Molecular mimicry sweetens the deal for MANagement of host mRNA translation and stress responses by Legionella pneumophila.

The Legionella pneumophila effector SidI inhibits host mRNA translation and must be regulated for intracellular replication. Subramanian et al.1 reveal the mechanism of SidI translation inhibition and how stress signaling in response to sustained SidI activity drives host cell death.

Correction: Effector-mediated subversion of proteasome activator (PA)28αß enhances host defense against Legionella pneumophila under inflammatory and oxidative stress conditions.

[This corrects the article DOI: 10.1371/journal.ppat.1011473.].

Effector-mediated subversion of proteasome activator (PA)28αß enhances host defense against Legionella pneumophila under inflammatory and oxidative stress conditions.

Legionella pneumophila is a natural pathogen of amoebae that causes Legionnaires' Disease in immunocompromised individuals via replication within macrophages. L. pneumophila virulence and intracellular replication hinges on hundreds of Dot/Icm-translocated effector proteins, which are essential for biogenesis of the replication-permissive Legionella-containing vacuole (LCV). However, effector activity can also enhance mammalian host defense via effector-triggered immunity. The L. pneumophila effector LegC4 is important for virulence in amoebae but enhances host defense against L. pneumophila in the mouse lung and, uniquely, within macrophages activated with either tumor necrosis factor (TNF) or interferon (IFN)-γ. The mechanism by which LegC4 potentiates cytokine-mediated host defense in macrophages is unknown. Here, we found that LegC4 enhances cytokine-mediated phagolysosomal fusion with Legionella-containing vacuole (LCV) and binds host proteasome activator (PA)28α, which forms a heterooligomer with PA28ß to facilitate ubiquitin-independent proteasomal degradation of oxidant-damaged (carbonylated) proteins. We found that oxidative stress was sustained in the presence of LegC4 and that the LegC4 restriction phenotype was relieved in PA28αß-deficient macrophages and in the lungs of mice in vivo. Our data also show that oxidative stress is sufficient for LegC4-mediated restriction in macrophages producing PA28αß. PA28αß has been traditionally associated with antigen presentation; however, our data support a novel mechanism whereby effector-mediated subversion of PA28αß enhances cell-autonomous host defense against L. pneumophila under inflammatory and oxidative stress conditions. This work provides a solid foundation to evaluate induced proteasome regulators as mediators of innate immunity.

Eat or Be Eaten: Strategies Used by Legionella to Acquire Host-Derived Nutrients and Evade Lysosomal Degradation.

To replicate within host cells, bacterial pathogens must acquire host-derived nutrients while avoiding degradative antimicrobial pathways. Fundamental insights into bacterial pathogenicity have been revealed by bacteria of the genus Legionella, which naturally parasitize free-living protozoa by establishing a membrane-bound replicative niche termed the Legionella-containing vacuole (LCV). Biogenesis of the LCV and intracellular replication rely on rapid evasion of the endocytic pathway and acquisition of host-derived nutrients, much of which is mediated by bacterial effector proteins translocated into host cells by a Dot/Icm type IV secretion system. Billions of years of co-evolution with eukaryotic hosts and broad host tropism have resulted in expansion of the Legionella genome to accommodate a massive repertoire of effector proteins that promote LCV biogenesis, safeguard the LCV from endolysosomal maturation, and mediate the acquisition of host nutrients. This minireview is focused on the mechanisms by which an ancient intracellular pathogen leverages effector proteins and hijacks host cell biology to obtain essential host-derived nutrients and prevent lysosomal degradation.

Intrabacterial Regulation of a Cytotoxic Effector by Its Cognate Metaeffector Promotes Legionella pneumophila Virulence.

