Differential Proteomic Analysis of Rickettsia prowazekii Propagated in Diverse Host Backgrounds

Abstract

The obligate intracellular growth of Rickettsia prowazekii places severe restrictions on the analysis of rickettsial gene expression. With a small genome, predicted to code for 835 proteins, identifying which proteins are differentially expressed in rickettsiae that are isolated from different hosts or that vary in virulence is critical to an understanding of rickettsial pathogenicity. We employed a liquid chromatography (LC)-linear trap quadrupole (LTQ)-Orbitrap mass spectrometer for simultaneous acquisition of quantitative mass spectrometry (MS)-only data and tandem mass spectrometry (MS-MS) sequence data. With the use of a combination of commercially available algorithms and in-house software, quantitative MS-only data and comprehensive peptide coverage generated from MS-MS were integrated, resulting in the assignment of peptide identities with intensity values, allowing for the differential comparison of complex protein samples. With the use of these protocols, it was possible to directly compare protein abundance and analyze changes in the total proteome profile of R. prowazekii grown in different host backgrounds. Total protein extracted from rickettsiae grown in murine, tick, and insect cell lines or hen egg yolk sacs was analyzed. Here, we report the fold changes, including an upregulation of shock-related proteins, in rickettsiae cultivated in tissue culture compared to the level for rickettsiae harvested from hen yolk sacs. The ability to directly compare, in a complex sample, differential rickettsial protein expression provides a snapshot of host-specific proteomic profiles that will help to identify proteins important in intracellular growth and virulence.Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracytoplasmic bacterium with a genome annotated to encode 835 proteins (6, 12). The small rickettsial genome reflects the evolution of specialized transporters that exploit complex metabolic intermediates found in the host cell cytoplasm (7,8,29,32,35), as well as a reductive evolutionary process resulting in the loss of biosynthetic pathway components (3,5,6). This reductive evolution has resulted in a pseudogene-riddled genome with a high proportion of noncoding DNA (4). Characterizing rickettsial protein expression under various conditions and environments is critical to our understanding of how R. prowazekii, with its relatively small protein repertoire, exploits the intracellular niche in host cells as diverse as those of the louse vector and the human host.Several groups have evaluated rickettsial protein expression using both gel-based and mass spectrometry (MS)-based methods (9,10,19,24,26,27). Although gel-based methods offer certain advantages, the use of mass spectrometry offers the opportunity to evaluate comprehensive differential protein expression in complex samples. Differential analysis of proteomes has been performed using methods that employ protein labeling (20), isotope coded affinity tagging (ICAT) (14), and isobaric tags for relative and absolute ...

Rickettsial genomics and the paradigm of genome reduction associated with increased virulence

“…Proteomic profile comparison of R. prowazekii grown in different cell lines, revealed an upregulation of stress-related proteins in L929 murine fibroblasts [63]. In addition, proteins involved in protein synthesis, especially enoyl-(acyl carrier protein) reductase, a protein involved in fatty acid biosynthesis, were highly expressed when grown in I. scapularis ISE6 cells, suggesting that this rickettsia has the ability to regulate differentially its proteome according to the host [63]. Using transcriptomic and proteomic analyses of virulent and avirulent R. prowazekii strains, we identified four phenotypes that differed in virulence depending on the regulation of anti-apoptotic genes or the interferon I pathway in host cells [53].…”

“…A proteomic analysis of Rickettsia parkeri revealed that 91 proteins, including mostly virulence-related surface proteins (OmpA, OmpB, b-peptide, RickA), were differentially expressed during human infection [62]. Proteomic profile comparison of R. prowazekii grown in different cell lines, revealed an upregulation of stress-related proteins in L929 murine fibroblasts [63]. In addition, proteins involved in protein synthesis, especially enoyl-(acyl carrier protein) reductase, a protein involved in fatty acid biosynthesis, were highly expressed when grown in I. scapularis ISE6 cells, suggesting that this rickettsia has the ability to regulate differentially its proteome according to the host [63].…”

An Ixodes scapularis cell line with a predominantly neuron-like phenotype

“…ISE6 cells have been successfully used to isolate into culture many tick-associated bacteria including members of the genera Rickettsia (Simser et al 2002; Pornwiroon et al 2006; Baldridge et al 2010), Anaplasma (Munderloh et al 1996a; Munderloh et al 1996b; Woldehiwet et al 2002; Munderloh et al 2003; Tate et al 2013), Ehrlichia (Munderloh et al 2009), Borrelia (Varela et al 2004), and Neoehrlichia (Munderloh et al 2007). Ixodes scapularis -derived cells have also been used to study vector-pathogen interactions, such as the expression of variant proteins of several Anaplasmataceae members in response to the tick environment (Nelson et al 2008; Kuriakose et al 2011; Chávez et al 2012), and differential expression of Rickettsia prowazekii genes during growth in mammalian and arthropod cells (Tucker et al 2011). Arboviruses and eukaryotes such as Babesia have also been propagated and studied in I. scapularis cells (Lawrie et al 2004; Garcia et al 2005; Ribeiro et al 2009).…”

Recent Molecular Insights into Rickettsial Pathogenesis and Immunity

“…A recent study comparing the proteome of R. prowazekii grown in different cell lines found differential expression of its proteins, and the most striking differences were observed in universal stress protein UspA-like nucleotide binding protein and deoxyuridine 5′-triphosphate nucleotidohydrolase, which are highly expressed when grown in murine fibroblast L929 cell line. In addition, enoyl-(acyl carrier protein) reductase, a protein involved in fatty acid biosynthesis, was highly expressed when grown in I. scapularis ISE6 cell line, indicating that Rickettsia have the inherent ability to differentially regulate its proteome depending on the host [169]. …”