Brucella targets the host ubiquitin-specific protease, Usp8, through the effector protein, TcpB, for facilitating infection of macrophages.

Brucella species are Gram-negative intracellular bacterial pathogens that cause the worldwide zoonotic disease brucellosis. Brucella can infect many mammals, including humans and domestic and wild animals. Brucella manipulates various host cellular processes to invade and multiply in professional and non-professional phagocytic cells. However, the host targets and their modulation by Brucella to facilitate the infection process remain obscure. Here, we report that the host ubiquitin-specific protease, USP8, negatively regulates the invasion of Brucella into macrophages through the plasma membrane receptor, CXCR4. Upon silencing or chemical inhibition of USP8, the membrane localization of the CXCR4 receptor was enriched, which augmented the invasion of Brucella into macrophages. Activation of USP8 through chemical inhibition of 14-3-3 protein affected the invasion of Brucella into macrophages. Brucella suppressed the expression of Usp8 at its early stage of infection in the infected macrophages. Furthermore, we found that only live Brucella could negatively regulate the expression of Usp8, suggesting the role of secreted effector protein of Brucella in modulating the gene expression. Subsequent studies revealed that the Brucella effector protein, TIR-domain containing protein from Brucella, TcpB, plays a significant role in downregulating the expression of Usp8 by targeting the cyclic-AMP response element-binding protein pathway. Treatment of mice with USP8 inhibitor resulted in enhanced survival of B. melitensis, whereas mice treated with CXCR4 or 14-3-3 antagonists showed a diminished bacterial load. Our experimental data demonstrate a novel role of Usp8 in the host defense against microbial intrusion. The present study provides insights into the microbial subversion of host defenses, and this information may ultimately help to develop novel therapeutic interventions for infectious diseases.

Immuno-profiling of Brucella proteins for developing improved vaccines and DIVA capable serodiagnostic assays for brucellosis.

Brucellosis remains a worldwide zoonotic disease with a serious impact on public health and livestock productivity. Controlling brucellosis in livestock is crucial for limiting human infections in the absence of effective human vaccines. Brucellosis control measures are majorly dependent on rigorous monitoring of disease outbreaks and mass vaccination of livestock. Live attenuated vaccines are available for livestock vaccination that play a vital role in brucellosis control programs in many countries. Even though the existing animal vaccines confer protection against brucellosis, they carry some drawbacks, including their infectivity to humans and interference with sero-monitoring. The available serodiagnostic assays for brucellosis depend on detecting anti-LPS antibodies in the serum. Since diagnosis plays a vital role in controlling brucellosis, developing improved serodiagnostic assays with enhanced specificity, sensitivity and DIVA capability is required. Therefore, it is essential to identify novel antigens for developing improved vaccines and serodiagnostic assays for brucellosis. In the present study, we performed a high throughput immunoprofiling of B. melitensis protein microarray using brucellosis-positive human and animal serum samples. The screening identified several serodominant proteins of Brucella that exhibited common or differential reactivity with sera from animals and humans. Subsequently, we cloned, expressed, and purified ten serodominant proteins, followed by analyzing their potential to develop next-generation vaccines and improved serodiagnostic assays for brucellosis. Further, we demonstrated the protective efficacy of one of the serodominant proteins against the B. melitensis challenge in mice. We found that the seroreactive protein, Dps (BMEI1980), strongly reacted with brucellosis-positive serum samples, but it did not react with sera from B. abortus S19-vaccinated cattle, indicating DIVA capability. A prototype lateral flow assay and indirect ELISA based on Dps protein exhibited high sensitivity, specificity, and DIVA capability. Thus, the present study identified promising candidates for developing improved vaccines and affordable, DIVA-capable serodiagnostic assays for animal and human brucellosis.

Outer membrane protein 25 of Brucella suppresses TLR-mediated expression of proinflammatory cytokines through degradation of TLRs and adaptor proteins.

