It is estimated that approximately one billion people are at risk of infection with obligate intracellular bacteria, but little is known about the underlying mechanisms that govern their life cycles. ...The difficulty in studying Chlamydia spp., Coxiella spp., Rickettsia spp., Anaplasma spp., Ehrlichia spp. and Orientia spp. is, in part, due to their genetic intractability. Recently, genetic tools have been developed; however, optimizing the genomic manipulation of obligate intracellular bacteria remains challenging. In this Review, we describe the progress in, as well as the constraints that hinder, the systematic development of a genetic toolbox for obligate intracellular bacteria. We highlight how the use of genetically manipulated pathogens has facilitated a better understanding of microbial pathogenesis and immunity, and how the engineering of obligate intracellular bacteria could enable the discovery of novel signalling circuits in host-pathogen interactions.
Ticks are obligate hematophagous parasites and are important vectors of a wide variety of pathogens. These pathogens include spirochetes in the genus
that cause Lyme disease, rickettsial pathogens, ...and tick-borne encephalitis virus, among others. Due to their prolonged feeding period of up to two weeks, hard ticks must counteract vertebrate host defense reactions in order to survive and reproduce. To overcome host defense mechanisms, ticks have evolved a large number of pharmacologically active molecules that are secreted in their saliva, which inhibits or modulates host immune defenses and wound healing responses upon injection into the bite site. These bioactive molecules in tick saliva can create a privileged environment in the host's skin that tick-borne pathogens take advantage of. In fact, evidence is accumulating that tick-transmitted pathogens manipulate tick saliva composition to enhance their own survival, transmission, and evasion of host defenses. We review what is known about specific and functionally characterized tick saliva molecules in the context of tick infection with the genus
, the intracellular pathogen
, and tick-borne encephalitis virus. Additionally, we review studies analyzing sialome-level responses to pathogen challenge.
The insect immune deficiency (IMD) pathway resembles the tumour necrosis factor receptor network in mammals and senses diaminopimelic-type peptidoglycans present in Gram-negative bacteria. Whether ...unidentified chemical moieties activate the IMD signalling cascade remains unknown. Here, we show that infection-derived lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl diacylglycerol (PODAG) stimulate the IMD pathway of ticks. The tick IMD network protects against colonization by three distinct bacteria, that is the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale. Cell signalling ensues in the absence of transmembrane peptidoglycan recognition proteins and the adaptor molecules Fas-associated protein with a death domain (FADD) and IMD. Conversely, biochemical interactions occur between x-linked inhibitor of apoptosis protein (XIAP), an E3 ubiquitin ligase, and the E2 conjugating enzyme Bendless. We propose the existence of two functionally distinct IMD networks, one in insects and another in ticks.
The E3 ubiquitin ligase X-linked inhibitor of apoptosis (XIAP) acts as a molecular rheostat for the immune deficiency (IMD) pathway of the tick Ixodes scapularis. How XIAP activates the IMD pathway ...in response to microbial infection remains ill defined. Here, we identified the XIAP enzymatic substrate p47 as a positive regulator of the I. scapularis IMD network. XIAP polyubiquitylates p47 in a lysine 63-dependent manner and interacts with the p47 ubiquitin-like (UBX) module. p47 also binds to Kenny (IKKγ/NEMO), the regulatory subunit of the inhibitor of nuclear factor (NF)- κB kinase complex. Replacement of the amino acid lysine to arginine within the p47 linker region completely abrogated molecular interactions with Kenny. Furthermore, mitigation of p47 transcription levels through RNA interference in I. scapularis limited Kenny accumulation, reduced phosphorylation of IKKβ (IRD5), and impaired cleavage of the NF-κB molecule Relish. Accordingly, disruption of p47 expression increased microbial colonization by the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agent Anaplasma phagocytophilum. Collectively, we highlight the importance of ticks for the elucidation of paradigms in arthropod immunology. Manipulating immune signaling cascades within I. scapularis may lead to innovative approaches to reducing the burden of tick-borne diseases.
