Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides

Peer reviewedPublisher PD

The Transcriptome of the Salivary Glands of Amblyomma aureolatum Reveals the Antimicrobial Peptide Microplusin as an Important Factor for the Tick Protection Against Rickettsia rickettsii Infection

The salivary glands (SG) of ixodid ticks play a pivotal role in blood feeding, producing both the cement and the saliva. The cement is an adhesive substance that helps the attachment of the tick to the host skin, while the saliva contains a rich mixture of antihemostatic, anti-inflammatory, and immunomodulatory substances that allow ticks to properly acquire the blood meal. The tick saliva is also a vehicle used by several pathogens to be transmitted to the vertebrate host, including various bacterial species from the genus Rickettsia. Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes the severe Rocky Mountain spotted fever. In Brazil, the dog yellow tick Amblyomma aureolatum is a vector of R. rickettsii. In the current study, the effects of an experimental infection with R. rickettsii on the global gene expression profile of A. aureolatum SG was determined by next-generation RNA sequencing. A total of 260 coding sequences (CDSs) were modulated by infection, among which 161 were upregulated and 99 were downregulated. Regarding CDSs in the immunity category, we highlight one sequence encoding one microplusin-like antimicrobial peptide (AMP) (Ambaur-69859). AMPs are important effectors of the arthropod immune system, which lack the adaptive response of the immune system of vertebrates. The expression of microplusin was confirmed to be significantly upregulated in the SG as well as in the midgut (MG) of infected A. aureolatum by a quantitative polymerase chain reaction preceded by reverse transcription. The knockdown of the microplusin expression by RNA interference caused a significant increase in the prevalence of infected ticks in relation to the control. In addition, a higher rickettsial load of one order of magnitude was recorded in both the MG and SG of ticks that received microplusin-specific dsRNA. No effect of microplusin knockdown was observed on the R. rickettsii transmission to rabbits. Moreover, no significant differences in tick engorgement and oviposition were recorded in ticks that received dsMicroplusin, demonstrating that microplusin knockdown has no effect on tick fitness. Further studies must be performed to determine the mechanism of action of this AMP against R. rickettsii

The Iron Stimulon of Xylella fastidiosa Includes Genes for Type IV Pilus and Colicin V-Like Bacteriocins▿ †

Xylella fastidiosa is the etiologic agent of a wide range of plant diseases, including citrus variegated chlorosis (CVC), a major threat to citrus industry. The genomes of several strains of this phytopathogen were completely sequenced, enabling large-scale functional studies. DNA microarrays representing 2,608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c were used to investigate transcript levels during growth with different iron availabilities. When treated with the iron chelator 2,2′-dipyridyl, 193 CDS were considered up-regulated and 216 were considered down-regulated. Upon incubation with 100 μM ferric pyrophosphate, 218 and 256 CDS were considered up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription-quantitative PCR. Several CDS involved with regulatory functions, pathogenicity, and cell structure were modulated under both conditions assayed, suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to functions of pili/fimbriae. We also investigated the contribution of the ferric uptake regulator Fur to the iron stimulon of X. fastidiosa. The promoter regions of the strain 9a5c genome were screened for putative Fur boxes, and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron stimulon of X. fastidiosa, and they present novel evidence for iron regulation of pathogenicity determinants

Image_1_The anal pore route is efficient to infect Amblyomma spp. ticks with Rickettsia rickettsii and allows the assessment of the role played by infection control targets.pdf

Adult Amblyomma sculptum and Amblyomma aureolatum ticks are partially refractory to Rickettsia rickettsii when fed on infected hosts, hindering the functional characterization of potentially protective targets in the bacterial acquisition. In the current study, we used the anal pore route to infect adult A. sculptum and A. aureolatum ticks with R. rickettsii and to assess the effects of the knockdown of microplusin in infection control. The anal pore route was efficient to infect both species, resulting in a prevalence of around 100% of infected ticks. Higher loads of R. rickettsii were detected in microplusin-silenced A. aureolatum in relation to the control, as previously obtained when microplusin-silenced ticks were fed on R. rickettsii-infected rabbits. This is the first report showing R. rickettsii infection through the anal pore in Amblyomma ticks, highlighting this route as a powerful tool to assess the role played by additional targets in the control of pathogens.</p

Table_1_The anal pore route is efficient to infect Amblyomma spp. ticks with Rickettsia rickettsii and allows the assessment of the role played by infection control targets.pdf

Adult Amblyomma sculptum and Amblyomma aureolatum ticks are partially refractory to Rickettsia rickettsii when fed on infected hosts, hindering the functional characterization of potentially protective targets in the bacterial acquisition. In the current study, we used the anal pore route to infect adult A. sculptum and A. aureolatum ticks with R. rickettsii and to assess the effects of the knockdown of microplusin in infection control. The anal pore route was efficient to infect both species, resulting in a prevalence of around 100% of infected ticks. Higher loads of R. rickettsii were detected in microplusin-silenced A. aureolatum in relation to the control, as previously obtained when microplusin-silenced ticks were fed on R. rickettsii-infected rabbits. This is the first report showing R. rickettsii infection through the anal pore in Amblyomma ticks, highlighting this route as a powerful tool to assess the role played by additional targets in the control of pathogens.</p

