Modulation of translation and induction of autophagy by bacterial exoproducts.

Autophagy is a catabolic process of paramount importance for cellular homeostasis during starvation. Generally, autophagy and translation are inversely regulated. Many kinds of stress lead to attenuation of translation via phosphorylation of eukaryotic translation initiation factor alpha (eIF2α). This response is conserved from yeast to man and can be either protective or detrimental depending on strength and duration of stress, and additional factors. During starvation or viral infection, phosphorylation of eIF2α is required for induction of autophagy. As exemplified here by α-hemolysin, a small pore-forming toxin (PFT) of Staphylococcus aureus and (S)-3-oxo-C12-homoserine lactone [(S)-3-oxo-C12-HSL], a quorum-sensing hormone of Pseudomonas aeruginosa, bacterial exoproducts may also impact translation and autophagy. Thereby, PFT and (S)-3-oxo-C12-HSL act differentially. Damage of the plasma membrane by PFT causes efflux of potassium, which leads to amino acid starvation and energy loss. This triggers amino acid-sensitive eIF2α-kinase GCN2, as well as energy sensor AMPK, and deactivates mTORC1. The output of this response, that is, transient metabolic reprogramming is an essential part of a defense program which enables cells to survive attack by a pore-forming agent. Thus, nutrient/energy sensors serve as sentinels of plasma membrane integrity. In contrast to PFT, (S)-3-oxo-C12-HSL does not cause acute loss of ATP or activation of GCN2, but also triggers phosphorylation of eIF2α and inhibits translation. This response appears not to depend on efflux of potassium and requires eIF2α-kinase PKR. Like α-toxin, (S)-3-oxo-C12-HSL increases lipidation of LC3 and accumulation of autophagosomes in cells. Apart from directly affecting host-cell viability, bacterial exoproducts might galvanize bystander cells to prepare for close combat with microbial offenders or inadvertently accommodate some of them.

A subunit of eukaryotic translation initiation factor 2α-phosphatase (CreP/PPP1R15B) regulates membrane traffic.

The constitutive reverter of eIF2α phosphorylation (CReP)/PPP1r15B targets the catalytic subunit of protein phosphatase 1 (PP1c) to phosphorylated eIF2α (p-eIF2α) to promote its dephosphorylation and translation initiation. Here, we report a novel role and mode of action of CReP. We found that CReP regulates uptake of the pore-forming Staphylococcus aureus α-toxin by epithelial cells. This function was independent of PP1c and translation, although p-eIF2α was involved. The latter accumulated at sites of toxin attack and appeared conjointly with α-toxin in early endosomes. CReP localized to membranes, interacted with phosphomimetic eIF2α, and, upon overexpression, induced and decorated a population of intracellular vesicles, characterized by accumulation of N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a lipid marker of exosomes and intralumenal vesicles of multivesicular bodies. By truncation analysis, we delineated the CReP vesicle induction/association region, which comprises an amphipathic α-helix and is distinct from the PP1c interaction domain. CReP was also required for exocytosis from erythroleukemia cells and thus appears to play a broader role in membrane traffic. In summary, the mammalian traffic machinery co-opts p-eIF2α and CReP, regulators of translation initiation.

Global functional analyses of cellular responses to pore-forming toxins.

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (∼0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs.

Pro-autophagic signal induction by bacterial pore-forming toxins.

Pore-forming toxins (PFT) comprise a large, structurally heterogeneous group of bacterial protein toxins. Nucleated target cells mount complex responses which allow them to survive moderate membrane damage by PFT. Autophagy has recently been implicated in responses to various PFT, but how this process is triggered is not known, and the significance of the phenomenon is not understood. Here, we show that S. aureus α-toxin, Vibrio cholerae cytolysin, streptolysin O and E. coli haemolysin activate two pathways leading to autophagy. The first pathway is triggered via AMP-activated protein kinase (AMPK). AMPK is a major energy sensor which induces autophagy by inhibiting the target of rapamycin complex 1 (TORC1) in response to a drop of the cellular ATP/AMP-ratio, as is also observed in response to membrane perforation. The second pathway is activated by the conserved eIF2α-kinase GCN2, which causes global translational arrest and promotes autophagy in response to starvation. The latter could be accounted for by impaired amino acid transport into target cells. Notably, PKR, an eIF2α-kinase which has been implicated in autophagy induction during viral infection, was also activated upon membrane perforation, and evidence was obtained that phosphorylation of eIF2α is required for the accumulation of autophagosomes in α-toxin-treated cells. Treatment with 3-methyl-adenine inhibited autophagy and disrupted the ability of cells to recover from sublethal attack by S. aureus α-toxin. We propose that PFT induce pro-autophagic signals through membrane perforation-dependent nutrient and energy depletion, and that an important function of autophagy in this context is to maintain metabolic homoeostasis.

Pore-forming toxins activate MAPK p38 by causing loss of cellular potassium.

