Loss of srf-3-encoded Nucleotide Sugar Transporter Activity in Caenorhabditis elegans Alters Surface Antigenicity and Prevents Bacterial Adherence
2004; Elsevier BV; Volume: 279; Issue: 29 Linguagem: Inglês
10.1074/jbc.m402429200
ISSN1083-351X
AutoresJörg Höflich, Patricia Berninsone, Christine Göbel, Maria J. Gravato‐Nobre, Brian Libby, Creg Darby, Samuel M. Politz, Jonathan Hodgkin, Carlos B. Hirschberg, Ralf Baumeister,
Tópico(s)Bacterial biofilms and quorum sensing
ResumoDuring the establishment of a bacterial infection, the surface molecules of the host organism are of particular importance, since they mediate the first contact with the pathogen. In Caenorhabditis elegans, mutations in the srf-3 locus confer resistance to infection by Microbacterium nematophilum, and they also prevent biofilm formation by Yersinia pseudotuberculosis, a close relative of the bubonic plague agent Yersinia pestis. We cloned srf-3 and found that it encodes a multitransmembrane hydrophobic protein resembling nucleotide sugar transporters of the Golgi apparatus membrane. srf-3 is exclusively expressed in secretory cells, consistent with its proposed function in cuticle/surface modification. We demonstrate that SRF-3 can function as a nucleotide sugar transporter in heterologous in vitro and in vivo systems. UDP-galactose and UDP-N-acetylglucosamine are substrates for SRF-3. We propose that the inability of Yersinia biofilms and M. nematophilum to adhere to the nematode cuticle is due to an altered glycoconjugate surface composition of the srf-3 mutant. During the establishment of a bacterial infection, the surface molecules of the host organism are of particular importance, since they mediate the first contact with the pathogen. In Caenorhabditis elegans, mutations in the srf-3 locus confer resistance to infection by Microbacterium nematophilum, and they also prevent biofilm formation by Yersinia pseudotuberculosis, a close relative of the bubonic plague agent Yersinia pestis. We cloned srf-3 and found that it encodes a multitransmembrane hydrophobic protein resembling nucleotide sugar transporters of the Golgi apparatus membrane. srf-3 is exclusively expressed in secretory cells, consistent with its proposed function in cuticle/surface modification. We demonstrate that SRF-3 can function as a nucleotide sugar transporter in heterologous in vitro and in vivo systems. UDP-galactose and UDP-N-acetylglucosamine are substrates for SRF-3. We propose that the inability of Yersinia biofilms and M. nematophilum to adhere to the nematode cuticle is due to an altered glycoconjugate surface composition of the srf-3 mutant. To counteract infections, all higher organisms have evolved sophisticated immunological defenses. Although adaptive immunity is specific to vertebrates, the mechanisms of innate immunity are ancient in evolutionary terms and have been highly conserved during evolution (1Hoffmann J.A. Kafatos F.C. Janeway C.A. Ezekowitz R.A. Science. 1999; 284: 1313-1318Crossref PubMed Scopus (2153) Google Scholar). This suggests that studying their role in diverse species may yield key insights into the evolutionary origins and molecular mechanisms of the mammalian innate immune system. Therefore, vertebrate as well as invertebrate model systems have been established to understand the underlying mechanisms of innate immunity. The use of Caenorhabditis elegans as a host model system for innate immunity was first demonstrated for the human opportunistic pathogen Pseudomonas aeruginosa (2Mahajan-Miklos S. Tan M.W. Rahme L.G. Ausubel F.M. Cell. 1999; 96: 47-56Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar). In the short time since then, C. elegans has been established as a model system for studying infection by a wide variety of pathogens (3Alegado R.A. Campbell M.C. Chen W.C. Slutz S.S. Tan M.W. Cell Microbiol. 