Central to Q fever pathogenesis is replication of the causative agent, Coxiella burnetii, within a phagolysosome-like parasitophorous vacuole (PV) in mononuclear phagocytes. C. burnetii modulates PV biogenesis and other host cell functions, such as apoptotic signaling, presumably via the activity of proteins delivered to the host cytosol by a Dot/Icm type IVB secretion system (T4BSS). In this study, we utilized a C. burnetii strain carrying IcmD inactivated by the Himar1 transposon to investigate the requirements for Dot/Icm function in C. burnetii parasitism of human THP-1 macrophage-like cells. The icmD::Tn mutant failed to secrete characterized T4BSS substrates, a defect that correlated with deficient replication, PV development, and apoptosis protection. Restoration of type IVB secretion and intracellular growth of the icmD::Tn mutant required complementation with icmD, -J, and -B, indicating a polar effect of the transposon insertion on downstream dot/icm genes. Induction of icmDJB expression at 1 day postinfection resulted in C. burnetii replication and PV generation. Collectively, these data prove that T4BSS function is required for productive infection of human macrophages by C. burnetii. However, illustrating the metabolic flexibility of C. burnetti, the icmD::Tn mutant could replicate intracellularly when sequestered in a PV generated by wild-type bacteria, where Dot/Icm function is provided in trans, and within a phenotypically similar PV generated by the protozoan parasite Leishmania amazonensis, where host cells are devoid of Dot/Icm T4BSS effector proteins.
We recently described acidified citrate cysteine medium (ACCM), which supports host cell-free (axenic) growth of Coxiella burnetii. After 6 days of incubation, greater than 3 logs of growth was achieved with the avirulent Nine Mile phase II (NMII) strain. Here, we describe modified ACCM and culture conditions that support improved growth of C. burnetii and their use in genetic transformation and pathogen isolation from tissue samples. ACCM was modified by replacing fetal bovine serum with methyl--cyclodextrin to generate ACCM-2. Cultivation of NMII in ACCM-2 with moderate shaking and in 2.5% oxygen yielded 4 to 5 logs of growth over 7 days. Similar growth was achieved with the virulent Nine Mile phase I and G isolates of C. burnetii. Colonies that developed after 6 days of growth in ACCM-2 agarose were approximately 0.5 mm in diameter, roughly 5-fold larger than those formed in ACCM agarose. By electron microscopy, colonies consisted primarily of the C. burnetii small cell variant morphological form. NMII was successfully cultured in ACCM-2 when medium was inoculated with as little as 10 genome equivalents contained in tissue homogenates from infected SCID mice. A completely axenic C. burnetii genetic transformation system was developed using ACCM-2 that allowed isolation of transformants in about 2 1/2 weeks. Transformation experiments demonstrated clonal populations in colonies and a transformation frequency of approximately 5 ؋ 10 ؊5 . Cultivation in ACCM-2 will accelerate development of C. burnetii genetic tools and provide a sensitive means of primary isolation of the pathogen from Q fever patients.Coxiella burnetii is a wide-ranging zoonotic pathogen that causes a debilitating influenza-like illness in humans called Q fever (20). Following infection of a eukaryotic host cell, this intracellular bacterium replicates exclusively within a phagolysosome-like parasitophorous vacuole (PV) (16). C. burnetii directs development of the PV (15, 24) and manipulates other host cell functions such as apoptotic signaling (18,29). The mechanisms by which C. burnetii modifies the host cell and causes disease are largely unknown. Indeed, lipopolysaccharide is the only defined virulence factor of C. burnetii with the full-length molecule of phase I bacteria required for full virulence (21).The obligate intracellular nature of C. burnetii has severely impeded development of genetic tools for virulence factor discovery. Beare et al. (3) recently genetically transformed C. burnetii to chloramphenicol resistance and mCherry red fluorescent protein expression using the mariner-based Himar1 transposon. However, a significant limitation of the procedure is the 8 to 12 weeks required for expansion and clonal isolation of individual transposon mutants using cell culture-based propagation of C. burnetii. Moreover, the micromanipulation method used for harvesting clonal C. burnetii from an individual PV is technically challenging.The recent description of a method for axenic (host cellfree) propagation of C. burnetii is a significant...
We developed a highly sensitive and specific LAMP assay for Escherichia coli. It does not require DNA extraction and can detect as few as 10 copies. It detected all 36 of 36 E. coli isolates and all 22 urine samples (out of 89 samples tested) that had E. coli. This assay is rapid, low in cost, and simple to perform.
Coxiella burnetii, the etiological agent of Q fever, is a small, Gram-negative, obligate intracellular bacterium. Replication of C. burnetii during infection has been shown to be increased by decreasing oxidative stress using p47 phox ؊/؊ and iNOS ؊/؊ mice in vivo and by pharmacologic inhibitors in vitro. Building upon this model, we investigated the role polymorphonuclear leukocytes (PMN) play in the control of infection, since NADPH oxidase-mediated release of reactive oxygen intermediates (ROI) is a primary bactericidal mechanism for these cells that is critical for early innate clearance. Earlier studies suggested that C. burnetii actively inhibited release of ROI from PMN through expression of an unidentified acid phosphatase (ACP). Recent genomic annotations identified one open reading frame (CBU0335) which may encode a Sec-and type II-dependent secreted ACP. To test this model, viable C. burnetii propagated in tissue culture host cells or axenic media, C. burnetii extracts, or purified recombinant ACP (rACP) was combined with human PMN induced with 4-phorbol 12-myristate 13-acetate (PMA). The release of ROI was inhibited when PMN were challenged with viable C. burnetii, C. burnetii extracts, or rACP but not when PMN were challenged with electron beam-inactivated C. burnetii. C. burnetii extracts and rACP were also able to inhibit PMA-induced formation of NADPH oxidase complex on PMN membranes, suggesting a molecular mechanism responsible for this inhibition. These data support a model in which C. burnetii eludes the primary ROI killing mechanism of activated PMN by secreting at least one acid phosphatase.Coxiella burnetii, the etiological agent of Q fever, is a small, Gram-negative, obligate intracellular bacterium. Human Q fever is most commonly acquired through aerosol inhalation of soil contaminated with livestock birth products (43). There are two distinct disease progressions associated with Q fever: acute and chronic infection. The acute infection presents as a flu-like illness often including severe periorbital headache, and clinical presentations have included the neurological, gastrointestinal, endocrine, and renal systems (34, 35). Chronic Q fever has been characterized by the development of endocarditis, with less common manifestations of hepatitis and osteoarticular or vascular infections (3). C. burnetii resides within specialized phagolysosomes (27) of host cells during infection, including macrophages (22, 26) and potentially polymorphonuclear leukocytes (PMN). The ability of C. burnetii to survive and replicate within the phagolysosome requires it to either inhibit or withstand degradative enzymes and an oxidative burst from PMNs that generate reactive oxygen intermediates (ROI), such as superoxide anions. Earlier studies suggested that PMN challenged with C. burnetii Nine Mile phase I (NMI) did not stimulate an oxidative burst (1). Additionally, C. burnetii extracts have acid phosphatase activity that can serve as an inhibitor of an oxidative burst in N-formyl-Met-Leu-Phe (fMLP)-stimulate...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.