Biology subjects

Valencia, L.

Publications and source records attributed to Valencia, L..

6 recordsLinked to original sources

bioRxiv2023

Tick extracellular vesicles impair epidermal homeostasis through immune-epithelial networks during hematophagy

Wound healing has been extensively studied through the lens of inflammatory disorders and cancer, but limited attention has been given to hematophagy and arthropod-borne diseases. Hematophagous ectoparasites, including ticks, subvert the wound healing response to maintain prolonged attachment and facilitate blood-feeding. Here, we unveil a strategy by which extracellular vesicles (EVs) ensure blood-feeding and arthropod survival in three medically relevant tick species. Through single cell RNA sequencing and murine genetics, we demonstrate that wildtype animals infested with EV-deficient Ixodes scapularis display a unique epidermal sub-population with a mesenchymal-like transcriptional program and an overrepresentation of pathways connected to wound healing. Furthermore, tick EVs inhibit proliferation and diminish the capacity of wound closure in keratinocytes. This occurrence was linked to phosphoinositide 3-kinase activity, keratinocyte growth factor 1 (KGF-1) and transforming growth factor {beta} (TGF-{beta}) levels. Collectively, we uncovered a strategy employed by a blood-feeding arthropod that disrupts the circuitry in cutaneous wound healing, contributing to ectoparasite fitness.

microbiology↗

bioRxiv2023

Rab27 in tick extracellular vesicle biogenesis and infection

BackgroundThe blacklegged tick, Ixodes scapularis, transmits most vector-borne diseases in the United States. It vectors seven pathogens of public health relevance, including the emerging human pathogen Anaplasma phagocytophilum. Nevertheless, it remains critically understudied when compared to other arthropod vectors. I. scapularis releases a variety of molecules that assist in the modulation of host responses. Recently, it was found that extracellular vesicles (EVs) carry several of these molecules and may impact microbial transmission to the mammalian host. EV biogenesis has been studied in mammalian systems and is relatively well understood, but the molecular players important for the formation and secretion of EVs in arthropods of public health relevance remain elusive. RabGTPases are among the major molecular players in mammalian EV biogenesis. They influence membrane identity and vesicle budding, uncoating, and motility. MethodsUsing BLAST, an in-silico pathway for EV biogenesis in ticks was re-constructed. We identified Rab27 for further study. EVs were collected from ISE6 tick cells after knocking down rab27 to examine its role in tick EV biogenesis. I. scapularis nymphs were injected with small interfering RNAs to knock down rab27 then fed on naive and A. phagocytophilum infected mice to explore the importance of rab27 in tick feeding and bacterial acquisition. ResultsOur BLAST analysis identified several of the proteins involved in EV biogenesis in ticks, including Rab27. We show that silencing rab27 in I. scapularis impacts tick fitness. Additionally, ticks acquire less A. phagocytophilum after rab27 silencing. Experiments in the tick ISE6 cell line show that silencing of rab27 causes a distinct range profile of tick EVs, indicating that Rab27 is needed to regulate EV biogenesis. ConclusionsRab27 is needed for successful tick feeding and may be important for acquiring A. phagocytophilum during a blood meal. Additionally, silencing rab27 in tick cells results in a shift of extracellular vesicle size. Overall, we have observed that Rab27 plays a key role in tick EV biogenesis and the tripartite interactions among the vector, the mammalian host, and a microbe it encounters.

microbiology↗

bioRxiv2023

Tick hemocytes have pleiotropic roles in microbial infection and arthropod fitness

Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here, we describe immune cells or hemocytes from the clinically relevant tick Ixodes scapularis using bulk and single cell RNA sequencing combined with depletion via clodronate liposomes, RNA interference, Clustered Regularly Interspaced Short Palindromic Repeats activation (CRISPRa) and RNA-fluorescence in situ hybridization (FISH). We observe molecular alterations in hemocytes upon tick infestation of mammals and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We predict distinct hemocyte lineages and reveal clusters exhibiting defined signatures for immunity, metabolism, and proliferation during hematophagy. Furthermore, we perform a mechanistic characterization of two I. scapularis hemocyte markers: hemocytin and astakine. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, which impacts blood feeding and molting behavior of ticks. Hemocytin specifically affects the c-Jun N-terminal kinase (JNK) signaling pathway, whereas astakine alters hemocyte proliferation in I. scapularis. Altogether, we uncover the heterogeneity and pleiotropic roles of hemocytes in ticks and provide a valuable resource for comparative biology in arthropods.

