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The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.

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Articles by Gary Splitter in JoVE

 JoVE Immunology and Infection

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells


JoVE 2250 11/09/2010

1, 2, 2

1Pathology and Laboratory Medicine, University of Wisconsin-Madison, 2Pathobiological Sciences, University of Wisconsin-Madison

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Other articles by Gary Splitter on PubMed

Susceptibility of IFN Regulatory Factor-1 and IFN Consensus Sequence Binding Protein-deficient Mice to Brucellosis

Journal of Immunology (Baltimore, Md. : 1950). Mar, 2002  |  Pubmed ID: 11859135

IFN-gamma is a key cytokine controlling Brucella infection, and the diverse functions of this cytokine are mediated by IFN regulatory factors (IRFs) such as IRF-1, IRF-2, and IFN consensus sequence binding protein (ICSBP). However, the roles of these three IRFs in Brucella infection have not been investigated. The infection of each IRF-deficient mouse strain provides an opportunity to determine not only the significance of each IRF molecule but also the crucial immune components necessary for host defense during in vivo infection, because respective IRF-deficient mouse strains contain unique immunodeficient phenotypes. Brucella abortus S2308-infected IRF-1-/- mice were dead within 2 wk postinfection, while IRF-2-/- mice contained less splenic Brucella CFU than wild-type mice at the early stage of infection. Infected ICSBP-/- mice maintained a plateau of splenic Brucella CFU throughout the infection. Additional infection of IL-12p40-, NO synthase 2-, and gp91(phox)-deficient mice indicates that these immune components are crucial for Brucella immunity and may contribute to the susceptibility of IRF-1-/- and ICSBP-/- mice. Immunologic and histopathological analyses of infected IRF-1-/- mice indicate that the absence of IL-12p40 induction and serious hepatic damage are involved in the death of IRF-1-/- mice. These results indicate that 1) IRF-1 and ICSBP are essential transcriptional factors for IFN-gamma-mediated protection against Brucella; 2) IL-12, reactive nitrogen intermediates, and reactive oxygen intermediates are crucial immune components against Brucella, and their absence may contribute to the susceptibility of IRF-1-/- and ICSBP-/- mice; and 3) hepatic damage caused by Brucella virulence contributes to the death of IRF-1-/- mice.

Encephalomyocarditis and Mengo Viruses Productively Infect Murine T-lymphocyte Cell Lines but Not Fresh Ex Vivo Derived T Lymphocytes

Viral Immunology. 2002  |  Pubmed ID: 11952137

Encephalomyocarditis virus (EMCV) and Mengo virus are highly virulent murine cardioviruses that are found in abundant quantities in the spleen and lymph nodes after infection. T lymphocytes are pivotal mediators of humoral and cellular immunity against cardioviral challenge, and are highly suspect candidates of EMCV and Mengo virus infection. We found T lymphocyte-like cell lines CTLL-2, EL-4, LY1+2/9, and LBRM33 were susceptible to productive viral infection and exhibited cytopathology after infection with virulent EMCV-R or attenuated Mengo virus strains vMC0 and vMC24. Flow cytometric analysis demonstrated progressive intracellular accumulation of viral proteins, such as the replication-dependent 3D viral polymerase, in EL-4 cells during infection. Conversely, freshly isolated and mitogen-stimulated CD4+ and CD8+ T cells were resistant to productive infection with these viruses, exhibiting no viral-induced cytopathic effects or intracellular presence of viral proteins. These data indicate that although T-lymphocyte-like tumor cell lines are highly susceptible to viral infection and cytopathic effects, primary/freshly isolated T cells are resistant to infection by EMCV-R or Mengo virus.

Molecular and Immunological Characterisation of Recombinant Brucella Abortus Glyceraldehyde-3-phosphate-dehydrogenase, a T- and B-cell Reactive Protein That Induces Partial Protection when Co-administered with an Interleukin-12-expressing Plasmid in a DNA Vaccine Formulation

Journal of Medical Microbiology. Aug, 2002  |  Pubmed ID: 12171297

To identify antigens of Brucella spp. that are potentially involved in stimulating a protective T-cell-mediated immune response, previous studies identified 10 clones from a Brucella abortus 2308 genomic library with primed lymphocytes as probes. One selected positive clone (182) contained an insert of 1.2 kb which was identified, sequenced and characterised. The deduced amino acid sequence of the open reading frame (ORF) revealed 82% and 81% identity to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes from Agrobacterium tumefaciens and Xanthobacter flavus, respectively. Southern blot analysis demonstrated that the gap gene is present in only one copy in the Brucella genome. B. abortus GAPDH was then expressed in Escherichia coli as a fusion protein with the maltose-binding protein (MBP). To demonstrate the functional activity of Brucella GAPDH, E. coli gap mutants were transformed with a Brucella pMAL-gap construct. Genetic complementation was achieved and as a result E. coli mutants were able to grow on glucose or other carbon source medium. The humoral and cellular immune responses to the recombinant (r) GAPDH were characterised. In Western blots, sera from naturally infected cattle and sheep showed antibody reactivity against rGAPDH. In response to in-vitro stimulation by rGAPDH, splenocytes from mice vaccinated with rGAPDH or B. abortus S19 were able to produce gamma-interferon and tumour necrosis factor-a but not interleukin (IL)-4. Furthermore, gap associated with murine IL-12 gene in a DNA vaccine formulation partially protected mice against experimental infection.

Identification and Characterization of a Brucella Abortus ATP-binding Cassette Transporter Homolog to Rhizobium Meliloti ExsA and Its Role in Virulence and Protection in Mice

Infection and Immunity. Sep, 2002  |  Pubmed ID: 12183550

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not fully understood yet. Furthermore, genes that allow Brucella to reach the intracellular niche and to interact with host cells need to be identified. Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATP-binding cassette (ABC) transporter of B. abortus strain S2308. The deduced amino acid sequence encoded by this gene exhibited 69 and 67% identity with the sequences of the ABC transporters encoded by the exsA genes of Rhizobium meliloti and Mesorhizobium loti, respectively. Additionally, B. abortus ExsA, like R. meliloti and M. loti ExsA, possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Furthermore, ortholog group analysis placed B. abortus ExsA in ortholog group 6 of ABC transporters more likely to be involved in bacterial pathogenesis. In R. meliloti, ExsA is an exopolysaccharide transporter essential for alfalfa root nodule invasion and establishment of infection. To test the role of ExsA in Brucella pathogenesis, an exsA deletion mutant was constructed. Replacement of the wild-type exsA by recombination was demonstrated by Southern blot analysis of Brucella genomic DNA. Decreased survival in mice of the Brucella DeltaexsA mutant compared to the survival of parental strain S2308 demonstrated that ExsA is critical for full bacterial virulence. Additionally, the B. abortus exsA deletion mutant was used as a live vaccine. Challenge experiments revealed that the exsA mutant strain induced superior protective immunity in BALB/c mice compared to the protective immunity induced by strain S19 or RB51.

