Examination of the Borrelia burgdorferi Transcriptome in Ixodes scapularis during Feeding

Abstract

Borrelia burgdorferi gene expression within the guts of engorging Ixodes scapularis ticks was examined by use of differential immunoscreening and differential expression with a customized amplified library. Fourteen chromosomal genes involved in energy metabolism, substrate transport, and signal transduction and 10 (4 chromosomal and 6 plasmid) genes encoding putative lipoproteins and periplasmic proteins were preferentially expressed in engorging ticks. These data demonstrate a new approach to the global analysis of B. burgdorferi genes that are preferentially expressed within the vector during feeding.Borrelia burgdorferi, the agent of Lyme disease, is transmitted when Ixodes ticks feed on mammals (5, 17). While the tick is engorging, the antigenic composition of B. burgdorferi changes dramatically (1,8,9,20,21). Analysis of differentially expressed genes will provide insights into the molecular changes that occur in spirochetes during tick feeding. Recent studies have focused on the expression profiles of only a few genes, such as ospA and ospC (12,15,20), erpT (10), bbk32 and bbk50 (11), and more recently rev, mlpA, erpa/i/n, and erpb/j/o (14), that are expressed in the tick vector. In order to systematically identify B. burgdorferi antigens that are differentially expressed during tick feeding, we have used an antibody-based approach, differential immunoscreening (24), and differential expression with a customized amplification library (DECAL), a new technique to examine the transcriptomes of prokaryotic genomes. DECAL was first developed and utilized for the global analysis of gene expression in Mycobacterium tuberculosis grown in vitro (2). The rationale for exploiting DECAL is based on the following factors: (i) the analysis can be performed with as little as 10 ng of total bacterial RNA and can detect differences in levels of gene expression that are as small as fourfold and (ii) the technique is ideally suited for investigations where contaminating host material is present.Differential immunoscreening of B. burgdorferi. Ixodes scapularis nymphs infected with B. burgdorferi strain N40 and either fed to repletion on C3H/HeN mice (9) or left unfed were dissected. The isolated guts were suspended in phosphate-buffered saline (10 l/tick gut) and homogenized. The number of spirochetes in each preparation was measured by direct immunofluorescence (9). Equivalent doses of B. burgdorferi (10 6 organisms) in complete Freund's adjuvant were used to immunize C3H/HeN mice after heat inactivating the spirochetes (1 h at 60°C). Control mice were immunized with gut extracts from uninfected I. scapularis nymphs. Ten days after the final booster injection, antisera against spirochetes from unfed and fed ticks were collected. A ZAP II-B. burgdorferi N40 expression library (10,000 plaques) was screened with antisera from unfed and fed ticks. Hybridizing plaques were visualized with a picoBlue immunoscreening kit (Stratagene, La Jolla, Calif.). About 100 clones hybridized to sera from both unfed and fed ticks. Seven positi...

Genetic and physiological characterization of the Borrelia burgdorferi ORF BB0374-pfs-metK-luxS operon

“…consistent with previous observations that B. burgdorferi produces maximal levels of DPD/AI-2 during exponential phase, and increased expression of luxS during the period of rapid growth accompanying transmission from feeding tick vectors to mammalian hosts (Babb et al, 2005;Narasimhan et al, 2002). Thus, high metabolic states are accompanied by increases in three enzymic activities: (1) MetK, which increases cellular levels of the only known borrelial methyl donor, SAM; (2) Pfs, which detoxifies the product of SAM-dependent methylation reactions; and (3) LuxS, which produces DPD and homocysteine.…”

“…Several studies have implicated the importance of RNA polymerase subunits s S and s N in B. burgdorferi transitional events, including transmission from tick to mammal Elias et al, 2000;Yang et al, 2000). Since the ORF BB0374-pfs-metK-luxS operon is induced during tick-to-mammal transmission (Narasimhan et al, 2002), we examined whether either s S or s N played a role in regulation of operon transcription. The s S subunit is induced as cultures approach stationary phase, and s S levels are dependent on s N Elias et al, 2000;Yang et al, 2000).…”

Live imaging reveals a biphasic mode of dissemination of Borrelia burgdorferi within ticks

“…Live imaging has revealed that this conception is an oversimplification. That Borrelia within unfed nymphal midguts are nonmotile is consistent with the quiescent metabolic state of organisms in this milieu (50)(51)(52)(53)(54) and is in line with observations made with crushed midguts dating back to the discovery of the Lyme spirochete as a tick-borne pathogen (49). On the other hand, our finding that the fed midgut is populated with replicating, but nonmotile, spirochetes was unexpected and runs counter to the prevailing notion, which equates metabolic activity with motility (58).…”

Effects of Environmental Changes on Expression of the Oligopeptide Permease ( opp ) Genes of Borrelia burgdorferi

“…After the meal is complete, within days the number of organisms drops precipitously and remains low until the next blood meal. Narasimhan et al have previously found that genes in the opp operon were differentially expressed in fed and unfed ticks but did not identify specific genes within the operon that accounted for the differences (13). We have found that expression of oppA-II and oppA-IV remains very low in the tick hosts and that oppA-III and oppA-V are up-regulated during periods of stable nutrient availability (unfed tick and mouse hosts).…”