More than fishing in the dark: PCR of a dispersed sequence produces simple but ultrasensitive Wolbachia detection

BMC Microbiology

Wolfgang J Miller²  Anders E Lind¹  Lisa Klasson¹  Daniela I Schneider² 

[1] Department of Molecular Evolution, Cell and Molecular Biology, Science for Life Laboratory, Biomedical Centre, Uppsala, Sweden;Laboratory of Genome Dynamics, Department of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University of Vienna, Waehringerstrasse 10, Vienna 1090, Austria

关键词: A-supergroup repeat motif (ARM);    Limit of detection;    High- and low-titer endosymbiont infection;    Hybrid;    Glossina;    Drosophila;    Wolbachia;   

Others  :  1141089
DOI  :  10.1186/1471-2180-14-121

 received in 2014-03-03, accepted in 2014-04-30,  发布年份 2014

【 摘 要 】

Background

Detecting intracellular bacterial symbionts can be challenging when they persist at very low densities. Wolbachia, a widespread bacterial endosymbiont of invertebrates, is particularly challenging. Although it persists at high titers in many species, in others its densities are far below the detection limit of classic end-point Polymerase Chain Reaction (PCR). These low-titer infections can be reliably detected by combining PCR with DNA hybridization, but less elaborate strategies based on end-point PCR alone have proven less sensitive or less general.

Results

We introduce a multicopy PCR target that allows fast and reliable detection of A-supergroup Wolbachia - even at low infection titers - with standard end-point PCR. The target is a multicopy motif (designated ARM: A-supergroup repeat motif) discovered in the genome of wMel (the Wolbachia in Drosophila melanogaster). ARM is found in at least seven other Wolbachia A-supergroup strains infecting various Drosophila, the wasp Muscidifurax and the tsetse fly Glossina. We demonstrate that end-point PCR targeting ARM can reliably detect both high- and low-titer Wolbachia infections in Drosophila, Glossina and interspecific hybrids.

Conclusions

Simple end-point PCR of ARM facilitates detection of low-titer Wolbachia A-supergroup infections. Detecting these infections previously required more elaborate procedures. Our ARM target seems to be a general feature of Wolbachia A-supergroup genomes, unlike other multicopy markers such as insertion sequences (IS).

