Tick Extracellular Vesicles Enable Arthropod Feeding and Promote Distinct Outcomes of Bacterial Infection

Extracellular vesicles are thought to facilitate pathogen transmission from arthropods to humans and other animals. Here, we reveal that pathogen spreading from arthropods to the mammalian host is multifaceted. Extracellular vesicles from Ixodes scapularis enable tick feeding and promote infection of the mildly virulent rickettsial agent Anaplasma phagocytophilum through the SNARE proteins Vamp33 and Synaptobrevin 2 and dendritic epidermal T cells. However, extracellular vesicles from the tick Dermacentor andersoni mitigate microbial spreading caused by the lethal pathogen Francisella tularensis. Collectively, we establish that tick extracellular vesicles foster distinct outcomes of bacterial infection and assist in vector feeding by acting on skin immunity. Thus, the biology of arthropods should be taken into consideration when developing strategies to control vector-borne diseases

Anaplasma phagocytophilum Transmission Activates Immune Pathways While Repressing Wound Healing in the Skin

Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis (HGA), is an obligate intracellular bacterium transmitted by the bite of black-legged ticks, Ixodes scapularis. The main host cells in vertebrates are neutrophils. However, the first site of entry is in the skin during tick feeding. Given that the initial responses within skin are a crucial determinant of disease outcome in vector-borne diseases, we used a non-biased approach to characterize the transcriptional changes that take place at the bite during I. scapularis feeding and A. phagocytophilum transmission. Experimentally infected ticks were allowed to feed for 3 days on C57BL/6J mice to allow bacterial transmission and establishment. Skin biopsies were taken from the attachment site of uninfected ticks and A. phagocytophilum-infected ticks. Skin without ticks (intact skin) was used as baseline. RNA was isolated and sequenced using next-generation sequencing (NGS). The differentially expressed genes were used to identify over-represented pathways by gene ontology (GO) and pathway enrichment (PE). Anaplasma phagocytophilum transmission resulted in the activation of interferon signaling and neutrophil chemotaxis pathways in the skin. Interestingly, it also led to the downregulation of genes encoding extracellular matrix (ECM) components, and upregulation of metalloproteinases, suggesting that A. phagocytophilum delays wound healing responses and may increase vascular permeability at the bite site

Expression patterns of Anaplasma marginale Msp2 variants change in response to growth in cattle, and tick cells versus mammalian cells

Antigenic variation of major surface proteins is considered an immune-evasive maneuver used by pathogens as divergent as bacteria and protozoa. Likewise, major surface protein 2 (Msp2) of the tick-borne pathogen, Anaplasma marginale, is thought to be involved in antigenic variation to evade the mammalian host immune response. However, this dynamic process also works in the tick vector in the absence of immune selection pressure. We examined Msp2 variants expressed during infection of four tick and two mammalian cell-lines to determine if the presence of certain variants correlated with specific host cell types. Anaplasma marginale colonies differed in their development and appearance in each of the cell lines (P<0.001). Using Western blots probed with two Msp2-monospecific and one Msp2-monoclonal antibodies, we detected expression of variants with differences in molecular weight. Immunofluorescence-assay revealed that specific antibodies bound from 25 to 60% of colonies, depending on the host cell-line (P<0.001). Molecular analysis of cloned variant-encoding genes demonstrated expression of different predominant variants in tick (V1) and mammalian (V2) cell-lines. Analysis of the putative secondary structure of the variants revealed a change in structure when A. marginale was transferred from one cell-type to another, suggesting that the expression of particular Msp2 variants depended on the cell-type (tick or mammalian) in which A. marginale developed. Similarly, analysis of the putative secondary structure of over 200 Msp2 variants from ticks, blood samples, and other mammalian cells available in GenBank showed the predominance of a specific structure during infection of a host type (tick versus blood sample), demonstrating that selection of a possible structure also occurred in vivo. The selection of a specific structure in surface proteins may indicate that Msp2 fulfils an important role in infection and adaptation to diverse host systems. Supplemental Abstract in Spanish (File S1) is provided