Image_2_The anal pore route is efficient to infect Amblyomma spp. ticks with Rickettsia rickettsii and allows the assessment of the role played by infection control targets.pdf

Adult Amblyomma sculptum and Amblyomma aureolatum ticks are partially refractory to Rickettsia rickettsii when fed on infected hosts, hindering the functional characterization of potentially protective targets in the bacterial acquisition. In the current study, we used the anal pore route to infect adult A. sculptum and A. aureolatum ticks with R. rickettsii and to assess the effects of the knockdown of microplusin in infection control. The anal pore route was efficient to infect both species, resulting in a prevalence of around 100% of infected ticks. Higher loads of R. rickettsii were detected in microplusin-silenced A. aureolatum in relation to the control, as previously obtained when microplusin-silenced ticks were fed on R. rickettsii-infected rabbits. This is the first report showing R. rickettsii infection through the anal pore in Amblyomma ticks, highlighting this route as a powerful tool to assess the role played by additional targets in the control of pathogens.</p

Virulence genes of Rickettsia rickettsii are differentially modulated by either temperature upshift or blood-feeding in tick midgut and salivary glands

Abstract Background Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever, is transmitted to humans by ticks. During tick feeding, R. rickettsii is exposed to both temperature elevation and components of the blood meal, which have previously been associated with the reactivation of its virulence. These environmental stimuli were also reported to modulate virulence genes of R. rickettsii infecting a set of organs of adult females of its natural vector, Amblyomma aureolatum. Methods In this study, we determined the effects of a temperature upshift, blood-feeding, and both stimuli simultaneously on the expression of 85 selected genes of R. rickettsii infecting either the midgut (MG) or salivary glands (SG) of male and female A. aureolatum by microfluidic high-throughput RT-qPCR. These two organs are key for acquisition of this bacterium by the tick and transmission to the vertebrate host, respectively. Results Data showed that these environmental stimuli exert distinct effects on rickettsial transcription depending on the colonized organ and gender of the vector. Temperature upshift induced the majority of differentially expressed genes of R. rickettsii in tick SG, including tRNA synthetases encoding genes. On the contrary, blood-feeding downregulated most of differentially expressed genes in both organs, but induced type IV secretion system components and OmpB in tick MG. The combined effects of both stimuli resulted in a merged gene expression profile representing features of each stimulus analyzed independently, but was more similar to the profile induced by blood-feeding. Conclusion The upregulation of the majority of differentially expressed genes in tick SG by temperature upshift suggests that this stimulus is important to prepare R. rickettsii for transmission to the vertebrate host. Blood-feeding, on the other hand, induced important virulence genes in the tick MG, which might be associated with colonization of the tick and transmission to the vertebrate host. The role of the proteins identified in this study must be addressed and might help to define future targets to block tick infection, thereby preventing RMSF. To our knowledge, this is the first transcriptional tissue-specific study of a virulent strain of R. rickettsii infecting a natural tick vector

The Distinct Transcriptional Response of the Midgut of Amblyomma sculptum and Amblyomma aureolatum Ticks to Rickettsia rickettsii Correlates to Their Differences in Susceptibility to Infection

Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain Spotted Fever (RMSF). In Brazil, two species of ticks in the genus Amblyomma, A. sculptum and A. aureolatum, are incriminated as vectors of this bacterium. Importantly, these two species present remarkable differences in susceptibility to R. rickettsii infection, where A. aureolatum is more susceptible than A. sculptum. In the current study, A. aureolatum and A. sculptum ticks were fed on suitable hosts previously inoculated with R. rickettsii, mimicking a natural infection. As control, ticks were fed on non-infected animals. Both midgut and salivary glands of all positively infected ticks were colonized by R. rickettsii. We did not observe ticks with infection restricted to midgut, suggesting that important factors for controlling rickettsial colonization were produced in this organ. In order to identify such factors, the total RNA extracted from the midgut (MG) was submitted to next generation RNA sequencing (RNA-seq). The majority of the coding sequences (CDSs) of A. sculptum differentially expressed by infection were upregulated, whereas most of modulated CDSs of A. aureolatum were downregulated. The functional categories that comprise upregulated CDSs of A. sculptum, for instance, metabolism, signal transduction, protein modification, extracellular matrix, and immunity also include CDSs of A. aureolatum that were downregulated by infection. This is the first study that reports the effects of an experimental infection with the highly virulent R. rickettsii on the gene expression of two natural tick vectors. The distinct transcriptional profiles of MG of A. sculptum and A. aureolatum upon infection stimulus strongly suggest that molecular factors in this organ are responsible for delineating the susceptibility to R. rickettsii. Functional studies to determine the role played by proteins encoded by differentially expressed CDSs in the acquisition of R. rickettsii are warranted and may be considered as targets for the development of strategies to control the tick-borne pathogens as well as to control the tick vectors