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.

dBrucella melitensis is a well-adapted zoonotic pathogen considered a scourge of mankind since recorded history. In some cases, initial infection leads to chronic and reactivating brucellosis, incurring significant morbidity and economic loss. The mechanism by which B. melitensis subverts adaptive immunological memory is poorly understood. Previous work has shown that Brucellaspecific CD8؉ T cells express gamma interferon (IFN-␥) and can transition to long-lived memory cells but are not polyfunctional. In this study, chronic infection of mice with B. melitensis led to CD8 ؉ T cell exhaustion, manifested by programmed cell death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) expression and a lack of IFN-␥ production. ؉ T cells increased after adoptive transfer in both challenged and unchallenged recipients. CD8 ؉ T cells of challenged recipients initially retained the stunted IFN-␥ production found prior to transfer, and cells from acutely infected mice were never seen to transition to either memory subset at all time points tested, up to 30 days post-primary infection, suggesting a delay in the generation of memory. Here we have identified defects in Brucella-responsive CD8 ؉ T cells that allow chronic persistence of infection. Brucellosis caused by Brucella melitensis has a high incidence in developing countries, and the World Health Organization considers brucellosis one of the seven neglected zoonoses, a group of diseases that contribute to the perpetuation of poverty (1, 2). Brucella has many mechanisms to survive and replicate in hostile host cells, including inducing the unfolded-protein response (UPR), hijacking host nutrients, and counteracting the effects of pH changes, among many others (3-6). The chronic, reactivating nature of Brucella infection, along with its stealthy intracellular life-style, makes infections difficult to clear and requires lengthy antibiotic treatment (7-9). CD8 ϩ T cells control intracellular infections by identifying and killing compromised host cells as a part of the adaptive immune response (10, 11). Recognition of nonself antigenic epitopes in the context of major histocompatibility complex (MHC) class I by cytotoxic T cells also leads to the release of effector molecules to increase local inflammation, thereby "raising the alert" of the host in response to intracellular infection (12). A subset of MHC class I-restricted epitopes of Brucella melitensis generated during infection has been characterized and can elicit specific CD8 ϩ T cells (13). These T cells have been shown to kill their target cells, release cytokines, and survive into the chronic phase of infection (7). Why, then, in the successful establishment of chronic brucellosis, do we see the highly evolved CD8 ϩ T cell arm of adaptive immunity fail to protect the host from long-term infection?Immunological memory is the ability of the host to mount a fast, effective secondary response to infection. CD8 ϩ T cell memory is derived from effectors generated during primary infection or vaccination, a small cohort of ...

f Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host-Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a ⌬bpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the ⌬bpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase ⌬cgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, ⌬bpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection. Brucella species are Gram-negative, facultative intracellular bacterial pathogens that cause brucellosis, the most prevalent zoonosis worldwide (1, 2). With more than 500,000 infections per year, the high incidence of brucellosis in southeastern Europe, the Mediterranean, South America, and Africa causes a major economic burden (2, 3). In animals, brucellosis is characterized by increased abortion, weak offspring, and decreased milk production. Brucella melitensis is the predominant cause of human brucellosis; however, B. abortus, B. suis, and B. canis can also infect humans (4). Human brucellosis is typically acquired by consuming contaminated milk products or via inhalation of aerosolized bacteria from occupational hazards (5). Human brucellosis is a debilitating disease in which most people initially experience a period of undulating fever which can progress to a chronic infection if untreated or if antibiotic treatment fails. Complications of chronic infections include liver damage, orchitis, endocarditis, and arthritis (1, 4).Brucella spp. have the ability to infect both professional and nonprofessional phagocytes (6). Because of this, Brucella spp. encounter varied environments both throughout the body and within a cell and must adapt accordingly. To date, few virulence factors have been identified in Brucella, and even less is known about how these virulence factors are regulated. Subsequently, little is known how Brucella adapts to its rapidly changing environments and how it alternates between acute and chronic virulence.The second messenger cyclic di-GMP (c-di-GMP...

Background: Atu027 is a liposomally formulated short interfering RNA with anti-metastatic activity, which silences the expression of protein kinase N3 (PKN3) in the vascular endothelium. This trial was designed to assess the safety, pharmacokinetics and efficacy of Atu027 in combination with gemcitabine in advanced pancreatic carcinoma (APC). Methods: In total, 23 patients (pts) with inoperable APC were randomly assigned to gemcitabine combined with two different Atu027 schedules (0.235 mg/kg once weekly vs. 0.235 mg/kg twice weekly). ClinicalTrials.gov Identifier: NCT01808638. Results: The treatment was well-tolerated. There were Grade 3 adverse events (AEs) in 9/11 pts (arm 1) and 11/12 pts (arm 2), while Grade 4 AEs were reported for two pts in each arm. The AEs were mainly laboratory abnormalities without clinical significance. The median progression-free survival reached statistical significance in patients who had metastatic disease (1.6 vs. 2.9 months, p = 0.025). Disease control during treatment was achieved in 4/11 pts (arm 1) and in 7/12 pts (arm 2). Pts in arm 1 experienced stable global health status while pts in arm 2 reported improvement. Conclusions: Combining Atu027 with gemcitabine is safe and well tolerated. In pts with metastatic APC, twice-weekly Atu027 is associated with significantly improved outcomes. Our clinical results support the significant involvement of the vascular endothelium in the spread of cancer, and thus the further investigation of its target role.