【 授权许可】

   
2014 Schneider et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

【 参考文献 】

• [1]Maiden MC, Bygraves JA, Feil E, Morelli G, Russell JE, Urwin R, Zhang Q, Zhou J, Zurth K, Caugant DA, Feavers IM, Achtman M, Spratt BG: Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A 1998, 95:3140-3145.
• [2]Paraskevopoulos C, Bordenstein SR, Wernegreen JJ, Werren JH, Bourtzis K: Toward a Wolbachia multilocus sequence typing system: discrimination of Wolbachia strains present in Drosophila species. Curr Microbiol 2006, 53:388-395.
• [3]Baldo L, Dunning Hotopp JC, Jolley KA, Bordenstein SR, Biber SA Choudhury RR, Hayashi C, Maiden MC, Tettelin H, Werren JH: Multilocus sequence typing system for the endosymbiont Wolbachia pipientis. Appl Environ Microbiol 2008, 72:7098-7110.
• [4]Zhou W, Rousset F, O'Neil S: Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proc Biol Sci 1998, 265:509-515.
• [5]Braig HR, Zhou W, Dobson SL, O'Neill SL: Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis. J Bacteriol 1998, 180:2373-2378.
• [6]Arthofer W, Riegler M, Schneider D, Krammer M, Miller WJ, Stauffer C: Hidden Wolbachia diversity in field populations of the European cherry fruit fly, Rhagoletis cerasi (Diptera, Tephritidae). Mol Ecol 2009, 18:3816-3830.
• [7]Masui S, Kamoda S, Sasaki T, Ishikawa H: The first detection of the insertion sequence ISW1 in the intracellular reproductive parasite Wolbachia. Plasmid 1999, 42:13-19.
• [8]Wu M, Sun LV, Vamathevan J, Riegler M, Deboy R, Brownlie JC, McGraw EA, Martin W, Esser C, Ahmadinejad N, Wiegand C, Madupu R, Beanan MJ, Brinkac LM, Daugherty SC, Durkin AS, Kolonay JF, Nelson WC, Mohamoud Y, Lee P, Berry K, Young MB, Utterback T, Weidman J, Nierman WC, Paulsen IT, Nelson KE, Tettelin H, O'Neill SL, Eisen JA: Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements. PLoS Biol 2004, 2:E69.
• [9]Riegler M, Sidhu M, Miller WJ, O'Neill SL: Evidence for a global Wolbachia replacement in Drosophila melanogaster. Curr Biol 2005, 15:1428-1433.
• [10]Cordaux R: ISWpi1 from Wolbachia pipientis defines a novel group of insertion sequences within the IS5 family. Gene 2008, 409:20-27.
• [11]Miller WJ, Ehrman L, Schneider D: Infectious speciation revisited: impact of symbiont-depletion on female fitness and mating behavior of Drosophila paulistorum. PLoS Pathog 2010, 6:e1001214.
• [12]Schneider DI, Garschall KI, Parker AG, Abd-Alla AM, Miller WJ: Global Wolbachia prevalence, titer fluctuations and their potential of causing cytoplasmic incompatibilities in tsetse flies and hybrids of Glossina morsitans subgroup species. J Invertebr Pathol 2013, 112(Suppl):S104-S115.
• [13]Riegler M, Iturbe-Ormaetxe I, Woolfit M, Miller WJ, O'Neill SL: Tandem repeat markers as novel diagnostic tools for high resolution fingerprinting of Wolbachia. BMC Microbiol 2012, 12(Suppl 1):S12. BioMed Central Full Text
• [14]Brelsfoard C, Tsiamis G, Falchetto M, Gomulski L, Telleria E, Alam U, Ntountoumis E, Swain M, Malacrida A, Bourtzis K, Aksoy S: Wolbachia symbiont genome sequence and extensive chromosomal insertions described from the tsetse fly Glossina morsitans. 2014. in press: doi:10.1371/journal.pntd.0002728
• [15]Stahlhut JK, Desjardins CA, Clark ME, Baldo L, Russell JA, Werren JH, Jaenike J: The mushroom habitat as an ecological arena for global exchange of Wolbachia. Mol Ecol 2010, 19:1940-1952.
• [16]Belda E, Moya A, Bentley S, Silva FJ: Mobile genetic element proliferation and gene inactivation impact over the genome structure and metabolic capabilities of Sodalis glossinidius, the secondary endosymbiont of tsetse flies. BMC Genom 2010, 11:449. BioMed Central Full Text
• [17]Oakeson KF, Gil R, Clayton AL, Dunn DM, von Niederhausern AC, Hamil C, Aoyagi A, Duval B, Baca A, Silva FJ, Vallier A, Jackson DG, Latorre A, Weiss RB, Heddi A, Moya A, Dale C: Genome degeneration and adaptation in a nascent stage of symbiosis. Genome Biol Evol 2014, 6:76-93.
