RecA and RadA Proteins of Brucella abortus Do Not Perform Overlapping Protective DNA Repair Functions following Oxidative Burst

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. Originally published Journal of Bacteriology, Vol. 188, No. 14, July 200

Genome Degradation in Brucella ovis Corresponds with Narrowing of Its Host Range and Tissue Tropism

Brucella ovis is a veterinary pathogen associated with epididymitis in sheep. Despite its genetic similarity to the zoonotic pathogens B. abortus, B. melitensis and B. suis, B. ovis does not cause zoonotic disease. Genomic analysis of the type strain ATCC25840 revealed a high percentage of pseudogenes and increased numbers of transposable elements compared to the zoonotic Brucella species, suggesting that genome degradation has occurred concomitant with narrowing of the host range of B. ovis. The absence of genomic island 2, encoding functions required for lipopolysaccharide biosynthesis, as well as inactivation of genes encoding urease, nutrient uptake and utilization, and outer membrane proteins may be factors contributing to the avirulence of B. ovis for humans. A 26.5 kb region of B. ovis ATCC25840 Chromosome II was absent from all the sequenced human pathogenic Brucella genomes, but was present in all of 17 B. ovis isolates tested and in three B. ceti isolates, suggesting that this DNA region may be of use for differentiating B. ovis from other Brucella spp. This is the first genomic analysis of a non-zoonotic Brucella species. The results suggest that inactivation of genes involved in nutrient acquisition and utilization, cell envelope structure and urease may have played a role in narrowing of the tissue tropism and host range of B. ovis

Spiroplasma spp. biofilm formation is instrumental for their role in the pathogenesis of plant, insect and animal diseases

Spiroplasma spp. are important phyto and insect pathogens, and candidate causal agent/s of transmissible spongiform encephalopathies (TSE) in man and animals. These filterable wall-less bacteria are widely distributed in nature with an unspecified environmental reservoir. In this study we showed by scanning electron microscopy that spiroplasma form biofilm on an assortment of hard surfaces including mica, nickel and stainless steel. Spiroplasma were stuck to the surfaces by fibrillar threads consistent with curli fibers (an amyloid protein found in bacterial biofilms). After a lengthy time in cultures (6. weeks), spiroplasma in biofilm bound to mica disks lost their spiral shapes and formed coccoid forms interconnected by long (\u3e 2 μm) branched membranous nanotubules, therein representing direct conjugate connections between the cells. The affinity of spiroplasma biofilms for mica and nickel, and the membrane communications suggest that soil could be a reservoir for these bacteria. The persistence of clay bound spiroplasma in soil could serve as the mechanism of lateral spread of TSEs by ingestion of soil by ruminants. Spiroplasma binding to stainless steel wire supports bacterial contamination of surgical instruments following surgery on dementia patients as a mechanism of iatrogenic transmission of TSEs, especially with resistance of spiroplasma in biofilms to drying or exposure to 50% glutaraldehyde. The discovery of biofilm formation by spiroplasma addresses questions regarding environmental persistence of these organisms in nature and suggests novel mechanisms of intercellular communication and transmission. © 2012 Elsevier Inc.

Brucella species lacking the major outer membrane protein Omp25 are attenuated in mice and protect against Brucella melitensis and Brucella ovis

To aid in the development of novel efficacious vaccines against brucellosis, Omp25 was examined as a potential candidate. To determine the role of Omp25 in virulence, mutants were created with Brucella abortus (BA25), Brucella melitensis (BM25), and Brucella ovis (BO25) which contain disruptions in the omp25 gene (Δomp25 mutants). Western immunoblot analysis and PCR verified that the Omp25 protein was not expressed and that the omp25 gene was disrupted in each strain. BALB/c mice infected with B. abortus BA25 or B. melitensis BM25 showed a significant decrease in mean CFU/spleen at 18 and 4 weeks post-infection, respectively, when compared to the virulent parental strain (P\u3c0.05, n=5). Mice infected with B. ovis BO25 had significantly lower mean CFU/spleen counts from 1 to 8 weeks post-infection, at which point the mutant was cleared from the spleens (P\u3c0.01, n=5). Murine vaccination with either BM25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in more than a 2log10 reduction in bacterial load following challenge with virulent B. melitensis (P\u3c0.01, n=5). Vaccination of mice with the B. ovis mutant resulted in clearance of the challenge strain and provided 2.5log10 greater protection against virulent B. ovis than vaccine strain Rev. 1. Based on these data, the B. melitensis and B. ovis Δomp25 mutants are interesting vaccine candidates that are currently under study in our laboratory for their safety and efficacy in small ruminants. © 2002 Elsevier Science B.V. All rights reserved

A Brucella melitensis high temperature requirement A (htrA) deletion mutant demonstrates a stress response defective phenotype in vitro and transient attenuation in the BALB/c mouse model

