Rocky mountain spotted fever is tick-borne infection. In endemic areas, the prevalence of the diseases is 15 per 100,000 persons. The disease is characterized by sudden onset of fever and headache. It is caused by gram-negative intracellular bacteria Rickettsia rickettsii. The bacteria have a high predisposition for the endothelial cell. Actin protein polymerization in the cells is important for the spread of bacteria from one cell to another. (Raoult & Parola, 2007)
The bacteria cause lymphohistiocytic vasculitis and necrosis of the infected cells. It typically takes 5-7 days from the day of tick bite to the onset of symptoms. Rashes can be seen in the wrist, ankle, palms, and soles. There is no preventive medicine for the disease. Severe cases are treated using 7 days' course of Doxycycline. Chloramphenicol is used in some patients. Immunofluorescent antibody assay is the standard diagnostic test. There is a fourfold increase in antibody titer from the acute phase to convalesce phase of the disease. Identifying rickettsia organism in blood or autopsy is also of diagnostic value. (Raoult & Parola, 2007)
Rickettsia ricketsii is some non-motile cocci. The bacterial genome codes for 680 proteins and the function of many of these proteins are unknown. Some of these proteins contribute to bacterial virulence. While most strains of Rickettsia ricketsii are susceptible to Doxycycline and Chloramphenicol, the lack of a convenient testing protocol has restricted the evaluation of the sensitivity of bacteria to other drugs. The protein that contributes to drug resistance in the bacteria is also not known. (Dantas-Torres, 2007)
Hypothesis: We hypothesis that the presence of a virulence factor is contributing to drug resistance in the bacteria. By using techniques that will identify the differential expression of the gene in the virulent and non-virulent strain of Rickettsia rickettssi, the genes that contribute to the virulence of the strain can be identified.
Method: The virulent and non-virulent strain will be inoculated separated in different guinea pigs (Ammerman, Beier-Sexton, & Azad, 2008). The endothelial samples from the guinea pig containing the rickettsii will be taken. Total RNA will be isolated and the RNA will be used for cDNA synthesis using a cDNA universal primer. cDNA subtraction will be done using the Clontech's Suppression Subtractive Hybridization (SSH) kit. The cDNA library from each sample will be cloned into a InsTAclone T-vector. The vector will be then inserted into E. coli (OmniMAX 2 T1 Phage-Resistant, Invitrogen). The vector has ampicillin resistance gene and the ampicillin selection media will be used to isolate colonies and individual bacteria. The bacteria that are selected by the ampicillin selective media contain plasmids. After inoculation into LB broth, plasmids will be isolated from each colony and individual plasmid will be subjected to EST sequencing. After removing the vector sequence from the data obtained, the ESTs will be submitted to EST database and a contig analysis will be performed. The Blast X analysis will reveal the differentially expressed transcripts. The transcripts will then be identified and their function will be explored using RNA-Seq data set. (MacFarlane & Singh, 2006)
Result: The differential expressed EST will be categorized based on their function using Blast2Go RNA-Seq data set. Expression of genes related to multidrug-resistant can be identified through this technique.
References
Ammerman, N., Beier-Sexton, M., & Azad, A. (2008). Laboratory Maintenance of Rickettsia rickettsii.Curr Protoc Microbiol., Nov, Unit–3A.5.
Dantas-Torres, F. (2007). Rocky Mountain spotted fever, 7(11), 724-732.
Hong Zhang,, Z., Jhaveri, D., Marshall, V., & Bauer, D. (2014). A Comparative Study of Techniques for Differential Expression Analysis on RNA-Seq Data. Plos One, 9(8), e103207.
MacFarlane, R. & Singh, U. (2006). Identification of Differentially Expressed Genes in Virulent and Nonvirulent Entamoeba Species: Potential Implications for Amebic Pathogenesis. Infect. Immun,74(1), 340-351.
Raoult, D. & Parola, P. (2007). Rickettsial diseases (pp. 96-110). New York: Informa Healthcare.
