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ABSTRACT Bacterial infection is a highly complex biological process involving a dynamic interaction between the invading microorganism and the host. Specifically, intracellular pathogens seize control over the host cellular processes including membrane dynamics, actin cytoskeleton, phosphoinositide metabolism, intracellular trafficking and immune defense mechanisms to promote their host colonization. To accomplish such challenging tasks, virulent bacteria deploy unique species-specific secreted effectors to evade and/or subvert cellular defense surveillance mechanisms to establish a replication niche. However, despite superficially similar infection strategies, diverse Rickettsia species utilize different effector repertoires to promote host colonization. This review will discuss our current understandings on how different Rickettsia species deploy their effector arsenal to manipulate host cellular processes to promote their intracytosolic life within the mammalian host. Rickettsiae deploy their effector arsenal to manipulate membrane dynamics, actin cytoskeleton, phosphoinositide metabolism, intracellular trafficking and immune defense mechanisms; to gain access, and promote their intracytosolic lifespan to ultimately expedite transmission.

Ticks and other arthropods often are hosts to nutrient providing bacterial endosymbionts, which contribute to their host's fitness by supplying nutrients such as vitamins and amino acids. It has been detected, in our lab, that Ixodes pacificus is host to Rickettsia species phylotype G021. This endosymbiont is predominantly present, and 100% maternally transmitted in I. pacificus. To study roles of phylotype G021 in I. pacificus, bioinformatic and molecular approaches were carried out. MUMmer genome alignments of whole genome sequence of I. scapularis, a close relative to I. pacificus, against completely sequenced genomes of R. bellii OSU85-389, R. conorii, and R. felis, identified 8,190 unique sequences that are homologous to Rickettsia sequences in the NCBI Trace Archive. MetaCyc metabolic reconstructions revealed that all folate gene orthologues (folA, folC, folE, folKP, ptpS) required for de novo folate biosynthesis are present in the genome of Rickettsia buchneri in I. scapularis. To examine the metabolic capability of phylotype G021 in I. pacificus, genes of the folate biosynthesis pathway of the bacterium were PCR amplified using degenerate primers. BLAST searches identified that nucleotide sequences of the folA, folC, folE, folKP, and ptpS genes possess 98.6%, 98.8%, 98.9%, 98.5% and 99.0% identity respectively to the corresponding genes of Rickettsia buchneri. Phylogenetic tree constructions show that the folate genes of phylotype G021 and homologous genes from various Rickettsia species are monophyletic. This study has shown that all folate genes exist in the genome of Rickettsia species phylotype G021 and that this bacterium has the genetic capability for de novo folate synthesis.

ABSTRACT The cat flea, Ctenocephalides felis, is an arthropod vector capable of transmitting several human pathogens including Rickettsia species. Earlier studies identified Rickettsia felis in the salivary glands of the cat flea and transmission of rickettsiae during arthropod feeding. The saliva of hematophagous insects contains multiple biomolecules with anticlotting, vasodilatory and immunomodulatory activities. Notably, the exact role of salivary factors in the molecular interaction between flea-borne rickettsiae and their insect host is still largely unknown. To determine if R. felis modulates gene expression in the cat flea salivary glands, cat fleas were infected with R. felis and transcription patterns of selected salivary gland-derived factors, including antimicrobial peptides and flea-specific antigens, were assessed. Salivary glands were microdissected from infected and control cat fleas at different time points after exposure and total RNA was extracted and subjected to reverse-transcriptase quantitative PCR for gene expression analysis. During the experimental 10-day feeding period, a dynamic change in gene expression of immunity-related transcripts and salivary antigens between the two experimental groups was detected. The data indicated that defensin-2 (Cf-726), glycine-rich antimicrobial peptide (Cf-83), salivary antigens (Cf-169 and Cf-65) and deorphanized peptide (Cf-75) are flea-derived factors responsive to rickettsial infection. This study demonstrated gene expression patterns of immunity-related and flea-specific transcripts in the salivary glands of the cat flea in response to Rickettsia felis infection.

Rickettsia are obligate intracellular bacteria often associated with ticks and best known for causing human diseases (rickettsiosis), including typhus fever and sporadic cases of serious infection. In this study, we conducted a large survey of ticks in French Guiana to understand the overall diversity of Rickettsia in this remote area largely covered by dense rainforests. Out of 819 individuals (22 tick species in six genera), 252 (30.8%) samples were positive for Rickettsia infection. Multilocus typing and phylogenetic analysis identified 19 Rickettsia genotypes, but none was 100% identical to already known Rickettsia species or strains. Among these 19 genotypes, we identified two validated Rickettsia species, Rickettsia amblyommatis (spotted fever group) and Rickettsia bellii (bellii group), and characterized a novel and divergent Rickettsia phylogenetic group, the guiana group. While some tick hosts of these Rickettsia genotypes are among the most common ticks to bite humans in French Guiana, their potential pathogenicity remains entirely unknown. However, we found a strong association between Rickettsia genotypes and their host tick species, suggesting that most of these Rickettsia genotypes may be nonpathogenic forms maintained through transovarial transmission.

Rickettsia species are an important cause of emerging infectious diseases in people and animals, and rickettsiosis is one of the oldest known vector-borne diseases. Laboratory diagnosis of Rickettsia is complex and time-consuming. This study was aimed at developing two quantitative real-time PCRs targeting ompB and ompA genes for the detection, respectively, of Rickettsia spp. and R. conorii DNA. Primers were designed following an analysis of Rickettsia gene sequences. The assays were optimized using SYBR Green and TaqMan methods and tested for sensitivity and specificity. This study allowed the development of powerful diagnostic methods, able to detect and quantify Rickettsia spp. DNA and differentiate R. conorii species.

