scholarly journals Molecular Insights Into Neutrophil Biology From the Zebrafish Perspective: Lessons From CD18 Deficiency

2021 ◽  
Vol 12 ◽  
Author(s):  
Almke Bader ◽  
Jincheng Gao ◽  
Thibaud Rivière ◽  
Bettina Schmid ◽  
Barbara Walzog ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Neutrophil Migration ◽  
Amino Acid Sequences ◽  
Sterile Inflammation ◽  
Type I ◽  
Hematopoietic Tissue ◽  
Sequence Alignments ◽  
Zebrafish Larvae ◽  
Inflamed Tissue

Neutrophils are key players in innate immunity and originate from the bone marrow of the adult mammalian organism. In mammals, mature neutrophils are released from the bone marrow into the peripheral blood where they circulate until their recruitment to sites of inflammation in a multistep adhesion cascade. Here, adhesion molecules of the β2 integrin family (CD11/CD18) are critically required for the initial neutrophil adhesion to the inflamed endothelium and several post-adhesion steps allowing their extravasation into the inflamed tissue. Within the mammalian tissue, interstitial neutrophil migration can occur widely independent of β2 integrins. This is in sharp contrast to neutrophil recruitment in zebrafish larvae (Danio rerio) where neutrophils originate from the caudal hematopoietic tissue and mainly migrate interstitially to sites of lesion upon the early onset of inflammation. However, neutrophils extravasate from the circulation to the inflamed tissue in zebrafish larvae at later-time points. Although zebrafish larvae are a widely accepted model system to analyze neutrophil trafficking in vivo, the functional impact of β2 integrins for neutrophil trafficking during acute inflammation is completely unknown in this model. In this study, we generated zebrafish with a genetic deletion of CD18, the β subunit of β2 integrins, using CRISPR/Cas9 technology. Sequence alignments demonstrated a high similarity of the amino acid sequences between zebrafish and human CD18 especially in the functionally relevant I-like domain. In addition, the cytoplasmic domain of CD18 harbors two highly conserved NXXF motifs suggesting that zebrafish CD18 may share functional properties of human CD18. Accordingly, CD18 knock-out (KO) zebrafish larvae displayed the key symptoms of patients suffering from leukocyte adhesion deficiency (LAD) type I due to defects in ITGB2, the gene for CD18. Importantly, CD18 KO zebrafish larvae showed reduced neutrophil trafficking to sites of sterile inflammation despite the fact that an increased number of neutrophils was detectable in the circulation. By demonstrating the functional importance of CD18 for neutrophil trafficking in zebrafish larvae, our findings shed new light on neutrophil biology in vertebrates and introduce a new model organism for studying LAD type I.

scholarly journals Stat2 loss disrupts damage signalling and is protective in acute pancreatitis

2019 ◽  
Author(s):  
Helen Heath ◽  
Gary Britton ◽  
Hiromi Kudo ◽  
George Renney ◽  
Malcolm Ward ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Pancreatitis ◽  
Type I Interferon ◽  
Pancreatic Tissue ◽  
Sterile Inflammation ◽  
Mitogen Activated Protein Kinase ◽  
Type I ◽  
Label Free ◽  
Bone Marrow Chimera ◽  
Interferon Signalling