Molecular and Cellular Interactions Between Brucella Abortus Antigens and Host Immune Responses

Veterinary Microbiology. Dec, 2002  |  Pubmed ID: 12414160

Host protection against Brucella abortus, is thought to be mediated primarily by a Th1 type immune response. Unfortunately, only few specific bacterial antigens involved in stimulating protective cellular immunity against Brucella are known. Therefore, identifying bacterial proteins that induce a T-lymphocyte mediated response is critical to determine Brucella immunity. Several library screening methods are discussed that have been used to identify Brucella proteins that stimulate T lymphocytes including cellular immunoblotting, Escherichia coli expressed Brucella proteins, green fluorescence reporter systems, and signature tagged mutagenesis. Future studies would likely examine how bacterial proteins expressed within host cells aid pathogen survival and/or induce host responses. Some of these newly identified bacterial gene products may serve as antigens to activate a protective host immune response. Also, identifying Brucella proteins expressed at particular times during infection will also yield insights into Brucella pathogenesis.

Virulence Criteria for Brucella Abortus Strains As Determined by Interferon Regulatory Factor 1-deficient Mice

Infection and Immunity. Dec, 2002  |  Pubmed ID: 12438380

Interferon regulatory factor 1-deficient (IRF-1(-/-)) mice infected with virulent Brucella abortus 2308 at 5 x 10(5) CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF-1(-/-) mice were unable to resolve infection and died. In contrast, IRF-1(-/-) mice survived when infected at 5 x 10(5) CFU with several attenuated Brucella strains (S19, RB51, cbp, and cyd). The survival of infected IRF-1(-/-) mice is likely a function of the level of virulence of each Brucella strain and the extent of retained immunity. Further, these findings suggest that adaptive immunity may be important to the survival of IRF-1(-/-) mice since attenuated Brucella strains can protect IRF-1(-/-) mice against lethal challenge with virulent Brucella: Using the IRF-1(-/-) mouse model, the following set of criteria were identified to define Brucella virulence: (i) the day of death for 50% of mice infected with 5 x 10(5)CFU of Brucella, (ii) the extent of liver toxicity, and (iii) the minimum immunizing dose of Brucella to protect against challenge with virulent S2308. Thus, IRF-1(-/-) mice are important to determining the level of Brucella virulence, to evaluating Brucella mutants for attenuation, and to investigating adaptive immunity in brucellosis.

Rough Lipopolysaccharide from Brucella Abortus and Escherichia Coli Differentially Activates the Same Mitogen-activated Protein Kinase Signaling Pathways for Tumor Necrosis Factor Alpha in RAW 264.7 Macrophage-like Cells

Infection and Immunity. Dec, 2002  |  Pubmed ID: 12438403

The intracellular, gram-negative pathogen Brucella abortus establishes chronic infections in host macrophages while downregulating cytokines such as tumor necrosis factor alpha (TNF-alpha). When producing TNF-alpha, Brucella abortus rough lipopolysaccharide (LPS) activates the same mitogen-activated protein kinase signaling pathways (ERK and JNK) as Escherichia coli LPS, but Brucella LPS is a much less potent agonist.

Molecular Host-pathogen Interaction in Brucellosis: Current Understanding and Future Approaches to Vaccine Development for Mice and Humans

Clinical Microbiology Reviews. Jan, 2003  |  Pubmed ID: 12525425

Brucellosis caused by Brucella spp. is a major zoonotic disease. Control of brucellosis in agricultural animals is a prerequisite for the prevention of this disease in human beings. Recently, Brucella melitensis was declared by the Centers for Disease Control and Prevention to be one of three major bioterrorist agents due to the expense required for the treatment of human brucellosis patients. Also, the economic agricultural loss due to bovine brucellosis emphasizes the financial impact of brucellosis in society. Thus, vaccination might efficiently solve this disease. Currently, B. abortus RB51 and B. melitensis REV.1 are used to immunize cattle and to immunize goats and sheep, respectively, in many countries. However, these genetically undefined strains still induce abortion and persistent infection, raising questions of safety and efficiency. In fact, the REV.1 vaccine is quite virulent and apparently unstable, creating the need for improved vaccines for B. melitensis. In addition, Brucella spp. may or may not provide cross-protection against infection by heterologous Brucella species, hampering the acceleration of vaccine development. This review provides our current understanding of Brucella pathogenesis and host immunity for the development of genetically defined efficient vaccine strains. Additionally, conditions required for an effective Brucella vaccine strain as well as the future research direction needed to investigate Brucella pathogenesis and host immunity are postulated.

Microarray Analysis of MRNA Levels from RAW264.7 Macrophages Infected with Brucella Abortus

Infection and Immunity. Mar, 2003  |  Pubmed ID: 12595423

Identification of host responses at the gene transcription level provides a molecular profile of the events that occur following infection. Brucella abortus is a facultative intracellular pathogen of macrophages that induces chronic infection in humans and domestic animals. Using microarray technology, the response of macrophages 4 h following B. abortus infection was analyzed to identify early intracellular infection events that occur in macrophages. Of the >6,000 genes, we identified over 140 genes that were reproducibly differentially transcribed. First, an increase in the transcription of a number of proinflammatory cytokines and chemokines, such as tumor necrosis factor alpha, interleukin-1beta (IL-1beta), IL-1alpha, and members of the SCY family of proteins, that may constitute a general host recruitment of antibacterial defenses was evident. Alternatively, Brucella may subvert newly arriving macrophages for additional intracellular infection. Second, transcription of receptors and cytokines associated with antigen presentation, e.g., major histocompatibility complex class II and IL-12p40, were not evident at this 4-h period of infection. Third, Brucella inhibited transcription of various host genes involved in apoptosis, cell cycling, and intracellular vesicular trafficking. Identification of macrophage genes whose transcription was inhibited suggests that Brucella utilizes specific mechanisms to target certain cell pathways. In conclusion, these data suggest that B. abortus can alter macrophage pathways to recruit additional macrophages for future infection while simultaneously inhibiting apoptosis and innate immune mechanisms within the macrophage, permitting intracellular survival of the bacterium. These results provide insights into the pathogenic strategies used by Brucella for long-term survival within a hostile environment.

