Construction of an expression plasmid (vector) encoding Brucella melitensis outer membrane protein,a candidate for DNA vaccine

Background:

DNA vaccination with plasmid encoding bacterial, viral, and parasitic immunogens has been shown to be an attractive method to induce efficient immune responses. Bacteria of the genus Brucella are facultative intracellular pathogens for which new and efficient vaccines are needed.

Methods:

To evaluate the use of a DNA immunization strategy for protection against brucellosis, a plasmid containing the DNA encoding the Brucella melitensis (B. melitensis) 31 kDa outer membrane protein, as a potent immunogenic target, was constructed.

Results:

The constructed plasmid, pcDNA3.1+omp31, was injected intramuscularly into mice and the expression of omp31 RNA was assessed by RT-PCR. The integrity of the pcDNA3.1+omp31 construct was confirmed with restriction analysis and sequencing. Omp31 mRNA expression was verified by RT-PCR.

Conclusion:

Our results indicate that the pcDNA3.1+omp31 eukaryotic expression vector expresses omp31 mRNA and could be useful as a vaccine candidate.Key Words: Brucella melitensis, omp31, DNA Vaccine, pcDNA3.1     

Identification of Brucella by MALDI-TOF mass spectrometry. Fast and reliable identification from agar plates and blood cultures

Background

MALDI-TOF mass spectrometry (MS) is a reliable method for bacteria identification. Some databases used for this purpose lack reference profiles for Brucella species, which is still an important pathogen in wide areas around the world. We report the creation of profiles for MALDI-TOF Biotyper 2.0 database (Bruker Daltonics, Germany) and their usefulness for identifying brucellae from culture plates and blood cultures.

Methodology/Principal Findings

We created MALDI Biotyper 2.0 profiles for type strains belonging to B. melitensis biotypes 1, 2 and 3; B. abortus biotypes 1, 2, 5 and 9; B. suis, B. canis, B ceti and B. pinnipedialis. Then, 131 clinical isolates grown on plate cultures were used in triplicate to check identification. Identification at genus level was always correct, although in most cases the three replicates reported different identification at species level. Simulated blood cultures were performed with type strains belonging to the main human pathogenic species (B. melitensis, B. abortus, B. suis and B. canis), and studied by MALDI-TOF MS in triplicate. Identification at genus level was always correct.

Conclusions/Significance

MALDI-TOF MS is reliable for Brucella identification to the genus level from culture plates and directly from blood culture bottles.     

Rapid Differential Diagnosis between Extrapulmonary Tuberculosis and Focal Complications of Brucellosis Using a Multiplex Real-Time PCR Assay

Background

Arduous to differ clinically, extrapulmonary tuberculosis and focal complications of brucellosis remain important causes of morbidity and mortality in many countries. We developed and applied a multiplex real-time PCR assay (M RT-PCR) for the simultaneous detection of Mycobacterium tuberculosis complex and Brucella spp.

Methodology

Conventional microbiological techniques and M RT-PCR for M. tuberculosis complex and Brucella spp were performed on 45 clinical specimens from patients with focal complications of brucellosis or extrapulmonary tuberculosis and 26 control samples. Fragments of 207 bp and 164 bp from the conserved region of the genes coding for an immunogenic membrane protein of 31 kDa of B. abortus (BCSP31) and the intergenic region SenX3-RegX3 were used for the identification of Brucella and M. tuberculosis complex, respectively.

Conclusions

The detection limit of the M RT-PCR was 2 genomes per reaction for both pathogens and the intra- and inter-assay coefficients of variation were 0.44% and 0.93% for Brucella and 0.58% and 1.12% for Mycobacterium. M RT-PCR correctly identified 42 of the 45 samples from patients with tuberculosis or brucellosis and was negative in all the controls. Thus, the overall sensitivity, specificity, PPV and NPV values of the M RT PCR assay were 93.3%, 100%, 100% and 89.7%, respectively, with an accuracy of 95.8% (95% CI, 91.1%–100%). Since M RT-PCR is highly reproducible and more rapid and sensitive than conventional microbiological tests, this technique could be a promising and practical approach for the differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.     

The Differential Interaction of Brucella and Ochrobactrum with Innate Immunity Reveals Traits Related to the Evolution of Stealthy Pathogens

Background

During evolution, innate immunity has been tuned to recognize pathogen-associated molecular patterns. However, some α-Proteobacteria are stealthy intracellular pathogens not readily detected by this system. Brucella members follow this strategy and are highly virulent, but other Brucellaceae like Ochrobactrum are rhizosphere inhabitants and only opportunistic pathogens. To gain insight into the emergence of the stealthy strategy, we compared these two phylogenetically close but biologically divergent bacteria.

