Abstract

   Rickettsial infections of the central nervous system (CNS) are
   manifested by severe neurological symptoms and represent a serious
   life-threatening condition. Despite the considerable health danger,
   only a few studies have been conducted focusing on the pathogenesis
   induced by Rickettsia sp. in CNS. To investigate the signaling pathways
   associated with the neurotoxic effects of rickettsiae, we employed an
   experimental model of cerebrocortical neurons combined with molecular
   profiling and comprehensive bioinformatic analysis. The cytopathic
   effect induced by Rickettsia akari and Rickettsia slovaca was
   demonstrated by decreased neuronal viability, structural changes in
   cell morphology, and extensive fragmentation of neurites in vitro.
   Targeted profiling revealed the deregulation of genes involved in the
   neuroinflammatory and neurotoxic cell response pathways. Although
   quantitative analysis showed differences in gene expression response,
   functional annotation revealed that the biological processes are
   largely shared between both Rickettsia species. The identified enriched
   pathways are associated with cytokine signaling, chemotaxis of immune
   cells, responses to infectious agents, interactions between neurons,
   endothelial and glial cells, and regulation of neuronal apoptotic
   processes. The findings of our study provide new insight into the
   etiopathogenesis of CNS infection and further expand the understanding
   of molecular signaling associated with neuroinvasive Rickettsia
   species.

   Keywords: infection, neuron, pathway, rickettsiae, signaling

1. Introduction

   Bacterial infection represents a major cause of suspected encephalitis
   or meningitis, with high mortality rates [[32]1]. However, current
   diagnostics can identify about half of all central nervous system (CNS)
   pathogens [[33]2,[34]3].

   Rickettsioses are devastating human infections [[35]4] caused by
   obligate intracellular bacteria of the genus Rickettsia. Rickettsiae
   are transmitted by various arthropods (ticks, mites, lice, and fleas)
   and are distributed worldwide [[36]5]. Rickettsial infections present
   with clinical manifestations ranging from mild febrile illness to
   life-threatening complications [[37]6], including endothelial barrier
   dysfunction in the brain and lungs [[38]7,[39]8].

   Acute neurological manifestations of rickettsial diseases include
   severe headache, behavioral abnormalities, meningismus, photophobia,
   amnesia, aphasia, visual and hearing loss, ataxia, seizures,
   encephalitis, and coma (reviewed in [[40]9]). These symptoms are most
   often reported in children and patients with delayed diagnosis
   [[41]10,[42]11]. Infection and associated neuroinflammation may be
   fatal [[43]12] or lead to lasting sequelae [[44]13,[45]14].

   Neurological symptomatology in rickettsial diseases occurs after
   bacterial dissemination through the bloodstream and is linked with
   infective vasculitis [[46]15,[47]16]. Previous studies have confirmed
   rickettsial invasion of vascular endothelial cells in the brain of
   human patients [[48]17,[49]18] and experimentally infected animal
   models [[50]19,[51]20]. Rickettsiae can breach the endothelial cells of
   the blood–brain barrier or the epithelial cells of blood–choroid
   barriers and are presumably recognized by antigen-presenting cells
   through binding to Toll-like receptors [[52]21,[53]22]. Rickettsiae
   affect preferentially small blood vessels and cause aggregates of
   microglial and mononuclear cells around brain capillaries. Thrombosis
   of small arteries, followed by focal necrosis of the CNS, has been
   reported as a consequence of rickettsial infection [[54]21,[55]23].

   Despite the considerable health danger, the molecular pathogenesis of
   rickettsial CNS infection is largely unknown. Moreover, in the context
   of etiopathogenesis of neurodegeneration, no detailed mechanisms have
   been described so far, although the pro-apoptotic effect of rickettsiae
   in neurons was demonstrated in vitro [[56]24,[57]25].

   Using a model of primary cerebrocortical neurons, molecular profiling,
   and integrated bioinformatic analysis, this study aimed to characterize
   the underlying signaling associated with rickettsial neuroinfection.

