Characterization of Flagellotropic, Chi-Like Salmonella Phages Isolated from Thai Poultry Farms.

Despite a wealth of knowledge on Salmonella phages worldwide, little is known about poultry-associated Salmonella phages from Thailand. Here, we isolated 108 phages from Thai poultry farms that infect Salmonellaenterica serovar Typhimurium. Phages STm101 and STm118 were identified as temperate Siphoviridae phages. Genome sequencing and analyses revealed these phages share approximately 96% nucleotide sequence similarity to phage SPN19, a member of the Chi-like virus genus. PCR amplification of the gene encoding capsid protein E of the Chi-like phage was positive for 50% of phage isolates, suggesting a predominance of this phage type among the sampled poultry farms. In addition to the flagella, two phages required the lipopolysaccharide to infect and lyse Salmonella. Furthermore, phylogenomic analysis demonstrated that phages STm101 and STm118 formed a monophyletic clade with phages isolated from Western countries, but not from closer isolated phages from Korea. However, further investigation and more phage isolates are required to investigate possible causes for this geographic distribution.

Disrupting the Amblyomma americanum (L.) CD147 receptor homolog prevents ticks from feeding to repletion and blocks spontaneous detachment of ticks from their host.

The CD147 receptor is a cell-surface glycoprotein in the IgG family that plays pivotal roles in intercellular interactions involved with numerous physiological and pathological processes such as extracellular matrix remodeling. We previously found an Amblyomma americanum (Aam) tick CD147 receptor homolog among genes that were up regulated in response to tick feeding stimuli. This study characterizes an AamCD147 receptor protein that is 72-83% conserved in other tick species and possess characteristic CD147 receptor sequence features: an extracellular (EC) region containing two IgG domains, a transmembrane and the cytoplasmic domains. Likewise, the AamCD147 EC domain folds into secondary structures that are consistent to the human homolog: an amino-terminus beta-barrel that is linked to 2-carboxy-terminus beta-sheets with consensus disulfide bonds conserved in each of the 2 domains. CD147 receptor signaling and regulatory mechanisms are putatively conserved in ticks as revealed by in silico analysis that show presence in the tick genome of CD147 receptor signaling protein homologs, cyclophilin (CyP) A and B, and chaperones that transport it to the plasma membrane, caveolin-1 and CyP60. The AamCD147 receptor has a dichotomous expression pattern of where it is up regulated in response to feeding in the salivary gland but remains constant at the midgut and ovary levels suggesting that it may regulate different functions in different tick organs. We speculate that biological functions of the AamCD147 receptor are essential to tick feeding success as revealed by RNAi-mediated silencing that caused ticks to obtain smaller blood meals, of which approximately 69% were below threshold to trigger spontaneous detachment of ticks from the host. These ticks showed unusual cuticle tenderness and assumed a reddish coloration, a phenomenon that has been attributed to tick midgut damage allowing red blood cells to leak into tick hemolymph. On the basis of the CD147 receptor being linked to tissue growth regulation in mammals, we speculate that silencing of the AamCD147 receptor blocked progression of the tick intermolt growth, a process that precedes tick engorgement and their spontaneous detachment of from the host to end feeding. The results are discussed in context of advances in tick molecular physiology.

Silencing of three Amblyomma americanum (L.) insulin-like growth factor binding protein-related proteins prevents ticks from feeding to repletion.

The insulin-like growth factor (IGF) binding proteins (IGFBP) family is the regulatory arm of the IGF signaling system that control mitogenic and anabolic actions of IGF peptide hormones. This study describes cloning and biological characterization of three Amblyomma americanum (L.) (Aam) proteins that show amino-terminal sequence and secondary structure similarity to the IGFBP superfamily. The three molecules here provisionally identified as AamIGFBP-rP1 and short (S) and long (L) AamIGFBP-rP6 are expressed in multiple tick organs and are responsive to tick feeding activity with the former being upregulated and the latter being downregulated. We show that they regulate tick physiological functions that may be related to A. americanum tick feeding success as revealed by RNAi-mediated dual silencing of AamIGFBP-rP6S and AamIGFBP-rP6L or AamIGFBP-rP1 alone, which caused a reduction in blood meal size compared to the controls. Additionally, in the case of AamIGFBP-rP1 silencing, 47% of ticks died while attempting to feed and those that did survive and spontaneously detached from the host failed to lay eggs. Although AamIGFBP-rP6S and AamIGFBP-rP6L show overall identities of 49% and 59%, respectively, to Rhipicephalus microplus C protein, the identity level jumps to ~84% when the comparison is restricted to first 70 amino acids of the mature protein. Similarly, the AamIGFBP-rP1 mature protein is ~72%, 87%, 88% and 92% identical to that of Ixodes scapularis S, R. microplus, R. appendiculatus N and A. variegatum F, respectively. The observed across-tick-species conservation suggests that the three molecules (AamIGFBP-rP1, AamIGFBP-rP6S and AamIGFBP-rP6L) represent target for development of vaccines to protect animals against multiple tick species. The data are discussed with reference to advances in tick molecular biology and the potential of the three proteins as targets for immunizing animals against tick feeding.

