Publications by authors named "Roop R"

92 Publications

The impact of consultant delivered service in emergency medicine: the Wrexham Model.

Emerg Med J 2012 May 13;29(5):366-71. Epub 2011 Apr 13.

Emergency Department, Wrexham Maelor Hospital, Betsi Cadwaladr University Health Board, Wrexham, UK.

Objective: Consultant based delivery of emergency service is perceived to add value. This study aims to demonstrate the impact of such a service model based on consultant working in a UK emergency department.

Methods: This retrospective study was based on the emergency department of a district general hospital. Activity data was analysed for 2009. Workload and admission rates were compared between consultants, middle grade doctors and senior house officers (SHOs). Admission rates were compared against two similar departments. Data from night shifts allowed consultant activity to be contrasted with middle grades and SHOs. Time spent in the department, admission rates, patients who left without treatment, discharged outright and clinic returns were used for comparison.

Results: Consultants often saw more patients than SHOs or middle grade doctors. This was on top of their traditional duties of senior opinion. On comparison of activity at night shifts, they admitted fewer (25.2% vs 30.3%, p=0.026), had fewer leaving without treatment (1.6% vs 5.1%, p<0.001), discharged more outright (59.8% vs 47.5%, p<0.001), referred fewer to clinic (5.7% vs 6.6%, p=0.49) and had a faster turnaround time (p<0.001: Priority 2, 3 and 4) for every triage category. Some of the comparisons were clinically but not statistically significant.

Conclusion: A consultant based service delivery offers many advantages. These cannot be matched by either junior or middle grades. This would be in addition to the consultants' supervisory role. Consultant expansion is urgently required to achieve this sustainably. A further study evaluating the cost benefits of this service model is now underway.
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http://dx.doi.org/10.1136/emj.2010.107797DOI Listing
May 2012

Comparative study of the roles of AhpC and KatE as respiratory antioxidants in Brucella abortus 2308.

J Bacteriol 2010 Oct 30;192(19):4912-22. Epub 2010 Jul 30.

Department of Microbiology and Immunology, East Carolina University School of Medicine, 600 Moye Boulevard, Greenville, NC 27834, USA.

Brucella strains are exposed to potentially toxic levels of H2O2 both as a consequence of their aerobic metabolism and through the respiratory burst of host phagocytes. To evaluate the relative contributions of the sole catalase KatE and the peroxiredoxin AhpC produced by these strains in defense against H2O2-mediated toxicity, isogenic katE, ahpC, and katE ahpC mutants were constructed and the phenotypic properties of these mutants compared with those of the virulent parental strain B. abortus 2308. The results of these studies indicate that AhpC is the primary detoxifier of endogenous H2O2 generated by aerobic metabolism. KatE, on the other hand, plays a major role in scavenging exogenous and supraphysiologic levels of H2O2, although this enzyme can play a supporting role in the detoxification of H2O2 of endogenous origin if AhpC is absent. B. abortus ahpC and katE mutants exhibit wild-type virulence in C57BL/6 and BALB/c mice, but the B. abortus ahpC katE double mutant is extremely attenuated, and this attenuation is not relieved in derivatives of C57BL/6 mice that lack NADPH oxidase (cybb) or inducible nitric oxide synthase (Nos2) activity. These experimental findings indicate that the generation of endogenous H2O2 represents a relevant environmental stress that B. abortus 2308 must deal with during its residence in the host and that AhpC and KatE perform compensatory roles in detoxifying this metabolic H2O2.
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http://dx.doi.org/10.1128/JB.00231-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944529PMC
October 2010

Subversion of innate immune responses by Brucella through the targeted degradation of the TLR signaling adapter, MAL.

J Immunol 2010 Jan 14;184(2):956-64. Epub 2009 Dec 14.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA.

Gram-negative bacteria belonging to the Brucella species cause chronic infections that can result in undulant fever, arthritis, and osteomyelitis in humans. Remarkably, Brucella sp. genomes encode a protein, named TcpB, that bears significant homology with mammalian Toll/IL-1 receptor domains and whose expression causes degradation of the phosphorylated, signal competent form of the adapter MyD88-adapter-like (MAL). This effect of TcpB is mediated through its box 1 region and has no effect on other TLR adapter proteins such as MyD88 or TIR-domain containing adapter protein-inducing IFNbeta. TcpB also does not affect a mutant, signal-incompetent form of MAL that cannot be phosphorylated. Interestingly, the presence of TcpB leads to enhanced polyubiquitination of MAL, which is likely responsible for its accelerated degradation. A Brucella abortus mutant lacking TcpB fails to reduce levels of MAL in infected macrophages. Therefore, TcpB represents a unique pathogen-derived molecule that suppresses host innate-immune responses by specifically targeting an individual adapter molecule in the TLR signaling pathway for degradation.
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http://dx.doi.org/10.4049/jimmunol.0902008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3644118PMC
January 2010

Survival of the fittest: how Brucella strains adapt to their intracellular niche in the host.

Med Microbiol Immunol 2009 Nov 22;198(4):221-38. Epub 2009 Sep 22.

Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA.

