scholarly journals Evaluation of Protection Afforded by Brucella abortus and Brucella melitensis Unmarked Deletion Mutants Exhibiting Different Rates of Clearance in BALB/c Mice

2006 ◽  
Vol 74 (7) ◽  
pp. 4048-4057 ◽  
Author(s):  
M. M. Kahl-McDonagh ◽  
T. A. Ficht
Keyword(s):  
Mouse Model ◽  
Brucella Abortus ◽  
Protective Immunity ◽  
Brucella Melitensis ◽  
Deletion Mutants ◽  
Heterologous Challenge ◽  
Current Vaccine ◽  
Protection Against Infection ◽  
Ideal Vaccine

ABSTRACT Research for novel Brucella vaccines has focused upon the development of live vaccine strains, which have proven more efficacious than killed or subunit vaccines. In an effort to develop improved vaccines, signature-tagged mutant banks were screened to identify mutants attenuated for survival. Mutants selected from these screens exhibited various degrees of attenuation characterized by the rate of clearance, ranging from a failure to grow in macrophages after 24 h of infection to a failure to persist in the mouse model beyond 8 weeks. Ideal vaccine candidates should be safe to the host, while evoking protective immunity. In the present work, we constructed unmarked deletion mutants of three gene candidates, manBA, virB2, and asp24, in both Brucella abortus and Brucella melitensis. The Δasp24 mutants, which persist for extended periods in vivo, are superior to current vaccine strains and to other deletion strains tested in the mouse model against homologous challenge infection after 12, 16, and 20 weeks postvaccination. The Δasp24 mutants also display superior protection compared to ΔmanBA and ΔvirB2 mutants against heterologous challenge in mice. From this study, a direct association between protection against infection and cytokine response was not apparent between all vaccine groups and, therefore, correlates of protective immunity will need to be considered further. A distinct correlation between persistence of the vaccine strain and protection against infection was corroborated.

scholarly journals Brucella abortus Δ rpoE1 confers protective immunity against wild type challenge in a mouse model of brucellosis

Vaccine ◽  
2016 ◽  
Vol 34 (42) ◽  
pp. 5073-5081 ◽  
Author(s):  
Jonathan W. Willett ◽  
Julien Herrou ◽  
Daniel M. Czyż ◽  
Jason X. Cheng ◽  
Sean Crosson
Keyword(s):  
Mouse Model ◽  
Brucella Abortus ◽  
Protective Immunity ◽  
Wild Type

scholarly journals WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System

2016 ◽  
Vol 198 (8) ◽  
pp. 1281-1293 ◽  
Author(s):  
Julien Herrou ◽  
Daniel M. Czyż ◽  
Jonathan W. Willett ◽  
Hye-Sook Kim ◽  
Gekleng Chhor ◽  
...  
Keyword(s):  
Stress Response ◽  
Mouse Model ◽  
Binding Site ◽  
Brucella Abortus ◽  
Chronic Infection ◽  
Content Type ◽  
General Stress Response ◽  
General Stress

ABSTRACTThe general stress response (GSR) system of the intracellular pathogenBrucella abortuscontrols the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required forB. abortussurvival under nonoptimal growth conditionsin vitroand for maintenance of chronic infection in anin vivomouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined.bab1_1070is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditionsin vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However,B. abortusWrbA-relatedprotein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductasein vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion ofwrpA(ΔwrpA) does not compromise cell survival under acute oxidative stressin vitroor attenuate infection in cell-based or mouse models. However, a ΔwrpAstrain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulatesB. abortusinteraction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose thatB. abortusWrpA represents a functionally distinct member of the diverse flavodoxin family.IMPORTANCEBrucella abortusis an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system ofB. abortuscontrols the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes remain uncharacterized. We presentin vitroandin vivofunctional and structural analyses of WrpA, whose expression is strongly induced by GSR under oxidative conditions. Though WrpA is structurally related to NADH:quinone oxidoreductases, it does not bind redox cofactors in solution, nor does it exhibit oxidoreductase activityin vitro. However, WrpA does affect spleen inflammation in a murine infection model. Our data provide evidence that WrpA forms a new functional class of WrbA/flavodoxin family proteins.

scholarly journals Complementation of Brucella abortus RB51 with a Functional wboA Gene Results in O-Antigen Synthesis and Enhanced Vaccine Efficacy but No Change in Rough Phenotype and Attenuation

2000 ◽  
Vol 68 (7) ◽  
pp. 3927-3932 ◽  
Author(s):  
Ramesh Vemulapalli ◽  
Yongqun He ◽  
Larissa S. Buccolo ◽  
Stephen M. Boyle ◽  
Nammalwar Sriranganathan ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Brucella Melitensis ◽  
Virulent Strain ◽  
Polymyxin B ◽  
Gamma Interferon ◽  
T Cell Response ◽  
Bacterial Cells ◽  
O Antigen