Methodology/Principal Findings

In contrast to Brucella abortus, Ochrobactrum anthropi did not replicate within professional and non-professional phagocytes and, whereas neutrophils had a limited action on B. abortus, they were essential to control O. anthropi infections. O. anthropi triggered proinflammatory responses markedly lower than Salmonella enterica but higher than B. abortus. In macrophages and dendritic cells, the corresponding lipopolysaccharides reproduced these grades of activation, and binding of O. anthropi lipopolysaccharide to the TLR4 co-receptor MD-2 and NF-κB induction laid between those of B. abortus and enteric bacteria lipopolysaccharides. These differences correlate with reported variations in lipopolysaccharide core sugars, sensitivity to bactericidal peptides and outer membrane permeability.

Conclusions/Significance

The results suggest that Brucellaceae ancestors carried molecules not readily recognized by innate immunity, so that non-drastic variations led to the emergence of stealthy intracellular parasites. They also suggest that some critical envelope properties, like selective permeability, are profoundly altered upon modification of pathogen-associated molecular patterns, and that this represents a further adaptation to the host. It is proposed that this adaptive trend is relevant in other intracellular α-Proteobacteria like Bartonella, Rickettsia, Anaplasma, Ehrlichia and Wolbachia.     

The Attenuated Brucella abortus Strain 19 Invades,Persists in,and Activates Human Dendritic Cells,and Induces the Secretion of IL-12p70 but Not IL-23

Background

Bacterial vectors have been proposed as novel vaccine strategies to induce strong cellular immunity. Attenuated strains of Brucella abortus comprise promising vector candidates since they have the potential to induce strong CD4⁺ and CD8⁺ T-cell mediated immune responses in the absence of excessive inflammation as observed with other Gram-negative bacteria. However, some Brucella strains interfere with the maturation of dendritic cells (DCs), which is essential for antigen-specific T-cell priming. In the present study, we investigated the interaction of human monocyte-derived DCs with the smooth attenuated B. abortus strain (S) 19, which has previously been employed successfully to vaccinate cattle.

Methodology/Principal findings

We first looked into the potential of S19 to hamper the cytokine-induced maturation of DCs; however, infected cells expressed CD25, CD40, CD80, and CD86 to a comparable extent as uninfected, cytokine-matured DCs. Furthermore, S19 activated DCs in the absence of exogeneous stimuli, enhanced the expression of HLA-ABC and HLA-DR, and was able to persist intracellularly without causing cytotoxicity. Thus, DCs provide a cellular niche for persisting brucellae in vivo as a permanent source of antigen. S19-infected DCs produced IL-12/23p40, IL-12p70, and IL-10, but not IL-23. While heat-killed bacteria also activated DCs, soluble mediators were not involved in S19-induced activation of human DCs. HEK 293 transfectants revealed cellular activation by S19 primarily through engagement of Toll-like receptor (TLR)2.

Conclusions/Significance

Thus, as an immunological prerequisite for vaccine efficacy, B. abortus S19 potently infects and potently activates (most likely via TLR2) human DCs to produce Th1-promoting cytokines.     

Extensive cell envelope modulation is associated with virulence in Brucella abortus

Brucella virulence is linked to components of the cell envelope and tightly connected to the function of the BvrR/BvrS sensory-regulatory system. To quantify the impact of BvrR/BvrS on cell envelope proteins, we performed a label-free mass spectrometry-based proteomic analysis of spontaneously released outer membrane fragments from four strains of Brucella abortus (wild type virulent, avirulent bvrR- and bvrS- mutants as well as reconstituted virulent bvrR+ (bvrR-/pbvrR+)). We identified 167 differentially expressed proteins, of which 25 were assigned to the outer membrane. Approximately half of the outer membrane proteins decreased in abundance, whereas half increased. Notably, expression of five Omp3 family proteins decreased whereas five lipoproteins increased in the mutant strains. In the periplasmic space, by contrast, approximately 80% of the 60 differentially expressed proteins were increased in at least one avirulent mutant. Periplasmic proteins are primarily involved in substrate uptake and transport, and a uniform increase in this class may indicate a nutritional stress response, possibly a consequence of defective outer membrane function. Virtually all proteins reverted to wild type levels in the reconstituted virulent bvrR+ strain. We propose that the wide changes in cell envelope protein expression relate to the markedly avirulent phenotype of bvrR- and bvrS- mutants and that Brucella virulence depends on regulatory networks involving cell envelope and metabolism rather than on discrete virulence factors. This model may be relevant to other alpha-Proteobacteria harboring BvrR/BvrS orthologous systems known to be essential for parasitism or endosymbiosis.     

Brucellosis vaccines: assessment of Brucella melitensis lipopolysaccharide rough mutants defective in core and O-polysaccharide synthesis and export

Background

The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines.

Methodology/Principal Findings

To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies.

Conclusions/Significance

The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.     

RpoS Regulates Essential Virulence Factors Remaining to Be Identified in Borrelia burgdorferi

Background

Since the RpoN-RpoS regulatory network was revealed in the Lyme disease spirochete Borrelia burgdorferi a decade ago, both upstream and downstream of the pathway have been intensively investigated. While significant progress has been made into understanding of how the network is regulated, most notably, discovering a relationship of the network with Rrp2 and BosR, only three crucial virulence factors, including outer surface protein C (OspC) and decorin-binding proteins (Dbps) A and B, are associated with the pathway. Moreover, for more than 10 years no single RpoS-controlled gene has been found to be critical for infection, raising a question about whether additional RpoS-dependent virulence factors remain to be identified.

