Rickettsia conorii, a member of the spotted fever group (SFG) of the genus Rickettsia and causative agent of Mediterranean spotted fever, is an obligate intracellular pathogen capable of infecting various mammalian cell types. SFG rickettsiae express two major immunodominant surface cell antigen (Sca) proteins, OmpB (Sca5) and OmpA (Sca0). While OmpB‐mediated entry has been characterized, the contribution of OmpA has not been well defined. Here we show OmpA expression in Escherichia coli is sufficient to mediate adherence to and invasion of non‐phagocytic human endothelial cells. A recombinant soluble C‐terminal OmpA protein domain (954–1735) with predicted structural homology to the Bordetella pertussis pertactin protein binds mammalian cells and perturbs R. conorii invasion by interacting with several mammalian proteins including β1 integrin. Using functional blocking antibodies, small interfering RNA transfection, and mouse embryonic fibroblast cell lines, we illustrate the contribution of α2β1 integrin as a mammalian ligand involved in R. conorii invasion of primary endothelial cells. We further demonstrate that OmpA‐mediated attachment to mammalian cells is in part dependent on a conserved non‐continuous RGD motif present in a predicted C‐terminal ‘pertactin’ domain in OmpA.Our results demonstrate that multiple adhesin–receptor pairs are sufficient in mediating efficient bacterial invasion of R. conorii.

Spotted fever group (SFG) rickettsiae are recognized as important agents of human tick-borne diseases worldwide, such as Mediterranean spotted fever (R. conorii) and Rocky Mountain spotted fever (R. rickettsii). Recent studies in several animal models have provided evidence of non-endothelial parasitism by pathogenic SFG Rickettsia species, suggesting that the interaction of rickettsiae with cells other than the endothelium may play an important role in pathogenesis of rickettsial diseases. These studies raise the hypothesis that the role of macrophages in rickettsial pathogenesis may have been underappreciated. Herein, we evaluated the ability of two SFG rickettsial species, R. conorii (a recognized human pathogen) and R. montanensis (a non-virulent member of SFG) to proliferate in THP-1 macrophage-like cells, or within non-phagocytic cell lines. Our results demonstrate that R. conorii was able to survive and proliferate in both phagocytic and epithelial cells in vitro. In contrast, R. montanensis was able to grow in non-phagocytic cells, but was drastically compromised in the ability to proliferate within both undifferentiated and PMA-differentiated THP-1 cells. Interestingly, association assays revealed that R. montanensis was defective in binding to THP-1-derived macrophages; however, the invasion of the bacteria that are able to adhere did not appear to be affected. We have also demonstrated that R. montanensis which entered into THP-1-derived macrophages were rapidly destroyed and partially co-localized with LAMP-2 and cathepsin D, two markers of lysosomal compartments. In contrast, R. conorii was present as intact bacteria and free in the cytoplasm in both cell types. These findings suggest that a phenotypic difference between a non-pathogenic and a pathogenic SFG member lies in their respective ability to proliferate in macrophage-like cells, and may provide an explanation as to why certain SFG rickettsial species are not associated with disease in mammals.

Lipids are a broad group of molecules required for cell maintenance and homeostasis. Various intracellular pathogens have developed mechanisms of modulating and sequestering host lipid processes for a large array of functions for both bacterial and host cell survival. Among the host cell lipid functions that intracellular bacteria exploit for infection are the modulation of host plasma membrane microdomains (lipid rafts) required for efficient bacterial entry; the recruitment of specific lipids for membrane integrity of intracellular vacuoles; and the utilization of host lipid droplets for the regulation of immune responses and for energy production through fatty acid β-oxidation and oxidative phosphorylation. The majority of published studies on the utilization of these host lipid pathways during infection have focused on intracellular bacterial pathogens that reside within a vacuole during infection and, thus, have vastly different requirements for host lipid metabolites when compared to those intracellular pathogens that are released into the host cytosol upon infection. Here we summarize the mechanisms by which intracellular bacteria sequester host lipid species and compare the modulation of host lipid pathways and metabolites during host cell infection by intracellular pathogens residing in either a vacuole or within the cytosol of infected mammalian cells. This review will also highlight common and unique host pathways necessary for intracellular bacterial growth that could potentially be targeted for therapeutic intervention.

