scholarly journals The Cat Flea (Ctenocephalides felis) Immune Deficiency Signaling Pathway Regulates Rickettsia typhi Infection

2017 ◽  
Vol 86 (1) ◽  
Author(s):  
Sherri A. Rennoll ◽  
Kristen E. Rennoll-Bankert ◽  
Mark L. Guillotte ◽  
Stephanie S. Lehman ◽  
Timothy P. Driscoll ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Deficiency ◽  
Ctenocephalides Felis ◽  
Spotted Fever Group ◽  
Rickettsia Typhi ◽  
Rickettsial Infection ◽  
Cat Flea ◽  
Content Type ◽  
Imd Pathway ◽  
Pathway Activation

ABSTRACTRickettsiaspecies are obligate intracellular bacteria with both conserved and lineage-specific strategies for invading and surviving within eukaryotic cells. One variable component ofRickettsiabiology involves arthropod vectors: for instance, typhus group rickettsiae are principally vectored by insects (i.e., lice and fleas), whereas spotted fever group rickettsiae are exclusively vectored by ticks. For flea-borneRickettsia typhi, the etiological agent of murine typhus, research on vertebrate host biology is facilitated using cell lines and animal models. However, due to the lack of any stable flea cell line or a published flea genome sequence, little is known regardingR. typhibiology in flea vectors that, importantly, do not suffer lethality due toR. typhiinfection. To address if fleas combat rickettsial infection, we characterized the cat flea (Ctenocephalides felis) innate immune response toR. typhi. Initially, we determined thatR. typhiinfectsDrosophilacells and increases antimicrobial peptide (AMP) gene expression, indicating immune pathway activation. While bioinformatics analysis of theC. felistranscriptome identified homologs to all of theDrosophilaimmune deficiency (IMD) and Toll pathway components, an AMP gene expression profile inDrosophilacells indicated IMD pathway activation upon rickettsial infection. Accordingly, we assessedR. typhi-mediated flea IMD pathway activationin vivousing small interfering RNA (siRNA)-mediated knockdown. Knockdown ofRelishandImdincreasedR. typhiinfection levels, implicating the IMD pathway as a critical regulator ofR. typhiburden inC. felis. These data suggest that targeting the IMD pathway could minimize the spread ofR. typhi, and potentially other human pathogens, vectored by fleas.

scholarly journals CRISPR/Cas9 Mutagenesis in Phlebotomus papatasi: the Immune Deficiency Pathway Impacts Vector Competence for Leishmania major

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Isabelle Louradour ◽  
Kashinath Ghosh ◽  
Ehud Inbar ◽  
David L. Sacks
Keyword(s):  
Immune Deficiency ◽  
Immune Response ◽  
Leishmania Major ◽  
Vector Competence ◽  
Targeted Mutagenesis ◽  
Sand Fly ◽  
Sand Flies ◽  
Phlebotomus Papatasi ◽  
Content Type ◽  
Imd Pathway

ABSTRACT Sand flies are the natural vectors for the Leishmania species that produce a spectrum of diseases in their mammalian hosts, including humans. Studies of sand fly/Leishmania interactions have been limited by the absence of genome editing techniques applicable to these insects. In this report, we adapted CRISPR (clustered regularly interspaced palindromic repeat)/Cas9 (CRISPR-associated protein 9) technology to the Phlebotomus papatasi sand fly, a natural vector for Leishmania major, targeting the sand fly immune deficiency (IMD) pathway in order to decipher its contribution to vector competence. We established a protocol for transformation in P. papatasi and were able to generate transmissible null mutant alleles for Relish (Rel), the only transcription factor of the IMD pathway. While the maintenance of a homozygous mutant stock was severely compromised, we were able to establish in an early generation their greater susceptibility to infection with L. major. Flies carrying different heterozygous mutant alleles variably displayed a more permissive phenotype, presenting higher loads of parasites or greater numbers of infective-stage promastigotes. Together, our data show (i) the successful adaptation of the CRISPR/Cas9 technology to sand flies and (ii) the impact of the sand fly immune response on vector competence for Leishmania parasites. IMPORTANCE Sand flies are the natural vectors of Leishmania parasites. Studies of sand fly/Leishmania interactions have been limited by the lack of successful genomic manipulation of these insects. This paper shows the first example of successful targeted mutagenesis in sand flies via adaptation of the CRISPR/Cas9 editing technique. We generated transmissible null mutant alleles of relish, a gene known to be essential for the control of immune response in other insects. In addition to the expected higher level of susceptibility to bacteria, the mutant flies presented higher loads of parasites when infected with L. major, showing that the sand fly immune response impacts its vector competence for this pathogen.

