Discordant Brucella melitensis antigens yield cognate CD8+ T cells in vivo.

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.

Genomic island 2 of Brucella melitensis is a major virulence determinant: functional analyses of genomic islands.

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.

Brucella: functional genomics and host-pathogen interactions.

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.

Isolation of Brucella abortus total RNA from B. abortus-infected murine RAW macrophages.

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.

Erythritol triggers expression of virulence traits in Brucella melitensis.

Erythritol is a four-carbon sugar preferentially utilized by Brucella spp. The presence of erythritol in the placentas of goats, cows, and pigs has been used to explain the localization of Brucella to these sites and the subsequent accumulation of large amounts of bacteria, eventually leading to abortion. Here we show that Brucella melitensis will also localize to an artificial site of erythritol within a mouse, providing a potential model system to study the pathogenesis of Brucella abortion. Immunohistological staining of the sites of erythritol within infected mice indicated a higher than expected proportion of extracellular bacteria. Ensuing experiments suggested intracellular B. melitensis was unable to replicate within macrophages in the presence of erythritol and that erythritol was able to reach the site of intracellular bacteria. The intracellular inhibition of growth was found to encourage the bacteria to replicate extracellularly rather than intracellularly, a particularly interesting development in Brucella pathogenesis. To determine the effect of erythritol on expression of B. melitensis genes, bacteria grown either with or without erythritol were analyzed by microarray. Two major virulence pathways were up-regulated in response to exposure to erythritol (the type IV secretion system VirB and flagellar proteins), suggesting a role for erythritol in virulence.

Differential expression of iron acquisition genes by Brucella melitensis and Brucella canis during macrophage infection.

Brucella spp. cause chronic zoonotic disease often affecting individuals and animals in impoverished economic or public health conditions; however, these bacteria do not have obvious virulence factors. Restriction of iron availability to pathogens is an effective strategy of host defense. For brucellae, virulence depends on the ability to survive and replicate within the host cell where iron is an essential nutrient for the growth and survival of both mammalian and bacterial cells. Iron is a particularly scarce nutrient for bacteria with an intracellular lifestyle. Brucella melitensis and Brucella canis share ~99% of their genomes but differ in intracellular lifestyles. To identify differences, gene transcription of these two pathogens was examined during infection of murine macrophages and compared to broth grown bacteria. Transcriptome analysis of B. melitensis and B. canis revealed differences of genes involved in iron transport. Gene transcription of the TonB, enterobactin, and ferric anguibactin transport systems was increased in B. canis but not B. melitensis during infection of macrophages. The data suggest differences in iron requirements that may contribute to differences observed in the lifestyles of these closely related pathogens. The initial importance of iron for B. canis but not for B. melitensis helps elucidate differing intracellular survival strategies for two closely related bacteria and provides insight for controlling these pathogens.

Brucella melitensis, B. neotomae and B. ovis elicit common and distinctive macrophage defense transcriptional responses.

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.

Unraveling Brucella genomics and pathogenesis in immunocompromised IRF-1-/- mice.

PROBLEM: 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. METHOD OF STUDY: Microarray of the B. melitensis genome allowed comparison of gene content from six Brucella species. Bioluminescent B. melitensis strains were developed using transposon mutagenesis permitting the study of pathogenic Brucella in mice. Monitoring bacterial dissemination as well as organ localization permits evaluating the role of genes and genomic islands in mutant bacteria. RESULTS: Comparative genomic analysis revealed 217 ORFs altered in five Brucella species and were often found in islands. Bioluminescent bacteria disseminated from the injection site to liver, spleen, inguinal lymph nodes, testes and submanibular region. CONCLUSIONS: Genomic islands contribute to Brucella pathogenicity. Biophotonic imaging suggests that Brucella dissemination in mice parallels acute and chronic infections of humans.

Microarray analysis of mRNA levels from RAW264.7 macrophages infected with Brucella abortus.

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.