Faculty Bibliography

The gut microbiome includes trillions of commensal bacteria that provide key benefits to the host including aiding digestion, promoting the development of the immune system, and protecting the epithelial barrier. However, an inappropriate immune response directed at members of the microbiome can also lead to diseases such as inflammatory bowel disease (IBD). For these reasons, the bacterial microbiome has received much attention as critical regulators of mammalian health and disease. Less is known about the impact of other intestinal inhabitants, such as viruses that are part of the enteric virome. We present evidence that an intestinal animal virus can influence host physiology in a manner similar to commensal bacteria. Although murine norovirus (MNV) can persistently infect mice without causing obvious signs of disease, we found that this virus induces intestinal pathologies in a mousemodel of IBD. Therefore, like colonization by commensal bacteria, MNV infection is typically harmless but can induce disease in a genetically susceptible host. Remarkably, we found that MNV infection reproduces beneficial functions of bacteria as well. Viral infection of germ-free mice or antibioticstreated mice reversed abnormalities in intestinal and immune development thatwere due to the depletion of bacteria. Moreover, MNV protected bacterially-deficient mice from chemical and infectious damage to the intestine. These finding indicate that viruses can perform functions that have been attributed to commensal bacteria, and has implications for how we view the role of non-bacterial members of the microbiome. The relationship between MNV, cytokine signaling, and other infectious entities will be discussed in the context of disease susceptibility

Defense strategies against infectious threats can be divided into resistance and tolerance mechanisms. Resistance mechanisms involve reduction of pathogen burden and include many established examples, one of them being the destruction of intracellular pathogens through autophagy (xenophagy). Tolerance mechanisms protect the host from damage caused by the pathogen or the immune response independent of pathogen load. The role of autophagy in maintaining homeostasis in response to environmental stress suggests that this pathway is involved in tolerance to a variety of infectious agents. However, demonstrating that autophagy promotes tolerance independent of its role in resistance has been a challenge, especially during infection by clinically relevant pathogens. We have found that autophagy protects against Staphylococcus aureus infection by maintaining tolerance toward a pore forming toxin secreted by the bacteria, alpha-toxin.

The success of Mycobacterium tuberculosis (Mtb) as a pathogen rests upon its ability to grow intracellularly in macrophages. Interferon-gamma (IFN-gamma) is critical in host defense against Mtb and stimulates macrophage clearance of Mtb through an autophagy pathway. Here we show that the host protein ubiquilin 1 (UBQLN1) promotes IFN-gamma-mediated autophagic clearance of Mtb. Ubiquilin family members have previously been shown to recognize proteins that aggregate in neurodegenerative disorders. We find that UBQLN1 can interact with Mtb surface proteins and associates with the bacilli in vitro. In IFN-gamma activated macrophages, UBQLN1 co-localizes with Mtb and promotes the anti-mycobacterial activity of IFN-gamma. The association of UBQLN1 with Mtb depends upon the secreted bacterial protein, EsxA, which is involved in permeabilizing host phagosomes. In autophagy-deficient macrophages, UBQLN1 accumulates around Mtb, consistent with the idea that it marks bacilli that traffic through the autophagy pathway. Moreover, UBQLN1 promotes ubiquitin, p62, and LC3 accumulation around Mtb, acting independently of the E3 ligase parkin. In summary, we propose a model in which UBQLN1 recognizes Mtb and in turn recruits the autophagy machinery thereby promoting intracellular control of Mtb. Thus, polymorphisms in ubiquilins, which are known to influence susceptibility to neurodegenerative illnesses, might also play a role in host defense against Mtb.

The mammalian virome includes diverse commensal and pathogenic viruses that evoke a broad range of immune responses from the host. Sustained viral immunomodulation is implicated in a variety of inflammatory diseases, but also confers unexpected benefits to the host. These outcomes of viral infections are often dependent on host genotype. Moreover, it is becoming clear that the virome is part of a dynamic network of microorganisms that inhabit the body. Therefore, viruses can be viewed as a component of the microbiome, and interactions with commensal bacteria and other microbial agents influence their behavior. This piece is a review of our current understanding of how the virome, together with other components of the microbiome, affects the function of the host immune system to regulate health and disease.

