scholarly journals Natural Antibody Contributes to Host Defense against an Attenuated Brucella abortus virB Mutant

2009 ◽  
Vol 77 (7) ◽  
pp. 3004-3013 ◽  
Author(s):  
Hortensia G. Rolán ◽  
Mariana N. Xavier ◽  
Renato L. Santos ◽  
Renée M. Tsolis
Keyword(s):  
Knockout Mice ◽  
Brucella Abortus ◽  
Reticuloendothelial System ◽  
Type Iv Secretion ◽  
Natural Antibody ◽  
Immune Functions ◽  
Phagocytic Cells ◽  
Type Iv ◽  
Early Expression

ABSTRACT Brucella abortus is an intracellular pathogen that persists within phagocytic cells of the reticuloendothelial system. To identify in vivo interactions between B. abortus and the host that lead to persistent infection, we studied the persistence of B. abortus and an isogenic virB mutant deficient in the VirB type IV secretion system (T4SS) in knockout mice. In contrast to control mice, mice lacking B cells (Igh6 −/−) were permissive for infection with the attenuated virB mutant. To determine the basis for this phenotype, we characterized immune functions of Igh6 −/− mice in the context of B. abortus infection. Igh6 −/− mice had greater numbers of extracellular bacteria in the spleen and increased early expression of proinflammatory cytokines during B. abortus infection. Further, a virB mutant, despite its wild-type level of survival, failed to elicit microgranuloma formation in the spleens of Igh6 −/− mice, suggesting a requirement for the T4SS to elicit this pathological change. Passive transfer of immunoglobulin G from naïve mice restored the ability of Igh6 −/− mice to control the persistence of the virB mutant by a complement-independent mechanism. Further, adoptive transfer of CD11b+ cells from C57BL/6 mice to Igh6 −/− mice restored the ability of the knockout mice to limit the replication of the virB mutant in the spleen, suggesting that the Igh6 − / − mutation affects phagocyte function and that phagocyte function can be restored by natural antibody.

scholarly journals VirB3 to VirB6 and VirB8 to VirB11, but Not VirB7, Are Essential for Mediating Persistence of Brucella in the Reticuloendothelial System

2008 ◽  
Vol 190 (13) ◽  
pp. 4427-4436 ◽  
Author(s):  
Andreas B. den Hartigh ◽  
Hortensia G. Rolán ◽  
Maarten F. de Jong ◽  
Renée M. Tsolis
Keyword(s):  
Brucella Abortus ◽  
Reticuloendothelial System ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Open Reading Frames ◽  
Type Iv Secretion System ◽  
Type Iv ◽  
Virulence Trait ◽  
Reading Frames

ABSTRACT The Brucella abortus virB locus contains 12 open reading frames, termed virB1 through virB12, which encode a type IV secretion system. Polar mutations in the virB locus markedly reduce the ability of B. abortus to survive in cultured macrophages or to persist in organs of mice. While a nonpolar deletion of the virB2 gene reduces survival in cultured macrophages and in organs of mice, a nonpolar deletion of virB1 only reduces survival in macrophages, whereas virB12 is dispensable for either virulence trait. Here we investigated the role of the remaining genes in the virB locus during survival in macrophages and virulence in mice. Mutants carrying nonpolar deletions of the virB3, virB4, virB5, virB6, virB7, virB8, virB9, virB10, or virB11 gene were constructed and characterized. All mutations reduced the ability of B. abortus to survive in J774A.1 mouse macrophage-like cells to a degree similar to that caused by a deletion of the entire virB locus. Deletion of virB3, virB4, virB5, virB6, virB8, virB9, virB10, or virB11 markedly reduced the ability of B. abortus to persist in the spleens of mice at 8 weeks after infection. Interestingly, deletion of virB7 did not reduce the ability of B. abortus to persist in spleens of mice. We conclude that virB2, virB3, virB4, virB5, virB6, virB8, virB9, virB10, and virB11 are essential for virulence of B. abortus in mice, while functions encoded by the virB1, virB7, and virB12 genes are not required for persistence in organs with this animal model.