ABSTRACT Brucella abortus RB51 is a stable rough, attenuated mutant vaccine strain derived from the virulent strain 2308. Recently, we demonstrated that the wboA gene in RB51 is disrupted by an IS711 element (R. Vemulapalli, J. R. McQuiston, G. G. Schurig, N. Srirauganathan, S. M. Halling, and S. M. Boyle, Clin. Diagn. Lab. Immunol. 6:760–764, 1999). Disruption of the wboA gene in smooth, virulent B. abortus, Brucella melitensis, and Brucella suis results in rough, attenuated mutants which fail to produce the O polysaccharide (O antigen). In this study, we explored whether the wboA gene disruption is responsible for the rough phenotype of RB51. We complemented RB51 with a functionalwboA gene, and the resulting strain was designated RB51WboA. Colony and Western blot analyses indicated that RB51WboA expressed the O antigen; immunoelectron microscopy revealed that the O antigen was present in the cytoplasm. Crystal violet staining, acryflavin agglutination, and polymyxin B sensitivity studies indicated that RB51WboA had rough phenotypic characteristics similar to those of RB51. Bacterial clearance studies of BALB/c mice indicated no increase in the survival ability of RB51WboA in vivo compared to that of RB51. Vaccination of mice with live RB51WboA induced antibodies to the O antigen which were predominantly of the immunoglobulin G2a (IgG2a) and IgG3 isotypes. After in vitro stimulation of splenocytes with killed bacterial cells, quantitation of gamma interferon in the culture supernatants indicated that RB51WboA immunization induced higher levels of gamma interferon than immunization with RB51. Mice vaccinated with RB51WboA were better protected against a challenge infection with the virulent strain 2308 than those vaccinated with RB51. These studies indicate that in addition to the disruption of the wboAgene there is at least one other mutation in RB51 responsible for its rough phenotype. These studies also suggest that the expressed O antigen in RB51WboA is responsible either directly or indirectly for the observed enhancement in the T-cell response.

scholarly journals Brucella abortus HtrA Functions as an Authentic Stress Response Protease but Is Not Required for Wild-Type Virulence in BALB/c Mice

2001 ◽  
Vol 69 (9) ◽  
pp. 5911-5913 ◽  
Author(s):  
Robert W. Phillips ◽  
R. Martin Roop
Keyword(s):  
Stress Response ◽  
Mouse Model ◽  
Brucella Abortus ◽  
Parental Strain ◽  
Biological Function ◽  
Critical Role ◽  
Wild Type ◽  
Secondary Line

ABSTRACT 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 htrAmutant, 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. abortusRWP11 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 Salmonellaand Yersinia proteins, Brucella HtrA does not play a critical role in virulence in the mouse model.

scholarly journals In VivoDifferences in the Virulence, Pathogenicity, and Induced Protective Immunity ofwboAMutants from Genetically Different Parent Brucella spp.

2012 ◽  
Vol 20 (2) ◽  
pp. 174-180 ◽  
Author(s):  
Zhen Wang ◽  
Jianrui Niu ◽  
Shuangshan Wang ◽  
Yanli Lv ◽  
Qingmin Wu
Keyword(s):  
Survival Time ◽  
Protective Immunity ◽  
Brucella Melitensis ◽  
Survival Times ◽  
Content Type ◽  
Virulence Assay ◽  
Pregnant Sheep ◽  
Or Genes ◽  
Rough Mutant

ABSTRACTTo explore the effects of the genetic background on the characteristics ofwboAgene deletion rough mutants generated from different parentBrucellasp. strains, we constructed the rough-mutant strainsBrucella melitensis16 M-MB6,B. abortus2308-SB6,B. abortusS19-RB6, andB. melitensisNI-NB6 and evaluated their survival, pathogenicity, and induced protective immunity in mice and sheep. In mice, the survival times of the four mutants were very different in the virulence assay, from less than 6 weeks forB. abortusS19-RB6 to 11 weeks forB. abortus2308-SB6 andB. melitensisNI-NB6. However,B. abortusS19-RB6 andB. melitensis16 M-MB6, with a shorter survival time in mice, offered better protection against challenges withB. abortus2308 in protection tests thanB. abortus2308-SB6 andB. melitensisNI-NB6. It seems that the induced protective immunity of each mutant might not be associated with its survival timein vivo. In the cross-protection assay, bothB. melitensis16 M-MB6 andB. abortusS19-RB6 induced greater protection against homologous challenges than heterologous challenges. When pregnant sheep were inoculated withB. abortusS19-RB6 andB. melitensis16 M-MB6,B. abortusS19-RB6 did not induce abortion, whereasB. melitensis16 M-MB6 did. These results demonstrated the differences in virulence, pathogenicity, and protective immunityin vivoin thewboAdeletion mutants from genetically different parentBrucellaspp. and also indicated that future rough vaccine strain development could be promising if suitable parentBrucellastrains and/or genes were selected.

