SUMMARY Coxiella burnetii is a gram-negative intracellular bacterium that forms a large, lysosome-like parasitophorous vacuole (PV) essential for bacterial replication. Host membrane lipids are critical for the formation and maintenance of this intracellular niche, yet the mechanisms by which Coxiella manipulates host cell lipid metabolism, trafficking, and signaling are unknown. ORP1L (oxysterol-binding protein (OSBP)-related protein 1, long) is a mammalian lipid-binding protein that plays a dual role in cholesterol-dependent endocytic trafficking as well as interactions between endosomes and the endoplasmic reticulum (ER). We found that ORP1L localized to the Coxiella PV within 12 hours of infection through a process requiring the Coxiella Dot/Icm Type 4B secretion system, which secretes effector proteins into the host cell cytoplasm where they manipulate trafficking and signaling pathways. The ORP1L N-terminal ankyrin repeats were necessary and sufficient for PV localization, indicating ORP1L binds a PV membrane protein. Strikingly, ORP1L simultaneously co-localized with the PV and ER, and electron microscopy revealed membrane contact sites between the PV and ER membranes. In ORP1L-depleted cells, PVs were significantly smaller than PVs from control cells. These data suggest ORP1L is specifically recruited by the bacteria to the Coxiella PV, where it influences PV membrane dynamics and interactions with the ER.

Cholesterol is a multifunctional lipid that plays important metabolic and structural roles in the eukaryotic cell. Despite having diverse lifestyles, the obligate intracellular bacterial pathogens Chlamydia, Coxiella, Anaplasma, Ehrlichia, and Rickettsia all target cholesterol during host cell colonization as a potential source of membrane, as well as a means to manipulate host cell signaling and trafficking. To promote host cell entry, these pathogens utilize cholesterol-rich microdomains known as lipid rafts, which serve as organizational and functional platforms for host signaling pathways involved in phagocytosis. Once a pathogen gains entrance to the intracellular space, it can manipulate host cholesterol trafficking pathways to access nutrient-rich vesicles or acquire membrane components for the bacteria or bacteria-containing vacuole. To acquire cholesterol, these pathogens specifically target host cholesterol metabolism, uptake, efflux, and storage. In this review, we examine the strategies obligate intracellular bacterial pathogens employ to manipulate cholesterol during host cell colonization. Understanding how obligate intracellular pathogens target and use host cholesterol provides critical insight into the host-pathogen relationship.

is an obligate intracellular bacterium and the etiological agent of Q fever. Successful host cell infection requires the type IVB secretion system (T4BSS), which translocates bacterial effector proteins across the vacuole membrane into the host cytoplasm, where they manipulate a variety of cell processes. To identify host cell targets of T4BSS effector proteins, we determined the transcriptome of murine alveolar macrophages infected with a T4BSS effector mutant. We identified a set of inflammatory genes that are significantly upregulated in T4BSS mutant-infected cells compared to mock-infected cells or cells infected with wild-type (WT) bacteria, suggesting that T4BSS effector proteins downregulate the expression of these genes. In addition, the interleukin-17 (IL-17) signaling pathway was identified as one of the top pathways affected by the bacteria. While previous studies demonstrated that IL-17 plays a protective role against several pathogens, the role of IL-17 during infection is unknown. We found that IL-17 kills intracellular in a dose-dependent manner, with the T4BSS mutant exhibiting significantly more sensitivity to IL-17 than WT bacteria. In addition, quantitative PCR confirmed the increased expression of IL-17 downstream signaling genes in T4BSS mutant-infected cells compared to WT- or mock-infected cells, including the proinflammatory cytokine genes ,, and , the chemokine genes and , and the antimicrobial protein gene We further confirmed that the T4BSS downregulates macrophage CXCL2/macrophage inflammatory protein 2 and CCL5/RANTES protein levels following IL-17 stimulation. Together, these data suggest that downregulates IL-17 signaling in a T4BSS-dependent manner in order to escape the macrophage immune response.

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Introduction: Patients with hematologic malignancies, including multiple myeloma (MM), experience worse SARS-CoV-2 infection outcomes and sub-optimal vaccine responses. Monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) precede MM and affect ~5% of individuals age >=50. We previously showed that individuals with MGUS and SMM exhibit immune dysregulation. Here, we investigate the immune response to SARS-CoV-2 vaccination in these asymptomatic but potentially immunocompromised individuals. Methods: The IMPACT study (IRB #20-332) is a prospective study at Dana-Farber Cancer Institute in collaboration with MMRF, which enrolled individuals nationwide with a diagnosed plasma cell dyscrasia and healthy individuals. As of October 2021, 3,005 individuals completed a questionnaire regarding prior infection or vaccination. We obtained 1,350 blood samples from 628 participants and analyzed anti-SARS-CoV-2 IgG antibody titer by ELISA. Results: 2,771 (92%) participants were fully vaccinated (2 doses BNT162b2 or mRNA-1273; 1 dose Ad26.COV2.s), 269 (9%) had received a 3rd mRNA vaccine dose, and 234 (8%) were unvaccinated. 1,387 (46%) and 1,093 (36%) participants received mRNA vaccines (BNT162b2 and mRNA-1273), and 139 (5%) participants received an adenovirus vector vaccine (Ad26.COV2.S). 34 (1%) individuals reported SARS-CoV-2 infection after full vaccination. We measured antibody titers in 201 MGUS, 223 SMM, 40 smoldering Waldenstrom macroglobulinemia (SWM), 64 MM, and 100 healthy controls. Multiple linear regression model estimated the association between various clinical variables and post-vaccination antibody titers. As previously reported, having MM was associated with low antibody titer (p < 0.001). Of note, having SMM, regardless of risk stratification by 2/20/20 criteria, was also associated with low antibody titers, indicating that even low-risk SMM patients have a poor response to vaccination. MGUS and SWM diagnoses were not significantly associated with antibody titers. Additionally, male sex (p < 0.010), elapsed time after vaccination (p < 0.001), and BNT162b2 vaccine (p < 0.001) were associated with low antibody titers. SARS-CoV-2 infection prior to vaccination was associated with high antibody titers. We identified 25 patients (6 MGUS, 10 SMM, 2 SWM, 7 MM) who submitted blood samples after both the 2nd and 3rd dose. In these patients we observed a significant increase in antibody titer after a 3rd dose (p = 0.002). We also observed that antibody titers of patients after a 3rd dose (13 MGUS, 12 SMM, 2 SWM, 31 MM) were comparable to that of healthy individuals after a 2nd dose (p = 0.833). Conclusion: Our data indicates that suboptimal response to SARS-CoV-2 does not only occur with MM and cancer patients receiving therapy but also in precursor asymptomatic patients including low-risk SMM. Citation Format: Yoshinobu Konishi, Romanos Sklavenitis-Pistofidis, Hong Yue, Federico Ferrari, Massimiliano Russo, Robert A. Redd, Jacqueline Perry, Erica Horowitz, Anna V. Justis, Nader A. Shayegh, Elizabeth D. Lightbody, Shohreh Varmeh, Radoslaw P. Nowak, Mark Hamilton, Daniel Auclair, Catherine R. Marinac, Lorenzo Trippa, Eric S. Fischer, Irene M. Ghobrial. Humoral SARS-CoV-2 response in asymptomatic precursor plasma cell dyscrasia patients: IMPACT study results [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5277.