Methodology/Principal Findings

The rpoS gene was deleted in B. burgdorferi; resulting mutants were modified to constitutively express all the known virulence factors, OspC, DbpA and DbpB. This genetic modification was unable to restore the rpoS mutant with infectivity.

Conclusions/Significance

The inability to restore the rpoS mutant with infectivity by simultaneously over-expressing all the three virulence factors allows us to conclude RpoS also regulates essential genes that remain to be identified in B. burgdorferi.     

Identification and Effect Decomposition of Risk Factors for Brucella Contamination of Raw Whole Milk in China

Background

Lack of clear risk factor identification is the main reason for the persistence of brucellosis infection in the Chinese population, and there has been little assessment of the factors contributing to Brucella contamination of raw whole milk. The purpose of this study was to identify risk factors affecting Brucella contamination of raw milk, and to evaluate effective measures for disease reduction in order to determine preventive strategies.

Methods and Findings

A nationwide survey was conducted and samples were obtained from 5211 cows corresponding to 25 sampling locations throughout 15 provinces in China. The prevalence of Brucella in the raw milk samples averaged 1.07% over the 15 Chinese provinces, while the prevalence of positive areas within these regions ranged from 0.23–3.84% among the nine provinces with positive samples. The survey examined factors that supposedly influence Brucella contamination of raw whole milk, such as management style, herd size, abortion rate, hygiene and disease control practices. A binary logistic regression analysis was carried out to determine the association between risk factors for Brucella and contamination of milk samples. Furthermore, a relative effect decomposition study was conducted to determine effective strategies for reducing the risk of Brucella contamination of raw whole milk. Our data indicate that disease prevention and control measures, abortion rate, and animal polyculture are the most important risk factors. Meanwhile, culling after quarantine was identified as an effective protective measure in the current Chinese dairy situation.

Conclusions

These results indicate that, although there is a low risk of contamination of milk with Brucella nationwide in China, there are individual regions where contamination is a significant problem. Controlling three factors–culling after quarantine, maintaining a low abortion rate, and avoiding mixing groups of cattle and small ruminants–could effectively reduce the risk of Brucella contamination of raw whole milk.     

Gene polymorphism analysis of Yersinia enterocolitica outer membrane protein A and putative outer membrane protein A family protein

Background

Yersinia enterocolitica outer membrane protein A (OmpA) is one of the major outer membrane proteins with high immunogenicity. We performed the polymorphism analysis for the outer membrane protein A and putative outer membrane protein A (p-ompA) family protein gene of 318 Y. enterocolitica strains.

Results

The data showed all the pathogenic strains and biotype 1A strains harboring ystB gene carried both ompA and p-ompA genes; parts of the biotype 1A strains not harboring ystB gene carried either ompA or p-ompA gene. In non-pathogenic strains (biotype 1A), distribution of the two genes and ystB were highly correlated, showing genetic polymorphism. The pathogenic and non-pathogenic, highly and weakly pathogenic strains were divided into different groups based on sequence analysis of two genes. Although the variations of the sequences, the translated proteins and predicted secondary or tertiary structures of OmpA and P-OmpA were similar.

Conclusions

OmpA and p-ompA gene were highly conserved for pathogenic Y. enterocolitica. The distributions of two genes were correlated with ystB for biotype 1A strains. The polymorphism analysis results of the two genes probably due to different bio-serotypes of the strains, and reflected the dissemination of different bio-serotype clones of Y. enterocolitica.     

Processing of Chlamydia abortus Polymorphic Membrane Protein 18D during the Chlamydial Developmental Cycle

Background

Chlamydia possess a unique family of autotransporter proteins known as the Polymorphic membrane proteins (Pmps). While the total number of pmp genes varies between Chlamydia species, all encode a single pmpD gene. In both Chlamydia trachomatis (C. trachomatis) and C. pneumoniae, the PmpD protein is proteolytically cleaved on the cell surface. The current study was carried out to determine the cleavage patterns of the PmpD protein in the animal pathogen C. abortus (termed Pmp18D).

Methodology/Principal Findings

Using antibodies directed against different regions of Pmp18D, proteomic techniques revealed that the mature protein was cleaved on the cell surface, resulting in a100 kDa N-terminal product and a 60 kDa carboxy-terminal protein. The N-terminal protein was further processed into 84, 76 and 73 kDa products. Clustering analysis resolved PmpD proteins into three distinct clades with C. abortus Pmp18D, being most similar to those originating from C. psittaci, C. felis and C. caviae.

Conclusions/Significance

This study indicates that C. abortus Pmp18D is proteolytically processed at the cell surface similar to the proteins of C. trachomatis and C. pneumoniae. However, patterns of cleavage are species-specific, with low sequence conservation of PmpD across the genus. The absence of conserved domains indicates that the function of the PmpD molecule in chlamydia remains to be elucidated.