Daniel A. Pensinger

The gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile, but the molecular basis of this colonization resistance is incompletely understood. A prominent class of gut microbiome-produced metabolites important for colonization resistance against C. difficile is short-chain fatty acids (SCFAs). In particular, one SCFA (butyrate) decreases the fitness of C. difficile in vitro and is correlated with C. difficile -inhospitable gut environments, both in mice and in humans. Here, we demonstrate that butyrate-dependent growth inhibition in C. difficile occurs under conditions where C. difficile also produces butyrate as a metabolic end product. Furthermore, we show that exogenous butyrate is internalized into C. difficile cells and is incorporated into intracellular CoA pools where it is metabolized in a reverse (energetically unfavorable) direction to crotonyl-CoA and ( S )−3-hydroxybutyryl-CoA and/or 4-hydroxybutyryl-CoA. This internalization of butyrate and reverse metabolic flow of a butyrogenic pathway(s) in C. difficile coincides with alterations in toxin release and sporulation. Together, this work highlights butyrate as a marker of a C. difficile -inhospitable environment to which C. difficile responds by releasing its diarrheagenic toxins and producing environmentally resistant spores necessary for transmission between hosts. These findings provide foundational data for understanding the molecular and genetic basis of how C. difficile growth is inhibited by butyrate and how butyrate alters C. difficile virulence in the face of a highly competitive and dynamic gut environment. IMPORTANCE: The gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile, but the molecular basis of this colonization resistance is incompletely understood, which hinders the development of novel therapeutic interventions for C. difficile infection (CDI). We investigated how C. difficile responds to butyrate, an end-product of gut microbiome community metabolism which inhibits C. difficile growth. We show that exogenously produced butyrate is internalized into C. difficile , which inhibits C. difficile growth by interfering with its own butyrate production. This growth inhibition coincides with increased toxin release from C. difficile cells and the production of environmentally resistant spores necessary for transmission between hosts. Future work to disentangle the molecular mechanisms underlying these growth and virulence phenotypes will likely lead to new strategies to restrict C. difficile growth in the gut and minimize its pathogenesis during CDI.

Bacterial pathogens must adapt to their host environment in order to cause disease. The cytosolic bacterial pathogen Listeria monocytogenes requires a highly conserved protein of unknown function, GlmR (previously known as YvcK), to survive in the host cytosol.

Clostridioides difficileis an opportunistic diarrheal pathogen responsible for significant morbidity and mortality worldwide. A disrupted (dysbiotic) gut microbiome, commonly engendered by antibiotic treatment, is the primary risk factor forC.difficileinfection, highlighting thatC.difficile–microbiome interactions are critical for determining the fitness of this pathogen. Here, we review short chain fatty acids (SCFAs): a major class of metabolites present in the gut, their production by the gut microbiome, and their impacts on the biology of the host and ofC.difficile. We use these observations to illustrate a conceptual model wherebyC.difficilesenses and responds to SCFAs as a marker of a healthy gut and tunes its virulence accordingly in order to maintain dysbiosis. Future work to learn the molecular mechanisms and genetic circuitry underlying the relationships betweenC.difficileand SCFAs will help to identify precision approaches, distinct from antibiotics and fecal transplant, for mitigating disease caused byC.difficileand will inform similar investigations into other gastrointestinal pathogens.

Listeria monocytogenes, the causative agent of listeriosis, is an intracellular pathogen that is exquisitely evolved to survive and replicate in the cytosol of eukaryotic cells. Eukaryotic cells typically restrict bacteria from colonising the cytosol, likely through a combination of cell autonomous defences, nutritional immunity, and innate immune responses including induction of programmed cell death. This suggests that L. monocytogenes and other professional cytosolic pathogens possess unique metabolic adaptations, not only to support replication but also to facilitate resistance to host‐derived stresses/defences and avoidance of innate immune activation. In this review, we outline our current understanding of L. monocytogenes metabolism in the host cytosol and highlight major metabolic processes which promote intracellular replication and survival.