Bacteria of the genus Rickettsia are transmitted from arthropod vectors and primarily infect cells of the mammalian endothelial system. Throughout this infectious cycle, the bacteria are exposed to the deleterious effects of serum complement. Using Rickettsia conorii, the etiologic agent of Mediterranean spotted fever (MSF), as a model rickettsial species, we have previously demonstrated that this class of pathogen interacts with human factor H to mediate partial survival in human serum. Herein, we demonstrate that R. conorii also interacts with the terminal complement complex inhibitor vitronectin (Vn). We further demonstrate that an evolutionarily conserved rickettsial antigen, Adr1/RC1281, interacts with human vitronectin and is sufficient to mediate resistance to serum killing when expressed at the outer-membrane of serum sensitive Escherichia coli. Adr1 is an integral outer-membrane protein whose structure is predicted to contain eight membrane-embedded β-strands and four ‘loop’ regions that are exposed to extracellular milieu. Site-directed mutagenesis of Adr1 revealed that at least two predicted ‘loop’ regions are required to mediate resistance to complement-mediatedkilling and vitronectin acquisition. These results demonstrate that rickettsial species have evolved multiple mechanisms to evade complement deposition and that evasion of killing in serum is an evolutionarily conserved virulence attribute for this genus of obligate intracellular pathogens.

We have previously reported that Rickettsia conorii and Rickettsia montanensis have distinct intracellular fates within THP-1 macrophages, suggesting that the ability to proliferate within macrophages may be a distinguishable factor between pathogenic and non-pathogenic Spotted fever group (SFG) members. To start unraveling the molecular mechanisms underlying the capacity (or not) of SFG Rickettsia to establish their replicative niche in macrophages, we have herein used quantitative proteomics by SWATH-MS to profile the alterations resulted by the challenge of THP-1 macrophages with R. conorii and R. montanensis. We show that the pathogenic, R. conorii, and the non-pathogenic, R. montanensis, member of SFG Rickettsia trigger differential proteomic signatures in macrophage-like cells upon infection. R. conorii specifically induced the accumulation of several enzymes of the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid β-oxidation, and glutaminolysis, as well as of several inner and outer membrane mitochondrial transporters. These results suggest a profound metabolic rewriting of macrophages by R. conorii toward a metabolic signature of an M2-like, anti-inflammatory activation program. Moreover, several subunits forming the proteasome and immunoproteasome are found in lower abundance upon infection with both rickettsial species, which may help bacteria to escape immune surveillance. R. conorii-infection specifically induced the accumulation of several host proteins implicated in protein processing and quality control in ER, suggesting that this pathogenic Rickettsia may be able to increase the ER protein folding capacity. This work reveals novel aspects of macrophage-Rickettsia interactions, expanding our knowledge of how pathogenic rickettsiae explore host cells to their advantage.

Despite their high degree of genomic similarity, different spotted fever group (SFG) Rickettsia are often associated with very different clinical presentations. For example, Rickettsia conorii causes Mediterranean spotted fever, a life-threatening disease for humans, whereas Rickettsia montanensis is associated with limited or no pathogenicity to humans. However, the molecular basis responsible for the different pathogenicity attributes are still not understood. Although killing microbes is a critical function of macrophages, the ability to survive and/or proliferate within phagocytic cells seems to be a phenotypic feature of several intracellular pathogens. We have previously shown that R. conorii and R. montanensis exhibit different intracellular fates within macrophage-like cells. By evaluating early macrophage responses upon insult with each of these rickettsial species, herein we demonstrate that infection with R. conorii results in a profound reprogramming of host gene expression profiles. Transcriptional programs generated upon infection with this pathogenic bacteria point toward a sophisticated ability to evade innate immune signals, by modulating the expression of several anti-inflammatory molecules. Moreover, R. conorii induce the expression of several pro-survival genes, which may result in the ability to prolong host cell survival, thus protecting its replicative niche. Remarkably, R. conorii-infection promoted a robust modulation of different transcription factors, suggesting that an early manipulation of the host gene expression machinery may be key to R. conorii proliferation in THP-1 macrophages. This work provides new insights into the early molecular processes hijacked by a pathogenic SFG Rickettsia to establish a replicative niche in macrophages, opening several avenues of research in host-rickettsiae interactions.