Brucella second messenger coordinates stages of infection
Authors: Splitter Gary A. , Banai Menachem , Harms Jerome S. 
Published: March 2022

Aim 1: To determine levels of this second messenger in: a) B. melitensiscyclic-dimericguanosinemonophosphate-regulating mutants (BMEI1448, BMEI1453, and BMEI1520), and b) B. melitensis16M (wild type) and mutant infections of macrophages and immune competent mice. (US lab primary) Aim 2: To determine proteomic differences between Brucelladeletion mutants BMEI1453 (high cyclic-dimericguanosinemonophosphate, chronic persistent state) and BMEI1520 (low cyclicdimericguanosinemonophosphate, acute virulent state) compared to wild type B. melitensisto identify the role of this second messenger in establishing the two polar states of brucellosis. (US lab primary with synergistic assistance from the Israel lab Aim 3: Determine the level of Brucellacyclic-dimericguanosinemonophosphate and transcriptional expression from naturally infected placenta. (Israel lab primary with synergistic assistance from the US lab). B. Background Brucellaspecies are Gram-negative, facultative intracellular bacterial pathogens that cause brucellosis, the most prevalent zoonosis worldwide. Brucellosis is characterized by increased abortion, weak offspring, and decreased milk production in animals. Humans are infected with Brucellaby consuming contaminated milk products or via inhalation of aerosolized bacteria from occupational hazards. Chronic human infections can result in complications such as liver damage, orchitis, endocarditis, and arthritis. Brucellaspp. have the ability to infect both professional and non-professional phagocytes. Because of this, Brucellaencounter varied environments both throughout the body and within a cell and must adapt accordingly. To date, few virulence factors have been identified in B. melitensisand even less is known about how these virulence factors are regulated. Subsequently, little is known about how Brucellaadapt to its rapidly changing environments, and how it alternates between acute and chronic virulence. Our studies suggest that decreased concentrations of cyclic dimericguanosinemonophosphate (c-di-GMP) lead to an acute virulent state and increased concentrations of c-di-GMP lead to persistent, chronic state of B. melitensisin a mouse model of infection. We hypothesize that B. melitensisuses c-di-GMP to transition from the chronic state of an infected host to the acute, virulent stage of infection in the placenta where the bacteria prepare to infect a new host. Studies on environmental pathogens such as Vibrio choleraeand Pseudomonas aeruginosasupport a mechanism where changes in c-di-GMP levels cause the bacterium to alternate between virulent and chronic states. Little work exists on understanding the role of c-di-GMP in dangerous intracellular pathogens, like Brucellathat is a frequent pathogen in Israeli domestic animals and U.S. elk and bison. Brucellamust carefully regulate virulence factors during infection of a host to ensure proper expression at appropriate times in response to host cues. Recently, the novel secondary signaling molecule c-di-GMP has been identified as a major component of bacterial regulation and we have identified c-di-GMP as an important signaling factor in B. melitensishost adaptation. C. Major conclusions, solutions, achievements 1. The B. melitensis1453 deletion mutant has increased c-di-GMP, while the 1520 deletion mutant has decreased c-di-GMP. 2. Both mutants grow similarly in in vitro cultures; however, the 1453 mutant has a microcolony phenotype both in vitro and in vivo 3. The 1453 mutant has increased crystal violet staining suggesting biofilm formation. 4. Scanning electron microscopy revealed an abnormal coccus appearance with in increased cell area. 5. Proteomic analysis revealed the 1453 mutant possessed increased production of proteins involved in cell wall processes, cell division, and the Type IV secretion system, and a decrease in proteins involved in amino acid transport/metabolism, carbohydrate metabolism, fatty acid production, and iron acquisition suggesting less preparedness for intracellular survival. 6. RNAseq analysis of bone marrow derived macrophages infected with the mutants revealed the host immune response is greatly reduced with the 1453 mutant infection. These findings support that microlocalization of proteins involved in c-di-GMP homeostasis serve a second messenger to B. melitensisregulating functions of the bacteria during infection of the host.