Brucella strains produce abortion and infertility in their natural hosts and a zoonotic disease in humans known as undulant fever. These bacteria do not produce classical virulence factors, and their capacity to successfully survive and replicate within a variety of host cells underlies their pathogenicity. Extensive replication of the brucellae in placental trophoblasts is associated with reproductive tract pathology in natural hosts, and prolonged persistence in macrophages leads to the chronic infections that are a hallmark of brucellosis in both natural hosts and humans. This review describes how Brucella strains have efficiently adapted to their intracellular lifestyle in the host.
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http://dx.doi.org/10.1007/s00430-009-0123-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814008PMC
November 2009

The manganese transporter MntH is a critical virulence determinant for Brucella abortus 2308 in experimentally infected mice.

Infect Immun 2009 Aug 1;77(8):3466-74. Epub 2009 Jun 1.

Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27834, USA.

The gene designated BAB1_1460 in the Brucella abortus 2308 genome sequence is predicted to encode the manganese transporter MntH. Phenotypic analysis of an isogenic mntH mutant indicates that MntH is the sole high-affinity manganese transporter in this bacterium but that MntH does not play a detectable role in the transport of Fe(2+), Zn(2+), Co(2+), or Ni(2+). Consistent with the apparent selectivity of the corresponding gene product, the expression of the mntH gene in B. abortus 2308 is repressed by Mn(2+), but not Fe(2+), and this Mn-responsive expression is mediated by a Mur-like repressor. The B. abortus mntH mutant MWV15 exhibits increased susceptibility to oxidative killing in vitro compared to strain 2308, and a comparative analysis of the superoxide dismutase activities present in these two strains indicates that the parental strain requires MntH in order to make wild-type levels of its manganese superoxide dismutase SodA. The B. abortus mntH mutant also exhibits extreme attenuation in both cultured murine macrophages and experimentally infected C57BL/6 mice. These experimental findings indicate that Mn(2+) transport mediated by MntH plays an important role in the physiology of B. abortus 2308, particularly during its intracellular survival and replication in the host.
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http://dx.doi.org/10.1128/IAI.00444-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2715675PMC
August 2009

Broad-host-range expression vectors with tightly regulated promoters and their use to examine the influence of TraR and TraM expression on Ti plasmid quorum sensing.

Appl Environ Microbiol 2008 Aug 7;74(16):5053-62. Epub 2008 Jul 7.

Department of Microbiology, University of Illinois at Urbana-Champaign, B103 Chemical and Life Sciences Laboratory, 601 South Goodwin Avenue, Urbana, IL 61801, USA.

Experiments requiring strong repression and precise control of cloned genes can be difficult to conduct because of the relatively high basal level of expression of currently employed promoters. We report the construction of a family of vectors that contain a reengineered lacI(q)-lac promoter-operator complex in which cloned genes are strongly repressed in the absence of inducer. The vectors, all based on the broad-host-range plasmid pBBR1, are mobilizable and stably replicate at moderate copy number in representatives of the alpha- and gammaproteobacteria. Each vector contains a versatile multiple cloning site that includes an NdeI site allowing fusion of the cloned gene to the initiation codon of lacZalpha. In each tested bacterium, a uidA reporter fused to the promoter was not expressed at a detectable level in the absence of induction but was inducible by 10- to 100-fold, depending on the bacterium. The degree of induction was controllable by varying the concentration of inducer. When the vector was tested in Agrobacterium tumefaciens, a cloned copy of the traR gene, the product of which is needed at only a few copies per cell, did not confer activity under noninducing conditions. We used this attribute of very tight and variably regulatable control to assess the relative amounts of TraR required to activate the Ti plasmid conjugative transfer system. We identified levels of induction that gave wild-type transfer frequencies, as well as levels that induced correspondingly lower frequencies of transfer. We also used this system to show that the antiactivator TraM sets the level of intracellular TraR required for tra gene activation.
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http://dx.doi.org/10.1128/AEM.01098-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519271PMC
August 2008

The AraC-like transcriptional regulator DhbR is required for maximum expression of the 2,3-dihydroxybenzoic acid biosynthesis genes in Brucella abortus 2308 in response to iron deprivation.

J Bacteriol 2008 Mar 21;190(5):1838-42. Epub 2007 Dec 21.

Department of Microbiology and Immunology, East Carolina University School of Medicine, 600 Moye Boulevard, Greenville, NC 27834, USA.

Phenotypic evaluation of isogenic mutants derived from Brucella abortus 2308 indicates that the AlcR homolog DhbR (2,3-dihydroxybenzoic acid [2,3-DHBA] biosynthesis regulator) modulates the expression of the genes involved in 2,3-DHBA production, employing 2,3-DHBA or brucebactin as a coinducer.
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http://dx.doi.org/10.1128/JB.01551-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2258684PMC
March 2008

Brucella abortus requires the heme transporter BhuA for maintenance of chronic infection in BALB/c mice.

Infect Immun 2007 Nov 20;75(11):5248-54. Epub 2007 Aug 20.

Department of Microbiology and Immunology, East Carolina University School of Medicine, 600 Moye Boulevard, Greenville, NC 27834, USA.