Legionella pneumophila is a natural pathogen of unicellular protozoa that can opportunistically infect macrophages and cause Legionnaires' Disease. Intracellular replication is driven by hundreds of bacterial effector proteins that are translocated into infected host cells by a Dot/Icm type IV secretion system. L. pneumophila effectors are temporally regulated in part by a unique family of translocated regulatory effectors, termed metaeffectors, which bind and modulate the function of a cognate effector in host cells. Regulation of the cytotoxic effector SidI by its cognate metaeffector, MesI, is critical for L. pneumophila virulence in natural and opportunistic hosts. MesI binds and negatively regulates SidI activity in vitro, but how impaired regulation of SidI impairs L. pneumophila intracellular replication is unclear. Using a chromosomally encoded inducible expression system, we found that SidI was toxic to L. pneumophila when uncoupled from MesI. SidI enzymatic activity was required for intrabacterial toxicity since L. pneumophila growth was unaffected by induced expression of a catalytically inactive sidI allele. We also found that MesI translocation into host cells was dispensable for intracellular replication and that MesI-deficient bacteria were rapidly degraded within host cells. These data suggest that MesI promotes L. pneumophila intracellular replication by regulating SidI within the bacterium and reveal a unique role for intrabacterial effector regulation by a translocated metaeffector in L. pneumophila virulence. IMPORTANCE Legionella pneumophila replicates within phagocytic host cells using hundreds of effector protein virulence factors, which canonically subvert the function of host proteins and pathways. L. pneumophila encodes a unique family of translocated effectors called metaeffectors, which bind and regulate the function of a cognate effector in host cells. The metaeffector MesI promotes L. pneumophila virulence by regulating the cytotoxic effector SidI; however, the MesI regulatory mechanism is poorly understood. We discovered a unique intrabacterial role for MesI in L. pneumophila virulence. When uncoupled from MesI, SidI was toxic to L. pneumophila in vitro and triggered robust bacterial degradation in host cells. Furthermore, translocation of MesI was dispensable for intracellular replication, demonstrating that intrabacterial regulation of SidI contributes to L. pneumophila virulence. These data show a novel and important role for translocated effector activity within the bacterium, which challenges the dogma that L. pneumophila effectors function exclusively within host cells.

Pathogenicity and Virulence of Legionella: Intracellular replication and host response.

Bacteria of the genus Legionella are natural pathogens of amoebae that can cause a severe pneumonia in humans called Legionnaires' Disease. Human disease results from inhalation of Legionella-contaminated aerosols and subsequent bacterial replication within alveolar macrophages. Legionella pathogenicity in humans has resulted from extensive co-evolution with diverse genera of amoebae. To replicate intracellularly, Legionella generates a replication-permissive compartment called the Legionella-containing vacuole (LCV) through the concerted action of hundreds of Dot/Icm-translocated effector proteins. In this review, we present a collective overview of Legionella pathogenicity including infection mechanisms, secretion systems, and translocated effector function. We also discuss innate and adaptive immune responses to L. pneumophila, the implications of Legionella genome diversity and future avenues for the field.

Affecting the Effectors: Regulation of Legionella pneumophila Effector Function by Metaeffectors.

Many bacterial pathogens utilize translocated virulence factors called effectors to successfully infect their host. Within the host cell, effector proteins facilitate pathogen replication through subversion of host cell targets and processes. Legionella pneumophila is a Gram-negative intracellular bacterial pathogen that relies on hundreds of translocated effectors to replicate within host phagocytes. Within this large arsenal of translocated effectors is a unique subset of effectors called metaeffectors, which target and regulate other effectors. At least one dozen metaeffectors are encoded by L. pneumophila; however, mechanisms by which they promote virulence are largely unknown. This review details current knowledge of L pneumophila metaeffector function, challenges associated with their identification, and potential avenues to reveal the contribution of metaeffectors to bacterial pathogenesis.

High-polyphenol extracts from Sorghum bicolor attenuate replication of Legionella pneumophila within RAW 264.7 macrophages.

