DDX5 inhibits inflammation by modulating m6A levels of TLR2/4 transcripts during bacterial infection.

DExD/H-box helicases are crucial regulators of RNA metabolism and antiviral innate immune responses; however, their role in bacteria-induced inflammation remains unclear. Here, we report that DDX5 interacts with METTL3 and METTL14 to form an m6A writing complex, which adds N6-methyladenosine to transcripts of toll-like receptor (TLR) 2 and TLR4, promoting their decay via YTHDF2-mediated RNA degradation, resulting in reduced expression of TLR2/4. Upon bacterial infection, DDX5 is recruited to Hrd1 at the endoplasmic reticulum in an MyD88-dependent manner and is degraded by the ubiquitin-proteasome pathway. This process disrupts the DDX5 m6A writing complex and halts m6A modification as well as degradation of TLR2/4 mRNAs, thereby promoting the expression of TLR2 and TLR4 and downstream NF-κB activation. The role of DDX5 in regulating inflammation is also validated in vivo, as DDX5- and METTL3-KO mice exhibit enhanced expression of inflammatory cytokines. Our findings show that DDX5 acts as a molecular switch to regulate inflammation during bacterial infection and shed light on mechanisms of quiescent inflammation during homeostasis.

RNA helicase DExD/H-box 5 modulates intestinal microbiota in mice.

The RNA helicase DExD/H-box (DDX) family of proteins plays a central role in host cellular RNA metabolism, including mRNA regulation, microRNA biogenesis, and ribosomal processing. DDX5, also known as p68, promotes viral replication and tumorigenesis. However, there have been no studies on the regulation of the intestinal microbiota by DDX family proteins. We constructed DDX5 knockout mice (Ddx5+/-) using CRISPR/CAS9 technology. Subsequently, DDX5 knockout mice were analyzed for PCR products, mRNA levels, protein expression, immunohistochemistry, and histopathological lesions. Fecal (n = 12) and ileum (n = 12) samples were collected from the Ddx5+/- and wild-type (Ddx5+/+) mice. The diversity, richness, and structural separation of the intestinal microbiota of the Ddx5+/- and Ddx5+/+ mice were determined by 16S rRNA sequencing and analysis. Ddx5+/- mice were successfully established, and the ileum had normal morphology, a clear layer of tissue structures, and neatly arranged cupped cells. DDX5 knockout mice did not exhibit adverse effects on the ileal tissue. Microbial diversity and abundance were not significantly different, but the microbial structure of the intestinal microbiota was clustered separately between Ddx5+/+ and Ddx5+/- mice. Furthermore, we found that the relative abundance of Akkermansia and Clostridium_sensu_stricto_1 in the Ddx5+/- mice was significantly lower than in the Ddx5+/+ mice. These analyses indicated specific interactions between the intestinal microbiota and DDX5 protein. Our results indicate that DDX5 has a significant effect on the composition of the intestinal microbiota in mice, suggesting its potential as a promising novel target for the treatment of inflammation and tumorigenesis in the intestine.

A LysR Transcriptional Regulator Manipulates Macrophage Autophagy Flux During Brucella Infection.

Brucella, the intracellular bacteria, have evolved subtle strategies to efficiently survive and replicate in macrophages. However, the virulence effector proteins involved are still unclear. LysR-type transcriptional regulators (lttrs) are the largest regulator family with diverse function in prokaryotes. However, very little is known about the role of LysR regulators in the Brucella spp. Here, a BSS2_II0858 gene, encoded as one of the LysR-type regulators, was studied. We successfully constructed a BSS2_II0858 deletion mutant, Δ0858, and complementation strain CΔ0858 in Brucella suis S2. The cell apoptosis induced by B. suis S2 and its derivatives were detected by flow cytometry. The autophagy was then assessed by immunoblot analysis using the IL3I/II and p62 makers. In addition, the autophagy flux was evaluated by double fluorescent labeling method for autophagy marker protein LC3. Our studies demonstrated that B. suis S2 and its derivatives inhibited the programmed cell death in early stage and promoted apoptosis in the later stage during infection in RAW264.7 cells. The BSS2_II0858 gene was found to play no role during apoptosis according to these results. Compared with the wild-type strain, Δ0858 mutant can stimulate the conversion of LC3-I to LC3-II and markedly inhibited the autophagy flux at early stage leading to obvious autophagosome accumulation. This study explored the function of BSS2_II0858 gene and may provide new insights for understanding the mechanisms involved in the survival of Brucella in macrophages.