2003; 5: 435-444Crossref PubMed Scopus (100) Google Scholar). Unlike P. aeruginosa, Yersinia pestis and Yersinia pseudotuberculosis do not colonize the intestinal tissues of C. elegans but instead generate a sticky biofilm on the exterior of the animal's head that impairs feeding, leading to growth delay or larval arrest (4Darby C. Hsu J.W. Ghori N. Falkow S. Nature. 2002; 417: 243-244Crossref PubMed Scopus (214) Google Scholar). This phenomenon is a model for bubonic plague transmission, because biofilm formation by Y. pestis requires putative polysaccharide biosynthetic genes that are also required for flea infection and transmission of the pathogen by flea bites (5Hinnebusch B.J. Perry R.D. Schwan T.G. Science. 1996; 273: 367-370Crossref PubMed Scopus (283) Google Scholar). A biofilm is defined as a community of bacteria enclosed in a self-produced exopolysaccharide matrix that adheres to a biotic or abiotic surface. Biofilm formation by pathogens is of great clinical importance, because bacteria embedded in biofilms have been shown to be more resistant to antibiotics, to components of the host immune system, and to removal by mechanical forces (6Costerton J.W. Stewart P.S. Greenberg E.P. Science. 1999; 284: 1318-1322Crossref PubMed Scopus (8939) Google Scholar). A novel C. elegans pathogen has been described recently (7Hodgkin J. Kuwabara P.E. Corneliussen B. Curr. Biol. 2000; 10: 1615-1618Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Microbacterium nematophilum adheres to the rectum of wild type animals, inducing a localized nonlethal response and causing a swelling of the underlying hypodermal tissue (deformed anal region (Dar) 1The abbreviations used are: Dar, deformed anal region; NST, nucleotide sugar transporter; FITC, fluorescein isothiocyanate; RT, reverse transcriptase; VSV, vesicular stomatitis virus; MDCK, Madin-Darby canine kidney; ER, endoplasmic reticulum; LPG, lipophosphoglycan; GFP, green fluorescent protein. phenotype). In the same study, several mutants with altered surface antigenicity (srf mutants) were found to be resistant to infection by M. nematophilum. Altering surface antigenicity is an important mechanism by which parasitic nematodes can evade the host immune system, and C. elegans has been used as a model to understand the factors required for changing the surface composition (8Blaxter M. Page A.P. Rudin W. Maizels R.M. Parasitol. Today. 1992; 8: 243-247Abstract Full Text PDF PubMed Scopus (158) Google Scholar, 9Politz S.M. Philipp M. Parasitol. Today. 1992; 8: 6-12Abstract Full Text PDF PubMed Scopus (96) Google Scholar). srf-1 was identified as a surface polymorphism in variants of C. elegans that failed to bind a polyclonal antiserum raised against the adult cuticle of wild type N2 worms (10Politz S.M. Chin K.J. Herman D.L. Genetics. 1987; 117: 467-476Crossref PubMed Google Scholar). Additional srf mutations were identified in a screen for mutants showing altered surface binding of antisera. It has been proposed that srf-3 animals have lost surface components, thereby exposing antigenic determinants that are hidden in wild-type animals (11Politz S.M. Philipp M. Estevez M. O'Brien P.J. Chin K.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2901-2905Crossref PubMed Scopus (39) Google Scholar). srf mutants were also found in a screen for ectopic binding by wheat germ agglutinin (12Link C.D. Silverman M.A. Breen M. Watt K.E. Dames S.A. Genetics. 