The Proteasome As a Lipopolysaccharide-binding Protein in Macrophages: Differential Effects of Proteasome Inhibition on Lipopolysaccharide-induced Signaling Events

Journal of Immunology (Baltimore, Md. : 1950). Aug, 2003  |  Pubmed ID: 12874245

We have developed a novel LPS probe using a highly purified and homogenous preparation of [(3)H] Escherichia coli LPS from the deep rough mutant, which contains a covalently linked, photoactivable 4-p-(azidosalicylamido)-butylamine group. This cross-linker was used to identify the LPS-binding proteins in membranes of the murine-macrophage-like cell line RAW 264.7. The alpha-subunit (PSMA1 C2, 29.5 kDa) and the beta-subunit (PSMB4 N3, 24.36 kDa) of the 20S proteasome complex were identified as LPS-binding proteins. This is the first report demonstrating LPS binding to enzymes such as the proteasome subunits. Functionally, LPS enhanced the chymotrypsin-like activity of the proteasome to degrade synthetic peptides in vitro and, conversely, the proteasome inhibitor lactacystin completely blocked the LPS-induced proteasome's chymotrypsin activity as well as macrophage TNF-alpha secretion and the expression of multiple inflammatory mediator genes. Lactacystin also completely blocked the LPS-induced expression of Toll-like receptor 2 mRNA. In addition, lactacystin dysregulated mitogen-activated protein kinase phosphorylation in LPS-stimulated macrophages, but failed to inhibit IL-1 receptor-associated kinase-1 activity. Importantly, lactacystin also prevented LPS-induced shock in mice. These data strongly suggest that the proteasome complex regulates the LPS-induced signal transduction and that it may be an important therapeutic target in Gram-negative sepsis.

Role of Toll-like Receptor 4 in Induction of Cell-mediated Immunity and Resistance to Brucella Abortus Infection in Mice

Infection and Immunity. Jan, 2004  |  Pubmed ID: 14688095

Initial host defense to bacterial infection is executed by innate immunity, and therefore the main goal of this study was to examine the contribution of Toll-like receptors (TLRs) during Brucella abortus infection. CHO reporter cell lines transfected with CD14 and TLRs showed that B. abortus triggers both TLR2 and TLR4. In contrast, lipopolysaccharide (LPS) and lipid A derived from Brucella rough (R) and smooth (S) strains activate CHO cells only through TLR4. Consistently, macrophages from C3H/HePas mice exposed to R and S strains and their LPS produced higher levels of tumor necrosis factor alpha (TNF-alpha) and interleukin-12 compared to C3H/HeJ, a TLR4 mutant mouse. The essential role of TLR4 for induction of proinflammatory cytokines was confirmed with diphosphoryl lipid A from Rhodobacter sphaeroides. Furthermore, to determine the contribution of TLR2 and TLR4 in bacterial clearance, numbers of Brucella were monitored in the spleen of C3H/HeJ, C3H/HePas, TLR2 knockout, and wild-type mice at 1, 3, and 6 weeks following B. abortus infection. Interestingly, murine brucellosis was markedly exacerbated at weeks 3 and 6 after infection in animals that lacked functional TLR4 (C3H/HeJ) compared to C3H/HePas that paralleled the reduced gamma interferon production by this mouse strain. Finally, by mass spectrometry analysis we found dramatic differences on the lipid A profiles of R and S strains. In fact, S lipid A was shown to be more active to trigger TLR4 than R lipid A in CHO cells and more effective in inducing dendritic cell maturation. In conclusion, these results indicate that TLR4 plays a role in resistance to B. abortus infection and that S lipid A has potent adjuvant activity.

Bovine Herpesvirus Tegument Protein VP22 Enhances Thymidine Kinase/ganciclovir Suicide Gene Therapy for Neuroblastomas Compared to Herpes Simplex Virus VP22

Journal of Virology. Apr, 2004  |  Pubmed ID: 15047837

Herpesvirus tegument protein VP22 can enhance the effect of therapeutic proteins in gene therapy, such as thymidine kinase (tk) and p53; however, the mechanism is unclear or controversial. In this study, mammalian expression vectors carrying bovine herpesvirus 1 (BHV-1) VP22 (BVP22) or herpes simplex virus type 1 (HSV-1) VP22 (HVP22) and equine herpesvirus type 4 (EHV-4) tk (Etk) were constructed in order to evaluate and compare the therapeutic potentials of BVP22 and HVP22 to enhance Etk/ganciclovir (Etk/GCV) suicide gene therapy for neuroblastomas by GCV cytotoxicity assays and noninvasive bioluminescent imaging in vitro and in vivo. BVP22 enhanced Etk/GCV cytotoxicity compared to that with HVP22 both in vitro and in vivo. However, assays utilizing a mixture of parental and stably transfected cells indicated that the enhancement was detected only in transfected cells. Thus, the therapeutic potential of BVP22 and HVP22 in Etk/GCV suicide gene therapy in this tumor system is not due to VP22 delivery of Etk into surrounding cells but rather is likely due to an enhanced intracellular effect.

Comparative Whole-genome Hybridization Reveals Genomic Islands in Brucella Species

Journal of Bacteriology. Aug, 2004  |  Pubmed ID: 15262941

Brucella species are responsible for brucellosis, a worldwide zoonotic disease causing abortion in domestic animals and Malta fever in humans. Based on host preference, the genus is divided into six species. Brucella abortus, B. melitensis, and B. suis are pathogenic to humans, whereas B. ovis and B. neotomae are nonpathogenic to humans and B. canis human infections are rare. Limited genome diversity exists among Brucella species. Comparison of Brucella species whole genomes is, therefore, likely to identify factors responsible for differences in host preference and virulence restriction. To facilitate such studies, we used the complete genome sequence of B. melitensis 16M, the species highly pathogenic to humans, to construct a genomic microarray. Hybridization of labeled genomic DNA from Brucella species to this microarray revealed a total of 217 open reading frames (ORFs) altered in five Brucella species analyzed. These ORFs are often found in clusters (islands) in the 16M genome. Examination of the genomic context of these islands suggests that many are horizontally acquired. Deletions of genetic content identified in Brucella species are conserved in multiple strains of the same species, and genomic islands missing in a given species are often restricted to that particular species. These findings suggest that, whereas the loss or gain of genetic material may be related to the host range and virulence restriction of certain Brucella species for humans, independent mechanisms involving gene inactivation or altered expression of virulence determinants may also contribute to these differences.

