The Retropepsin-Type Protease APRc as a Novel Ig-Binding Protein and Moonlighting Immune Evasion Factor of Rickettsia

Many Rickettsia organisms are pathogenic to humans, causing severe infections, like Rocky Mountain spotted fever and Mediterranean spotted fever. However, immune evasion mechanisms and pathogenicity determinants in rickettsiae are far from being resolved.

Determination of NK cell immunophenotype and cytokine profile in patients with Rocky Mountain spotted fever

The intracellular pathogen Rickettsia rickettsii causes tick-borne spotted fever (also called Rocky Mountain spotted fever (RMSF) and is increasingly recognized as an emerging cause of febrile illness in Mexico. However, little is known about the early immune responses to infection. Four RMSF pediatric patients on acute phase and eight healthy controls from Chihuahua, Mexico were recruited. The natural killer cell (NK) immunophenotype and the cytokine profile in peripheral blood were characterized by flow cytometry. The population of cytotoxic NK cells expressing NKG2D was significantly decreased in patients on 3rd day of hospitalization compared to the first sampling on admission. Interleukins IL-6, IL-8, and IL-10 levels were significantly increased in patients upon admission compared to controls. This study shows that circulating NK cells are numerically decreased, while cytokines induce a pro-inflammatory process in patients.

672. Diagnosis of Rocky Mountain Spotted Fever Using Plasma Metagenomic Next-Generation Sequencing

Background Rocky mountain spotted fever (RMSF), caused by Rickettsia rickettsii, incurs significant morbidity and mortality, especially in children. Early in the course of illness, standard diagnostic tests are of limited sensitivity, and diagnosis is often based on clinical symptoms and local epidemiology. The diagnosis can be missed in areas where RMSF is not endemic, and a delay in initiation of therapy may lead to poor clinical outcomes. Plasma metagenomic next-generation sequencing (mNGS), with turnaround times approaching 48 hours, may be a useful adjunctive tool in the diagnosis of RMSF. Methods We describe four children hospitalized with RMSF between January 1, 2017 to May 15, 2021 at a tertiary children’s hospital in southern California. All had plasma mNGS and rickettsial serologic testing as part of clinical care. Results mNGS detected Rickettsia rickettsii in all 4 patients. Only 2 subjects had positive serologic testing initially and required repeat testing in the convalescent stage to confirm RMSF. The mean turnaround time for mNGS was 2.75 days, which was comparable to serologic testing. Antibiotic therapy was changed in three subjects as a result of the plasma mNGS result. Conclusion Plasma mNGS may be a useful diagnostic modality early in the disease course of RMSF. Disclosures Lauge Farnaes, MD, PhD, Cardea Bio (Advisor or Review Panel member)IDbyDNA (Employee)

265. Rocky Mountain Spotted Fever Encephalopathy

Background Rocky mountain spotted fever (RMSF) is a rickettsial disease with incidence of 11 per million and is rarely associated with encephalopathy. Here we discuss a patient with RMSF encephalopathy, highlighting the natural course. Methods A 54 year old man with history of hypertension and chronic progressive external ophthalmoplegia, presented with waxing and waning confusion, headache, slurred speech, agitation and difficulty swallowing. He was afebrile and hemodynamically stable. Investigations showed leukocytosis of 15400 and mild transaminitis. Computed-tomography (CT) head was unremarkable. Lumbar puncture revealed normal pressure. Cerebrosopinal fluid (CSF) analysis was notable for WBC 7, glucose 76 and moderately elevated total protein 114. Urine drug screen was negative. Blood, fungal, and CSF cultures were sent and empiric therapy with vancomycin, ceftriaxone, ampicillin and acyclovir commenced, for suspected encephalitis. High dose solumedrol 1gm/day was given due to suspicion of autoimmune encephalitis. MRI brain showed cerebral atrophy. There was slight abnormal FLAIR/T2 signal within the medial aspect of the temporal lobes, right more than left. Results Occupational history revealed that he was a logger by profession, which steered our focus on tick borne diseases. Extensive serologic evaluation was requested and RMSF IgG titres came back positive at 1:512. Doxycycline was added, while ampicillin and ceftriaxone were discontinued. With doxycycline, patient made remarkable recovery and was discharged home well. However, he returned within 48 hours with recurring encephalopathy. His clinical presentation remained convincing for RMSF encephalitis, with the natural course of the illness spanning over weeks, with waxing and waning symptoms. Patient was managed with IV doxycycline for 72 hours following which he returned to his baseline mental status. Figure 1. MRI findings Figure 2. Serological investigations Figure 3. CSF studies Conclusion Patient’s occupation played a pivotal role in establishing diagnosis. In RMSF, IgM and IgG antibodies appear 7 to 10 days after the onset of the illness, and a fourfold rise in IgG is diagnostic of seroconversion and recent illness. Patient’s waxing and waning symptoms, persisting for weeks and remarkable response to doxycycline, are typical features of RMSF encephalitis. Disclosures All Authors: No reported disclosures