ABSTRACTSeverity of sterile inflammation, as seen in acute pancreatitis, is determined by damage-sensing receptors, signalling cascades and cytokine production. Stat2 is a type I interferon signalling mediator that also has interferon-independent roles in murine lipopolysaccharide-induced NF-κB-mediated sepsis. However its role in sterile inflammation is unknown. We hypothesised that Stat2 determines severity of non-infective inflammation in the pancreas.Wild type (WT) and Stat2−/− mice were injected intraperitoneally with cerulein or L-arginine. Specific cytokine-blocking antibodies were used in some experiments. Pancreata and blood were harvested 1h and 24h after the final dose of cerulein and up to 96h post L-arginine. Whole-tissue phosphoproteomic changes were assessed using label-free mass spectrometry. Tissue-specific Stat2 effects were studied in WT/Stat2−/− bone-marrow chimera and using Cre-lox recombination to delete Stat2 in pancreatic and duodenal homeobox 1(Pdx1)-expressing cells.Stat2−/− mice were protected from cerulein- and L-arginine-induced pancreatitis. Protection was independent of type I interferon signalling. Stat2−/− mice had lower cytokine levels including TNFα and IL-10 and reduced NF-kB nuclear localisation in pancreatic tissue compared to WT. Inhibition of TNFα improved (inhibition of IL-10 worsened) cerulein-induced pancreatitis in WT but not Stat2−/− mice. Phosphoproteomics showed down-regulation of mitogen-activated protein kinase (MAPK) mediators but accumulation of Ser412-phosphorylated Tak1. Stat2 deletion in Pdx1-expressing acinar cells (Stat2flox/Pdx1-cre) reduced pancreatic TNFα expression, but not histological injury or serum amylase. WT/Stat2−/− bone-marrow chimera were protected from pancreatitis irrespective of host or recipient genotype.Stat2 loss results in disrupted signalling in pancreatitis, upstream of NF-κB in non-acinar and/or bone marrow derived cells.

Morphological and Functional Characteristics of Three-Dimensional Engineered Bone-Ligament-Bone Constructs Following Implantation

2009 ◽  
Vol 131 (10) ◽  
Author(s):  
Jinjin Ma ◽  
Kristen Goble ◽  
Michael Smietana ◽  
Tatiana Kostrominova ◽  
Lisa Larkin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Type I Collagen ◽  
Three Dimensional ◽  
Functionally Graded ◽  
Neonatal Rat ◽  
Ligament Injury ◽  
Type I ◽  
Tangent Moduli

The incidence of ligament injury has recently been estimated at 400,000/year. The preferred treatment is reconstruction using an allograft, but outcomes are limited by donor availability, biomechanical incompatibility, and immune rejection. The creation of an engineered ligament in vitro solely from patient bone marrow stromal cells (has the potential to greatly enhance outcomes in knee reconstructions. Our laboratory has developed a scaffoldless method to engineer three-dimensional (3D) ligament and bone constructs from rat bone marrow stem cells in vitro. Coculture of these two engineered constructs results in a 3D bone-ligament-bone (BLB) construct with viable entheses, which was successfully used for medial collateral ligament (MCL) replacement in a rat model. 1 month and 2 month implantations were applied to the engineered BLBs. Implantation of 3D BLBs in a MCL replacement application demonstrated that our in vitro engineered tissues grew and remodeled quickly in vivo to an advanced phenotype and partially restored function of the knee. The explanted 3D BLB ligament region stained positively for type I collagen and elastin and was well vascularized after 1 and 2 months in vivo. Tangent moduli of the ligament portion of the 3D BLB 1 month explants increased by a factor of 2.4 over in vitro controls, to a value equivalent to those observed in 14-day-old neonatal rat MCLs. The 3D BLB 1 month explants also exhibited a functionally graded response that closely matched native MCL inhomogeneity, indicating the constructs functionally adapted in vivo.

Isolation of a Common Natural Type-I Interferon Producing Cell and Dendritic Cell Progenitor Population in Mouse Bone Marrow.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1653-1653
Author(s):  
Nobuyuki Onai ◽  
Aya Onai ◽  
Markus G. Manz
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Type I Interferon ◽  
Receptor Expression ◽  
Mouse Bone Marrow ◽  
Type I ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Gm Csf

