Molecular dissection of cytosolic innate immune activation during bacterial infection.

It was first recognized several decades ago by Janeway and Medzhitov that there are pattern recognition receptors on the surface of mammalian cells that recognize microbial-associated molecular patterns. More recently, it has been discovered that there are similar “receptors” that monitor the sanctity of the host cell cytosol and respond to “danger” signals by initiating innate immune responses. The Monack Lab studies the molecular mechanisms of intracellular recognition of bacterial pathogens by comparing and contrasting infections with intracellular Salmonella and Francisella. One of the lab’s most notable discoveries was demonstrating that the type I Interferon and the inflammasome innate immune pathways are sequentially linked and that this 2-tiered response is a gauge of the “danger” level before commitment to cell death.

Specifically, the Monack Lab has shown that cytosolic Francisella elicit a type I IFN response within an hour of infection. This results in up-regulation of many innate immune genes including AIM2, a host cytosolic receptor that binds dsDNA, which is a cytosolic “danger signal.” The dsDNA - AIM2 interaction triggers inflammasome assembly and caspase-1 activation. Active caspase-1 cleaves pro-inflammatory cytokines and mediates macrophage death, which eliminates the intracellular pathogen niche. Importantly, none of these pathways are important for Salmonella, which might reflect the fact that Salmonella does not replicate in the cytosol of macrophages. 

Intracellular Salmonella stimulates caspase-1 activation by a different pathway compared to Francisella. The Monack Lab has shown that translocation of flagellin through Salmonella’s type 3 secretion system triggers a very rapid macrophage death that requires an intracellular scaffold protein called NLRC4/Ipaf that binds and activates caspase-1, which then leads to cytokine activation and cell death. The Monack Lab has shown in mouse models of infection that the caspase-1-dependent maturation of proinflammatory cytokines and macrophage death are important for innate immune defenses against Francisella and Salmonella. The Monack Lab also elucidated a caspase-11-dependent cell death that is critical in eliminating Salmonella’s intracellular niche. Collectively, the Monack Lab’s work is key to increasing our understanding of how caspase-1- and caspase-11-dependent innate immune mechanisms detect bacterial pathogens.

More recently, the Monack Lab interrogated the caspase-11 pathway by conducting a genome-wide CRISPR/Cas9 screen in macrophages to identify host factors that impact LPS-dependent activation of caspase-11.  This led to the discovery of pathways that amplify caspase-11-dependent cell death.  Importantly, in a mouse model of endotoxic shock where uncontrolled caspase-11 activation leads to death, her lab has shown that these new amplification pathways of the caspase-11 pathway increase severity and pathologies during sepsis.


Identification of novel Salmonella virulence determinants that mediate persistent infection.

The lifestyle of Salmonella enterica serovar Typhimurium during acute infections in the gastrointestinal tract and within macrophages in systemic tissues has been intensely studied. However, the mechanisms that lead to bacterial survival, systemic dissemination, and transmission during persistent infections are less well understood.

Through genetic screens, we have discovered genes required for long-term survival in a mouse model of Salmonella infection. We have shown during colonization of systemic sites that Salmonella injects effector proteins through the type 3-secretion system encoded on Salmonella Pathogenicity Island 2 that mediate inhibition of dendritic cell (DC) chemotaxis. We have also shown that this inhibition of DC chemotaxis contributes to Salmonella persistence by dampening adaptive immune responses. In addition, we have shown that there are nutritional and virulence mechanisms that mediate intraspecies competition in the distal gut that influence transmission to naïve hosts.

 


Characterization of immune states that mediate tolerance and influence disease transmission.

We have shown that oral infection of 129SvJ mice with Salmonella results in 20-30% of the mice being supershedders (shed >108 CFU/g feces), which rapidly transmit infection. Although supershedder mice develop colitis, they remain asymptomatic. Importantly, both supershedder and non-supershedder hosts carry identical pathogen burdens across all tissues except the intestinal tract. Our results strongly suggest that tolerance mechanisms play a role in the maintenance of the asymptomatic supershedder state. What is tolerance in the context of our model? An infected host can fight pathogenic infection by two distinct processes, resistance and tolerance. Resistance encompasses a diverse set of mechanisms employed by the host to control pathogen invasion and replication. Tolerance, on the other hand, employs different mechanisms that help the host organism tolerate the damage caused by the pathogenic infection and the resulting immune response.

We have been testing the hypothesis that tolerance mechanisms play a role in the maintenance of the asymptomatic supershedder state. Although very little is known about the full spectrum of tolerance mechanisms, the few studies in animals suggest that since pathogens and immunopathology can potentially affect almost any physiological process, tolerance is not restricted to a single protective pathway. Thus, we are taking broad unbiased approaches to map the crosstalk between the host immune state and pathogen during asymptomatic carriage. Our initial studies have shown that supershedder hosts have a unique dampened immune state that not only facilitates disease transmission but also confers tolerance to antibiotic-induced disturbances of the microbial communities in the gastrointestinal tract.