Toll-like receptors (TLRs) are essential components of innate immunity that serves as the first line of defense against the invaded microorganisms. However, successful infectious pathogens subvert TLR signaling to suppress the activation of innate and adaptive responses. Brucella species are infectious intracellular bacterial pathogens causing the worldwide zoonotic disease, brucellosis, that impacts economic growth of many countries. Brucella species are considered as stealthy bacterial pathogens as they efficiently evade or suppress host innate and adaptive immune responses for their chronic persistence. However, the bacterial effectors and their host targets for modulating the immune responses remain obscure. Brucella encodes various outer membrane proteins (Omps) that facilitate their invasion, intracellular replication, and immunomodulation. Outer membrane protein 25 (Omp25) of Brucella plays an important role in the immune modulation through suppression of proinflammatory cytokines. However, the mechanism and the signaling pathways that are targeted by Omp25 to attenuate the production of proinflammatory cytokines remain obscure. Here, we report that Omp25 and its variants, viz. Omp25b, Omp25c, and Omp25d, suppress production of proinflammatory cytokines that are mediated by various TLRs. Furthermore, we demonstrate that Omp25 and its variants promote enhanced ubiquitination and degradation of TLRs and their adaptor proteins to attenuate the expression of proinflammatory cytokines. Targeting multiple TLRs and adaptor proteins enables Omp25 to effectively suppress the expression of proinflammatory cytokines that are induced by diverse pathogen-associated molecular patterns. This can contribute to the defective adaptive immune response and the chronic persistence of Brucella in the host.

Host F-Box Protein 22 Enhances the Uptake of Brucella by Macrophages and Drives a Sustained Release of Proinflammatory Cytokines through Degradation of the Anti-Inflammatory Effector Proteins of Brucella.

Brucella species are intracellular bacterial pathogens, causing the worldwide zoonotic disease brucellosis. Brucella invades professional and nonprofessional phagocytic cells, followed by resisting intracellular killing and establishing a replication permissive niche. Brucella also modulates the innate and adaptive immune responses of the host for its chronic persistence. The complex intracellular cycle of Brucella depends in a major way on multiple host factors, but limited information is available on host and bacterial proteins that play an essential role in the invasion, intracellular replication, and modulation of host immune responses. By employing a small interfering RNA (siRNA) screening, we identified a role for the host protein FBXO22 in the Brucella-macrophage interaction. FBXO22 is the key element in the SCF E3 ubiquitination complex, where it determines the substrate specificity for ubiquitination and degradation of various host proteins. Downregulation of FBXO22 by siRNA or the CRISPR-Cas9 system resulted in diminished uptake of Brucella into macrophages, which was dependent on NF-κB-mediated regulation of phagocytic receptors. FBXO22 expression was upregulated in Brucella-infected macrophages, which resulted in induction of phagocytic receptors and enhanced production of proinflammatory cytokines through NF-κB. Furthermore, we found that FBXO22 recruits the effector proteins of Brucella, including the anti-inflammatory proteins TcpB and OMP25, for degradation through the SCF complex. We did not observe any role for another F-box-containing protein of the SCF complex, ß-TrCP, in the Brucella-macrophage interaction. Our findings unravel novel functions of FBXO22 in host-pathogen interaction and its contribution to pathogenesis of infectious diseases.

The neurosteroid pregnenolone promotes degradation of key proteins in the innate immune signaling to suppress inflammation.

Pregnenolone is a steroid hormone precursor that is synthesized in various steroidogenic tissues, in the brain, and in lymphocytes. In addition to serving as the precursor for other steroid hormones, pregnenolone exerts its own effect as an anti-inflammatory molecule to maintain immune homeostasis in various inflammatory conditions. Pregnenolone and its metabolic derivatives have been shown to have beneficial effects in the brain, including enhancing memory and learning, reversing depressive disorders, and modulating cognitive functions. A decreased level of pregnenolone has been observed in neuroinflammatory diseases, which emphasizes its role in neuroprotection and neuroregeneration. Although the anti-inflammatory property of pregnenolone was recognized several decades ago, its mechanism of action remains unknown. Here we report that pregnenolone promotes ubiquitination and degradation of the TLR2/4 adaptor protein TIRAP and TLR2 in macrophages and microglial cells. Pregnenolone and its metabolites suppressed the secretion of tumor necrosis factor α and interleukin-6 mediated through TLR2 and TLR4 signaling. Pregnenolone has been reported to induce activation of cytoplasmic linker protein 170, and this protein has recently been shown to promote targeted degradation of TIRAP. We observed enhanced degradation of TIRAP and TLR4 suppression by cytoplasmic linker protein 170 in the presence of pregnenolone. Our experimental data reveal novel nongenomic targets of pregnenolone and provide important leads to understand its role in restoring immune homeostasis in various inflammatory conditions.

