scholarly journals Novel small RNAs expressed by Bartonella bacilliformis under multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host

2020 ◽  
Author(s):  
Shaun Wachter ◽  
Linda D. Hicks ◽  
Rahul Raghavan ◽  
Michael F. Minnick
Keyword(s):  
Life Cycle ◽  
Rna Sequencing ◽  
Vascular Endothelial Cells ◽  
Sand Fly ◽  
Sand Flies ◽  
Bartonella Bacilliformis ◽  
Phlebotomine Sand Flies ◽  
Group I

AbstractBartonella bacilliformis, the etiological agent of Carrión’s disease, is a Gram-negative, facultative intracellular alphaproteobacterium. Carrión’s disease is an emerging but neglected tropical illness endemic to Peru, Colombia, and Ecuador. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant and repeated environmental shifts during its life cycle, including changes in pH and temperature. In most bacteria, small non-coding RNAs (sRNAs) serve as effectors that may post-transcriptionally regulate the stress response to such changes. However, sRNAs have not been characterized in B. bacilliformis, to date. We therefore performed total RNA-sequencing analyses on B. bacilliformis grown in vitro then shifted to one of ten distinct conditions that simulate various environments encountered by the pathogen during its life cycle. From this, we identified 160 sRNAs significantly expressed under at least one of the conditions tested. sRNAs included the highly-conserved tmRNA, 6S RNA, RNase P RNA component, SRP RNA component, ffH leader RNA, and the alphaproteobacterial sRNAs αr45 and speF leader RNA. In addition, 153 other potential sRNAs of unknown function were discovered. Northern blot analysis was used to confirm the expression of eight novel sRNAs. We also characterized a Bartonellabacilliformisgroup I intron (BbgpI) that disrupts an un-annotated tRNACCUArg gene and determined that the intron splices in vivo and self-splices in vitro. Furthermore, we demonstrated the molecular targeting of Bartonellabacilliformissmall RNA 9(BbsR9) to transcripts of the ftsH, nuoF, and gcvT genes, in vitro.Author summaryB. bacilliformis is a bacterial pathogen that is transmitted between humans by phlebotomine sand flies. Bacteria often express sRNAs to fine-tune the production of proteins involved in a wide array of biological processes. We cultured B. bacilliformis in vitro under standard conditions then shifted the pathogen for a period of time to ten distinct environments, including multiple temperatures, pH levels, and infections of human blood and human vascular endothelial cells. After RNA-sequencing, a manual transcriptome search identified 160 putative sRNAs, including seven highly-conserved sRNAs and 153 novel potential sRNAs. We then characterized two of the novel sRNAs, BbgpI and BbsR9. BbgpI is a group I intron (ribozyme) that self-splices and disrupts an unannotated gene coding for a transfer RNA (tRNACCUArg). BbsR9 is an intergenic sRNA expressed under conditions that simulate the sand fly. We found that BbsR9 targets transcripts of the ftsH, nuoF, and gcvT genes. Furthermore, we determined the specific sRNA-mRNA interactions responsible for BbsR9 binding to its target mRNAs through in vitro mutagenesis and binding assays.

scholarly journals Novel small RNAs expressed by Bartonella bacilliformis under multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host

2020 ◽  
Vol 14 (11) ◽  
pp. e0008671
Author(s):  
Shaun Wachter ◽  
Linda D. Hicks ◽  
Rahul Raghavan ◽  
Michael F. Minnick
Keyword(s):  
Life Cycle ◽  
Northern Blot Analysis ◽  
Sand Fly ◽  
Bartonella Bacilliformis ◽  
Phlebotomine Sand Flies ◽  
Non Coding Rnas ◽  
Srp Rna ◽  
Multiple Conditions

