Elena Rydkina

Rickettsia raoultii sp. nov., a spotted fever group rickettsia associated with Dermacentor ticks in Europe and Russia

We describe the characterization of a novel Rickettsia species cultivated from Dermacentor ticks collected in Russia and France, for which we propose the name Rickettsia raoultii sp. nov. Using multigene sequencing, we demonstrated that five rickettsial isolates from Dermacentor silvarum, Dermacentor reticulatus, Dermacentor marginatus and Dermacentor nuttalli ticks were classified within this novel spotted fever rickettsia species. This rickettsia also exhibited a serotype distinct from previously described Rickettsia species. The type strain of Rickettsia raoultii sp. nov. is strain Khabarovsk(T) (=CSUR R3(T) =ATCC VR-1596(T)).

The geographical segregation of human lice preceded that of Pediculus humanus capitis and Pediculus humanus humanus

In order to investigate human-louse phylogeny, we partially sequenced two nuclear (18S rRNA and EF-1 alpha) and one mitochondrial (COI) genes from 155 Pediculus from different geographical origins. The phylogenetic analysis of 18S rRNA and EF-1 alpha sequences showed that human lice were classified into lice from Sub-Saharan Africa and lice from other areas. In both clusters, head and body lice were clearly grouped into two separate clusters. Our results indicate that the earliest divergence within human pediculidae occurred between African lice and other lice, and the divergence between head and body lice was not the result from a single event.

Rickettsia rickettsii Infection of Cultured Human Endothelial Cells Induces Heme Oxygenase 1 Expression

ABSTRACT Existing evidence suggests that oxidative insults and antioxidant defense mechanisms play a critical role in the host cell response during infection of endothelial cells by Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever. Heme oxygenase (HO), a rate-limiting enzyme in the pathway for heme catabolism, protects against oxidant damage in a variety of stress situations. Here, we report on the expression of the inducible and constitutive HO isozymes, HO-1 and HO-2, during R. rickettsii infection of endothelial cells. Steady-state levels for HO-1 mRNA were increased two- to threefold, as early as 4 h postinfection, whereas HO-2 mRNA was not affected. Induction of HO-1 mRNA was dependent on the dose of infection and occurred in a time-dependent manner, reaching maximal levels at 4 to 7 h. The increase in HO-1 mRNA occurred at the level of trancription as it was blocked by the transcriptional inhibitors, actinomycin D and α-amanitin. The eukaryotic protein synthesis inhibitor, cycloheximide, caused a >50% reduction in the infection-induced increase in HO-1 mRNA level, suggesting its dependence on de novo protein synthesis of host cell. The uptake of viable organisms appeared to be necessary, since inactivation of R. rickettsii by heat or formalin fixation, or incubation of cells with cytochalasin B to prevent entry resulted in marked inhibition of HO-1 response. N-Acetyl-l-cysteine, a known oxidant scavenger, inhibited the HO-1 induction by R. rickettsii. Finally, Western analysis with a specific monoclonal antibody revealed higher levels of HO-1 protein (∼32 kDa), confirming that changes in HO-1 mRNA levels were followed by increases in the levels of protein. The findings indicate that R. rickettsii infection induces HO-1 expression in host endothelial cells and suggest an important role for this enzyme in cellular response to infection, possibly by serving a protective function against oxidative injury.

Infection of Human Endothelial Cells with Spotted Fever Group Rickettsiae Stimulates Cyclooxygenase 2 Expression and Release of Vasoactive Prostaglandins

ABSTRACT 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-PGF1α and PGE2. 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-κB Activation during Rickettsia rickettsii Infection of Endothelial Cells Involves the Activation of Catalytic IκB Kinases IKKα and IKKβ and Phosphorylation-Proteolysis of the Inhibitor Protein IκBα

