A polyyne toxin produced by an antagonistic bacterium blinds and lyses a Chlamydomonad alga

Algae are key contributors to global carbon fixation and form the basis of many food webs. In nature, their growth is often supported or suppressed by microorganisms. The bacterium Pseudomonas protegens Pf-5 arrests the growth of the green unicellular alga Chlamydomonas reinhardtii, deflagellates the alga by the cyclic lipopeptide orfamide A, and alters its morphology [P. Aiyar et al., Nat. Commun. 8, 1756 (2017)]. Using a combination of Raman microspectroscopy, genome mining, and mutational analysis, we discovered a polyyne toxin, protegencin, which is secreted by P. protegens, penetrates the algal cells, and causes destruction of the carotenoids of their primitive visual system, the eyespot. Together with secreted orfamide A, protegencin thus prevents the phototactic behavior of C. reinhardtii. A mutant of P. protegens deficient in protegencin production does not affect growth or eyespot carotenoids of C. reinhardtii. Protegencin acts in a direct and destructive way by lysing and killing the algal cells. The toxic effect of protegencin is also observed in an eyeless mutant and with the colony-forming Chlorophyte alga Gonium pectorale. These data reveal a two-pronged molecular strategy involving a cyclic lipopeptide and a conjugated tetrayne used by bacteria to attack select Chlamydomonad algae. In conjunction with the bloom-forming activity of several chlorophytes and the presence of the protegencin gene cluster in over 50 different Pseudomonas genomes [A. J. Mullins et al., bioRxiv [Preprint] (2021). https://www.biorxiv.org/content/10.1101/2021.03.05.433886v1 (Accessed 17 April 2021)], these data are highly relevant to ecological interactions between Chlorophyte algae and Pseudomonadales bacteria.

Cyanobacteria and algae in the aquatic environment contribute about 50% to global CO₂ fixation (1). As primary producers, they are fundamental to food webs (2, 3). Algal activities can also influence biogeochemical processes as exemplified recently with regards to the Greenland ice sheet (4). In nature, algae are usually associated with other microbes that influence their fitness through mutualistic or antagonistic interactions (3, 5, 6), and the exchange of natural products can play a central role in these interactions (7, 8). Despite their ecological importance, the interactions of algae with microorganisms are still poorly understood at the molecular level, especially relative to our understanding of higher plant–microbe interactions (8).In recent years, the biciliate green alga Chlamydomonas reinhardtii (Fig. 1 A and B), for which a large molecular toolkit is available (9–11), has become a model for studying the molecular interactions between unicellular algae and microbes (7). C. reinhardtii occurs mainly in wet soil ecosystems (7) and can establish mutualistic carbon–nitrogen metabolic exchange mechanisms with fungi (12) or bacteria such as Methylobacterium spp (13). Moreover, algal–bacterial consortia have been used to mutualistically enhance natural hydrogen production of C. reinhardtii (14). However, C. reinhardtii can also be prone to attacks by antagonistic bacteria. For example, the soil bacterium Streptomyces iranensis releases the algicide azalomycin F, which is toxic for C. reinhardtii unless the alga protects itself among the mycelia of the fungus Aspergillus nidulans (15). In a previous study, we showed that another soil bacterium, Pseudomonas protegens Pf-5, known to produce a wide variety of secondary metabolites (16), can inhibit the growth of C. reinhardtii (17). Specifically, P. protegens Pf-5 releases orfamide A, a cyclic lipopeptide that causes a spike in cytosolic Ca²⁺ and rapid loss of cilia (historically known as flagella). However, an orfamide A–null mutant of P. protegens Pf-5 is still able to prevent C. reinhardtii cultures from growing for several days (17), leading us to hypothesize that at least another bacterial secondary metabolite is involved in the antagonism of P. protegens Pf-5 on C. reinhardtii. Here, we report the discovery of an unusual bacterial polyyne, protegencin, that plays a key role in the algicidal activity of P. protegens Pf-5: it causes destruction of the carotenoids within the eyespot, a primitive visual system (18, 19) (Fig. 1 A and B), and lysis of the algal cells.Open in a separate windowFig. 1.Raman microspectroscopy of C. reinhardtii highlights the loss of eyespot carotenoids and an unidentified compound in bacterial coculture. (A) Simplified scheme of C. reinhardtii (modified from ref. 7, which is licensed under CC BY 4.0) with an enlarged eyespot area; data from (18, 19). CG, carotenoid-rich lipid globules; D4R, D4 rootlet; ChRs, channelrhodopsins. (B) Brightfield microscopic image of C. reinhardtii. The arrowheads highlight a cilium (gray), the pyrenoid (blue), and the eyespot (orange). (C) Raman spectroscopy of a representative C. reinhardtii cell grown in monoculture. An algal overnight culture in TAP was fixed with 4% formaldehyde and embedded in 0.5% TAP agarose for single cell analysis. The color-coded Raman spectra display the individual cluster spectra found by k-means cluster analysis of a typical algal cell. Raman marker bands are labeled green (cell compounds), blue (starch), and orange (carotenoids). Note that phenylalanine and carotenoids share a marker band at 1,004 cm⁻¹ (SI Appendix, Table S1). The black spectrum (1*) represents the vector-normalized background generated from areas with low content of detectable biological material. All other displayed cluster spectra are background corrected and represent clusters of components rich in cell compounds (spectra 2 to 7), starch (spectra 4 and 5), or carotenoids (spectra 6 and 7). Additional spectra are shown in SI Appendix, Fig. S2. (D) Color-coded spatial distribution of Raman spectral components of a representative C. reinhardtii cell (Left) as shown in C. Green, orange, and blue false color maps represent Raman intensities of cell compounds, carotenoids, and starch, respectively. The composite red-green-blue (RGB) image (Right) is created by overlaying the normalized sums of the marker band regions associated with cell compounds (green), starch (blue), or carotenoids (red). The opacity in each pixel is proportional to the overall intensity in each pixel. The arrowheads highlight the starch sheets around the pyrenoid (blue) and the eyespot (orange). (E) Raman spectra of a representative C. reinhardtii cell after overnight cocultivation with P. protegens (algae:bacteria 1:1,000) in TAP medium. See C for further details. (F) Spatial distribution of the Raman spectroscopic clusters, integrated intensities and composite RGB image of a representative C. reinhardtii cell after overnight cocultivation with P. protegens. See D for details. (Scale bars: 3 μm in B, D, and F.) (C–F) Exemplary cells were taken from the 16-h series (Fig. 2A).     