1992; 131: 867-881Crossref PubMed Google Scholar). srf-4, srf-8, and srf-9 have extensive pleiotropic defects and show no resistance to infection by M. nematophilum, suggesting that the mutations are involved in biological processes distinct from that of other srf genes. In contrast, srf-2, srf-3, and srf-5, which all show no visible alterations in morphology or behavior, are resistant to infection by M. nematophilum (7Hodgkin J. Kuwabara P.E. Corneliussen B. Curr. Biol. 2000; 10: 1615-1618Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Here we show that the resistance of srf-3 animals to infection by M. nematophilum and to biofilm formation by Y. pseudotuberculosis is due to the failure of the bacteria or their secreted products to adhere to the animals' cuticle. We cloned srf-3 and found that it codes for a nucleotide sugar transporter (NST) capable of translocating UDP-galactose and UDP-N-acetylglucosamine into the Golgi apparatus lumen. Furthermore, the observed expression pattern supports a function of SRF-3 in cuticle and surface modification. The results presented here should facilitate further study of the mechanisms and the genetic network underlying pathogen resistance of srf mutants. Strain Maintenance and Genetics—All C. elegans strains were grown as described (13Wood W.B. The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Transgenic animals were constructed as described (14Mello C.C. Kramer J.M. Stinchcomb D. Ambros V. EMBO J. 1991; 10: 3959-3970Crossref PubMed Scopus (2446) Google Scholar). Injection of dsRNA was done as described (15Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Nature. 1998; 391: 806-811Crossref PubMed Scopus (11794) Google Scholar). Staining for β-galactosidase activity was done as previously described (16Mounsey A. Molin L. Hope I.A. Hope I.A. C. elegans: A Practical Approach. Oxford University Press, Oxford1999: 181-199Google Scholar). The following strains were used: LGII rrf-3(pk1426) (17Simmer F. Tijsterman M. Parrish S. Koushika S.P. Nonet M.L. Fire A. Ahringer J. Plasterk R.H. Curr. Biol. 2002; 12: 1317-1319Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar); LGIII unc-119(ed4) (18Maduro M. Pilgrim D. Genetics. 1995; 141: 977-988Crossref PubMed Google Scholar); LG IV dpy-20(e1282) unc-30(e191), srf-3(yj10), unc-31(e169) srf-3(yj10) (19, Libby, B. J. (1998) Molecular Genetic Analysis of srf-6, a Gene Involved in Surface Antigen Switching in Caenorhabdit is Elegans. Ph.D. thesis, Worcester Polytechnic InstituteGoogle Scholar), unc-31(e169) srf-3(yj10) lev-1(e211) (19, Libby, B. J. (1998) Molecular Genetic Analysis of srf-6, a Gene Involved in Surface Antigen Switching in Caenorhabdit is Elegans. Ph.D. thesis, Worcester Polytechnic InstituteGoogle Scholar); SU93 (jcIs1) (20Mohler W.A. Simske J.S. Williams-Masson E.M. Hardin J.D. White J.G. Curr. Biol. 1998; 8: 1087-1090Abstract Full Text Full Text PDF PubMed Google Scholar), and sDf22/nT1 IV +/nT1V (21Clark D.V. Rogalski T.M. Donati L.M. Baillie D.L. Genetics. 1988; 119: 345-353Crossref PubMed Google Scholar). The following yeast strains were used: Saccharomyces cerevisiae, PRY225 (ura3–52, lys2–801am, ade2–1020c, his3, leu2, trp1Δ1); Kluyveromyces lactis, KL3 (Mat a, uraA, mnn2–2, arg–K+, pKD1+). Mapping of srf-3 was carried out as follows. From unc-31(e169) srf-3(yj10) +/++ lev-1(e211) parents, 13 Lev Srf Unc and three Lev Unc recombinants were isolated. From unc-31(e169) srf-3(yj10) lev-1(e211)/+ + + parents, zero Lev Srf non-Unc and seven Lev non-Srf non-Unc recombinants were isolated. This placed srf-3 0.24 map units to the right of unc-31. The srf-3 alleles br6, e2680, e2689, e2789, and e2797 were identified first by assignment to linkage group IV and then by complementation tests. All alleles except br6 were tested for complementation of the reference allele srf-3(yj10), using both the visible Bus phenotype and the Srf phenotype as judged by FITC-wheat germ agglutinin (Vector Laboratories) binding (12Link C.D. Silverman M.A. Breen M. Watt K.E. Dames S.A. Genetics. 