The Lone Star tick, Amblyomma americanum, is endemic to the southeastern United States and capable of transmitting pathogenic diseases and causing non-pathogenic conditions. To remain firmly attached to the host, the tick secretes a proteinaceous matrix termed the cement cone which hardens around the tick’s mouthparts to assist in the attachment of the tick as well as to protect the mouthparts from the host immune system. Cement cones collected from ticks on a host are commonly contaminated with host skin and hair making analysis of the cone difficult. To reduce the contamination found in the cement cone, we have adapted an artificial membrane feeding system used to feed long mouthpart ticks. Cones collected from in vivo and membrane fed ticks are analyzed to determine changes in the cone morphology. Comparisons of the cement cones using light microscopy shows similar structures and color however using scanning electron microscopy the cones have drastically different structures. The in vivo cones contain fibrils, sheets, and are heavily textured whereas cones from membrane fed ticks are remarkably smooth with no distinct structures. Analysis of the secondary protein structures using FTIR-ATR show both in vivo and membrane fed cement cones contain β sheets but only in vivo cement cones contain helical protein structures. Additionally, proteomic analysis using LC–MS/MS identifies many proteins including glycine rich proteins, metalloproteases, and protease inhibitors. Proteomic analysis of the cones identified both secreted and non-secreted tick proteins. Artificial membrane feeding is a suitable model for increased collection of cement cones for proteomic analysis however, structurally there are significant differences.

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.

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.

Rickettsia are significant sources of tick-borne diseases in humans worldwide. In North America, two species in the spotted fever group of Rickettsia have been conclusively associated with disease of humans: Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, and Rickettsia parkeri, the cause of R. parkeri rickettsiosis. Previous work in our lab demonstrated non-endothelial parasitism by another pathogenic SFG Rickettsia species, Rickettsia conorii, within THP-1-derived macrophages, and we have hypothesized that this growth characteristic may be an underappreciated aspect of rickettsial pathogenesis in mammalian hosts. In this work, we demonstrated that multiple other recognized human pathogenic species of Rickettsia, including R. rickettsii, R. parkeri, Rickettsia africae, and Rickettsiaakari can grow within target endothelial cells as well as within PMA-differentiated THP-1 cells. In contrast, Rickettsia bellii, a Rickettsia species not associated with disease of humans, and R. rickettsii strain Iowa, an avirulent derivative of pathogenic R. rickettsii, could invade both cell types but proliferate only within endothelial cells. Further analysis revealed that similar to previous studies on R. conorii, other recognized pathogenic Rickettsia species could grow within the cytosol of THP-1-derived macrophages and avoided localization with two different markers of lysosomal compartments; LAMP-2 and cathepsin D. R. bellii, on the other hand, demonstrated significant co-localization with lysosomal compartments. Collectively, these findings suggest that the ability of pathogenic rickettsial species to establish a niche within macrophage-like cells could be an important factor in their ability to cause disease in mammals. These findings also suggest that analysis of growth within mammalian phagocytic cells may be useful to predict the pathogenic potential of newly isolated and identified Rickettsia species.

Rickettsia conorii is a Gram-negative, cytosolic intracellular bacterium that has classically been investigated in terms of endothelial cell infection. However, R. conorii and other human pathogenic Rickettsia species have evolved mechanisms to grow in various cell types, including macrophages, during mammalian infection. During infection of these phagocytes, R. conorii shifts the host cell's overall metabolism towards an antiinflammatory M2 response, metabolically defined by an increase in host lipid metabolism and oxidative phosphorylation. Lipid metabolism has more recently been identified as a key regulator of host homeostasis through modulation of immune signalling and metabolism. Intracellular pathogens have adapted mechanisms of hijacking host metabolic pathways including host lipid catabolic pathways for various functions required for growth and survival. In the present study, we hypothesised that alterations of host lipid droplets initiated by lipid catabolic pathways during R. conorii infection is important for bacterial survival in macrophages. Herein, we determined that host lipid droplet modulation is initiated early during R. conorii infection, and these alterations rely on active bacteria and lipid catabolic pathways. We also find that these lipid catabolic pathways are essential for efficient bacterial survival. Unlike the mechanisms used by other intracellular pathogens, the catabolism of lipid droplets induced by R. conorii infection is independent of upstream host peroxisome proliferator-activated receptor-alpha (PPARα) signalling. Inhibition of PPARɣ signalling and lipid droplet accumulation in host cells cause a significant decrease in R. conorii survival suggesting a negative correlation with lipid droplet production and R. conorii survival. Together, these results strongly suggest that the modulation of lipid droplets in macrophage cells infected by R. conorii is an important and underappreciated aspect of the infection process.