ABSTRACT Rocky Mountain spotted fever, a systemic tick-borne illness caused by the obligate intracellular bacterium Rickettsia rickettsii, is associated with widespread infection of the vascular endothelium. R. rickettsii infection induces a biphasic pattern of the nuclear factor-κB (NF-κB) activation in cultured human endothelial cells (ECs), characterized by an early transient phase at 3 h and a late sustained phase evident at 18 to 24 h. To elucidate the underlying mechanisms, we investigated the expression of NF-κB subunits, p65 and p50, and IκB proteins, IκBα and IκBβ. The transcript and protein levels of p50, p65, and IκBβ remained relatively unchanged during the course of infection, but Ser-32 phosphorylation of IκBα at 3 h was significantly increased over the basal level in uninfected cells concomitant with a significant increase in the expression of IκBα mRNA. The level of IκBα mRNA gradually returned toward baseline, whereas that of total IκBα protein remained lower than the corresponding controls. The activities of IKKα and IKKβ, the catalytic subunits of IκB kinase (IKK) complex, as measured by in vitro kinase assays with immunoprecipitates from uninfected and R. rickettsii-infected ECs, revealed significant increases at 2 h after infection. The activation of IKK and early phase of NF-κB response were inhibited by heat treatment and completely abolished by formalin fixation of rickettsiae. The IKK inhibitors parthenolide and aspirin blocked the activities of infection-induced IKKα and IKKβ, leading to attenuation of nuclear translocation of NF-κB. Also, increased activity of IKKα was evident later during the infection, coinciding with the late phase of NF-κB activation. Thus, activation of catalytic components of the IKK complex represents an important upstream signaling event in the pathway for R. rickettsii-induced NF-κB activation. Since NF-κB is a critical regulator of inflammatory genes and prevents host cell death during infection via antiapoptotic functions, selective inhibition of IKK may provide a potential target for enhanced clearance of rickettsiae and an effective strategy to reduce inflammatory damage to the host during rickettsial infections.

Activation of p38 stress‐activated protein kinase during Rickettsia rickettsii infection of human endothelial cells: role in the induction of chemokine response

Rickettsia rickettsii, a Gram‐negative and obligate intracellular bacterium, preferentially infects the vascular endothelium during human infections leading to inflammation and dysfunction. The aim of this study was to determine whether R. rickettsii infection of endothelial cells (EC) activates p38 and/or c‐jun N‐terminal kinases (JNK) mitogen‐activated protein (MAP) kinase, key regulatory proteins that control the response to inflammatory stimuli. We show that infection of cultured human EC results in the dose‐dependent activation of p38, as assessed by increased phosphorylation and activity, without affecting the status of JNK. Rickettsia inactivation by heat or formaldehyde abolished the activation of p38 kinase and inhibition of cellular invasion by infection at low temperature, pre‐treatment of host EC with cytochalasin D, or pre‐incubation of rickettsiae with an irreversible phospholipase inhibitor led to a diminished p38 phosphorylation, suggesting requirement of invasion by viable rickettsiae for this host cell response. SB 203580, a p38‐specific inhibitor, had no effect on infection‐induced activation of the ubiquitous transcriptional regulator nuclear factor‐kappa B, but effectively reduced the expression and secretion of important chemoattractant cytokines interleukin (IL)‐8 and monocyte chemoattractant protein (MCP)‐1 by R. rickettsii‐infected EC. Selective inhibition of p38 activity may be exploited as an anti‐inflammatory target to prevent rickettsial vasculitis and to develop new and improved chemotherapeutic agents.

Rickettsia rickettsii Infection of Human Macrovascular and Microvascular Endothelial Cells Reveals Activation of Both Common and Cell Type-Specific Host Response Mechanisms

ABSTRACT Although inflammation and altered barrier functions of the vasculature, due predominantly to the infection of endothelial cell lining of small and medium-sized blood vessels, represent salient pathological features of human rickettsioses, the interactions between pathogenic rickettsiae and microvascular endothelial cells remain poorly understood. We have investigated the activation of nuclear transcription factor-kappa B (NF-κB) and p38 mitogen-activated protein (MAP) kinase, expression of heme oxygenase 1 (HO-1) and cyclooxygenase 2 (COX-2), and secretion of chemokines and prostaglandins after Rickettsia rickettsii infection of human cerebral, dermal, and pulmonary microvascular endothelial cells in comparison with pulmonary artery cells of macrovascular origin. NF-κB and p38 kinase activation and increased HO-1 mRNA expression were clearly evident in all cell types, along with relatively similar susceptibility to R. rickettsii infection in vitro but considerable variations in the intensities/kinetics of the aforementioned host responses. As expected, the overall activation profiles of macrovascular endothelial cells derived from human pulmonary artery and umbilical vein were nearly identical. Interestingly, cerebral endothelial cells displayed a marked refractoriness in chemokine production and secretion, while all other cell types secreted various levels of interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in response to infection. A unique feature of all microvascular endothelial cells was the lack of induced COX-2 expression and resultant inability to secrete prostaglandin E2 after R. rickettsii infection. Comparative evaluation thus yields the first experimental evidence for the activation of both common and unique cell type-specific host response mechanisms in macrovascular and microvascular endothelial cells infected with R. rickettsii, a prototypical species known to cause Rocky Mountain spotted fever in humans.

