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ABSTRACT: A large number of hypothetical genes potentially encoding small proteins of unknown function are annotated in the Brucella abortus genome. Individual deletion of 30 of these genes identified four mutants, in BAB1_0355, BAB2_0726, BAB2_0470, and BAB2_0450 that were highly attenuated for infection. BAB2_0726, an YbgT-family protein located at the 3' end of the cydAB genes encoding cytochrome bd ubiquinal oxidase, was designated cydX. A B. abortus cydX mutant lacked cytochrome bd oxidase activity, as shown by increased sensitivity to H(2)O(2), decreased acid tolerance and increased resistance to killing by respiratory inhibitors. The C terminus, but not the N terminus, of CydX was located in the periplasm, suggesting that CydX is an integral cytoplasmic membrane protein. Phenotypic analysis of the cydX mutant, therefore, suggested that CydX is required for full function of cytochrome bd oxidase, possibly via regulation of its assembly or activity.
Frontiers in cellular and infection microbiology. 01/2012; 2:47.
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ABSTRACT: Many types of cells migrate directionally in direct current (DC) electric fields (EFs), a phenomenon termed galvanotaxis or electrotaxis. The directional sensing mechanisms responsible for this response to EFs, however, remain unknown. Exposing cells to an EF causes changes in plasma membrane potentials (V(m)). Exploiting the ability of Dictyostelium cells to tolerate drastic V(m) changes, we investigated the role of V(m) in electrotaxis and, in parallel, in chemotaxis. We used three independent factors to control V(m): extracellular pH, extracellular [K(+)], and electroporation. Changes in V(m) were monitored with microelectrode recording techniques. Depolarized V(m) was observed under acidic (pH 5.0) and alkaline (pH 9.0) conditions as well as under higher extracellular [K(+)] conditions. Electroporation permeabilized the cell membrane and significantly reduced the V(m), which gradually recovered over 40 min. We then recorded the electrotactic behaviors of Dictyostelium cells with a defined V(m) using these three techniques. The directionality (directedness of electrotaxis) was quantified and compared to that of chemotaxis (chemotactic index). We found that a reduced V(m) significantly impaired electrotaxis without significantly affecting random motility or chemotaxis. We conclude that extracellular pH, [K(+)], and electroporation all significantly affected electrotaxis, which appeared to be mediated by the changes in V(m). The initial directional sensing mechanisms for electrotaxis therefore differ from those of chemotaxis and may be mediated by changes in resting V(m).
Eukaryotic Cell 07/2011; 10(9):1251-6. · 3.60 Impact Factor
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ABSTRACT: Damage to the respiratory epithelium is one of the most critical steps to many life-threatening diseases, such as acute respiratory distress syndrome and chronic obstructive pulmonary disease. The mechanisms underlying repair of the damaged epithelium have not yet been fully elucidated. Here we provide experimental evidence suggesting a novel mechanism for wound repair: endogenous electric currents. It is known that the airway epithelium maintains a voltage difference referred to as the transepithelial potential. Using a noninvasive vibrating probe, we demonstrate that wounds in the epithelium of trachea from rhesus monkeys generate significant outward electric currents. A small slit wound produced an outward current (1.59 μA/cm(2)), which could be enhanced (nearly doubled) by the ion transport stimulator aminophylline. In addition, inhibiting cystic fibrosis transmembrane conductance regulator (CFTR) with CFTR(Inh)-172 significantly reduced wound currents (0.17 μA/cm(2)), implicating an important role of ion transporters in wound induced electric potentials. Time-lapse video microscopy showed that applied electric fields (EFs) induced robust directional migration of primary tracheobronchial epithelial cells from rhesus monkeys, towards the cathode, with a threshold of <23 mV/mm. Reversal of the field polarity induced cell migration towards the new cathode. We further demonstrate that application of an EF promoted wound healing in a monolayer wound healing assay. Our results suggest that endogenous electric currents at sites of tracheal epithelial injury may direct cell migration, which could benefit restitution of damaged airway mucosa. Manipulation of ion transport may lead to novel therapeutic approaches to repair damaged respiratory epithelium.