Comparison of Bovine Leukemia Virus (BLV) and CMV Promoter-driven Reporter Gene Expression in BLV-infected and Non-infected Cells

Genetic Vaccines and Therapy. Aug, 2004  |  Pubmed ID: 15327692

BACKGROUND: Viral promoters are used in mammalian expression vectors because they generally have strong activity in a wide variety of cells of differing tissues and species. METHODS: The utility of the BLV LTR/promoter (BLVp) for use in mammalian expression vectors was investigated through direct comparison to the CMV promoter (CMVp). Promoter activity was measured using luciferase assays of cell lines from different tissues and species stably transduced with BLVp or CMVp driven luciferase vectors including D17, FLK, BL3.1 and primary bovine B cells. Cells were also modified through the addition of BLV Tax expression vectors and/or BLV infection as well as treatment with trichostatin A (TSA). RESULTS: Results indicate the BLV promoter, while having low basal activity compared to the CMV promoter, can be induced to high-levels of activity similar to the CMV promoter in all cells tested. Tax or BLV infection specifically enhanced BLVp activity with no effect on CMVp activity. In contrast, the non-specific activator, TSA, enhanced both BLVp and CMVp activity. CONCLUSION: Based on these data, we conclude the BLV promoter could be very useful for transgene expression in mammalian expression vectors.

Denitrification Genes Regulate Brucella Virulence in Mice

Journal of Bacteriology. Sep, 2004  |  Pubmed ID: 15342571

Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world. Genome sequencing of B. suis and B. melitensis revealed that both are complete denitrifiers. To learn more about the role of denitrification in these animal pathogens, a study of the role of denitrification in the closely related B. neotomae was undertaken. In contrast to B. suis and B. melitensis, it was found that B. neotomae is a partial denitrifier that can reduce nitrate to nitrite but no further. Examination of the B. neotomae genome showed that a deletion in the denitrification gene cluster resulted in complete loss of nirV and the partial deletion of nirK and nnrA. Even though the nor operon is intact, a norC-lacZ promoter fusion was not expressed in B. neotomae. However, the norC-lacZ fusion was expressed in the related denitrifier Agrobacterium tumefaciens, suggesting that the lack of expression in B. neotomae is due to inactivation of NnrA. A narK-lacZ promoter fusion was found to exhibit nitrate-dependent expression consistent with the partial denitrifier phenotype. Complementation of the deleted region in B. neotomae by using nirK, nirV, and nnrA from B. melitensis restored the ability of B. neotomae to reduce nitrite. There was a significant difference in the death of IRF-1-/- mice when infected with B. neotomae containing nirK, nirV, and nnrA and those infected with wild-type B. neotomae. The wild-type strain killed all the infected mice, whereas most of the mice infected with B. neotomae containing nirK, nirV, and nnrA survived.

The Role of Integrase/recombinase XerD and Monofunctional Biosynthesis Peptidoglycan Transglycosylase Genes in the Pathogenicity of Brucella Abortus Infection in Vitro and in Vivo

Microbial Pathogenesis. Nov, 2004  |  Pubmed ID: 15519045

Brucella abortus clones identified previously using a green fluorescence protein reporter system after 4h macrophage infection provided insight regarding possible genes involved in early host-pathogen interaction. Among identified genes were an integrase/recombinase (xerD) gene involved in cell division, and a monofunctional biosynthesis peptidoglycan transglycosylase (mtgA) gene that catalyzes the final stages of the peptidoglycan membrane synthesis. Here, we evaluate the in vitro and in vivo survival of B. abortus xerD and mtgA insertional mutants. B. abortus xerD::kan and B. abortus mtgA::kan demonstrated no significant growth defects in broth culture when compared to the parental strain, S2308. Also, neither gene was required for B. abortus S2308 replication in RAW 264.7 macrophages. However, experimental evidence using interferon regulatory factor 1 knockout mice, a mouse strain highly susceptible to virulent Brucella, revealed that mice infected with B. abortus xerD::kan or B. abortus mtgA::kan survived longer than mice infected with S2308. Additionally, in immunocompetent BALB/c mice, B. abortus xerD::kan had a significantly lower level of bacterial survival when compared to S2308. Together, these results suggest that B. abortus xerD and mtgA genes play a role during the initial phase of infection in mice.

Isolation of Brucella Abortus Total RNA from B. Abortus-infected Murine RAW Macrophages

Journal of Microbiological Methods. Mar, 2005  |  Pubmed ID: 15649540

Brucella is a Gram-negative facultative bacterium that persists intracellularly in macrophages. However, the intracellular survival mechanisms used by Brucella are not fully understood. Isolation of Brucella RNA from infected macrophages has been challenging, and the inability to isolate sufficient Brucella RNA from infected macrophages has contributed to the failure in understanding bacterial transcriptional events. We describe the isolation of sufficient Brucella abortus RNA from its infective host cell environment using osmotic lysis and RNase and DNase digestion. This method takes advantage of the B. abortus cell envelope that protects bacterial RNA and DNA. The cell envelope of B. abortus was digested using SDS/proteinase K (PK) that, importantly, inhibits any residual RNase after digesting macrophage RNA permitting the extraction of B. abortus RNA. In our experiments, 4.5 microg of RNA was routinely isolated from 1 ml bacterial culture and 2-9 microg of bacterial RNA from infected macrophages without detectable host cell RNA or DNA contamination. The method is rapid and uses inexpensive, commonly available reagents. Total bacterial RNA was isolated in quantities sufficient for RT-PCR and microarray analysis.

Bovine Herpesvirus VP22 Induces Apoptosis in Neuroblastoma Cells by Upregulating the Expression Ratio of Bax to Bcl-2

Human Gene Therapy. Jan, 2005  |  Pubmed ID: 15703493

Herpesvirus tegument protein VP22 has been shown to have biotherapeutic potential in tumor gene therapy. Some studies indicate that VP22 may enhance the transfer efficiency of therapeutic proteins by delivering them to more cells while trafficking. Our previous study showed that bovine herpesvirus VP22 (BVP22) enhanced equine herpesvirus thymidine kinase-ganciclovir (Etk-GCV) suicide gene therapy by an unknown intracellular effect. In this study, the interaction between BVP22 and host tumor cells was studied in neuroblastoma NXS2 cells. Cell cycle analysis was performed to determine whether BVP22 possesses biotherapeutic potential by altering the cell cycle, making cells more sensitive to therapeutic genes. As a result, the cell cycle was not affected by the transfection of BVP22 into NXS2 cells. However, cytotoxicity induced by BVP22 was observed in NXS2 cells on the second and third days after transient transfection. Further, analyses of caspase-3 activity and apoptosis suggested that BVP22 induces apoptosis in host tumor cells by upregulating the expression ratio of Bax to Bcl-2.