1992; 131: 867-881Crossref PubMed Google Scholar). srf-3(br6) was tested for complementation of yj10 in the Yersinia biofilm formation assay. For cloning srf-3 by rescue, we injected srf-3(e2789) unc-30(e191)/+ animals with cosmids or subclones, using co-injected pBY1153 (sel-12::egfp) at a concentration of 25 ng/μl as a transformation marker. The progeny of transgenic Unc animals were grown and tested for the Dar phenotype on plates that had been seeded with a mixture of 99.9% Escherichia coli OP 50 and 0.1% M. nematophilum CBX102. All injection mixes were supplemented to a DNA concentration of 100 ng/μl with Bluescript II SK-(Stratagene). Biofilm formation was assayed as follows. Five adult worms per plate were allowed to lay eggs for 2–4 h on plates containing Y. pseudotuberculosis YPIII. After removing the worms, the plates were incubated at 20 °C for 2 days or 15 °C for 4 days, and the number of L4 larvae was compared with the number of total worms. Because the biofilm blocks feeding and causes larval arrest or growth delay, this assay serves as an indirect measurement of biofilm attachment. Plasmid Construction—To identify the open reading frame sufficient to provide srf-3 activity, the rescuing cosmid M02B1 (line 1 at the top of Fig. 3B) was digested with PacI, and the 27-kb backbone was religated, creating the plasmid pBY1453 (line 6). A 4.6-kb (line 2) and a 7-kb (line 3) PstI fragment of M02B1 were ligated into BluescriptII SK-(Stratagene), generating the plasmids pBY1451 and pBY1452, respectively. A 13-kb AatII fragment derived from M02B1 was ligated into Litmus28 (New England Biolabs), creating the plasmid pBY1454 (line 4). pBY1508 was constructed by deleting 3.2 kb with Eco52I, which removes the ORF M02B1.1 in pBY1454 (line 7). pBY1454 was digested with SapI to generate pBY1509, which carries a 4.2-kb deletion in the ORF ZK896.9 (line 8). To tag SRF-3 at the N terminus with GFP, 2.5-kb of srf-3 promoter sequence was PCR-amplified with primers that introduced a SalI site upstream and an EcoRV immediately downstream of the ATG. This PCR product was inserted into pPD118.15 (SalI- and Acc65I-blunted), creating the plasmid pBY1603. The SRF-3 coding region plus 1.7-kb of untranslated sequence were PCR-amplified and cloned via NheI and AatII into pBY1603, resulting in the plasmid pBY1605. To tag SRF-3 at the C terminus with β-galactosidase carrying a nuclear localization signal, the srf-3 genomic region, including 2.5-kb of promoter sequence, was PCR-amplified. The primers were designed so that the stop codon was removed, and SalI sites were introduced at both ends of the PCR product. The PCR product was inserted into pPD95.57, creating pBY1907. For heterologous expression of srf-3, the cDNA was fused by PCR mutagenesis with a sequence encoding an 11-amino acid VSV-G tag and cloned into BluescriptII SK-, creating the plasmid pBY1820 (VSV tag at the N terminus, VSVSRF-3). From pBY1820 the cDNA was cloned via XbaI to pcDNA3.1, creating pBY1823 (VSVSRF-3). To ligate VSVSRF-3 to p426GPD, pBY1820 was digested with XbaI, and p426GPD was digested with SpeI; fragments were ligated to create pBY1822 (VSVSRF-3). For generating constructs allowing expression in K. lactis, SRF-3 was fused with a VSV-G tag at the C terminus by PCR mutagenesis and cloned into BluescriptII SK-(pBY1819). SRF-3VSV was PCR-amplified with primers introducing SalI sites immediately upstream of the ATG and downstream of the stop codon. The PCR product was ligated to XhoI-digested pE4 vector, resulting in the plasmid pBY1866 (SRF-3VSV). The co-injection marker pBY1153 was cloned as follows. The sel-12 cDNA in pBY895 (22Wittenburg N. Eimer S. Lakowski B. Rohrig S. Rudolph C. Baumeister R. Nature. 