Polyphenols derived from a variety of plants have demonstrated antimicrobial activity against diverse microbial pathogens. Legionella pneumophila is an intracellular bacterial pathogen that opportunistically causes a severe inflammatory pneumonia in humans, called Legionnaires' Disease, via replication within macrophages. Previous studies demonstrated that tea polyphenols attenuate L. pneumophila intracellular replication within mouse macrophages via increased tumor necrosis factor (TNF) production. Sorghum bicolor is a sustainable cereal crop that thrives in arid environments and is well-suited to continued production in warming climates. Sorghum polyphenols have anticancer and antioxidant properties, but their antimicrobial activity has not been evaluated. Here, we investigated the impact of sorghum polyphenols on L. pneumophila intracellular replication within RAW 264.7 mouse macrophages. Sorghum high-polyphenol extract (HPE) attenuated L. pneumophila intracellular replication in a dose-dependent manner but did not impair either bacterial replication in rich media or macrophage viability. Moreover, HPE treatment enhanced both TNF and IL-6 secretion from L. pneumophila infected macrophages. Thus, polyphenols derived from sorghum enhance macrophage restriction of L. pneumophila, likely via increased pro-inflammatory cytokine production. This work reveals commonalities between plant polyphenol-mediated antimicrobial activity and provides a foundation for future evaluation of sorghum as an antimicrobial agent.

The Legionella pneumophila Metaeffector Lpg2505 (MesI) Regulates SidI-Mediated Translation Inhibition and Novel Glycosyl Hydrolase Activity.

Legionella pneumophila, the etiological agent of Legionnaires' disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function to regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and Lpg2505 is not mutually exclusive, and the proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyl hydrolase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505 MesI (metaeffector of SidI). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.

Mechanisms of Effector-Mediated Immunity Revealed by the Accidental Human Pathogen Legionella pneumophila.

Many Gram-negative bacterial pathogens employ translocated virulence factors, termed effector proteins, to facilitate their parasitism of host cells and evade host anti-microbial defenses. However, eukaryotes have evolved to detect effector-mediated virulence strategies through a phenomenon termed effector-triggered immunity (ETI). Although ETI was discovered in plants, a growing body of literature demonstrates that metazoans also utilize effector-mediated immunity to detect and clear bacterial pathogens. This mini review is focused on mechanisms of effector-mediated immune responses by the accidental human pathogen Legionella pneumophila. We highlight recent advancements in the field and discuss the future prospects of harnessing effectors for the development of novel therapeutics, a critical need due to the prevalence and rapid spread of antibiotic resistance.

Potentiation of Cytokine-Mediated Restriction of Legionella Intracellular Replication by a Dot/Icm-Translocated Effector.

Legionella pneumophila is ubiquitous in freshwater environments, where it replicates within unicellular protozoa. However, L. pneumophila is also an accidental human pathogen that can cause Legionnaires' disease in immunocompromised individuals by uncontrolled replication within alveolar macrophages. To replicate within eukaryotic phagocytes, L. pneumophila utilizes a Dot/Icm type IV secretion system to translocate a large arsenal of over 300 effector proteins directly into host cells. In mammals, translocated effectors contribute to innate immune restriction of L. pneumophila We found previously that the effector LegC4 is important for L. pneumophila replication within a natural host protist but is deleterious to replication in a mouse model of Legionnaires' disease. In the present study, we used cultured mouse primary macrophages to investigate how LegC4 attenuates L. pneumophila replication. We found that LegC4 enhanced restriction of L. pneumophila replication within macrophages activated with tumor necrosis factor (TNF) or interferon gamma (IFN-γ). In addition, expression of legC4 was sufficient to restrict Legionella longbeachae replication within TNF- or IFN-γ-activated macrophages. Thus, this study demonstrates that LegC4 contributes to L. pneumophila clearance from healthy hosts by potentiating cytokine-mediated host defense mechanisms.IMPORTANCELegionella spp. are natural pathogens of protozoa and accidental pathogens of humans. Innate immunity in healthy individuals effectively controls Legionella infection due in part to rapid and robust production of proinflammatory cytokines resulting from detection of Dot/Icm-translocated substrates, including effectors. Here, we demonstrate that the effector LegC4 enhances proinflammatory host restriction of Legionella by macrophages. These data suggest that LegC4 may augment proinflammatory signaling or antimicrobial activity of macrophages, a function that has not previously been observed for another bacterial effector. Further insight into LegC4 function will likely reveal novel mechanisms to enhance immunity against pathogens.