Bartonella bacilliformis, the etiological agent of Carrión’s disease, is a Gram-negative, facultative intracellular alphaproteobacterium. Carrión’s disease is an emerging but neglected tropical illness endemic to Peru, Colombia, and Ecuador. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant and repeated environmental shifts during its life cycle, including changes in pH and temperature. In most bacteria, small non-coding RNAs (sRNAs) serve as effectors that may post-transcriptionally regulate the stress response to such changes. However, sRNAs have not been characterized in B. bacilliformis, to date. We therefore performed total RNA-sequencing analyses on B. bacilliformis grown in vitro then shifted to one of ten distinct conditions that simulate various environments encountered by the pathogen during its life cycle. From this, we identified 160 sRNAs significantly expressed under at least one of the conditions tested. sRNAs included the highly-conserved tmRNA, 6S RNA, RNase P RNA component, SRP RNA component, ffH leader RNA, and the alphaproteobacterial sRNAs αr45 and speF leader RNA. In addition, 153 other potential sRNAs of unknown function were discovered. Northern blot analysis was used to confirm the expression of eight novel sRNAs. We also characterized a Bartonella bacilliformis group I intron (BbgpI) that disrupts an un-annotated tRNACCUArg gene and determined that the intron splices in vivo and self-splices in vitro. Furthermore, we demonstrated the molecular targeting of Bartonella bacilliformis small RNA 9 (BbsR9) to transcripts of the ftsH, nuoF, and gcvT genes, in vitro.

scholarly journals Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections

2018 ◽  
Author(s):  
Tom Beneke ◽  
François Demay ◽  
Edward Hookway ◽  
Nicole Ashman ◽  
Heather Jeffery ◽  
...  
Keyword(s):  
Life Cycle ◽  
Large Scale ◽  
Gene Knockout ◽  
Sand Fly ◽  
Relative Fitness ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Label Free ◽  
Flagellar Proteins

AbstractThe protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite’s life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single mutant lacking the central pair protein PF16 showed that these paralysed promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies.Author SummaryLeishmania are protozoan parasites, transmitted between mammals by the bite of phlebotomine sand flies. Promastigote forms in the sand fly have a long flagellum, which is motile and used for anchoring the parasites to prevent clearance with the digested blood meal remnants. To dissect flagellar functions and their importance in life cycle progression, we generated here a comprehensive list of >300 flagellar proteins and produced a CRISPR-Cas9 gene knockout library of 100 mutant Leishmania. We studied their behaviour in vitro before examining their fate in the sand fly Lutzomyia longipalpis. Measuring mutant swimming speeds showed that about half behaved differently compared to the wild type: a few swam faster, many slower and some were completely paralysed. We also found a group of uncoordinated swimmers. To test whether flagellar motility is required for parasite migration from the fly midgut to the foregut from where they reach the next host, we infected sand flies with a mixed mutant population. Each mutant carried a unique tag and tracking these tags up to nine days after infection showed that paralysed and uncoordinated Leishmania were rapidly lost from flies. These data indicate that directional swimming is important for successful colonisation of sand flies.

scholarly journals Phlebotomine sand flies (Diptera: Psychodidae) of the Maghreb region: A systematic review of distribution, morphology, and role in the transmission of the pathogens

2022 ◽  
Vol 16 (1) ◽  
pp. e0009952
Author(s):  
Kamal Eddine Benallal ◽  
Rafik Garni ◽  
Zoubir Harrat ◽  
Petr Volf ◽  
Vít Dvorak
Keyword(s):  
Google Earth ◽  
Sand Fly ◽  
Distribution Data ◽  
Sand Flies ◽  
Bartonella Bacilliformis ◽  
Phlebotomine Sand Flies ◽  
Distribution Maps ◽  
Vector Borne Diseases ◽  
Animal Pathogens ◽  
Species Specific

Background Phlebotomine sand flies (Diptera: Psychodidae) are important vectors of various human and animal pathogens such as Bartonella bacilliformis, Phlebovirus, and parasitic protozoa of the genus Leishmania, causative agent of leishmaniases that account among most significant vector-borne diseases. The Maghreb countries Mauritania, Morocco, Algeria, Tunisia, and Libya occupy a vast area of North Africa and belong to most affected regions by these diseases. Locally varying climatic and ecological conditions support diverse sand fly fauna that includes many proven or suspected vectors. The aim of this review is to summarize often fragmented information and to provide an updated list of sand fly species of the Maghreb region with illustration of species-specific morphological features and maps of their reported distribution. Materials and methods The literature search focused on scholar databases to review information on the sand fly species distribution and their role in the disease transmissions in Mauritania, Morocco, Algeria, Tunisia, and Libya, surveying sources from the period between 1900 and 2020. Reported distribution of each species was collated using Google Earth, and distribution maps were drawn using ArcGIS software. Morphological illustrations were compiled from various published sources. Results and conclusions In total, 32 species of the genera Phlebotomus (Ph.) and Sergentomyia (Se.) were reported in the Maghreb region (15 from Libya, 18 from Tunisia, 23 from Morocco, 24 from Algeria, and 9 from Mauritania). Phlebotomus mariae and Se. africana subsp. asiatica were recorded only in Morocco, Ph. mascitti, Se. hirtus, and Se. tiberiadis only in Algeria, whereas Ph. duboscqi, Se. dubia, Se. africana africana, Se. lesleyae, Se. magna, and Se. freetownensis were reported only from Mauritania. Our review has updated and summarized the geographic distribution of 26 species reported so far in Morocco, Algeria, Tunisia, and Libya, excluding Mauritania from a detailed analysis due to the unavailability of accurate distribution data. In addition, morphological differences important for species identification are summarized with particular attention to closely related species such as Ph. papatasi and Ph. bergeroti, Ph. chabaudi, and Ph. riouxi, and Se. christophersi and Se. clydei.