Mitogen activated protein kinase (MAPK) p38 has emerged as a survival protein in cells that are attacked by bacterial toxins forming small membrane pores. Activation of p38 by pore forming toxins (PFT) has been attributed to osmotic stress, but here we show that loss of K+ is likely to be the critical parameter. Several lines of evidence support this conclusion: first, osmoprotection did not prevent p38-phosphorylation in alpha-toxin-loaded cells. Second, treatment of cells with a K+ ionophore, or simple incubation in K+-free medium sufficed to cause robust p38-phosphorylation. Third, media containing high [K+] prevented p38-activation by Staphylococcus aureus alpha-toxin, Vibrio cholerae cytolysin (VCC), Streptolysin O (SLO), or Escherichia coli hemolysin (HlyA), but did not impair activation by H2O2. Fourth, potential roles of LPS, TLR4, or calcium-influx were ruled out. Therefore, we propose that PFT trigger the p38 MAPK-pathway by causing loss of cellular K+.

Elimination of a bacterial pore-forming toxin by sequential endocytosis and exocytosis.

Staphylococcus aureus alpha-toxin is the archetype of bacterial pore forming toxins and a key virulence factor secreted by the majority of clinical isolates of S. aureus. Toxin monomers bind to target cells and oligomerize to form small beta-barrel pores in the plasma membrane. Many nucleated cells are able to repair a limited number of lesions by unknown, calcium-independent mechanisms. Here we show that cells can internalize alpha-toxin, that uptake is essential for cellular survival, and that pore-complexes are not proteolytically degraded, but returned to the extracellular milieu in the context of exosome-like structures, which we term toxosomes.

Ammonia triggers photodamage of photosystem II in the cyanobacterium Synechocystis sp. strain PCC 6803.

Ammonia has long been known to be toxic for many photosynthetic organisms; however, the target for its toxicity remains elusive. Here, we show that in the cyanobacterium Synechocystis sp. strain PCC 6803, ammonia triggers a rapid photodamage of photosystem II (PSII). Whereas wild-type cells can cope with this damage by turning on the FtsH2-dependent PSII repair cycle, the FtsH2-deficient mutant is highly sensitive and loses PSII activity at millimolar concentration of ammonia. Ammonia-triggered PSII destruction is light dependent and occurs already at low photon fluence rates. Experiments with monochromatic light showed that ammonia-promoted PSII photoinhibition is executed by wavebands known to directly destroy the manganese cluster in the PSII oxygen-evolving complex, suggesting that the oxygen-evolving complex may be a direct target for ammonia toxicity.

Structural analysis of the PP2C phosphatase tPphA from Thermosynechococcus elongatus: a flexible flap subdomain controls access to the catalytic site.

The homologue of the phosphoprotein PII phosphatase PphA from Thermosynechococcus elongatus, termed tPphA, was identified and its structure was resolved in two different space groups, C222(1) and P4(1)2(1)2, at a resolution of 1.28 and 3.05 A, respectively. tPphA belongs to a large and widely distributed subfamily of Mg(2+)/Mn(2+)-dependent phosphatases of the PPM superfamily characterized by the lack of catalytic and regulatory domains. The core structure of tPphA shows a high degree of similarity to the two PPM structures identified so far. In contrast to human PP2C, but similar to Mycobacterium tuberculosis phosphatase PstP, the catalytic centre exhibits a third metal ion in addition to the dinuclear metal centre universally conserved in all PPM members. The fact that the third metal is only liganded by amino acids, which are universally conserved in all PPM members, implies that the third metal could be general for all members of this family. As a specific feature of tPphA, a flexible subdomain, previously recognized as a flap domain, could be revealed. Comparison of different structural isomers of tPphA as well as site-specific mutagenesis implied that the flap domain is involved in substrate binding and catalytic activity. The structural arrangement of the flap domain was accompanied by a large side-chain movement of an Arg residue (Arg169) at the basis of the flap. Mutation of this residue strongly impaired protein stability as well as catalytic activity, emphasizing the importance of this amino acid for the regional polysterism of the flap subdomain and confirming the assumption that flap domain flexibility is involved in catalysis.

Signal transduction protein PII phosphatase PphA is required for light-dependent control of nitrate utilization in synechocystis sp. strain PCC 6803.

Signal transduction protein P(II) is dephosphorylated in Synechocystis sp. strain PCC 6803 by protein phosphatase PphA. To determine the impact of PphA-mediated P(II) dephosphorylation on physiology, the phenotype of a PphA-deficient mutant was analyzed. Mutants lacking either PphA or P(II) were impaired in efficient utilization of nitrate as the nitrogen source. Under conditions of limiting photosystem I (PSI)-reduced ferredoxin, excess reduction of nitrate along with impaired reduction of nitrite occurred in P(II) signaling mutants, resulting in excretion of nitrite to the medium. This effect could be reversed by increasing the level of PSI-reduced ferredoxin. We present evidence that nonphosphorylated P(II) controls the utilization of nitrate in response to low light intensity by tuning down nitrate uptake to meet the actual reduction capacity. This control mechanism can be bypassed by exposing cells to excess levels of nitrate. Uncontrolled nitrate uptake leads to light-dependent nitrite excretion even in wild-type cells, confirming that nitrate uptake controls nitrate utilization in response to limiting photon flux densities.