BtpB inhibits innate inflammatory responses in goat alveolar macrophages through the TLR/NF-κB pathway and NLRP3 inflammasome during Brucella infection.

Brucella species are infectious facultative intracellular pathogens. They have evolved multiple strategies to thwart immune responses and replicate in macrophages for chronic persistence in the host. As a Brucella effector, BtpB is transferred into target cells through the type IV secretion system. BtpB, a Toll/interleukin-1 receptor domain-containing protein, blocks host innate immune responses by interfering with Toll-like receptor signaling. However, the intracellular targets and their activated downstream pathways remain unclear. In this study, we constructed a strain of Brucella suis S2 with a deletion in the gene for BtpB, ΔbtpB, and the complemented strain, C-ΔbtpB with a restored copy of the btpB gene. The bacterial growth curves and stress resistance results showed that BtpB did not affect B. suis S2 growth. Infection of alveolar macrophages with WT and ΔbtpB strains showed that BtpB inhibited TLR2 and TLR4 expression and attenuated NLRP3 inflammasome activation. BtpB also attenuated secretion of the Brucella-induced proinflammatory cytokines, IL-1ß, IL-6, and TNF-α, in alveolar macrophages while up-regulating IL-10 expression. In general, the results confirmed that BtpB specifically inhibits TLR2/TLR4 and disrupts NLRP3 signaling pathways to inhibit host immune responses in early Brucella infections.

A Brucella Omp16 Conditional Deletion Strain Is Attenuated in BALB/c Mice.

Brucella spp. are facultative intracellular pathogens that invade, survive and proliferate in numerous phagocytic and non-phagocytic cell types, thereby leading to human and animal brucellosis. Outer membrane proteins (Omps) are major immunogenic and protective antigens that are implicated in Brucella virulence. A strain deleted of the omp16 gene has not been obtained which suggests that the Omp16 protein is vital for Brucella survival. Nevertheless, we previously constructed an omp16 conditional deletion strain of Brucella, ΔOmp16. Here, the virulence and immune response elicted by this strain were assessed in a mouse model of infection. Splenomegaly was significantly reduced at two weeks post-infection in ΔOmp16-infected mice compared to infection with the parental strain. The bacterial load in the spleen also was significantly decreased at this post-infection time point in ΔOmp16-infected mice. Histopathological changes in the spleen were observed via hematoxylineosin staining and microscopic examination which showed that infection with the ΔOmp16 strain alleviated spleen histopathological alterations compared to mice infected with the parental strain. Moreover, the levels of humoral and cellular immunity were similar in both ΔOmp16-infected mice and parental strain-infected mice. The results overall show that the virulence of ΔOmp16 is attenuated markedly, but that the immune responses mediated by the deletion and parental strains in mice are indistinguishable. The data provide important insights that illuminate the pathogenic strategies adopted by Brucella.

An ArsR Transcriptional Regulator Facilitates Brucella sp. Survival via Regulating Self and Outer Membrane Protein.

The arsenic acid-resistant (ArsR) family transcriptional regulators are widely distributed in microorganisms, including in the facultative intracellular pathogen Brucella spp. ArsR proteins are implicated in numerous biological processes. However, the specific roles of ArsR family members in Brucella remain obscure. Here, we show that ArsR6 (BSS2_RS07325) is required for Brucella survival both under heat, oxidative, and osmotic stress and in a murine infection model in vivo. RNA-seq and ChIP-seq reveal that 34 potential target genes for ArsR6 protein were identified, among which eight genes were up-regulated and 26 genes were down-regulated, including outer membrane protein 25D (Omp25D). ArsR6 autoregulates its own expression to maintain bacterial intracellular Cu/Ni homeostasis to benefit bacterial survival in hostile environments. Moreover, ArsR6 also regulates the production of virulence factor Omp25D, which is important for the survival of Brucella under stress conditions. Significantly, Omp25D deletion strain attenuated in a murine infection model in vivo. Altogether, our findings reveal a unique mechanism in which the ArsR family member ArsR6 autoregulates its expression and also modulates Omp25D expression to maintain metal ion homeostasis and virulence in Brucella.