The Legionella pneumophila effector RavY contributes to a replication-permissive vacuolar environment during infection

2021 ◽  
Author(s):  
Luying Liu ◽  
Craig R. Roy
Keyword(s):  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Effector Protein ◽  
Intracellular Replication ◽  
Phagocytic Cells ◽  
Host Molecules ◽  
Type Iv ◽  
Legionnaires Disease

Legionella pneumophila is the causative agent of Legionnaires’ Disease and is capable replicating inside phagocytic cells such as mammalian macrophages. The Dot/Icm type IV secretion system is a L. pneumophila virulence factor that is essential for successful intracellular replication. During infection, L. pneumophila builds a replication permissive vacuole by recruiting multiple host molecules and hijacking host cellular signaling pathways, a process mediated by the coordinated functions of multiple Dot/Icm effector proteins. RavY is a predicted Dot/Icm effector protein found to be important for optimal L. pneumophila replication inside host cells. Here, we demonstrate that RavY is a Dot/Icm-translocated effector protein that is dispensable for axenic replication of L. pneumophila , but critical for optimal intracellular replication of the bacteria. RavY is not required for avoidance of endosomal maturation, nor does RavY contribute to the recruitment of host molecules found on replication-permissive vacuoles, such as ubiquitin, RAB1a, and RTN4. Vacuoles containing L. pneumophila ravY mutants promote intracellular survival but limit replication. The replication defect of the L. pneumophila ravY mutant was complemented when the mutant was in the same vacuole as wild type L. pneumophila . Thus, RavY is an effector that is essential for promoting intracellular replication of L. pneumophila once the specialized vacuole has been established.

scholarly journals STUDIES ON TUBERCULIN FEVER

1970 ◽  
Vol 131 (3) ◽  
pp. 483-498 ◽  
Author(s):  
William J. Hall ◽  
Lorraine Francis ◽  
Elisha Atkins
Keyword(s):  
Mononuclear Cells ◽  
Reticuloendothelial System ◽  
Passive Transfer ◽  
Host Cells ◽  
Endogenous Pyrogen ◽  
Phagocytic Cells ◽  
Lymph Node Cells ◽  
Intermediate Substance

Utilizing techniques of passive transfer, we have investigated the factors responsible for production of fever when tuberculin is given intravenously to specifically sensitized rabbits. The ability to develop a febrile response to tuberculin could be passively transferred to normal recipients with viable mononuclear cells from peritoneal exudates, spleen, or lymph nodes of donor rabbits sensitized with BCG. Sensitivity was usually apparent 48 hr after transfer, maximal at 7 to 14 days, and rapidly declined thereafter. Granulocytes and nonviable, sonicated, mononuclear cells from similarly sensitized donors were unable to transfer this form of reactivity. Passive transfer of reactivity was also effected with plasma and serum, suggesting that the reaction of antibody with antigen contained in tuberculin is one of the initial steps by which the host cells are activated to release the endogenous pyrogen (EP) that mediates this form of hypersensitivity fever. An intravenous infusion of granulocytes, as well as of several types of mononuclear cells from sensitized donors, made most recipients responsive to the pyrogenic effect of old tuberculin (OT) given 2 hr later. Some of these passively transferred cells, such as the granulocyte and alveolar macrophage, may be activated in vivo by OT, as they are in vitro. However, in the case of splenic and lymph node cells that cannot be activated by OT to produce EP in vitro, it seems likely that an intravenous injection of OT causes these transferred, sensitized cells to liberate an intermediate substance that either directly, or in association with antigen, activates the host's normal cells to produce EP. In support of previous suggestions that leukocytes of several types, as well as phagocytic cells of the reticuloendothelial system, serve as potential sources of EP in tuberculin-induced fever, evidence was presented that OT also activates both granulocytes and mononuclear cells from sterile exudates of BCG-sensitized donors to produce EP in vitro.

scholarly journals Mice Lacking Components of Adaptive Immunity Show Increased Brucella abortus virB Mutant Colonization