scholarly journals Molecular Detection of Bartonella sp. in Psathyromyia shannoni and Lutzomyia cruciata From Northeastern Mexico

2021 ◽  
Vol 2 ◽  
Author(s):  
Yokomi N. Lozano-Sardaneta ◽  
Nadia Joselyne Soto-Olguín ◽  
Jorge J. Rodríguez-Rojas ◽  
Sokani Sánchez-Montes ◽  
Eduardo A. Rebollar-Téllez ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Molecular Detection ◽  
Citrate Synthase ◽  
Phylogenetic Reconstruction ◽  
Sand Fly ◽  
Sand Flies ◽  
Light Traps ◽  
Bartonella Bacilliformis ◽  
Phlebotomine Sand Flies ◽  
Northeastern Mexico

Phlebotomine sand flies are vectors of Leishmania spp., Bartonella bacilliformis, and several arboviruses worldwide. In Mexico, the presence of Bartonella species is associated sporadically with arthropods and little is known on the diversity of insects that could be incriminated with its transmission. The aim of this study was to perform a molecular detection of Bartonella DNA in sand fly species collected in northeastern Mexico. Sand flies were collected at the states of Nuevo Leon and Tamaulipas from June to August 2010, using 16 light traps per night. Sand fly species were morphologically identified, and for Bartonella detection, we amplified ~378 bp of the citrate synthase gene (gltA). DNA sequences were compared in a phylogenetic reconstruction based on maximum likelihood. A total of 532 specimens from seven sand fly species were morphologically identified, where 11 specimens from Tamaulipas tested positive for the presence of a new lineage of Bartonella sp. associated with Psathyromyia shannoni and Lutzomyia cruciata. This work represents the second record of Bartonella-associated with sand flies outside of the endemic area of Carrion’s disease. More studies are necessary to understand their life cycle, transmission dynamics, and their relationship with sand fly species.

Effects of Rest and Exercise on Cardiac Blood Volume Determinations

1997 ◽  
Vol 36 (08) ◽  
pp. 259-264
Author(s):  
N. Topuzović
Keyword(s):  
Radionuclide Ventriculography ◽  
Left Ventricular ◽  
Peak Exercise ◽  
Volume Determination ◽  
Group I ◽  
Lv Volumes ◽  
Group Ii ◽  
Lv Volume

Summary Aim: The purpose of this study was to investigate the changes in blood activity during rest, exercise and recovery, and to assess its influence on left ventricular (LV) volume determination using the count-based method requiring blood sampling. Methods: Forty-four patients underwent rest-stress radionuclide ventriculography; Tc-99m-human serum albumin was used in 13 patients (Group I), red blood cells was labeled using Tc-99m in 17 patients (Group II) in vivo, and in 14 patients (Group III) by modified in vivo/in vitro method. LV volumes were determined by a count-based method using corrected count rate in blood samples obtained during rest, peak exercise and after recovery. Results: In group I at stress, the blood activity decreased by 12.6 ± 5.4%, p <0.05, as compared to the rest level, and increased by 25.1 ± 6.4%, p <0.001, and 12.8 ± 4.5%, p <0.05, above the resting level in group II and III, respectively. This had profound effects on LV volume determinations if only one rest blood aliquot was used: during exercise, the LV volumes significantly decreased by 22.1 ± 9.6%, p <0.05, in group I, whereas in groups II and III it was significantly overestimated by 32.1 ± 10.3%, p <0.001, and 10.7 ± 6.4%, p <0.05, respectively. The changes in blood activity between stress and recovery were not significantly different for any of the groups. Conclusion: The use of only a single blood sample as volume aliquot at rest in rest-stress studies leads to erroneous estimation of cardiac volumes due to significant changes in blood radioactivity during exercise and recovery.

