Microarray analysis of gene expression changes in feeding female and male lone star ticks, Amblyomma americanum (L).

A collection of EST clones from female tick Amblyomma americanum salivary glands was hybridized to RNA from different feeding stages of female tick salivary glands and from unfed or feeding adult male ticks. In the female ticks, the expression patterns changed dramatically upon starting feeding, then changed again towards the end of feeding. On beginning feeding, genes possibly involved in survival on the host increased in expression as did many housekeeping genes. As feeding progressed, some of the survival genes were downregulated, while others were upregulated. When the tick went into the rapid feeding phase, many of the survival genes were downregulated, while a number of transport-associated genes and genes possibly involved in organ degeneration increased. In the males, the presence of females during feeding made a small difference, but feeding made a larger difference. Males showed clear differences from females in expression, as well. Protein synthesis genes were expressed more in all male groups than in the partially fed females, while the putative secreted genes involved in avoiding host defenses were expressed less.

The sodium-dependent D-glucose transport protein of Helicobacter pylori.

Helicobacter pylori is a gram-negative pathogenic microaerophile with a particular tropism for the mucosal surface of the gastric epithelium. Despite its obligatory microaerophilic character, it can metabolize D-glucose and/or D-galactose in both oxidative and fermentative pathways via a Na(+)-dependent secondary active transport, a glucokinase and enzymes of the pentose phosphate pathway. We have assigned the Na(+)-dependent transport of glucose to the protein product of the H. pylori 1174 gene. The gene was heterologously expressed in a glucose transport-deficient Escherichia coli strain, where transport activities of radiolabelled D-glucose, D-galactose and 2-deoxy-D-glucose were restored, consistent with the expected specificity of the hexose uptake system in H. pylori. D-mannose was also identified as a substrate. The HP1174 transport protein was purified and reconstituted into proteoliposomes, where sodium dependence of sugar transport activity was demonstrated. Additionally the tryptophan/tyrosine fluorescence of the purified protein showed quenching by 2-deoxy-D-glucose, D-mannose, D-glucose or D-galactose in the presence of sodium ions. This is the first reported purification and characterization of an active glucose transport protein member of the TC 2.1.7 subgroup of the Major Facilitator Superfamily, constituting the route for entry of sugar nutrients into H. pylori. A model is derived of its three-dimensional structure as a paradigm of the family.

Gene expression in male tick salivary glands is affected by feeding in the presence of females.

In this study, we have compared gene expression in the salivary glands of male Dermacentor andersoni ticks fed in the presence or absence of females to that in unfed males. We have established that the patterns of expression are different using arbitrarily primed polymerase chain reaction following reverse transcription of RNA (RAP-PCR) and differential display. Our results indicate a significant difference in salivary gland gene expression between fed and unfed males and also between males fed in the presence of females versus males fed in the absence of females. This difference in expression was partially confirmed by hybridization of probes made from mRNA from the three groups of ticks against the cloned gene fragments. Gene fragments for septin, utrophin, eukaryotic chain release factor 3, and elongation factor 2 were identified in this study, though the majority of fragments were not similar to anything in the databases.

Application of RNA interference in tick salivary gland research.

Ticks are obligate ectoparasites that feed on a variety of hosts including mammals, birds and reptiles. Prolonged attachment on the host and an ability to transmit a wide variety of pathogens are the special features of tick feeding. Salivary glands are the major route for secretion of excess fluid, several proteins, and factors that counteract the host immune response and hence play a significant role in the success of tick feeding. RNA interference (RNAi) enables scientists to silence genes encoding proteins in an absolutely sequence specific manner at the mRNA level. This technique has already been successfully employed in analyzing roles of proteins of important functions or in assigning roles to several proteins of unknown functions in a variety of animals. In this review, we outline the process of RNAi and the applicability of RNAi in tick salivary gland research.

Amblyomma americanum salivary gland homolog of nSec1 is essential for saliva protein secretion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins assemble in tight core complexes which promote fusion of carrier vesicles with target compartments. Members of this class of proteins are expressed in all eukaryotic cells and distributed in distinct subcellular compartments. All vesicle transport mechanisms known to date have an essential requirement for a member of the Sec1 protein family, including the nSec1 in regulated exocytosis. A homolog of nSec1 was cloned and sequenced from the salivary glands of partially fed female ticks. Double-stranded RNA was used to specifically reduce the amount of nSec1 mRNA and protein in female adult tick salivary glands. This reduction was accompanied by a decrease in anticoagulant protein release by the glands and by abnormalities in feeding by dsRNA treated ticks. We report the efficacy of double-stranded RNA-mediated interference in "knocking down" nSec1 both in vivo and in vitro in tick salivary glands and the applicability of this technique for studying the mechanism of exocytosis in tick salivary glands.

Amblyomma americanum salivary glands: double-stranded RNA-mediated gene silencing of synaptobrevin homologue and inhibition of PGE2 stimulated protein secretion.