2000; 406: 306-309Crossref PubMed Scopus (86) Google Scholar) was replaced with EGFP coding sequence on a SmaI/NotI fragment from pEGFP-N1 (Clontech). The resulting construct, pBY1153 (sel-12::egfp), drives expression of egfp under the control of the sel-12 promoter. To express the srf-3 cDNA under the control of the srf-3 regulatory sequences, the 3′-untranslated region was amplified with PCR using the primers 5′-GATCTCGAGATTCTCGTCTGTATATTATAGTGACGCT-3′ and 5′-GATCCTCGAGTGTGGCCTGGAAATTGGAGG-3′ and cloned with XhoI into BluescriptII SK-. The srf-3 promoter without an ATG was amplified with the primers 5′-GATCTCTAGAATTTCCTTTTGATTGTGAAGATTC-3′ and 5′-GATCGAGCTCGCTTTCCCCAAACATTTTTAG-3′ and then cloned with XbaI/SacI to the construct containing the 3′-untranslated region, resulting in plasmid pBY1818. The srf-3 cDNA obtained from the RT-PCR was ligated to pBY1818 with HindIII, generating pBY1865. Cloning of the srf-3 cDNA—To confirm the exon-intron boundaries of srf-3, three independent reverse transcription reactions (primer RT1, AAATTATAAAAGCAGCAGA; RT2, TGATAATAAATCACAGATTC; RT3, GCATTTTTTAAGCGTCACTA) were performed on RNA prepared from a population of mixed staged N2 animals. PCR was performed using the primers 5′-GACTAAGCTTATGAAGACGGCAATTTTGAT-3′ and 5′-GATCAAGCTTTTACAGACAAAACGCCTCTT-3′, and the cDNAs were cloned and sequenced. RNA preparation was done with the Qiagen RNAeasy kit according to the manufacturer's instructions. SL1 and SL2 splicing was tested by PCR using specific primers 5′-ACGTGGATCCGGTTTAATTACCCAAGTTTGAG and ACGTGGATCCGGTTTTAACCCAGTTACTCAAG, respectively. Molecular Biology—DNA sequences of srf-3 alleles were determined directly from PCR-amplified DNA. The site of the Tc1 insertion in the e2789 allele was identified by the DNA sequence of a PCR product obtained with Tc1-specific primers combined with SRF-3-specific primers. For the analysis of SRF-3 localization, 5 ng/μl pBY1605 was co-injected with 50 ng/μl pDP#MM016B (unc-119 rescuing plasmid (18Maduro M. Pilgrim D. Genetics. 1995; 141: 977-988Crossref PubMed Google Scholar)) into unc-119(ed4) animals, and 5 ng/μl pBY1907 was injected with 25 ng/μl pRF4 (dominant rol-6 marker (14Mello C.C. Kramer J.M. Stinchcomb D. Ambros V. EMBO J. 1991; 10: 3959-3970Crossref PubMed Scopus (2446) Google Scholar)) into wild type worms. For each injection, three independent lines were analyzed for GFP expression or β-galactosidase staining, respectively. For heterologous expression of SRF-3, the cDNA was fused in frame with a single VSV-G tag coding sequence by PCR mutagenesis and cloned into pcDNA3.1 (Invitrogen) for expression in mammalian cell lines, into pG426 (23Mumberg D. Muller R. Funk M. Gene (Amst.). 1995; 156: 119-122Crossref PubMed Scopus (1594) Google Scholar) for expression in S. cerevisiae, and into pE4 (24Guillen E. Abeijon C. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7888-7892Crossref PubMed Scopus (81) Google Scholar) for expression in K. lactis. General molecular biology methods were as described (25Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Yeast transformations were done using the LiAc/PEG method (26Gietz R.D. Woods R.A. Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2077) Google Scholar). For detection of the VSV-tagged SRF-3, an anti-VSV antibody from Roche Applied Science was used. Syto13 staining of worms was carried out as previously described (7Hodgkin J. Kuwabara P.E. Corneliussen B. Curr. Biol. 2000; 10: 1615-1618Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), using dye purchased from Molecular Labs (Leiden, The Netherlands). Nucleotide Sugar Transport Assay—The theoretical basis for the translocation assay of nucleotide sugars into Golgi apparatus enriched vesicles has been described previously (27Perez M. Hirschberg C.B. Methods Enzymol. 