PPARalpha agonist fenofibrate suppresses tumor growth through direct and indirect angiogenesis inhibition

Angiogenesis and inflammation are central processes through which the tumor microenvironment influences tumor growth. We have demonstrated recently that peroxisome proliferator-activated receptor (PPAR)alpha deficiency in the host leads to overt inflammation that suppresses angiogenesis via excess production of thrombospondin (TSP)-1 and prevents tumor growth. Hence, we speculated that pharmacologic activation of PPARalpha would promote tumor growth. Surprisingly, the PPARalpha agonist fenofibrate potently suppressed primary tumor growth in mice. This effect was not mediated by cancer-cell-autonomous antiproliferative mechanisms but by the inhibition of angiogenesis and inflammation in the host tissue. Although PPARalpha-deficient tumors were still susceptible to fenofibrate, absence of PPARalpha in the host animal abrogated the potent antitumor effect of fenofibrate. In addition, fenofibrate suppressed endothelial cell proliferation and VEGF production, increased TSP-1 and endostatin, and inhibited corneal neovascularization. Thus, both genetic abrogation of PPARalpha as well as its activation by ligands cause tumor suppression via overlapping antiangiogenic pathways. These findings reveal the potential utility of the well tolerated PPARalpha agonists beyond their use as lipid-lowering drugs in anticancer therapy. Our results provide a mechanistic rationale for evaluating the clinical benefits of PPARalpha agonists in cancer treatment, alone and in combination with other therapies.     