The gene annotated BAB2_1150 in the Brucella abortus 2308 genome sequence is predicted to encode a homolog of the well-characterized heme transporter ShuA of Shigella dysenteriae and accordingly has been given the designation bhuA (Brucella heme utilization). Phenotypic analysis of an isogenic bhuA mutant derived from B. abortus 2308 verified that there is a link between BhuA and the ability of the parent strain to use heme as an iron source in in vitro assays. Maximum expression of bhuA in B. abortus 2308 is observed during stationary phase when this strain in cultivated in low-iron minimal medium, and a comparison of the growth characteristics of the B. abortus bhuA mutant and 2308 in this medium suggested that heme serves as an important iron source for the parent strain during stationary phase. The B. abortus bhuA mutant HR1703 exhibits significant attenuation in cultured murine macrophages compared to strain 2308, and unlike its parent strain, the B. abortus bhuA mutant is unable to maintain a chronic spleen infection in experimentally infected BALB/c mice. These experimental findings suggest that heme and/or heme-containing proteins represent important iron sources for B. abortus 2308 during its residence in the mammalian host and that BhuA is required for efficient utilization of these iron sources.
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http://dx.doi.org/10.1128/IAI.00460-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2168305PMC
November 2007

Brucella abortus bacA mutant induces greater pro-inflammatory cytokines than the wild-type parent strain.

Microbes Infect 2007 Jan 11;9(1):55-62. Epub 2006 Dec 11.

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA.

The inner-membrane protein BacA affects Brucella LPS structure. A bacA deletion mutant of Brucella abortus, known as KL7 (bacA(mut)-KL7), is attenuated in BALB/c mice and protects against challenge. Thus, bacA mutation was a candidate for incorporation into live attenuated vaccines. We assessed bacA(mut)-KL7 in 2 additional mouse strains: the more resistant C57BL/6 that produces interferon-gamma throughout the infection and the highly susceptible interferon-gamma-deficient C57BL/6 in which brucellae exhibit continual exponential growth. While it was hypothesized that bacA(mut)-KL7 would exhibit even greater attenuation relative to its parent strain B. abortus 2308 in C57BL/6 mice than it did in BALB/c mice, this was not the case. Moreover, it was more pathogenic in C57BL/6 interferon-gamma-deficient mice than 2308 causing abscesses and wasting even though the splenic loads of bacA(mut)-KL7 were significantly lower. These 2 observations were correlated, respectively, with an ability of IFNgamma-activated macrophages to equivalently control strains 2308 and bacA(mut)-KL7 and the ability of bacA(mut)-KL7 organism and its LPS to induce greater amounts of pro-inflammatory cytokines than 2308. We conclude that attenuation properties of bacA mutation are dependent upon the nature of the host but more importantly that bacterial gene deletion can result in increased host pathology without an increase in bacterial load, crucial considerations for vaccine design.
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http://dx.doi.org/10.1016/j.micinf.2006.10.008DOI Listing
January 2007

RecA and RadA proteins of Brucella abortus do not perform overlapping protective DNA repair functions following oxidative burst.

J Bacteriol 2006 Jul;188(14):5187-95

Department of Biology, P.O. Box 42451, University of Louisiana, Lafayette, LA 70504-2451, USA.

Very little is known about the role of DNA repair networks in Brucella abortus and its role in pathogenesis. We investigated the roles of RecA protein, DNA repair, and SOS regulation in B. abortus. While recA mutants in most bacterial species are hypersensitive to UV damage, surprisingly a B. abortus recA null mutant conferred only modest sensitivity. We considered the presence of a second RecA protein to account for this modest UV sensitivity. Analyses of the Brucella spp. genomes and our molecular studies documented the presence of only one recA gene, suggesting a RecA-independent repair process. Searches of the available Brucella genomes revealed some homology between RecA and RadA, a protein implicated in E. coli DNA repair. We considered the possibility that B. abortus RadA might be compensating for the loss of RecA by promoting similar repair activities. We present functional analyses that demonstrated that B. abortus RadA complements a radA defect in E. coli but could not act in place of the B. abortus RecA. We show that RecA but not RadA was required for survival in macrophages. We also discovered that recA was expressed at high constitutive levels, due to constitutive LexA cleavage by RecA, with little induction following DNA damage. Higher basal levels of RecA and its SOS-regulated gene products might protect against DNA damage experienced following the oxidative burst within macrophages.
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http://dx.doi.org/10.1128/JB.01994-05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1539968PMC
July 2006

The Brucella abortus xthA-1 gene product participates in base excision repair and resistance to oxidative killing but is not required for wild-type virulence in the mouse model.

J Bacteriol 2006 Feb;188(4):1295-300

Department of Microbiology and Immunology, East Carolina University School of Medicine, 600 Moye Boulevard, Greenville, North Carolina 27834, USA.

Exonuclease III, encoded by the xthA gene, plays a central role in the base excision pathway of DNA repair in bacteria. Studies with Escherichia coli xthA mutants have also shown that exonuclease III participates in the repair of oxidative damage to DNA. An isogenic xthA-1 mutant (designated CAM220) derived from virulent Brucella abortus 2308 exhibited increased sensitivity to the alkylating agent methyl methanesulfonate (MMS) compared to the parent strain. In contrast, 2308 and the isogenic xthA-1 mutant displayed similar levels of resistance to the DNA cross-linker mitomycin C. These phenotypic properties are those that would be predicted for a strain defective in base excision repair. The B. abortus xthA-1 mutant also displayed reduced resistance to killing by H2O2 and the ONOO(-)-generating compound 3-morpholinosydnonimine (SIN-1) compared to strain 2308, indicating that the xthA-1 gene product participates in protecting B. abortus 2308 from oxidative damage. Introducing a plasmid-borne copy of the parental xthA-1 gene into CAM220 restored wild-type resistance of this mutant to MMS, H2O2, and SIN-1. Although the B. abortus xthA-1 mutant exhibited increased sensitivity to oxidative killing compared to the parental strain in laboratory assays, CAM220 and 2308 displayed equivalent spleen colonization profiles in C57BL/6 [corrected] mice through 8 weeks postinfection and equivalent intracellular survival and replication profiles in cultured murine macrophages. Thus, although the xthA-1 gene product participates in base excision repair and resistance to oxidative killing in B. abortus 2308, XthA-1 is not required for wild-type virulence of this strain in the mouse model.
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http://dx.doi.org/10.1128/JB.188.4.1295-1300.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1367252PMC
February 2006

Opsonized virulent Brucella abortus replicates within nonacidic, endoplasmic reticulum-negative, LAMP-1-positive phagosomes in human monocytes.