1987; 138: 709-715Crossref PubMed Scopus (36) Google Scholar). The nucleotide sugar transport assay and analysis of the samples was carried out as previously described (28Berninsone P. Hwang H.Y. Zemtseva I. Horvitz H.R. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3738-3743Crossref PubMed Scopus (116) Google Scholar). To determine radioactivity, liquid scintillation spectrometry was used. Radionucleotides were purchased from PerkinElmer Life Sciences and American Radiolabelled Chemicals (St. Louis, MO). Generation of Stable Madin-Darby Canine Kidney (MDCK) RCAr Transfectants and Determination of Ricin Resistance—MDCK RCAr cells were transfected in OPTI-MEM (Invitrogen) medium with 1 μg of plasmid DNA using Lipofectin (Invitrogen) for 6 h and then grown for 72 h in complete medium (minimal essential medium containing 10% fetal calf serum and antibiotics). Cells were trypsinized and plated at low density in complete medium containing Geneticin (G418; 0.4 mg/ml). Surviving cells were cloned and grown at 30 °C at variable concentrations of ricin (RCA II (E. Y. Laboratories Inc., San Mateo, CA)) in microtiter plates to determine resistance. Cell survival was determined by staining with methylene blue in 50% methanol. Stained plates were photographed with white light illumination. Cell Surface Labeling of K. lactis—K. lactis cells transformed with pE4-srf-3vsv or vector alone were grown at 30 °C in SCM-URA medium and then washed three times with 0.9% NaCl, 0.5 mm CaCl2. Approximately 5 A600 of cells was resuspended in 100 μl of 0.5 mg/ml GSII-FITC (EY Laboratories) in 0.9% NaCl, 0.5 mm CaCl2 and incubated for 1 h at 30 °C with shaking. Samples were washed three times and resuspended in 0.9% NaCl, 0.5 mm CaCl2. Fluorescence at 535 nm was measured with a Tecan microplate reader. Sequences of primers used for cloning of the constructs presented in this study can be obtained upon request. srf-3 Animals Are Resistant to Infection by M. nematophilum and to Biofilm Formation by Y. pseudotuberculosis—Wild-type worms grown on plates containing M. nematophilum CBX102 become constipated due to a postanal swelling and, as a consequence, feed less. This results in a 20% slower growth rate compared with a resistant srf-2 mutant grown under the same conditions (7Hodgkin J. Kuwabara P.E. Corneliussen B. Curr. Biol. 2000; 10: 1615-1618Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). When grown on M. nematophilum, srf-3(yj10) animals were also found to be resistant to infection. The tails of the srf-3 animals grown on this pathogen were indistinguishable from tails of animals grown on E. coli OP50, the normal food, indicating that srf-3 mutants strongly suppressed the Dar phenotype (Fig. 1). This resistance was not allele-specific, since additional srf-3 alleles were isolated in mut-7 and ethylmethanesulfonate screens for mutants resistant to M. nematophilum infection (Bus, bacterial unswollen). 2M. J. Gravato-Nobre, unpublished data. With the notable exception of e2797, all srf-3 alleles tested did not exhibit a Dar phenotype when grown under standard conditions (20 °C) on plates containing M. nematophilum (Table I and Fig. 1). At 15 and 20 °C, srf-3(e2797) animals showed a Dar phenotype that was weaker than that exhibited by wild-type animals, but at 25 °C, animals were indistinguishable from other srf-3 alleles. Thus, the Bus phenotype of e2797 is temperature-sensitive.Table IPenetrance of Dar phenotype in N2 and srf-3 animals on plates containing M. nematophilumTWild type (N2)yj10e2789e2680e2797e2689br6%%%%%%%25 °C10000(n = 322)(n = 238)(n = 279)20 °C1000001900(n = 403)(n = 543)(n = 