2. Materials and Methods

2.1. Rickettsial Culture and Purification

   The bacterial species used in the study were Rickettsia akari, strain
   MK (Kaplan, ATCC VR-148) isolated from a patient’s blood in New York,
   NY, USA [[58]26], and Rickettsia slovaca, strain 13-B (ATCC VR-1639),
   isolated from a Dermacentor marginatus tick in Slovakia [[59]27].
   Rickettsiae were propagated in confluent monolayers of the Vero cell
   line (Cercopithecus aethiops kidney cells, ATCC CCL-81), maintained in
   an RPMI 1640 medium (Lonza, Basel, Switzerland) supplemented with 2 mM
   L-glutamine (Lonza, Basel, Switzerland), 25 mM HEPES (Sigma-Aldrich,
   St. Louis, MO, USA), and 2.5% heat-inactivated fetal bovine serum
   (HyClone, Logan, UT, USA) at 34 °C in a humidified 5% CO[2] incubator.
   Rickettsiae were purified by a modified isopycnic density gradient
   centrifugation protocol described previously [[60]28] and stored in an
   SPG buffer (0.218 M sucrose, 3.76 mM KH[2]PO[4], 7.1 mM K[2]HPO[4], and
   4.9 mM potassium glutamate) at −80 °C for further experiments.

2.2. Infection of Primary Neurons and Viability of Host Cells

   Rat cerebrocortical neurons were prepared according to a published
   method [[61]29]. Briefly, the brains of anesthetized rats (18-day-old
   embryos) were dissected in a chilled L-15 medium free of L-glutamine
   (PAA, Leonding, Austria). After removal of meninges, cortical tissue
   was isolated, minced into small pieces, and treated with a mixture of
   Accutase (2 mL/10 brains) and DNAse I (1.5 ng/mL) for 10 min at 37 °C.
   The enzymatic treatment was stopped with fetal calf serum, and the
   suspension was centrifuged at 17× g for 3 min. The supernatant was
   discarded and replaced with 5 mL of culture medium. The cells were
   plated in 100 µL onto poly-D-lysine-coated 96-well plates at a density
   of 70,000/cm^2. The medium for embryonic neurons (Neurobasal medium,
   Life Technologies, Carlsbad, CA, USA) was supplemented with B27 (2%),
   0.5 mM L-glutamine, and Pen/Strep. Antibiotics were washed out 24 h
   before the experiment. Neuronal cultures were treated with a cell
   division inhibitor (2.5 µM cytosine β-D-arabino-furanoside) for 3–5
   days in vitro to stop the proliferation of glial and non-neuronal
   cells. The cells were maintained in a humidified atmosphere of 5% CO[2]
   at 37 °C, and the medium was replaced every 3–4 days.

   Infection of primary neurons with two Rickettsia spp. was performed as
   previously reported [[62]25]. Briefly, the number of rickettsial genome
   equivalents was calculated by quantitative PCR (qPCR) using
   genus-specific primers and TaqMan probe amplifying a portion of the
   rpsL gene as described [[63]30]. The infectivity and quantity of
   purified rickettsiae were confirmed by plaque assay [[64]31,[65]32].

   Neuron survival following infection with Rickettsia spp. was
   determined, as published previously [[66]25]. Briefly, monolayers of
   cerebrocortical neurons, cultured in 96-well plates (8 × 10^4/cm^2) for
   eight days in vitro, were inoculated with R. akari and R. slovaca at a
   multiplicity of infection (MOI) of 1, 5, 10, and 20. After incubation
   at 34 °C for 24 h, the intracellular ATP was measured using
   CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI,
   USA) according to the manufacturer’s instructions. The generated
   luminescent signal (a.u.), proportional to the amount of ATP present in
   cell culture, was measured using a Fluoroscan Ascent FL luminometer
   (MTX Lab Systems, Vienna, VA, USA). The experiment was performed in
   hexaplicate, and data were analyzed using Prism 9 (GraphPad Software,
   San Diego, CA, USA). For statistical analysis, a one-way ANOVA with
   Dunnett’s multiple-comparison test was performed. A p-value of <0.05
   was considered significantly different.

   For gene expression profiling, rat primary neurons cultured in 6-well
   plates (1 × 10^5/cm^2) for eight days in vitro were inoculated with R.
   akari at MOI 2.5 and 5 and R. slovaca at MOI 10 and 20. Following the
   infection for 24 h, cells were harvested from the culture plates for
   subsequent RNA extraction.