Pathogenic, but not non-pathogenic, Rickettsia manipulate inflammasome-dependent IL-1 responses to facilitate their replication and host dissemination

Rickettsia species (spp.) are strict obligate intracellular bacteria, symbiotic in their arthropod vector, and some being pathogenic in their mammalian host, including humans. One critical feature of these stealthy group of pathogens is their ability to manipulate hostile cytosolic environments to their benefits. Although our understanding of Rickettsia cell biology and pathogenesis are evolving, the mechanisms of host innate immune defense evasion by pathogenic Rickettsia spp. remains to be elucidated. Here, we showed that disease severity in wild-type (WT) C57BL/6J mice infected with R. typhi- (etiologic agent of murine typhus) and R. rickettsii (etiologic agent of Rocky Mountain Spotted Fever), but not with non-pathogenic R. montanensis, correlated with levels of bacterial burden as detected in the spleens, as well as the serum concentrations of pro-inflammatory cytokine IL-1α and to a lesser extent IL-1β. Antibody-mediated neutralization of IL-1α confirmed a key role in controlling mortality rates and bacterial burdens of rickettsiae-infected WT mice. As macrophages are a primary source of both IL-1α and IL-1β cytokines, we determined the mechanism of the anti-rickettsial activities using bone-marrow-derived macrophages. We found that pathogenic R. typhi and R. rickettsii, but not non-pathogenic R. montanensis, induced autophagy, and avoided autophagolysosomal destruction, while simultaneously eluded pro-IL-1α induction and benefited from the dampening of IL-1α secretion, via Caspase-11-Gsdmd-dependent mechanism, to facilitate intracytosolic replication. Adoptative transfer experiments identified that IL-1α secretion by macrophages was critical for controlling rickettsiosis in WT mice. In sum, we identified a previously unappreciated pathway by which pathogenic, unlike non-pathogenic, rickettsiae preferentially target the Caspase-11-Gsdmd-IL-1α signaling axis in macrophages, possibly via an autophagy-dependent mechanism, to support their replication and dissemination within the host.Significance StatementCurrently, no vaccines are available to prevent rickettsioses, and tick- and flea-borne rickettsial infections in humans are on the rise globally. In fact, the insufficient understanding of how pathogenic Rickettsia species circumvent host immune defense mechanisms has significantly hindered the development of more effective therapeutics. Here, we identified a previously unappreciated role for the Caspase-11-Gsdmd-IL-1α signaling axis, to limiting the survival and dissemination of pathogenic R. rickettsia and R. typhi species in macrophages and wild-type C57BL/6J mice. Adoptative transfer studies further identified IL-1α-secreting macrophages as critical mediators in controlling rickettsial infection in WT mice. Collectively, these findings provide insight into the potential mechanism of how pathogenic, but not non-pathogenic Rickettsia spp., become ubiquitinated, induce autophagy and benefit from the dampening of the Caspase-11-Gsdmd-mediated release of IL-1α to support host colonization.

Nitric Oxide Inhibition of Rickettsia rickettsii

Rickettsia rickettsii , the causative agent of Rocky Mountain spotted fever, is an enzootic, obligate intracellular bacterial pathogen. Nitric oxide (NO) synthesized by the inducible nitric oxide synthase (iNOS) is a potent antimicrobial component of innate immunity and has been implicated in the control of virulent Rickettsia spp. in diverse cell types. In this study, we examined the antibacterial role of NO on R. rickettsii . Our results indicate that NO challenge dramatically reduces R. rickettsii adhesion through the disruption of bacterial energetics. Additionally, NO-treated R. rickettsii were unable to synthesize protein or replicate in permissive cells. Activated, NO-producing macrophages restricted R. rickettsii infections, but inhibition of iNOS ablated the inhibition of bacterial growth. These data indicate that NO is a potent anti-rickettsial effector of innate immunity that targets energy generation in these pathogenic bacteria to prevent growth and subversion of infected host cells.