scholarly journals Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

scholarly journals Phlebotomine Sand Fly Fauna andLeishmaniaInfection in the Vicinity of the Serra do Cipó National Park, a Natural Brazilian Heritage Site

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Rosana Silva Lana ◽  
Érika Monteiro Michalsky ◽  
Consuelo Latorre Fortes-Dias ◽  
João Carlos França-Silva ◽  
Fabiana de Oliveira Lara-Silva ◽  
...  
Keyword(s):  
National Park ◽  
Sand Fly ◽  
Sand Flies ◽  
Local Data ◽  
Phlebotomine Sand Flies ◽  
Serra Do Cipó ◽  
Heritage Site ◽  
One Year ◽  
The One ◽  
Favorable Conditions

In the New World, the leishmaniases are primarily transmitted to humans through the bites ofLeishmania-infectedLutzomyia(Diptera: Psychodidae) phlebotomine sand flies. Any or both of two basic clinical forms of these diseases are endemic to several cities in Brazil—the American cutaneous leishmaniasis (ACL) and the American visceral leishmaniasis (AVL). The present study was conducted in the urban area of a small-sized Brazilian municipality (Jaboticatubas), in which three cases of AVL and nine of ACL have been reported in the last five years. Jaboticatubas is an important tourism hub, as it includes a major part of the Serra do Cipó National Park. Currently, no local data is available on the entomological fauna or circulatingLeishmania. During the one-year period of this study, we captured 3,104 phlebotomine sand flies belonging to sixteenLutzomyiaspecies. In addition to identifying incriminated or suspected vectors of ACL with DNA of the etiological agent of AVL and vice versa, we also detectedLeishmaniaDNA in unexpectedLutzomyiaspecies. The expressive presence of vectors and naturalLeishmaniainfection indicates favorable conditions for the spreading of leishmaniases in the vicinity of the Serra do Cipó National Park.

scholarly journals A sand fly salivary protein acts as a neutrophil chemoattractant

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anderson B. Guimaraes-Costa ◽  
John P. Shannon ◽  
Ingrid Waclawiak ◽  
Jullyanna Oliveira ◽  
Claudio Meneses ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Calcium Influx ◽  
Parasite Burden ◽  
Salivary Protein ◽  
Sand Fly ◽  
Human Neutrophils ◽  
Chemotactic Protein ◽  
The Impact

AbstractApart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P < 0.0001) and increases parasite burden (P < 0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.

scholarly journals Fibronectin Adsorption on Electrospun Synthetic Vascular Grafts Attracts Endothelial Progenitor Cells and Promotes Endothelialization in Dynamic In Vitro Culture

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 778 ◽  
Author(s):  
Ruben Daum ◽  
Dmitri Visser ◽  
Constanze Wild ◽  
Larysa Kutuzova ◽  
Maria Schneider ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Cell Activation ◽  
Vascular Endothelial Cells ◽  
Endothelial Progenitor ◽  
Advanced Therapy Medicinal Product ◽  
Endothelial Colony Forming Cells ◽  
Advanced Therapy ◽  
Custom Made

Appropriate mechanical properties and fast endothelialization of synthetic grafts are key to ensure long-term functionality of implants. We used a newly developed biostable polyurethane elastomer (TPCU) to engineer electrospun vascular scaffolds with promising mechanical properties (E-modulus: 4.8 ± 0.6 MPa, burst pressure: 3326 ± 78 mmHg), which were biofunctionalized with fibronectin (FN) and decorin (DCN). Neither uncoated nor biofunctionalized TPCU scaffolds induced major adverse immune responses except for minor signs of polymorph nuclear cell activation. The in vivo endothelial progenitor cell homing potential of the biofunctionalized scaffolds was simulated in vitro by attracting endothelial colony-forming cells (ECFCs). Although DCN coating did attract ECFCs in combination with FN (FN + DCN), DCN-coated TPCU scaffolds showed a cell-repellent effect in the absence of FN. In a tissue-engineering approach, the electrospun and biofunctionalized tubular grafts were cultured with primary-isolated vascular endothelial cells in a custom-made bioreactor under dynamic conditions with the aim to engineer an advanced therapy medicinal product. Both FN and FN + DCN functionalization supported the formation of a confluent and functional endothelial layer.

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