2.3. Immunofluorescence Assay (IFA)

   Cell cultures of rat cerebrocortical neurons were infected with R.
   akari at MOI 5 and R. slovaca at MOI 20. After 36 h, IFA was performed
   according to a published protocol [[67]33]. As negative controls,
   infected neurons were probed with secondary antibodies only, and
   uninfected cells treated with both primary and secondary antibodies
   were included. Images were acquired by a Zeiss LSM 510 META confocal
   fluorescent microscope. The contrast was adjusted by LSM Image Browser
   software (Zeiss, Jena, Germany). The final images with legends were
   created in CorelDRAW version 24.0.0.301 (Alludo, Ottawa, ON, Canada).

2.4. RNA Extraction

   Total RNA was isolated using the RNeasy Mini Kit according to the
   manufacturer’s recommendations, including the DNase I digestion step
   (Qiagen, Hilden, Germany). RNA was eluted into 40 µL RNase-free water.
   The integrity of isolated RNA samples was determined with an Agilent
   2100 Bioanalyzer using an RNA 6000 Nano Labchip kit (Agilent
   Technologies, Waldbronn, Germany). For qPCR analysis, high-quality RNA
   samples were used (RNA integrity number = 6.4 to 8.8). Synthesis of the
   first strand cDNA was carried out using the High Capacity cDNA Reverse
   Transcription Kit (Applied Biosystems, Foster City, CA, USA) according
   to the manufacturer’s protocol. Briefly, 10 µL of the 2× reverse
   transcription master mix was combined with an RNA sample (1 µg/10 µL),
   and cDNA was synthesized under the following conditions: 25 °C for 10
   min, 37 °C for 120 min, 85 °C for 5 min, and 4 °C for 1 min.

2.5. Gene Expression Profiling Using Real-Time PCR

   Gene expression profiling was performed using the predesigned TaqMan
   gene expression assays (Applied Biosystems, Foster City, CA, USA). A
   list of analyzed genes is provided in [68]Supplementary Spreadsheet S1.
   The composition of the qPCR reaction (25 µL) was as follows: 12.5 µL 2×
   TaqMan gene expression master mix; 1.25 µL 20× target FAM-labelled
   TaqMan primer assay; 10.25 µL nuclease-free H[2]O and 1 µL cDNA sample
   (50 ng/µL). PCR reactions were performed in triplicates under the
   following conditions: 50 °C for 2 min, 95 °C for 10 min, followed by 40
   cycles of 95 °C for 15 s and 60 °C for 1 min. Comparative cycle
   threshold (2^−ΔΔCt) analysis was performed to compare gene expression
   between control (uninfected) and Rickettsia-infected rat primary
   neuronal cells using the eukaryotic 18S rRNA (Hs99999901_s1), Actin
   beta (Rn00667869_m1), and glyceraldehyde-3-phosphate dehydrogenase
   (Rn99999916_s1) as endogenous reference genes. Results are expressed as
   a fold change of the mRNA level in infected cells compared with
   uninfected control primary neurons. Genes with a fold change ≥1.5 were
   defined as differentially expressed.

2.6. Pathway Enrichment Analysis (PEA)

   Human orthologs for deregulated genes were used to query the Enrichr
   tool with a Bioplanet 2019 pathway library (KEGG, BioCarta, Reactome,
   WikiPathways, NCI-Nature, Science Signaling, and NetPath databases) and
   Gene Ontology (GO) Biological process 2021 library
   ([69]https://maayanlab.cloud/Enrichr accessed on 24 August 2022)
   [[70]34]. Using the Enrichr platform, the input gene set was queried
   against libraries of annotated gene sets with the goal of identifying
   significantly overlapping genes. These genes were further annotated for
   their affiliation to signaling pathways (Bioplanet 2019 library) and
   biological processes (Gene Ontology 2021 library), and enrichment
   results were calculated. The bioinformatic analysis included genes with
   an average ≥1.5-fold up-regulation and ≤−1.5-fold down-regulation to
   identify significantly enriched pathways for R. akari (15 genes) and R.
   slovaca (11 genes) separately. Enriched pathways and GO biological
   processes are ranked by the q-value (adjusted p-value calculated using
   the Benjamini–Hochberg method, q < 0.05 considered significant) and
   odds ratio. To visualize both common and unique pathways and biological
   processes among rickettsia species, we used NAViGaTOR ver. 3.0.11
   [[71]35]. Top enriched terms are highlighted by statistical
   significance (pink, blue, and grey refer to low-to-higher q-value) and
   odds ratio (node size). Final graphs were exported in SVG format and
   post-processed in CorelDRAW version 24.0.0.301 to produce the final
   images with legends (Alludo, Ottawa, ON, Canada). A comprehensive list
   of enriched pathways and GO biological processes for each species is
   provided in [72]Supplementary Spreadsheet S1.

