Genetic manipulation of an<i>Ixodes scapularis</i>cell line

Singh, Nisha; Rolandelli, Agustin; O’Neal, Anya J.; Butler, L. Rainer; Samaddar, Sourabh; Laukaitis-Yousey, Hanna J.; Butnaru, Matthew; Mohr, Stephanie E.; Perrimon, Norbert; Pedra, Joao H. F.

doi:10.1101/2023.09.08.556855

Cited by 3 publications

(1 citation statement)

References 43 publications

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“…With recent advancements in the technology available to the vector-borne disease community, such as Clustered Regularly Interspaced Palindromic Repeats (CRISPR) in ticks [30], and ectopic expression in tick cells [31], future work can aim to unravel Rab27’s exact mechanism of action as well as its interaction with important tick-borne pathogens, such as A. phagocytophilum [32]. Furthermore, it is unclear whether Rab27 is important for tick fitness in other species.…”

Section: Discussionmentioning

confidence: 99%

Rab27 in tick extracellular vesicle biogenesis and infection

Butler,

Singh,

Marnin

et al. 2023

Preprint

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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 pathogenAnaplasma phagocytophilum. Nevertheless, it remains critically understudied when compared to other arthropod vectors.I. scapularisreleases 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, anin-silicopathway for EV biogenesis in ticks was re-constructed. We identified Rab27 for further study. EVs were collected from ISE6 tick cells after knocking downrab27to examine its role in tick EV biogenesis.I. scapularisnymphs were injected with small interfering RNAs to knock downrab27then fed on naïve andA. phagocytophiluminfected mice to explore the importance ofrab27in tick feeding and bacterial acquisition.ResultsOur BLAST analysis identified several of the proteins involved in EV biogenesis in ticks, including Rab27. We show that silencingrab27inI. scapularisimpacts tick fitness. Additionally, ticks acquire lessA. phagocytophilumafterrab27silencing. Experiments in the tick ISE6 cell line show that silencing ofrab27causes 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 acquiringA. phagocytophilumduring a blood meal. Additionally, silencingrab27in 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.

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Section: Discussionmentioning

confidence: 99%

Rab27 in tick extracellular vesicle biogenesis and infection

Butler,

Singh,

Marnin

et al. 2023

Preprint

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Pathogenic Rickettsia spp. as emerging models for bacterial biology

Sit,

Lamason

2024

J Bacteriol

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Our understanding of free-living bacterial models like Escherichia coli far outpaces that of obligate intracellular bacteria, which cannot be cultured axenically. All obligate intracellular bacteria are host-associated, and many cause serious human diseases. Their constant exposure to the distinct biochemical niche of the host has driven the evolution of numerous specialized bacteriological and genetic adaptations, as well as innovative molecular mechanisms of infection. Here, we review the history and use of pathogenic Rickettsia species, which cause an array of vector-borne vascular illnesses, as model systems to probe microbial biology. Although many challenges remain in our studies of these organisms, the rich pathogenic and biological diversity of Rickettsia spp. constitutes a unique backdrop to investigate how microbes survive and thrive in host and vector cells. We take a bacterial-focused perspective and highlight emerging insights that relate to new host–pathogen interactions, bacterial physiology, and evolution. The transformation of Rickettsia spp. from pathogens to models demonstrates how recalcitrant microbes may be leveraged in the lab to tap unmined bacterial diversity for new discoveries. Rickettsia spp. hold great promise as model systems not only to understand other obligate intracellular pathogens but also to discover new biology across and beyond bacteria.

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The role of Rab27 in tick extracellular vesicle biogenesis and pathogen infection

Butler,

Singh,

Marnin

et al. 2024

Parasites Vectors

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Background The blacklegged tick, Ixodes scapularis, transmits most vector-borne diseases in the US. It vectors seven pathogens of public health relevance, including the emerging human pathogen Anaplasma phagocytophilum. Nevertheless, it remains critically understudied compared to other arthropod vectors. Ixodes 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. Methods Using 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. Ixodes scapularis nymphs were injected with small interfering RNAs to knock down rab27 and then fed on naïve and A. phagocytophilum-infected mice to explore the importance of rab27 in tick feeding and bacterial acquisition. Results Our 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. Conclusions Rab27 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. Graphical Abstract

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Genetic manipulation of anIxodes scapulariscell line

Cited by 3 publications

References 43 publications

Rab27 in tick extracellular vesicle biogenesis and infection

Rab27 in tick extracellular vesicle biogenesis and infection

Pathogenic Rickettsia spp. as emerging models for bacterial biology

The role of Rab27 in tick extracellular vesicle biogenesis and pathogen infection

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