a-protocol-to-infect-caenorhabditis-elegans-with-salmonella

A Protocol to Infect <em>Caenorhabditis elegans</em> with <em>Salmonella typhimurium</em>

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

Florida Atlantic University

The daf-9 gene functions to integrate transforming growth factor-beta and insulin-like signaling pathways to regulate Caenorhabditis elegans larval development. Mutations in daf-9 result in transient dauer-like larval arrest, abnormal reproductive development, molting defects and increased adult longevity. The phenotype is sterol-dependent, and dependent on the activity of DAF-12, a nuclear hormone receptor. Genetic tests show that daf-9 is upstream of daf-12 in the genetic pathways for larval development and adult longevity. daf-9 encodes a cytochrome P450 related to those involved in biosynthesis of steroid hormones in mammals. We propose that it specifies a step in the biosynthetic pathway for a DAF-12 ligand, which might be a steroid. The surprising cellular specificity of daf-9 expression (predominantly in two sensory neurons) supports a previously unrecognized role for these cells in neuroendocrine control of larval development, reproduction and life span.

DAF-9, a cytochrome P450 regulating C. elegans larval development and adult longevity.

The highly conserved target-of-rapamycin (TOR) protein kinases control cell growth in response to nutrients and growth factors. In mammals, TOR has been shown to interact with raptor to relay nutrient signals to downstream translation machinery. We report that in C. elegans, mutations in the genes encoding CeTOR and raptor result in dauer-like larval arrest, implying that CeTOR regulates dauer diapause. The daf-15 (raptor) and let-363 (CeTOR) mutants shift metabolism to accumulate fat, and raptor mutations extend adult life span. daf-15 transcription is regulated by DAF-16, a FOXO transcription factor that is in turn regulated by daf-2 insulin/IGF signaling. This is a new mechanism that regulates the TOR pathway. Thus, DAF-2 insulin/IGF signaling and nutrient signaling converge on DAF-15 (raptor) to regulate C. elegans larval development, metabolism and life span.

The TOR pathway interacts with the insulin signaling pathway to regulate C. elegans larval development, metabolism and life span.

Expanded polyglutamine (polyQ) proteins aggregate intracellularly in Huntington's disease and other neurodegenerative disorders. The lysosomal degradation pathway, autophagy, is known to promote clearance of polyQ protein aggregates in cultured cells. Moreover, basal autophagy in neuronal cells in mice prevents neurodegeneration by suppressing the accumulation of abnormal intracellular proteins. However, it is not yet known whether autophagy genes play a role in vivo in protecting against disease caused by mutant aggregate-prone, expanded polyQ proteins. To examine this question, we used two models of polyQ-induced toxicity in C. elegans, including the expression of polyQ40 aggregates in muscle and the expression of a human huntingtin disease fragment containing a polyQ tract of 150 residues (Htn-Q150) in ASH sensory neurons. Here, we show that genetic inactivation of autophagy genes accelerates the accumulation of polyQ40 aggregates in C. elegans muscle cells and exacerbates polyQ40-induced muscle dysfunction. Autophagy gene inactivation also increases the accumulation of Htn-Q150 aggregates in C. elegans ASH sensory neurons and results in enhanced neurodegeneration. These data provide in vivo genetic evidence that autophagy genes suppress the accumulation of polyQ aggregates and protect cells from disease caused by polyQ toxicity.

Autophagy genes protect against disease caused by polyglutamine expansion proteins in Caenorhabditis elegans.

Dietary restriction extends life span in diverse species including Caenorhabditis elegans. However, the downstream cellular targets regulated by dietary restriction are largely unknown. Autophagy, an evolutionary conserved lysosomal degradation pathway, is induced under starvation conditions and regulates life span in insulin signaling C. elegans mutants. We now report that two essential autophagy genes (bec-1 and Ce-atg7) are required for the longevity phenotype of the C. elegans dietary restriction mutant (eat-2(ad1113) animals. Thus, we propose that autophagy mediates the effect, not only of insulin signaling, but also of dietary restriction on the regulation of C. elegans life span. Since autophagy and longevity control are highly conserved from C. elegans to mammals, a similar role for autophagy in dietary restriction-mediated life span extension may also exist in mammals.

Autophagy is required for dietary restriction-mediated life span extension in C. elegans.