RNA-Seq Analysis Reveals the Role of Omp16 in Brucella-Infected RAW264.7 Cells.

Brucellosis is an endemic zoonotic infectious disease in the majority of developing countries, which causes huge economic losses. As immunogenic and protective antigens at the surface of Brucella spp., outer membrane proteins (Omps) are particularly attractive for developing vaccine and could have more relevant role in host-pathogen interactions. Omp16, a homolog to peptidoglycan-associated lipoproteins (Pals), is essential for Brucella survival in vitro. At present, the functions of Omp16 have been poorly studied. Here, the gene expression profile of RAW264.7 cells infected with Brucella suis vaccine strain 2 (B. suis S2) and ΔOmp16 was analyzed by RNA-seq to investigate the cellular response immediately after Brucella entry. The RNA-sequence analysis revealed that a total of 303 genes were significantly regulated by B. suis S2 24 h post-infection. Of these, 273 differentially expressed genes (DEGs) were upregulated, and 30 DEGs were downregulated. These DEGs were mainly involved in innate immune signaling pathways, including pattern recognition receptors (PRRs), proinflammatory cytokines, and chemokines by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. In ΔOmp16-infected cells, the expression of 52 total cells genes was significantly upregulated and that of 9 total cells genes were downregulated compared to B. suis S2-infected RAW264.7 cells. The KEGG pathway analysis showed that several upregulated genes were proinflammatory cytokines and chemokines, such as interleukin (IL)-6, IL-11, IL-12ß, C-C motif chemokine (CCL2), and CCL22. All together, we clearly demonstrate that ΔOmp16 can alter macrophage immune-related pathways to increase proinflammatory cytokines and chemokines, which provide insights into illuminating the Brucella pathogenic strategies.

Integrated Proteomic and Transcriptomic Analyses Reveal the Roles of Brucella Homolog of BAX Inhibitor 1 in Cell Division and Membrane Homeostasis of Brucella suis S2.

BAX inhibitor 1 (BI-1) is an evolutionarily conserved transmembrane protein first identified in a screening process for human proteins that suppress BAX-induced apoptosis in yeast cells. Eukaryotic BI-1 is a cytoprotective protein that suppresses cell death induced by multiple stimuli in eukaryotes. Brucella, the causative agent of brucellosis that threatens public health and animal husbandry, contains a conserved gene that encodes BI-1-like protein. To explore the role of the Brucella homolog of BI-1, BrBI, in Brucella suis S2, we constructed the brbI deletion mutant strain and its complemented strain. brbI deletion altered the membrane properties of Brucella suis S2 and decreased its resistance to acidic pH, H2O2, polymyxin B, and lincomycin. Additionally, deleting brbI led to defective growth, cell division, and viability in Brucella suis S2. We then revealed the effect of brbI deletion on the physiological characteristics of Brucella suis S2 via integrated transcriptomic and proteomic analyses. The integrated analysis showed that brbI deletion significantly affected the expression of multiple genes at the mRNA and/or protein levels. Specifically, the affected divisome proteins, FtsB, FtsI, FtsL, and FtsQ, may be the molecular basis of the impaired cell division of the brbI mutant strain, and the extensively affected membrane proteins and transporter-associated proteins were consistent with the phenotype of the membrane properties' alterations of the brbI mutant strain. In conclusion, our results revealed that BrBI is a bacterial cytoprotective protein involved in membrane homeostasis, cell division, and stress resistance in Brucella suis S2.

Omp16, a conserved peptidoglycan-associated lipoprotein, is involved in Brucella virulence in vitro.