3. Results

3.1. Neuronal Damage Induced by R. akari and R. slovaca

   The neurotoxic effect of rickettsiae was elucidated using an
   experimental model of rat embryonal neurons. Cerebrocortical primary
   neurons were infected in vitro with two different bacterial species of
   the genus Rickettsia, namely R. akari and R. slovaca. First, to observe
   cell culture viability in response to rickettsial infection, we
   measured the intracellular concentration of ATP in an inoculation
   dose-dependent manner. Rickettsiae induced a statistically significant
   decrease in the viability of neurons 24 h post-infection. In the case
   of R. akari, infection resulted in the loss of 83.7, 87.3, and 97.5%
   ATP in host cells when inoculated with MOI 5, 10, and 20, respectively,
   and profoundly compromised neuronal survival. Significant differences
   in cell viability were also detected after infection with R. slovaca at
   MOI 10 and 20, which resulted in the loss of 24.3 and 61.4% ATP,
   respectively, compared to uninfected control cultures ([73]Figure 1a).

Figure 1.

   [74]Figure 1
   [75]Open in a new tab

   The cytopathic effect of rickettsiae in primary cerebrocortical
   neurons. (a) Primary rat neuronal cultures were incubated with R. akari
   and R. slovaca at MOI 1, 5, 10, and 20. Both Rickettsia spp. induced a
   statistically significant decrease in host cell viability 24 h
   post-infection compared to uninfected control (CTRL) neurons (one-way
   ANOVA, ** p < 0.01, *** p < 0.001; values are displayed as empty dots,
   n = 6, columns represent mean with 95% confidence interval). (b) Light
   microscopy of cerebrocortical primary neurons infected with R. akari at
   MOI 2.5 and 5 and R. slovaca at MOI 10 and 20. The cytopathic effect of
   rickettsiae is represented by structural changes in cell morphology,
   extensive fragmentation of neurites, and neuronal loss. Scale bar
   represents 50 μm.

   Based on the results of the ATP cell viability assay, we next infected
   the cerebrocortical neurons for gene expression profiling with R. akari
   at MOI 2.5 and 5 and R. slovaca at MOI 10 and 20. After 24 h
   post-infection, the primary neuronal culture was observed for induced
   cytopathic effect microscopically ([76]Figure 1b). We recorded evident
   phenotypic changes in neurons after infection with both rickettsiae,
   showing strong destruction of cell morphology when compared to
   uninfected control cultures. Furthermore, we visualized R. akari and R.
   slovaca intracellularly in cerebrocortical neurons 36 h post-infection
   by IFA. Bacteria were localized inside the neurons, as well as in cell
   neurites ([77]Figure 2 upper panels). Uninfected control cells labeled
   with both primary and secondary antibodies and infected cells probed
   with only secondary antibodies showed no positive signal ([78]Figure 2
   lower panels).

Figure 2.

   [79]Figure 2
   [80]Open in a new tab

   Detection of rickettsiae by indirect IFA. Infected neurons were labeled
   with anti-R. akari and anti-R. slovaca rabbit primary antibodies,
   followed by Alexa Fluor 488 conjugated goat anti-rabbit secondary
   antibodies (upper panels, green signal). No signal was detected in
   uninfected neurons or in control staining using only secondary
   antibodies (lower panels). Cell nuclei were stained with DAPI (blue
   signal). To show the overall shape of the neurons, we applied
   differential interference contrast (DIC). Scale bar represents 10 μm.