• [18]Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, Salzberg SL: Versatile and open software for comparing large genomes. Genome Biol 2004, 5:R12. BioMed Central Full Text
• [19]Jeyaprakash A, Hoy MA: Long PCR improves Wolbachia DNA amplification: wsp sequences found in 76% of sixty-three arthropod species. Insect Mol Biol 2000, 9:393-405.
• [20]Hanner R, Fugate M: Branchiopod phylogenetic reconstruction from 12S rDNA sequence data. J Crustacean Biol 1997, 17:74-183.
• [21]Augustinos AA, Santos-Garcia D, Dionyssopoulou E, Moreira M, Papapanagiotou A, Scarvelakis M, Doudoumis V, Ramos S, Aguiar AF, Borges PA, Khadem M, Latorre A, Tsiamis G, Bourtzis K: Detection and characterization of Wolbachia infections in natural populations of aphids: is the hidden diversity fully unraveled? PLoS One 2011, 6:e28695.
• [22]Klasson L, Westberg J, Sapountzis P, Näslund K, Lutnaes Y, Darby AC, Veneti Z, Chen L, Braig HR, Garrett R, Bourtzis K, Andersson SG: The mosaic genome structure of the Wolbachia wRi strain infecting Drosophila simulans. Proc Natl Acad Sci U S A 2009, 106:5725-5730.
• [23]Elegaard KM, Klasson L, Näslund K, Bourtzis K, Andersson SG: Comparative genomics of Wolbachia and the bacterial species concept. PLoS Genet 2013, 9:e1003381.
• [24]Salzberg SL, Dunning Hotopp JC, Delcher AL, Pop M, Smith DR, Eisen MB, Nelson WC: Serendipitous discovery of Wolbachia genomes in multiple Drosophila species. Genome Biol 2005, 6:R23. Erratum in. Genome Biol 2005, 6:402. BioMed Central Full Text
• [25]Siozios S, Cestaro A, Kaur R, Pertot I, Rota-Stabelli O, Anfora G: Draft Genome Sequence of the Wolbachia Endosymbiont of Drosophila suzukii. Genome Announc 2013, 1:e00032-13. doi:10.1128/genomeA.00032-13
• [26]Kent BN, Salichos L, Gibbons JG, Rokas A, Newton IL, Clark ME, Bordenstein SR: Complete bacteriophage transfer in a bacterial endosymbiont (Wolbachia) determined by targeted genome capture. Genome Biol Evol 2011, 3:209-218.
• [27]Klasson L, Walker T, Sebaihia M, Sanders MJ, Quail MA, Lord A, Sanders S, Earl J, O'Neill SL, Thomson N, Sinkins SP, Parkhill J: Genome evolution of Wolbachia strain wPip from the Culex pipiens group. Mol Biol Evol 2008, 25:1877-1887.
• [28]Darby AC, Armstrong SD, Bah GS, Kaur G, Hughes MA, Kay SM, Koldkjær P, Rainbow L, Radford AD, Blaxter ML, Tanya VN, Trees AJ, Cordaux R, Wastling JM, Makepeace BL: Analysis of gene expression from the Wolbachia genome of a filarial nematode supports both metabolic and defensive roles within the symbiosis. Genome Res 2012, 22:2467-2477.
• [29]Desjardins CA, Cerqueira GC, Goldberg JM, Chandler M, Mahillon J: Insertion sequences revisited. In Edited by Craig NL, Craigie R, Gellert M, Lambowitz AM. Edited by Mobile DNAII. Washington, DC: American Society of Microbiology; 2002:305-366.
• [30]Foster J, Ganatra M, Kamal I, Ware J, Makarova K, Ivanova N, Bhattacharyya A, Kapatral V, Kumar S, Posfai J, Vincze T, Ingram J, Moran L, Lapidus A, Omelchenko M, Kyrpides N, Ghedin E, Wang S, Goltsman E, Joukov V, Ostrovskaya O, Tsukerman K, Mazur M, Comb D, Koonin E, Slatko B: The Wolbachia genome of Brugia malayi: endosymbiont evolution within a human pathogenic nematode. PLoS Biol 2005, 3:E121.
• [31]Ehrman L, Powell JR: The Drosophila willistoni species group. Ashburner, Carson, Thompson 1981-1986, 193-225.
• [32]Miller WJ, Riegler M: Evolutionary dynamics of wAu-like Wolbachia variants in Neotropical Drosophila species. Appl Environ Microbiol 2006, 72:826-835.
• [33]Kidwell MG, Novy JB: Hybrid dysgenesis in Drosophila melanogaster: sterility resulting from gonadal dysgenesis in the P-M system. Genetics 1979, 92:1127-1140.
• [34]Poinsot D, Montchamp-Moreau C, Merçot H: Wolbachia segregation rate in Drosophila simulans naturally bi-infected cytoplasmic lineages. Heredity (Edinb) 2000, 85(Pt 2):191-198.