Infect Immun 2005 Jun;73(6):3702-13

Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932, USA.

Cells in the Brucella spp. are intracellular pathogens that survive and replicate within host monocytes. Brucella maintains persistent infections in animals despite the production of high levels of anti-Brucella-specific antibodies. To determine the effect of antibody opsonization on the ability of Brucella to establish itself within monocytes, the intracellular trafficking of virulent Brucella abortus 2308 and attenuated hfq and bacA mutants was followed in the human monocytic cell line THP-1. Early trafficking events of B. abortus 2308-containing phagosomes (BCP) were indistinguishable from those seen for control particles (heat-killed B. abortus 2308, live Escherichia coli HB101, or latex beads). All phagosomes transiently communicated the early-endosomal compartment and rapidly matured into LAMP-1(+), cathepsin D(+), and acidic phagosomes. By 2 h postinfection, however, the number of cathepsin D(+) BCP was significantly lower for live B. abortus 2308-infected cells than for either Brucella mutant strains or control particles. B. abortus 2308 persisted within these cathepsin D(-), LAMP-1(+), and acidic vesicles; however, at the onset of intracellular replication, the numbers of acidic B. abortus 2308 BCP decreased while remaining cathepsin D(-) and LAMP-1(+). In contrast to B. abortus 2308, the isogenic hfq and bacA mutants remained in acidic, LAMP-1(+) phagosomes and failed to initiate intracellular replication. Notably, markers specific for the host endoplasmic reticulum were absent from the BCPs throughout the course of the infection. Thus, opsonized B. abortus in human monocytes survives within phagosomes that remain in the endosomal pathway and replication of virulent B. abortus 2308 within these vesicles corresponds with an increase in intraphagosomal pH.
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http://dx.doi.org/10.1128/IAI.73.6.3702-3713.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1111828PMC
June 2005

Role of HdeA in acid resistance and virulence in Brucella abortus 2308.

Vet Microbiol 2005 May;107(3-4):307-12

Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27834, USA.

Two-dimensional gel electrophoretic analysis of cell lysates suggests that stationary phase production of wild-type levels of an ortholog of the low pH dependent chaperone HdeA in Brucella abortus 2308 during growth in a minimal medium requires the presence of the RNA binding protein Hfq. Although mutational analysis demonstrated that HdeA contributes to acid resistance in this bacterium, this protein is not required for wild-type virulence in the BALB/c mouse model. These experimental findings indicate that the brucellae rely upon additional gene products to resist the acidic conditions they encounter in the phagosomal compartment of host macrophages.
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http://dx.doi.org/10.1016/j.vetmic.2005.01.018DOI Listing
May 2005

The Brucella abortus Cu,Zn superoxide dismutase is required for optimal resistance to oxidative killing by murine macrophages and wild-type virulence in experimentally infected mice.

Infect Immun 2005 May;73(5):2873-80

Department of Microbiology and Immunology, East Carolina University School of Medicine, 600 Moye Boulevard, Greenville, NC 27834, USA.

Two-dimensional gel electrophoretic analysis of cell lysates from Brucella abortus 2308 and the isogenic hfq mutant Hfq3 revealed that the RNA binding protein Hfq (also known as host factor I or HF-I) is required for the optimal stationary phase production of the periplasmic Cu,Zn superoxide dismutase SodC. An isogenic sodC mutant, designated MEK2, was constructed from B. abortus 2308 by gene replacement, and the sodC mutant exhibited much greater susceptibility to killing by O(2)(-) generated by pyrogallol and the xanthine oxidase reaction than the parental 2308 strain supporting a role for SodC in protecting this bacterium from O(2)(-) of exogenous origin. The B. abortus sodC mutant was also found to be much more sensitive to killing by cultured resident peritoneal macrophages from C57BL6J mice than 2308, and the attenuation displayed by MEK2 in cultured murine macrophages was enhanced when these phagocytes were treated with gamma interferon (IFN-gamma). The attenuation displayed by the B. abortus sodC mutant in both resting and IFN-gamma-activated macrophages was alleviated, however, when these host cells were treated with the NADPH oxidase inhibitor apocynin. Consistent with its increased susceptibility to killing by cultured murine macrophages, the B. abortus sodC mutant also displayed significant attenuation in experimentally infected C57BL6J mice compared to the parental strain. These experimental findings indicate that SodC protects B. abortus 2308 from the respiratory burst of host macrophages. They also suggest that reduced SodC levels may contribute to the attenuation displayed by the B. abortus hfq mutant Hfq3 in the mouse model.
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http://dx.doi.org/10.1128/IAI.73.5.2873-2880.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1087332PMC
May 2005

Role of catalase in the virulence of Brucella melitensis in pregnant goats.