Abstract Most type-I interferon producing cells (IPCs) and dendritic cells (DCs) are non-dividing cells with a short in vivo half-live of several days, and thus need to be continuously replaced. A common differentiation pathway for IPCs and DCs, and accordingly, the existence of common IPC and DC progenitors remains controversial. Flt3-ligand (Flt3L) is a non-redundant cytokine for in vivo IPC and DC development: IPC and DC differentiation potential is confined to Flt3+-hematopoietic progenitors; Flt3L KO mice show massively reduced IPCs and DCs. In contrast to Flt3, the “myeloid” cytokines GM-CSF and M-CSF seem to be less relevant in steady-state IPC and DC differentiation, however, they might be critically important in inflammatory conditions. To identify a candidate common IPC and DC progenitor population, we evaluated Flt3 and “myeloid” cytokine receptor expression in mouse bone marrow. We found that c-kitintlin− cells contained a Flt3+M-CSFR+ fraction that in Flt3L supplemented cultures gave rise to about 95% pure CD11c+MHC class II+ cells, consisting of both CD11c+B220+ IPCs and CD11c+B220− DCs, at a efficiency comparable to that of hematopoietic stem cells. In the presence of GM-CSF, Flt3+M-CSFR+c-kitintlin− cells gave rise to CD11c+CD11b+ DCs but not CD11c−CD11b+ macrophages/monocytes. Furthermore, Flt3+M-CSFR+c-kitintlin− cells possessed very poor, if any activity in myeloid colony forming assays, and lacked pre-B cell colony forming activity. In both, lethally and sub-lethally irradiated mice, transferred Flt3+M-CSFR+c-kitintlin− cells differentiated into CD11c+B220+ IPCs, CD11c+CD8α+, and CD11c+CD8α− conventional DC subsets, while no other hematopoietic cells were detectable. In vivo reconstitution and CFSE-labeling experiments showed that Flt3+M-CSFR+c-kitintlin− cells extensively proliferate in the lethally irradiated mice, reaching peak progeny levels of IPC and DC at day 10 after transplantation, indicating high proliferative, but limited self-renewal capacity of these cells. Quantitative RT-PCR analysis revealed high expression of DC and IPC-development affiliated genes (such as PU.1, STAT3, GM-CSFR, and CX3CR1), but no lymphoid- and erythroid-development affiliated gene transcription. These data suggest the existence of common developmental intermediates for both IPCs and DCs in mouse bone marrow, and thus might provide new insights into the regulation of IPC and DC differentiation in steady-state and inflammation.

Type I interferons produced by dendritic cells promote their phenotypic and functional activation

Blood ◽  
2002 ◽  
Vol 99 (9) ◽  
pp. 3263-3271 ◽  
Author(s):  
Maria Montoya ◽  
Giovanna Schiavoni ◽  
Fabrizio Mattei ◽  
Ion Gresser ◽  
Filippo Belardelli ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
T Cell ◽  
Bone Marrow Cells ◽  
Type I Interferons ◽  
Type I ◽  
Type I Ifn ◽  
Type I Ifns

Abstract Resting dendritic cells (DCs) are resident in most tissues and can be activated by environmental stimuli to mature into potent antigen-presenting cells. One important stimulus for DC activation is infection; DCs can be triggered through receptors that recognize microbial components directly or by contact with infection-induced cytokines. We show here that murine DCs undergo phenotypic maturation upon exposure to type I interferons (type I IFNs) in vivo or in vitro. Moreover, DCs either derived from bone marrow cells in vitro or isolated from the spleens of normal animals express IFN-α and IFN-β, suggesting that type I IFNs can act in an autocrine manner to activate DCs. Consistent with this idea, the ability to respond to type I IFN was required for the generation of fully activated DCs from bone marrow precursors, as DCs derived from the bone marrow of mice lacking a functional receptor for type I IFN had reduced expression of costimulatory and adhesion molecules and a diminished ability to stimulate naive T-cell proliferation compared with DCs derived from control bone marrow. Furthermore, the addition of neutralizing anti–IFN-α/β antibody to purified splenic DCs in vitro partially blocked the “spontaneous” activation of these cells, inhibiting the up-regulation of costimulatory molecules, secretion of IFN-γ, and T-cell stimulatory activity. These results show that DCs both secrete and respond to type I IFN, identifying type I interferons as autocrine DC activators.

scholarly journals Generation of Mouse-Zebrafish Hematopoietic Tissue Chimeric Embryos for Hematopoiesis and Host-Pathogen Interaction Studies

2017 ◽  
Author(s):  
Margarita Parada-Kusz ◽  
Anne Clatworthy ◽  
Elliott J. Hagedorn ◽  
Cristina Penaranda ◽  
Anil V. Nair ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Hematopoietic Cell ◽  
Confocal Imaging ◽  
Hematopoietic Tissue ◽  
Simple Method ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Marrow Cells