Resistance and tolerance are two defense strategies employed by the host against microbial threats. Autophagy-mediated degradation of bacteria has been extensively described as a major resistance mechanism. Here we find that the dominant function of autophagy proteins during infections with the epidemic community-associated methicillin-resistant Staphylococcus aureus USA300 is to mediate tolerance rather than resistance. Atg16L1 hypomorphic mice (Atg16L1HM), which have reduced autophagy, were highly susceptible to lethality in both sepsis and pneumonia models of USA300 infection. Autophagy confers protection by limiting the damage caused by alpha-toxin, particularly to endothelial cells. Remarkably, Atg16L1HM mice display enhanced survival rather than susceptibility upon infection with alpha-toxin-deficient S. aureus. These results identify an essential role for autophagy in tolerance to Staphylococcal disease and highlight how a single virulence factor encoded by a pathogen can determine whether a given host factor promotes tolerance or resistance.

Viruses affect host physiology beyond causing acute disease, thereby giving rise to the concept that the virome is a component of the microbiome. However, the role of the enteric virome is understudied relative to the fast-paced research examining commensal bacteria in the intestine. In this article, I discuss our recent work on murine norovirus indicating that an animal virus in the intestine can provide many of the signals to the host that have been attributed to commensal bacteria. Our findings suggest that the surge in microbiome research should incorporate examination of the enteric virome.

Mutations that alter virulence and antibiotic susceptibility arise and persist during Staphylococcus aureus bacteremia. However, an experimental system demonstrating transmission following bacteremia has been lacking, and thus implications of within-host adaptation for between-host transmission are unknown. We report that S. aureus disseminates to the gastrointestinal tract of mice following intravenous injection and readily transmits to cohoused naive mice. Both intestinal dissemination and transmission were linked to the production of virulence factors based on gene deletion studies of the sae and agr two-component systems. Furthermore, antimicrobial selection for antibiotic-resistant S. aureus displaced susceptible S. aureus from the intestine of infected hosts, which led to the preferential transmission and dominance of antibiotic-resistant bacteria among cohoused untreated mice. These findings establish an animal model to investigate gastrointestinal dissemination and transmission of S. aureus and suggest that adaptation during the course of systemic infection has implications beyond the level of a single host.

Autophagy is a process whereby a double-membrane structure (autophagosome) engulfs unnecessary cytosolic proteins, organelles, and invading pathogens and delivers them to the lysosome for degradation. We examined the fate of cytosolic Salmonella targeted by autophagy and found that autophagy-targeted Salmonella present in the cytosol of HeLa cells correlates with intracellular bacterial replication. Real-time analyses revealed that a subset of cytosolic Salmonella extensively associates with autophagy components p62 and/or LC3 and replicates quickly, whereas intravacuolar Salmonella shows no or very limited association with p62 or LC3 and replicates much more slowly. Replication of cytosolic Salmonella in HeLa cells is significantly decreased when autophagy components are depleted. Eventually, hyperreplication of cytosolic Salmonella potentiates cell detachment, facilitating the dissemination of Salmonella to neighboring cells. We propose that Salmonella benefits from autophagy for its cytosolic replication in HeLa cells. IMPORTANCE As a host defense system, autophagy is known to target a population of Salmonella for degradation and hence restricting Salmonella replication. In contrast to this concept, a recent report showed that knockdown of Rab1, a GTPase required for autophagy of Salmonella, decreases Salmonella replication in HeLa cells. Here, we have reexamined the fate of Salmonella targeted by autophagy by various cell biology-based assays. We found that the association of autophagy components with cytosolic Salmonella increases shortly after initiation of intracellular bacterial replication. Furthermore, through a live-cell imaging method, a subset of cytosolic Salmonella was found to be extensively associated with autophagy components p62 and/or LC3, and they replicated quickly. Most importantly, depletion of autophagy components significantly reduced the replication of cytosolic Salmonella in HeLa cells. Hence, in contrast to previous reports, we propose that autophagy facilitates Salmonella replication in the cytosol of HeLa cells.