Brucella abortus nitric oxide metabolite regulates inflammasome activation and IL-1ß secretion in murine macrophages.

NLRP3 inflammasome is a protein complex crucial to caspase-1 activation and IL-1ß and IL-18 maturation. This receptor participates in innate immune responses to different pathogens, including the bacteria of genus Brucella. Our group recently demonstrated that Brucella abortus-induced IL-1ß secretion involves NLRP3 inflammasome and it is partially dependent on mitochondrial ROS production. However, other factors could be involved, such as P2X7-dependent potassium efflux, membrane destabilization, and cathepsin release. Moreover, there is increasing evidence that nitric oxide acts as a modulator of NLRP3 inflammasome. The aim of this study was to unravel the mechanism of NLRP3 inflammasome activation induced by B. abortus, as well as the involvement of bacterial nitric oxide (NO) as a modulator of this inflammasome pathway. We demonstrated that NO produced by B. abortus can be used by the bacteria to modulate IL-1ß secretion in infected murine macrophages. Additionally, our results suggest that B. abortus-induced IL-1ß secretion depends on a P2X7-independent potassium efflux, lysosomal acidification, cathepsin release, mechanisms clearly associated to NLRP3 inflammasome. In summary, our results help to elucidate the molecular mechanisms of NLRP3 activation and regulation during an intracellular bacterial infection.

TLR9 is required for MAPK/NF-κB activation but does not cooperate with TLR2 or TLR6 to induce host resistance to Brucella abortus.

Brucella abortus is a Gram-negative intracellular bacterial pathogen that causes a zoonosis of worldwide occurrence, leading to undulant fever in humans and abortion in domestic animals. B. abortus is recognized by several pattern-recognition receptors triggering pathways during the host innate immune response. Therefore, here, we determined the cooperative role of TLR9 with TLR2 or TLR6 receptors in sensing Brucella Furthermore, we deciphered the host innate immune response against B. abortus or its DNA, emphasizing the role of TLR9-MAPK/NF-κB signaling pathways in the production of proinflammatory cytokines. TLR9 is required for the initial host control of B. abortus, but this TLR was dispensable after 6 wk of infection. The susceptibility of TLR9(-/-)-infected animals to Brucella paralleled with lower levels of IFN-γ produced by mouse splenocytes stimulated with this pathogen compared with wild-type cells. However, no apparent cooperative interplay was observed between TLR2-TLR9 or TLR6-TLR9 receptors to control infection. Moreover, B. abortus or its DNA induced activation of MAPK/NF-κB pathways and production of IL-12 and TNF-α by macrophages partially dependent on TLR9 but completely dependent on MyD88. In addition, B. abortus-derived CpG oligonucleotides required TLR9 to promote IL-12 and TNF-α production by macrophages. By confocal microscopy, we demonstrated that TLR9 redistributed and colocalized with lysosomal-associated membrane protein-1 upon Brucella infection. Thus, B. abortus induced TLR9 traffic, leading to cell signaling activation and IL-12 and TNF-α production. Although TLR9 recognized Brucella CpG motifs, our results suggest a new pathway of B. abortus DNA-activating macrophages independent of TLR9.

Mutant Brucella abortus membrane fusogenic protein induces protection against challenge infection in mice.

Brucella species can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of the Brucella membrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed that B. abortus Δmfp::kan and Δomp19::kan deletion mutant strains have reduced persistence in vivo in C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kan or Δomp19::kan strain expressing green fluorescent protein (GFP) approximately 80% or 65% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments. B. abortus Δomp19::kan was attenuated in vivo but had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kan strain had a lower virulence in these same mouse models. Furthermore, Δmfp::kan and Δomp19::kan strains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kan strain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kan strain induced protection similar to that of RB51. Thus, these results demonstrate that Brucella Mfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kan mutant may serve as a potential vaccine candidate in future studies.

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.

Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi.

The oxidative lesion 8-oxoguanine (8-oxoG) is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1). This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1), the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1(-/-) (CD138) to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H(2)O(2)). Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H(2)O(2) of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER.

DNA polymerase beta from Trypanosoma cruzi is involved in kinetoplast DNA replication and repair of oxidative lesions.

Specific DNA repair pathways from Trypanosoma cruzi are believed to protect genomic DNA and kinetoplast DNA (kDNA) from mutations. Particular pathways are supposed to operate in order to repair nucleotides oxidized by reactive oxygen species (ROS) during parasite infection, being 7,8-dihydro-8-oxoguanine (8oxoG) a frequent and highly mutagenic base alteration. If unrepaired, 8oxoG can lead to cytotoxic base transversions during DNA replication. In mammals, DNA polymerase beta (Polß) is mainly involved in base excision repair (BER) of oxidative damage. However its biological role in T. cruzi is still unknown. We show, by immunofluorescence localization, that T. cruzi DNA polymerase beta (Tcpolß) is restricted to the antipodal sites of kDNA in replicative epimastigote and amastigote developmental stages, being strictly localized to kDNA antipodal sites between G1/S and early G2 phase in replicative epimastigotes. Nevertheless, this polymerase was detected inside the mitochondrial matrix of trypomastigote forms, which are not able to replicate in culture. Parasites over expressing Tcpolß showed reduced levels of 8oxoG in kDNA and an increased survival after treatment with hydrogen peroxide when compared to control cells. However, this resistance was lost after treating Tcpolß overexpressors with methoxiamine, a potent BER inhibitor. Curiously, a presumed DNA repair focus containing Tcpolß was identified in the vicinity of kDNA of cultured wild type epimastigotes after treatment with hydrogen peroxide. Taken together our data suggest participation of Tcpolß during kDNA replication and repair of oxidative DNA damage induced by genotoxic stress in this organelle.