ABSTRACTXenografts of the hematopoietic system are extremely useful as disease models and for translational research. Zebrafish xenografts have been widely used to monitor blood cancer cell dissemination and homing due to the optical clarity of embryos and larvae, which allow unrestricted in vivo visualization of migratory events. To broaden the scope of xenotransplantation studies in zebrafish, we have developed a technique that transiently generates hematopoietic tissue chimeras by transplanting murine bone marrow cells into zebrafish blastulae. This procedure leads to mammalian cell integration into the fish developmental hematopoietic program. Monitoring zebrafish chimeras at different time points post fertilization using in vivo time-lapse and confocal imaging showed murine cell co-localization with developing primitive and definitive hematopoietic tissues, intravasation into fish circulation, and dynamic hematopoietic cell-vascular endothelial and hematopoietic cell-niche interactions. Immunohistochemistry assays performed in chimeric animals showed that, after engraftment, murine cells expressed antigens related to i) hematopoietic stem and progenitor cells, ii) active cell proliferation, and iii) myeloid cell lineages. Lastly, xenografted zebrafish larvae infected with Klebsiella pneumoniae showed murine immune cells trafficking to bacterial foci and interacting with bacterial cells. Overall, these results show that mammalian bone marrow cells xenografted in zebrafish integrate into the host hematopoietic system revealing highly conserved molecular mechanisms of hematopoiesis between zebrafish and mammals. In addition, this procedure introduces a useful and simple method that improves and broadens the scope of hematopoietic tissue xenotransplantation studies in zebrafish.

scholarly journals The LTB4–BLT1 axis regulates actomyosin and β2-integrin dynamics during neutrophil extravasation

2020 ◽  
Vol 219 (10) ◽  
Author(s):  
Bhagawat C. Subramanian ◽  
Nicolas Melis ◽  
Desu Chen ◽  
Weiye Wang ◽  
Devorah Gallardo ◽  
...  
Keyword(s):  
Neutrophil Migration ◽  
Extracellular Vesicle ◽  
Leukotriene B4 ◽  
Cell Polarization ◽  
Sterile Inflammation ◽  
Β2 Integrin ◽  
Neutrophil Extravasation ◽  
Neutrophil Response ◽  
Muscle Myosin

The eicosanoid leukotriene B4 (LTB4) relays chemotactic signals to direct neutrophil migration to inflamed sites through its receptor BLT1. However, the mechanisms by which the LTB4–BLT1 axis relays chemotactic signals during intravascular neutrophil response to inflammation remain unclear. Here, we report that LTB4 produced by neutrophils acts as an autocrine/paracrine signal to direct the vascular recruitment, arrest, and extravasation of neutrophils in a sterile inflammation model in the mouse footpad. Using intravital subcellular microscopy, we reveal that LTB4 elicits sustained cell polarization and adhesion responses during neutrophil arrest in vivo. Specifically, LTB4 signaling coordinates the dynamic redistribution of non-muscle myosin IIA and β2-integrin, which facilitate neutrophil arrest and extravasation. Notably, we also found that neutrophils shed extracellular vesicles in the vascular lumen and that inhibition of extracellular vesicle release blocks LTB4-mediated autocrine/paracrine signaling required for neutrophil arrest and extravasation. Overall, we uncover a novel complementary mechanism by which LTB4 relays extravasation signals in neutrophils during early inflammation response.

scholarly journals The LTB4-BLT1 axis regulates actomyosin and β2 integrin dynamics during neutrophil extravasation

2019 ◽  
Author(s):  
Bhagawat C. Subramanian ◽  
Nicolas Melis ◽  
Desu Chen ◽  
Weiye Wang ◽  
Devorah Gallardo ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Neutrophil Migration ◽  
Cell Polarization ◽  
Sterile Inflammation ◽  
Paracrine Signaling ◽  
Neutrophil Extravasation ◽  
Actomyosin Cytoskeleton ◽  
Neutrophil Response ◽  
Muscle Myosin

