Inhibiting autophagy increases the efficacy of low-dose photodynamic therapy.

Rupture and permeabilization of endocytic vesicles can be triggered by various causes, such as pathogenic invasions, amyloid proteins, and silica crystals leading to cell death and degeneration. A cellular quality control process, called lysophagy was recently described to target damaged lysosomes for autophagic sequestration within isolation membranes in order to protect the cell from the consequences of lysosomal leakage. This protective process, however, might interfere with treatment conditions, such as photodynamic therapy (PDT) and the intracellular drug delivery method photochemical internalization (PCI). PCI-induced permeabilization of endosomes and lysosomes is purposely triggered to release drugs that are sequestered in these organelles into the cytosol in order to synergistically kill cancer cells. Here, we show that photochemical treatment with the PCI-photosensitizer TPCS2a/fimaporfin results in both induction of autophagy and inhibition of the autophagic flux. The autophagic response is accompanied by recruitment of ubiquitin (Ubq), p62, and microtubule-associated protein 1A/1B-light chain 3 (LC3) to damaged vesicles, marked by Galectin 3 (Gal3). Furthermore, ultrastructural analysis revealed a homogenously thick p62-positive layer surrounding these permeabilized vesicles. Although p62 seems to be important during the selective autophagic sequestration, we show that its presence is not essential for the effective removal of damaged vesicles or the recovery of the lysosomal content. An active autophagic response and the presence of p62, however, is important for cancer cells to survive low-dose TPCS2a-PDT. Thus, targeting both p62 and autophagy together and independently, in a light-controlled/PCI based delivery of cancer therapeutics could increase the effectiveness of the treatment regime.

Development of resistance to photodynamic therapy (PDT) in human breast cancer cells is photosensitizer-dependent: Possible mechanisms and approaches for overcoming PDT-resistance.

Here we report on the induction of resistance to photodynamic therapy (PDT) in the ABCG2-high human breast cancer cell line MA11 after repetitive PDT, using either Pheophorbide A (PhA) or di-sulphonated meso-tetraphenylchlorin (TPCS2a) as photosensitizer. Resistance to PhA-PDT was associated with enhanced expression of the efflux pump ABCG2. TPCS2a-PDT-resistance was neither found to correspond with lower TPCS2a-accumulation nor reduced generation of reactive oxygen species (ROS). Cross-resistance to chemotherapy (doxorubicin) or radiotherapy was not observed. TPCS2a-PDT-resistant cells acquired a higher proliferation capacity and an enhanced expression of EGFR and ERK1/2. p38 MAPK was found to be a death-signalling pathway in the MA11 cells post TPCS2a-PDT, contrasting the MA11/TR cells in which PDT generated a sustained phosphorylation of p38 that had lost its death-mediated signalling, and an abrogated activation of its downstream effector MAPKAPK2. No difference in apoptosis, necrosis or autophagy responses was found between the treated cell lines. Development of TPCS2a-PDT resistance in the MDA-MB-231 cell line was also established, however, p38 MAPK did not play a role in the PDT-resistance. MCF-7 cells did not develop TPCS2a-PDT-resistance. Photochemical internalisation (PCI) of 1 pM of EGF-saporin induced equal strong cytotoxicity in both MA11 and MA11/TR cells. In conclusion, loss of p38 MAPK-inducing death signalling is the main mechanism of resistance to TPCS2a-PDT in the MA11/TR cell line. This work provides mechanistic knowledge of intrinsic and acquired PDT-resistance which is dependent on choice of photosensitizer, and suggests PCI as a rational therapeutic intervention for the elimination of PDT-resistant cells.

Microscopic Visualisation of Zoonotic Arbovirus Replication in Tick Cell and Organ Cultures Using Semliki Forest Virus Reporter Systems.

Ticks are vectors and reservoirs of many arboviruses pathogenic for humans or domestic animals; in addition, during bloodfeeding they can acquire and harbour pathogenic arboviruses normally transmitted by other arthropods such as mosquitoes. Tick cell and organ cultures provide convenient tools for propagation and study of arboviruses, both tick-borne and insect-borne, enabling elucidation of virus-tick cell interaction and yielding insight into the mechanisms behind vector competence and reservoir potential for different arbovirus species. The mosquito-borne zoonotic alphavirus Semliki Forest virus (SFV), which replicates well in tick cells, has been isolated from Rhipicephalus, Hyalomma, and Amblyomma spp. ticks removed from mammalian hosts in East Africa; however nothing is known about any possible role of ticks in SFV epidemiology. Here we present a light and electron microscopic study of SFV infecting cell lines and organ cultures derived from African Rhipicephalus spp. ticks. As well as demonstrating the applicability of these culture systems for studying virus-vector interactions, we provide preliminary evidence to support the hypothesis that SFV is not normally transmitted by ticks because the virus does not infect midgut cells.

Ixodes scapularis and Ixodes ricinus tick cell lines respond to infection with tick-borne encephalitis virus: transcriptomic and proteomic analysis.