464)(n = 395)(n = 243)(n = 460)(n = 135)15 °C100022(n = 183)(n = 253)(n = 385) Open table in a new tab We also examined the progeny of wild type and various srf-3 alleles on plates containing M. nematophilum (Table II). When grown on a lawn of E. coli OP50, the number of progeny of all tested srf-3 alleles was in the range of or slightly lower than that of wild type. In contrast, on plates containing 0.1% M. nematophilum, the brood size of srf-3 mutants was unaffected, whereas the number of progeny produced by wild type worms was reduced by 50% (Table II).Table IIFecundity of N2 and different srf-3 alleles on plates containing standard OP50 as food source and on plates containing M. nematophilumStrainE. coli OP50E. coli OP50 + 0.1% M. nematophilumN2310 ± 05 (n = 15)144 ± 09 (n = 16)srf-3(yj10)285 ± 09 (n = 20)260 ± 07 (n = 20)srf-3(e2798)248 ± 09 (n = 18)259 ± 06 (n = 20)srf-3(e2680)262 ± 20 (n = 17)241 ± 19 (n = 20)srf-3(e2797)272 ± 08 (n = 18)231 ± 13 (n = 20)srf-3(e2689)253 ± 12 (n = 18)295 ± 08 (n = 20) Open table in a new tab All srf-3 alleles were also resistant to biofilm formation by Y. pseudotuberculosis. When we grew wild-type animals on a lawn of Y. pseudotuberculosis, a close relative of the plague agent Y. pestis, the heads of the animals became surrounded by a biofilm (Fig. 1), which blocks feeding and inhibits larval development (4Darby C. Hsu J.W. Ghori N. Falkow S. Nature. 2002; 417: 243-244Crossref PubMed Scopus (214) Google Scholar, 29Joshua G.W. Karlyshev A.V. Smith M.P. Isherwood K.E. Titball R.W. Wren B.W. Microbiology. 2003; 149: 3221-3229Crossref PubMed Scopus (105) Google Scholar). No bacteria could be detected in the intestine or in other interior tissues, suggesting that biofilm production represents the only sign of infection by this bacterial species. In contrast, all srf-3 alleles were resistant to Yersinia biofilm formation (Fig. 1 and Table III), including e2797 which, as with M. nematophilum, displayed a temperature-sensitive phenotype. A novel srf-3 allele, br6, was identified in a direct screen for animals resistant to Yersinia biofilm formation. 3C. Darby, unpublished results.Table IIIGrowth of N2 animals and srf-3 animals on Y. pseudotuberculosisTWild type (N2)yj10e2789e2680e2797e2689br6%%%%%%%25 °C7.9 (n = 764)56.7 (n = 716)99.5 (n = 664)20 °C3.4 (n = 641)99.7 (n = 481)100 (n = 680)99.8 (n = 528)1.4 (n = 452)100 (n = 725)99.4 (n = 436)15 °C0 (n = 395)1.8 (n = 365)98.9 (n = 201) Open table in a new tab M. nematophilum and Y. pseudotuberculosis Biofilms Cannot Adhere to the Cuticle of srf-3 Animals—There are two obvious mechanisms that would result in the resistance of C. elegans to infection by these diverse bacterial species. First, adherence to the cuticle surface could be inhibited in the mutant, preventing colonization by M. nematophilum and biofilm formation by Yersinia. Second, bacterial adherence might not be prevented, but alteration of secondary (signaling) mechanisms could prevent anal swelling or the generation of a biofilm. For example, it was recently shown that inactivation of a MAPKK pathway can alter the C. elegans immune response (30Kim D.H. Feinbaum R. Alloing G. Emerson F.E. Garsin D.A. Inoue H. Tanaka-Hino M. Hisamoto N. Matsumoto K. Tan M.W. Ausubel F.M. Science. 2002; 297: 623-626Crossref PubMed Scopus (592) Google Scholar). To distinguish between these possibilities for M. nematophilum, srf-3 mutants and wild-type worms were grown on plates containing the pathogen and subsequently incubated with the dye Syto13, a nucleic acid vital stain, under conditions that stain bacteria preferentially. In wild-type C. elegans, fluorescent M. nematophilum that have infected the anal region are clearly visible. In contrast, srf-3 mutants do not show any staining, indicating that M. nematophilum is not able to adhere to the cuticle of srf-3 mutants (Fig. 2). In the case of Yersinia, bacteria do not adhere directly even to wild-type animals; adherence is always mediated by biofilm polysaccharide (4Darby C. Hsu J.W. Ghori N. Falkow S. Nature. 