scholarly journals Assessment of Persistence of Bartonella henselae in Ctenocephalides felis

2013 ◽  
Vol 79 (23) ◽  
pp. 7439-7444 ◽  
Author(s):  
Emilie Bouhsira ◽  
Michel Franc ◽  
Henri-Jean Boulouis ◽  
Philippe Jacquiet ◽  
Isabelle Raymond-Letron ◽  
...  
Keyword(s):  
Bartonella Henselae ◽  
Membrane System ◽  
Ctenocephalides Felis ◽  
Artificial Membrane ◽  
Cat Flea ◽  
Infected Blood ◽  
Content Type ◽  
Entire Life ◽  
Kinetics Of ◽  
Fastidious Bacterium

ABSTRACTBartonella henselae(Rhizobiales:Bartonellaceae) is a Gram-negative fastidious bacterium of veterinary and zoonotic importance. The cat fleaCtenocephalides felis(Siphonaptera: Pulicidae) is the main recognized vector ofB. henselae, and transmission among cats and humans occurs mainly through infected flea feces. The present study documents the use of a quantitative molecular approach to follow the daily kinetics ofB. henselaewithin the cat flea and its excreted feces after exposure to infected blood for 48 h in an artificial membrane system.B. henselaeDNA was detected in both fleas and feces for the entire life span of the fleas (i.e., 12 days) starting from 24 h after initiation of the blood meal.

scholarly journals RalF-Mediated Activation of Arf6 Controls Rickettsia typhi Invasion by Co-Opting Phosphoinositol Metabolism

2016 ◽  
Vol 84 (12) ◽  
pp. 3496-3506 ◽  
Author(s):  
Kristen E. Rennoll-Bankert ◽  
M. Sayeedur Rahman ◽  
Mark L. Guillotte ◽  
Stephanie S. Lehman ◽  
Magda Beier-Sexton ◽  
...  
Keyword(s):  
Spotted Fever ◽  
Host Cells ◽  
Spotted Fever Group ◽  
Nucleotide Exchange ◽  
Rickettsia Typhi ◽  
Content Type ◽  
Adp Ribosylation ◽  
Nucleotide Exchange Factor ◽  
Sec7 Domain ◽  
Phosphatidylinositol 4

Rickettsiae are obligate intracellular pathogens that induce their uptake into nonphagocytic cells; however, the events instigating this process are incompletely understood. Importantly, diverse Rickettsia species are predicted to utilize divergent mechanisms to colonize host cells, as nearly all adhesins and effectors involved in host cell entry are differentially encoded in diverse Rickettsia species. One particular effector, RalF, a Sec7 domain-containing protein that functions as a guanine nucleotide exchange factor of ADP-ribosylation factors (Arfs), is critical for Rickettsia typhi (typhus group rickettsiae) entry but pseudogenized or absent from spotted fever group rickettsiae. Secreted early during R. typhi infection, RalF localizes to the host plasma membrane and interacts with host ADP-ribosylation factor 6 (Arf6). Herein, we demonstrate that RalF activates Arf6, a process reliant on a conserved Glu within the RalF Sec7 domain. Furthermore, Arf6 is activated early during infection, with GTP-bound Arf6 localized to the R. typhi entry foci. The regulation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which generates PI(4,5)P 2 , by activated Arf6 is instrumental for bacterial entry, corresponding to the requirement of PI(4,5)P 2 for R. typhi entry. PI(3,4,5)P 3 is then synthesized at the entry foci, followed by the accumulation of PI(3)P on the short-lived vacuole. Inhibition of phosphoinositide 3-kinases, responsible for the synthesis of PI(3,4,5)P 3 and PI(3)P, negatively affects R. typhi infection. Collectively, these results identify RalF as the first bacterial effector to directly activate Arf6, a process that initiates alterations in phosphoinositol metabolism critical for a lineage-specific Rickettsia entry mechanism.

scholarly journals Microbiota-Induced Changes in Drosophila melanogaster Host Gene Expression and Gut Morphology