Nuclear and Mitochondrial Localization Signals Overlap Within Bovine Herpesvirus 1 Tegument Protein VP22

The Journal of Biological Chemistry. Apr, 2005  |  Pubmed ID: 15705574

VP22, a tegument protein of bovine herpesvirus 1, accumulates in the nucleus of infected and transiently transfected cells. Previous studies indicated a possible regulatory function of VP22 within nuclei, but how VP22 enters nuclei is unknown. Despite the abundance of basic residues within this protein, no classic nuclear localization signal (NLS) motif has been identified. To identify the signal directing nuclear accumulation, a series of truncations, internal deletions, and point mutations were constructed. Fluorescence microscopy of cells transfected with VP22 constructs indicated that a sequence of 103 residues is necessary and sufficient for nuclear localization. This NLS sequence is conformation-sensitive in contrast to a classical sequential NLS. Energy depletion assays and co-immunoprecipitation suggested that this NLS sequence also binds histone H4, resulting in nuclear retention of VP22. In addition, a mitochondrial targeting sequence was identified at the C-terminal 49 amino acids, which overlapped the sequence required for nuclear targeting. Our findings demonstrate the diversity of VP22 protein to localize within the cell and provide the opportunity for VP22 to direct cargo specifically to different subcellular compartments.

Enhancement of Antigen Acquisition by Dendritic Cells and MHC Class II-restricted Epitope Presentation to CD4+ T Cells Using VP22 DNA Vaccine Vectors That Promote Intercellular Spreading Following Initial Transfection

Journal of Leukocyte Biology. Aug, 2005  |  Pubmed ID: 15857936

Induction of immune responses against microbial antigens using DNA is an attractive strategy to mimic the immunity induced by live vaccines. Although DNA vaccines are efficacious in murine models, the requirement for multiple immunizations using high doses in outbred animals and humans has hindered deployment. This requirement is, in part, a result of poor vaccine spreading and suboptimal DC transfection efficiency. Incorporation of a signal that directs intercellular spreading of a DNA-encoded antigen is proposed to mimic live vaccine spreading and increase dendritic cell (DC) presentation. Bovine herpes virus 1 tegument protein, BVP22, is capable of trafficking to surrounding cells. To test the hypothesis that BVP22 enhances spreading and antigen presentation to CD4+ T cells, a DNA construct containing BVP22, fused in-frame to a sequence encoding a T cell epitope of Anaplasma marginale, was generated. A construct with reversed BVP22 sequence served as a negative control. Immunocytometric analysis of transfected primary keratinocytes, human embryonic kidney 293, COS-7, and Chinese hamster ovary cells showed that BVP22 enhanced intercellular spreading by > or = 150-fold. Flow cytometric analysis of antigen-presenting cells (APCs) positively selected from cocultures of transfected cells and APCs showed that 5% of test APCs were antigen-positive, compared with 0.6% of control APCs. Antigen-specific CD4+ T cell proliferation demonstrated that BVP22 enhanced DC antigen presentation by > or = 20-fold. This first report of the ability of BVP22 to increase DNA-encoded antigen acquisition by DCs and macrophages, with subsequent enhancement of major histocompatibility complex class II-restricted CD4+ T cell responses, supports incorporating a spreading motif in a DNA vaccine to target CD4+ T cell-dependent immunity in outbred animals.

Temporal Analysis of Pathogenic Events in Virulent and Avirulent Brucella Melitensis Infections

Cellular Microbiology. Oct, 2005  |  Pubmed ID: 16153245

Despite progress in mouse models of brucellosis, much remains unknown regarding Brucella dissemination and tissue localization. Here, we report the dynamics of Brucella infection in individual mice using bioluminescent Brucella melitensis. Bioluminescent imaging of infected interferon regulatory factor-1 knockout (IRF-1(-/-)) mice identified acute infection in many tissues. Brucella was found to replicate in the salivary glands of IRF-1(-/-) and wild-type C57BL/6 mice suggesting a previously unknown tissue preference. Establishing a niche in this region may have relevance in humans where infection can result from ingestion of few bacteria. Sublethal infection of IRF-1(-/-) mice resulted in chronic Brucella localization in tail joints, an infection parallel to osteoarticular brucellosis in humans. Importantly, bioluminescent imaging rapidly identified attenuated EZ::TN/lux mutants in infected mice and revealed differences in dissemination, thereby defining the contribution of Brucella genes to virulence and tissue localization. Surprisingly, a virB mutant, though defective in persistence, disseminated similarly to virulent Brucella, suggesting bacterial spread is independent of VirB proteins that are important for intracellular survival. Together, our results reveal kinetics of acute and chronic Brucella infection in individual mice that parallels human infection as well as readily identified attenuated bacteria. Our approach facilitates identifying virulence determinants that may control tissue specific replication and may help develop therapeutics to overcome Brucella-induced chronic debilitating conditions.

Unraveling Brucella Genomics and Pathogenesis in Immunocompromised IRF-1-/- Mice

American Journal of Reproductive Immunology (New York, N.Y. : 1989). Dec, 2005  |  Pubmed ID: 16305661

Brucellosis causes abortion in domestic animals and Malta fever in humans. Comparison of Brucella species genomes may reveal potential virulence mechanisms. Engineering bioluminescent Brucella would permit monitoring bacterial dissemination.

Attenuated Bioluminescent Brucella Melitensis Mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) Confer Protection in Mice

Infection and Immunity. May, 2006  |  Pubmed ID: 16622231

In vivo bioluminescence imaging is a persuasive approach to investigate a number of issues in microbial pathogenesis. Previously, we have applied bioluminescence imaging to gain greater insight into Brucella melitensis pathogenesis. Endowing Brucella with bioluminescence allowed direct visualization of bacterial dissemination, pattern of tissue localization, and the contribution of Brucella genes to virulence. In this report, we describe the pathogenicity of three attenuated bioluminescent B. melitensis mutants, GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091), and the dynamics of bioluminescent virulent bacterial infection following vaccination with these mutants. The virB4, galE, and BMEI1090-BMEI1091 mutants were attenuated in interferon regulatory factor 1-deficient (IRF-1(-/-)) mice; however, only the GR019 (virB4) mutant was attenuated in cultured macrophages. Therefore, in vivo imaging provides a comprehensive approach to identify virulence genes that are relevant to in vivo pathogenesis. Our results provide greater insights into the role of galE in virulence and also suggest that BMEI1090 and downstream genes constitute a novel set of genes involved in Brucella virulence. Survival of the vaccine strain in the host for a critical period is important for effective Brucella vaccines. The galE mutant induced no changes in liver and spleen but localized chronically in the tail and protected IRF-1(-/-) and wild-type mice from virulent challenge, implying that this mutant may serve as a potential vaccine candidate in future studies and that the direct visualization of Brucella may provide insight into selection of improved vaccine candidates.

