Faculty Bibliography

Necroptosis is a RIP1-dependent programmed cell death (PCD) pathway that is distinct from apoptosis. Downstream effector pathways of necroptosis include formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS), both of which depend on glycolysis. This suggests that increased cellular glucose may prime necroptosis. Here we show that exposure to hyperglycemic levels of glucose enhances necroptosis in primary red blood cells (RBCs), Jurkat T cells, and U937 monocytes. Pharmacologic or siRNA inhibition of RIP1 prevented the enhanced death, confirming it as RIP1-dependent necroptosis. Hyperglycemic enhancement of necroptosis depends upon glycolysis with AGEs and ROS playing a role. Total levels of RIP1, RIP3, and mixed lineage kinase domain-like (MLKL) proteins were increased following treatment with high levels of glucose in Jurkat and U937 cells and was not due to transcriptional regulation. The observed increase in RIP1, RIP3, and MLKL protein levels suggests a potential positive feedback mechanism in nucleated cell types. Enhanced PCD due to hyperglycemia was specific to necroptosis as extrinsic apoptosis was inhibited by exposure to high levels of glucose. Hyperglycemia resulted in increased infarct size in a mouse model of brain hypoxia-ischemia injury. The increased infarct size was prevented by treatment with nec-1s, strongly suggesting that increased necroptosis accounts for exacerbation of this injury in conditions of hyperglycemia. This work reveals that hyperglycemia represents a condition in which cells are extraordinarily susceptible to necroptosis, that local glucose levels alter the balance of PCD pathways, and that clinically relevant outcomes may depend on glucose-mediated effects on PCD.

Bacterial vaginosis (BV) affects almost a quarter of US women, making it a condition of major public health relevance. Key questions remain regarding the etiology of BV, mechanisms for its association with poor reproductive health outcomes, and reasons for high rates of treatment failure. New model systems are required to answer these remaining questions, elucidate the complex host-microbe and microbe-microbe interactions, and develop new, effective interventions. In this review, we cover the strengths and limitations of in vitro and in vivo model systems to study these complex intercellular interactions. Furthermore, we discuss advancements needed to maximize the translational utility of the model systems. As no single model can recapitulate all of the complex physiological and environmental conditions of the human vaginal microenvironment, we conclude that combinatorial approaches using in vitro and in vivo model systems will be required to address the remaining fundamental questions surrounding the enigma that is BV.

BACKGROUND: Next-generation sequencing of transposon-genome junctions from a saturated bacterial mutant library (Tn-seq) is a powerful tool that permits genome-wide determination of the contribution of genes to fitness of the organism under a wide range of experimental conditions. We report development, testing, and results from a Tn-seq system for use in Streptococcus agalactiae (group B Streptococcus; GBS), an important cause of neonatal sepsis. METHODS: Our method uses a Himar1 mini-transposon that inserts at genomic TA dinucleotide sites, delivered to GBS on a temperature-sensitive plasmid that is subsequently cured from the bacterial population. In order to establish the GBS essential genome, we performed Tn-seq on DNA collected from three independent mutant libraries-with at least 135,000 mutants per library-at serial 24 h time points after outgrowth in rich media. RESULTS: After statistical analysis of transposon insertion density and distribution, we identified 13.5 % of genes as essential and 1.2 % as critical, with high levels of reproducibility. Essential and critical genes are enriched for fundamental cellular housekeeping functions, such as acyl-tRNA biosynthesis, nucleotide metabolism, and glycolysis. We further validated our system by comparing fitness assignments of homologous genes in GBS and a close bacterial relative, Streptococcus pyogenes, which demonstrated 93 % concordance. Finally, we used our fitness assignments to identify signal transduction pathway components predicted to be essential or critical in GBS. CONCLUSIONS: We believe that our baseline fitness assignments will be a valuable tool for GBS researchers and that our system has the potential to reveal key pathogenesis gene networks and potential therapeutic/preventative targets.

Staphylococcus aureus, like other bacterial pathogens, scavenges host iron for growth through incompletely understood mechanisms. In this issue of Cell Host & Microbe, Spaan et al. (2015) demonstrate that two Staphylococcus aureus leukotoxins, HlgAB and LukED, target the Duffy antigen receptor for chemokines on erythrocytes, resulting in lysis and iron release.

