The role of NLRP3 and AIM2 in inflammasome activation during Brucella abortus infection.

The innate immune system is essential for the detection and elimination of bacterial pathogens. Upon inflammasome activation, caspase-1 cleaves pro-IL-1ß and pro-IL-18 to their mature forms IL-1ß and IL-18, respectively, and the cell undergoes inflammatory death termed pyroptosis. Here, we reviewed recent findings demonstrating that Brucella abortus ligands activate NLRP3 and AIM2 inflammasomes which lead to control of infection. This protective effect is due to the inflammatory response caused by IL-1ß and IL-18 rather than cell death. Brucella DNA is sensed by AIM2 and bacteria-induced mitochondrial reactive oxygen species is detected by NLRP3. However, deregulation of pro-inflammatory cytokine production can lead to immunopathology. Nervous system invasion by bacteria of the genus Brucella results in an inflammatory disorder termed neurobrucellosis. Herein, we discuss the mechanism of caspase-1 activation and IL-1ß secretion in glial cells infected with B. abortus. Our results demonstrate that the ASC inflammasome is indispensable for inducing the activation of caspase-1 and secretion of IL-1ß upon infection of astrocytes and microglia with Brucella. Moreover, our results demonstrate that secretion of IL-1ß by Brucella-infected glial cells depends on NLRP3 and AIM2 and leads to neurobrucellosis. Further, the inhibition of the host cell inflammasome as an immune evasion strategy has been described for bacterial pathogens. We discuss here that the bacterial type IV secretion system VirB is required for inflammasome activation in host cells during infection. Taken together, our results indicate that Brucella is sensed by ASC inflammasomes mainly NLRP3 and AIM2 that collectively orchestrate a robust caspase-1 activation and pro-inflammatory response.

Innate immune sensing of nucleic acids from mycobacteria.

Endosomal and cytosolic receptors engage recognition of mycobacterial-derived nucleic acids (MyNAs). In contrast, virulent mycobacteria may utilize nucleic acid recognition pathways to escape the host immune system. This short review will summarize the mechanisms by which MyNAs are sensed and how they influence host protective responses.

Brucella abortus DNA is a major bacterial agonist to activate the host innate immune system.

Immunity against Brucella abortus depends on the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Signaling pathways triggered by Brucella DNA involves TLR9, AIM2 and possibly STING and MAVS. Herein, we review the advances in B. abortus DNA sensing by host innate immune receptors and the progress in this field.

Recognition of Legionella pneumophila nucleic acids by innate immune receptors.

Innate immune receptors evolved to sense conserved molecules that are present in microbes or are released during non-physiological conditions. Activation of these receptors is essential for early restriction of microbial infections and generation of adaptive immunity. Among the conserved molecules sensed by innate immune receptors are the nucleic acids, which are abundantly contained in all infectious organisms including virus, bacteria, fungi and parasites. In this review we focus in the innate immune proteins that function to sense nucleic acids from the intracellular bacterial pathogen Legionella pneumophila and the importance of these processes to the outcome of the infection.

Oral immunization of mice with recombinant Lactococcus lactis expressing Cu,Zn superoxide dismutase of Brucella abortus triggers protective immunity.

Brucella infections mainly occur through mucosal surfaces. Thus, the development of mucosal administered vaccines could be instrumental for the control of brucellosis. Here, we evaluated the usefulness of recombinant Lactococcus lactis secreting Brucella abortus Cu-Zn superoxide dismutase (SOD) as oral antigen delivery system, when administered alone or in combination with L. lactis expressing IL-12. To this end, mice were vaccinated by oral route with L. lactis NZ9000 transformed with pSEC derivatives encoding for SOD (pSEC:SOD) and IL-12 (pSEC:scIL-12). In animals receiving L. lactis pSEC:SOD alone, anti-SOD-specific IgM antibodies were detected in sera at day 28 post-vaccination, together with an IgG2a dominated IgG response. SOD-specific sIgA was also detected in nasal and bronchoalveolar lavages. In addition, T-cell-proliferative responses upon re-stimulation with either recombinant SOD or crude Brucella protein extracts were observed up to 6 months after the last boost, suggesting the induction of long term memory. Vaccinated animals were also protected against challenge with the virulent B. abortus 2308 strain. Responses were mildly improved when L. lactis pSEC:SOD was co-administered with L. lactis pSEC:scIL-12. These results indicated that vaccines based on lactococci-derived live carriers are promising interventions against B. abortus infections.

Extracellular DNA: a major proinflammatory component of Pseudomonas aeruginosa biofilms.