CGHScan: Finding Variable Regions Using High-density Microarray Comparative Genomic Hybridization Data

BMC Genomics. 2006  |  Pubmed ID: 16638145

Comparative genomic hybridization can rapidly identify chromosomal regions that vary between organisms and tissues. This technique has been applied to detecting differences between normal and cancerous tissues in eukaryotes as well as genomic variability in microbial strains and species. The density of oligonucleotide probes available on current microarray platforms is particularly well-suited for comparisons of organisms with smaller genomes like bacteria and yeast where an entire genome can be assayed on a single microarray with high resolution. Available methods for analyzing these experiments typically confine analyses to data from pre-defined annotated genome features, such as entire genes. Many of these methods are ill suited for datasets with the number of measurements typical of high-density microarrays.

Brucella: Functional Genomics and Host-pathogen Interactions

Animal Health Research Reviews / Conference of Research Workers in Animal Diseases. Jun-Dec, 2006  |  Pubmed ID: 17389050

Brucellosis is a zoonotic disease caused by a number of Brucella species and is characterized by chronic macrophage infection. However, genes that may contribute to intracellular survival of the Brucella species are not well studied. This review presents, first, genomic islands that are present or absent in various Brucella species that may help establish Brucella infection and survival strategies. Second, the alteration in macrophage transcription by Brucella to permit its long-term survival within this hostile intracellular environment. A large number of macrophage gene transcripts are altered following Brucella infection indicating that Brucella is not a silent invader of host cells. Macrophage transcript levels associated with inflammation, apoptosis, signal transduction and vesicular intracellular trafficking are altered during Brucella infection, and likely contribute to intracellular survival of Brucella. Lastly, the host-pathogen interaction events associated with Brucella infection in living mice visualized in real-time using biophotonic imaging. Mice are often used to evaluate Brucella infections; however, Brucella dissemination and pathogenesis is poorly understood in mice. Biophotonic imaging of Brucella infections revealed sites of bacterial localization similar to human infections and different patterns of infection by attenuated or virulent Brucella.

Blue-light-activated Histidine Kinases: Two-component Sensors in Bacteria

Science (New York, N.Y.). Aug, 2007  |  Pubmed ID: 17717187

Histidine kinases, used for environmental sensing by bacterial two-component systems, are involved in regulation of bacterial gene expression, chemotaxis, phototaxis, and virulence. Flavin-containing domains function as light-sensory modules in plant and algal phototropins and in fungal blue-light receptors. We have discovered that the prokaryotes Brucella melitensis, Brucella abortus, Erythrobacter litoralis, and Pseudomonas syringae contain light-activated histidine kinases that bind a flavin chromophore and undergo photochemistry indicative of cysteinyl-flavin adduct formation. Infection of macrophages by B. abortus was stimulated by light in the wild type but was limited in photochemically inactive and null mutants, indicating that the flavin-containing histidine kinase functions as a photoreceptor regulating B. abortus virulence.

DNA Vaccine Construct Incorporating Intercellular Trafficking and Intracellular Targeting Motifs Effectively Primes and Induces Memory B- and T-cell Responses in Outbred Animals

Clinical and Vaccine Immunology : CVI. Mar, 2007  |  Pubmed ID: 17215335

We developed a vaccine construct in which a BVP22 domain and an invariant-chain major histocompatibility complex class II-targeting motif capable of enhancing dendritic cell antigen uptake and presentation were fused to a sequence encoding a B- and T-cell antigen from the Anaplasma marginale major surface protein 1a and tested whether this construct would prime and expand immune responses in outbred calves. A single inoculation with this construct effectively primed the immune responses, as demonstrated by a significant enhancement of CD4(+) T-cell proliferation compared to that in calves identically inoculated but inoculated with a DNA construct lacking the targeting domains and compared to that in calves inoculated with an empty vector. These proliferative responses were mirrored by priming and expansion of gamma interferon-positive CD4(+) T cells and immunoglobulin G responses against the linked B-cell epitope. Priming by the single immunization induced memory that underwent rapid recall following reexposure to the antigen. These results demonstrate that DNA vaccines targeting key intercellular and intracellular events significantly enhance priming and expansion and support the feasibility of single-dose DNA immunization in outbred populations.

DNA Recognition Properties of the Cell-to-cell Movement Protein (MP) of Soybean Isolate of Mungbean Yellow Mosaic India Virus (MYMIV-Sb)

Virus Research. Feb, 2008  |  Pubmed ID: 17949843

Bipartite geminiviruses possess two movement proteins (NSP and MP), which mediate the intra- and intercellular movement. In order to accomplish the transport process the movement proteins interact with viral nucleic acids in a sequence non-specific manner. To investigate the nucleic acid recognition properties of MP of MYMIV-Sb, the protein was expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP) and purified in native condition. Gel mobility shift assay was employed for analyzing the DNA recognition properties of purified MBP-MP fusion protein. The analyses demonstrated the sequence non-specific binding of MYMIV-Sb MP to both ds and ssDNA and its high affinity for ssDNA. MP of MYMIV-Sb did not show any specificity towards various forms of plasmid DNA but displayed size selection towards linear dsDNA.

Putative Quorum-sensing Regulator BlxR of Brucella Melitensis Regulates Virulence Factors Including the Type IV Secretion System and Flagella

Journal of Bacteriology. May, 2008  |  Pubmed ID: 18310341

Brucella melitensis is an intracellular pathogen that establishes a replicative niche within macrophages. While the intracellular lifestyle of Brucella is poorly understood and few virulence factors have been identified, components of a quorum-sensing pathway in Brucella have recently been identified. The LuxR-type regulatory protein, VjbR, and an N-acylhomoserine lactone signaling molecule are both involved in regulating expression of the virB-encoded type IV secretion system. We have identified a second LuxR-type regulatory protein (BlxR) in Brucella. Microarray analysis of a blxR mutant suggests that BlxR regulates the expression of a number of genes, including those encoding the type IV secretion system and flagella. Confirming these results, deletion of blxR in B. melitensis reduced the transcriptional activities of promoters for the virB operon, flagellar genes, and another putative virulence factor gene, bopA. Furthermore, our data suggested that both BlxR and VjbR are positively autoregulated and cross-regulate the expression of each other. The blxR deletion strain exhibited reduced growth in macrophages, similar to that observed for a vjbR deletion strain. However, unlike the vjbR deletion, the blxR deletion did not fully attenuate virulence in mice. More strikingly, bioluminescent imaging revealed that dissemination of the blxR mutant was similar to that of wild-type B. melitensis, while the vjbR mutant was defective for systemic spread in IRF-1(-/-) mice, suggesting that these regulators are not functionally redundant but that they converge in a common pathway regulating bacterial processes.

