Host skin immunity to arthropod vector bites: from mice to humans

Infections caused by vector-borne pathogens impose a significant burden of morbidity and mortality in a global scale. In their quest for blood, hematophagous arthropods penetrate the host skin and may transmit pathogens by the bite. These pathogens are deposited along with saliva and a complex mixture of vector derived factors. Hematophagous arthopod vectors have evolved a complex array of adaptations to modulate the host immune response at the bite site with the primary goal to improve blood feeding, which have been exploited throughout evolution by these pathogens to enhance infection establishment in the host. While this paradigm has been firmly established in mouse models, comparable data from human studies are scarce. Here we review how the host skin immune response to vector bites in animal models is hijacked by microbes to promote their pathogenesis. We mainly explored four distinct vector-pathogen pairs of global health importance: sand flies and Leishmania parasites, Ixodes scapularis ticks and Borrelia burgdorferi, Aedes aegypti mosquitoes and arboviruses, and Anopheles gambiae mosquitos and Plasmodium parasites. Finally, we outline how critical it is for the field of vector biology to shift from rodent models to clinical studies focused on the interface of vector-pathogen-host immune system to push further the frontiers of knowledge of the field

Light chain 2 is a Tctex-type related axonemal dynein light chain that regulates directional ciliary motility in \u3ci\u3eTrypanosoma brucei\u3c/i\u3e

Flagellar motility is essential for the cell morphology, viability, and virulence of pathogenic kinetoplastids. Trypanosoma brucei flagella beat with a bending wave that propagates from the flagellum’s tip to its base, rather than base-to-tip as in other eukaryotes. Thousands of dynein motor proteins coordinate their activity to drive ciliary bending wave propagation. Dynein-associated light and intermediate chains regulate the biophysical mechanisms of axonemal dynein. Tctex-type outer arm dynein light chain 2 (LC2) regulates flagellar bending wave propagation direction, amplitude, and frequency in Chlamydomonas reinhardtii. However, the role of Tctex-type light chains in regulating T. brucei motility is unknown. Here, we used a combination of bioinformatics, in-situ molecular tagging, and immunofluorescence microscopy to identify a Tctex-type light chain in the procyclic form of T. brucei (TbLC2). We knocked down TbLC2 expression using RNAi in both wild-type and FLAM3, a flagellar attachment zone protein, knockdown cells and quantified TbLC2’s effects on trypanosome cell biology and biophysics. We found that TbLC2 knockdown reduced the directional persistence of trypanosome cell swimming, induced an asymmetric ciliary bending waveform, modulated the bias between the base-to-tip and tip-to-base beating modes, and increased the beating frequency. Together, our findings are consistent with a model of TbLC2 as a down-regulator of axonemal dynein activity that stabilizes the forward tip-to-base beating ciliary waveform characteristic of trypanosome cells. Our work sheds light on axonemal dynein regulation mechanisms that contribute to pathogenic kinetoplastids’ unique tip-to-base ciliary beating nature and how those mechanisms underlie dynein-driven ciliary motility more generally

Antibodies to Aedes aegypti D7L salivary proteins as a new serological tool to estimate human exposure to Aedes mosquitoes

IntroductionAedes spp. are the most prolific mosquito vectors in the world. Found on every continent, they can effectively transmit various arboviruses, including the dengue virus which continues to cause outbreaks worldwide and is spreading into previously non-endemic areas. The lack of widely available dengue vaccines accentuates the importance of targeted vector control strategies to reduce the dengue burden. High-throughput tools to estimate human-mosquito contact and evaluate vector control interventions are lacking. We propose a novel serological tool that allows rapid screening of human cohorts for exposure to potentially infectious mosquitoes. MethodsWe tested 563 serum samples from a longitudinal pediatric cohort study previously conducted in Cambodia. Children enrolled in the study were dengue-naive at baseline and were followed biannually for dengue incidence for two years. We used Western blotting and enzyme-linked immunosorbent assays to identify immunogenic Aedes aegypti salivary proteins and measure total anti-Ae. aegypti IgG. ResultsWe found a correlation (rs=0.86) between IgG responses against AeD7L1 and AeD7L2 recombinant proteins and those to whole salivary gland homogenate. We observed seasonal fluctuations of AeD7L1+2 IgG responses and no cross-reactivity with Culex quinquefasciatus and Anopheles dirus mosquitoes. The baseline median AeD7L1+2 IgG responses for young children were higher in those who developed asymptomatic versus symptomatic dengue. DiscussionThe IgG response against AeD7L1+2 recombinant proteins is a highly sensitive and Aedes specific marker of human exposure to Aedes bites that can facilitate standardization of future serosurveys and epidemiological studies by its ability to provide a robust estimation of human-mosquito contact in a high-throughput fashion

Light chain 2 is a Tctex-type related axonemal dynein light chain that regulates directional ciliary motility in Trypanosoma brucei.