Vet Microbiol 2004 Aug;102(1-2):111-5

Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport 71130-3932, USA.

An isogenic katE mutant derived from virulent Brucella melitensis 16M displays hypersensitivity to hydrogen peroxide in disk sensitivity assays but retains the capacity to colonize pregnant goats and induce abortion. These experimental findings indicate that although the sole periplasmic catalase of Brucella melitensis functions as an antioxidant, this enzyme does not play a critical role in virulence in the natural host.
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http://dx.doi.org/10.1016/j.vetmic.2004.05.009DOI Listing
August 2004

Intact purine biosynthesis pathways are required for wild-type virulence of Brucella abortus 2308 in the BALB/c mouse model.

Infect Immun 2004 Aug;72(8):4911-7

Department of Microbiology and Immunology, East Carolina University School of Medicine, 600 Moye Blvd., Greenville, NC 27858-4354, USA.

Brucella abortus 2308 derivatives with mini-Tn5 insertions in purE, purL, and purD display significant attenuation in the BALB/c mouse model, while isogenic mutants with mini-Tn5 insertions in pheA, trpB, and dagA display little or no attenuation in cultured murine macrophages or mice. These experimental findings confirm the importance of the purine biosynthesis pathways for the survival and replication of the brucellae in host macrophages. In contrast to previous reports, however, these results indicate that exogenous tryptophan and phenylalanine are available for use by the brucellae in the phagosomal compartment.
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http://dx.doi.org/10.1128/IAI.72.8.4911-4917.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC470684PMC
August 2004

Adaptation of the Brucellae to their intracellular niche.

Mol Microbiol 2004 May;52(3):621-30

Department of Microbiology and Immunology, East Carolina University School of Medicine, 600 Moye Boulevard, Greenville, NC 27858-4354, USA.

Members of the bacterial genus Brucella are facultative intracellular pathogens that reside predominantly within membrane-bound compartments within two host cell types, macrophages and placental trophoblasts. Within macrophages, the brucellae route themselves to an intracellular compartment that is favourable for survival and replication, and they also appear to be well-adapted from a physiological standpoint to withstand the environmental conditions encountered during prolonged residence in this intracellular niche. Much less is known about the interactions of the Brucella with placental trophoblasts, but experimental evidence suggests that these bacteria use an iron acquisition system to support extensive intracellular replication within these host cells that is not required for survival and replication in host macrophages. Thus, it appears that the brucellae rely upon the products of distinct subsets of genes to adapt successfully to the environmental conditions encountered within the two cell types within which they reside in their mammalian hosts.
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http://dx.doi.org/10.1111/j.1365-2958.2004.04017.xDOI Listing
May 2004

Similarity to peroxisomal-membrane protein family reveals that Sinorhizobium and Brucella BacA affect lipid-A fatty acids.

Proc Natl Acad Sci U S A 2004 Apr 24;101(14):5012-7. Epub 2004 Mar 24.

Biology Department, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

Sinorhizobium meliloti, a legume symbiont, and Brucella abortus, a phylogenetically related mammalian pathogen, both require the bacterial-encoded BacA protein to establish chronic intracellular infections in their respective hosts. We found that the bacterial BacA proteins share sequence similarity with a family of eukaryotic peroxisomal-membrane proteins, including the human adrenoleukodystrophy protein, required for the efficient transport of very-long-chain fatty acids out of the cytoplasm. This insight, along with the increased sensitivity of BacA-deficient mutants to detergents and cell envelope-disrupting agents, led us to discover that BacA affects the very-long-chain fatty acid (27-OHC28:0 and 29-OHC30:0) content of both Sinorhizobium and Brucella lipid A. We discuss models for how BacA function affects the lipid-A fatty-acid content and why this activity could be important for the establishment of chronic intracellular infections.
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http://dx.doi.org/10.1073/pnas.0307137101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC387365PMC
April 2004

Brucella stationary-phase gene expression and virulence.

Annu Rev Microbiol 2003 1;57:57-76. Epub 2003 May 1.

Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27858-4354, USA.

The capacity of the Brucella spp. to establish and maintain long-term residence in the phagosomal compartment of host macrophages is critical to their ability to produce chronic infections in their mammalian hosts. The RNA binding protein host factor I (HF-I) encoded by the hfq gene is required for the efficient translation of the stationary-phase sigma factor RpoS in many bacteria, and a Brucella abortus hfq mutant displays a phenotype in vitro, which suggests that it has a generalized defect in stationary-phase physiology. The inability of the B. abortus hfq mutant to survive and replicate in a wild-type manner in cultured murine macrophages, and the profound attenuation displayed by this strain and its B. melitensis counterpart in experimentally infected animals indicate that stationary-phase physiology plays an essential role in the capacity of the brucellae to establish and maintain long-term intracellular residence in host macrophages. The nature of the Brucella HF-I-regulated genes that have been identified to date suggests that the corresponding gene products contribute to the remarkable capacity of the brucellae to resist the harsh environmental conditions they encounter during their prolonged residence in the phagosomal compartment.
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http://dx.doi.org/10.1146/annurev.micro.57.030502.090803DOI Listing
February 2004

Production of the siderophore 2,3-dihydroxybenzoic acid is required for wild-type growth of Brucella abortus in the presence of erythritol under low-iron conditions in vitro.

Infect Immun 2003 May;71(5):2927-832

Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932, USA.