3.2. Rickettsiae Induce Deregulation of Gene Expression in Primary
Cerebrocortical Neurons

   In order to investigate the molecular signaling associated with the
   neurotoxic effects of rickettsiae, we performed a targeted analysis of
   key regulatory genes involved in the neuroinflammatory and neurotoxic
   cell response pathways. Specifically, the infection of primary
   cortico-hippocampal neurons with R. akari resulted in increased
   expression of pro-inflammatory chemokines and cytokines (↑Ccl2, ↑Ccl3,
   ↑Ccl4, ↑Il1b), pathogen infection and innate immunity responsive genes
   (↑Il6, ↑Tlr4, ↑Tgfb1), deregulation of apoptosis and cell survival
   regulatory genes (↑Bak1, ↑Bdnf, ↑Birc3, ↓Casp3, ↑Ucp1, ↑Ucp2), and
   genes involved in signal transduction (↓Jun, ↑Map3k1). The toxic effect
   of R. slovaca on primary neurons was associated with the up-regulation
   of Ccl2, Ccl3, Ccl4, Il1b, Tlr4, and Birc3 genes and down-regulation of
   Ucp1, Hk2, Ngf, Casp3, and Jun. Quantitative analysis showed
   differences in the gene expression response between the tested species.
   In particular, R. akari induced the up-regulation of Bak1, Bdnf, Il6,
   Map3k1, Tgfb1, and Ucp2, while the expression of these genes was not
   altered after R. slovaca infection. We found a specific down-regulation
   of Hk2 and Ngf together with an opposite pattern of Ucp1 expression in
   R. slovaca compared to R. akari, suggesting several distinct molecular
   pathways associated with neuronal toxicity of these Rickettsia spp.
   Overall, the magnitude of altered gene expression corresponded to the
   MOI of individual pathogen species. Moreover, R. akari showed a higher
   neurotoxic effect compared to R. slovaca, which was represented by a
   greater number of deregulated genes at lower MOI ([81]Figure 3).
   Comprehensive gene expression profiles are shown in [82]Supplementary
   Spreadsheet S1.

Figure 3.

   [83]Figure 3
   [84]Open in a new tab

   Deregulated genes in Rickettsia-infected neurons in vitro. Quantitative
   profiling revealed overexpression of pro-inflammatory and
   pathogen-responsive genes after R. akari and R. slovaca infection of
   rat-primary neurons. The neurotoxic effect of both bacterial species
   was also associated with the deregulation of genes involved in signal
   transduction, apoptosis, and cell survival. The dotted line indicates
   the gene expression threshold (≥1.5-fold for up-regulation and
   ≤−1.5-fold for down-regulation compared to uninfected controls).
   Columns represent the mean log[10]-fold change of gene expression in
   infected neurons with a 95% confidence interval (n = 3), and color
   indicates the MOI of individual Rickettsia species.

3.3. Signaling Pathways Involved in Response to Rickettsial Infection in
Primary Cerebrocortical Neurons

   To elucidate the functional annotation of signaling pathways associated
   with rickettsia-induced neurotoxicity, we performed integrated
   bioinformatic analysis using the Enrichr tool with the Bioplanet 2019
   pathway library. Overall, we identified 243 significantly enriched
   pathways for R. akari and 224 significantly enriched pathways for R.
   slovaca. Our analysis revealed that the majority of identified enriched
   pathways are shared between both rickettsiae (182 pathways), and only a
   fraction of enriched pathways are unique for either of the tested
   species (R. akari: 61 pathways, R. slovaca: 42 pathways). The group of
   shared enriched pathways associates with pro-inflammatory signaling via
   TNFα, IL-1, IL-2, and IL-5, microbial infection signaling, as well as
   regulation of apoptosis, senescence, and autophagy. A panel of top
   enriched pathways specific to R. akari includes DNA damage response,
   host–pathogen interactions, TGFβ, chemokine, and cytokine signaling.
   Enriched pathways specific to R. slovaca are mostly related to
   pro-apoptotic signaling, activation of programmed cell death, and
   suppressed cell survival pathways ([85]Figure 4). These data are
   consistent with the decreased cell viability and reduced neuronal
   survival observed after rickettsial infection of cerebrocortical
   neurons in vitro.

Figure 4.

