Isolate-Dependent Differences in Clinical, Pathological, and Transcriptional Profiles following In Vitro and In Vivo Infections with Rickettsia rickettsii.

Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever (RMSF), a life-threatening tick-borne disease that affects humans and various animal species, has been recognized in medicine and science for more than 100 years. Isolate-dependent differences in virulence of R. rickettsii have been documented for many decades; nonetheless, the specific genetic and phenotypic factors responsible for these differences have not been characterized. Using in vivo and in vitro methods, we identified multiple phenotypic differences among six geographically distinct isolates of R. rickettsii, representing isolates from the United States, Costa Rica, and Brazil. Aggregate phenotypic data, derived from growth in Vero E6 cells and from clinical and pathological characteristics following infection of male guinea pigs (Cavia porcellus), allowed separation of these isolates into three categories: nonvirulent (Iowa), mildly virulent (Sawtooth and Gila), and highly virulent (Sheila SmithT, Costa Rica, and Taiaçu). Transcriptional profiles of 11 recognized or putative virulence factors confirmed the isolate-dependent differences between mildly and highly virulent isolates. These data corroborate previous qualitative assessments of strain virulence and suggest further that a critical and previously underappreciated balance between bacterial growth and host immune response could leverage strain pathogenicity. Also, this work provides insight into isolate-specific microbiological factors that contribute to the outcome of RMSF and confirms the hypothesis that distinct rickettsial isolates also differ phenotypically, which could influence the severity of disease in vertebrate hosts.

Experimental Infection of Amblyomma aureolatum Ticks with Rickettsia rickettsii

We experimentally infected Amblyomma aureolatumticks with the bacterium Rickettsia rickettsii, the etiologicagent of Rocky Mountain spotted fever (RMSF). These ticksare a vector for RMSF in Brazil. R. rickettsii was effi cientlyconserved by both transstadial maintenance and vertical(transovarial) transmission to 100% of the ticks through4 laboratory generations. However, lower reproductive performance and survival of infected females was attributedto R. rickettsii infection. Therefore, because of the highsusceptibility of A. aureolatum ticks to R. rickettsii infection,the deleterious effect that the bacterium causes in theseticks may contribute to the low infection rates (<1%) usuallyreported among fi eld populations of A. aureolatum ticksin RMSF-endemic areas of Brazil. Because the numberof infected ticks would gradually decrease after eachgeneration, it seems unlikely that A. aureolatum ticks couldsustain R. rickettsii infection over multiple successivegenerations solely by vertical transmission...

Plasmids and rickettsial evolution: insight from Rickettsia felis.

BACKGROUND: The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG) or spotted fever group (SFG) rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF) that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG) rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives) also occur in AG (but not SFG) rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFdelta, is an artifact of the original genome assembly. CONCLUSION/SIGNIFICANCE: Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG) rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in the acquirement of virulence traits in pathogenic strains, and the likely origin of plasmids within the rickettsial tree.

Infection of human endothelial cells with spotted Fever group rickettsiae stimulates cyclooxygenase 2 expression and release of vasoactive prostaglandins.

Rickettsiae, a diverse group of obligately intracellular gram-negative bacteria, include etiologic agents of the spotted fever and typhus groups of diseases. Rocky Mountain spotted fever and boutonneuse fever, due to Rickettsia rickettsii and R. conorii, respectively, are characterized by widespread infection of the vascular endothelium, microvascular injury, and vasculitis. Cultured human endothelial cells (EC) are highly susceptible to infection and respond by altering the expression of adhesion molecules, regulatory cytokines, and the antioxidant enzyme heme oxygenase (HO). In the vasculature, HO regulates the cyclooxygenase (COX) enzymes, among which the inducible isozyme COX-2 facilitates the synthesis of prostaglandins (PGs). Using in vitro and ex vivo models of infection, we demonstrate here that R. rickettsii infection of human EC causes robust induction of COX-2 mRNA and protein expression but has no apparent effect on the constitutive COX-1 isoform. Cells infected with viable rickettsiae consistently displayed significantly increased secretion of 6-keto-PGF(1alpha) and PGE(2). R. rickettsii-induced COX-2 was sensitive to inhibitors of de novo transcription and the pyridinylimidazole-based compound SB 203580, suggesting that this transcriptional host cell response involves signaling through p38 mitogen-activated protein kinase. PG production by infected cells was abrogated by NS 398 (a selective COX-2 inhibitor) and indomethacin (a pan-COX inhibitor). Immunohistochemical staining of sections of infected umbilical cords and corresponding uninfected controls revealed comparatively more intense and abundant staining for COX-2 in infected endothelia. Induction of the endothelial COX-2 system and the resultant enhanced release of vasoactive PGs may contribute to the regulation of inflammatory responses and vascular permeability changes during spotted fever rickettsioses.

