Arthropod Organic Anion Transporting Polypeptides in Tick-Borne Bacterial and Viral Infections

Vector-borne diseases (VBDs) are human illness transmitted by an arthropod vector. World Health Organization (WHO) estimates that VBD has a huge impact worldwide that is responsible for affecting a billion people and causes 700,000 deaths annually. In recent years, there has been a continuous increase in the incidences of tick-borne diseases such as Lyme diseases and human anaplasmosis as reported by Center for Disease Control and Prevention. Very few reliable VBD control strategies have emerged till now. Transmission-blocking vaccines can provide effective management of VBDs but requires identification and characterization of novel vector-pathogen conserved molecules that play a significant role in pathogen survival and transmission from the vector host. This work describes two studies identifying the role of conserved tick organic anion transporting polypeptides (OATPs) in bacterial and viral infections. In the first study, the results show that A. phagocytophilum modulates a specific tick host organic anion transporting polypeptide (isoatp4056) and kynurenine aminotransferase (kat), a gene responsible for the production of metabolite xanthurenic acid (XA) from tryptophan catabolism pathway, for its survival in Ixodes scapularis ticks. Silencing of isoatp4056 expression using RNA interference revealed that this gene has no effect on bacterial acquisition from the murine host, but affects bacterial survival in tick cells. Furthermore, silencing of gene expression for either kat alone or with isoatp4056 affected both bacterial survival and expression of isoatp4056. Exogenous addition of XA revealed increased isoatp4056 expression and bacterial burden in tick salivary gland and ticks cells. By using electrophoretic mobility shift assays (EMSA), the study provides evidence that both XA and A. phagocytophilum influences regulation of isoatp4056 gene. The second part of the study focuses on characterization of the role of these conserved organic anion transporting polypeptides in association with Lyme disease agent Borrelia burgdorferi and tick-borne Langat virus (LGTV), a viral pathogen closely related to tick-borne encephalitis virus (TBEV). Quantitative Real-Time PCR (QRT-PCR), data show that B. burgdorferi has no impact on the arthropod oatps gene expression in unfed nymphal ticks. Similarly, synchronous LGTV infection of unfed ticks (nymphs) revealed no impact on the expression of tick OATPs. However, specific OATPs were significantly downregulated upon LGTV infection in ticks cells at 24 h but not at 72 h post infection (p.i.). Furthermore, OATP inhibitor (SPZ) treatment followed by LGTV infection of tick cells showed significant reduction of LGTV loads, expression of kat gene, and several OATP genes. Bioinformatic characterization of medically important arthropod vectors including ticks, mosquitos, and lice revealed presence of several post-translational modifications sites such as glycosylation, phosphorylation and myristoylation sites. This set of studies provides a novel understanding on the manipulation of conserved OATPs, tryptophan pathway byproduct XA and kat, by a rickettsial pathogen for its survival in the tick vector host. In addition, these studies provide evidence on the role of tick OATPs in vector-tick-borne virus interactions. Collectively, this study provides compelling evidence on the involvement of arthropod OATPs in tick interactions with intracellular bacteria and viruses

An Efficient Microinjection Method to Generate Human Anaplasmosis Agent \u3ci\u3eAnaplasma Phagocytophilum\u3c/i\u3e-Infected Ticks

Ticks are important vectors that transmit several pathogens including human anaplasmosis agent, Anaplasma phagocytophilum. This bacterium is an obligate intracellular rickettsial pathogen. An infected reservoir animal host is often required for maintenance of this bacterial colony and as a source for blood to perform needle inoculations in naïve animals for tick feeding studies. In this study, we report an efficient microinjection method to generate A. phagocytophilum-infected ticks in laboratory conditions. The dense-core (DC) form of A. phagocytophilum was isolated from in vitro cultures and injected into the anal pore of unfed uninfected Ixodes scapularis nymphal ticks. These ticks successfully transmitted A. phagocytophilum to the murine host. The bacterial loads were detected in murine blood, spleen, and liver tissues. In addition, larval ticks successfully acquired A. phagocytophilum from mice that were previously infected by feeding with DC-microinjected nymphal ticks. Transstadial transmission of A. phagocytophilum from larvae to nymphal stage was also evident in these ticks. Taken together, our study provides a timely, rapid, and an efficient method not only to generate A. phagocytophilum-infected ticks but also provides a tool to understand acquisition and transmission dynamics of this bacterium and perhaps other rickettsial pathogens from medically important vectors

Comparison of ropivacaine alone or with dexamethasone as an adjuvant for reducing pain during positioning for neuraxial blockade with ultrasound-guided fascia iliaca compartment block