Ixodes scapularis tick serine proteinase inhibitor (serpin) gene family; annotation and transcriptional analysis.

BACKGROUND: Serine proteinase inhibitors (Serpins) are a large superfamily of structurally related, but functionally diverse proteins that control essential proteolytic pathways in most branches of life. Given their importance in the biology of many organisms, the concept that ticks might utilize serpins to evade host defenses and immunizing against or disrupting their functions as targets for tick control is an appealing option. RESULTS: A sequence homology search strategy has allowed us to identify at least 45 tick serpin genes in the Ixodes scapularis genome that are structurally segregated into 32 intronless and 13 intron-containing genes. Nine of the intron-containing serpins occur in a cluster of 11 genes that span 170 kb of DNA sequence. Based on consensus amino acid residues in the reactive center loop (RCL) and signal peptide scanning, 93% are putatively inhibitory while 82% are putatively extracellular. Among the 11 different amino acid residues that are predicted at the P1 sites, 16 sequences possess basic amino acid (R/K) residues. Temporal and spatial expression analyses revealed that 40 of the 45 serpins are differentially expressed in salivary glands (SG) and/or midguts (MG) of unfed and partially fed ticks. Ten of the 38 serpin genes were expressed from six to 24 hrs of feeding while six and fives genes each are predominantly or exclusively expressed in either MG and SG respectively. CONCLUSION: Given the diversity among tick species, sizes of tick serpin families are likely to be variable. However this study provides insight on the potential sizes of serpin protein families in ticks. Ticks must overcome inflammation, complement activation and blood coagulation to complete feeding. Since these pathways are regulated by serpins that have basic residues at their P1 sites, we speculate that I. scapularis may utilize some of the serpins reported in this study to manipulate host defense. We have discussed our data in the context of advances on the molecular physiology of I. scapularis. Although the paper is descriptive, this study provides the first step toward a comprehensive understanding of serpins in tick physiology.

Molecular and biological characterization of the Amblyomma americanum organic anion transporter polypeptide.

The organic anion transporting polypeptides (Oatps in rodents and other organism; OATPs in human) are Na(+)-independent transporters that shuttle a wide range of endogenous and xenobotic amphipathic compounds across plasma membranes. We previously discovered an Amblyomma americanum tick (Aam) Oatp cDNA among genes that were upregulated or induced in ticks that were stimulated to start feeding. In this study, we have characterized a 2860 bp full-length cDNA that encode a 724 amino acid putative protein. Bioinformatics and hydropathy analyses revealed that, in addition to the kazal-type serine proteinase inhibitor motif, AamOatp possess typical features that characterize the Oatp/OATP protein family, including 12 transmembrane (TM) domains, the consensus amino acid motif D-X-RW-(I,V)-GAWW-X-G-(F,L)-L and 11 consensus cysteine residues in the large extracellular domain between TM9 and TM10. AamOatp is constitutively and ubiquitously expressed, as determined by RT-PCR amplification of the transcript, in all organs of ticks that fed for 1-7 days. Analysis of the normalized transcript abundance revealed that from days 1 to 5 of feeding, AamOatp mRNA expression in the midgut (MG) was 60-80-fold higher than levels found in the salivary gland (SG), ovary (OV) and carcass (CA). By contrast, by day 7 of feeding, the AamOatp mRNA was 60-80-fold more strongly expressed in the OV than in the SG, MG and CA. These data strongly indicate that changing physiological needs during the tick feeding process influences transcriptional regulation of AamOatp. Our data also show that RNAi-mediated suppression of the AamOatp caused ticks to obtain smaller blood meals, which consequently resulted in ticks laying fewer eggs. The results are discussed in the context of AamOatp as a potential pharmacological or anti-tick vaccine target.

Molecular and expression analysis of a family of the Amblyomma americanum tick Lospins.