immunology↗

bioRxiv2021

The AMIGO1 adhesion protein modulates movement of Kv2.1 voltage sensors

Kv2 voltage-gated potassium channels are modulated by AMIGO neuronal adhesion proteins. Here, we identify steps in the conductance activation pathway of Kv2.1 channels that are modulated by AMIGO1 using voltage clamp recordings and spectroscopy of heterologously expressed Kv2.1 and AMIGO1 in mammalian cell lines. AMIGO1 speeds early voltage sensor movements and shifts the gating charge-voltage relationship to more negative voltages. The gating charge-voltage relationship indicates that AMIGO1 exerts a larger energetic effect on voltage sensor movement than apparent from the midpoint of the conductance-voltage relationship. When voltage sensors are detained at rest by voltage sensor toxins, AMIGO1 has a greater impact on the conductance-voltage relationship. Fluorescence measurements from voltage sensor toxins bound to Kv2.1 indicate that with AMIGO1, the voltage sensors enter their earliest resting conformation, yet this conformation is less stable upon voltage stimulation. We conclude that AMIGO1 modulates the Kv2.1 conductance activation pathway by destabilizing the earliest resting state of the voltage sensors. Statement of SignificanceKv2 potassium channels activate a potassium conductance that shapes neuronal action potentials. The AMIGO family of adhesion proteins modulate activation of Kv2 conductances, yet, which activation steps are modified is unknown. This study finds that AMIGO1 destabilizes the earliest resting conformation of the Kv2.1 voltage sensors to promote activation of channel conductance.

biophysics↗

bioRxiv2020

A bimodular PKS platform that expands the biological design space

Traditionally engineered to produce novel bioactive molecules, Type I modular polyketide synthases (PKSs) could be engineered as a new biosynthetic platform for the production of de novo fuels, commodity chemicals, and specialty chemicals. Previously, our investigations manipulated the first module of the lipomycin PKS to produce short chain ketones, 3-hydroxy acids, and saturated, branched carboxylic acids. Building upon this work, we have expanded to multi-modular systems by engineering the first two modules of lipomycin to generate unnatural polyketides as potential biofuels and specialty chemicals in Streptomyces albus. First, we produce 20.6 mg/L of the ethyl ketone, 4,6 dimethylheptanone through a reductive loop exchange in LipPKS1 and a ketoreductase knockouts in LipPKS2. We then show that an AT swap in LipPKS1 and a reductive loop exchange in LipPKS2 can produce the potential fragrance 3-isopropyl-6-methyltetrahydropyranone. Highlighting the challenge of maintaining product fidelity, in both bimodular systems we observed side products from premature hydrolysis in the engineered first module and stalled dehydration in reductive loop exchanges. Collectively, our work expands the biological design space and moves the field closer to the production of “designer” biomolecules.HighlightsEngineered lipomycin module 1 and module 2 to produce unnatural polyketides as valuable bio-based chemicalsA reductive loop swap and ketoreductase knockout used to produce 20 mg/mL of a novel ethyl ketone, a gasoline replacementAn acyltransferase swap and reductive loop swap successfully produced δ-lactone, a potential fragrant compoundIncomplete reduction and premature hydrolysis observed in engineered modulesCompeting Interest StatementJ.D.K. has a financial interest in Amyris, Lygos, Demetrix, Napigen, Maple Bio, Berkeley Brewing Sciences, Ansa Biotech and Apertor Labs.View Full Text

synthetic biology↗

bioRxiv2019

Chemoinformatic-guided engineering of polyketide synthases

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge in PKS design is engineering a partially reductive module to produce a saturated {beta}-carbon through a reductive loop exchange. In this work, we sought to establish that chemoinformatics, a field traditionally used in drug discovery, could provide a viable strategy to reductive loop exchanges. We first introduced a set of donor reductive loops of diverse genetic origin and chemical substrate structures into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with atom pair chemical similarity between the substrate of the donor reductive loops and recipient LipPKS1, reaching a titer of 165 mg/L of short chain fatty acids produced by Streptomyces albus J1074 harboring these engineered PKSs. Expanding this method to larger intermediates requiring bimodular communication, we introduced reductive loops of divergent chemosimilarity into LipPKS2 and determined triketide lactone production. Collectively, we observed a statistically significant correlation between atom pair chemosimilarity and production, establishing a new chemoinformatic method that may aid in the engineering of PKSs to produce desired, unnatural products.

biochemistry↗