Journal of Applied Physiology 06/2011; 111(4):1031-41. · 3.75 Impact Factor
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ABSTRACT: Survival and replication inside host cells by Brucella spp. requires a type IV secretion system (T4SS), encoded by the virB locus. However, the identity of the molecules secreted by the T4SS has remained elusive. We hypothesized that proteins translocated by the T4SS would be co-regulated with the virB operon. The LuxR family regulator VjbR, known to regulate virB, bound a fragment of the virB promoter containing an 18 bp palindromic motif (virB promoter box), showing that VjbR regulated the virB operon directly. To identify virB co-regulated genes, we searched the Brucella suis 1330 and B. abortus 2308 genomes for genes with an upstream virB promoter box. One hundred and forty-four promoters in the two genomes contained the virB promoter box, including those of fliC encoding flagellin and cgs encoding cyclic beta-glucan synthetase. Thirteen of these proteins were tested for VirB-dependent translocation into macrophages using a beta-lactamase reporter assay. This analysis resulted in the identification of the proteins encoded by BAB1_1652 (VceA) and BR1038/BAB1_1058 (VceC) as novel protein substrates of the Brucella T4SS. VceC could also be translocated by the Legionella pneumophila Dot/Icm T4SS into host cells. Our results suggest that VjbR co-ordinates expression of the T4SS and at least two of its secreted substrates.
Molecular Microbiology 11/2008; 70(6):1378-96. · 5.01 Impact Factor
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Karla L Lightfield,
Jenny Persson,
Sky W Brubaker,
Chelsea E Witte,
Jakob von Moltke,
Eric A Dunipace,
Thomas Henry, Yao-Hui Sun,
Dragana Cado,
William F Dietrich,
Denise M Monack,
Renée M Tsolis,
Russell E Vance
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ABSTRACT: Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes and what they sense remain poorly defined. Here we demonstrate that 35 amino acids of the carboxyl terminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in the intracellular sensor Naip5. These mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Our data clarify the molecular basis for the cytosolic response to flagellin.
Nature Immunology 09/2008; 9(10):1171-8. · 26.01 Impact Factor
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ABSTRACT: Bacterial flagellins are potent inducers of innate immunity. Three signaling pathways have been implicated in the sensing of flagellins; these involve toll-like receptor 5 (TLR5) and the cytosolic proteins Birc1e/Naip5 and Ipaf. Although the structural basis of TLR5-flagellin interaction is known, little is known about how flagellin enters the host cell cytosol to induce signaling via Birc1e/Naip5 and Ipaf. Here we demonstrate for the first time the translocation of bacterial flagellin into the cytosol of host macrophages by the vacuolar pathogen, Salmonella enterica serotype Typhimurium. Translocation of flagellin into the host cell cytosol was directly demonstrated using beta-lactamase reporter constructs. Flagellin translocation required the Salmonella Pathogenicity Island 1 Type III secretion system (SPI-1 T3SS) but not the flagellar T3SS.
