studies by others have tracked the origin of B-lineage leukemias in elderly mice to the mouse B-1a pre/pro-B lineage, which lacks the TdT activity that diversifies the VH-D-JH Ig heavy chain joints found in the early-B or B-2 lineage. Here, we show a similar divergence in human B-cell development pathways between the Pax5+TdT− pre/pro-B differentiation pathway that gives rise to infant B-lineage leukemias and the early-B pathway.

Modulation of Host Autophagy during Bacterial Infection: Sabotaging Host Munitions for Pathogen Nutrition.

Cellular homeostasis requires the balanced regulation of anabolic and catabolic processes. While anabolic metabolism consumes energy to build up cellular components, catabolic processes break down organic matters in order to provide energy for the cell and its anabolic processes. Autophagy is a highly conserved and regulated catabolic process by which the eukaryotic cell degrades unnecessary, undesirable, or dysfunctional cellular components, including organelles (1–3). Autophagy is induced by a variety of extra-and intracellular stress stimuli, such as nutrient starvation, oxidative stress, or accumulation of damaged organelles or toxic protein aggregates. Initiation of autophagy first leads to the formation of cup-shaped structures known as phagophores that engulf the undesirable or damaged cellular components. Subsequent elongation of phagophores form double-membrane vesicles called autophagosomes, which deliver their cargo to lysosomes where the content is degraded and recycled (1–3). Autophagy plays a central role in quality control of organelles and proteins, and additionally is a key mechanism to maintain cellular energy levels and nutrient homeostasis during starvation, promoting the recycling and salvage of cellular nutrients. Furthermore, the cellular autophagic machinery is also used to remove invading intracellular pathogens, a process called xenophagy (1, 2). In this case, phagophores engulf invading microbes forming autophagosomes and steering them toward lysosomal degradation. Thus, xenophagy is an innate immune mechanism against bacterial infection that has been shown to be essential to restrict intracellular growth of many bacteria such as Salmonella enterica serovar Typhimurium (4), Mycobacterium tuberculosis (5, 6), Listeria monocytogenes (7), or Group A Streptococcus (8).

Legionella pneumophila restrains autophagy by modulating the host's sphingolipid metabolism

ABSTRACT Sphingolipids are bioactive molecules playing a key role as membrane components, but they are also central regulators of many intracellular processes including macroautophagy/autophagy. In particular, sphingosine-1-phosphate (S1P) is a critical mediator that controls the balance between sphingolipid-induced autophagy and cell death. S1P levels are adjusted via S1P synthesis, dephosphorylation or degradation, catalyzed by SGPL1 (sphingosine-1-phosphate lyase 1). Intracellular pathogens are able to modulate many different host cell pathways to allow their replication. We have found that infection of eukaryotic cells with the human pathogen Legionella pneumophila triggers a change in the host cell sphingolipid metabolism and specifically affects the levels of sphingosine. Indeed, L. pneumophila secretes a protein highly homologous to eukaryotic SGPL1 (named LpSPL). We solved the crystal structure of LpSPL and showed that it encodes lyase activity, targets the hosts sphingolipid metabolism, and plays a role in starvation-induced autophagy during L. pneumophila infection to promote intracellular survival.

Legionella longbeachae Is Immunologically Silent and Highly Virulent In Vivo

Background Legionella longbeachae (Llo) and Legionella pneumophila (Lpn) are the most common pneumonia-causing agents of the genus. Although both species can be lethal to humans and are highly prevalent, little is known about the molecular pathogenesis of Llo infections. In murine models of infection, Lpn infection is self-limited, whereas Llo infection is lethal. Methods We used mouse macrophages, human macrophages, human epithelial cells, and mouse infections in vivo to evaluate multiple parameters of the infection. Results We determined that the Llo Dot/Icm secretion system is critical for virulence. Different than Lpn, Llo disseminates and the animals develop a severe pulmonary failure, as demonstrated by lung mechanics and blood oxygenation assays. As compared to Lpn, Llo is immunologically silent and fails to trigger the production of cytokines in human pulmonary epithelial cells and in mouse and human macrophages. Infections in Tnfr1-/-, Ifng-/-, and Il12p40-/- mice supported the participation of cytokines for the resistance phenotype. Conclusions Both Lpn and Llo require the Dot/Icm system for pathogenesis, but the infection outcome is strikingly different. Llo is immunologically silent, highly virulent, and lethal. The differences reported herein may reflect unappreciated clinical differences in patients infected with Lpn or Llo.

