Thomas P. Burke

Lipopolysaccharide (LPS) is highly immunostimulatory, yet it is evolutionarily conserved among many obligate intracellular bacteria for unknown reasons. We report a forward genetic screen to identify factors required for survival of the tick-borne obligate cytosolic pathogen Rickettsia parkeri in primary macrophages. The most critical factors were WecA and RmlD, which synthesize O-antigen, the outermost layer of LPS. wecA and rmlD mutants grew at similar rates to wild type bacteria in epithelial cells, yet in macrophages they were targeted by guanylate binding proteins (GBPs) and they hyperactivated inflammasomes. Survival of O-antigen-deficient mutants was restored >1,000-fold in macrophages lacking Caspases-1 and -11, interferon signaling, and nitric oxide production, suggesting a multifaceted role for O-antigen in protecting against innate immunity. O-antigen was essential for causing disease in mice and protected R. parkeri against complement in vitro . Despite O-antigen being known as a major target of antibodies, mice immunized with O-antigen-deficient mutants were protected from a lethal rechallenge, suggesting that protection can be elicited independently of O-antigen-targeting antibodies. Together, these findings help resolve a paradox as to why obligate cytosolic bacteria evolutionarily maintain LPS despite it being immunostimulatory, which is that it serves as a multifunctional shield against innate immunity. Significance: Eukaryotic innate immune systems evolved to detect conserved microbial structures as danger signals of infection. Intracellular pathogens, in turn, evolved to hide from innate immunity, yet these mechanisms remain incompletely understood. Here, we performed an unbiased forward genetic screen in macrophages that identified lipopolysaccharide O-antigen as a critical virulence determinant in the tick-borne obligate cytosolic pathogen Rickettsia parkeri . We found that O-antigen shields the bacteria from multiple innate immune defenses, including guanylate-binding proteins, inflammasomes, nitric oxide, and complement. These findings reveal why a highly immunostimulatory molecule such as lipopolysaccharide is maintained by an obligate intracellular pathogen and establish O-antigen as a central determinant of Rickettsia cytosolic survival with implications for vaccine development.

Burke TP, Engström P, Tran CJ, Langohr IM, Glasner DR, Espinosa DA, Harris E, Welch MD. 2021. Interferon receptor-deficient mice are susceptible to eschar-associated rickettsiosis. eLife 10:e67029. doi: 10.7554/eLife.67029.

Intracellular bacteria rely on eukaryotic metabolites for their fitness and pathogenesis. Yet, the mechanisms of how host metabolites promote bacterial physiology and immune evasion are often unclear. Here, we employed obligate cytosolic Rickettsia parkeri, which parasitizes over fifty host metabolites, to investigate bacterial utilization of host glutathione (GSH). We observed that GSH depletion impaired R. parkeri intracellular survival. Super-resolution microscopy revealed that GSH depletion caused bacterial chaining in the host cytosol, prohibiting proper actin-based motility and cell-to-cell spread. GSH was especially critical for bacterial survival in primary macrophages, where it enabled R. parkeri to evade ubiquitylation and antibacterial autophagy. Cell division and survival defects were restored by supplementing N-acetylcysteine, suggesting that GSH serves as a cysteine source for R. parkeri. Together, these data suggest that Rickettsia requires GSH as a nutrient source to promote cell division, actin-based motility, evasion of antibacterial autophagy, and intracellular survival. This knowledge contributes to the expanding paradigm that GSH plays diverse roles in the pathogenesis of intracellular bacteria and represents a potential target for host-acting therapeutics.

Arthropod-borne pathogens cause serious human infections, yet they only cause limited disease in rodent reservoirs. Wild type mice resist infection by tick-borne Rickettsia parkeri, which causes spotted fever in humans, and it remains unclear why humans are vulnerable. Here, we report that whereas mouse type I interferon (IFN-I) or interferon-γ (IFN-γ) dramatically restrict R. parkeri in macrophages, human interferons do not. Differential RNA-seq revealed a significant induction of nitric oxide synthase 2 ( Nos2, encoding inducible nitric oxide synthase, iNOS) in infected mouse but not human macrophages upon interferon treatment. Chemical iNOS inhibition or Nos2 deletion restored IFN-γ-mediated restriction in mouse cells. Human cells treated with cytokine cocktails or with iNOS cofactors and substrates were still unable to restrict R. parkeri . In vivo , whereas wild type mice restricted R. parkeri , infected Nos2 ^-/- mice developed mild skin eschars, recapitulating a key human disease manifestation. Together, our findings suggest that there is a threshold of NO production required to restrict R. parkeri, which mouse cells reach but human cells do not, and this is a key explanation for why humans develop tick-borne rickettsial diseases while rodents can be tolerant, asymptomatic reservoirs. Differences in NO abundance may provide an evolutionary explanation for human susceptibility to pathogens that propagate themselves in rodent reservoirs.