Screening Targeted Legionella pneumophila Mutant Libraries In Vivo Using INSeq.

Legionella pneumophila is an intracellular bacterial pathogen that can cause a severe inflammatory pneumonia in humans called Legionnaires' disease, which results from bacterial replication within alveolar macrophages. L. pneumophila replication within macrophages is dependent on hundreds of individual protein virulence factors. Understanding how these virulence factors contribute to disease in an animal model is important to reveal aspects of host-pathogen interactions. High-throughput sequencing (HTS)-based screens using transposon (Tn) mutagenesis are powerful approaches to identify bacterial genes important for host-pathogen interactions. Since large libraries of Tn mutants are at risk of bottleneck effects, phenotypic screening of smaller numbers of targeted mutants is an effective alternative. Insertion sequencing (INSeq) is a method that enables production of targeted Tn mutant libraries and has been used successfully to identify L. pneumophila virulence phenotypes. In this chapter, a protocol is described for using INSeq to generate an arrayed L. pneumophila Tn mutant library and for subsequent screening of targeted mutant pools in a mouse model of infection.

Multiple Legionella pneumophila effector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries.

Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires' disease. A single strain of L. pneumophila encodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number of L. pneumophila effectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhanced L. pneumophila in the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute to L. pneumophila virulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis.

Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.

During infection of macrophages, the pathogenic bacterium Legionella pneumophila secretes effector proteins that induce the conversion of the plasma membrane-derived vacuole into an endoplasmic reticulum (ER)-like replicative vacuole. These ER-like vacuoles are ultimately fused with the ER, where the pathogen replicates. Here we show that the L. pneumophila effector Lpg1137 is a serine protease that targets the mitochondria and their associated membranes. Lpg1137 binds to and cleaves syntaxin 17, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein that is known to participate in the regulation of mitochondrial dynamics through interaction with the mitochondrial fission factor Drp1 in fed cells and in autophagy through interaction with Atg14L and other SNAREs in starved cells. Cleavage of syntaxin 17 inhibits not only autophagy but also staurosporine-induced apoptosis occurring in a Bax, Drp1-dependent manner. Thus, L. pneumophila can shut down ER-mitochondria communication through cleavage of syntaxin 17.

Acyl Histidines: New N-Acyl Amides from Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative gammaproteobacterial pathogen that infects and intracellularly replicates in human macrophages and a variety of protozoa. L. pneumophila encodes an orphan biosynthetic gene cluster (BGC) that contains isocyanide-associated biosynthetic genes and is upregulated during infection. Because isocyanide-functionalized metabolites are known to harbor invertebrate innate immunosuppressive activities in bacterial pathogen-insect interactions, we used pathway-targeted molecular networking and tetrazine-based chemoseletive ligation chemistry to characterize the metabolites from the orphan pathway in L. pneumophila. We also assessed their intracellular growth contributions in an amoeba and in murine bone-marrow-derived macrophages. Unexpectedly, two distinct groups of aromatic amino acid-derived metabolites were identified from the pathway, including a known tyrosine-derived isocyanide and a family of new N-acyl-l-histidine metabolites.

Quantitative Mass Spectrometry Identifies Novel Host Binding Partners for Pathogenic Escherichia coli Type III Secretion System Effectors.

Enteropathogenic and enterohemorrhagic Escherichia coli cause enteric diseases resulting in significant morbidity and mortality worldwide. These pathogens remain extracellular and translocate a set of type III secreted effector proteins into host cells to promote bacterial virulence. Effectors manipulate host cell pathways to facilitate infection by interacting with a variety of host targets, yet the binding partners and mechanism of action of many effectors remain elusive. We performed a mass spectrometry screen to identify host targets for a library of effectors. We found five known effector targets and discovered four novel interactions. Interestingly, we identified multiple effectors that interacted with the microtubule associated protein, ensconsin. Using co-immunoprecipitations, we confirmed that NleB1 and EspL interacted with ensconsin in a region that corresponded to its microtubule binding domain. Ensconsin is an essential cofactor of kinesin-1 that is required for intracellular trafficking, and we demonstrated that intracellular trafficking was severely disrupted during wild type EPEC infections but not during infections with ΔnleB1 or ΔespL mutants. Our findings demonstrate the efficacy of quantitative proteomics for identifying effector-host protein interactions and suggest that vesicular trafficking is a crucial cellular process that may be targeted by NleB1 and EspL through their interaction with ensconsin.

