Novel Pyoverdine Inhibitors Mitigate Pseudomonas aeruginosa Pathogenesis

Pseudomonas aeruginosa is a clinically important pathogen that causes a variety of infections, including urinary, respiratory, and other soft-tissue infections, particularly in hospitalized patients with immune defects, cystic fibrosis, or significant burns. Antimicrobial resistance is a substantial problem in P. aeruginosa treatment due to the inherent insensitivity of the pathogen to a wide variety of antimicrobial drugs and its rapid acquisition of additional resistance mechanisms. One strategy to circumvent this problem is the use of anti-virulent compounds to disrupt pathogenesis without directly compromising bacterial growth. One of the principle regulatory mechanisms for P. aeruginosa’s virulence is the iron-scavenging siderophore pyoverdine, as it governs in-host acquisition of iron, promotes expression of multiple virulence factors, and is directly toxic. Some combination of these activities renders pyoverdine indispensable for pathogenesis in mammalian models. Here we report identification of a panel of novel small molecules that disrupt pyoverdine function. These molecules directly act on pyoverdine, rather than affecting its biosynthesis. The compounds reduce the pathogenic effect of pyoverdine and improve the survival of Caenorhabditis elegans when challenged with P. aeruginosa by disrupting only this single virulence factor. Finally, these compounds can synergize with conventional antimicrobials, forming a more effective treatment. These compounds may help to identify, or be modified to become, viable drug leads in their own right. Finally, they also serve as useful tool compounds to probe pyoverdine activity

Pseudomonas aeruginosa Disrupts Caenorhabditis elegans Iron Homeostasis, Causing a Hypoxic Response and Death

SummaryThe opportunistic pathogen Pseudomonas aeruginosa causes serious human infections, but effective treatments and the mechanisms mediating pathogenesis remain elusive. Caenorhabditis elegans shares innate immune pathways with humans, making it invaluable to investigate infection. To determine how P. aeruginosa disrupts host biology, we studied how P. aeruginosa kills C. elegans in a liquid-based pathogenesis model. We found that P. aeruginosa-mediated killing does not require quorum-sensing pathways or host colonization. A chemical genetic screen revealed that iron chelators alleviate P. aeruginosa-mediated killing. Consistent with a role for iron in P. aeruginosa pathogenesis, the bacterial siderophore pyoverdin was required for virulence and was sufficient to induce a hypoxic response and death in the absence of bacteria. Loss of the C. elegans hypoxia-inducing factor HIF-1, which regulates iron homeostasis, exacerbated P. aeruginosa pathogenesis, further linking hypoxia and killing. As pyoverdin is indispensable for virulence in mice, pyoverdin-mediated hypoxia is likely to be relevant in human pathogenesis

DAF-16/FOXO employs the chromatin remodeller SWI/SNF to promote stress resistance and longevity

Organisms are constantly challenged by stresses and privations and require adaptive responses for their survival. The transcription factor DAF-16/FOXO is central nexus in these responses, but despite its importance little is known about how it regulates its target genes. Proteomic identification of DAF-16/FOXO binding partners in Caenorhabditis elegans and their subsequent functional evaluation by RNA interference (RNAi) revealed several candidate DAF-16/FOXO cofactors, most notably the chromatin remodeller SWI/SNF. DAF-16/FOXO and SWI/SNF form a complex and globally colocalize at DAF-16/FOXO target promoters. We show that specifically for gene-activation, DAF-16/FOXO depends on SWI/SNF, facilitating SWI/SNF recruitment to target promoters, in order to activate transcription by presumed remodelling of local chromatin. For the animal, this translates into an essential role of SWI/SNF for DAF-16/FOXO-mediated processes, i.e. dauer formation, stress resistance, and the promotion of longevity. Thus we give insight into the mechanisms of DAF-16/FOXO-mediated transcriptional regulation and establish a critical link between ATP-dependent chromatin remodelling and lifespan regulation

Stimulation of Host Immune Defenses by a Small Molecule Protects C. elegans from Bacterial Infection

The nematode Caenorhabditis elegans offers currently untapped potential for carrying out high-throughput, live-animal screens of low molecular weight compound libraries to identify molecules that target a variety of cellular processes. We previously used a bacterial infection assay in C. elegans to identify 119 compounds that affect host-microbe interactions among 37,214 tested. Here we show that one of these small molecules, RPW-24, protects C. elegans from bacterial infection by stimulating the host immune response of the nematode. Using transcriptome profiling, epistasis pathway analyses with C. elegans mutants, and an RNAi screen, we show that RPW-24 promotes resistance to Pseudomonas aeruginosa infection by inducing the transcription of a remarkably small number of C. elegans genes (∼1.3% of all genes) in a manner that partially depends on the evolutionarily-conserved p38 MAP kinase pathway and the transcription factor ATF-7. These data show that the immunostimulatory activity of RPW-24 is required for its efficacy and define a novel C. elegans–based strategy to identify compounds with activity against antibiotic-resistant bacterial pathogens

Coordinated Regulation of Intestinal Functions in C. elegans by LIN-35/Rb and SLR-2

LIN-35 is the sole C. elegans representative of the pocket protein family, which includes the mammalian Retinoblastoma protein pRb and its paralogs p107 and p130. In addition to having a well-established and central role in cell cycle regulation, pocket proteins have been increasingly implicated in the control of critical and diverse developmental and cellular processes. To gain a greater understanding of the roles of pocket proteins during development, we have characterized a synthetic genetic interaction between lin-35 and slr-2, which we show encodes a C2H2-type Zn-finger protein. Whereas animals harboring single mutations in lin-35 or slr-2 are viable and fertile, lin-35; slr-2 double mutants arrest uniformly in early larval development without obvious morphological defects. Using a combination of approaches including transcriptome profiling, mosaic analysis, starvation assays, and expression analysis, we demonstrate that both LIN-35 and SLR-2 act in the intestine to regulate the expression of many genes required for normal nutrient utilization. These findings represent a novel role for pRb family members in the maintenance of organ function. Our studies also shed light on the mechanistic basis of genetic redundancy among transcriptional regulators and suggest that synthetic interactions may result from the synergistic misregulation of one or more common targets