A conserved insulin-like pathway modulates both aging and pathogen resistance in Caenorhabditis elegans. However, the specific innate effector functions that mediate this pathogen resistance are largely unknown. Autophagy, a lysosomal degradation pathway, plays a role in controlling intracellular bacterial pathogen infections in cultured cells, but less is known about its role at the organismal level. We examined the effects of autophagy gene inactivation on Salmonella enterica Serovar Typhimurium (Salmonella typhimurium) infection in 2 model organisms, Caenorhabditis elegans and Dictyostelium discoideum. In both organisms, genetic inactivation of the autophagy pathway increases bacterial intracellular replication, decreases animal lifespan, and results in apoptotic-independent death. In C. elegans, genetic knockdown of autophagy genes abrogates pathogen resistance conferred by a loss-of-function mutation, daf-2(e1370), in the insulin-like tyrosine kinase receptor or by over-expression of the DAF-16 FOXO transcription factor. Thus, autophagy genes play an essential role in host defense in vivo against an intracellular bacterial pathogen and mediate pathogen resistance in long-lived mutant nematodes.

Autophagy genes protect against Salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance.

Aging is a process in which individuals undergo an exponential decline in vitality, leading to death. In the last two decades, the study of the molecular regulation of aging in model organisms, particularly in C. elegans, has greatly expanded our knowledge of aging. Multiple longevity pathways, such as insulin-like growth factor signaling, TOR signaling, dietary restriction and mitochondrial activity, control aging in C. elegans. Recent genetic studies indicate that autophagy, an evolutionary conserved lysosomal degradation pathway, interacts with various longevity signals in the regulation of C. elegans life span. Here, we review the current progress in understanding the role of autophagy in the regulation of C. elegans life span.

Autophagy and longevity: lessons from C. elegans.

Efficient apoptotic corpse clearance is essential for metazoan development and adult tissue homeostasis. Several autophagy proteins have been previously shown to function in apoptotic cell clearance; however, it remains unknown whether autophagy genes are essential for efficient apoptotic corpse clearance in the developing embryo. Here we show that, in Caenorhabditis elegans embryos, loss-of-function mutations in several autophagy genes that act at distinct steps in the autophagy pathway resulted in increased numbers of cell corpses and delayed cell corpse clearance. Further analysis of embryos with a null mutation in bec- 1, the C. elegans ortholog of yeast VPS30/ATG6/mammalian beclin 1 (BECN1), revealed normal phosphatidylserine exposure on dying cells. Moreover, the corpse clearance defects of bec- 1(ok691) embryos were rescued by BEC-1 expression in engulfing cells, and bec- 1(ok691) enhanced corpse clearance defects in nematodes with simultaneous mutations in the engulfment genes, ced- 1, ced- 6 or ced- 12. Together, these data demonstrate that autophagy proteins play an important role in cell corpse clearance during nematode embryonic development, and likely function in parallel to known pathways involved in corpse removal.

Autophagy genes function in apoptotic cell corpse clearance during C. elegans embryonic development.

Salmonella typhimurium infects both intestinal epithelial cells and macrophages. Autophagy is a lysosomal degradation pathway that is present in all eukaryotes. Autophagy has been reported to limit the Salmonella replication in Caenorhabditis elegans and in mammals. However, it is unknown whether intestinal autophagy activity plays a role in host defense against Salmonella infection in C. elegans. In this study, we inhibited the autophagy gene bec-1 in different C. elegans tissues and examined the survival of these animals following Salmonella infection. Here we show that inhibition of the bec-1 gene in the intestine but not in other tissues confers susceptibility to Salmonella infection, which is consistent with recent studies in mice showing that autophagy is involved in clearance of Salmonella in the intestinal epithelial cells. Therefore, the intestinal autophagy activity is essential for host defense against Salmonella infection from C. elegans to mice, perhaps also in humans.

Article	Year
DAF-9, a cytochrome P450 regulating C. elegans larval development and adult longevity. Development (Cambridge, England)\| PubMed ID: 11782415	2002
The TOR pathway interacts with the insulin signaling pathway to regulate C. elegans larval development, metabolism and life span. Development (Cambridge, England)\| PubMed ID: 15253933	2004
Autophagy genes protect against disease caused by polyglutamine expansion proteins in Caenorhabditis elegans. Autophagy\| PubMed ID: 17172799	2007
Autophagy is required for dietary restriction-mediated life span extension in C. elegans. Autophagy\| PubMed ID: 17912023	2007
Autophagy genes protect against Salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19667176	2009
Autophagy and longevity: lessons from C. elegans. Advances in experimental medicine and biology\| PubMed ID: 20886756	2010
Autophagy genes function in apoptotic cell corpse clearance during C. elegans embryonic development. Autophagy\| PubMed ID: 23108454	2013
Intestinal autophagy activity is essential for host defense against Salmonella typhimurium infection in Caenorhabditis elegans. Developmental and comparative immunology\| PubMed ID: 24674884	2014

Kailiang Jia

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