Signal transduction and Rickettsial infection of tick cells

Spotted fever group (SFG) Rickettsia are obligate intracellular bacteria that are carried by ticks. One such tick, Dermacentor variabilis is a vector for the etiologic agent of Rocky Mountain spotted fever, R. rickettsii. These ticks also carry a non-pathogenic R. montanensis, the agent used in this study. Interestingly, field data collected from infected D. variabilis throughout the United States revealed that the majority of Rickettsia in ticks are non-pathogenic species such as R. montanensis. Although ticks serve as both vector and reservoir hosts for SFG Rickettsia, many questions regarding tick-Rickettsia interaction remain unresolved. Therefore, the overall goal of this research was to study the relationship between ticks and Rickettsia, specifically examining the molecular mechanisms of rickettsial infection of tick host. As SFG Rickettsia can move between vertebrate and invertebrate hosts, the hypothesis is that conserved mechanisms are utilized for invasion of both types of host cell. Biochemical inhibition assays revealed that the tick molecules, PI 3-kinase, protein tyrosine kinases, Src, FAK, Rho GTPase Rac1, N-WASP, Arp2/3 complex, actin, and V-ATPase are important for R. montanensis invasion. Further studies were executed to molecularly and functionally characterize the tick molecules, Arp2/3 complex and V-ATPase, which are central to rickettsial internalization. Full length cDNA of Arp2/3 complex subunits and V-ATPase from D. variabilis were isolated. Transcriptional profiles of Arp2/3 complex subunits and V-ATPase showed greater expression of the mRNA in tick ovaries compared to midgut and salivary glands. In addition, to gain insight into rickettsial invasion in nature, Arp2/3 complex inhibition assays were performed in tick tissues. The results demonstrated the involvement of Arp2/3 complex in rickettsial entry into midgut, ovary, and salivary glands. The tick molecules identified in this study may provide novel points of intervention in the transmission of tick-borne rickettsial diseases

Molecular Characterization and Tissue-Specific Gene Expression of Dermacentor variabilis α-catenin in Response to Rickettsial Infection

Alpha catenin is a cytoskeleton protein that acts as a regulator of actin rearrangement by forming an E-cadherin adhesion complex. In Dermacentor variabilis, a putative α-catenin (Dvα-catenin) was previously identified as differentially regulated in ovaries of ticks chronically infected with Rickettsia montanensis. To begin characterizing the role(s) of Dvα-catenin during rickettsial infection, the full-length Dvα-catenin cDNA was cloned and analysed. Comparative sequence analysis demonstrates a 3069-bp cDNA with a 2718-bp open reading frame with a sequence similar to Ixodes scapularis α-catenin. A portion of Dvα-catenin is homologous to the vinculin-conserved domain containing a putative actin-binding region and β-catenin-binding and -dimerization regions. Quantitative reverse-transcription PCR analysis demonstrated that Dvα-catenin is predominantly expressed in tick ovaries and is responsive to tick feeding. The tissue-specific gene expression analysis of ticks exposed to Rickettsia demonstrates that Dvα-catenin expression was significantly downregulated 12 h after exposure to R. montanensis, but not in Rickettsia amblyommii-exposed ovaries, compared with Rickettsia-unexposed ticks. Studying tick-derived molecules associated with rickettsial infection will provide a better understanding of the transmission dynamics of tick-borne rickettsial diseases

Interaction of Rickettsia felis with histone H2B facilitates the infection of a tick cell line

Haematophagous arthropods are the primary vectors in the transmission of Rickettsia, yet the molecular mechanisms mediating the rickettsial infection of arthropods remain elusive. This study utilized a biotinylated protein pull-down assay together with LC-MS/MS to identify interaction between Ixodes scapularis histone H2B and Rickettsia felis. Co-immunoprecipitation of histone with rickettsial cell lysate demonstrated the association of H2B with R. felis proteins, including outer-membrane protein B (OmpB), a major rickettsial adhesin molecule. The rickettsial infection of tick ISE6 cells was reduced by approximately 25 % via RNA-mediated H2B-depletion or enzymic treatment of histones. The interaction of H2B with the rickettsial adhesin OmpB suggests a role for H2B in mediating R. felis internalization into ISE6 cells

Pyruvate kinase is post-translationally regulated by sirtuin 2 in Aedes aegypti mosquitoes

We previously demonstrated that Aedes aegypti pyruvate kinase (AaPK) plays a key role in the regulation of both carbon and nitrogen metabolism in mosquitoes. To further elucidate whether AaPK can be post-translationally regulated by Ae. aegypti sirtuin 2 (AaSirt2), an NAD+-dependent deacetylase that catalyzes the removal of acetyl groups from acetylated lysine residues, we conducted a series of analysis in non-starved and starved female mosquitoes. Transcriptional and protein profiles of AaSirt2, analyzed by qPCR and western blots, indicated that the AaSirt2 is differentially modulated in response to sugar or blood feeding in mosquito tissues dissected at different times during the first gonotrophic cycle. We also found that AaSirt2 is localized in both cytosolic and mitochondrial cellular compartments of fat body and thorax. Multiple lysine-acetylated proteins were detected by western blotting in both cellular compartments. Furthermore, western blotting of immunoprecipitated proteins provided evidence that AaPK is lysine-acetylated and bound with AaSirt2 in the cytosolic fractions of fat body and thorax from non-starved and starved females. In correlation with these results, we also discovered that RNAi-mediated knockdown of AaSirt2 in the fat body of starved females significantly decreased AaPK protein abundance. Notably, survivorship of AaSirt2-deficient females maintained under four different nutritional regimens was not significantly affected. Taken together, our data reveal that AaPK is post-translationally regulated by AaSirt2.Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Tick Arp2 subunit multiple sequence alignment and identification of conserved ATP binding sites.

