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

Mediterranean spotted fever is a reemerging acute tick-borne infection produced by the α-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. conorii-infected 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.

Vector Tick Transmission Model of Spotted Fever Rickettsiosis.

Many aspects of rickettsial infections have been characterized, including pathogenic and immune pathways and mechanisms of rickettsial survival within the vertebrate host and tick vector. However, very few studies are focused on the complex pathogen-vector-host interactions during tick feeding. Therefore, our objective was to develop a tick transmission model of the spotted fever group of rickettsial infections to study the initial events in disease development. The most appropriate strain of mouse was identified for evaluation as a transmission model, and the course of infection, bacterial levels, histopathologic changes, and antibody response during tick transmission in mice infested with Amblyomma maculatum ticks carrying the emerging pathogen, Rickettia parkeri, were studied. Results showed distinct clinical signs in C3H/HeN mice infected intravenously, leading to selection of this mouse strain for tick transmission studies. Active infection of animals was observed after tick vector transmission. The bacteria disseminated systemically and spread to several organs at 24 hours after tick attachment, with peak bacterial load at day 6 after tick attachment. Skin, lung, and liver showed the greatest pathologic changes, with inflammatory cellular infiltration and necrosis. These findings indicate the feasibility of using murine infection with R. parkeri by A. maculatum tick transmission as a model to study different aspects of the spotted fever group of rickettsial disease establishment.

Atg5 Supports Rickettsia australis Infection in Macrophages In Vitro and In Vivo.

Rickettsiae can cause life-threatening infections in humans. Macrophages are one of the initial targets for rickettsiae after inoculation by ticks. However, it remains poorly understood how rickettsiae remain free in macrophages prior to establishing their infection in microvascular endothelial cells. Here, we demonstrated that the concentration of Rickettsia australis was significantly greater in infected tissues of Atg5flox/flox mice than in the counterparts of Atg5flox/flox Lyz-Cre mice, in association with a reduced level of interleukin-1ß (IL-1ß) in serum. The greater concentration of R. australis in Atg5flox/flox bone marrow-derived macrophages (BMMs) than in Atg5flox/flox Lyz-Cre BMMs in vitro was abolished by exogenous treatment with recombinant IL-1ß. Rickettsia australis induced significantly increased levels of light chain 3 (LC3) form II (LC3-II) and LC3 puncta in Atg5-competent BMMs but not in Atg5-deficient BMMs, while no p62 turnover was observed. Further analysis found the colocalization of LC3 with a small portion of R. australis and Rickettsia-containing double-membrane-bound vacuoles in the BMMs of B6 mice. Moreover, treatment with rapamycin significantly increased the concentrations of R. australis in B6 BMMs compared to those in the untreated B6 BMM controls. Taken together, our results demonstrate that Atg5 favors R. australis infection in mouse macrophages in association with a suppressed level of IL-1ß production but not active autophagy flux. These data highlight the contribution of Atg5 in macrophages to the pathogenesis of rickettsial diseases.

MyD88 Mediates Instructive Signaling in Dendritic Cells and Protective Inflammatory Response during Rickettsial Infection.

Spotted fever group rickettsiae cause potentially life-threatening infections throughout the world. Several members of the Toll-like receptor (TLR) family are involved in host response to rickettsiae, and yet the mechanisms by which these TLRs mediate host immunity remain incompletely understood. In the present study, we found that host susceptibility of MyD88(-/-)mice to infection with Rickettsia conorii or Rickettsia australis was significantly greater than in wild-type (WT) mice, in association with severely impaired bacterial clearance in vivo R. australis-infected MyD88(-/-)mice showed significantly lower expression levels of gamma interferon (IFN-γ), interleukin-6 (IL-6), and IL-1ß, accompanied by significantly fewer inflammatory infiltrates of macrophages and neutrophils in infected tissues, than WT mice. The serum levels of IFN-γ, IL-12, IL-6, and granulocyte colony-stimulating factor were significantly reduced, while monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, and RANTES were significantly increased in infected MyD88(-/-)mice compared to WT mice. Strikingly, R. australis infection was incapable of promoting increased expression of MHC-II(high)and production of IL-12p40 in MyD88(-/-)bone marrow-derived dendritic cells (BMDCs) compared to WT BMDCs, although costimulatory molecules were upregulated in both types of BMDCs. Furthermore, the secretion levels of IL-1ß by Rickettsia-infected BMDCs and in the sera of infected mice were significantly reduced in MyD88(-/-)mice compared to WT controls, suggesting that in vitro and in vivo production of IL-1ß is MyD88 dependent. Taken together, our results suggest that MyD88 signaling mediates instructive signals in DCs and secretion of IL-1ß and type 1 immune cytokines, which may account for the protective inflammatory response during rickettsial infection.