Cytoplasmic Linker Protein CLIP170 Negatively Regulates TLR4 Signaling by Targeting the TLR Adaptor Protein TIRAP.

Cytoplasmic linker protein 170 (CLIP170) is a CAP-Gly domain-containing protein that is associated with the plus end of growing microtubules and implicated in various cellular processes, including the regulation of microtubule dynamics, cell migration, and intracellular transport. Our studies revealed a previously unrecognized property and role of CLIP170. We identified CLIP170 as one of the interacting partners of Brucella effector protein TcpB that negatively regulates TLR2 and TLR4 signaling. In this study, we demonstrate that CLIP170 interacts with the TLR2 and TLR4 adaptor protein TIRAP. Furthermore, our studies revealed that CLIP170 induces ubiquitination and subsequent degradation of TIRAP to negatively regulate TLR4-mediated proinflammatory responses. Overexpression of CLIP170 in mouse macrophages suppressed the LPS-induced expression of IL-6 and TNF-α whereas silencing of endogenous CLIP170 potentiated the levels of proinflammatory cytokines. In vivo silencing of CLIP170 in C57BL/6 mice by CLIP170-specific small interfering RNA enhanced LPS-induced IL-6 and TNF-α expression. Furthermore, we found that LPS modulates the expression of CLIP170 in mouse macrophages. Overall, our experimental data suggest that CLIP170 serves as an intrinsic negative regulator of TLR4 signaling that targets TIRAP.

The Brucella effector protein TcpB induces degradation of inflammatory caspases and thereby subverts non-canonical inflammasome activation in macrophages.

The inflammasome contains intracellular receptors that recognize various pathogen-associated molecular patterns and play crucial roles in innate immune responses to invading pathogens. Non-canonical inflammasome activation is mediated by caspase-4/11, which recognizes intracellular LPS and promotes pyroptosis and secretion of proinflammatory cytokines. Brucella species are infectious intracellular pathogens that replicate in professional and non-professional phagocytic cells and subvert immune responses for chronic persistence in the host. The Brucella effector protein TcpB suppresses Toll-like receptor 2 (TLR2)- and TLR4-mediated innate immune responses by targeted degradation of the Toll/interleukin-1 receptor (TIR) domain-containing adaptor protein. TcpB is a cell-permeable protein with multiple functions, and its intracellular targets other than TIR domain-containing adaptor protein remain unclear. Here, we report that TcpB induces ubiquitination and degradation of the inflammatory caspases 1, 4, and 11. Furthermore, in both mouse and human macrophages, TcpB attenuated LPS-induced non-canonical inflammasome activation and suppressed pyroptosis and secretion of IL-1α and IL-1ß induced by intracellular LPS delivery. The intact TIR domain was essential for TcpB to subvert the non-canonical inflammasome activation as a TcpB(G158A) mutant failed to suppress pyroptotic cell death and inflammatory responses. Brucella-infected macrophages exhibited minimal pyroptosis but secreted IL-1ß, which was suppressed by TcpB. We also demonstrated that TcpB protein can efficiently attenuate Salmonella enterica serovar Typhimurium-induced pyroptosis and proinflammatory cytokine secretion in macrophages. Because TcpB suppresses both TLR4- and caspase-4/11-mediated inflammation, TcpB might be a candidate target for developing drugs against LPS-induced septicemia.

Genetic Characterization and Comparative Genome Analysis of Brucella melitensis Isolates from India.

Brucellosis is the most frequent zoonotic disease worldwide, with over 500,000 new human infections every year. Brucella melitensis, the most virulent species in humans, primarily affects goats and the zoonotic transmission occurs by ingestion of unpasteurized milk products or through direct contact with fetal tissues. Brucellosis is endemic in India but no information is available on population structure and genetic diversity of Brucella spp. in India. We performed multilocus sequence typing of four B. melitensis strains isolated from naturally infected goats from India. For more detailed genetic characterization, we carried out whole genome sequencing and comparative genome analysis of one of the B. melitensis isolates, Bm IND1. Genome analysis identified 141 unique SNPs, 78 VNTRs, 51 Indels, and 2 putative prophage integrations in the Bm IND1 genome. Our data may help to develop improved epidemiological typing tools and efficient preventive strategies to control brucellosis.