2007 ◽  
Vol 75 (6) ◽  
pp. 2965-2973 ◽  
Author(s):  
Hortensia García Rolán ◽  
Renée M. Tsolis
Keyword(s):  
Adaptive Immunity ◽  
Brucella Abortus ◽  
Mononuclear Phagocytes ◽  
Wild Type ◽  
Adaptive Immune ◽  
Type Iv ◽  
Absolute Requirement ◽  
The Relationship

ABSTRACT The Brucella abortus type IV secretion system (T4SS), encoded by the virB genes, is essential for survival in mononuclear phagocytes in vitro. In the mouse model, a B. abortus virB mutant was initially able to colonize the spleen at the level of the wild type for approximately 3 to 5 days, which coincided with the development of adaptive immunity. To investigate the relationship between survival in macrophages cultivated in vitro and persistence in tissues in vivo, we tested the ability of mutant mice lacking components of adaptive immunity to eliminate the virB mutant from the spleen during a mixed infection with the B. abortus wild type. Ifng −/− or β 2 m −/− mice were able to clear the virB mutant to the same degree as control mice. However, spleens of Rag1 −/− mice and Igh6 −/− mice were more highly colonized by the virB mutant than control mice after 14 to 21 days, suggesting that, in these mice, there is not an absolute requirement for the T4SS to mediate persistence of B. abortus in the spleen. Macrophages isolated from Igh6 −/− mice killed the virB mutant to the same extent as macrophages from control mice, showing that the reduced ability of these mice to clear the virB mutant from the spleen does not correlate with diminished macrophage function in vitro. These results show that in the murine model host, the T4SS is required for persistence beyond 3 to 5 days after infection and suggest that the T4SS may contribute to evasion of adaptive immune mechanisms by B. abortus.

scholarly journals Identification of Genes Required for Chronic Persistence of Brucella abortus in Mice

2000 ◽  
Vol 68 (7) ◽  
pp. 4102-4107 ◽  
Author(s):  
Priscilla C. Hong ◽  
Renée M. Tsolis ◽  
Thomas A. Ficht
Keyword(s):  
Brucella Abortus ◽  
Chronic Infection ◽  
Type Iv Secretion ◽  
Virulence Determinants ◽  
Type Iv ◽  
Transposon Insertion ◽  
Lipopolysaccharide Biosynthesis ◽  
Comparison Of The Results ◽  
Transposon Insertions

ABSTRACT The genetic basis for chronic persistence of Brucella abortus in lymphoid organs of mice, cows, and humans is currently unknown. We identified B. abortus genes involved in chronic infection, by assessing the ability of 178 signature-tagged mutants to establish and maintain persistent infection in mice. Each mutant was screened for its ability to colonize the spleens of mice at 2 and 8 weeks after inoculation. Comparison of the results from both time points identified two groups of mutants attenuated for chronic infection in mice. The first group was not recovered at either 2 or 8 weeks postinfection and was therefore defective in establishing infection. Mutants in this group carried transposon insertions in genes involved in lipopolysaccharide biosynthesis (wbkA), in aromatic amino acid biosynthesis, and in type IV secretion (virB1 and virB10). The second group, which was recovered at wild-type levels 2 weeks postinfection but not 8 weeks postinfection was able to establish infection but was unable to maintain chronic infection. One mutant in this group carried a transposon insertion in a gene with homology to gcvB ofMycobacterium tuberculosis, encoding glycine dehydrogenase, an enzyme whose activity is increased during the state of nonreplicating persistence. These results suggest that some mechanisms for long-term persistence may be shared among chronic intracellular pathogens. Furthermore, identification of two groups of genes, those required for initiating infection and those required only for long-term persistence, suggests that B. abortus uses distinct sets of virulence determinants to establish and maintain chronic infection in mice.

scholarly journals In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center at the Channel Entrance

2021 ◽  
Author(s):  
Pratick Khara ◽  
Peter J. Christie ◽  
Bo Hu
Keyword(s):  
Cell Envelope ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Membrane Complex ◽  
Type Iv ◽  
Inner Membrane Complex ◽  
Cytoplasmic Complex

Bacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as ‘minimized’ or ‘expanded’ based on whether assembly requires only a core set of signature subunits or additional system-specific components. The prototypical ‘minimized’ systems mediating Agrobacterium tumefaciens T-DNA transfer and conjugative transfer of plasmids pKM101 and R388 are built from 12 subunits generically named VirB1-VirB11 and VirD4. In this study, we visualized the pKM101-encoded T4SS in the native context of the bacterial cell envelope by in situ cryoelectron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCCexhibits 14-fold symmetry and resembles that of the T4SSR388, a large substructure of which was previously purified and analyzed by negative-stain electron microscopy (nsEM). The IMC of the in situ T4SSpKM101 machine is highly symmetrical and exhibits 6-fold symmetry, dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMCclosely resembles equivalent regions of three ‘expanded’ T4SSs previously visualized by in situ CryoET, but strikingly differs from the IMC of the purified T4SSR388 whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers.  Together, our findings support a unified architectural model for all T4SSs assembled in vivo regardless of their classification as ‘minimized’ or ‘expanded’: the signature VirB4-like ATPases invariably are arranged as central hexamers of dimers at the entrances to the T4SS channels.

scholarly journals In Vivo Structures of the Helicobacter pylori cag Type IV Secretion System

Cell Reports ◽  
2018 ◽  
Vol 23 (3) ◽  
pp. 673-681 ◽  
Author(s):  
Yi-Wei Chang ◽  
Carrie L. Shaffer ◽  
Lee A. Rettberg ◽  
Debnath Ghosal ◽  
Grant J. Jensen
Keyword(s):  
Helicobacter Pylori ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Type Iv

scholarly journals Brucella abortus Infection of Placental Trophoblasts Triggers Endoplasmic Reticulum Stress-Mediated Cell Death and Fetal Loss via Type IV Secretion System-Dependent Activation of CHOP

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Mariana X. Byndloss ◽  
April Y. Tsai ◽  
Gregory T. Walker ◽  
Cheryl N. Miller ◽  
Briana M. Young ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Brucella Abortus ◽  
Type Iv Secretion ◽  
Content Type ◽  
Unfolded Protein ◽  
Type Iv ◽  
Pregnant Mice ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACT Subversion of endoplasmic reticulum (ER) function is a feature shared by multiple intracellular bacteria and viruses, and in many cases this disruption of cellular function activates pathways of the unfolded protein response (UPR). In the case of infection with Brucella abortus, the etiologic agent of brucellosis, the unfolded protein response in the infected placenta contributes to placentitis and abortion, leading to pathogen transmission. Here we show that B. abortus infection of pregnant mice led to death of infected placental trophoblasts in a manner that depended on the VirB type IV secretion system (T4SS) and its effector VceC. The trophoblast death program required the ER stress-induced transcription factor CHOP. While NOD1/NOD2 expression in macrophages contributed to ER stress-induced inflammation, these receptors did not play a role in trophoblast death. Both placentitis and abortion were independent of apoptosis-associated Speck-like protein containing a caspase activation and recruitment domain (ASC). These studies show that B. abortus uses its T4SS to induce cell-type-specific responses to ER stress in trophoblasts that trigger placental inflammation and abortion. Our results suggest further that in B. abortus the T4SS and its effectors are under selection as bacterial transmission factors. IMPORTANCE Brucella abortus infects the placenta of pregnant cows, where it replicates to high levels and triggers abortion of the calf. The aborted material is highly infectious and transmits infection to both cows and humans, but very little is known about how B. abortus causes abortion. By studying this infection in pregnant mice, we discovered that B. abortus kills trophoblasts, which are important cells for maintaining pregnancy. This killing required an injected bacterial protein (VceC) that triggered an endoplasmic reticulum (ER) stress response in the trophoblast. By inhibiting ER stress or infecting mice that lack CHOP, a protein induced by ER stress, we could prevent death of trophoblasts, reduce inflammation, and increase the viability of the pups. Our results suggest that B. abortus injects VceC into placental trophoblasts to promote its transmission by abortion.

scholarly journals Enterococcus faecalis PcfC, a Spatially Localized Substrate Receptor for Type IV Secretion of the pCF10 Transfer Intermediate