ABSTRACTThe eicosanoid Leukotriene B4 (LTB4) relays chemotactic signals to direct neutrophil migration to inflamed sites through its receptor BLT1. However, the mechanisms by which the LTB4-BLT1 axis relays chemotactic signals during intravascular neutrophil response to inflammation remain unclear. Here, we report that LTB4 produced by neutrophils acts as an autocrine/paracrine signal to direct the vascular recruitment, arrest and extravasation of neutrophils in a sterile inflammation model in the mouse footpad. Using Intravital Subcellular Microscopy (ISMic), we reveal that LTB4 elicits sustained cell polarization and adhesion responses during neutrophil arrest in vivo. Specifically, LTB4 signaling coordinates the dynamic redistribution of non-muscle Myosin IIA (NMIIA) and β2-integrin (Itgb2), which facilitate neutrophil arrest and extravasation. Notably, we also found that neutrophils shed extracellular vesicles (EVs) in the vascular lumen, and that inhibition of EV release blocks LTB4-mediated autocrine/paracrine signaling required for neutrophil arrest and extravasation. Overall, we uncover a novel complementary mechanism by which LTB4 relays extravasation signals in neutrophils during early inflammation response.SUMMARYNeutrophils arrest and extravasate from the blood vessels in response to infection and injury. Using intravital subcellular microscopy, Subramanian et al. identify a role for extracellular vesicles-based autocrine/paracrine LTB4-BLT1 signaling in promoting the re-arrangement of actomyosin cytoskeleton and β2-integrin during neutrophil extravasation in live animals.

scholarly journals Mitofusin 2 regulates neutrophil adhesive migration and the actin cytoskeleton

2020 ◽  
Vol 133 (17) ◽  
pp. jcs248880 ◽  
Author(s):  
Wenqing Zhou ◽  
Alan Y. Hsu ◽  
Yueyang Wang ◽  
Ramizah Syahirah ◽  
Tianqi Wang ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Neutrophil Migration ◽  
Neutrophil Infiltration ◽  
Neutrophil Function ◽  
Mitochondrial Outer Membrane ◽  
Hematopoietic Tissue ◽  
Mitofusin 2 ◽  
Sheer Stress ◽  
Intracellular Ca

ABSTRACTNeutrophils rely on glycolysis for energy production. How mitochondria regulate neutrophil function is not fully understood. Here, we report that mitochondrial outer membrane protein Mitofusin 2 (MFN2) regulates neutrophil homeostasis and chemotaxis in vivo. Mfn2-deficient neutrophils are released from the hematopoietic tissue, trapped in the vasculature in zebrafish embryos, and not capable of chemotaxis. Consistent with this, human neutrophil-like cells that are deficient for MFN2 fail to arrest on activated endothelium under sheer stress or perform chemotaxis on 2D surfaces. Deletion of MFN2 results in a significant reduction of neutrophil infiltration to the inflamed peritoneal cavity in mice. Mechanistically, MFN2-deficient neutrophil-like cells display disrupted mitochondria–ER interaction, heightened intracellular Ca2+ levels and elevated Rac activation after chemokine stimulation. Restoring a mitochondria–ER tether rescues the abnormal Ca2+ levels, Rac hyperactivation and chemotaxis defect resulting from MFN2 depletion. Finally, inhibition of Rac activation restores chemotaxis in MFN2-deficient neutrophils. Taken together, we have identified that MFN2 regulates neutrophil migration via maintaining the mitochondria–ER interaction to suppress Rac activation, and uncovered a previously unrecognized role of MFN2 in regulating cell migration and the actin cytoskeleton.This article has an associated First Person interview with the first authors of the paper.

Erk1 Plays Critical Role in Macrophage Development

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 517-517 ◽  
Author(s):  
Yongzheng He ◽  
Karl Staser ◽  
Steven D Rhodes ◽  
Xiaohua Wu ◽  
Ping Zhang ◽  
...  
Keyword(s):  
Bone Mineral Density ◽  
Bone Marrow ◽  
Bone Mineral ◽  
Type I ◽  
Mineral Density ◽  
Macrophage Differentiation ◽  
Fold Reduction ◽  
The Impact