Journal of Biological Chemistry 12/2007; 282(47):33897-901. · 4.77 Impact Factor
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Manuela Raffatellu, Yao-Hui Sun,
R Paul Wilson,
Quynh T Tran,
Daniela Chessa,
Helene L Andrews-Polymenis,
Sara D Lawhon,
Josely F Figueiredo,
Renée M Tsolis,
L Garry Adams,
Andreas J Bäumler
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ABSTRACT: Salmonella enterica serotype Typhi is a strictly human adapted pathogen that does not cause disease in nonprimate vertebrate hosts, while Salmonella enterica serotype Typhimurium is a broad-host-range pathogen. Serotype Typhi lacks some of the proteins (effectors) exported by the invasion-associated type III secretion system that are required by serotype Typhimurium for eliciting fluid secretion and inflammation in bovine ligated ileal loops. We investigated whether the remaining serotype Typhi effectors implicated in enteropathogenicity (SipA, SopB, and SopD) are functionally exchangeable with their serotype Typhimurium homologues. Serotype Typhi elicited fluid accumulation in bovine ligated ileal loops at levels similar to those elicited by a noninvasive serotype Typhimurium strain (the sipA sopABDE2 mutant) or by sterile culture medium. However, introduction of the cloned serotype Typhi sipA, sopB, and sopD genes complemented the ability of a serotype Typhimurium sipA sopABDE2 mutant to elicit fluid secretion in bovine ligated ileal loops. Introduction of the cloned serotype Typhi sipA, sopB, and sopD genes increased the invasiveness of a serotype Typhimurium sipA sopABDE2 mutant for human colon carcinoma epithelial (HT-29 and T84) cells and bovine kidney (MDBK) cells. Translational fusions between the mature TEM-1 beta-lactamase reporter and SipA or SopD demonstrated that serotype Typhi translocates these effectors into host cells. We conclude that the inability of serotype Typhi to cause fluid accumulation in bovine ligated ileal loops is not caused by a functional alteration of its SipA, SopB, and SopD effector proteins with respect to their serotype Typhimurium homologues.
Infection and Immunity 01/2006; 73(12):7817-26. · 4.16 Impact Factor
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ABSTRACT: This unit provides protocols for growth of Brucella abortus on solid or in liquid media and for long-term storage of laboratory stocks. Two issues affecting the culture and storage of isolates of this slow-growing bacterium are emphasized: contamination of cultures and outgrowth of attenuated variants lacking a complete lipopolysaccharide. Laboratories planning to work with B. abortus should be aware that Biosafety Level 3 facilities are required. Furthermore, this organism is classified in the U.S. as a Select Agent, which restricts its use to laboratories registered with the U.S. government's Select Agent programs.
Current protocols in microbiology 11/2005; Chapter 3:Unit 3B.1.
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ABSTRACT: The Brucella abortus virB operon, consisting of 11 genes, virB1 to virB11, and two putative genes, orf12 (virB12) and orf13, encodes a type IV secretion system (T4SS) that is required for intracellular replication and persistent infection in the mouse model. This study was undertaken to determine whether orf12 (virB12) encodes an essential part of the T4SS apparatus. The virB12 gene was found to encode a 17-kDa protein, which was detected in vitro in B. abortus grown to stationary phase. Mice infected with B. abortus 2308 produced an antibody response to the protein encoded by virB12, showing that this gene is expressed during infection. Expression of virB12 was not required for survival in J774 macrophages. VirB12 was also dispensable for the persistence of B. abortus, B. melitensis, and B. suis in mice up to 4 weeks after infection, since deletion mutants lacking virB12 were recovered from splenic tissue at wild-type levels. These results show that VirB12 is not essential for the persistence of the human-pathogenic Brucella spp. in the mouse and macrophage models of infection.
Infection and Immunity 10/2005; 73(9):6048-54. · 4.16 Impact Factor
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ABSTRACT: We have identified genes required for competence of Neisseria meningitidis, a naturally transformable human pathogen. Although not comprehensive, our analysis identified competence-defective mutants with transposon insertions in genes not previously implicated in this process in Neisseria.