Legionella pneumophila CsrA regulates a metabolic switch from amino acid to glycerolipid metabolism

Legionella pneumophila CsrA plays a crucial role in the life-stage-specific expression of virulence phenotypes and metabolic activity. However, its exact role is only partly known. To elucidate how CsrA impacts L. pneumophila metabolism we analysed the CsrA depended regulation of metabolic functions by comparative 13C-isotopologue profiling and oxygen consumption experiments of a L. pneumophila wild-type (wt) strain and its isogenic csrA− mutant. We show that a csrA− mutant has significantly lower respiration rates when serine, alanine, pyruvate, α-ketoglutarate or palmitate is the sole carbon source. By contrast, when grown in glucose or glycerol, no differences in respiration were detected. Isotopologue profiling uncovered that the transfer of label from [U-13C3]serine via pyruvate into the citrate cycle and gluconeogenesis was lower in the mutant as judged from the labelling patterns of protein-derived amino acids, cell-wall-derived diaminopimelate, sugars and amino sugars and 3-hydroxybutyrate derived from polyhydroxybutyrate (PHB). Similarly, the incorporation of [U-13C6]glucose via the glycolysis/Entner–Doudoroff (ED) pathway but not via the pentose phosphate pathway was repressed in the csrA− mutant. On the other hand, fluxes due to [U-13C3]glycerol utilization were increased in the csrA− mutant. In addition, we showed that exogenous [1,2,3,4-13C4]palmitic acid is efficiently used for PHB synthesis via 13C2-acetyl-CoA. Taken together, CsrA induces serine catabolism via the tricarboxylic acid cycle and glucose degradation via the ED pathway, but represses glycerol metabolism, fatty acid degradation and PHB biosynthesis, in particular during exponential growth. Thus, CsrA has a determining role in substrate usage and carbon partitioning during the L. pneumophila life cycle and regulates a switch from amino acid usage in replicative phase to glycerolipid usage during transmissive growth.

MAMs are attractive targets for bacterial repurposing of the host cell: MAM-functions might be key for undermining an infected cell

Pathogenic bacteria frequently target the endoplasmic reticulum (ER) and mitochondria in order to exploit host functions. ER‐mitochondria inter‐organelle communication is topologically sub‐compartmentalized at mitochondria‐associated ER membranes (MAMs). MAMs are specific membranous microdomains with unique regulatory functions such as lipid synthesis and trafficking, calcium homeostasis, mitochondrial morphology, inflammasome activation, autophagosome formation, and apoptosis. These important cellular processes are all modulated by pathogens to subvert host functions and promote infection, thus it is tempting to assume that pathogenic bacteria target MAMs to subvert these different pathways in their hosts. First lines of evidence that support this hypothesis come from Legionella pneumophila. This intracellular bacterium secretes an effector that exhibits sphingosine‐1 phosphate lyase activity (LpSpl) that seems to target MAMs to modulate the autophagy response to infection. Here we thus propose the concept that MAMs could be targeted by pathogenic bacteria to undermine key host cellular processes.

Sustained Interleukin-1β Exposure Modulates Multiple Steps in Glucocorticoid Receptor Signaling, Promoting Split-Resistance to the Transactivation of Prominent Anti-Inflammatory Genes by Glucocorticoids

Clinical treatment with glucocorticoids (GC) can be complicated by cytokine-induced glucocorticoid low-responsiveness (GC-resistance, GCR), a condition associated with a homogeneous reduction in the expression of GC-receptor- (GR-) driven anti-inflammatory genes. However, GR level and phosphorylation changes modify the expression of individual GR-responsive genes differently. As sustained IL-1β exposure is key in the pathogenesis of several major diseases with prevalent GCR, we examined GR signaling and the mRNA expression of six GR-driven genes in cells cultured in IL-1β and afterwards challenged with GC. After a GC challenge, sustained IL-1β exposure reduced the cytoplasmic GR level, GRSer203 and GRSer211 phosphorylation, and GR nuclear translocation and led to selective GCR in the expression of the studied genes. Compared to GC alone, in a broad range of GC doses plus sustained IL-1β, FKBP51 mRNA expression was reduced by 1/3, TTP by 2/3, and IRF8 was completely knocked down. In contrast, high GC doses did not change the expression of GILZ and DUSP1, while IGFBP1 was increased by 5-fold. These effects were cytokine-selective, IL-1β dose- and IL-1R1-dependent. The integrated gain and loss of gene functions in the “split GCR” model may provide target cells with a survival advantage by conferring resistance to apoptosis, chemotherapy, and GC.

Metabolic reprogramming of host cells upon bacterial infection: Why shift to a Warburg‐like metabolism?

The finding that the Warburg effect observed in proliferating cancer cells is also observed during immune responses renewed the interest in the study of metabolic reprogramming of immune cells, a field of investigation called immunometabolism. However, the specific mechanisms and processes underlying metabolic changes of host cells upon bacterial infection remain poorly understood. Several recent reports have reported that mammalian cells infected with intracellular bacteria have an altered metabolism that resembles the Warburg effect seen in cancer cells. In this Review, we will summarize current knowledge on metabolic reprogramming and discuss putative causes underlying the preferential remodelling of host cells to Warburg‐like metabolic programs during infection by intracellular bacteria.