We previously demonstrated that extracellular bacterial DNA activates neutrophils through a CpG- and TLR9-independent mechanism. Biofilms are microbial communities enclosed in a polymeric matrix that play a critical role in the pathogenesis of many infectious diseases. Because extracellular DNA is a key component of biofilms of different bacterial species, the aim of this study was to determine whether it plays a role in the ability of biofilms to induce human neutrophil activation. We found that degradation of matrix extracellular DNA with DNase I markedly reduced the capacity of Pseudomonas aeruginosa biofilms to induce the release of the neutrophil proinflammatory cytokines IL-8 and IL-1beta (>75%); reduced the upregulation of neutrophil activation markers CD18, CD11b, and CD66b (p < 0.001); reduced the number of bacteria phagocytosed per neutrophil contacting the biofilm; and reduced the production of neutrophil extracellular traps. Consistent with these findings, we found that biofilms formed by the lasI rhlI P. aeruginosa mutant strain, exhibiting a very low content of matrix extracellular DNA, displayed a lower capacity to stimulate the release of proinflammatory cytokines by neutrophils, which was not decreased further by DNase I treatment. Together, our findings support that matrix extracellular DNA is a major proinflammatory component of P. aeruginosa biofilms.

Identification by genomic immunization of a pool of DNA vaccine candidates that confer protective immunity in mice against Neisseria meningitidis serogroup B.

We have shown previously that expression library immunization is viable alternative approach to induce protective immunity against Neisseria meningitidis serogroup B. In this study we report that few rounds of library screening allow identification of protective pools of defined antigens. A previously reported protective meningococcal library (L8, with 600 clones) was screened and two sub-libraries of 95 clones each were selected based on the induction of bactericidal and protective antibodies in BALB/c mice. After sequence analysis of each clone within these sub-libraries, we identified a pool of 20 individual antigens that induced protective immune responses in mice against N. meningitidis infection, and the observed protection was associated with the induction of bactericidal antibodies. Our studies demonstrate for the first time that ELI combined with sequence analysis is a powerful and efficient tool for identification of candidate antigens for use in a meningococcal vaccine.

Immunization of mice with a Mycobacterium tuberculosis genomic expression library results in lower bacterial load in lungs after challenge with BCG.

Tuberculosis is a serious infectious disease in many developing countries. The lack of an effective vaccine for preventing this disease has stimulated the search for new vaccine candidates against Mycobacterium tuberculosis. In the present work, the construction of a genomic expression library of M. tuberculosis in a eukaryotic expression vector was carried out. Immunization of Balb/c mice with a plasmid DNA pool from this library (containing 8360 clones) induced a significant IgG antibody response. Immunized mice were challenged by intratracheal route with 10(5) cfu of non-pathogenic Mycobacterium bovis BCG and were sacrificed 21 days post-challenge. Mice immunized with the genomic expression library showed a significant reduction of viable bacteria in lungs and less pulmonary tissue damage. Granulomas were not observed and the lungs had a more discrete perivascular inflammatory cell infiltrate compared to control mice. Results suggest that the genomic expression library contains genes encoding proteins that are protective against M. tuberculosis infection.

Immunoresponse of Coho salmon immunized with a gene expression library from Piscirickettsia salmonis.

We have used the expression library immunization technology to study the protection of Coho salmon Oncorhynchus kisutch to the infection with Piscirickettsia salmonis. Purified DNA from this bacterium was sonicated and the fragments were cloned in the expression vector pCMV-Bios. Two libraries were obtained containing 22,000 and 28,000 colonies and corresponding to approximately 8 and 10 times the genome of the pathogen, respectively. On average, the size of the inserts ranged between 300 and 1,000 bp. The plasmid DNA isolated from one of these libraries was purified and 20 micrograms were injected intramuscularly into 60 fish followed by a second dose of 10 micrograms applied 40 days later. As control, fish were injected with the same amount of DNA of the vector pCMV-Bios without insert. The titer of IgM anti-P. salmonis of vaccinated fish, evaluated 60 days post-injection, was significantly higher than that of the control group injected with the vector alone. Moreover, this response was specific against P. salmonis antigens, since no cross reaction was detected with Renibacterium salmoninarum and Yersinia ruckeri. The vaccinated and control fish were challenged 60 days after the second dose of DNA with 2.5 x 10(7) P. salmonis corresponding to 7.5 times the LD50. At 30 days post-challenge, 100% mortality was obtained with the control fish while 20% of the vaccinated animals survived. All surviving fish exhibited a lower bacterial load in the kidney than control fish. The expression library was also tested in Balb/c mice and it was found that the humoral immune response was specific to P. salmonis and it was dependent on the amount of DNA injected.