15-Deoxy-Δ12,14-prostaglandin J2 inhibits macrophage colonization by Salmonella enterica serovar Typhimurium.

15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) is an anti-inflammatory downstream product of the cyclooxygenase enzymes. It has been implicated to play a protective role in a variety of inflammatory mediated diseases, including rheumatoid arthritis, neural damage, and myocardial infarctions. Here we show that 15d-PGJ2 also plays a role in Salmonella infection. Salmonella enterica Typhimurium is a Gram-negative facultative intracellular pathogen that is able to survive and replicate inside phagocytic immune cells, allowing for bacterial dissemination to systemic sites. Salmonella species cause a wide range of morbidity and mortality due to gastroenteritis and typhoid fever. Previously we have shown that in mouse models of typhoid fever, Salmonella infection causes a major perturbation in the prostaglandin pathway. Specifically, we saw that 15d-PGJ2 production was significantly increased in both liver and feces. In this work we show that 15d-PGJ2 production is also significantly increased in macrophages infected with Salmonella. Furthermore, we show that the addition of 15d-PGJ2 to Salmonella infected RAW264.7, J774, and bone marrow derived macrophages is sufficient to significantly reduce bacterial colonization. We also show evidence that 15d-PGJ2 is reducing bacterial uptake by macrophages. 15d-PGJ2 reduces the inflammatory response of these infected macrophages, as evidenced by a reduction in the production of cytokines and reactive nitrogen species. The inflammatory response of the macrophage is important for full Salmonella virulence, as it can give the bacteria cues for virulence. The reduction in bacterial colonization is independent of the expression of Salmonella virulence genes SPI1 and SPI2, and is independent of the 15d-PGJ2 ligand PPAR-γ. 15d-PGJ2 also causes an increase in ERK1/2 phosphorylation in infected macrophages. In conclusion, we show here that 15d-PGJ2 mediates the outcome of bacterial infection, a previously unidentified role for this prostaglandin.

Bacterial effector interplay: a new way to view effector function.

Bacterial pathogens are dependent on virulence factors to efficiently colonize and propagate within their hosts. Many Gram-negative bacterial pathogens rely on specialized proteinaceous secretion systems that inject virulence factors, termed effectors, directly into host cells. These bacterial effector proteins perform various functions within host cells; however, regulation of their function within the host cell is highly enigmatic. It is becoming increasingly apparent that many of these effectors directly influence and regulate each other and their mechanisms within the host cell. We discuss the emerging theme of bacterial effector interplay impacting infection and the importance of investigating this topic.

The type III system-secreted effector EspZ localizes to host mitochondria and interacts with the translocase of inner mitochondrial membrane 17b.

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) are attaching and effacing (A/E) bacterial pathogens that cause severe diarrheal disease worldwide. To cause disease, A/E pathogens require a type III secretion system, which facilitates transport of bacterial effector proteins directly into infected host cells. One of these effector proteins translocated by the type III secretion system, EspZ, is essential for A/E pathogen infection and functions to prevent rapid death of EPEC-infected cells. We further investigated the mechanism of EspZ-mediated protection of infected host cells and found that a severe decrease in host mitochondrial membrane potential (Δψ(m)) occurs concurrently with host cell lysis during infection with EPEC lacking EspZ (ΔespZ). It was also demonstrated that EspZ localizes to host cell mitochondria and interacts with the translocase of inner mitochondrial membrane 17b (TIM17b). In addition, host cell cytotoxicity was exacerbated in the absence of TIM17b during wild-type (WT) EPEC infection. The findings of this study together provide the first evidence that EspZ localizes to host mitochondria and that TIM17b contributes to protection against rapid cell death during EPEC infection.