Obstacles to bacterial survival and replication in the cytosol of host cells, and the mechanisms used by bacterial pathogens to adapt to this niche are not well understood. Listeria monocytogenes is a well-studied Gram-positive foodborne pathogen that has evolved to invade and replicate within the host cell cytosol; yet the mechanisms by which it senses and responds to stress to survive in the cytosol are largely unknown. To assess the role of the L. monocytogenes penicillin-binding-protein and serine/threonine associated (PASTA) kinase PrkA in stress responses, cytosolic survival and virulence, we constructed a ΔprkA deletion mutant. PrkA was required for resistance to cell wall stress, growth on cytosolic carbon sources, intracellular replication, cytosolic survival, inflammasome avoidance and ultimately virulence in a murine model of Listeriosis. In Bacillus subtilis and Mycobacterium tuberculosis, homologues of PrkA phosphorylate a highly conserved protein of unknown function, YvcK. We found that, similar to PrkA, YvcK is also required for cell wall stress responses, metabolism of glycerol, cytosolic survival, inflammasome avoidance and virulence. We further demonstrate that similar to other organisms, YvcK is directly phosphorylated by PrkA, although the specific site(s) of phosphorylation are not highly conserved. Finally, analysis of phosphoablative and phosphomimetic mutants of YvcK in vitro and in vivo demonstrate that while phosphorylation of YvcK is irrelevant to metabolism and cell wall stress responses, surprisingly, a phosphomimetic, nonreversible negative charge of YvcK is detrimental to cytosolic survival and virulence in vivo. Taken together our data identify two novel virulence factors essential for cytosolic survival and virulence of L. monocytogenes. Furthermore, our data demonstrate that regulation of YvcK phosphorylation is tightly controlled and is critical for virulence. Finally, our data suggest that yet to be identified substrates of PrkA are essential for cytosolic survival and virulence of L. monocytogenes and illustrate the importance of studying protein phosphorylation in the context of infection. Infection with intracellular pathogens causes a majority of the global infectious disease associated mortality. A number of intracellular pathogens must directly access the host cytosol in order to cause disease; however, non-cytosol adapted bacteria do not survive or replicate upon access to the cytosol. The mechanisms cytosolic pathogens use to adapt to this niche are largely unknown. The model cytosolic bacterial pathogen Listeria monocytogenes contains a single penicillin-binding-protein and serine/threonine associated (PASTA) kinase, PrkA. In other bacteria, PASTA kinases bind cell wall fragments and phosphorylate downstream effectors involved in cell wall synthesis, central metabolism, virulence, cell division, and biofilm formation. We demonstrate that in L. monocytogenes, PrkA is required for cell wall homeostasis, growth under nutrient limiting conditions, survival and replication in host cells, and virulence in vivo. Furthermore, we identify a highly conserved protein of unknown function, YvcK, as a PrkA substrate. We demonstrate that L. monocytogenes YvcK is similarly required for cell wall stress responses, growth on glycerol, cytosolic survival and virulence in vivo. Surprisingly, a phosphomimetic, nonreversible negative charge at the phosphorylation sites on YvcK inactivates functions of the protein related to intracellular survival and virulence, suggesting that the identification of PASTA kinase substrates phosphorylated during infection will be critical to our understanding of this central regulator metabolism, cell wall homeostasis and ultimately virulence.

Significance: The small nucleotide cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) recently emerged as a ubiquitous signaling molecule among bacteria, with essential roles in both bacterial physiology and host–pathogen interactions. Bacterial mutants with abnormal c-di-AMP levels exhibit growth and virulence defects, reflecting the importance of regulating c-di-AMP synthesis and degradation for normal signal transduction and adaptation to changing environments. Previously documented phosphodiesterases hydrolyze c-di-AMP via the DHH-DHHA1 domain, but they are not present in all c-di-AMP synthesizing species. We identified a previously unrecognized class of His-Asp -domain phosphodiesterases that are widespread across several taxonomic groups. Furthermore, for the bacterial pathogen Listeria monocytogenes , phosphodiesterase mutants exhibit enhanced host inflammation, growth defects inside host cells, and significantly attenuated virulence in a murine model of infection.