BACKGROUND: Ixodid ticks are important vectors of a wide variety of viral, bacterial and protozoan pathogens of medical and veterinary importance. Although several studies have elucidated tick responses to bacteria, little is known about the tick response to viruses. To gain insight into the response of tick cells to flavivirus infection, the transcriptomes and proteomes of two Ixodes spp cell lines infected with the flavivirus tick-borne encephalitis virus (TBEV) were analysed. METHODS: RNA and proteins were isolated from the Ixodes scapularis-derived cell line IDE8 and the Ixodes ricinus-derived cell line IRE/CTVM19, mock-infected or infected with TBEV, on day 2 post-infection (p.i.) when virus production was increasing, and on day 6 p.i. when virus production was decreasing. RNA-Seq and mass spectrometric technologies were used to identify changes in abundance of, respectively, transcripts and proteins. Functional analyses were conducted on selected transcripts using RNA interference (RNAi) for gene knockdown in tick cells infected with the closely-related but less pathogenic flavivirus Langat virus (LGTV). RESULTS: Differential expression analysis using DESeq resulted in totals of 43 and 83 statistically significantly differentially-expressed transcripts in IDE8 and IRE/CTVM19 cells, respectively. Mass spectrometry detected 76 and 129 statistically significantly differentially-represented proteins in IDE8 and IRE/CTVM19 cells, respectively. Differentially-expressed transcripts and differentially-represented proteins included some that may be involved in innate immune and cell stress responses. Knockdown of the heat-shock proteins HSP90, HSP70 and gp96, the complement-associated protein Factor H and the protease trypsin resulted in increased LGTV replication and production in at least one tick cell line, indicating a possible antiviral role for these proteins. Knockdown of RNAi-associated proteins Argonaute and Dicer, which were included as positive controls, also resulted in increased LGTV replication and production in both cell lines, confirming their role in the antiviral RNAi pathway. CONCLUSIONS: This systems biology approach identified several molecules that may be involved in the tick cell innate immune response against flaviviruses and highlighted that ticks, in common with other invertebrate species, have other antiviral responses in addition to RNAi.

Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells.

Anaplasma phagocytophilum is an emerging zoonotic pathogen that causes human granulocytic anaplasmosis. These intracellular bacteria establish infection by affecting cell function in both the vertebrate host and the tick vector, Ixodes scapularis. Previous studies have characterized the tick transcriptome and proteome in response to A. phagocytophilum infection. However, in the postgenomic era, the integration of omics datasets through a systems biology approach allows network-based analyses to describe the complexity and functionality of biological systems such as host-pathogen interactions and the discovery of new targets for prevention and control of infectious diseases. This study reports the first systems biology integration of metabolomics, transcriptomics, and proteomics data to characterize essential metabolic pathways involved in the tick response to A. phagocytophilum infection. The ISE6 tick cells used in this study constitute a model for hemocytes involved in pathogen infection and immune response. The results showed that infection affected protein processing in endoplasmic reticulum and glucose metabolic pathways in tick cells. These results supported tick-Anaplasma co-evolution by providing new evidence of how tick cells limit pathogen infection, while the pathogen benefits from the tick cell response to establish infection. Additionally, ticks benefit from A. phagocytophilum infection by increasing survival while pathogens guarantee transmission. The results suggested that A. phagocytophilum induces protein misfolding to limit the tick cell response and facilitate infection but requires protein degradation to prevent ER stress and cell apoptosis to survive in infected cells. Additionally, A. phagocytophilum may benefit from the tick cell's ability to limit bacterial infection through PEPCK inhibition leading to decreased glucose metabolism, which also results in the inhibition of cell apoptosis that increases infection of tick cells. These results support the use of this experimental approach to systematically identify cell pathways and molecular mechanisms involved in tick-pathogen interactions. Data are available via ProteomeXchange with identifier PXD002181.

Identification and Characterization of Anaplasma phagocytophilum Proteins Involved in Infection of the Tick Vector, Ixodes scapularis.

Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was used to characterize A. phagocytophilum proteome during rickettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10-15% and 65-71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophilum-infected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface proteins during A. phagocytophilum infection in ticks. Characterization of Anaplasma proteome contributes information on host-pathogen interactions and provides targets for development of novel control strategies for pathogen infection and transmission.

Ability of the Encephalitic Arbovirus Semliki Forest Virus To Cross the Blood-Brain Barrier Is Determined by the Charge of the E2 Glycoprotein.