Role of Base Excision Repair in Listeria monocytogenes DNA Stress Survival During Infections

2020 ◽  
Author(s):  
Juan Zhang ◽  
Shuyi Wang ◽  
Tjakko Abee ◽  
Stijn van der Veen
Keyword(s):  
Listeria Monocytogenes ◽  
Mouse Model ◽  
Base Excision Repair ◽  
Bacterial Infections ◽  
Excision Repair ◽  
Deletion Mutants ◽  
Dna Glycosylases ◽  
Repair Mechanisms ◽  
Base Excision

Abstract Background Base excision repair (BER), consisting mostly of lesion-specific DNA glycosylases and apurinic/apyrimidinic (AP) endonucleases, is one of the most important DNA repair mechanisms for repair of single nucleobase lesions generated by reactive oxygen and nitrogen species as part of an immune response against bacterial infections. However, few studies have addressed the contribution of BER to bacterial virulence and Listeria monocytogenes BER has thus far remained completely uncharacterized. Methods Analysis of the L. monocytogenes EGDe genome identified 7 DNA glycosylases (MutM, MutY, Nth, Tag, Mpg, Ung, and Ung2) and 2 apurinic/apyrimidinic endonucleases (Xth and Nfo) as part of BER. Markerless in-frame deletion mutants were generated for all 9 genes, and mutants were tested for DNA damage survival, mutagenesis, and the ability to colonize a mouse model of infection. Results Distinct lesion-specific phenotypes were identified for all deletion mutants. Importantly, the Δnth, ΔmutY, and Δnfo mutants were significantly attenuated for virulence in the mouse model and showed much lower colonization of the liver and spleen or were unable to compete with the wild-type strain during in vivo competition assays. Conclusions Our results highlight the importance of BER for L. monocytogenes virulence and survival of DNA-damaging insults during host colonization.

scholarly journals Vaccination with Brucella abortus Recombinant In Vivo-Induced Antigens Reduces Bacterial Load and Promotes Clearance in a Mouse Model for Infection

PLoS ONE ◽  
2011 ◽  
Vol 6 (3) ◽  
pp. e17425 ◽  
Author(s):  
Jake E. Lowry ◽  
Dale D. Isaak ◽  
Jack A. Leonhardt ◽  
Giulia Vernati ◽  
Jessie C. Pate ◽  
...  
Keyword(s):  
Mouse Model ◽  
Brucella Abortus ◽  
Bacterial Load

scholarly journals Attenuated Signature-Tagged Mutagenesis Mutants ofBrucella melitensis Identified during the Acute Phase ofInfection inMice

2003 ◽  
Vol 71 (12) ◽  
pp. 7053-7060 ◽  
Author(s):  
P. Lestrate ◽  
A. Dricot ◽  
R.-M. Delrue ◽  
C. Lambert ◽  
V. Martinelli ◽  
...  
Keyword(s):  
Mouse Model ◽  
Molecular Mechanisms ◽  
Brucella Melitensis ◽  
Functional Protein ◽  
Transposon Insertion ◽  
Gene Coding ◽  
Insertion Sites ◽  
Mouse Model Of Infection

ABSTRACT For this study, we screened 1,152 signature-tagged mutagenesis mutants of Brucella melitensis 16M in a mouse model of infection and found 36 of them to be attenuated in vivo. Molecular characterization of transposon insertion sites showed that for four mutants, the affected genes were only present in Rhizobiaceae. Another mutant contained a disruption in a gene homologous to mosA, which is involved in rhizopine biosynthesis in some strains of Rhizobium, suggesting that this sugar may be involved in Brucella pathogenicity. A mutant was disrupted in a gene homologous to fliF, a gene potentially coding for the MS ring, a basal component of the flagellar system. Surprisingly, a mutant was affected in the rpoA gene, coding for the essentialα -subunit of the RNA polymerase. This disruption leaves a partially functional protein, impaired for the activation of virB transcription, as demonstrated by the absence of induction of the virB promoter in the Tn5::rpoA background. The results presented here highlight the fact that the ability of Brucella to induce pathogenesis shares similarities with the molecular mechanisms used by both Rhizobium and Agrobacterium to colonize their hosts.

scholarly journals Effect of P39 Gene Deletion in LiveBrucella Vaccine Strains on Residual Virulence and Protective Activity in Mice

1998 ◽  
Vol 66 (11) ◽  
pp. 5561-5564 ◽  
Author(s):  
Anne Tibor ◽  
Isabelle Jacques ◽  
Laurence Guilloteau ◽  
Jean-Michel Verger ◽  
Maggy Grayon ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Gene Deletion ◽  
Brucella Melitensis ◽  
Gene Replacement ◽  
Deletion Mutants ◽  
Protective Activity ◽  
Mutant Strains ◽  
Virulent Challenge ◽  
Residual Virulence

ABSTRACT The 39-kilodalton protein (P39) has previously been shown to be an immunodominant protein in Brucella infections. P39 gene deletion mutants of vaccine strains Brucella abortus S19 and Brucella melitensis Rev.1 were constructed by gene replacement. This deletion did not significantly modify the residual virulence of both vaccine strains in CD-1 mice. CD-1 mice vaccinated with the parent or mutant strains were protected against a virulent challenge. Mutant vaccine strains devoid of P39 could provide a means for differentiating vaccinated from infected animals.

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