O'Rourke, Delia et al. (2015) International Worm Meeting "Using bacterial pathogens to explore the nematode surface coat."

Stroud, Dave, Hodgkin, Jonathan, O'Rourke, Delia

[International Worm Meeting, 2015]

The surface of nematodes is poorly characterized, despite its critical roles in structural integrity, locomotion and protection from environmental stresses, as well in constituting a major interface with the mammalian immune system in parasitic nematodes. We have used bacterial pathogens that naturally adhere to the surface of C.elegans to identify mutants with altered surface biochemistry. We have thereby identified many genes involved in surface coat synthesis, which encode a variety of proteins implicated in glycosylation (glycosyl transferases and sugar transporters) and GPI anchorage. Mutant worms resistant to a virulent infection by one pathogen, Leucobacter Verde2, exhibit a trade off in being hypersensitive to the related "worm-star" pathogen Leucobacter Verde 1. Selections for resistance to Verde1 reveal new mutant classes. Many mutants affect the gene AGMO-1 (alklyglycerol monooxygenase), or enzymes required for the synthesis of its co-factor tetrahydrobiopterin. AGMO-1 is uniquely required for the breakdown of ether lipids, suggesting that these unusual lipids play an important role in the worm surface. To complement these genetic approaches we are characterizing surface-related lipids and proteins, in both C. elegans and parasitic nematodes.

O'Rourke, Delia M. et al. (2009) International Worm Meeting "Investigation of four C-type lectin genes: clec-17, 60, 70 and 86."

O'Rourke, Delia M., Hodgkin, Jonathan

[International Worm Meeting, 2009]

Many genes encoding C-type lectins (clecs) are induced following infection of C.elegans with either Gram positive or Gram negative bacteria, as part of the worm''s innate immune response. Different infections induce both shared and infection specific clec genes (H. Schulenburg 2008). The function of these genes is currently unclear: they may be involved in pathogen recognition and they may also have a role as antibacterial effectors. We are using a number of approaches to study four clecs induced in response to infection by Microbacterium nematophilum, clec-17, clec-60, clec-70 and clec-86 (O''Rourke et al 2006). RNAi knockdown or genetic knockouts of clec-17, 60 and 86 affect C.elegans defense against M.nematophilum. We are constructing reporters to examine where and when the clec promoters are activated, making TAP TAG vectors and raising antibodies to examine protein location, post-translational modification and to identify interacting proteins. We are also attempting to express these clecs in heterologous systems with the aim of obtaining pure protein for structural analysis and for in vitro assays of antibacterial activity. We will present progress and data from these approaches. Schulenburg et al Immunology, 2008, 213(237-250) O''Rourke et al Genome Res, 2006, 16(1005-1016).

O'Rourke, Delia et al. (2015) International Worm Meeting "Weird gene structure: the 5' region of mRNA for the essential glycosyltransferase BUS-8 encodes a cis-acting regulatory peptide."

Stroud, Dave, O'Rourke, Delia, Hodgkin, Jonathan

[International Worm Meeting, 2015]

The bus-8 gene encodes a diverged homolog of the conserved bifunctional glycosyltransferase ALG2, which probably acts in N-linked glycosylation. As previously published (Partridge et al. 2008, PMID: 18395708), bus-8 has essential functions in embryogenesis and in maintaining cuticle integrity, as well as affecting drug sensitivity, pathogen susceptibility, locomotion and mate recognition. Most bus-8 transcripts contain two 5' exons (exons 1 and 2) that are out of frame with the rest of the coding sequence (exons 3-10). A missense mutation in exon 1, lj22, fails to complement missense alleles in exons 5 and 10, and has similar phenotypic properties. This suggested that a long form of BUS-8 could be made from all ten exons by translational frameshifting. However, mass-spectrometry data obtained using purified C-terminally-tagged BUS-8 constructs revealed peptides only from exons 3-10 and none from exons 1 and 2, so translational frameshifting is unlikely. Moreover, transgenes expressing only exons 1 and 2 partially rescued the lj22 mutation, but did not rescue other bus-8 mutations. Therefore, it seems that exons 1 and 2 can act on their own, as a 72aa peptide, modifying the activity of the BUS-8 protein encoded by exons 3-10. Most bus-8 mutants, including lj22, are killed by Leucobacter Verde1, unlike wildtype worms; this sensitivity allows strong selection for suppressors. Verde1 selections carried out with different bus-8 alleles have yielded both extragenic and intragenic suppressors, whose sequences and properties add further complexity. The unusual gene structure of bus-8 seems to be conserved in other rhabditids at least, and bus-8 homologs are found in more distant nematode genomes (Brugia, Onchocerca). Why nematodes apparently need both bus-8 and a less diverged ALG2 homolog (algn-2 in C. elegans) is mysterious, as is the role of the regulatory peptide.