Mechanisms of host cell exit by the intracellular bacterium Chlamydia

The mechanisms that mediate the release of intracellular bacteria from cells are poorly understood, particularly for those that live within a cellular vacuole. The release pathway of the obligate intracellular bacterium Chlamydia from cells is unknown. Using a GFP-based approach to visualize chlamydial inclusions within cells by live fluorescence videomicroscopy, we identified that Chlamydia release occurred by two mutually exclusive pathways. The first, lysis, consisted of an ordered sequence of membrane permeabilizations: inclusion, nucleus and plasma membrane rupture. Treatment with protease inhibitors abolished inclusion lysis. Intracellular calcium signaling was shown to be important for plasma membrane breakdown. The second release pathway was a packaged release mechanism, called extrusion. This slow process resulted in a pinching of the inclusion, protrusion out of the cell within a cell membrane compartment, and ultimately detachment from the cell. Treatment of Chlamydia-infected cells with specific pharmacological inhibitors of cellular factors demonstrated that extrusion required actin polymerization, neuronal Wiskott-Aldrich syndrome protein, myosin II and Rho GTPase. The participation of Rho was unique in that it functioned late in extrusion. The dual nature of release characterized for Chlamydia has not been observed as a strategy for intracellular bacteria.     

Etomidate suppresses adrenocortical function by inhibition of 11 beta-hydroxylation

We studied the effect of short term infusion of the imidazole-derived anesthetic agent etomidate on plasma concentrations of ACTH, cortisol, and the cortisol-precursors 11-desoxycortisol and 17-hydroxyprogesterone. During the infusion of etomidate, a significant increase in the peripheral concentration of ACTH occurred, while plasma cortisol concentrations decreased. After the end of the infusion, cortisol concentrations further decreased, while the concentrations of desoxycortisol and 17-hydroxyprogesterone increased. Furthermore, in in vitro experiments with isolated rat pituitary and adrenal cells, etomidate did not affect corticotropin-releasing hormone-induced ACTH secretion from pituitary cells, whereas ACTH-stimulated corticosterone secretion from adrenal cells was inhibited by addition of etomidate in concentrations which occur in plasma during and after infusion of the drug. These results lead to the conclusion that etomidate inhibits adrenal 11 beta-hydroxylation.     

Modulation of host innate and adaptive immune defenses by cytomegalovirus: timing is everything

Abstract. Loewendorf A, Benedict CA (La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA). Modulation of host innate and adaptive immune defenses by cytomegalovirus: timing is everything (Symposium). J Intern Med 2010; 267 : 483–501. Human cytomegalovirus (HCMV) (HHV‐5, a β‐herpesvirus) causes the vast majority of infection‐related congenital birth defects, and can trigger severe disease in immune suppressed individuals. The high prevalence of societal infection, the establishment of lifelong persistence and the growing number of immune‐related diseases where HCMV is touted as a potential promoter is slowly heightening public awareness to this virus. The millions of years of co‐evolution between CMV and the immune system of its host provides for a unique opportunity to study immune defense strategies, and pathogen counterstrategies. Dissecting the timing of the cellular and molecular processes that regulate innate and adaptive immunity to this persistent virus has revealed a complex defense network that is shaped by CMV immune modulation, resulting in a finely tuned host–pathogen relationship.     

Experimental metastasis inhibition by pretreatment of the host

In an experimental murine metastasis model host pretreatment protocol (HPP) was tested to abrogate lung colonization of tumor cells. The stimulation of the host defense by lentinan or TP4, and the PGI2 administration was effective in the case of the immunosensitive low metastatic tumor. The modulation of the host cells and/or the extracellular matrix by the glycosaminoglycan biosynthesis blocking agent KL-103--but not by the degradation inhibitor suramin--inhibited the lung colonization of the highly metastatic immunoresistant tumor variant. In combination with the cytotoxic antiproliferative agents these non-toxic drugs could be useful in new protocols to prevent tumor dissemination.     

An antibiotic recipe for an arrhythmic disaster

We describe the case of a patient who developed torsade de pointes during temporary pacemaker insertion after administration of intravenous erythromycin. The case highlights the dangers of administering drugs that prolong the QT interval in patients with complete atrioventricular block, and we discuss the underlying pathophysiological recipe that can lead to a potential arrhythmic disaster.     

An uncommon infection in an uncommon host

We present the case of an international traveller who was eventually diagnosed with amoebiasis and autosomal dominant polycystic kidney disease (ADPKD) after she presented with recurring fevers. The patient was also diagnosed with non-amoebic bacterial hepatitis. Positron emission tomography (PET) can play an important role in locating the source of infection in patients with ADPKD when hepatic or renal infection is suspected. It can also be used to document clinical resolution of infection in difficult cases. Hepatic parenchymal infections in ADPKD patients may warrant a prolonged course of rotating antibiotics.     

Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation

Hepatitis C virus (HCV) RNA replication depends on viral protein association with intracellular membranes, but the influence of membrane composition on viral replication is unclear. We report that HCV RNA replication and assembly of the viral replication complex require geranylgeranylation of one or more host proteins. In cultured hepatoma cells, HCV RNA replication was disrupted by treatment with lovastatin, an inhibitor of 3-hydroxy-3-methyglutaryl CoA reductase, or with an inhibitor of protein geranylgeranyl transferase I, each of which induced the dissolution of the HCV replication complex. Viral replication was not affected by treatment of cells with an inhibitor of farnesyl transferase. When added to lovastatin-treated cells, geranylgeraniol, but not farnesol, restored replication complex assembly and viral replication. Inasmuch as the HCV genome does not encode a canonical geranylgeranylated protein, the data suggest the involvement of a geranylgeranylated host protein in HCV replication. Inhibition of its geranylgeranylation affords a therapeutic strategy for treatment of HCV infection.     

Profiling early infection responses: Pseudomonas aeruginosa eludes host defenses by suppressing antimicrobial peptide gene expression

Insights into the host factors and mechanisms mediating the primary host responses after pathogen presentation remain limited, due in part to the complexity and genetic intractability of host systems. Here, we employ the model Drosophila melanogaster to dissect and identify early host responses that function in the initiation and progression of Pseudomonas aeruginosa pathogenesis. First, we use immune potentiation and genetic studies to demonstrate that flies mount a heightened defense against the highly virulent P. aeruginosa strain PA14 when first inoculated with strain CF5, which is avirulent in flies; this effect is mediated via the Imd and Toll signaling pathways. Second, we use whole-genome expression profiling to assess and compare the Drosophila early defense responses triggered by the PA14 vs. CF5 strains to identify genes whose expression patterns are different in susceptible vs. resistant host-pathogen interactions, respectively. Our results identify pathogenesis- and defense-specific genes and uncover a previously undescribed mechanism used by P. aeruginosa in the initial stages of its host interaction: suppression of Drosophila defense responses by limiting antimicrobial peptide gene expression. These results provide insights into the genetic factors that mediate or restrict pathogenesis during the early stages of the bacterial-host interaction to advance our understanding of P. aeruginosa-human infections.     

Adenosine monophosphate-activated protein kinase suppresses vascular smooth muscle cell proliferation through the inhibition of cell cycle progression

Vascular smooth muscle cell (VSMC) proliferation is a critical event in the development and progression of vascular diseases, including atherosclerosis. We investigated whether the activation of adenosine monophosphate-activated protein kinase (AMPK) could suppress VSMC proliferation and inhibit cell cycle progression. Treatment of human aortic smooth muscle cells (HASMCs) or isolated rabbit aortas with the AMPK activator 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) induced phosphorylation of AMPK and acetyl Co-A carboxylase. AICAR significantly inhibited HASMC proliferation induced by both platelet-derived growth factor-BB (PDGF-BB) and fetal calf serum (FCS). Treatment with AICAR inhibited the phosphorylation of retinoblastoma gene product (Rb) induced by PDGF-BB or FCS, and increased the expression of cyclin-dependent kinase inhibitor p21(CIP) but not that of p27(KIP). Pharmacological inhibition of AMPK or overexpression of dominant negative-AMPK inhibited both the suppressive effect of AICAR on cell proliferation and the phosphorylation of Rb, suggesting that the effect of AICAR is mediated through the activation of AMPK. Cell cycle analysis in HASMCs showed that AICAR significantly increased cell population in G0/G1-phase and reduced that in S- and G2/M-phase, suggesting AICAR induced cell cycle arrest. AICAR increased both p53 protein and Ser-15 phosphorylated p53 in HASMCs, which were blocked by inhibition of AMPK. In isolated rabbit aortas, AICAR also increased Ser-15 phosphorylation and protein expression of p53 and inhibited Rb phosphorylation induced by FCS. These data suggest for the first time that AMPK suppresses VSMC proliferation via cell cycle regulation by p53 upregulation. Therefore, AMPK activation in VSMCs may be a therapoietic target for the prevention of vascular diseases.