Production of the siderophore 2,3-dihyroxybenzoic acid (2,3-DHBA) is required for the wild-type virulence of Brucella abortus in cattle. A possible explanation for this requirement was uncovered when it was determined that a B. abortus dhbC mutant (BHB1) defective in 2,3-DHBA production displays marked growth restriction in comparison to its parent strain, B. abortus 2308, when cultured in the presence of erythritol under low-iron conditions. This phenotype is not displayed when these strains are cultured under low-iron conditions in the presence of other readily utilizable carbon and energy sources. The addition of either exogenous 2,3-DHBA or FeCl(3) relieves this growth defect, suggesting that the inability of the B. abortus dhbC mutant to display wild-type growth in the presence of erythritol under iron-limiting conditions is due to a defect in iron acquisition. Restoring 2,3-DHBA production to the B. abortus dhbC mutant by genetic complementation abolished the erythritol-specific growth defect exhibited by this strain in low-iron medium, verifying the relationship between 2,3-DHBA production and efficient growth in the presence of erythritol under low-iron conditions. The positive correlation between 2,3-DHBA production and growth in the presence of erythritol was further substantiated by the observation that the addition of erythritol to low-iron cultures of B. abortus 2308 stimulated the production of 2,3-DHBA by increasing the transcription of the dhbCEBA operon. Correspondingly, the level of exogenous iron needed to repress dhbCEBA expression in B. abortus 2308 was also greater when this strain was cultured in the presence of erythritol than that required when it was cultured in the presence of any of the other readily utilizable carbon and energy sources tested. The tissues of the bovine reproductive tract are rich in erythritol during the latter stages of pregnancy, and the ability to metabolize erythritol is thought to be important to the virulence of B. abortus in pregnant ruminants. Consequently, the experimental findings presented here offer a plausible explanation for the attenuation of the B. abortus 2,3-DHBA-deficient mutant BHB1 in pregnant ruminants.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC153232PMC
http://dx.doi.org/10.1128/IAI.71.5.2927-2932.2003DOI Listing
May 2003

Interactions between Brucella melitensis and human phagocytes: bacterial surface O-Polysaccharide inhibits phagocytosis, bacterial killing, and subsequent host cell apoptosis.

Infect Immun 2003 Apr;71(4):2110-9

Department of Bacterial Diseases, Walter Reed Army Institute of Research, Bldg. 503, Room 2N57, Washington, D.C. 20307-5100, USA.

Brucellae are gram-negative intracellular pathogens that survive and multiply within host phagocytic cells. Smooth organisms present O-polysaccharides (OPS) on their surface. The wboA gene, which codes for the enzyme glycosyl transferase, is essential for the assembly of O-chain in Brucella. Deletion of wboA in smooth, virulent B. melitensis 16M results in a rough mutant designated WRR51. Unlike B. abortus, both smooth and rough strains of B. melitensis are resistant to complement-mediated killing. To determine the role of surface OPS in the interactions of B. melitensis with monocytes/macrophages (M/M), 16M and WRR51 were transformed with the plasmid pBBR1MCS-6y encoding green fluorescent protein, and the transformants were used to infect human mononuclear phagocytes with and without fresh human serum as a source of complement. Human monocytes were cultured in the presence of macrophage colony-stimulating factor to allow their differentiation into macrophages during the course of infection. Intracellular bacteria were easily visualized using fluorescence microscopy. Infection in M/M, identified by surface staining and fate of infected phagocytes, was quantitated by flow cytometry. Rough bacteria were internalized, with no requirement for opsonization by serum, at a higher rate than smooth organisms. Smooth B. melitensis survived and multiplied for at least 6 days inside M/M, but rough organisms were eliminated by death of the infected cells. In human monocytes cultured for 1 day without serum in order to trigger the apoptotic pathway, infection by rough brucellae accelerated phagocyte death; smooth brucellae inhibited apoptosis. This study suggests that the presence of surface OPS on live B. melitensis benefits the bacterium by preventing the death of macrophages, Brucella's preferred target for intracellular replication.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC152029PMC
http://dx.doi.org/10.1128/IAI.71.4.2110-2119.2003DOI Listing
April 2003

Genetic organization and iron-responsive regulation of the Brucella abortus 2,3-dihydroxybenzoic acid biosynthesis operon, a cluster of genes required for wild-type virulence in pregnant cattle.

Infect Immun 2003 Apr;71(4):1794-803

Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USA.

Brucella abortus reportedly produces the monocatechol siderophore 2,3-dihydroxybenzoic acid (2,3-DHBA) in response to iron limitation. Nucleotide sequence analysis of the cloned DHBA biosynthesis locus from virulent B. abortus 2308 and genetic complementation of defined Escherichia coli mutants were used to identify the B. abortus genes (designated dhbC, -B, and -A) responsible for synthesis of this siderophore. Reverse transcriptase PCR analysis of total RNA with dhb-specific primers demonstrated that dhbC, -B, and -A are transcribed as components of an operon, together with dhbE, a functional homolog of the Escherichia coli entE gene. Homologs of the E. coli entD and Vibrio cholerae vibH genes were also detected in the flanking regions immediately adjacent to the B. abortus dhbCEBA operon, suggesting that B. abortus has the genetic capacity to produce a more complex 2,3-DHBA-based siderophore. Slot blot hybridization experiments and primer extension analysis showed that transcription of the B. abortus dhbCEBA operon originates from two iron-regulated promoters located upstream of dhbC. Consistent with their iron-dependent regulation, both of the dhbCEBA promoter sequences contain typical consensus Fur-binding motifs. Although previously published studies have shown that 2,3-DHBA production is not required for the establishment and maintenance of chronic spleen infection by B. abortus in mice, experimental infection of pregnant cattle with the B. abortus dhbC mutant BHB1 clearly showed that production of this siderophore is essential for wild-type virulence in the natural ruminant host.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC152065PMC
http://dx.doi.org/10.1128/IAI.71.4.1794-1803.2003DOI Listing
April 2003

Seeking a niche: putative contributions of the hfq and bacA gene products to the successful adaptation of the brucellae to their intracellular home.

