Browsing by Subject "Salmonella enterica"

As the current SARS-CoV-2 pandemic highlights, infectious diseases outcomes range from asymptomatic cases to prolonged morbidity and death. We can predict these outcomes using risk factors like age and BMI; however, we currently lack the capacity to generate more fine-grained, or even individualized, outcome predications. In my thesis, I increased mechanistic insight into how Salmonella enterica (S. enterica) generates an equally diverse set of outcomes by unraveling how genetic diversity in both Salmonellae and humans impact infection.

S. enterica is a very diverse pathogen with thousands of serovars. In their core genome, all S. enterica serovars carry two molecular syringes called type-three secretions systems (T3SS), which inject bacterial proteins called effectors into host cells. These effectors create a hospitable niche inside host cells for the replicating bacteria. Outside of the core genome, each serovar carries hundreds to thousands of different genes, which means each serovar packs a unique genetic toolset. We discovered that only a handful of non-typhoidal serovars carry an effector called SarA—an effector that activates the host transcription factor STAT3 to render host cells and mice more permissive to S. enterica replication through an anti-inflammatory transcriptional program.

Following this discovery, I determined that SarA activates STAT3 by mimicking the active cytokine co-receptor gp130. Specifically, a 40 amino acid stretch in SarA is homologous to the STAT3-binding portion of gp130’s cytoplasmic tail. We dubbed this stretch the GBS (gp130-binding of STAT3) sequence. By generating chimeric gp130-SarA proteins, I determined that the two GBS are functionally interchangeable. In fact, the SarA GBS binds STAT3 with even greater affinity when measured with isothermal titration calorimetry. This results in SarA driving prolonged and more robust STAT3 signaling than cytokine-gp130 signaling. This continues a reoccurring theme in bacterial pathogenesis: effectors evolve to be more effective than their mammalian counterparts. This is due to selective pressure driving effectors toward supraphysiological responses that enable replication and dissemination, whereas host signaling is pressed toward a measured and regulated response that promotes homeostasis.

In complementary studies exploring how human genetics contribute to variable outcomes, we infected hundreds of genotyped human cell lines from around the world and measured S. enterica serovar Typhi (S. Typhi) replication inside these human cells as a proxy for virulence in a whole person. We then associated this quantitative outcome with more than 10 million genetic markers in the genotyped cell lines to identify specific loci that associate with significantly more or less intracellular S. Typhi replication. One of these loci regulates expression of the divalent cation channel gene MCOLN2. By deleting the MCOLN2 gene, I confirmed mucolipin-2 (MCOLN2 or TRPML2) is a host factor that reduces S. Typhi replication inside human immune cells. Further, MCOLN2-/- mice have increased Salmonella burden.

To determining how mucolipin-2 restricts S. Typhi replication, I used dual RNA- seq of host and bacterial transcripts, which allows the intracellular S. Typhi to serve as a probe that reports how intracellular conditions change when MCOLN2 is removed. These results indicated that mucolipin-2 reduces Mg2+ availability to the bacteria. Repleting Mg2+ during in vitro infection only increases S. Typhi replication when mucolipin-2 is present, demonstrating that Mg2+ addition overcomes MCOLN2- dependent restriction.

Identifying these variable bacterial and host factors can improve the targeting of therapeutics—especially as next generation sequencing becomes more common clinically. In the long-term, elucidating how these genetic variants modulate the outcomes of Salmonella infection teaches us how severe outcomes occur, and hopefully, how to avoid or mitigate them.