Flagellar motility is essential for the cell morphology, viability, and virulence of pathogenic kinetoplastids. Trypanosoma brucei flagella beat with a bending wave that propagates from the flagellum's tip to its base, rather than base-to-tip as in other eukaryotes. Thousands of dynein motor proteins coordinate their activity to drive ciliary bending wave propagation. Dynein-associated light and intermediate chains regulate the biophysical mechanisms of axonemal dynein. Tctex-type outer arm dynein light chain 2 (LC2) regulates flagellar bending wave propagation direction, amplitude, and frequency in Chlamydomonas reinhardtii. However, the role of Tctex-type light chains in regulating T. brucei motility is unknown. Here, we used a combination of bioinformatics, in-situ molecular tagging, and immunofluorescence microscopy to identify a Tctex-type light chain in the procyclic form of T. brucei (TbLC2). We knocked down TbLC2 expression using RNAi in both wild-type and FLAM3, a flagellar attachment zone protein, knockdown cells and quantified TbLC2's effects on trypanosome cell biology and biophysics. We found that TbLC2 knockdown reduced the directional persistence of trypanosome cell swimming, induced an asymmetric ciliary bending waveform, modulated the bias between the base-to-tip and tip-to-base beating modes, and increased the beating frequency. Together, our findings are consistent with a model of TbLC2 as a down-regulator of axonemal dynein activity that stabilizes the forward tip-to-base beating ciliary waveform characteristic of trypanosome cells. Our work sheds light on axonemal dynein regulation mechanisms that contribute to pathogenic kinetoplastids' unique tip-to-base ciliary beating nature and how those mechanisms underlie dynein-driven ciliary motility more generally

LC2 KD and WT/LC2 OE do not show cell clustering defects.

Representative images of LC2 KD and WT/LC2 OE cell in the culture flask using phase-contrast microscopy 72 hours post-induction when we did not shake the flasks. We observed no cell clustering. The scale bars represent 10 μm. (EPS)</p

LC2 knockdown causes mislocalization of kinetoplast and cell division defects.

A. Representative images of uninduced, FLAM3 KD, FLAM3-LC2 KD, and FLAM3-LC2 KD/LC2 OE cells cultures in the culture flask using phase-contrast microscopy 72 hours post-induction when we did not shake (top) and shook (bottom) the flasks. Major clusters of cells are indicated (red arrows). The scale bars represent 10 μm. B. Representative DAPI stained images for classification of cells as having x kinetoplasts (xK) and y nuclei (yN). 1K 1N refers to cells with one kinetoplast normally localized to one nucleus (left). MK MN refers to cells classified as having multiple (M>2) mislocalized (closer to each other) kinetoplasts and nuclei (right), likely resulting from incomplete kinetoplast migration and/or incomplete cytokinesis. The scale bar represents 5 μm and both images in this panel have the same scale. C. Occurrence frequency of one kinetoplast and one nucleus, normally localized within the cell (1K 1N) and the occurrence frequency of the multi-kinetoplast, multi-nucleus (MK MN) classification, as described in panel B., in uninduced and induced (72 hours post-induction) LC2 KD, FLAM3 KD, FLAM3-LC2 KD, FLAM3-LC2 KD/LC2 OE, and WT/LC2 OE cells. N = 101, 192, 122, 111, 72, and 70 total classified cells of each strain, respectively. Other classifications, e.g., 1K 2N, 2K 1N, and 2K 2N, which likely include cells undergoing cell division, account for the percentages not represented. The error bars represent the statistical counting error. ** = p-value D. DIC microscopy image of fixed FLAM3-LC2 KD cells, including a representative amorphous clump of cells with multiple detached cilia (red arrow). (TIF)</p

FLAM3-LC2 KD double knockdown cells exhibit ciliary beating locked to a reverse beating over an extensive period showing a longer dwell time in reverse beating mode.

The movie was recorded using a high-speed CMOS camera and a 60x objective at 200 fps with an exposure time of 250 μs and played back at 45 fps. Scale bar = 10 μm. (AVI)</p

Homology modeled structure of TbLC2 using CrLC2 as a template generated by SWISS-MODEL and used and modified under the creative commons license CC BY-SA 4.0.

(PDB)</p

FLAM3-LC2 KD double knockdown cells exhibit altered ciliary beating leading to frequent cell reorientation.

A typical freely swimming FLAM3-LC2 KD cell showing frequent reversals in ciliary beat mode and futile swimming motility with low directionality. The movie was recorded at 45 fps and played back at the same frame rate. The scale bar = 10 μm in both movies. (AVI)</p

LC2 knockdown cells exhibiting t high directional persistence motility.

The movie was recorded using phase-contrast microscopy with a 10x objective at 45 fps and played back at the same frame rate. The scale bar represents 10 μm. (AVI)</p