Transformation of Anaplasma marginale

The tick-borne pathogen, Anaplasma marginale, has a complex life cycle involving ruminants and ixodid ticks. It causes bovine anaplasmosis, a disease with significant economic impact on cattle farming worldwide. The obligate intracellular growth requirement of the bacteria poses a challenging obstacle to their genetic manipulation, a problem shared with other prokaryotes in the genera Anaplasma, Ehrlichia, and Rickettsia. Following our successful transformation of the human anaplasmosis agent, A. phagocytophilum, we produced plasmid constructs (a transposon bearing plasmid, pHimarAm-trTurboGFP-SS, and a transposase expression plasmid, pET28Am-trA7) designed to mediate random insertion of the TurboGFP and spectinomycin/streptomycin resistance genes by the Himar1 allele A7 into the A. marginale chromosome. In these trans constructs, expression of the fluorescent and the selectable markers on the transposon, and expression of the transposase are under control of the A. marginale tr promoter. Constructs were co-electroporated into A. marginale St. Maries purified from tick cell culture, and bacteria incubated for 2 months under selection with a combination of spectinomycin and streptomycin. At that time, ≤1% of tick cells contained colonies of brightly fluorescent Anaplasma, which eventually increased to infect about 80–90% of the cells. Cloning of the insertion site in E. coli and DNA sequence analyses demonstrated insertion of the entire plasmid pHimarAm-trTurboGFP-SS encoding the transposon in frame into the native tr region of A. marginale in an apparent single homologous crossover event not mediated by the transposase. Transformants are fastidious and require longer subculture intervals than wild type A. marginale. This result suggests that A. marginale, as well as possibly other species of Anaplasma and Ehrlichia, can be transformed using a strategy of homologous recombination

Antibodies used to serologically determine the variation of Msp2 in the different tick and mammalian cell lines.

<p>Antibodies used to serologically determine the variation of Msp2 in the different tick and mammalian cell lines.</p

Donor allele usage in Msp2 variant production by <i>A. marginale</i> during growth in vivo and in vitro.

<p>Each color represents the usage of one of seven specific donor alleles (i.e. pseudogenes) during infection of a particular cell culture system, and during acute or chronic infection in cattle. Allele usage was assessed to determine its preferential use for generation of new variants (5 A–D) and to determine its overall representation among all variants in a population (5 E–H). The frequency of use of each allele in the production of variants was calculated from the total number of variants that contained sequences from a specific donor allele divided by the total number of variants. This represented the preferential usage of an allele for the production of variants and is shown in (A), for tick cell cultures; (B), mammalian cell cultures, C), acutely infected bovine PA344; and (D), chronically infected bovine PA291. Overall allele usage in a population was calculated from the total number of clones that comprised DNA sequence from any specific allele, divided by the total number of clones recovered from each system. This provided an estimate of the use of an allele in any variant, whether major or minor, recovered from a population. (E), Overall usage of variants in tick cell cultures; (F), in mammalian cell cultures; (G), in the acutely infected bovine PA344; and (H), in the chronically infected bovine 291. As an example, comparison of corresponding pie charts A and E demonstrates that allele P1 is infrequently found in variants from tick cell cultures, although it is recovered in a large number of clones. Whereas, a comparison of pie charts B and F shows that allele TTV106/E6F7 is recovered from mammalian cell cultures about as often as alleles 2 and 1, although segments of TTV106/E6F7 are recovered in the majority of different clones.</p

Growth rates differences during infection of various tick and mammalian cell lines.

*<p>IC/day = Infected cells increase per day.</p

Differences in colony morphology and percentages of infection during in vitro replication in cell lines.

<p>Giemsa stained preparations show the colony morphology of <i>A. marginale</i> VA in (A) Vero cells, (B) DAE100T cells, (C) BME26 cells, (D) ISE6 cells, (E) RF/6A cells, and (F) IDE12 cells. Arrows point to the <i>A. marginale</i> VA colonies growing in each cell line. Cell nuclei stained in lighter red-purple color and marked with the letter n. Bar = 20 µm. (G) Increase in the percent of infected tick and mammalian cells during a 12-day period. The solid red line with red circles represent the infection rate of ISE6 cells at each time point. The solid blue line and blue circles represent IDE12. Solid black line and triangles represent to the infection rates in BME26 cells, whereas the green squares and line represent to the values for the infection rates in Vero cells. The linear regression calculated from the percent infected cells over time is shown as a dotted line in the color corresponding to the cell line represented.Bars represent the standard deviation for each time point.</p

Primers used for the amplification of <i>A. marginale</i> pseudogenes.

<p>Primers used for the amplification of <i>A. marginale</i> pseudogenes.</p