Status of research and development of vaccines for chikungunya.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that during the last decade has significantly expanded its geographical range and caused large outbreaks of human disease around the world. Although mortality rates associated with CHIKV outbreaks are low, acute and chronic illnesses caused by CHIKV represent a significant burden of disease largely affecting low and middle income countries. This report summarizes the current status of vaccine development for CHIKV.

Research and development of Zika virus vaccines.

Zika virus (ZIKV) is a member of the family Flaviviridae, genus Flavivirus, and is transmitted by Aedes sp. mosquitoes. There are three genetic lineages of ZIKV: the East African, West African and Asian lineages. Until recently, Zika fever (ZF) has normally been considered a rare, mild febrile disease, but reports since 2012 have shown potentially severe complications associated with ZIKV infection, including microcephaly and Guillain-Barré syndrome. There are no licensed vaccines for ZIKV; however, many vaccine platforms/approaches that have been utilised for other flavivirus vaccines are being applied to ZIKV. Given the current outbreak of ZIKV in the Americas with its associated risks to pregnancy, we summarise what is known about the virus, how knowledge of currently licensed flavivirus vaccines can be applied to ZIKV vaccine development and the assessments of potential challenges for ZIKV vaccine testing and evaluation.

Rickettsia australis Activates Inflammasome in Human and Murine Macrophages.

Rickettsiae actively escape from vacuoles and replicate free in the cytoplasm of host cells, where inflammasomes survey the invading pathogens. In the present study, we investigated the interactions of Rickettsia australis with the inflammasome in both mouse and human macrophages. R. australis induced a significant level of IL-1ß secretion by human macrophages, which was significantly reduced upon treatment with an inhibitor of caspase-1 compared to untreated controls, suggesting caspase-1-dependent inflammasome activation. Rickettsia induced significant secretion of IL-1ß and IL-18 in vitro by infected mouse bone marrow-derived macrophages (BMMs) as early as 8-12 h post infection (p.i.) in a dose-dependent manner. Secretion of these cytokines was accompanied by cleavage of caspase-1 and was completely abrogated in BMMs deficient in caspase-1/caspase-11 or apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), suggesting that R. australis activate the ASC-dependent inflammasome. Interestingly, in response to the same quantity of rickettsiae, NLRP3-/- BMMs significantly reduced the secretion level of IL-1ß compared to wild type (WT) controls, suggesting that NLRP3 inflammasome contributes to cytosolic recognition of R. australis in vitro. Rickettsial load in spleen, but not liver and lung, of R. australis-infected NLRP3-/- mice was significantly greater compared to WT mice. These data suggest that NLRP3 inflammasome plays a role in host control of bacteria in vivo in a tissue-specific manner. Taken together, our data, for the first time, illustrate the activation of ASC-dependent inflammasome by R. australis in macrophages in which NLRP3 is involved.

Rickettsia massiliae and Rickettsia conorii Israeli Spotted Fever Strain Differentially Regulate Endothelial Cell Responses.

Rickettsiae primarily target microvascular endothelial cells. However, it remains elusive how endothelial cell responses to rickettsiae play a role in the pathogenesis of rickettsial diseases. In the present study, we employed two rickettsial species with high sequence homology but differing virulence to investigate the pathological endothelial cell responses. Rickettsia massiliae is a newly documented human pathogen that causes a mild spotted fever rickettsiosis. The "Israeli spotted fever" strain of R. conorii (ISF) causes severe disease with a mortality rate up to 30% in hospitalized patients. At 48 hours post infection (HPI), R. conorii (ISF) induced a significant elevation of IL-8 and IL-6 while R. massiliae induced a statistically significant elevated amount of MCP-1 at both transcriptional and protein synthesis levels. Strikingly, R. conorii (ISF), but not R. massiliae, caused a significant level of cell death or injury in HMEC-1 cells at 72 HPI, demonstrated by live-dead cell staining, annexin V staining and lactate dehydrogenase release. Monolayers of endothelial cells infected with R. conorii (ISF) showed a statistically significant decrease in electrical resistance across the monolayer compared to both R. massiliae-infected and uninfected cells at 72 HPI, suggesting increased endothelial permeability. Interestingly, pharmacological inhibitors of caspase-1 significantly reduced the release of lactate dehydrogenase by R. conorii (ISF)-infected HMEC-1 cells, which suggests the role of caspase-1 in mediating the death of endothelial cells. Taken together, our data illustrated that a distinct proinflammatory cytokine profile and endothelial dysfunction, as evidenced by endothelial cell death/injury and increased permeability, are associated with the severity of rickettsial diseases.