We describe a case of fetal parvovirus B19 infection resulting in preterm birth and leading to hydrops fetalis requiring multiple in utero transfusions. The infant developed chronic postnatal anemia responsive to intravenous immunoglobulin therapy. Serum viral load decreased after immunoglobulin treatment but remained detectable for over 1 year.

The contribution of specific factors to bacterial virulence is generally investigated through creation of genetic "knockouts" that are then compared to wild-type strains or complemented mutants. This paradigm is useful to understand the effect of presence vs. absence of a specific gene product but cannot account for concentration-dependent effects, such as may occur with some bacterial toxins. In order to assess threshold and dose-response effects of virulence factors, robust systems for tunable expression are required. Recent evidence suggests that the folding free energy (DeltaG) of the 5' end of mRNA transcripts can have a significant effect on translation efficiency and overall protein abundance. Here we demonstrate that rational alteration of 5' mRNA folding free energy by introduction of synonymous mutations allows for predictable changes in pneumolysin (PLY) expression by Streptococcus pneumoniae without the need for chemical inducers or heterologous promoters. We created a panel of isogenic S. pneumoniae strains, differing only in synonymous (silent) mutations at the 5' end of the PLY mRNA that are predicted to alter DeltaG. Such manipulation allows rheostat-like control of PLY production and alters the cytotoxicity of whole S. pneumoniae on primary and immortalized human cells. These studies provide proof-of-principle for further investigation of mRNA DeltaG manipulation as a tool in studies of bacterial pathogenesis.

We conducted an open-label crossover trial to test whether proton pump inhibitors (PPIs) affect the gastrointestinal microbiome to facilitate Clostridium difficile infection (CDI). Twelve healthy volunteers each donated 2 baseline fecal samples, 4 weeks apart (at weeks 0 and 4). They then took PPIs for 4 weeks (40 mg omeprazole, twice daily) and fecal samples were collected at week 8. Six individuals took the PPIs for an additional 4 weeks (from week 8 to 12) and fecal samples were collected from all subjects at week 12. Samples were analyzed by 16S rRNA gene sequencing. We found no significant within-individual difference in microbiome diversity when we compared changes during baseline vs changes on PPIs. There were, however, significant changes during PPI use in taxa associated with CDI (increased Enterococcaceae and Streptococcaceae, decreased Clostridiales) and taxa associated with gastrointestinal bacterial overgrowth (increased Micrococcaceae and Staphylococcaceae). In a functional analysis, there were no changes in bile acids on PPIs, but there was an increase in genes involved in bacterial invasion. These alterations could provide a mechanism by which PPIs predispose to CDI. ClinicalTrials.gov:NCT01901276.

Mature erythrocytes (red blood cells (RBCs)) undergo the programmed cell death (PCD) pathway of necroptosis in response to bacterial pore-forming toxins (PFTs) that target human CD59 (hCD59) but not hCD59-independent PFTs. Here, we investigate the biochemical mechanism of RBC necroptosis with a focus on the mechanism of induction and the minimal requirements for such RBC death. Binding or crosslinking of the hCD59 receptor led to Syk-dependent induction of vesiculated morphology (echinocytes) that was associated with phosphorylation of Band 3 and was required for Fas ligand (FasL) release. FasL-dependent phosphorylation of receptor-interacting protein kinase 1 (RIP1) in combination with plasma membrane pore formation was required for execution of RBC necroptosis. RIP1 phosphorylation led to the phosphorylation of RIP3, which was also critical for RBC necroptosis. Notably, RBC necroptosis was mediated by FasL and not by other candidate inducers, including tumor necrosis factor alpha (TNF-alpha) and TNF-related apoptosis-inducing ligand (TRAIL). Other types of RBC damage, such as eryptotic damage, failed to induce necroptosis when combined with hCD59 crosslinking. This work sheds light on the requirements for this recently discovered PCD in RBCs and provides a clear picture of the biochemical mechanism of induction of RBC necroptosis.