High-Throughput Genetic Screen Reveals that Early Attachment and Biofilm Formation Are Necessary for Full Pyoverdine Production by Pseudomonas aeruginosa

Pseudomonas aeruginosa is a re-emerging, multidrug-resistant, opportunistic pathogen that threatens the lives of immunocompromised patients, patients with cystic fibrosis, and those in critical care units. One of the most important virulence factors in this pathogen is the siderophore pyoverdine. Pyoverdine serves several critical roles during infection. Due to its extremely high affinity for ferric iron, pyoverdine gives the pathogen a significant advantage over the host in their competition for iron. In addition, pyoverdine can regulate the production of multiple bacterial virulence factors and perturb host mitochondrial homeostasis. Inhibition of pyoverdine biosynthesis decreases P. aeruginosa pathogenicity in multiple host models. To better understand the regulation of pyoverdine production, we developed a high-throughput genetic screen that uses the innate fluorescence of pyoverdine to identify genes necessary for its biosynthesis. A substantial number of hits showing severe impairment of pyoverdine production were in genes responsible for early attachment and biofilm formation. In addition to genetic disruption of biofilm, both physical and chemical perturbations also attenuated pyoverdine production. This regulatory relationship between pyoverdine and biofilm is particularly significant in the context of P. aeruginosa multidrug resistance, where the formation of biofilm is a key mechanism preventing access to antimicrobials and the immune system. Furthermore, we demonstrate that the biofilm inhibitor 2-amino-5,6-dimethylbenzimidazole effectively attenuates pyoverdine production and rescues Caenorhabditis elegans from P. aeruginosa-mediated pathogenesis. Our findings suggest that targeting biofilm formation in P. aeruginosa infections may have multiple therapeutic benefits and that employing an unbiased, systems biology-based approach may be useful for understanding the regulation of specific virulence factors and identifying novel anti-virulence therapeutics or new applications for existing therapies for P. aeruginosa infections

An In Vitro Cell Culture Model for Pyoverdine-Mediated Virulence

Pseudomonas aeruginosa is a multidrug-resistant, opportunistic pathogen that utilizes a wide-range of virulence factors to cause acute, life-threatening infections in immunocompromised patients, especially those in intensive care units. It also causes debilitating chronic infections that shorten lives and worsen the quality of life for cystic fibrosis patients. One of the key virulence factors in P. aeruginosa is the siderophore pyoverdine, which provides the pathogen with iron during infection, regulates the production of secreted toxins, and disrupts host iron and mitochondrial homeostasis. These roles have been characterized in model organisms such as Caenorhabditis elegans and mice. However, an intermediary system, using cell culture to investigate the activity of this siderophore has been absent. In this report, we describe such a system, using murine macrophages treated with pyoverdine. We demonstrate that pyoverdine-rich filtrates from P. aeruginosa exhibit substantial cytotoxicity, and that the inhibition of pyoverdine production (genetic or chemical) is sufficient to mitigate virulence. Furthermore, consistent with previous observations made in C. elegans, pyoverdine translocates into cells and disrupts host mitochondrial homeostasis. Most importantly, we observe a strong correlation between pyoverdine production and virulence in P. aeruginosa clinical isolates, confirming pyoverdine’s value as a promising target for therapeutic intervention. This in vitro cell culture model will allow rapid validation of pyoverdine antivirulents in a simple but physiologically relevant manner

A conserved mitochondrial surveillance pathway is required for defense against <i>Pseudomonas aeruginosa</i>

<div><p>All living organisms exist in a precarious state of homeostasis that requires constant maintenance. A wide variety of stresses, including hypoxia, heat, and infection by pathogens perpetually threaten to imbalance this state. Organisms use a battery of defenses to mitigate damage and restore normal function. Previously, we described a <i>Caenorhabditis elegans-Pseudomonas aeruginosa</i> assay (Liquid Killing) in which toxicity to the host is dependent upon the secreted bacterial siderophore pyoverdine. Although pyoverdine is also indispensable for virulence in mammals, its cytological effects are unclear. We used genetics, transcriptomics, and a variety of pathogen and chemical exposure assays to study the interactions between <i>P</i>. <i>aeruginosa</i> and <i>C</i>. <i>elegans</i>. Although <i>P</i>. <i>aeruginosa</i> can kill <i>C</i>. <i>elegans</i> through at least 5 different mechanisms, the defense responses activated by Liquid Killing are specific and selective and have little in common with innate defense mechanisms against intestinal colonization. Intriguingly, the defense response utilizes the phylogenetically-conserved ESRE (Ethanol and Stress Response Element) network, which we and others have previously shown to mitigate damage from a variety of abiotic stresses. This is the first report of this networks involvement in innate immunity, and indicates that host innate immune responses overlap with responses to abiotic stresses. The upregulation of the ESRE network in <i>C</i>. <i>elegans</i> is mediated in part by a family of bZIP proteins (including ZIP-2, ZIP-4, CEBP-1, and CEBP-2) that have overlapping and unique functions. Our data convincingly show that, following exposure to <i>P</i>. <i>aeruginosa</i>, the ESRE defense network is activated by mitochondrial damage, and that mitochondrial damage also leads to ESRE activation in mammals. This establishes a role for ESRE in a phylogenetically-conserved mitochondrial surveillance system important for stress response and innate immunity.</p></div