Summary: Bacterial pathogens that invade the eukaryotic cytosol are distinctive tools for fighting cancer, as they preferentially target tumors and can deliver cancer antigens to MHC-I. Cytosolic bacterial pathogens have undergone extensive preclinical development and human clinical trials, yet the molecular mechanisms by which they are detected by innate immunity in tumors is unclear. We report that intratumoral delivery of phylogenetically distinct cytosolic pathogens, including Listeria, Rickettsia, and Burkholderia species, elicited anti-tumor responses in established, poorly immunogenic melanoma and lymphoma in mice. We were surprised to observe that although the bacteria required entry to the cytosol, the anti-tumor responses were largely independent of the cytosolic sensors cGAS/STING and instead required TLR signaling. Combining pathogens with TLR agonists did not enhance anti-tumor efficacy, while combinations with STING agonists elicited profound, synergistic anti-tumor effects with complete responses in >80% of mice after a single dose. Small molecule TLR agonists also synergistically enhanced the anti-tumor activity of STING agonists. The anti-tumor effects were diminished in Rag2 -deficient mice and upon CD8 T cell depletion. Mice cured from combination therapy developed immunity to cancer rechallenge that was superior to STING agonist monotherapy. Together, these data provide a framework for enhancing the efficacy of microbial cancer therapies and small molecule innate immune agonists, via the co-activation of STING and TLRs.

Summary: Spotted fever group Rickettsia obligately reside in the cytosol where they parasitize over fifty metabolites from their hosts. However, the role for metabolite acquisition in pathogenesis remains unclear. Here, we find that depletion of the abundant low molecular weight thiol glutathione led to an impaired ability of Rickettsia parkeri to form plaques. Super-resolution microscopy revealed that glutathione depletion with buthionine sulfoximine (BSO) in endothelial or epithelial cells led to the formation of bacterial chains that increased in length over time. Chained bacteria had fewer actin-tails and were impeded for their ability to spread from cell to cell. Glutathione depletion also caused an increased frequency of colocalization between the bacterial surface and polyubiquitin. R. parkeri was significantly more restricted upon glutathione depletion in primary macrophages than in epithelial cells in a mechanism that avoided activating the inflammasome and the production of type I interferon. Together, these data suggest that host glutathione is critical for rickettsial septation, actin-based motility, avoiding ubiquitylation, and survival in immune cells.

Rickettsia species of the spotted fever group are arthropod-borne obligate intracellular bacteria that can cause mild to severe human disease. These bacteria invade host cells, replicate in the cell cytosol, and spread from cell to cell. To access the host cytosol and avoid immune detection, they escape membrane-bound vacuoles by expressing factors that disrupt host membranes. Here, we show that a patatin-like phospholipase A2 enzyme (Pat1) facilitates Rickettsia parkeri infection by promoting escape from host membranes and cell-cell spread. Pat1 is important for infection in a mouse model and, at the cellular level, is crucial for efficiently escaping from single and double membrane-bound vacuoles into the host cytosol, and for avoiding host galectins that mark damaged membranes. Pat1 is also important for avoiding host polyubiquitin, preventing recruitment of autophagy receptor p62, and promoting actin-based motility and cell-cell spread.

Spotted fever group Rickettsia species are arthropod-borne obligate intracellular bacteria that can cause mild to severe human disease. These bacteria invade host cells, replicate in the cell cytosol, and then spread from cell to cell. To access the host cytosol and avoid detection by immune surveillance mechanisms, these pathogens must have evolved efficient ways to escape membrane-bound vacuoles. Although Rickettsia are predicted to express factors that disrupt host membranes, little is known about how and when these proteins function during infection. Here, we investigated the role of a Rickettsia patatin-like phospholipase A2 enzyme (Pat1) during host cell infection by characterizing a Rickettsia parkeri mutant with a transposon insertion in the pat1 gene. We show that Pat1 is important for infection in a mouse model and in host cells. We further show that Pat1 is critical for efficiently escaping from the single and double membrane-bound vacuoles into the host cytosol, and for avoiding host galectins that mark damaged membranes. In the host cytosol, Pat1 is important for avoiding host polyubiquitin, preventing recruitment of autophagy receptor p62, and promoting actin-based motility and cell-cell spread. Our results show that Pat1 plays critical roles in escaping host membranes and promoting cell-cell spread during R. parkeri infection and suggest diverse roles for patatin-like phospholipases in facilitating microbial infection. Importance: Spotted fever group Rickettsia are bacteria that reside in ticks and can be transmitted to mammalian hosts, including humans. Severe disease is characterized by high fever, headache, and rash, and results in occasional mortality despite available treatment. Rickettsia interact with host cell membranes while invading cells, escaping into the cytosol, and evading cellular defenses. Bacterial phospholipase enzymes have been proposed as critical factors for targeting host cell membranes, however the specific roles of rickettsial phospholipases are not well defined. We investigated the contribution of one conserved patatin-like phospholipase, Pat1, in Rickettsia parkeri . We observed that Pat1 is important for virulence in an animal model. Moreover, Pat1 plays critical roles in host cells by facilitating access to the cell cytosol, inhibiting detection by host defense pathways, and promoting cell-cell spread. Our study indicates that Pat1 performs several critical functions, suggesting a broad role for phospholipases throughout the Rickettsia lifecycle.