Vet Microbiol 2002 Dec;90(1-4):349-63

Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27858-4354, USA.

Long-term residence of the brucellae in the phagosomal compartment of host macrophages is essential to their ability to produce disease in both natural and experimental hosts. Correspondingly, the Brucella spp. appear to be well adapted to resist the multiple environmental stresses they encounter in their intracellular home. This brief review will focus on the contributions of the hfq and bacA gene products to this adaptation. Studies with Brucella hfq mutants suggest that stationary phase physiology is critical for successful long-term residence in host macrophages. Analysis of Brucella bacA mutants, on the other hand, reveal very striking parallels between the strategies employed by the rhizobia to establish and maintain protracted intracellular residence in their plant host and those used by the brucellae during their long-term survival in the phagosomal compartment of host macrophages.
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http://dx.doi.org/10.1016/s0378-1135(02)00220-1DOI Listing
December 2002

Deficiency of a Sinorhizobium meliloti BacA mutant in alfalfa symbiosis correlates with alteration of the cell envelope.

J Bacteriol 2002 Oct;184(20):5625-32

Biology Department, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.

The BacA protein is essential for the long-term survival of Sinorhizobium meliloti and Brucella abortus within acidic compartments in plant and animal cells, respectively. Since both the S. meliloti and B. abortus bacA mutants have an increased resistance to bleomycin, it was hypothesized that BacA was a transporter of bleomycin and bleomycin-like compounds into the bacterial cell. However, our finding that the S. meliloti bacA mutant also has an increased sensitivity to detergents, a hydrophobic dye, ethanol, and acid pH supported a model in which BacA function affects the bacterial cell envelope. In addition, an S. meliloti lpsB mutant that is defective at a stage in infection of the host similar to that found for a bacA mutant is also sensitive to the same agents, and the carbohydrate content of its lipopolysaccharide (LPS) is altered. However, analysis of crude preparations of the bacA mutant LPS suggested that, unlike that for LpsB, BacA function did not affect the carbohydrate composition of the LPS. Rather, we found that at least one function of BacA is to affect the distribution of LPS fatty acids, including a very-long-chain fatty acid thought to be unique to the alpha-proteobacteria, including B. abortus.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC139620PMC
http://dx.doi.org/10.1128/JB.184.20.5625-5632.2002DOI Listing
October 2002

Major histocompatibility complex class I and II expression on macrophages containing a virulent strain of Brucella abortus measured using green fluorescent protein-expressing brucellae and flow cytometry.

FEMS Immunol Med Microbiol 2002 Jul;33(3):191-200

Department of Veterinary and Animal Sciences, Paige Laboratory, University of Massachusetts, Amherst, MA 01003, USA.

Immune responses appropriate for control of an intracellular pathogen are generated in mice infected with Brucella abortus, shown by the ability of T cells to adoptively transfer resistance to naive mice. The infection nevertheless persists for months. It was hypothesized that one factor in maintaining the infection despite the presence of immune T cells was suboptimal expression of major histocompatibility complex (MHC) molecules on macrophages containing brucellae. This would allow B. abortus to elude detection by the host's immune system. To test this, B. abortus organisms expressing green fluorescent protein (GFP-Brucella) were constructed and three-color flow cytometry used to evaluate MHC expression on macrophages following in vitro or in vivo infection. When infected in vitro, the levels of MHC class I and class II expression on J774 macrophages containing GFP-Brucella were the same or higher than on macrophages without GFP-Brucella in the same cultures. Similarly, the MHC expression was higher on GFP(+) peritoneal exudate cells following infection or phagocytosis of heat-killed GFP-Brucella than it was on uninfected peritoneal exudate cells. Following in vivo infection of mice the level of MHC class I and II expression on GFP(+) cells in their spleens (the main site of infection) also tended to be as high as or higher than that on the GFP-negative cells. The only in vivo GFP(+) cells that showed a decreased MHC expression was a population of splenic Mac1(+) cells recovered from interferon-gamma gene-disrupted mice at the time of their death due to an overwhelming number of bacteria per spleen. Overall, it was concluded that decreased MHC expression is not a general principle associated with brucella infection of macrophages and thus not likely to contribute to maintenance of the chronic infection.
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http://dx.doi.org/10.1111/j.1574-695X.2002.tb00590.xDOI Listing
July 2002

Brucella abortus siderophore 2,3-dihydroxybenzoic acid (DHBA) facilitates intracellular survival of the bacteria.

Microb Pathog 2002 May;32(5):239-48

Department of Veterinary and Animal Sciences, Paige Laboratory, University of Massachusetts, Amherst, MA 01003, USA.