<p>Multiple sequence comparison by log-expectation (MUSCLE) software was used to create a sequence alignment of Arp2 subunits from <i>D. variabilis</i>, <i>D. melanogaster</i>, <i>M. musculus</i>, <i>H. sapiens</i>, and <i>S. cerevisiae</i>. Identical and similar amino acids are highlighted in black and grey, respectively. Conserved ATP binding sites predicted by the NsitePred web server are underlined.</p

Effect of Arp2/3 complex inhibitor on <i>R. montanensis</i> invasion of <i>D. variabilis</i> tissues.

<p>Tick tissues including midgut, ovary, and salivary glands were dissected out prior to infection with <i>R. montanensis</i> (8×10⁷ per tissue). After 1 h, rickettsiae were removed and the tissues were washed once with PBS and rickettsiae and tick cells were quantified by qPCR. The experiments were performed in quadruplicate for each treatment group and the results were the combination of the three independent experiments. The asterisk indicates a significant difference between treatment and inhibitor vehicle control.</p

Percent identity of <i>Dv</i>Arp2/3 complex subunits compared to the corresponding subunits of Arp2/3 complex from different organisms.

<p>Percent identity of <i>Dv</i>Arp2/3 complex subunits compared to the corresponding subunits of Arp2/3 complex from different organisms.</p

Novel Identification of <i>Dermacentor variabilis</i> Arp2/3 Complex and Its Role in Rickettsial Infection of the Arthropod Vector

<div><p>Tick-borne spotted fever group (SFG) <i>Rickettsia</i> species must be able to infect both vertebrate and arthropod host cells. The host actin-related protein 2/3 (Arp2/3) complex is important in the invasion process and actin-based motility for several intracellular bacteria, including SFG <i>Rickettsia</i> in <i>Drosophila</i> and mammalian cells. To investigate the role of the tick Arp2/3 complex in tick-<i>Rickettsia</i> interactions, open reading frames of all subunits of the protein including Arp2, Arp3, ARPC1, ARPC2, ARPC3, ARPC4, and ARPC5 were identified from <i>Dermacentor variabilis</i>. Amino acid sequence analysis showed variation (ranging from 25–88%) in percent identity compared to the corresponding subunits of the complex from <i>Drosophila melanogaster</i>, <i>Mus musculus</i>, <i>Homo sapiens</i>, and <i>Saccharomyces cerevisiae</i>. Potential ATP binding sites were identified in <i>D. variabilis</i> (<i>Dv</i>) Arp2 and Arp3 subunits as well as five putative WD (Trp-Asp) motifs which were observed in <i>Dv</i>ARPC1. Transcriptional profiles of all subunits of the <i>Dv</i>Arp2/3 complex revealed greater mRNA expression in both <i>Rickettsia</i>-infected and -uninfected ovary compared to midgut and salivary glands. In response to <i>R. montanensis</i> infection of the tick ovary, the mRNA level of only <i>Dv</i>ARPC4 was significantly upregulated compared to uninfected tissues. Arp2/3 complex inhibition bioassays resulted in a decrease in the ability of <i>R. montanensis</i> to invade tick tissues with a significant difference in the tick ovary, indicating a role for the Arp2/3 complex in rickettsial invasion of tick cells. Characterization of tick-derived molecules associated with rickettsial infection is imperative in order to better comprehend the ecology of tick-borne rickettsial diseases.</p></div

Schematic diagram represented the structure of <i>Dv</i>ARPC1 subunit with putative WD domains characteristic of ARPC1 subunit.

<p>Numbers correspond to amino acids of the protein sequence determined from the novel <i>D. variabilis</i> ARPC1 ORF. Shaded black regions are putative WD domains predicted by SMART software.</p

Transcriptional profile of Arp2/3 complex (all subunits) in <i>D. variabilis</i> tissues.

<p><i>R. montanensis</i> was used to infect tick midgut, ovary, and salivary glands (8×10⁷ rickettsiae per tissue) for 1 h. After removal of rickettsiae, tick tissues were washed and collected by low-speed centrifugation. Total RNA was then extracted from the tissues and the levels of <i>Dv</i>Arp2/3 complex mRNA were measured by qRT-PCR. <i>Dv</i>GAPDH mRNA was used to normalize the differences among samples. Data shown are mean (±SEM) relative expression from two independent experiments. The asterisk denotes a significant difference between treatment groups (unexposed- or <i>Rickettsia-</i>exposed group) in the same tissue. For each subunit, different letters above bars represents significance differences between tissues.</p