UNLABELLED: Semliki Forest virus (SFV) provides a well-characterized model system to study the pathogenesis of virus encephalitis. Several studies have used virus derived from the molecular clone SFV4. SFV4 virus does not have the same phenotype as the closely related L10 or the prototype virus from which its molecular clone was derived. In mice, L10 generates a high-titer plasma viremia, is efficiently neuroinvasive, and produces a fatal panencephalitis, whereas low-dose SFV4 produces a low-titer viremia, rarely enters the brain, and generally is avirulent. To determine the genetic differences responsible, the consensus sequence of L10 was determined and compared to that of SFV4. Of the 12 nucleotide differences, six were nonsynonymous; these were engineered into a new molecular clone, termed SFV6. The derived virus, SFV6, generated a high-titer viremia and was efficiently neuroinvasive and virulent. The phenotypic difference mapped to a single amino acid residue at position 162 in the E2 envelope glycoprotein (lysine in SFV4, glutamic acid in SFV6). Analysis of the L10 virus showed it contained different plaque phenotypes which differed in virulence. A lysine at E2 247 conferred a small-plaque avirulent phenotype and glutamic acid a large-plaque virulent phenotype. Viruses with a positively charged lysine at E2 162 or 247 were more reliant on glycosaminoglycans (GAGs) to enter cells and were selected for by passage in BHK-21 cells. Interestingly, viruses with the greatest reliance on binding to GAGs replicated to higher titers in the brain and more efficiently crossed an in vitro blood-brain barrier (BBB). IMPORTANCE: Virus encephalitis is a major disease, and alphaviruses, as highlighted by the recent epidemic of chikungunya virus (CHIKV), are medically important pathogens. In addition, alphaviruses provide well-studied experimental systems with extensive literature, many tools, and easy genetic modification. In this study, we elucidate the genetic basis for the difference in phenotype between SFV4 and the virus stocks from which it was derived and correct this by engineering a new molecular clone. We then use this clone in one comprehensive study to demonstrate that positively charged amino acid residues on the surface of the E2 glycoprotein, mediated by binding to GAGs, determine selective advantage and plaque size in BHK-21 cells, level of viremia in mice, ability to cross an artificial BBB, efficiency of replication in the brain, and virulence. Together with studies on Sindbis virus (SINV), this study provides an important advance in understanding alphavirus, and probably other virus, encephalitis.

Induction and suppression of tick cell antiviral RNAi responses by tick-borne flaviviruses.

Arboviruses are transmitted by distantly related arthropod vectors such as mosquitoes (class Insecta) and ticks (class Arachnida). RNA interference (RNAi) is the major antiviral mechanism in arthropods against arboviruses. Unlike in mosquitoes, tick antiviral RNAi is not understood, although this information is important to compare arbovirus/host interactions in different classes of arbovirus vectos. Using an Ixodes scapularis-derived cell line, key Argonaute proteins involved in RNAi and the response against tick-borne Langat virus (Flaviviridae) replication were identified and phylogenetic relationships characterized. Analysis of small RNAs in infected cells showed the production of virus-derived small interfering RNAs (viRNAs), which are key molecules of the antiviral RNAi response. Importantly, viRNAs were longer (22 nucleotides) than those from other arbovirus vectors and mapped at highest frequency to the termini of the viral genome, as opposed to mosquito-borne flaviviruses. Moreover, tick-borne flaviviruses expressed subgenomic flavivirus RNAs that interfere with tick RNAi. Our results characterize the antiviral RNAi response in tick cells including phylogenetic analysis of genes encoding antiviral proteins, and viral interference with this pathway. This shows important differences in antiviral RNAi between the two major classes of arbovirus vectors, and our data broadens our understanding of arthropod antiviral RNAi.

Tick capillary feeding for the study of proteins involved in tick-pathogen interactions as potential antigens for the control of tick infestation and pathogen infection.

BACKGROUND: Ticks represent a significant health risk to animals and humans due to the variety of pathogens they can transmit during feeding. The traditional use of chemicals to control ticks has serious drawbacks, including the selection of acaricide-resistant ticks and environmental contamination with chemical residues. Vaccination with the tick midgut antigen BM86 was shown to be a good alternative for cattle tick control. However, results vary considerably between tick species and geographic location. Therefore, new antigens are required for the development of vaccines controlling both tick infestations and pathogen infection/transmission. Tick proteins involved in tick-pathogen interactions may provide good candidate protective antigens for these vaccines, but appropriate screening procedures are needed to select the best candidates. METHODS: In this study, we selected proteins involved in tick-Anaplasma (Subolesin and SILK) and tick-Babesia (TROSPA) interactions and used in vitro capillary feeding to characterize their potential as antigens for the control of cattle tick infestations and infection with Anaplasma marginale and Babesia bigemina. Purified rabbit polyclonal antibodies were generated against recombinant SUB, SILK and TROSPA and added to uninfected or infected bovine blood to capillary-feed female Rhipicephalus (Boophilus) microplus ticks. Tick weight, oviposition and pathogen DNA levels were determined in treated and control ticks. RESULTS: The specificity of purified rabbit polyclonal antibodies against tick recombinant proteins was confirmed by Western blot and against native proteins in tick cell lines and tick tissues using immunofluorescence. Capillary-fed ticks ingested antibodies added to the blood meal and the effect of these antibodies on tick weight and oviposition was shown. However, no effect was observed on pathogen DNA levels. CONCLUSIONS: These results highlighted the advantages and some of the disadvantages of in vitro tick capillary feeding for the characterization of candidate tick protective antigens. While an effect on tick weight and oviposition was observed, the effect on pathogen levels was not evident probably due to high tick-to-tick variations among other factors. Nevertheless, these results together with previous results of RNA interference functional studies suggest that these proteins are good candidate vaccine antigens for the control of R. microplus infestations and infection with A. marginale and B. bigemina.

Gene silencing in tick cell lines using small interfering or long double-stranded RNA.

Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system.