Genomic Island 2 of Brucella Melitensis is a Major Virulence Determinant: Functional Analyses of Genomic Islands

Journal of Bacteriology. Sep, 2008  |  Pubmed ID: 18641138

Brucella genomic islands (GIs) share similarities in their genomic organization to pathogenicity islands from other bacteria and are likely acquired by lateral gene transfer. Here, we report the identification of a GI that is important for the pathogenicity of Brucella melitensis. The deletion of GI-1, GI-5, or GI-6 did not affect bacterial growth in macrophages as well as their virulence in interferon regulatory factor 1-deficient (IRF-1(-/-)) mice, suggesting that these islands do not contribute to Brucella virulence. However, the deletion of GI-2 resulted in the attenuation of bacterial growth in macrophages and virulence in IRF-1(-/-) mice. The GI-2 mutant also displayed a rough lipopolysaccharide (LPS) phenotype indicated by acriflavin agglutination, suggesting that in vitro and in vivo attenuation is a result of LPS alteration. Further, systematic analysis of the entire GI-2 revealed two open reading frames (ORFs), BMEI0997 and I0998, that encode hypothetical sugar transferases and contribute to LPS alteration, as the deletion of either of these ORFs resulted in a rough phenotype similar to that of the GI-2 mutant. Complementation analyses indicated that in addition to I0997 and I0998, I0999 is required to restore the smooth LPS in the GI-2 mutant as well as its full in vitro and in vivo virulence. The I0999 sequence analysis suggested that it might function as a transporter to help facilitate the transport or linking of the O antigen to the LPS. Our study also indicated that the rough LPS resulting from the GI-2 deletion may affect pathogen-associated molecular pattern recognition by Toll-like receptors.

TBK1 Does Not Play a Role in the Control of in Vitro Burkholderia Pseudomallei Growth

Transactions of the Royal Society of Tropical Medicine and Hygiene. Dec, 2008  |  Pubmed ID: 19121697

Burkholderia pseudomallei, the causative agent of melioidosis, is an important intracellular pathogen in tropical regions. TANK-binding kinase (TBK1), part of the pathway that induces transcription of Type I interferon genes, has been demonstrated to play an important role in controlling intracellular bacterial infections. To investigate the role of tbk1 in protecting against B. pseudomallei we developed tbk1-deficient cell lines by using shRNA for transient knockdown of the tbk1 gene in HeLa and RAW 264.7 cells. In tbk1-deficient RAW cells, the replication of invasive and non-invasive Escherichia coli was significantly increased at 48 h after infection compared with wild-type cells. The result was confirmed using Brucella melitensis in tbk1-deficient HeLa cells, which demonstrated a >1.5-2.0 log higher bacterial count at 6-48 h after infection compared to wild-type cells. By contrast, the growth of Burkholderia pseudomallei expressing either typical (A2) or atypical (G207) lipopolysaccharide was not significantly different between the tbk1-deficient and control cells. These results suggest that the tbk1 gene and its activation may be able to control invasive E. coli, non-invasive E. coli and B. melitensis growth but may not be able to control B. pseudomallei infection. The role of the tbk1 gene in proinflammatory cytokine induction and bacterial intracellular infection needs further investigation to identify mechanistic differences among the life cycles of various intracellular bacteria.

Evaluation of Recombinant Invasive, Non-pathogenic Eschericia Coli As a Vaccine Vector Against the Intracellular Pathogen, Brucella

Journal of Immune Based Therapies and Vaccines. 2009  |  Pubmed ID: 19126207

ABSTRACT:

Brucella TIR Domain-containing Protein Mimics Properties of the Toll-like Receptor Adaptor Protein TIRAP

The Journal of Biological Chemistry. Apr, 2009  |  Pubmed ID: 19196716

Toll-like receptors (TLRs) play essential roles in the activation of innate immune responses against microbial infections. TLRs and downstream adaptor molecules contain a conserved cytoplasmic TIR domain. TIRAP is a TIR domain-containing adaptor protein that recruits the signaling adaptor MyD88 to a subset of TLRs. Many pathogenic microorganisms subvert TLR signaling pathways to suppress host immune responses to benefit their survival and persistence. Brucella encodes a TIR domain-containing protein (TcpB) that inhibits TLR2- and TLR4-mediated NF-kappaB activation. Sequence analysis indicated a moderate level of similarity between TcpB and the TLR adaptor molecule TIRAP. We found that TcpB could efficiently block TIRAP-induced NF-kappaB activation. Subsequent studies revealed that by analogy to TIRAP, TcpB interacts with phosphoinositides through its N-terminal domain and colocalizes with the plasma membrane and components of the cytoskeleton. Our findings suggest that TcpB targets the TIRAP-mediated pathway to subvert TLR signaling. In vivo mouse studies indicated that TcpB-deficient Brucella is defective in systemic spread at the early stages of infection.

Brucella Regulators: Self-control in a Hostile Environment

Trends in Microbiology. Aug, 2009  |  Pubmed ID: 19660951

Brucella is an important zoonotic pathogen for which no human vaccine exists. In an infected host, Brucella resides in macrophages but must coordinate expression of multiple virulence factors for successful cell entry and trafficking to acquire this replicative niche. Brucella responds to environmental signals to regulate virulence strategies that circumvent or blunt the host immune response. The Brucella quorum sensing system is a nexus of control for several Brucella virulence factors including flagellar genes and the type IV secretion system. Other sensory transduction systems, such as BvrRS and the newly described LOV-HK, sense environmental factors to control virulence. Here, we examine the contributions of various regulatory systems to Brucella virulence.