New human rickettsial pathogens have been discovered, and long-known rickettsiae of undetermined pathogenicity have been demonstrated to cause illness. Disease associated with Rickettsia slovaca has unique clinical manifestations, including prominent lymphadenopathy without fever and rash. Rickettsial genomes are highly conserved, with reductive evolution leading to a small genome that relies on the host cell for many biosynthetic functions. Advances in the evaluation of the pathogenesis of rickettsial disease include identification of rickettsial adhesins, a host cell receptor, signaling elements associated with entry of rickettsiae by induced phagocytosis, rickettsial enzymes mediating phagosomal escape, and host actin-based rickettsial cell-to-cell spread. Disruption of adherens junctions of infected endothelial cells likely plays a role in the critical pathophysiologic mechanism: increased microvascular permeability. Production of reactive oxygen species by infected endothelium injures these cells. However, disseminated intravascular coagulation rarely occurs. Immunity is mediated by reactive cytokine-activated rickettsicidal nitrogen and oxygen species and by clearance of rickettsiae by cytotoxic CD8 T cells.

Bacterial Sec7-domain-containing proteins (RalF) are known only from species of Legionella and Rickettsia, which have facultative and obligate intracellular lifestyles, respectively. L. pneumophila RalF, a type IV secretion system (T4SS) effector, is a guanine nucleotide exchange factor (GEF) of ADP-ribosylation factors (Arfs), activating and recruiting host Arf1 to the Legionella-containing vacuole. In contrast, previous in vitro studies showed R. prowazekii (Typhus Group) RalF is a functional Arf-GEF that localizes to the host plasma membrane and interacts with the actin cytoskeleton via a unique C-terminal domain. As RalF is differentially encoded across Rickettsia species (e.g., pseudogenized in all Spotted Fever Group species), it may function in lineage-specific biology and pathogenicity. Herein, we demonstrate RalF of R. typhi (Typhus Group) interacts with the Rickettsia T4SS coupling protein (RvhD4) via its proximal C-terminal sequence. RalF is expressed early during infection, with its inactivation via antibody blocking significantly reducing R. typhi host cell invasion. For R. typhi and R. felis (Transitional Group), RalF ectopic expression revealed subcellular localization with the host plasma membrane and actin cytoskeleton. Remarkably, R. bellii (Ancestral Group) RalF showed perinuclear localization reminiscent of ectopically expressed Legionella RalF, for which it shares several structural features. For R. typhi, RalF co-localization with Arf6 and PI(4,5)P2 at entry foci on the host plasma membrane was determined to be critical for invasion. Thus, we propose recruitment of PI(4,5)P2 at entry foci, mediated by RalF activation of Arf6, initiates actin remodeling and ultimately facilitates bacterial invasion. Collectively, our characterization of RalF as an invasin suggests that, despite carrying a similar Arf-GEF unknown from other bacteria, different intracellular lifestyles across Rickettsia and Legionella species have driven divergent roles for RalF during infection. Furthermore, our identification of lineage-specific Arf-GEF utilization across some rickettsial species illustrates different pathogenicity factors that define diverse agents of rickettsial diseases.

The recently sequenced Rickettsia felis genome revealed an unexpected plasmid carrying several genes usually associated with DNA transfer, suggesting that ancestral rickettsiae might have been endowed with a conjugation apparatus. Here we present the genome sequence of Rickettsia bellii, the earliest diverging species of known rickettsiae. The 1,552,076 base pair-long chromosome does not exhibit the colinearity observed between other rickettsia genomes, and encodes a complete set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25, an obligate symbiont of amoebae. The genome exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. We sought and observed sex pili-like cell surface appendages for R. bellii. We also found that R. bellii very efficiently multiplies in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic \"melting pot\" where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to the intracellular lifestyle within eukaryotic cells.

Intracerebral microhemorrhages (CMHs) are small foci of hemorrhages in the cerebrum. Acute infections induced by some intracellular pathogens, including rickettsia, can result in CMHs. Annexin a2 (ANXA2) has been documented to play a functional role during intracellular bacterial adhesion. Here we report that ANXA2-knockout (KO) mice are more susceptible to CMHs in response to rickettsia and Ebola virus infections, suggesting an essential role of ANXA2 in protecting vascular integrity during these intracellular pathogen infections. Proteomic analysis via mass spectrometry of whole brain lysates and brain-derived endosomes from ANXA2-KO and wild-type (WT) mice post-infection with R. australis revealed that a variety of significant proteins were differentially expressed, and the follow-up function enrichment analysis had identified several relevant cell-cell junction functions. Immunohistology study confirmed that both infected WT and infected ANXA2-KO mice were subjected to adherens junctional protein (VE-cadherin) damages. However, key blood-brain barrier (BBB) components, tight junctional proteins ZO-1 and occludin, were disorganized in the brains from R. australis-infected ANXA2-KO mice, but not those of infected WT mice. Similar ANXA2-KO dependent CMHs and fragments of ZO-1 and occludin were also observed in Ebola virus-infected ANXA2-KO mice, but not found in infected WT mice. Overall, our study revealed a novel role of ANXA2 in the formation of CMHs during R. australis and Ebola virus infections; and the underlying mechanism is relevant to the role of ANXA2-regulated tight junctions and its role in stabilizing the BBB in these deadly infections.

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.