NF-kappaB activation suppresses host cell apoptosis during Rickettsia rickettsii infection via regulatory effects on intracellular localization or levels of apoptogenic and anti-apoptotic proteins.

Rickettsia rickettsii, a gram-negative and obligate intracellular bacterium, is the causative agent of Rocky Mountain spotted fever. In human infections, the primary target of R. rickettsii infection is vascular endothelium. Our laboratory has shown that activation of nuclear transcription factor-kappa B (NF-kappaB) during R. rickettsii infection of cultured human endothelial cells protects against apoptosis by preventing the activation of apical caspases-8 and -9, and the effector caspase-3. To understand upstream signaling mechanisms, we have determined the effect of NF-kappaB blockade on the status of different Bcl-2 (B-cell lymphoma 2) proteins in this study. Quantitative analysis following TUNEL and Hoechst staining confirmed that infection of endothelial cells with R. rickettsii for 6 h in the presence of a specific NF-kappaB inhibitor, MG132, resulted in induction of apoptosis. Infection-induced apoptosis of EC was associated with decreased level of Bid and accumulation of Bad, while cytosolic level of Bax remained relatively unchanged. Further, the cellular levels of apoptosis antagonist Bcl-2 were found to be down-regulated and apoptogenic mitochondrial proteins Smac and cytochrome c were released into cytoplasm. These results implicate an important regulatory role for NF-kappaB in controlling the intracellular levels and/or localization of pro- as well as anti-apoptotic proteins of Bcl-2 family, the intricate balance of which is a critical determinant of downstream signaling mechanisms governing cell fate during intracellular infection.

Interleukin-1 alpha production during Rickettsia rickettsii infection of cultured endothelial cells: potential role in autocrine cell stimulation.

Rickettsia rickettsii infection results in numerous responses by cultured endothelial cells, among them a rapid, transient increase in steady-state levels of tissue factor mRNA (L.A. Sporn, P.J. Haidaris, R.-J. Shi, Y. Nemerson, D.J. Silverman, and V.J. Marder, Blood 83:1527-1534, 1994). In this study, production of interleukin-1 (IL-1) was measured during infection and its potential role in autocrine cell stimulation was investigated. A fivefold increase in levels of IL-1 alpha antigen was measured in cell lysate samples by enzyme-linked immunosorbent assay at 18 h of infection. The majority of IL-1 alpha remained cell associated, as no significant increase was detected in culture medium. No IL-1 beta antigen was detected in cell lysates or culture medium from either control or infected cultures. A dramatic increase in the levels of IL-1 alpha mRNA occurred following infection, as measured by reverse transcriptase PCR, which revealed the appearance of the expected 421-kb product with RNA extracted from cells infected for 4 h and no detectable product from control cell samples. The presence of functional, cell-associated IL-1 alpha activity in infected cells was confirmed, following disruption, by the ability of the infected cells to induce tissue factor expression in target endothelial cells. Such induction was eliminated by pretreatment of the disrupted cell samples with neutralizing antibodies against IL-1 alpha but not against IL-1 beta. To investigate whether endogenously produced IL-1 participates in the stimulation of tissue factor expression, neutralizing antibodies against IL-1 or the IL-1 receptor antagonist were added to culture medium during infection. Both anti-IL-1 alpha and the IL-1 receptor antagonist resulted in approximately 40% inhibition of tissue factor expression, thus implicating IL-1 alpha in autocrine cell stimulation.

Rocky mountain spotted fever in childhood.

Experience with 138 cases of Rocky Mountain spotted fever indicates that the major clinical features of characteristic rash, fever, and tick bite, in combination with low serum sodium concentration and thrombocytopenia, are helpful in recognizing this serious and potentially lethal infectious disease.