Brucella Melitensis, B. Neotomae and B. Ovis Elicit Common and Distinctive Macrophage Defense Transcriptional Responses

Experimental Biology and Medicine (Maywood, N.J.). Dec, 2009  |  Pubmed ID: 19934366

Brucella spp. establish an intracellular replicative niche in macrophages, while macrophages attempt to eliminate the bacteria by innate defense mechanisms. Brucella spp. possess similar genomes yet exhibit different macrophage infections. Few B. melitensis and B. neotomae enter macrophages with intracellular adaptation occurring over 4-8 hr. Conversely, B. ovis are readily ingested by macrophages and exhibit a persistent plateau of infection. Evaluating early macrophage interaction with Brucella spp. allows discovery of host entry and intracellular translocation mechanisms. Microarray analysis of macrophage transcriptional response following a 4 hr infection by different Brucella spp. revealed common macrophage genes altered in expression compared to uninfected macrophages. Macrophage infection with three different Brucella spp. provokes a common innate immune theme with increased transcript levels of chemokines and defense response genes and decreased transcript levels of GTPase signaling and cytoskeletal function that may affect trafficking of Brucella containing vesicles. For example, transcript levels of genes associated with chemotaxis (IL-1beta, MIP-1alpha), cytokine regulation (Socs3) and defense (Fas, Tnf) were increased, while transcript levels of genes associated with vesicular trafficking (Rab3d) and lysosomal associated enzymes (prosaposin) were decreased. Genes with altered macrophage transcript levels among Brucella spp. infections may correlate with species specific host defenses and intracellular survival strategies. Depending on the infecting Brucella species, gene ontology categorization identified genes differentially involved in cell growth and maintenance, endopeptidase inhibitor activity and G-protein mediated signaling. Examples of decreased gene expression in B. melitensis infection but not other Brucella spp. were growth arrest (Gas2), immunoglobulin receptor (FcgammarI) and chemokine receptor (Cxcr4) genes, suggesting opposing effects on intracellular functions.

Biochemical and Functional Analysis of TIR Domain Containing Protein from Brucella Melitensis

Biochemical and Biophysical Research Communications. Jun, 2010  |  Pubmed ID: 20471373

Toll/interleukin-1 like receptors are evolutionarily conserved proteins in eukaryotes that play crucial role in pathogen recognition and innate immune responses. Brucella are facultative intracellular bacterial pathogens causing brucellosis in animal and human hosts. Brucella behave as a stealthy pathogen by evading the immune recognition or suppressing the TLR signaling cascades. Brucella encode a TIR domain containing protein, TcpB, which suppresses NF-kappaB activation as well as pro-inflammatory cytokine secretion mediated by TLR2 and TLR4 receptors. TcpB targets the TIRAP mediated pathway to suppress TLR signaling. With the objective of detailed characterization, we have over expressed and purified TcpB from Brucella melitensis in native condition. The purified protein exhibited lipid-binding properties and cell permeability. NF-kappaB inhibition property of endogenous TcpB has also been demonstrated. The data provide insight into the mechanism of action of TcpB in the intracellular niche of Brucella.

Discordant Brucella Melitensis Antigens Yield Cognate CD8+ T Cells in Vivo

Infection and Immunity. Jan, 2010  |  Pubmed ID: 19884330

Brucella spp. are intracellular bacteria that cause the most frequent zoonosis in the world. Although recent work has advanced the field of Brucella vaccine development, there remains no safe human vaccine. In order to produce a safe and effective human vaccine, the immune response to Brucella spp. requires greater understanding. Induction of Brucella-specific CD8+ T cells is considered an important aspect of the host response; however, the CD8+ T-cell response is not clearly defined. Discovering the epitope containing antigens recognized by Brucella-specific CD8+ T cells and correlating them with microarray data will aid in determining proteins critical for vaccine development that cover a kinetic continuum during infection. Developing tools to take advantage of the BALB/c mouse model of Brucella melitensis infection will help to clarify the correlates of immunity and improve the efficacy of this model. Two H-2(d) CD8+ T-cell epitopes have been characterized, and a group of immunogenic proteins have provoked gamma interferon production by CD8+ T cells. RYCINSASL and NGSSSMATV induced cognate CD8+ T cells after peptide immunization that showed specific killing in vivo. Importantly, we found by microarray analysis that the genes encoding these epitopes are differentially expressed following macrophage infection, further emphasizing that these discordant genes may play an important role in the pathogenesis of B. melitensis infection.

Modulation of Microtubule Dynamics by a TIR Domain Protein from the Intracellular Pathogen Brucella Melitensis

The Biochemical Journal. Oct, 2011  |  Pubmed ID: 21692747

TIR (Toll/interleukin-1 receptor) domain-containing proteins play a crucial role in innate immunity in eukaryotes. Brucella is a highly infectious intracellular bacterium that encodes a TIR domain protein (TcpB) to subvert host innate immune responses to establish a beneficial niche for pathogenesis. TcpB inhibits NF-κB (nuclear factor κB) activation and pro-inflammatory cytokine secretions mediated by TLR (Toll-like receptor) 2 and TLR4. In the present study, we have demonstrated that TcpB modulates microtubule dynamics by acting as a stabilization factor. TcpB increased the rate of nucleation as well as the polymerization phases of microtubule formation in a similar manner to paclitaxel. TcpB could efficiently inhibit nocodazole- or cold-induced microtubule disassembly. Microtubule stabilization by TcpB is attributed to the BB-loop region of the TIR domain, and a point mutation affected the microtubule stabilization as well as the TLR-suppression properties of TcpB.

Brucella Melitensis Cyclic Di-GMP Phosphodiesterase BpdA Controls Expression of Flagellar Genes

Journal of Bacteriology. Oct, 2011  |  Pubmed ID: 21856843

Brucella melitensis encounters a variety of conditions and stimuli during its life cycle--including environmental growth, intracellular infection, and extracellular dissemination--which necessitates flexibility of bacterial signaling to promote virulence. Cyclic-di-GMP is a bacterial secondary signaling molecule that plays an important role in adaptation to changing environments and altering virulence in a number of bacteria. To investigate the role of cyclic-di-GMP in B. melitensis, all 11 predicted cyclic-di-GMP-metabolizing proteins were separately deleted and the effect on virulence was determined. Three of these cyclic-di-GMP-metabolizing proteins were found to alter virulence. Deletion of the bpdA and bpdB genes resulted in attenuation of virulence of the bacterium, while deletion of the cgsB gene produced a hypervirulent strain. In a Vibrio reporter system to monitor apparent alteration in levels of cyclic-di-GMP, both BpdA and BpdB displayed a phenotype consistent with cyclic-di-GMP-specific phosphodiesterases, while CgsB displayed a cyclic-di-GMP synthase phenotype. Further analysis found that deletion of bpdA resulted in a dramatic decrease in flagellar promoter activities, and a flagellar mutant showed similar phenotypes to the bpdA and bpdB mutant strains in mouse models of infection. These data indicate a potential role for regulation of flagella in Brucella melitensis via cyclic-di-GMP.

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