Quantitative Proteomics of the Endothelial Secretome Identifies RC0497 as Diagnostic of Acute Rickettsial Spotted Fever Infections

Abstract

Mediterranean spotted fever is a reemerging acute tick-borne infection produced by the a-proteobacterium, Rickettsia conorii. Rickettsia conorii infects vascular endothelial cells producing disseminated plasma leakage, manifesting as nonspecific fever, headache, and maculopapular rash. Because there are no available tests of early infection, Mediterranean spotted fever is often undiagnosed and untreated, resulting in significant mortality. To address this critical need, we have applied a quantitative proteomics pipeline for analyzing the secretome of primary human umbilical vein endothelial cells. Of the 104 proteins whose abundance changed significantly in the R. conoriieinfected human umbilical vein endothelial cells' secretome, 46 proteins were up-regulated: 45 were host secreted proteins (including cytokines), and 1 was a rickettsial protein, the putative N-acetylmuramoyl-L-alanine amidase RC0497. Proteins with sequence highly homologous to RC0497 were found to be shared by many species of the spotted fever group rickettsiae, but not typhus group rickettsiae. Quantitative targeted proteomics studies of plasma from a mouse model of sublethal and lethal R. conorii identified RC0497 in the blood, and its circulating levels were proportionally associated with infection outcome. Finally, the presence of RC0497 in the serum samples from a cohort of humans presenting with acute rickettsioses was confirmed. The detection of RC0497 has the potential to be a sensitive and specific marker for acute rickettsial spotted rickettsioses. (Am J Pathol 2020, 190: 306e322; https://doi.

Comparative transcriptomic analysis of Rickettsia conorii during in vitro infection of human and tick host cells

“…Recently, we have characterized RC0497 as an ampD domain containing N-acteylmuramoyl-L-alanine amidase involved in peptidoglycan hydrolysis and demonstrated its localization on the septal regions in dividing bacteria and on the membranes of vesicles protruding from the rickettsial cell wall [28]. A simultaneous study has further revealed that RC0497 is also secreted into the culture supernatants during infection of endothelial cells in vitro and is readily detectable in the serum of infected patients, projecting it as a promising candidate for the design and development of rapid diagnostics [29]. Additionally, heat shock chaperone (groEL), cold shock protein of CSP family (cspA), CarD like transcriptional regulator, and anti-toxins (vapB and anti-toxin of relE) were ubiquitously expressed in both infected HMECs and tick cells.…”

LPPR5 Expression in Glioma Affects Growth, Vascular Architecture, and Sunitinib Resistance

“…Analysis was carried out with Fiji 1.53c (ImageJ, NIH, Bethesda, MA, USA), where we evaluated the positive or double-positive cells or respective nuclei in the tumor area and normalized the results as positives per standard field of view. Image correlation of the DAPI and LPPR5 signal was performed with an automated CellProfiler 2.2 pipeline (Broad Institute, Boston, FL, USA) [ 54 , 55 ]. In this setting, correlation is defined as the correlation between a pair of images I and J, calculated as Pearson’s correlation coefficient.…”

Endothelial Cells as a Key Cell Type for Innate Immunity: A Focused Review on RIG-I Signaling Pathway

“…Lastly, the use of biotechnological advances such as quantitative proteomic analysis of the endothelial secretome ( 74 ) and single cell RNA-sequencing of infected blood vessels will elucidate differential proteins/targets in ECs subject to bacterial and viral infection.…”