Efficacy of DNA-hsp65 vaccination for tuberculosis varies with method of DNA introduction in vivo.

A DNA vaccine codifying the mycobacterial hsp65 can prevent infection with Mycobacterium tuberculosis in a prophylactic setting and also therapeutically reduce the number of bacteria in infected mice. The protective mechanism is thought to be related to Th1-mediated events that result in bacterial killing. To determine the best method of hsp65 introduction for vaccination efficacy against tuberculosis (TB), we evaluated the immunogenicity and protection of DNA-hsp65 administered by gene gun bombardment or intramuscular (i.m.) injection of naked DNA. Immunization by gene gun induced immune response with plasmid doses 100-fold lower than those required for intramuscular immunization. However, in contrast to intramuscular immunization, which was protective in these studies, gene gun immunization did not protect BALB/c mice against challenge infection.

Bacterial DNA activates human neutrophils by a CpG-independent pathway.

Bacterial DNA stimulates macrophages, monocytes, B lymphocytes, NK cells, and dendritic cells in a CpG-dependent manner. In this work we demonstrate that bacterial DNA, but not mammalian DNA, induces human neutrophil activation as assessed by L-selectin shedding, CD11b upregulation, and stimulation of cellular shape change, IL-8 secretion, and cell migration. Induction of these responses is not dependent on the presence of unmethylated CpG motifs, as neutrophil stimulatory properties were neither modified by CpG-methylation of bacterial DNA nor reproduced by oligonucleotides bearing CpG motifs. We found that human neutrophils express Toll-like receptor (TLR) 9 mRNA. However, as expected for a CpG-independent mechanism, activation does not involve a TLR9-dependent signaling pathway; neutrophil stimulation was not prevented by immobilization of bacterial DNA or by wortmannin or chloroquine, two agents that inhibit TLR9 signaling. Of note, both single-stranded and double-stranded DNA were able to induce activation, suggesting that neutrophils might be activated by bacterial DNA at inflammatory foci even in the absence of conditions required to induce DNA denaturation. Our findings provide the first evidence that neutrophils might be alerted to the presence of invading bacteria through recognition of its DNA via a novel mechanism not involving CpG motifs.

Phenotypic and molecular characterization of isolates of Ornithobacterium rhinotracheale from Peru.

Twenty-five isolates of Ornithobacterium rhinotracheale were examined by agar gel precipitation, immunoperoxidase assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot analysis, and a polymerase chain reaction. All of the isolates were identified as serotype A. Protein profiles of whole cell extracts were similar for all the isolates, and a polypeptide with a molecular weight of 33 kD was a major component, being present in all the isolates. In the main, proteins of 33, 42, 52, and 66 kD were recognized in immunoblots with sera from chickens naturally exposed to O. rhinotracheale. A modified polymerase chain reaction assay identified O. rhinotracheale DNA from all the isolates and tracheal swabs, producing amplicons of 784 bp, and distinguished O. rhinotracheale from bacterial agents causing similar clinical signs.

Antibody response in mice immunized with a plasmid DNA encoding the colonization factor antigen I of enterotoxigenic Escherichia coli.

The colonization factor antigen I (CFA/I) is one of the most epidemiologically relevant enterotoxigenic Escherichia coli (ETEC) fimbrial adhesins, which mediates the binding to human small intestine epithelium. A recombinant eukaryotic expression plasmid, pRECFA, encoding the CFA/I protein fused to the glycoprotein D of herpes simplex type 1 virus, was used to generate an antibody response in a murine model following intramuscular inoculation of purified DNA. Eukaryotic cells (BHK-21) transfected with pRECFA expressed the CFA/I protein in vitro, as revealed by Western blot and immunofluorescence microscopy. Administration of a single pRECFA 100-microg dose induced a long-term CFA/I-specific antibody response in BALB/c mice composed mainly of IgG and, to a lesser extent, IgA isotypes. The major CFA/I-specific IgG subclass was IgG2a, suggesting a Th-1-type immune response. A second dose with the same amount of purified DNA, given 2 weeks later, caused a booster effect on the immunoglobulin levels, but did not qualitatively alter the isotypes and subclasses of the induced antibody response. Immunization with different amounts of purified DNA and/or number of doses showed that maximal transient CFA/I-specific antibody levels could be obtained after two 100-microg doses of pRECFA given 2 weeks apart, but long-term antibody levels were similar.