Determination of the tick cells proteome in response to Rickettsia rickettsii infection, the etiologic agent of spotted fever

Author(s)

Larissa Almeida Martins

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2018-11-27

Examining board members

Andréa Cristina Fogaça; Beatriz Simonsen Stolf Carboni; Pedro Lagerblad de Oliveira; Adriana Rios Lopes Rocha

• Advisor: Andréa Cristina Fogaça

Abstract

Rocky Mountain spotted fever, known in Brazil as Brazilian spotted fever (FMB), is the most severe rickettsial disease that affects humas. The disease is caused by Rickettsia rickettsii, an obligate intracellular -proteobacterium transmitted by the bite of different species of ticks. Despite the high rates of FMB lethality, its prophylaxis is based exclusively on the practice of avoiding contact with the vector ticks. In this context, the identification and characterization of the proteins involved in the interactions between R. rickettsii and ticks is important and may help to identify potential targets for disease control. To this end, the use of an in vitro system (cell line) is fundamental because it allows that analyzes can be carried out throughout the infection and with a greater control of the experimental conditions than the in vivo experiments, besides not involving the use of vertebrate animals. Thus, the general objective of this research project was to identify the protein (proteome) of Rhipicephalus microplus tick cells (BME26 line) infected or not by R. rickettsii. Proteins extracted from BME26 cells at a starting point of infection (6 h) and in the exponential phase of bacterial growth (48 h) were processed and analyzed by liquid chromatography coupled to in-tandem mass spectrometry (LC-MS/MS). As a control, proteins extracted from uninfected cells at 6 and 48 h were also processed and analyzed. A total of 1,119 proteins were identified, of which 1,111 corresponded to tick proteins and only eight to bacterial proteins, which were discarded from subsequent analyzes. After 6 h of infection, 163 proteins were upregulated and 159 were downregulated. In 48 h, 95 proteins were upregulated and 65 were downregulated. Among the proteins modulated in response to R. rickettsii infection, apoptosis negative regulatory proteins were downregulated at the 6 h point and upregulated at the 48 h point. Apoptosis is a programmed cell death process, which is activated by different stimuli, including infections. After the stimulus recognition, a number of factors are activated, including the key enzymes of process, called caspases, culminating in cell death. As the arthropod immune system is simpler than the vertebrate immune system, apoptosis is an important mechanism of infection control. Therefore, we evaluated the fragmentation of genomic DNA (DNAg) and the phosphatidylserine exposure on the outer surface of the plasma membrane, two typical characteristics of cells in apoptosis, as well as the activity of caspase 3, in BME26 cells infected or not by R. rickettsii. DNAg fragmentation was observed only in uninfected cells after 96 h. In addition, caspase-3 activity was significantly lower in infected cells than in control cells. In contrast, inhibition of the caspase-3 activity was not observed when cells were stimulated with heat-killed rickettsiae, suggesting that factors produced by R. rickettsii are required for such effect. Phosphedidylserine exposure was also markedly higher in infected cells than in uninfected or in cells stimulated with heat-killed bacteria. Chemical inhibition of apoptosis with Z-DEVD Fmk, a caspase-3 inhibitor, favored the growth of R. rickettsii in BME cells26. On the other hand, staurosporin treatment, a classic activator of apoptosis, inhibited bacterial growth. Taken together, these data show that the process of apoptosis in BME26 cells is controlled by R. rickettsii, which is important for its proliferation. This is the first report on the inhibition of apoptosis in tick cells by bacteria of the genus Rickettsia. Future studies should be carried out to identify the rickettsial proteins, as well as their vector ticks, involved in the control of apoptosis, and may help to better understand the vector-pathogen interaction. (AU)

• FAPESP's process: 14/05855-7 - Determination of the proteome of Rickettsia rickettsii, the etiological agent of Brazilian Spotted Fever
• Grantee: Larissa Almeida Martins
• Support Opportunities: Scholarships in Brazil - Doctorate