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Nichole A. Broderick ◽  
Nicolas Buchon ◽  
Bruno Lemaitre
Keyword(s):  
Gene Expression ◽  
Drosophila Melanogaster ◽  
Fruit Fly ◽  
Cell Types ◽  
Wild Type ◽  
Gut Morphology ◽  
Content Type ◽  
Imd Pathway ◽  
Host Physiology ◽  
Complex Relationships

ABSTRACT To elucidate mechanisms underlying the complex relationships between a host and its microbiota, we used the genetically tractable model Drosophila melanogaster. Consistent with previous studies, the microbiota was simple in composition and diversity. However, analysis of single flies revealed high interfly variability that correlated with differences in feeding. To understand the effects of this simple and variable consortium, we compared the transcriptome of guts from conventionally reared flies to that for their axenically reared counterparts. Our analysis of two wild-type fly lines identified 121 up- and 31 downregulated genes. The majority of these genes were associated with immune responses, tissue homeostasis, gut physiology, and metabolism. By comparing the transcriptomes of young and old flies, we identified temporally responsive genes and showed that the overall impact of microbiota was greater in older flies. In addition, comparison of wild-type gene expression with that of an immune-deficient line revealed that 53% of upregulated genes exerted their effects through the immune deficiency (Imd) pathway. The genes included not only classic immune response genes but also those involved in signaling, gene expression, and metabolism, unveiling new and unexpected connections between immunity and other systems. Given these findings, we further characterized the effects of gut-associated microbes on gut morphology and epithelial architecture. The results showed that the microbiota affected gut morphology through their impacts on epithelial renewal rate, cellular spacing, and the composition of different cell types in the epithelium. Thus, while bacteria in the gut are highly variable, the influence of the microbiota at large has far-reaching effects on host physiology. IMPORTANCE The guts of animals are in constant association with microbes, and these interactions are understood to have important roles in animal development and physiology. Yet we know little about the mechanisms underlying the establishment and function of these associations. Here, we used the fruit fly to understand how the microbiota affects host function. Importantly, we found that the microbiota has far-reaching effects on host physiology, ranging from immunity to gut structure. Our results validate the notion that important insights on complex host-microbe relationships can be obtained from the use of a well-established and genetically tractable invertebrate model.

scholarly journals MyD88 Mediates Instructive Signaling in Dendritic Cells and Protective Inflammatory Response during Rickettsial Infection

2016 ◽  
Vol 84 (4) ◽  
pp. 883-893 ◽  
Author(s):  
Jeremy Bechelli ◽  
Claire Smalley ◽  
Xuemei Zhao ◽  
Barbara Judy ◽  
Patricia Valdes ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Inflammatory Response ◽  
Spotted Fever ◽  
Host Susceptibility ◽  
Rickettsia Conorii ◽  
Spotted Fever Group ◽  
Rickettsial Infection ◽  
Content Type ◽  
Ifn Γ

Spotted fever group rickettsiae cause potentially life-threatening infections throughout the world. Several members of the Toll-like receptor (TLR) family are involved in host response to rickettsiae, and yet the mechanisms by which these TLRs mediate host immunity remain incompletely understood. In the present study, we found that host susceptibility of MyD88−/−mice to infection withRickettsia conoriiorRickettsia australiswas significantly greater than in wild-type (WT) mice, in association with severely impaired bacterial clearancein vivo.R. australis-infected MyD88−/−mice showed significantly lower expression levels of gamma interferon (IFN-γ), interleukin-6 (IL-6), and IL-1β, accompanied by significantly fewer inflammatory infiltrates of macrophages and neutrophils in infected tissues, than WT mice. The serum levels of IFN-γ, IL-12, IL-6, and granulocyte colony-stimulating factor were significantly reduced, while monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, and RANTES were significantly increased in infected MyD88−/−mice compared to WT mice. Strikingly,R. australisinfection was incapable of promoting increased expression of MHC-IIhighand production of IL-12p40 in MyD88−/−bone marrow-derived dendritic cells (BMDCs) compared to WT BMDCs, although costimulatory molecules were upregulated in both types of BMDCs. Furthermore, the secretion levels of IL-1β byRickettsia-infected BMDCs and in the sera of infected mice were significantly reduced in MyD88−/−mice compared to WT controls, suggesting thatin vitroandin vivoproduction of IL-1β is MyD88 dependent. Taken together, our results suggest that MyD88 signaling mediates instructive signals in DCs and secretion of IL-1β and type 1 immune cytokines, which may account for the protective inflammatory response during rickettsial infection.