Abstract Abstract 517 Extracellular signal-regulated kinase (ERK 1 and 2) are widely expressed and are involved in the regulation of meiosis, mitosis, and postmitotic functions in multiple cell lineages, including T cells, B cells and osteoblasts. Macrophages are capable of differentiating into osteoclasts, which resorb bone. Abnormal osteoclast development and functions underlie certain diseases, especially skeletal defects. Altered ERK1/2 signaling has been found in several genetic diseases with skeletal phenotypes, including Noonan syndrome, polycystic kidney disease and serious developmental disorders such as cardio-facio-cutaneous syndrome. These clinical findings suggest the importance of the ERK MAPK pathway in human skeletal development. In the present study, we examined the consequence of Erk1 and Erk2 disruption in modulating macrophage development in the murine system. We found that deletion of Erk1 reduced macrophage progenitor numbers. Erk1−/− bone marrow mononuclear cells (BMMNCs) had significant reduction in osteoclast formation as compared to wildtype BMMNCs. In addition, Erk1−/− macrophages; the osteoclast progenitors, had a two-three fold reduction in migration and a two-fold reduction in αv ß3 mediated adhesion as compared to WT macrophages as evaluated by transwell and adhesion assay, respectively. These in vitro data demonstrate that Erk1 positively regulates macrophage differentiation into osteoclasts. To evaluate the impact of deficiency of Erk1 in vivo, we examined bone mineral density and trabecular microarchitecture in the distal femoral metaphysis by dual-energy X-ray absorptiometry (DEXA) with a Lunar Piximus densitometer and a high-resolution desktop microcomputed tomography imaging system (μCT-20; Scanco Medical AG, Basserdorf, Switzerland), respectively. Erk1−/− mice displayed elevated bone mineral density and increased trabecular bone formation as compared to WT mice. Histomorphometric analysis indicated that the Erk1−/− femur had significant reduction in osteoclast numbers as determined by tartrate resistant acid phosphatase staining, an osteoclast specific staining, as compared to femur of wildtype and Erk2−/− mice. Most importantly, Erk1−/− plasma had reduced C-terminal telopeptide of type I collagen, indicating less bone resorption in vivo. These data suggest that the impaired macrophage differentiation and osteoclast bone resorptive activity play an important role in increased bone mass in Erk1−/− mice. Finally, to verify that the macrophage-osteoclast lineage is a key cell lineage for the phenotypic changes in vivo in Erk1−/− mice, we performed bone marrow transplantation. WT mice reconstituted long-term with Erk1−/− hematopoietic stem cells demonstrated increased bone mineral density as compared to WT and Erk2−/− stem cell recipients, implicating marrow autonomous, Erk1-dependent macrophage differentiation and osteoclast bioactivity in vivo. Collectively, our in vitro and in vivo data demonstrate isoform-specific Erk function in macrophage while providing rationale for the development of a specific inhibitor for Erk1 that might be used for the treatment of dysplastic and erosive bone diseases. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Molecular and Functional Analysis of the lepB Gene, Encoding a Type I Signal Peptidase from Rickettsia rickettsii and Rickettsia typhi

2003 ◽  
Vol 185 (15) ◽  
pp. 4578-4584 ◽  
Author(s):  
M. Sayeedur Rahman ◽  
Jason A. Simser ◽  
Kevin R. Macaluso ◽  
Abdu F. Azad
Keyword(s):  
Spotted Fever ◽  
Rocky Mountain ◽  
Amino Acid Sequences ◽  
Signal Peptidase ◽  
Type I ◽  
Rocky Mountain Spotted Fever ◽  
Rickettsia Typhi ◽  
Rickettsia Rickettsii ◽  
Signal Peptidase I

ABSTRACT The type I signal peptidase lepB genes from Rickettsia rickettsii and Rickettsia typhi, the etiologic agents of Rocky Mountain spotted fever and murine typhus, respectively, were cloned and characterized. Sequence analysis of the cloned lepB genes from R. rickettsii and R. typhi shows open reading frames of 801 and 795 nucleotides, respectively. Alignment analysis of the deduced amino acid sequences reveals the presence of highly conserved motifs that are important for the catalytic activity of bacterial type I signal peptidase. Reverse transcription-PCR and Northern blot analysis demonstrated that the lepB gene of R. rickettsii is cotranscribed in a polycistronic message with the putative nuoF (encoding NADH dehydrogenase I chain F), secF (encoding protein export membrane protein), and rnc (encoding RNase III) genes in a secF-nuoF-lepB-rnc cluster. The cloned lepB genes from R. rickettsii and R. typhi have been demonstrated to possess signal peptidase I activity in Escherichia coli preprotein processing in vivo by complementation assay.

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