2002; 417: 243-244Crossref PubMed Scopus (214) Google Scholar). There is also no evidence for signaling between Yersinia and C. elegans. 4Tan, L., and Darby, C. (2004) J. Bacteriol. 186, in press. In summary, resistance against two unrelated bacterial strains that use different pathogenic strategies can be conferred by mutations in a single factor, srf-3. Molecular Identification of srf-3—In order to identify the mechanism that confers resistance against bacterial infection, we cloned srf-3. Initial mapping placed srf-3 on chromosome IV to the right of unc-22 (11Politz S.M. Philipp M. Estevez M. O'Brien P.J. Chin K.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2901-2905Crossref PubMed Scopus (39) Google Scholar). Three-factor crosses mapped srf-3 between unc-31 and lev-1, near unc-31 (Fig. 3A). Injection of cosmids from this region into srf-3 mutants revealed that M02B1 rescued the resistance to infection in transgenic off-spring of injected worms (data not shown; see also “Experimental Procedures”). A subclone with only one open reading frame, M02B1.1, was sufficient to rescue susceptibility to infection by M. nematophilum and biofilm formation of Y. pseudotuberculosis in all srf-3 alleles tested (see Fig. 3B). RT-PCR and subsequent sequencing identified a 987-bp srf-3 cDNA (Fig. 4 for exon-intron boundaries; see “Experimental Procedures” for details). In C. elegans, the 5′-end of many mRNAs begins with a 22-nucleotide splice leader sequence added by trans splicing (31Blumenthal T. Steward K. Riddle D.L. C. elegans II. Cold Spring Harbor Laboratory Press, Plainview, NY1997: 117-146Google Scholar). The srf-3 transcript we found was exclusively splice leader sequence 1 (SL-1)-spliced and therefore most likely includes the true 5′-end of the mRNA. Injection of double-stranded RNA prepared from this cDNA into rrf-3(pk1426), a strain with increased sensitivity to RNA interference, rendered the progeny resistant to infection by M. nematophilum. Furthermore, expression of the srf-3 cDNA under the control of 2.5 kb of 5′ and 3′ genomic elements restored M. nematophilum infection in srf-3 animals (data not shown), indicating that the isolated cDNA codes for a functional protein and suggesting that the construct contains the entire regulatory region of the gene. We subsequently sequenced all six srf-3 alleles. All of them carried mutations in the open reading frame (Fig. 4). The mutations observed in yj10, br6, e2789, and e2689 would lead to a severe truncation of the encoded protein and are most likely null alleles. This is supported by the fact that, when srf-3(e2789) was placed over a deficiency, the srf-3(e2789)/sDf22 phenotype was not more severe than that of the homozygous srf-3(e2789) mutant. srf-3 encodes a 328-amino acid type III transmembrane protein that, as shown in an alignment with proteins from Drosophila melanogaster, Homo sapiens, and Schizosaccharomyces pombe (Fig. 4B), is similar to NSTs. These proteins function as antiporters, exchanging a nucleotide sugar for the corresponding nucleoside monophosphate in the lumen of the endoplasmic reticulum (ER) or the Golgi apparatus. Transport of nucleotide sugars into the Golgi apparatus lumen provides the donor substrate for glycosyltransferases and is therefore necessary for the subsequent addition of sugars to proteins, lipids, and glycosaminoglycans (32Hirschberg C.B. Robbins P.W. Abei
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