scholarly journals Reference gene selection and RNA preservation protocol in the cat flea, Ctenocephalides felis, for gene expression studies

Parasitology ◽  
2016 ◽  
Vol 143 (12) ◽  
pp. 1532-1542 ◽  
Author(s):  
CATRIONA H. MCINTOSH ◽  
JOHN BAIRD ◽  
ERICH ZINSER ◽  
DEBRA J. WOODS ◽  
EWAN M. CAMPBELL ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Animal Health ◽  
Companion Animals ◽  
Ctenocephalides Felis ◽  
Pest Species ◽  
Cat Flea ◽  
Rna Quality ◽  
Expression Studies ◽  
Gene Expression Studies

SUMMARYThe cat flea, Ctenocephalides felis, is a major pest species on companion animals thus of significant importance to the animal health industry. The aim of this study was to develop sampling and storage protocols and identify stable reference genes for gene expression studies to fully utilize the growing body of molecular knowledge of C. felis. RNA integrity was assessed in adult and larvae samples, which were either pierced or not pierced and stored in RNAlater at ambient temperature. RNA quality was maintained best in pierced samples, with negligible degradation evident after 10 days. RNA quality from non-pierced samples was poor within 3 days. Ten candidate reference genes were evaluated for their stability across four group comparisons (developmental stages, genders, feeding statuses and insecticide-treatment statuses). Glyceraldehyde 3 phosphate dehydrogenase (GAPDH), 60S ribosomal protein L19 (RPL19) and elongation factor-1α (Ef) were ranked highly in all stability comparisons, thus are recommended as reference genes under similar conditions. Employing just two of these three stable reference genes was sufficient for accurate normalization. Our results make a significant contribution to the future of gene expression studies in C. felis, describing validated sample preparation procedures and reference genes for use in this common pest.

scholarly journals Modulation of Chicken Intestinal Immune Gene Expression by Small Cationic Peptides as Feed Additives during the First Week Posthatch

2013 ◽  
Vol 20 (9) ◽  
pp. 1440-1448 ◽  
Author(s):  
Michael H. Kogut ◽  
Kenneth J. Genovese ◽  
Haiqi He ◽  
Christina L. Swaggerty ◽  
Yiwei Jiang
Keyword(s):  
Gene Expression ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Feed Additives ◽  
Feed Additive ◽  
Cationic Peptides ◽  
Type I ◽  
Immune Gene ◽  
Content Type ◽  
Functional Efficiency

ABSTRACTWe have been investigating modulation strategies tailored around the selective stimulation of the host's immune system as an alternative to direct targeting of microbial pathogens by antibiotics. One such approach is the use of a group of small cationic peptides (BT) produced by a Gram-positive soil bacterium,Brevibacillus texasporus. These peptides have immune modulatory properties that enhance both leukocyte functional efficiency and leukocyte proinflammatory cytokine and chemokine mRNA transcription activitiesin vitro. In addition, when provided as a feed additive for just 4 days posthatch, BT peptides significantly induce a concentration-dependent protection against cecal and extraintestinal colonization bySalmonella entericaserovar Enteritidis. In the present studies, we assessed the effects of feeding BT peptides on transcriptional changes on proinflammatory cytokines, inflammatory chemokines, and Toll-like receptors (TLR) in the ceca of broiler chickens with and withoutS. Enteritidis infection. After feeding a BT peptide-supplemented diet for the first 4 days posthatch, chickens were then challenged withS. Enteritidis, and intestinal gene expression was measured at 1 or 7 days postinfection (p.i.) (5 or 11 days of age). Intestinal expression of innate immune mRNA transcripts was analyzed by quantitative real-time PCR (qRT-PCR). Analysis of relative mRNA expression showed that a BT peptide-supplemented diet did not directly induce the transcription of proinflammatory cytokine, inflammatory chemokine, type I/II interferon (IFN), or TLR mRNA in chicken cecum. However, feeding the BT peptide-supplemented diet primed cecal tissue for increased (P≤ 0.05) transcription of TLR4, TLR15, and TLR21 upon infection withS. Enteritidis on days 1 and 7 p.i. Likewise, feeding the BT peptides primed the cecal tissue for increased transcription of proinflammatory cytokines (interleukin 1β [IL-1β], IL-6, IL-18, type I and II IFNs) and inflammatory chemokine (CxCLi2) in response toS. Enteritidis infection 1 and 7 days p.i. compared to the chickens fed the basal diet. These small cationic peptides may prove useful as alternatives to antibiotics as local immune modulators in neonatal poultry by providing prophylactic protection againstSalmonellainfections.