Siderophores are low molecular weight molecules that allow bacteria to acquire iron from host cell proteins. 2,3-dihydroxybenzoic acid (DHBA) is the only known siderophore produced by the intracellular pathogen Brucella abortus. Here its role in virulence was assessed by evaluating the ability of a mutant with a disruption of the entC gene to survive and replicate in vitro in murine and bovine cells and in vivo in resistant and susceptible murine hosts. It was hypothesized that DHBA is vital for bacterial virulence by its ability to chelate intracellular iron thereby preventing generation of anti-bacterial hydroxyl radicals via the Haber-Weiss reaction, to scavenge reactive oxygen intermediates and for acquisition of iron needed for nutritional purposes. The data showed DHBA played a significant role for bacterial survival in host cells after infection including in murine macrophages cultured in the presence and absence of exogenous interferon-gamma (IFN-gamma) and in bovine trophoblasts supplemented with erythritol. In severely iron-depleted conditions, DHBA was also found to be essential for growth in murine macrophages. Despite these deficiencies, the absence of DHBA had no long-term significant effect on the number of CFU recovered in vivo from either the Brucella-resistant C57BL/6 mice or Brucella-susceptible IFN-gamma knock-out C57BL/6 mice.
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http://dx.doi.org/10.1006/mpat.2002.0500DOI Listing
May 2002

An examination of nucleus accumbens cell firing during extinction and reinstatement of water reinforcement behavior in rats.

Brain Res 2002 Mar;929(2):226-35

Department of Psychology, The University of North Carolina at Chapel Hill, CB# 3270, Davie Hall, Chapel Hill, NC 27599-3270, USA.

Electrophysiological recording procedures were used to examine nucleus accumbens (Acb) cell firing in rats (n = 13) during water reinforcement sessions consisting of three phases. During phase one (maintenance), a lever press resulted in water reinforcement (fixed ratio 1; 0.05 ml/press) paired with an auditory stimulus (0.5 s). Of 128 Acb neurons recorded during maintenance, 40 cells (31%) exhibited one of three types of neuronal firing patterns described previously [J. Neurosci. 14 (12) (1994) 7735-7746; J. Neurosci. 20 (11) (2000) 4255-4266]. Briefly, Acb neurons exhibited increases in firing rate within seconds preceding the reinforced response (type PR) or increases (type RFe) or decreases (type RFi) in activity seconds following response completion. In phase two (extinction), subsequent lever pressing had no programmed consequences (i.e., water reinforcement and the auditory stimulus were not presented). After 30 min of no responding, animals were given water reinforcement/auditory stimulus 'primes' to reestablish lever pressing behavior during the third phase (reinstatement). Results indicated that all types of phasic neurons (PR, RFe and RFi) exhibited an attenuated firing rate during extinction, and in some cases recovery of patterned discharges were observed during reinstatement. No significant changes in cell firing were observed for any cell type during presentation of the stimulus prime used to reestablish operant responding following extinction. These findings are discussed in terms of how Acb neurons process information related to 'natural' reinforcers versus drugs of abuse.
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http://dx.doi.org/10.1016/s0006-8993(01)03396-0DOI Listing
March 2002

Accumbens activity during a multiple schedule for water and sucrose reinforcement in rats.

Synapse 2002 Mar;43(4):223-6

Department of Psychology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3270, USA.

Electrophysiological recording procedures were used to examine nucleus accumbens (Acb) cell firing during operant responding for water reinforcement vs. a highly palatable sweet solution (0.6 M sucrose). Rats (n = 8) were trained on a multiple schedule to press one lever for water (fixed ratio 1, FR1; 15 min) followed by a 20-sec timeout period (chamber dark, levers retracted), and extension of a second spatially distinct lever that the animals pressed for sucrose reinforcement (FR1; 15 min). Of 84 cells, 55 neurons (65%) displayed one of three types of patterned discharges (increases or decreases in firing rate) immediately before or following the sucrose- or water-reinforced response. The major finding of this report was that the majority of these neurons (36/55 cells; 65%) showed similar types of neuronal firing patterns across the two reinforcer conditions. The remaining cells (19/55; 35%) exhibited patterned activity specific to responding for one reinforcer only (water or sucrose). These findings are discussed with respect to how Acb neurons encode goal-directed behaviors for "natural" reinforcers including food, water, and a palatable sweet solution.
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http://dx.doi.org/10.1002/syn.10041DOI Listing
March 2002

Brucella abortus HtrA functions as an authentic stress response protease but is not required for wild-type virulence in BALB/c mice.

Infect Immun 2001 Sep;69(9):5911-3

Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932, USA.

A second mutation has recently been identified in the previously described Brucella abortus htrA mutant PHE1. As a result of this finding, a new B. abortus htrA mutant, designated RWP11, was constructed to evaluate the biological function of the Brucella HtrA protease. RWP11 is more sensitive to oxidative killing in vitro and less resistant to killing by cultured murine neutrophils and macrophages than the virulent parental strain 2308 but is not attenuated in BALB/c mice through 4 weeks postinfection. The in vitro phenotype of B. abortus RWP11 is consistent with the proposed function of bacterial HtrA proteases as components of a secondary line of defense against oxidative damage. The in vivo phenotype of this mutant, however, indicates that, unlike the corresponding Salmonella and Yersinia proteins, Brucella HtrA does not play a critical role in virulence in the mouse model.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC98712PMC
http://dx.doi.org/10.1128/IAI.69.9.5911-5913.2001DOI Listing
September 2001
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