Interactions of Rickettsia rickettsii with Endothelial Nuclear Factor‐κB in a “Cell‐Free” System

Vascular endothelial cells (EC) are the primary targets of in vivo infection with Rickettsia rickettsii , an obligate intracellular, gram-negative bacterial organism, and the etiological agent for Rocky Mountain spotted fever. Although infection of cultured human endothelial cells with R. rickettsii culminates in extensive membrane damage and necrotic death, 1 it has been established that the endothelial cell is not simply injured by infection, but undergoes a series of responses including functional changes characteristic of an “activated” phenotype. 2 In brief, the endothelial cells display procoagulant and proinflammatory properties during the course of infection, which likely contribute to the manifestation of disease symptoms. Nuclear factor-kappaB (NFκ B) is a dimeric transcription factor composed of homoand heterodimers of the Rel family of proteins (Rel A or p65, Rel B, c-Rel, NFκ B1 or p50, and NFκ B2 or p52). It is now known that NFκ B is present in the cytoplasm of virtually all eukaryotic cells as an inactive complex, bound to one of the members of I κ B (inhibitors of NFκ B) proteins. Stimulus-induced signaling cascades lead to the proteolysis of I κ B and release of active NFκ B, which translocates to the nucleus, binds to κ B enhancer sequences via the DNA-binding domain, and regulates the transcription of specific genes. 3 Our laboratory has shown that NFκ B governs important regulatory functions during R. rickettsii infection of cultured human EC, resulting in reprogramming of gene expression and suppression of apoptotic host cell death. 4,5

Rickettsia rickettsii Infection Protects Human Microvascular Endothelial Cells against Staurosporine-Induced Apoptosis by a cIAP2-Independent Mechanism

BACKGROUND Manipulation of host cell death is an important determinant of the outcome of an infection. Here, we investigate whether Rickettsia rickettsii-infected host endothelial cells resist the effects of staurosporine, a potent inducer of apoptosis, and we explore the mechanisms underlying the anti-apoptotic effect of infection. METHODS Human microvascular endothelial cells infected with R. rickettsii for 24 or 48 h were challenged with staurosporine. The extent of apoptosis was evaluated with flow cytometry. mRNA and protein expression levels were determined by use of microarray or polymerase chain reaction and immunoblotting, respectively. RESULTS Staurosporine-induced apoptosis in endothelial cells infected for 24 and 48 h was significantly reduced, compared with simultaneously treated uninfected cells. A microarray of human genes involved in apoptosis and polymerase chain reaction analyses revealed increased steady-state mRNA expression of cIAP2 (a member of the inhibitor-of-apoptosis family of proteins) at 24 h after infection. The levels of cIAP2 protein (+/-SD) in infected cells were 3.5 +/- 1.7 -fold and 2.3 +/- 1.2 -fold higher than that in uninfected control cells at 24 and 48 h after infection. Nucleofection of human-specific cIAP2-targeted siRNA resulted in inhibition of protein expression by > or = 50% but had no effect on infection-induced protection against apoptosis. CONCLUSIONS R. rickettsii-induced expression of cIAP2 in host endothelial cells is likely not a major contributor to protection against staurosporine-induced cell death.

Activation of p38 mitogen-activated protein kinase module facilitates in vitro host cell invasion by Rickettsia rickettsii.

Interactions with target membrane molecules and the resultant internalization into host cells are, in general, the first and foremost steps for subsequent host responses and pathogenesis of infectious micro-organisms. For pathogenic bacteria belonging to both spotted fever and typhus subgroups of Rickettsia species, which are known for dependence on the nutrientrich environment of the host cytoplasm and fastidious growth requirements, target cell invasion is absolutely critical for subsequent intracellular replication and intercellular spread. Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, primarily affects the endothelial cell lining of smalland medium-sized vessels leading to disseminated intracellular infection of the vasculature and, consequently, characteristic pathological features during human infections correspond to vascular inflammation, damage and dysfunction (Walker, 2007).