Slowly burning down the house: the role of calcium in spotted fever rickettsiosis pathogenesis

ABSTRACT Spotted fever group rickettsiae (SFGR) infect microvascular endothelial cells, increasing vascular permeability, a major pathophysiological effect with unclear underlying factors. To accurately model this disease feature, we used a microvascular barrier model that continuously monitors transendothelial electrical resistivity in brain microvascular endothelial cell barriers. Using this model, we previously showed that Rickettsia parkeri -infected microvascular endothelial cells treated with voltage-gated L-, T-, and N-type calcium channel blocker (VGCCb) benidipine delayed in vitro vascular permeability. This study aimed to investigate whether host calcium signaling modulation and metabolic stress with SFGR infection are primary drivers of rickettsial fitness and the subsequent loss of vascular integrity. We hypothesize that SFGR infections, while consuming host cell energy resources such as ATP, lead to modulation of microvascular endothelial cell calcium transport that enhances bacterial fitness, prefacing collateral barrier dysfunction. We confirmed that benidipine, the L-type VGCCb nifedipine, and the membrane-active divalent ion chelator DP-b99 have dose-dependent effects on vascular permeability, as measured by electric cell-substrate impedance sensing, which result in delayed rickettsial growth, as measured by qPCR, without direct bactericidal activity. During R. parkeri infection, intracellular calcium concentrations increased and ATP concentrations decreased simultaneously; these effects were mitigated by benidipine. VGCCb and DP-b99 treatment sustained VE-cadherin junctions, seen with immunofluorescence microscopy, similar to uninfected microvascular endothelial cells, corresponding to barrier integrity preservation. In addition to delayed rickettsial growth, VGCCbs and DP-b99 inhibited rickettsial actin tail formation, reduced cell-to-cell spread, and increased bacterial length by impacting FtsZ-ring localization, evaluated by immunofluorescence microscopy and plaque assay. Furthermore, bacterial protein synthesis is required, as replicating but not heat-killed R. parkeri caused microvascular endothelial cell barrier dysfunction, and a subinhibitory doxycycline dose delayed R. parkeri -induced microvascular endothelial cell dysfunction without impacting R. parkeri growth. Limiting calcium access disrupts R. parkeri growth by interrupting bacterial elongation and division, actin tail formation, and cell-to-cell spread, mitigating R. parkeri -induced microvascular endothelial cell barrier permeability in vitro . This suggests that rickettsial interference with host cell metabolism triggers calcium entry and accessibility, key in rickettsial fitness, and pathogenicity. IMPORTANCE This work investigated pathogen- and host-associated factors contributing to SFGR disease pathogenesis, as measured by vascular permeability. Our findings demonstrate that SFGR disrupts endothelial cell homeostasis by causing intracellular calcium influx, a process that is critical for rickettsial fitness and pathogenesis. Rickettsial growth and cell-to-cell spread are dependent on host cell calcium, underscoring the essential role of Ca² + access and linking bacterial fitness to calcium-dependent host processes. Although blocking calcium channels suppresses immune cell activation in vitro and increases mortality in an SFGR murine model in vivo , these findings warrant further investigation into whether selectively targeting endothelial cell-specific calcium signaling could reduce SFGR-associated vascular permeability without compromising innate immune responses. Such an approach may improve SFGR-related morbidity and mortality. Elucidating the role of calcium signaling in the obligate intracellular lifestyle of rickettsiae provides deeper insight into fundamental rickettsial biology and the mechanisms driving severe disease.