Vector-borne diseases cause over 700,000 deaths annually and represent 17% of all infectious illnesses worldwide. This public health menace highlights the importance of understanding how arthropod ...vectors, microbes and their mammalian hosts interact. Currently, an emphasis of the scientific enterprise is at the vector-host interface where human pathogens are acquired and transmitted. At this spatial junction, arthropod effector molecules are secreted, enabling microbial pathogenesis and disease. Extracellular vesicles manipulate signaling networks by carrying proteins, lipids, carbohydrates and regulatory nucleic acids. Therefore, they are well positioned to aid in cell-to-cell communication and mediate molecular interactions. This Review briefly discusses exosome and microvesicle biogenesis, their cargo, and the role that nanovesicles play during pathogen spread, host colonization and disease pathogenesis. We then focus on the role of extracellular vesicles in dictating microbial pathogenesis and host immunity during transmission of vector-borne pathogens.
Ticks and tick-borne diseases are a significant economic hindrance for livestock production and a menace to public health. The expansion of tick populations into new areas, the occurrence of ...acaricide resistance to synthetic chemical treatments, the potentially toxic contamination of food supplies, and the difficulty of applying chemical control in wild-animal populations have created greater interest in developing new tick control alternatives. Plant compounds represent a promising avenue for the discovery of such alternatives. Several plant extracts and secondary metabolites have repellent and acaricidal effects. However, very little is known about their mode of action, and their commercialization is faced with multiple hurdles, from the determination of an adequate formulation to field validation and public availability. Further, the applicability of these compounds to control ticks in wild-animal populations is restrained by inadequate delivery systems that cannot guarantee accurate dosage delivery at the right time to the target animal populations. More work, financial support, and collaboration with regulatory authorities, research groups, and private companies are needed to overcome these obstacles. Here, we review the advancements on known plant-derived natural compounds with acaricidal potential and discuss the road ahead toward the implementation of organic control in managing ticks and tick-borne diseases.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Antigenic variation of major surface proteins is considered an immune-evasive maneuver used by pathogens as divergent as bacteria and protozoa. Likewise, major surface protein 2 (Msp2) of the ...tick-borne pathogen, Anaplasma marginale, is thought to be involved in antigenic variation to evade the mammalian host immune response. However, this dynamic process also works in the tick vector in the absence of immune selection pressure. We examined Msp2 variants expressed during infection of four tick and two mammalian cell-lines to determine if the presence of certain variants correlated with specific host cell types. Anaplasma marginale colonies differed in their development and appearance in each of the cell lines (P<0.001). Using Western blots probed with two Msp2-monospecific and one Msp2-monoclonal antibodies, we detected expression of variants with differences in molecular weight. Immunofluorescence-assay revealed that specific antibodies bound from 25 to 60% of colonies, depending on the host cell-line (P<0.001). Molecular analysis of cloned variant-encoding genes demonstrated expression of different predominant variants in tick (V1) and mammalian (V2) cell-lines. Analysis of the putative secondary structure of the variants revealed a change in structure when A. marginale was transferred from one cell-type to another, suggesting that the expression of particular Msp2 variants depended on the cell-type (tick or mammalian) in which A. marginale developed. Similarly, analysis of the putative secondary structure of over 200 Msp2 variants from ticks, blood samples, and other mammalian cells available in GenBank showed the predominance of a specific structure during infection of a host type (tick versus blood sample), demonstrating that selection of a possible structure also occurred in vivo. The selection of a specific structure in surface proteins may indicate that Msp2 fulfils an important role in infection and adaptation to diverse host systems. Supplemental Abstract in Spanish (File S1) is provided.