O'Rourke, Delia M. et al. (2011) International Worm Meeting "Mutations in bus-10, encoding a putative membrane protein, alter both susceptibility and resistance to different bacterial infections."

Hodgkin, Jonathan, O'Rourke, Delia M., Price, Rebecca, Gravato-Nobre, Maria, Lillios, Nicholas

[International Worm Meeting, 2011]

C. elegans is used extensively to study host-pathogen interactions. We have conducted genetic screens to identify mutations that cause resistance to infection by the nematode-specific Gram-positive bacterial pathogen, Microbacterium nematophilum. This bacterium attaches to the rectal and post-anal cuticle and causes slow growth and a defensive swelling of the rectal epithelial cells. Many of the mutations (representing at least 20 different genes) isolated in our screens alter the cuticle of the worm and prevent attachment of bacteria and the establishment of an infection. We used whole genome sequencing to identify one of these mutants, bus-10, as ZK596.3. This gene encodes a 322 amino acid nematode-specific protein, predicted to be integral to the membrane. We have characterised several large deletions of the gene as well as multiple transposon insertions derived from a mut-7 induced mutagenesis screen, in which bus-10 was found to be a conspicuous hot-spot for transposon-induced mutation (>30 bus-10 alleles recovered). Two EMS-chemically induced alleles were identified as stop mutations in exons 2 and 3. Using a bicistronic construct with the bus-10 promoter and gene linked to the gpd2/3 sequence and TagRFPT, we demonstrate that a transgenic worm strain expressing ZK596.3 can rescue the Bus-10 phenotype. We find that the bus-10 promoter is active in many tissues, most notably in the seam cells, rectal gland cell, rectal valve and the excretory gland. In addition to preventing attachment of M. nematophilum, bus-10 mutants are also strikingly altered in the response to two other pathogens, Leucobacter strains Verde1 and Verde2. Whereas wild type worms survive infection by Verde 1, but are killed by infections with Verde2, bus-10 worms, in contrast, are killed by infection with Verde1, but survive infection with Verde2. We have use the differential susceptibility of bus-10 infection by Verde1 to conduct a suppressor screen for mutagenised bus-10 worms that can survive on Verde1. This screen identified 8 suppressors, and initial mapping followed by complementation assays suggest that we have identified 8 alleles of one extragenic suppressor gene. We will present further data on the identification and properties of this suppressor gene and propose a model to explain how the interactions of bus-10 and its suppressor result in the striking differential responses of these worms to infection by two different Leucobacter strains.

O'Rourke, Delia M. et al. (2013) International Worm Meeting "Does the C.elegans glycosylation gene bus-8 undergo translational frameshifting to generate different protein isoforms?"

Partridge, Frederick A, Pavlyukovskyy, Mark, Cullen, Martin, Stroud, Dave, Hodgkin, Jonathan, O'Rourke, Delia M.

[International Worm Meeting, 2013]

We have previously shown that C. elegans with missense mutations in bus-8 (a nematode-specific glycosyltransferase) are resistant to infections by M.nematophilum and Leucobacter Verde2, but hypersensitive to infection by Leucobacter Verde1. bus-8 worms are also drug-sensitive, skiddy, defective in mate recognition and bleach-sensitive due to changes in the surface coat of the worm. bus-8 is additionally essential for ventral enclosure during embryonic morphogenesis and is expressed in cells underneath the ventral epidermis in the embryo and post-embryonically in seam cells (Partridge et al. 2008). One bus-8 allele, lj22, is a point mutant in the 5'UTR that contains a conserved putative ORF out of frame with the downstream bus-8 gene. EST and RNA seq data indicate that the ORF is transcribed and spliced with the downstream bus-8 transcript. The 5'UTR ORF encodes a possible transmembrane domain that could localise the BUS-8 glycosyltransferase to membrane compartments. We hypothesize that this region is translated with the rest of the gene (which would require +1 translational frameshifting). To investigate this we constructed a TAP-tagged version of BUS-8 and transformed mutant bus-8 worms with this construct. TAP-tagged BUS-8 is functional as it can rescue both mild and severe bus-8 mutations. We have purified BUS-8 using the tags and used mass spectrometry analysis to look for peptides corresponding to the 5' UTR encoded ORF. We have also made a number of transgenic worms to investigate the functional roles of the BUS-8 isoforms. We have used genomic mutations in bus-8 that affect only the 5'UTR (lj22) or the main part of the gene (e2883, tm1410) with transgenic constructs containing modified versions of bus-8. In this way we can investigate which isoforms of BUS-8 can rescue the bus-8 phenotypes. The sensitivity of both bus-8 (lj22) and e2883 to Verde1 infection has allowed us to conduct suppressor screens. Using this approach we have identified 5 suppressors for each allele, which are currently being characterised. Partridge et al (2008) Dev Biol 317 549.