Bacterial stress response proteins of the high temperature requirement A (HtrA) family are serine proteases which appear to play an important role in scavenging oxidatively damaged proteins from the cell before they reach toxic levels. An isogenic htrA deletion mutant, designated RWP5, was constructed from virulent Brucella melitensis 16M via gene replacement to determine whether the B. melitensis HtrA protein functions as a stress response protein, and to evaluate the contribution of this protein to virulence. Unlike the parental strain, RWP5 would not form isolated colonies on solid media at 40°C or grow on Schaedler agar without blood supplementation. RWP5 also grew poorly in broth culture in contrast to 16M. The B. melitensis htrA mutant was significantly more sensitive (P \u3c 0.001) to killing by H2O2 and puromycin than the parental strain, and a significant reduction (P \u3c 0.001) in the number of RWP5 recovered from the spleens and livers of experimentally infected BALB/c mice was observed at one week post infection compared to 16M. However, by 3 weeks post-infection and continuing thereafter through to 20 weeks post-infection, the levels of RWP5 and 16M recovered from the spleens and livers of experimentally infected mice were similar. In vitro and in vivo evaluation of RWP5 reisolates obtained from the spleens of mice at 4 and 16 weeks post-infection demonstrated that mouse passage did not significantly alter these characteristic in vitro and in vivo properties of RWP5. These results support a stress response function for the B. melitensis HtrA protein and suggest that this protein contributes to the pathogenesis of B. melitensis early in infection. The basis for the recovery of RWP5 at later time points in infected mice is presently unknown; however, the results presented here suggest that it is not caused by a stable genetic change resulting from mouse passage

An indirect ELISA to detect the serologic response of elk (Cervus elaphus nelsoni) inoculated with Brucella abortus strain RB51

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to identify elk (Cervus elaphus nelsoni) with Brucella abortus strain RB51 (RB51)-specific antibodies using a mouse monoclonal antibody specific for bovine IgG1. This test was relatively easy to perform, accurate, and easily reproducible; therefore it could be standardized for use between laboratories. In addition, we attempted to compensate for inherent variabilities encountered when comparing ELISA readings from multiple samples taken from many animals over time. Optical density (OD) readings for each sample were converted into a percent positivity value for analysis. A negative cutoff value was determined above which a sample was considered to have a significantly elevated anti-RB51 antibody level. Pre- and postvaccination sera from 64 6-8 mo old elk, divided into four groups (females subcutaneously inoculated with saline (control animals), females ballistically inoculated with RB51, females subcutaneously inoculated with RB51, and males subcutaneously inoculated with RB51) were used. All serum samples were collected between 27 April and 15 November 1995. Values for all saline controls were appropriately below the negative cutoff value. All subcutaneously and ballistically inoculated elk were serologically positive to RB51 for at least two sampling periods during the study. The difference in percent positivity values for the ballistically compared to the subcutaneously inoculated groups was not statistically significant at 8, 10, 14, or 18 wk postvaccination. This suggests that processing RB51 into lactose based pellets and ballistically inoculating elk with these pellets does not alter the detectable elk antibody response. Also, inoculated and control animals can be accurately identified with ELISA at 4-8 weeks postvaccination

In vitro and in vivo phenotypes resulting from deletion of the high temperature requirement A (htrA) gene from the bovine vaccine strain Brucella abortus S19

An htrA deletion mutant was created in the bovine vaccine strain, B. abortus S19, by replacing the majority of the htrA gene with a kanamycin resistance gene. Antibiotic selection for a double crossover event yielded kanamycin-resistant, ampicillin-sensitive colonies confirmed by Southern and western blot analysis to be HtrA deficient. The B. abortus S19 htrA mutant was significantly more susceptible than the parental strain to killing by H2O2 (P \u3c 0.001) and O2- generated by the redox cycling agent paraquat (P \u3c 0.05) in disk sensitivity assays. Deletion of the htrA gene from S19 produced a bimodal effect on the spleen colonization profile of this strain in BALB/c mice. At one week post-infection, the B. abortus S19 htrA mutant colonized the spleens of experimentally infected BALB/c mice at significantly lower levels (P \u3c 0.01) than the parental strain. Enhanced clearance (P \u3c 0.05) was also observed at later time points, i.e. 4 and 7 weeks post infection, however at 2 and 3 weeks post infection, the mutant and parental strains colonized the mice at equivalent levels. The temporal development of specific delayed type hypersensitivity and antibody responses in BALB/c mice infected with the mutant or parental strain were equivalent. These results suggest that the htrA gene product contributes to successful host colonization by S19. However, deletion of this gene does not radically alter the overall, characteristic spleen colonization profile of this vaccine strain in the BALB/c mouse model, nor compromise the capacity of this strain to elicit Brucella specific cellular or humoral immune responses in this experimental host

Evaluation of immune response to orally administered Sterne strain 34F2 anthrax vaccine

Present animal vaccines against Bacillus anthracis infection are capable of inducing protective immunity. However, due to the route of administration, the vaccine has limited or no use in wildlife especially in rural areas of the world. Hence, an oral vaccine is needed for controlling this disease. For proof of concept we used the commercially available Sterne strain 34F2 vaccine mixed with oral scarifying agents. Although the immunological response as measured by ELISA in this group was not equal to the parenterally inoculated animals, the results indicate that the oral administration of this vaccine with oropharyngeal mucosa scarifying agents mixed with feed can induce immune responses in goats. © 2007 Elsevier Ltd. All rights reserved