2008 ◽  
Vol 190 (10) ◽  
pp. 3632-3645 ◽  
Author(s):  
Yuqing Chen ◽  
Xiaolin Zhang ◽  
Dawn Manias ◽  
Hye-Jeong Yeo ◽  
Gary M. Dunny ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Transmembrane Domain ◽  
Type Iv Secretion ◽  
Nucleoside Triphosphate ◽  
Mutant Proteins ◽  
Type Iv ◽  
Accessory Factor ◽  
Processed Form

ABSTRACT Upon sensing of peptide pheromone, Enterococcus faecalis efficiently transfers plasmid pCF10 through a type IV secretion (T4S) system to recipient cells. The PcfF accessory factor and PcfG relaxase initiate transfer by catalyzing strand-specific nicking at the pCF10 origin of transfer sequence (oriT). Here, we present evidence that PcfF and PcfG spatially coordinate docking of the pCF10 transfer intermediate with PcfC, a membrane-bound putative ATPase related to the coupling proteins of gram-negative T4S machines. PcfC and PcfG fractionated with the membrane and PcfF with the cytoplasm, yet all three proteins formed several punctate foci at the peripheries of pheromone-induced cells as monitored by immunofluorescence microscopy. A PcfC Walker A nucleoside triphosphate (NTP) binding site mutant (K156T) fractionated with the E. faecalis membrane and also formed foci, whereas PcfC deleted of its N-terminal putative transmembrane domain (PcfCΔN103) distributed uniformly throughout the cytoplasm. Native PcfC and mutant proteins PcfCK156T and PcfCΔN103 bound pCF10 but not pcfG or ΔoriT mutant plasmids as shown by transfer DNA immunoprecipitation, indicating that PcfC binds only the processed form of pCF10 in vivo. Finally, purified PcfCΔN103 bound DNA substrates and interacted with purified PcfF and PcfG in vitro. Our findings support a model in which (i) PcfF recruits PcfG to oriT to catalyze T-strand nicking, (ii) PcfF and PcfG spatially position the relaxosome at the cell membrane to stimulate substrate docking with PcfC, and (iii) PcfC initiates substrate transfer through the pCF10 T4S channel by an NTP-dependent mechanism.

scholarly journals Gene Discovery through Genomic Sequencing of Brucella abortus

2001 ◽  
Vol 69 (2) ◽  
pp. 865-868 ◽  
Author(s):  
Daniel O. Sánchez ◽  
Ruben O. Zandomeni ◽  
Silvio Cravero ◽  
Ramiro E. Verdún ◽  
Ester Pierrou ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Genomic Sequence ◽  
Rhizobium Meliloti ◽  
Type Iv Secretion ◽  
Type Iii ◽  
Type Iv ◽  
Close Phylogenetic Relationship ◽  
Serovar Typhimurium ◽  
Genes Encoding ◽  
Expect Value

ABSTRACT Brucella abortus is the etiological agent of brucellosis, a disease that affects bovines and human. We generated DNA random sequences from the genome of B. abortus strain 2308 in order to characterize molecular targets that might be useful for developing immunological or chemotherapeutic strategies against this pathogen. The partial sequencing of 1,899 clones allowed the identification of 1,199 genomic sequence surveys (GSSs) with high homology (BLAST expect value < 10−5) to sequences deposited in the GenBank databases. Among them, 925 represent putative novel genes for the Brucella genus. Out of 925 nonredundant GSSs, 470 were classified in 15 categories based on cellular function. Seven hundred GSSs showed no significant database matches and remain available for further studies in order to identify their function. A high number of GSSs with homology toAgrobacterium tumefaciens and Rhizobium meliloti proteins were observed, thus confirming their close phylogenetic relationship. Among them, several GSSs showed high similarity with genes related to nodule nitrogen fixation, synthesis of nod factors, nodulation protein symbiotic plasmid, and nodule bacteroid differentiation. We have also identified severalB. abortus homologs of virulence and pathogenesis genes from other pathogens, including a homolog to both the Shda gene fromSalmonella enterica serovar Typhimurium and the AidA-1 gene from Escherichia coli. Other GSSs displayed significant homologies to genes encoding components of the type III and type IV secretion machineries, suggesting that Brucella might also have an active type III secretion machinery.

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