Delia O'Rourke et al. (2007) International Worm Meeting "C-type lectins: elucidating their function in worm immunity."

Delia O'Rourke, Jonathan Hodgkin

[International Worm Meeting, 2007]

The nematode pathogen Microbacterium nematophilum infects C. elegans by attaching to the rectal and post-anal cuticle, which results in slowed growth and a characteristic tail swelling called the Dar response. The swelling is a protective host response and is induced by the ERK MAP kinase pathway (Nicholas et al 2004). Previously we investigated the genomic response of C.elegans to this infection, using microarrays to examine gene expression changes following infection. We found induction of genes encoding a number of putative defence proteins, including ten C-type lectins (ORourke et al 2006). C-type lectin genes (clec) are also induced by other infections in C. elegans including Serratia marcescens (Mallo et al 2002) and Pseudomonas aeruginosa PA14 (Shapira et al 2006, Troemel et al 2006), with each infection inducing a specific set of lectin genes. Their function in the immune response could be to act as antibacterial effectors and /or as pathogen recognition molecules. At least some of the clec genes are expressed in intestinal cells, consistent with a function in defence. We are now using a number of approaches to explore the role of clec genes in this host:pathogen interaction. We have shown that RNAi of clec-60, clec-17 and clec-86 affect C. elegans defence against M. nematophilum and we will present data on the effects of knockdown or knock out of the remaining seven M. nem-induced clec genes. We plan to express some of the induced clecs in the yeast Pichia pastoris, in order to study the CLEC proteins in isolation and conduct assays for bacterial binding and antibacterial activity. Within the worm, we are using TAP TAG technology to examine CLEC location (are these proteins secreted into the gut lumen?), to examine post-translational modifications and to identify interacting proteins. We will present progress and data from these experiments.

Delia O'Rourke et al. (2002) European Worm Meeting "Molecular cloning of C. elegans bus-6 using snip/SNP mapping"

Maria Gravato-Nobre, Jonathan Hodgkin, Delia O'Rourke

[European Worm Meeting, 2002]

Microbacterium nematophilum is a novel bacterial pathogen of C elegans (Hodgkin et al, 2000). This pathogen adheres to the rectum and post-anal region of the worm causing swelling of the underlying tissues, severe constipation and slow growth rates. Affected worms are described as having a Dar (Deformed Anal Region) phenotype. To define the molecular events involved in this host-pathogen interaction, we have used the mutator strain, mut-7, and EMS mutagenesis to screen for mutant worms that are resistant to infection. These worms exhibit a Bus (Bacterially UnSwollen) phenotype. In order to identify the bus loci we initially used transposon insertion display (TID) to try to clone the bus mutants isolated in the mut-7 screen. However, TID analysis of 14 alleles of 8 bus loci failed to identify any of the bus genes. We are now using conventional mapping techniques and are reducing our intervals using snip/SNP mapping (Wicks et al., 2001). In this poster we will present data on our progress in the mapping of one of the bus mutants, bus-6. bus-6 adult worms show no gross changes in surface lectin staining (Reindert Nijland) but have slight changes in larvae, similar to bus-1 worms. Microbacterium nematophilum do not adhere to bus-6 worms, suggesting that bus-6 may encode a protein that facilitates attachment of the bacteria to the cuticle. bus-6 has been mapped to LGV, between the dpy-11 and unc-42 markers. We will describe our progress in narrowing this interval using both deficiencies and snip/SNPs.

Delia O'Rourke et al. (2001) International C. elegans Meeting "Molecular cloning of C. elegans bus genes using Transposon Insertion Display."