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ehrlichia ruminantium is a tick-borne intracellular pathogen of ruminants that causes heartwater, a disease present in Sub-saharan Africa, islands in the Indian Ocean and the Caribbean, inducing ...significant economic losses. At present, three avirulent strains of E. ruminantium (Gardel, Welgevonden and Senegal isolates) have been produced by a process of serial passaging in mammalian cells in vitro, but unfortunately their use as vaccines do not offer a large range of protection against other strains, possibly due to the genetic diversity present within the species. So far no genetic basis for virulence attenuation has been identified in any E. ruminantium strain that could offer targets to facilitate vaccine production. Virulence attenuated Senegal strains have been produced twice independently, and require many fewer passages to attenuate than the other strains. We compared the genomes of a virulent and attenuated Senegal strain and identified a likely attenuator gene, ntrX, a global transcription regulator and member of a two-component system that is linked to environmental sensing. This gene has an inverted partial duplicate close to the parental gene that shows evidence of gene conversion in different E. ruminantium strains. The pseudogenisation of the gene in the avirulent Senegal strain occurred by gene conversion from the duplicate to the parent, transferring a 4 bp deletion which is unique to the Senegal strain partial duplicate amongst the wild isolates. We confirmed that the ntrX gene is not expressed in the avirulent Senegal strain by RT-PCR. The inverted duplicate structure combined with the 4 bp deletion in the Senegal strain can explain both the attenuation and the faster speed of attenuation in the Senegal strain relative to other strains of E. ruminantium. Our results identify nrtX as a promising target for the generation of attenuated strains of E. ruminantium by random or directed mutagenesis that could be used for vaccine production.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The blacklegged tick, Ixodes scapularis, transmits most vector-borne diseases in the US. It vectors seven pathogens of public health relevance, including the emerging human pathogen Anaplasma ...phagocytophilum. Nevertheless, it remains critically understudied compared to other arthropod vectors. Ixodes scapularis releases a variety of molecules that assist in the modulation of host responses. Recently, it was found that extracellular vesicles (EVs) carry several of these molecules and may impact microbial transmission to the mammalian host. EV biogenesis has been studied in mammalian systems and is relatively well understood, but the molecular players important for the formation and secretion of EVs in arthropods of public health relevance remain elusive. RabGTPases are among the major molecular players in mammalian EV biogenesis. They influence membrane identity and vesicle budding, uncoating, and motility.
Using BLAST, an in silico pathway for EV biogenesis in ticks was re-constructed. We identified Rab27 for further study. EVs were collected from ISE6 tick cells after knocking down rab27 to examine its role in tick EV biogenesis. Ixodes scapularis nymphs were injected with small interfering RNAs to knock down rab27 and then fed on naïve and A. phagocytophilum-infected mice to explore the importance of rab27 in tick feeding and bacterial acquisition.
Our BLAST analysis identified several of the proteins involved in EV biogenesis in ticks, including Rab27. We show that silencing rab27 in I. scapularis impacts tick fitness. Additionally, ticks acquire less A. phagocytophilum after rab27 silencing. Experiments in the tick ISE6 cell line show that silencing of rab27 causes a distinct range profile of tick EVs, indicating that Rab27 is needed to regulate EV biogenesis.
Rab27 is needed for successful tick feeding and may be important for acquiring A. phagocytophilum during a blood meal. Additionally, silencing rab27 in tick cells results in a shift of extracellular vesicle size. Overall, we have observed that Rab27 plays a key role in tick EV biogenesis and the tripartite interactions among the vector, the mammalian host, and a microbe it encounters.
The Ixodes scapularis embryo-derived cell line ISE6 is the most widely utilized tick-derived cell line due to its susceptibility to a wide variety of tick- and non-tick-vectored pathogens. Little is ...known about its tissue origin or biological background. Protein expression of ISE6 cells was compared with that of another I. scapularis-derived cell line, IDE12, and dissected tick synganglia. Results demonstrated the presence of a neuronal marker protein, type 3 β-tubulin, in all three samples, as well as other shared and unique neuronal and immune response-associated proteins. Of neuronal proteins shared between the two cell lines, ISE6 expressed several in significantly greater quantities than IDE12. Stimulation of ISE6 cells by in vivo exposure to the hemocoel environment in unfed larval and molting nymphal ticks, but not unfed nymphal ticks, resulted in the development of neuron-like morphologic characteristics in the implanted cells.
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