Serine proteinase inhibitors (serpins) are a family of structurally similar but functionally diverse proteins that regulate several important proteolytic cascades in most branches of life. We have characterized 17 Amblyomma americanum serpin cDNAs here named as ;Lospins' (L; an acronym for Lone Star tick serpin) that possess three beta-sheets, eight alpha-helices and a reactive center loop consistent with the consensus serpin superfamily secondary structures. Visual inspection of deduced amino acid sequences revealed two patterns of basic residues: (i) (86)DKSRVLKAYKRL(97) in L5 and L13-16 and (ii) (158)VRDKTRGKI(166) in all Lospins, which are similar to consensus glycosaminoglycan (GAG) binding sites (XBnXmBX, where X and B are non-basic and basic residues, n=1 or 2 and m=1, 2 or 3). On three-dimensional models, the two putative GAG binding sites mapped onto alpha-helices D and F, respectively, with calculation of electrostatic surface potentials revealing basic patches on L5 and L13-16 models that are comparable to the heparin-binding site on antithrombin. RT-PCR expression analysis of 15 selected genes showed that the majority (11/15) of the Lospins were ubiquitously expressed in the midgut, ovary and salivary glands. On a neighbor-joining phylogeny guide tree, 15 serpins from other ticks and 17 Lospins from this study, a total of 32 tick serpin sequences, segregated into five groups with Lospins in groups A and D being conserved across tick species. The discovery of Lospins in this study sets the framework for future studies to understand the role of serpins in tick physiology.

The molecular basis of the Amblyomma americanum tick attachment phase.

Towards discovery of molecular signaling cascades that trigger and/or facilitate the tick attachment and formation of its feeding lesion, suppressive subtractive hybridization, high throughput sequencing and validation of differential expression by cDNA dot blot hybridization were performed on Amblyomma americanum ticks that had attained appetence and were exposed to feeding stimuli. This approach allowed for identification of 40 genes that are up regulated before ticks begin to penetrate the host skin. Based on BLAST and secondary structure homology searches as well as motif scan analyses, provisional identification was assigned to approximately 38% (15/40) of the identified genes that have been classified into 6 groups: Ligand binding (2 insulin-like growth-factor binding, lipocalin/histamine binding), immune responsive (tumor necrosis receptor associated factor 6, Microplusin-like antimicrobial), stress response proteins (Heat shock protein [HSP] 90, HSP40, 78 kDa glucose regulated protein [GRP78]), transporter polypeptides (ABC transporter and organic anion transporter polypeptide [contains Kazal-type serine proteinase inhibitor domain]) and enzymes/regulators (extracellular matrix metaloprotease inducer, chitinase), extracellular matrix-like proteins (tropoelastin, flagelliform silk protein). Sixty-two percent (25/40) of genes that did not show similarity to known proteins are classified as orphans. BLASTN homology search against the tick EST database revealed that 50% (20/40) of candidate genes are conserved in other ticks suggesting that molecular events underlying the A. americanum tick attachment phase may be conserved in other tick species. Consistent with the general assumption that genes that are up regulated in ticks before they started to penetrate host skin represented the tick's molecular preparedness to evade host defense during the attachment phase, real time RT-PCR analyses data demonstrated that the majority of the tested genes (9/11) were highly expressed during the first 24 h of feeding. Identification of genes in this study provides the framework for future studies to elucidate molecular signaling cascades that regulate early molecular events during the tick attachment phase.

Steady-state cleavage kinetics for dengue virus type 2 ns2b-ns3(pro) serine protease with synthetic peptides.

The N-terminal part of the NS3 protein from dengue virus contains a trypsin-like serine protease responsible for processing the nonstructural region of the viral polyprotein. Enzymatic activity of the NS2B-NS3(pro) precursor incorporating a full-length NS2B cofactor of dengue virus type 2 was examined by using synthetic dodecamer peptide substrates encompassing native cleavage sequences of the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 polyprotein junctions. Cleavage of the dansylated substrates was monitored by a HPLC-based assay and kinetic parameters for K(1M), k(cat) and k(cat)/K(m) were obtained. The data presented here show that NS2B-NS3(pro) expressed in recombinant E. coli can be renatured to an active protease which reacts in the absence of microsomal membranes with all 4 substrate peptides, albeit the molecule does not exhibit autoproteolytic processing at the NS2B/NS3 site. A marked difference in cleavage efficiency was found for the NS2B/NS3 substrate and the remaining 3 peptides based on the NS2A/NS2B, NS3/NS4A and NS4A/NS5 cleavage sites.

In vitro determination of dengue virus type 2 NS2B-NS3 protease activity with fluorescent peptide substrates.

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1 M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2BNS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.