Protein phosphatase PphA from Synechocystis sp. PCC 6803: the physiological framework of PII-P dephosphorylation.

The phosphorylated signal transduction protein P(II) (P(II)-P) in the cyanobacterium Synechocystis sp. strain PCC 6803 is dephosphorylated by PphA, a protein phosphatase of the 2C family (PP2C). In this study, the physiological conditions of P(II)-P dephosphorylation were investigated with respect to the in vivo specificity of P(II)-P towards PphA and the cellular abundance of PphA in cells growing under different nitrogen regimes. Furthermore, the consequences of impaired P(II)-P dephosphorylation with respect to short-term inhibition of glutamine synthetase (GS) were studied. With a contribution of approximately 15 % of total Mn(2+)-dependent p-nitrophenyl phosphate hydrolysis activity, PphA has only a minor impact on the total PP2C activity in Synechocystis extracts. Nevertheless, residual P(II)-P dephosphorylation in PphA-deficient cells could only be observed after prolonged incubation in the presence of ammonium. The abundance of PphA correlates with the phosphorylation state of P(II) under nitrogen-replete conditions and is specifically enhanced by nitrite. Regulation of pphA expression operates at the post-transcriptional level. In the presence of nitrate/nitrite, PphA is present in molar excess over P(II)-P, enabling the cells to rapidly dephosphorylate P(II)-P in response to changing environmental conditions. A PphA-deficient mutant is not impaired in short-term inhibition of GS activity following ammonium treatment. Down-regulation of GS occurs by induction of gif genes (encoding GS inactivating factors 7 and 17), which is controlled by NtcA-mediated gene repression. Thus, impaired P(II)-P dephosphorylation does not affect ammonium-prompted inactivation of NtcA.

P signalling in unicellular cyanobacteria: analysis of redox-signals and energy charge.

This communication presents a short outline of the current knowledge on the molecular basis of P(II) signal transduction in unicellular cyanobacteria with respect to the perception of environmental stimuli. First, the general characteristics of the P(II) signalling system in unicellular cyanobacteria are presented, the hallmark of which is modification by serine-phosphorylation, as compared to the paradigmatic P(II) signal transduction system in proteobacteria, which is based on tyrosyl-uridylylation. Then, the focus is turned on the signals controlling P(II) phosphorylation state. Recently, the cellular phosphatase (termed PphA), which specifically dephosphorylates phosphorylated P(II) (P(II)-P) was identified in Synechocystis sp. strain PCC 6803. With the availability of a PphA-deficient mutant and the purified components for in vitro assay of PphA mediated P(II)-P dephosphorylation, novel insights into the signals, to which P(II)-P dephosphorylation responds, can be obtained. Here we present an investigation of the response of P(II)-P dephosphorylation towards treatments that affect the redox-balance of the cells. Furthermore, a possible role of varying ATP/ADP ratios on P(II)-P dephosphorylation was examined. From these studies, together with previous investigations, we conclude that P(II)-P dephosphorylation specifically responds to changes in the levels of central metabolites of carbon metabolism, in particular 2-oxoglutarate.

The novel protein phosphatase PphA from Synechocystis PCC 6803 controls dephosphorylation of the signalling protein PII.

The family of PII signal transduction proteins consists of one of the most highly conserved signalling proteins in nature. The cyanobacterial PII homologue transmits signals on the nitrogen and carbon status of the cells through phosphorylation of a seryl residue. Recently, we identified a protein phosphatase 2C (PP2C) homologue from the cyanobacterium Synechocystis PCC 6803, termed PphA, to be the cellular phospho-PII (PII-P) phosphatase. In this investigation, we characterized the enzymatic properties of PphA and investigated the regulation of its catalytic activity towards PII-P. PphA dephosphorylates phosphocasein and PII-P with similar efficiency in a strictly Mg2+- or Mn2+-dependent reaction. Low-molecular-weight phosphorylated molecules are poor substrates for PphA. Its reactivity towards PII-P, but not towards phosphocasein, is inhibited by various nucleotides, suggesting that this effect is based on specific properties of the PII protein. The inhibitory effect of ATP can be strongly enhanced by the addition of 2-oxoglutarate or oxaloacetate. At low concentrations of 2-oxoglutarate, changes in the ATP levels within the physiological range affect the degree of PII-Pase inhibition, whereas at 2-oxoglutarate levels beyond 0.1 mM, inhibition is almost complete at very low ATP levels. This suggests that PII dephosphorylation is not only sensitive to 2-oxoglutarate and oxaloacetate levels, it also integrates signals from the energy charge of the cells under specific cellular conditions.