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.

The pore-forming protein Cry5B elicits the pathogenicity of Bacillus sp. against Caenorhabditis elegans.

The soil bacterium Bacillus thuringiensis is a pathogen of insects and nematodes and is very closely related to, if not the same species as, Bacillus cereus and Bacillus anthracis. The defining characteristic of B. thuringiensis that sets it apart from B. cereus and B. anthracis is the production of crystal (Cry) proteins, which are pore-forming toxins or pore-forming proteins (PFPs). Although it is known that PFPs are important virulence factors since their elimination results in reduced virulence of many pathogenic bacteria, the functions by which PFPs promote virulence are incompletely understood. Here we study the effect of Cry proteins in B. thuringiensis pathogenesis of the nematode Caenorhabditis elegans. We find that whereas B. thuringiensis on its own is not able to infect C. elegans, the addition of the PFP Cry protein, Cry5B, results in a robust lethal infection that consumes the nematode host in 1-2 days, leading to a "Bob" or bag-of-bacteria phenotype. Unlike other infections of C. elegans characterized to date, the infection by B. thuringiensis shows dose-dependency based on bacterial inoculum size and based on PFP concentration. Although the infection process takes 1-2 days, the PFP-instigated infection process is irreversibly established within 15 minutes of initial exposure. Remarkably, treatment of C. elegans with Cry5B PFP is able to instigate many other Bacillus species, including B. anthracis and even "non-pathogenic" Bacillus subtilis, to become lethal and infectious agents to C. elegans. Co-culturing of Cry5B-expressing B. thuringiensis with B. anthracis can result in lethal infection of C. elegans by B. anthracis. Our data demonstrate that one potential property of PFPs is to sensitize the host to bacterial infection and further that C. elegans and probably other roundworms can be common hosts for B. cereus-group bacteria, findings with important ecological and research implications.

WWP-1 is a novel modulator of the DAF-2 insulin-like signaling network involved in pore-forming toxin cellular defenses in Caenorhabditis elegans.

Pore-forming toxins (PFTs) are the single largest class of bacterial virulence factors. The DAF-2 insulin/insulin-like growth factor-1 signaling pathway, which regulates lifespan and stress resistance in Caenorhabditis elegans, is known to mutate to resistance to pathogenic bacteria. However, its role in responses against bacterial toxins and PFTs is as yet unexplored. Here we reveal that reduction of the DAF-2 insulin-like pathway confers the resistance of Caenorhabditis elegans to cytolitic crystal (Cry) PFTs produced by Bacillus thuringiensis. In contrast to the canonical DAF-2 insulin-like signaling pathway previously defined for aging and pathogenesis, the PFT response pathway diverges at 3-phosphoinositide-dependent kinase 1 (PDK-1) and appears to feed into a novel insulin-like pathway signal arm defined by the WW domain Protein 1 (WWP-1). In addition, we also find that WWP-1 not only plays an important role in the intrinsic cellular defense (INCED) against PFTs but also is involved in innate immunity against pathogenic bacteria Pseudomonas aeruginosa and in lifespan regulation. Taken together, our data suggest that WWP-1 and DAF-16 function in parallel within the fundamental DAF-2 insulin/IGF-1 signaling network to regulate fundamental cellular responses in C. elegans.

Discovery of a highly synergistic anthelmintic combination that shows mutual hypersusceptibility.

The soil-transmitted helminths or nematodes (hookworms, whipworms, and Ascaris) are roundworms that infect more than 1 billion of the poorest peoples and are leading causes of morbidity worldwide. Few anthelmintics are available for treatment, and only one is commonly used in mass drug administrations. New anthelmintics are urgently needed, and crystal (Cry) proteins made by Bacillus thuringiensis are promising new candidates. Combination drug therapies are considered the ideal treatment for infectious diseases. Surprisingly, little work has been done to define the characteristics of anthelmintic combinations. Here, by means of quantitative assays with wild-type and mutants of the roundworm Caenorhabditis elegans, we establish a paradigm for studying anthelmintic combinations using Cry proteins and nicotinic acetylcholine receptor (nAChR) agonists, e.g., tribendimidine and levamisole. We find that nAChR agonists and Cry proteins, like Cry5B and Cry21A, mutually display what is known in the HIV field as hypersusceptibility--when the nematodes become resistant to either class, they become hypersensitive to the other class. Furthermore, we find that when Cry5B and nAChR agonists are combined, their activities are strongly synergistic, producing combination index values as good or better than seen with antitumor, anti-HIV, and insecticide combinations. Our study provides a powerful means by which anthelmintic combination therapies can be examined and demonstrate that the combination of nAChR agonists and Cry proteins has excellent properties and is predicted to give improved cure rates while being recalcitrant to the development of parasite resistance.

Hypoxia and the hypoxic response pathway protect against pore-forming toxins in C. elegans.

