The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response

Abstract

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...

The Emerging Roles of STING in Bacterial Infections

“…B. abortus DNA is the major bacterial PAMP inducing type I IFN response, with STING playing an important role in this process [86]. Surprisingly, elevated levels of c-di-GMP due to loss of a specific bacterial phosphodiesterase did not lead to an increased STING response [87]. However, lack of STING renders macrophages inefficient to kill Brucella , resulting in increased bacterial burden [87].…”

“…Surprisingly, elevated levels of c-di-GMP due to loss of a specific bacterial phosphodiesterase did not lead to an increased STING response [87]. However, lack of STING renders macrophages inefficient to kill Brucella , resulting in increased bacterial burden [87]. Thus, Brucella DNA remains a major bacterial component inducing IFNβ and there is no data about the importance of other bacterial CDNs in its physiology.…”

Genomic analysis of the original Elberg Brucella melitensis Rev.1 vaccine strain reveals insights into virulence attenuation

“…Hydroxypyruvate reductase metabolizes the toxic intermediate glyoxylate – a small and very reactive dicarboxylic acid molecule synthesized by most eukaryotes and prokaryotes [16]. Notably, the hydroxypyruvate reductase of the attenuated B. melitensis Δ bpdA phosphodiesterase mutant, which produces excess c-di-GMP, was shown to be downregulated, emphasizing the relevance of this enzyme in Brucella virulence [17]. …”

Brucella suppress innate immunity by down-regulating STING expression in macrophages

“…Given the requirement for STING in the control of chronic infection, it was surprising to note significant 101 STING (Tmem173) mRNA down-regulation in bone marrow-derived macrophages (BMDMs) infected 102 with wild type 16M Brucella melitensis at 24h (published RNAseq data set in Khan et al, 2016). To 103 via RT-qPCR ( Figure 3D).…”