Delia O'Rourke, Jonathan Hodgkin, Maria Gravato-Nobre, Hannah Nicholas

[International C. elegans Meeting, 2001]

Microbacterium nematophilum is a novel bacterial pathogen of C elegans (Hodgkin et al , 2000). This pathogen adheres to the rectum and post-anal region of the worm causing swelling of the underlying tissues, severe constipation and slow growth rates. Affected worms are described as having a Dar (Deformed Anal Region) phenotype. To define the molecular events involved in this host: pathogen interaction, we have used the mutator strain, mut-7, to activate C. elegans transposons and screen for mutant worms that are resistant to infection and exhibit a Bus (Bacterially UnSwollen) phenotype. Mutations in at least 8 bus genes have been obtained from this screen. To identify, clone and analyse the bus loci we have chosen to use the transposon insertion display (TID) technique, which has been previously used to identify C. elegans genes ( Wicks et al ., 2000). Southern blots of genomic DNA from bus and wt strains were probed with transposon specific probes. bus recombinants exhibiting unique Tc1 bands that co-segregated with the mutant phenotype were picked for TID analysis. Our initial probing of Southern blots with transposon probes illustrated the importance of multifactor crosses in obtaining strains with clean backgrounds before proceeding to TID. We will describe our use of TID and our progress in the molecular cloning of several of the bus genes. Hodgkin, J. Kuwabara, P. E. Corneliussen, B. Current Biology 2000 10 ;1615-1618 A novel bacterial pathogen, Microbacterium nematophilum , induces morphological change in the nematode C. elegans. Wicks SR, de Vries CJ, van Luenen HG, Plasterk RH Dev Biol 2000 May 15;221(2):295-307. CHE-3, a cytosolic dynein heavy chain, is required for sensory cilia structure and function in Caenorhabditis elegans .

Delia M O'Rourke et al. (2003) International Worm Meeting "Analysis of gene expression changes in C. elegans following infection with Microbacterium nematophilum"

Delia M O'Rourke, Joanne Staines, Patricia Kuwabara, Jonathan Hodgkin

[International Worm Meeting, 2003]

Microbacterium nematophilum is a novel bacterial pathogen of C. elegans (Hodgkin et al.,2000). This pathogen adheres to the rectum and post-anal region of the worm causing swelling of the underlying tissues, severe constipation and slow growth rates. Affected worms are described as having a Dar (Deformed Anal Region) phenotype.To define the molecular events involved in this host: pathogen interaction we have conducted a genome wide analysis of gene expression changes that occur during an infection. We infected a synchronised population of larval C. elegans in liquid culture before harvesting the worms and extracting RNA at two time points. Our control sample was an identical experiment using an avirulent mutant of the pathogen isolated by T. Akimkina and S. Curnock.We will present data from this microarray experiment, illustrating which genes are significantly induced or repressed. We will also examine the expression of genes previously shown to be important in the worm's response to infection by this and other pathogens.Hodgkin, J., Kuwabara, P. E. and Corneliussen, B. Current Biology 2000 10 ;1615-1618

Delia M O'Rourke et al. (2005) International Worm Meeting "Analysis of gene expression changes in C. elegans following infection with Microbacterium nematophilum."

Delia M O'Rourke, Maria Demidova, Jonathan Hodgkin, Richard Mott, Dilair Baban

[International Worm Meeting, 2005]

Microbacterium nematophilum is a bacterial pathogen of C. elegans (Hodgkin et al, 2000) that adheres to the rectum and post-anal region of the worm causing swelling of the underlying hypodermal tissue, mild constipation and slow growth rates. Affected worms are described as having a Dar (Deformed Anal Region) phenotype. C. elegans responds to this infection in part through activation of an ERK MAP kinase cascade which mediates tail swelling and prevents severe constipation (Nicholas and Hodgkin, 2004). To identify the downstream transcriptional changes that occur in C. elegans during this host/pathogen interaction we have conducted a genome wide analysis of gene expression using Affymetrix gene chips. We infected a synchronised population of larval C. elegans in liquid culture for 6hrs before harvesting the worms and extracting RNA. Our control sample was an identical experiment using an avirulent M. nematophilum generated in our laboratory by Tanya Akimkina and Steve Curnock. Analysis of data from triplicate microarray experiments has identified a number of statistically significant gene clusters whose expression changes upon infection. Clusters containing up-regulated genes are located on chromosomes IV and V and include C-type lectins, lysozyme-like proteins and proteins containing metridin-like ShK toxin and DUF141 domains. An RNAi feeding screen of these and other induced genes has identified a number that affect the Dar response. The microarray data suggest that although similar functional domains are found in proteins induced by both M. nematophilum and other pathogens, the response of C. elegans to M. nematophilum is specific. Hodgkin, J. Kuwabara, P. E. Corneliussen, B. Current Biology 200010 ;1615-1618 Nicholas, HR, Hodgkin, J. Current Biology 2004 14 ;1256-1261