Pore-forming toxins (PFTs) are by far the most abundant bacterial protein toxins and are important for the virulence of many important pathogens. As such, cellular responses to PFTs critically modulate host-pathogen interactions. Although many cellular responses to PFTs have been recorded, little is understood about their relevance to pathological or defensive outcomes. To shed light on this important question, we have turned to the only genetic system for studying PFT-host interactions-Caenorhabditis elegans intoxication by Crystal (Cry) protein PFTs. We mutagenized and screened for C. elegans mutants resistant to a Cry PFT and recovered one mutant. Complementation, sequencing, transgenic rescue, and RNA interference data demonstrate that this mutant eliminates a gene normally involved in repression of the hypoxia (low oxygen response) pathway. We find that up-regulation of the C. elegans hypoxia pathway via the inactivation of three different genes that normally repress the pathway results in animals resistant to Cry PFTs. Conversely, mutation in the central activator of the hypoxia response, HIF-1, suppresses this resistance and can result in animals defective in PFT defenses. These results extend to a PFT that attacks mammals since up-regulation of the hypoxia pathway confers resistance to Vibrio cholerae cytolysin (VCC), whereas down-regulation confers hypersusceptibility. The hypoxia PFT defense pathway acts cell autonomously to protect the cells directly under attack and is different from other hypoxia pathway stress responses. Two of the downstream effectors of this pathway include the nuclear receptor nhr-57 and the unfolded protein response. In addition, the hypoxia pathway itself is induced by PFT, and low oxygen is protective against PFT intoxication. These results demonstrate that hypoxia and induction of the hypoxia response protect cells against PFTs, and that the cellular environment can be modulated via the hypoxia pathway to protect against the most prevalent class of weapons used by pathogenic bacteria.

Post-translational control of the Streptomyces lividans ClgR regulon by ClpP.

It has been shown previously that expression of the Streptomyces lividans clpP1P2 operon, encoding proteolytic subunits of the Clp complex, the clpC1 gene, encoding the ATPase subunit, and the lon gene, encoding another ATP-dependent protease, are all activated by ClgR. The ClgR regulon also includes the clgR gene itself. It is shown here that the degradation of ClgR and Lon is ClpP1/P2-dependent and that the two C-terminal alanines of these new substrates are involved in their stability. The ClpC1 protein, which does not end with two alanines, is also accumulated in a clpP1P2 mutant. The results presented here support the idea that ClpP1/P2 ensure post-translational control of ClgR regulon members, including ClgR itself.

Effect of SsrA (tmRNA) tagging system on translational regulation in Streptomyces.

ssrA genes encoding tmRNA with transfer and messenger RNA functions are ubiquitous in bacteria. In a process called trans-translation, tmRNA enters a stalled ribosome and allows release of the original mRNA, then tmRNA becomes the template for translation of a short tag that signals for proteolytic degradation. We provide here the first evidences that the tmRNA tagging system (ssrA and cohort smpB) is active in Streptomyces. Transcription of the genes was shown and construction of a genetic probe allowed detection of a tmRNA-tagged peptide. Obtention of ssrA and smpB mutants of Streptomyces lividans showed that the ssrA system is dispensable in Streptomyces. Morphologies of the mutants colonies were similar to the wild type, thus tmRNA-mediated tagging does not seem to have, under conditions used, a significant effect in the Streptomyces differentiation.

ClgR, a novel regulator of clp and lon expression in Streptomyces.

The clp genes encoding the Clp proteolytic complex are widespread among living organisms. Five clpP genes are present in Streptomyces. Among them, the clpP1 clpP2 operon has been shown to be involved in the Streptomyces growth cycle, as a mutation blocked differentiation at the substrate mycelium step. Four Clp ATPases have been identified in Streptomyces coelicolor (ClpX and three ClpC proteins) which are potential partners of ClpP1 ClpP2. The clpC1 gene appears to be essential, since no mutant has yet been obtained. clpP1 clpP2 and clpC1 are important for Streptomyces growth, and a study of their regulation is reported here. The clpP3 clpP4 operon, which has been studied in Streptomyces lividans, is induced in a clpP1 mutant strain, and regulation of its expression is mediated via PopR, a transcriptional regulator. We report here studies of clgR, a paralogue of popR, in S. lividans. Gel mobility shift assays and DNase I footprinting indicate that ClgR binds not only to the clpP1 and clpC1 promoters, but also to the promoter of the Lon ATP-dependent protease gene and the clgR promoter itself. ClgR recognizes the motif GTTCGC-5N-GCG. In vivo, ClgR acts as an activator of clpC1 gene and clpP1 operon expression. Similarly to PopR, ClgR degradation might be ClpP dependent and could be mediated via recognition of the two carboxy-terminal alanine residues.

The lon gene, encoding an ATP-dependent protease, is a novel member of the HAIR/HspR stress-response regulon in actinomycetes.

Members of a family of ATP-dependent proteases related to Lon from Escherichia coli are present in most prokaryotes and eukaryotes. These proteases are generally reported to be heat induced, and various regulatory systems have been described. The authors cloned and disrupted the lon gene and studied the regulation of its expression in Streptomyces lividans. lon is negatively regulated by the HspR/HAIR repressor/operator system, suggesting that Lon is produced concomitantly with the other members of this regulon, DnaK and ClpB. The lon mutant grew more slowly than the wild-type and spore germination was impaired at high temperature. Nevertheless its cell cycle was not greatly affected and it sporulated normally.