   [86]Figure 4
   [87]Open in a new tab

   Enriched pathways associated with rickettsial infection. Pathway
   enrichment analysis identified a dominant neuronal response that is
   shared between R. akari and R. slovaca. Top over-represented enriched
   pathways for each species, as revealed by the Enrichr tool and the
   Bioplanet pathway library, are closely related to infectious disease,
   apoptosis, inflammation, and neuronal survival signaling. Node size
   corresponds to the odds ratio, and color represents the significance of
   pathway enrichment (pink, blue, and grey refer to low-to-higher
   q-value). Pathway names are listed for the top 30 enriched pathways in
   each group, while the grey circles represent the remaining
   significantly enriched pathways (152 shared pathways, 31 R.
   akari-specific pathways, 12 R. slovaca-specific pathways).

   Bioinformatic analysis linked deregulated genes to biological processes
   involved in the neuronal response to R. akari and R. slovaca infection
   in vitro. Most of the top over-represented GO biological processes are
   shared between both species, including dominant cellular responses to
   infectious agents, cytokine signaling, and chemotaxis of immune cells.
   Unique biological processes identified by enrichment analysis also
   highlighted similar neuroinflammatory responses following R. akari and
   R. slovaca infection, including glial cell proliferation and migration,
   macrophage chemotaxis, and regulation of neuronal apoptotic processes
   ([88]Figure 5). A comprehensive list of GO biological processes is
   provided in [89]Supplementary Spreadsheet S1.

Figure 5.

   [90]Figure 5
   [91]Open in a new tab

   Biological response of cerebrocortical neurons to rickettsial infection
   in vitro. Bioinformatic analysis revealed a significant overlap of
   biological processes involved in neuronal signaling after infection
   with Rickettsia spp. Deregulated genes associated with
   neuroinflammatory response mechanisms, activation of immune and glial
   cells together with regulation of apoptotic processes, and
   host–pathogen defense responses. The scheme visualizes the top 50
   significant GO biological processes for each species (34 shared
   processes, 16 R. akari-specific processes, 16 R. slovaca-specific
   processes). Node size corresponds to the odds ratio, and color
   represents the significance of biological process enrichment (pink,
   blue, and grey refer to low-to-higher q-value). The number in
   parentheses refers to the GO identifier.

4. Discussion

   Neurological manifestations of rickettsioses include behavioral
   abnormalities, meningoencephalitis, focal neurological deficits,
   stroke, seizures, and coma. Interestingly, it is not clear why only a
   subgroup of patients, e.g., 40% of those with spotted fever
   rickettsioses, develops neurologic sequelae [[92]36,[93]37,[94]38]. We
   have previously shown that rickettsial infection is associated with
   extensive destruction of neuronal morphology, fragmentation of
   neurites, and decreased viability. However, the observed difference in
   the cytopathic effect of tested rickettsiae was not correlated with the
   viability of purified bacteria, which may have influenced previously
   published data. The present analysis confirmed the infectivity and
   quantity of rickettsiae by plaque assay, providing a better
   characterization of isolated bacteria in comparison to previous results
   [[95]25].

   Here we explored two genetically different clusters of rickettsiae (R.
   akari and R. slovaca), aiming to characterize the specific signaling
   pathways associated with their neurotoxic effects. Using targeted gene
   profiling and comprehensive bioinformatic analysis, we identified key
   molecular mechanisms linked to rickettsial neuroinfection.

   Our findings indicate dominant neuroinflammatory and pro-apoptotic
   signaling, activation of programmed cell death, and suppressed cell
   survival pathways in neurons after the infection. The comparison of R.
   akari and R. slovaca revealed that the neuronal response to these
   pathogens is largely shared, suggesting very similar signaling.

   At the cell level, since rickettsiae are obligate intracellular
   bacteria, a recent hypothesis includes two ways of intrusion, direct or
   indirect neuroinvasion or immunologically mediated neuronal damage
   [[96]9]. In spite of the clinical importance and neurological
   consequences of rickettsial brain infection, including
   neuroinflammation and neurotoxicity, very little research was performed
   in order to reveal underlying molecular mechanisms induced by
   rickettsiae in neurons. It was reported that massive activation of
   microglia after rickettsial infection results in neurotoxicity and
   inflammation of the CNS, leading to the death of infected mice
   [[97]12]. However, in the context of the published data, we suppose
   that microglia migrate to the site of neuronal damage and exert
   neuroprotective processes, as this is their authentic function in a
   diseased brain [[98]39]. The recent review summarizes the pathogenesis
   of Rickettsia-infected mice and presents a set of upregulated genes
   coding for specific cytokines and indicates affected brain cells
   [[99]14].