Protein secretion into the saliva from the tick salivary glands is due to exocytosis of vesicular membrane bound granular material regulated by SNARE complex proteins after salivary gland stimulation by PGE2 [Insect Biochem. Mol. Biol. 32 (2002) 1711]. Proteins associated with vesicles (v-SNAREs) are essential components of the exocytotic process. Synaptobrevin is a key v-SNARE in all secreting cells studied to date. A vesicle-associated synaptobrevin cDNA fragment homologue from the salivary glands of partially fed lone star tick females was cloned and sequenced. Double-stranded (ds) RNA interference (RNAi) is an effective method to silence specific gene expression. The functional role of synaptobrevin in protein secretion in partially fed tick salivary glands was studied with an in vitro RNAi method. Incubation of isolated salivary glands with double-stranded RNA (dsRNA) transcribed from a tick salivary gland synaptobrevin cDNA fragment resulted in decreased expression of the transcript, a reduction in the level of synaptobrevin protein and inhibition of PGE2 stimulated anticoagulant protein secretion by isolated salivary glands. We demonstrate the applicability of RNAi for studying individual steps in the mechanism of PGE2 stimulated exocytosis in the salivary glands of ixodid ticks.

Collection and characterisation of bacterial membrane proteins.

A general strategy for the amplified expression in Escherichia coli of membrane transport and receptor proteins from other bacteria is described. As an illustration we report the cloning of the putative alpha-ketoglutarate membrane transport gene from the genome of Helicobacter pylori, overexpression of the protein tagged with RGS(His)6 at the C-terminus, and its purification in mg quantities. The retention of structural and functional integrity was verified by circular dichroism spectroscopy and reconstitution of transport activity. This strategy for overexpression and purification is extended to additional membrane proteins from H. pylori and from other bacteria.

Sugar metabolism by Brucellae.

The metabolic capabilities of the species of Brucella were originally of interest as a means of distinguishing them from each other and from other genera. Certain unusual characteristics, especially erythritol utilization, were studied in the hopes they would shed light on the pathogenicity. With the advent of modern genetic methods and genomic sequencing, it is now possible to get a good idea of the total capabilities of the organism and to do tests to confirm these deductions. Brucella appears to be a fairly normal member of the alpha-proteobacteria, but with some differences. A few questions remain, such as whether Brucella uses the Entner-Doudoroff pathway. Some of the genes in carbohydrate utilization have been shown to be important in virulence.

Comparison of differentially expressed genes in the salivary glands of male ticks, Amblyomma americanum and Dermacentor andersoni.

Genes expressed differentially in the salivary glands of unfed and fed male ticks, Amblyomma americanum (L.), were identified, cloned and sequenced, and some were compared with those expressed in the salivary glands of Dermacentor andersoni. Total protein and RNA increased sixfold in the salivary glands of fed male A. americanum, while in fed male D. andersoni salivary glands, RNA increased approximately 3.5 times. Feeding D. andersoni in the presence of females increased total RNA by 25% over those fed in the absence of females. Complementary DNAs were synthesized from RNA obtained from unfed and fed ticks and amplified using RNA arbitrarily primed polymerase chain reaction (RAP-PCR) with three different primers in separate reactions. Differential display showed clear banding differences between the fed and the unfed ticks in A. americanum and D. andersoni. Sixty-one cDNA fragments that appeared to be from differentially expressed genes in A. americanum were isolated, cloned and sequenced. Hybridization reactions with labeled cDNA probes confirmed the differential expression of many of the genes in unfed and fed ticks' salivary glands; however, many of the bands contained more than one fragment and some of the fragments isolated from apparently differential bands were not specific. Sequences for 28 of the cDNA fragments (150-600 nucleotides in length) demonstrated similarity to genes in the databases, but nine of these were similar to sequences of unknown function. Some of the gene fragments identified may be important to tick feeding or tick salivary gland physiology, including a histamine-binding protein, an organic ion transporter, an apoptosis inhibitor, a cathepsin-B-like cysteine protease, proteins involved in gene regulation and several proteins involved in protein synthesis. Cross-hybridization of identified cDNAs from A. americanum with cDNA probes synthesized from D. andersoni total RNA did not show significant similarity between the two species.

RNA interference: applicability in tick research.

The rapid development of new genetic tools has boosted the gene discovery machinery. RNA interference (RNAi), a gene silencing process, has been recently used in several eukaryotic organisms to elucidate the function(s) of unknown genes and biochemical pathways. We used the dsRNA technique in Amlyomma americanum female ticks to test the applicability of the RNAi approach in ticks. Incubation of tick salivary glands (TSGs) in vitro and in vivo injection into whole female ticks with histamine binding protein (HBP) dsRNA led to a reduction in the HBP transcripts in the dsRNA treated groups. The dsRNA-injected ticks had a profound difference in their feeding pattern compared to control ticks that might reflect an increase in local histamine concentrations at the feeding sites. To our knowledge, this is the first RNAi study in ticks. In conclusion, RNAi can be applied in ticks and might be used to test the function of key proteins crucial for avoiding host defense at the tick-host interface.

Brucella abortus strain 2308 putative glucose and galactose transporter gene: cloning and characterization.

The gene for the putative transporter for glucose and galactose from Brucella abortus strain 2308 was isolated by functional complementation of Escherichia coli strains lacking either glucose or galactose transport systems. The same two plasmid clones were isolated from each screen. These clones restored glucose and galactose transport to the respective E. coli strains. The sequence of the 1806 bp overlap between these two plasmids was determined. A 1242 bp ORF whose disruption eliminated complementation of both E. coli strains showed 36% identity with the E. coli fucP gene encoding a fucose transporter. These two transporters are members of the major facilitator superfamily, in which they represent a previously undescribed family. In addition, an incomplete gene similar to E. coli hisG was found. One of the plasmids complemented E. coli hisG mutants.