Studying host-pathogen interaction events in living mice visualized in real time using biophotonic imaging.

Despite progress in mouse models of bacterial pathogens, studies are often limited by evaluating infections in an individual organ or tissue or at a given time. Here we present a technique to engineer the pathogen, e.g., Brucella melitensis, with a bioluminescent marker permitting analysis of living bacteria in real time during the infectious process from acute to chronic infection. Using this bioluminescent approach, tissue preference, differences between virulent and mutant bacteria, as well as the response of the bacteria to host metabolites can provide extraordinary data enhancing our understanding of host-pathogen interactions.

Draft Genome Sequence of the Field Isolate Brucella melitensis Strain Bm IND1 from India.

Brucella spp. are facultative intracellular bacterial pathogens causing the zoonotic disease brucellosis. Here, we report the draft genome sequence of the Brucella melitensis strain from India designated Bm IND1, isolated from stomach contents of an aborted goat fetus.

Brucella induces an unfolded protein response via TcpB that supports intracellular replication in macrophages.

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.

Active evasion of CTL mediated killing and low quality responding CD8+ T cells contribute to persistence of brucellosis.

Brucellosis is a common zoonotic disease that remains endemic in many parts of the world. Dissecting the host immune response during this disease provides insight as to why brucellosis is often difficult to resolve. We used a Brucella epitope specific in vivo killing assay to investigate the ability of CD8+ T cells to kill targets treated with purified pathogenic protein. Importantly, we found the pathogenic protein TcpB to be a novel effector of adaptive immune evasion by inhibiting CD8+ T cell killing of Brucella epitope specific target cells in mice. Further, BALB/c mice show active Brucella melitensis infection beyond one year, many with previously unreported focal infection of the urogenital area. A fraction of CD8+ T cells show a CD8+ Tmem phenotype of LFA-1hi, CD127hi, KLRG-1lo during the course of chronic brucellosis, while the CD8+ T cell pool as a whole had a very weak polyfunctional cytokine response with diminished co-expression of IFN-γ with TNFα and/or IL-2, a hallmark of exhaustion. When investigating the expression of these 3 cytokines individually, we observed significant IFN-γ expression at 90 and 180 days post-infection. TNFα expression did not significantly exceed or fall below background levels at any time. IL-2 expression did not significantly exceeded background, but, interestingly, did fall significantly below that of uninfected mice at 180 days post-infection. Brucella melitensis evades and blunts adaptive immunity during acute infection and our findings provide potential mechanisms for the deficit observed in responding CD8+ T cells during chronic brucellosis.

Modulation of host microtubule dynamics by pathogenic bacteria.

The eukaryotic cytoskeleton is a vulnerable target of many microbial pathogens during the course of infection. Rearrangements of host cytoskeleton benefit microbes in various stages of their infection cycle such as invasion, motility, and persistence. Bacterial pathogens deliver a number of effector proteins into host cells for modulating the dynamics of actin and microtubule cytoskeleton. Alteration of the actin cytoskeleton is generally achieved by bacterial effectors that target the small GTPases of the host. Modulation of microtubule dynamics involves direct interaction of effector proteins with the subunits of microtubules or recruiting cellular proteins that affect microtubule dynamics. This review will discuss effector proteins from animal and human bacterial pathogens that either destabilize or stabilize host micro-tubules to advance the infectious process. A compilation of these research findings will provide an overview of known and unknown strategies used by various bacterial effectors to modulate the host microtubule dynamics. The present review will undoubtedly help direct future research to determine the mechanisms of action of many bacterial effector proteins and contribute to understanding the survival strategies of diverse adherent and invasive bacterial pathogens.

Modulation of microtubule dynamics by a TIR domain protein from the intracellular pathogen Brucella melitensis.

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.

Biochemical and functional analysis of TIR domain containing protein from Brucella melitensis.

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.

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

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.

DNA recognition properties of the cell-to-cell movement protein (MP) of soybean isolate of Mungbean yellow mosaic India virus (MYMIV-Sb).

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.