Brucella, the bacterial agent of common zoonotic brucellosis, primarily infects specific animal species. The Brucella outer membrane proteins (Omps) are particularly attractive for developing vaccine and improving diagnostic tests and are associated with the virulence of smooth Brucella strains. Omp16 is a homologue to peptidoglycan-associated lipoproteins (Pals), and an omp16 mutant has not been generated in any Brucella strain until now. Very little is known about the functions and pathogenic mechanisms of Omp16 in Brucella. Here, we confirmed that Omp16 has a conserved Pal domain and is highly conserved in Brucella. We attempted to delete omp16 in Brucella suis vaccine strain 2 (B. suis S2) without success, which shows that Omp16 is vital for Brucella survival. We acquired a B. suis S2 Omp16 mutant via conditional complementation. Omp16 deficiency impaired Brucella outer membrane integrity and activity in vitro. Moreover, inactivation of Omp16 decreased bacterial intracellular survival in macrophage RAW 264.7 cells. B. suis S2 and its derivatives induced marked expression of IL-1ß, IL-6, and TNF-a mRNA in Raw 264.7 cells. Whereas inactivation of Omp16 in Brucella enhanced IL-1ß and IL-6 expression in Raw 264.7 cells. Altogether, these findings show that the Brucella Omp16 mutant was obtained via conditional complementation and confirmed that Omp16 can maintain outer membrane integrity and be involved in bacterial virulence in Brucella in vitro and in vivo. These results will be important in uncovering the pathogenic mechanisms of Brucella.

VceC Mediated IRE1 Pathway and Inhibited CHOP-induced Apoptosis to Support Brucella Replication in Goat Trophoblast Cells.

The effectors of the type IV secretion system (T4SS) of bacteria play important roles in mediating bacterial intracellular proliferation and manipulating host-related pathway responses to bacterial infection. Brucella Spp. inhibit the apoptosis of host cells to benefit their own intracellular proliferation. However, the underlying mechanisms between T4SS effectors and Brucella-inhibited apoptosis in goat trophoblast cells remain unclear. Here, based on Brucella suis vaccine strain 2, the VceC was deleted by allelic exchange. We show that ΔVceC was able to infect and proliferate to high titers in goat trophoblast cells (GTCs) and increase C/EBP-homologous protein (CHOP)-mediated apoptosis. GRP78 expression decreased upon ΔVceC infection. In addition, we discovered that the inositolrequiring enzyme 1 (IRE1) pathway was inhibited in this process. Changing endoplasmic reticulum (ER) stress affected Brucella intracellular replication in GTCs. The replication of ΔVceC was more sensitive under the different ERstress conditions in the GTC line after treatment with ER stress inhibitors 4 phenyl butyric acid (4-PBA) or ER stress activator Tm. Together, our findings show that VceC has a protective effect on the intracellular persistence of Brucella infection, and inhibits ER stress-induced apoptosis in the CHOP pathway. The present work provides new insights for understanding the mechanism of VceC in the establishment of chronic Brucella infection.

Brucella induces unfolded protein response and inflammatory response via GntR in alveolar macrophages.

Brucella is an intracellular bacterium that causes the zoonosis brucellosis worldwide. Alveolar macrophages (AM) constitute the main cell target of inhaled Brucella. Brucella thwarts immune surveillance and evokes endoplasmic reticulum (ER) stress to replicate in macrophages via virulence factors. The GntR regulators family was concentrated as an important virulence factor in controlling virulence and intracellular survival of Brucella. However, the detailed underlying mechanism for the host-pathogen interaction is poorly understood. In this study the BSS2_II0438 mutant (ΔGntR) was constructed. The type IV secretion system (T4SS) virulence factor genes (VirB2, VirB6, and VirB8) were down-expression in ΔGntR. ΔGntR could infect and proliferate to high titers in GAMs without a significant difference compared with the parental strain. ΔGntR infection increased the expression of ER stress marker genes GRP78, ATF6, and PERK in the early stages of its intracellular cycle but decreased the expression of these genes in the late stages. ΔGntR increased greatly the number of Brucella CFUs in the inactive ER stress state in GAMs. Meanwhile, ΔGntR infection increased the levels of IFN-γ, IL-1ß, and TNF-α, indicating ΔGntR could induce the secretion of inflammatory but not anti-inflammatory cytokines IL-10. Taken together, our results clarified the role of the GntR in B. suis. S2 virulence expression and elucidated that GntR is potentially involved in the signaling pathway of the Brucella-induced UPR and inflammatory response in GAMs.