Background: Comparison of ropivacaine alone or with dexamethasone as an adjuvant for reducing pain during positioning for neuraxial blockade with ultrasound-guided fascia iliaca compartment block.Methods: In this double-blinded study, a total of 60 patients between 18 to 80 years of age, undergoing surgery for hip fracture were enrolled. Patients in Group A received 40 ml of 0.25% ropivacaine +2 ml saline and patients in Group B received 40 ml of 0.25% ropivacaine +8 mg dexamethasone. USG guided FICB and postoperative monitoring was done by the chief investigator who was unaware of group allotted and drug administered.Results: There is no significant difference in the heart rate between the two groups after 30 min of the block. The variation of systolic blood pressure of both the group for the first 30 min after giving FICB block was not significant (p>0.05). The absolute value of diastolic blood pressure (DBP) was significantly lower in Group B compared to group A just before the block, a variation of DBP with time was not significant. There was a gradual improvement of pain score from mean 6.7 in Group A and 6.6 in Group B at 0 min to score of 2 at the end of 30 min in both the group. This improvement was achieved earlier in Group B compared to Group A, although the difference was not significant (p>0.05). Vital parameters like HR, SBP, DBP, SpO2 values were similar in both the groups. No patients in either group required any interventions both pre-operatively and pos-operatively. Time of rescue analgesia was noted with the VAS score was significantly more in Group B (p≤0.004). The incidence of hematoma, accidental intravascular injection, convulsion, and paresthesia were nil in both groups.Conclusions: Although both the groups had comfortable and pain-free positioning for administering spinal anaesthesia before surgery. USG guided FICB is easy to perform block and give excellent analgesia for positioning and mobilization of hip fracture patients pre and post-operatively both, and dexamethasone as an adjuvant to 0.25%ropivavaine prolong its local anesthetic effect significantly

Double Anus in an \u3ci\u3eIxodes scapularis\u3c/i\u3e Nymph, a Medically Important Tick Vector

Background: Ixodes scapularis ticks are medically important arthropod vectors that transmit several pathogens to humans. The observations of morphological abnormalities, including nanism, missing leg, extra leg, and gynandromorphism, have been reported in these ticks. In this study, we report the presence of two anuses in a laboratory-reared I. scapularis nymph. Results: Larval ticks were allowed to feed on mice and to molt to nymphs. Two anuses were observed in one of the freshly molted nymphs. Stereo and scanning electron microscopy confirmed the presence of two anuses in one nymph within a single anal groove. Conclusions: This report confirms the rare occurrence of double anus in I. scapularis

Repression of Tick microRNA-133 Induces Organic Anion Transporting Polypeptide Expression Critical for \u3ci\u3eAnaplasma phagocytophilumsurvival\u3c/i\u3e in the Vector and Transmission to the Vertebrate Host

The microRNAs (miRNAs) are important regulators of gene expression. In this study, we provide evidence for the first time to show that rickettsial pathogen Anaplasma phagocytophilum infection results in the down-regulation of tick microRNA-133 (miR-133), to induce Ixodes scapularis organic anion transporting polypeptide (isoatp4056) gene expression critical for this bacterial survival in the vector and for its transmission to the vertebrate host. Transfection studies with recombinant constructs containing transcriptional fusions confirmed binding of miR-133 to isoatp4056 mRNA. Treatment with miR-133 inhibitor resulted in increased bacterial burden and isoatp4056 expression in ticks and tick cells. In contrast, treatment with miR-133 mimic or pre-mir-133 resulted in dramatic reduction in isoatp4056 expression and bacterial burden in ticks and tick cells. Moreover, treatment of ticks with pre-mir-133 affected vector-mediated A. phagocytophilum infection of murine host. These results provide novel insights to understand impact of modulation of tick miRNAs on pathogen colonization in the vector and their transmission to infect the vertebrate host

Arthropod Transcriptional Activator Protein-1 (AP-1) Aids Tick-Rickettsial Pathogen Survival in the Cold

Ixodes scapularis ticks transmit several pathogens to humans including rickettsial bacterium, Anaplasma phagocytophilum. Here, we report that A. phagocytophilum uses tick transcriptional activator protein-1 (AP-1) as a molecular switch in the regulation of arthropod antifreeze gene, iafgp. RNAi-mediated silencing of ap-1 expression significantly affected iafgp gene expression and A. phagocytophilum burden in ticks upon acquisition from the murine host. Gel shift assays provide evidence that both the bacterium and AP-1 influences iafgp promoter and expression. The luciferase assays revealed that a region of approximately 700 bp upstream of the antifreeze gene is sufficient for AP-1 binding to promote iafgp gene expression. Furthermore, survival assays revealed that AP- 1-deficient ticks were more susceptible to cold in comparison to the mock controls. In addition, this study also indicates arthropod AP-1 as a global regulator for some of the tick genes critical for A. phagocytophilum survival in the vector. In summary, our study defines a novel mode of arthropod signaling for the survival of both rickettsial pathogen and its medically important vector in the cold