Journal of Bacteriology 06/2005; 187(9):3273-6. · 3.83 Impact Factor
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ABSTRACT: Neisseria meningitidis is an important cause of septicaemia and meningitis. To cause disease, the bacterium must acquire essential nutrients for replication in the systemic circulation, while avoiding exclusion by host innate immunity. Here we show that the utilization of carbon sources by N. meningitidis determines its ability to withstand complement-mediated lysis, through the intimate relationship between metabolism and virulence in the bacterium. The gene encoding the lactate permease, lctP, was identified and disrupted. The lctP mutant had a reduced growth rate in cerebrospinal fluid compared with the wild type, and was attenuated during bloodstream infection through loss of resistance against complement-mediated killing. The link between lactate and complement was demonstrated by the restoration of virulence of the lctP mutant in complement (C3(-/-))-deficient animals. The underlying mechanism for attenuation is mediated through the sialic acid biosynthesis pathway, which is directly connected to central carbon metabolism. The findings highlight the intimate relationship between bacterial physiology and resistance to innate immune killing in the meningococcus.
Journal of Experimental Medicine 06/2005; 201(10):1637-45. · 13.85 Impact Factor
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ABSTRACT: The Brucella abortus virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The products of the first two genes of the virB operon, virB1 and virB2, are predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon. In order to determine whether VirB1 and VirB2 are required for the function of the T4SS apparatus, we constructed and characterized nonpolar deletion mutations of virB1 and virB2. Both mutants were shown to be nonpolar, as demonstrated by their ability to express the downstream gene virB5 during stationary phase of growth in vitro. Both VirB1 and VirB2 were essential for intracellular replication in J774 macrophages. The nonpolar virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection. In contrast, the nonpolar virB1 mutant persisted at wild-type levels, showing that the function of VirB1 is dispensable in the mouse model of persistent infection.
Infection and Immunity 10/2004; 72(9):5143-9. · 4.16 Impact Factor
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ABSTRACT: Meningococcal disease is a life-threatening infection caused by Neisseria meningitidis. Currently, there are no vaccines to prevent infection with serogroup B N. meningitidis strains, the leading cause of meningococcal meningitis in Europe and North America. Here we describe the construction and characterization of two attenuated serogroup B N. meningitidis strains, YH102 (MC58deltasia deltarfaF) and YH103 (MC58deltasia deltametH). Both strains are markedly attenuated in their capacity to cause bacteremia in rodent models and have a reduced ability to survive in a human whole-blood assay. Immunization of adult mice with these strains leads to the development of bactericidal antibodies and confers sterilizing protection against challenge with homologous live bacteria. Furthermore, we show that the strains confer protection against infection by other serogroups. Use of the attenuated strains in animals with gene knockouts or after depletion of immunological effectors could be used to define the basis of protection, and human volunteer studies could be undertaken to examine the immune response following exposure to this important human pathogen.
Infection and Immunity 02/2004; 72(1):345-51. · 4.16 Impact Factor
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ABSTRACT: The Brucella abortus virB locus is required for establishing chronic infection in the mouse. Using in vitro and in vivo models, we investigated whether virB is involved in evasion of the bactericidal activity of NADPH oxidase and the inducible nitric oxide synthase (iNOS) in macrophages. Elimination of NADPH oxidase or iNOS activity in macrophages in vitro increased recovery of wild-type B. abortus but not recovery of a virB mutant. In mice lacking either NADPH oxidase or iNOS, however, B. abortus infected and persisted to the same extent as it did in congenic C57BL/6 mice up until 60 days postinfection, suggesting that these host defense mechanisms are not critical for limiting bacterial growth in the mouse. A virB mutant did not exhibit increased survival in either of the knockout mouse strains, indicating that this locus does not contribute to evasion of nitrosative or oxidative killing mechanisms in vivo.
Infection and Immunity 10/2002; 70(9):4826-32. · 4.16 Impact Factor
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ABSTRACT: Numerous pathogenic bacteria contain luxS, which is required for autoinducer-2 production. Here, we demonstrate that Neisseria meningitidis contains a functional copy of luxS that is necessary for full meningococcal virulence; strains with a luxS deletion are defective for bacteremia, a prerequisite of meningococcal pathogenesis.
Infection and Immunity 05/2002; 70(4):2245-8. · 4.16 Impact Factor