scholarly journals Methanobactin from Methylocystis sp. Strain SB2 Affects Gene Expression and Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

2015 ◽  
Vol 81 (7) ◽  
pp. 2466-2473 ◽  
Author(s):  
Muhammad Farhan Ul-Haque ◽  
Bhagyalakshmi Kalidass ◽  
Alexey Vorobev ◽  
Bipin S. Baral ◽  
Alan A. DiSpirito ◽  
...  
Keyword(s):  
Gene Expression ◽  
Methane Monooxygenase ◽  
Particulate Methane Monooxygenase ◽  
Methylosinus Trichosporium Ob3b ◽  
Methylosinus Trichosporium ◽  
Monooxygenase Activity ◽  
Content Type ◽  
Soluble Methane Monooxygenase ◽  
A Subunit ◽  
Membrane Bound

ABSTRACTMethanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced byMethylosinus trichosporiumOB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. WhenMethylosinus trichosporiumOB3b was grown in the presence of 1 μM CuCl2, expression ofmmoX, encoding a subunit of the hydroxylase component of sMMO, was very low.mmoXexpression increased, however, when methanobactin fromMethylocystissp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated withM. trichosporiumOB3b. IfM. trichosporiumOB3b was grown in the absence of CuCl2, themmoXexpression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure ofMethylosinus trichosporiumOB3b to SB2-Mb had no effect on expression ofmbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl2.mbnAexpression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl2. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin “piracy” may be commonplace.

scholarly journals Developmentally Regulated Oscillations in the Expression of UV Repair Genes in a Soilborne Plant Pathogen Dictate UV Repair Efficiency and Survival

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Shira Milo-Cochavi ◽  
Sheera Adar ◽  
Shay Covo
Keyword(s):  
Gene Expression ◽  
Fusarium Oxysporum ◽  
Plant Pathogen ◽  
Uv Light ◽  
Sun Exposure ◽  
The Sun ◽  
Repair Gene ◽  
Repair Capacity ◽  
Content Type ◽  
Repair Genes

ABSTRACT The ability to withstand UV damage shapes the ecology of microbes. While mechanisms of UV tolerance were extensively investigated in microorganisms regularly exposed to the sun, far less is known about UV repair of soilborne microorganisms. Fusarium oxysporum is a soilborne fungal plant pathogen that is resistant to UV light. We hypothesized that its UV repair capacity is induced to deal with irregular sun exposure. Unlike the SOS paradigm, our analysis revealed only sporadic increases and even decreases in UV repair gene expression following UVC irradiation or exposure to visible light. Strikingly, a major factor determining the expression of UV repair genes was the developmental status of the fungus. At the early stages of germination, the expression of photolyase increased while the expression of UV endonuclease decreased, and then the trend was reversed. These gene expression oscillations were dependent on cell cycle progression. Consequently, the contribution of photoreactivation to UV repair and survival was stronger at the beginning of germination than later when a filament was established. F. oxysporum germinates following cues from the host. Early on in germination, it is most vulnerable to UV; when the filament is established, the pathogen is protected from the sun because it is already within the host tissue. IMPORTANCE Fusarium oxysporum infects plants through the roots and therefore is not exposed to the sun regularly. However, the ability to survive sun exposure expands the distribution of the population. UV from the sun is toxic and mutagenic, and to survive sun exposure, fungi encode several DNA repair mechanisms. We found that Fusarium oxysporum has a gene expression program that activates photolyase at the first hours of germination when the pathogen is not established in the plant tissue. Later on, the expression of photolyase decreases, and the expression of a light-independent UV repair mechanism increases. We suggest a novel point of view to a very fundamental question of how soilborne microorganisms defend themselves against sudden UV exposure.

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