   In the present study, we confirmed that the bacteria are clearly
   localized inside the neuronal cell bodies and in cell neurites. As a
   consequence of the intraneuronal pathogenic activity of rickettsiae, we
   found that the identified biological processes are associated with
   increased migration and chemotaxis of macrophages, lymphocytes, and
   natural killer cells, suggesting that neuronal signaling attracts
   phagocytic and immune cells to sites of rickettsial neuroinfection.

   The spreading of rickettsiae throughout the body leads to the infection
   of the vascular endothelium of the small and medium-sized blood vessels
   [[100]40]. Thus, most of the clinical symptoms of rickettsial diseases
   are associated with the infection of endothelial cells [[101]41]. Our
   analysis revealed that neuronal infection is associated with identical
   signaling pathways, as observed in Rickettsia-infected endothelial
   cells. In particular, NF-κB-mediated regulation of apoptotic pathways
   [[102]42], represented by the balance between pro- (↑Bak1) and
   anti-apoptotic (↑Birc3) signals and prevention of caspase activation
   (↓Casp3) was observed. Mitochondrial role in apoptotic signaling in
   neurons was observed concluding the importance of this signaling as it
   was described in Rickettsia-infected endothelial cells [[103]43].
   Furthermore, infected neurons overexpressed a panel of pro-inflammatory
   chemokines, cytokines, and innate immunity-responsive genes, such as
   Il1b, Il6, Ccl2, Ccl3, and Ccl4 that were previously reported in
   Rickettsia-infected endothelium [[104]44]. Typical host response to
   bacterial infection is mediated via TLR4/lipopolysaccharide signaling
   that triggers pro-inflammatory reactions. Mechanisms involving TLR4 and
   MyD88 were previously reported in dendritic cells, playing a critical
   role in eradication of the invading bacteria and host protection in
   vivo [[105]45,[106]46]. Immune responses via multiple TLRs and NLRs
   recognizing endotoxins from R. akari were also documented
   [[107]47,[108]48].

   Based on the data, we conclude that rickettsial neuroinfection induces
   innate immunity pathways involving TLRs with its downstream signals.
   Furthermore, the bioinformatic analysis highlighted significant
   crosstalk between neuronal, glial, and endothelial cells, the main
   components of the blood–brain-barrier. Positive regulation of vascular
   endothelial growth factor production, together with the regulatory
   pathways of migration and proliferation of glial cells, suggests that
   rickettsial neuroinfection may also activate reparative mechanisms
   necessary for the neuronal protection, re-establishment of homeostasis
   and integrity of the blood–brain-barrier. These pro-survival signals at
   the background of observed neuronal cell death in vitro are not
   surprising, as the rickettsiae manipulate host cell death mechanisms
   using several strategies (reviewed in [[109]48]).

   The findings of our study are consistent with published observations
   and further expand the understanding of molecular signaling associated
   with neuroinvasive Rickettsia species. Nevertheless, we acknowledge
   several limiting aspects that need to be considered. In particular, the
   analysis explored two specific Rickettsia species targeting only a
   subset of regulatory mechanisms that may be affected after infection of
   neuronal cells. Recent research in non-neuronal cells has identified
   impairment of many other regulatory pathways involved in
   Rickettsia–host interactions, such as cell adhesion, autophagy,
   cytoskeleton, oxidative stress, or regulation by non-coding RNAs
   [[110]48,[111]49], which were not investigated in more detail in this
   analysis. We are also aware that host–pathogen interactions may be
   cell-type and species-dependent, as the signaling mechanism may differ
   between the diverse hosts [[112]43,[113]50]. Therefore, further studies
   are warranted to explore the broad complexity of signaling pathways
   after rickettsial infection of neurons considering these aspects.

5. Conclusions

   We report a focused signature of molecular signaling associated with
   the neurotoxic effects of R. akari and R. slovaca, highlighting a
   shared neuroinflammatory and pro-apoptotic signaling, activation of
   programmed cell death, and suppressed cell survival pathways in vitro.
   A complex understanding of neuronal responses may help to elucidate the
   pathogenesis of rickettsial neuroinfections, leading to the
   identification of potential prognostic markers and improving the design
   of treatment regimens in the future.

Acknowledgments