Rickettsial Pathogen Perturbs Tick Circadian Gene to Infect the Vertebrate Host

Ixodes scapularis is a medically important tick that transmits several microbes to humans, including rickettsial pathogen Anaplasma phagocytophilum. In nature, these ticks encounter several abiotic factors including changes in temperature, humidity, and light. Many organisms use endogenously generated circadian pathways to encounter abiotic factors. In this study, we provide evidence for the first time to show that A. phagocytophilum modulates the arthropod circadian gene for its transmission to the vertebrate host. We noted a circadian oscillation in the expression of arthropod clock, bmal1, period and timeless genes when ticks or tick cells were exposed to alternate 12 h light: 12 h dark conditions. Moreover, A. phagocytophilum significantly modulates the oscillation pattern of expression of these genes. In addition, increased levels of clock and bmal1 and decreased expression of Toll and JAK/STAT pathway immune genes such as pelle and jak, respectively, were noted during A. phagocytophilum transmission from ticks to the vertebrate host. RNAi-mediated knockdown of clock gene expression in ticks resulted in the reduced expression of jak and pelle that increased bacterial transmission from ticks to the murine host. Furthermore, clock-deficient ticks fed late and had less engorgement weights. These results indicate an important role for circadian modulation of tick gene expression that is critical for arthropod blood feeding and transmission of pathogens from vector to the vertebrate host

Human Rickettsial Pathogen Modulates Arthropod Organic Anion Transporting Polypeptide and Tryptophan Pathway for Its Survival in Ticks

The black-legged tick Ixodes scapularis transmits the human anaplasmosis agent, Anaplasma phagocytophilum. In this study, we show that A. phagocytophilum specifically up-regulates I. scapularis organic anion transporting polypeptide, isoatp4056 and kynurenine amino transferase (kat), a gene involved in the production of tryptophan metabolite xanthurenic acid (XA), for its survival in ticks. RNAi analysis revealed that knockdown of isoatp4056 expression had no effect on A. phagocytophilum acquisition from the murine host but affected the bacterial survival in tick cells. Knockdown of the expression of kat mRNA alone or in combination with isoatp4056 mRNA significantly affected A. phagocytophilum survival and isoatp4056 expression in tick cells. Exogenous addition of XA induces isoatp4056 expression and A. phagocytophilum burden in both tick salivary glands and tick cells. Electrophoretic mobility shift assays provide further evidence that A. phagocytophilum and XA influences isoatp4056 expression. Collectively, this study provides important novel information in understanding the interplay between molecular pathways manipulated by a rickettsial pathogen to survive in its arthropod vector

Ticks Elicit Variable Fibrinogenolytic Activities Upon Feeding on Hosts With Different Immune Backgrounds

Ticks secrete several anti-hemostatic factors in their saliva to suppress the host innate and acquired immune defenses against infestations. Using Ixodes scapularis ticks and age-matched mice purchased from two independent commercial vendors with two different immune backgrounds as a model, we show that ticks fed on immunodeficient animals demonstrate decreased fibrinogenolytic activity in comparison to ticks fed on immunocompetent animals. Reduced levels of D-dimer (fibrin degradation product) were evident in ticks fed on immunodeficient animals in comparison to ticks fed on immunocompetent animals. Increased engorgement weights were noted for ticks fed on immunodeficient animals in comparison to ticks fed on immunocompetent animals. Furthermore, the LC-MS/MS and quantitative real-time-PCR analysis followed by inhibitor and antibody-blocking assays revealed that the arthropod HSP70-like molecule contributes to differential fibrinogenolysis during tick feeding. Collectively, these results not only indicate that ticks elicit variable fibrinogenolysis upon feeding on hosts with different immune backgrounds but also provide insights for the novel role of arthropod HSP70-like molecule in fibrinogenolysis during blood feeding

Potential industrial applications of yeast capable of fermenting high gravity cane molasses despite physiological stress

149-152Two Saccharomyces cerevisiae isolates were studied for their ability to ferment cane molasses of high gravity. Supplements like soybean meal (SM), groundnut meal (GM) or castor oil meal (CM) (@2.5%) were found to be quite effective in enabling the yeast to ferment molasses of high gravity (35-40° brix). Ethanol production efficiency was increased by 45-50% with supplementation of any of these additives to concentrated molasses. The viability of yeast also improved by 24-25% in high gravity molasses. In concentrated worts with no supplement, trehalose content of yeast was increased significantly but little increase was reported in medium supplemented with SM, GM or CM, indicating the stress relieving effect of these supplements. Glycerol content did not vary with increase in the concentration of sugars in the medium. There was also no effect on invertase activity of yeast while fermenting the concentrated molasses worts