While β-lactam antibiotics are a critical part of the antimicrobial arsenal, they are frequently compromised by various resistance mechanisms, including changes in penicillin binding proteins of the bacterial cell wall. Genetic deletion of the p enicillin binding protein a nd s erine/ t hreonine kinase- a ssociated protein (PASTA) kinase in methicillin-resistant Staphylococcus aureus (MRSA) has been shown to restore β-lactam susceptibility. However, the mechanism remains unclear, and whether pharmacologic inhibition would have the same effect is unknown. In this study, we found that deletion or pharmacologic inhibition of the PASTA kinase in Listeria monocytogenes by the nonselective kinase inhibitor staurosporine results in enhanced susceptibility to both aminopenicillin and cephalosporin antibiotics. Resistance to vancomycin, another class of cell wall synthesis inhibitors, or antibiotics that inhibit protein synthesis was unaffected by staurosporine treatment. Phosphorylation assays with purified kinases revealed that staurosporine selectively inhibited the PASTA kinase of L. monocytogenes (PrkA). Importantly, staurosporine did not inhibit a L. monocytogenes kinase without a PASTA domain (Lmo0618) or the PASTA kinase from MRSA (Stk1). Finally, inhibition of PrkA with a more selective kinase inhibitor, AZD5438, similarly led to sensitization of L. monocytogenes to β-lactam antibiotics. Overall, these results suggest that pharmacologic targeting of PASTA kinases can increase the efficacy of β-lactam antibiotics.

RNA has been recognized as a drug target for cancer therapy, as evidenced by the ongoing clinical trials of RNAi and antisense therapies. An alternative approach that circumvents the delivery and stability issues of those drugs is to harness the activity of naturally occurring RNA degradation enzymes. Human ribonuclease (RNase) variants have been generated with diminished binding to their natural inhibitor inside cells, which allows the new proteins to kill cancer cells. One of these RNase variants, referred to as QBI-139, demonstrated efficacy against multiple tumor types in xenograft models of human cancer and was selected for an ongoing first in human Phase I clinical trial. Non-small cell lung and ovarian cancer are two of the indications where QBI-139 has shown significant efficacy as a single agent in xenograft models. In planning for the next stage of clinical trials, the impact of combinations of QBI-139 plus standard of care agents in these diseases was determined both in vitro and in vivo. QBI-139 showed additive and synergistic activity in combination with cisplatin and docetaxel, which are standard of care for non-small cell lung and ovarian cancer, respectively. Fixed ratios of QBI-139 and the combination drug based on their EC50 values were tested. The Combination Index (CI) was determined using median effect analysis. Synergy was demonstrated with QBI-139 in combination with cisplatin against non-small cell lung cancer (A549 and H1975). An additive effect of QBI-139 and cisplatin was seen in ovarian (Ovcar-3) cancer. In ovarian cancer (SK-OV-3), a combination of docetaxel and QBI-139 was synergistic with QBI-139. The benefit of the combinations were repeated in xenograft models of both cancers and were signifcat relative to each agent alone. Non-small cell lung and ovarian cancer represent critical unmet needs and new, differentiated treatment approaches such as QBI-139 combination therapy are critical. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1838. doi:1538-7445.AM2012-1838

RNA has been recognized as a drug target for cancer therapy, as evidenced by the ongoing clinical trials of RNAi and antisense therapies. An alternative approach that circumvents the delivery and stability issues of RNAi and antisense is to harness the activity of naturally occurring enzymes that degrade RNA. The ribonuclease approach is partially validated by objective clinical responses seen in clinical trials of ribonuclease (RNase) discovered in frog eggs. Our research has focused on creating variants of human RNases with the ability of frog RNase to evade the human RNase inhibitor. Diminished binding of mammalian RNases to the inhibitor have been shown to endow the RNase with the ability to kill cancer cells. One of the RNase variants, called QBI-139, demonstrated particularly strong anti-cancer activity against a variety of tumor types in in vivo models. QBI-139 is differentiated from the frog RNase because QBI-139 is 95 % identical to a native human RNase, while the frog RNase is only 20% similar. This difference may be what has led to a significantly improved tolerability profile for QBI-139 across species. QBI-139 was selected for clinical development and is now in a Phase I trial of solid tumors. In charting the clinical development path for QBI-139, the ability of the RNase to work in combination with a variety of standard of care agents had to be evaluated. Based in the single agent activity of QBI-139 in colon, non-small cell lung, ovarian and pancreatic cancers, the potency of QBI-139 in combination with the standard of care agents for these indications was explored. In in vitro screening, QBI-139 has demonstrated additive and synergistic activity in combination with cisplatin, 5-FU and docetaxel. The treatments were evaluated using fixed ratios of QBI-139 and the combination drug based on their EC50 values. The Combination Index (CI) was determined using median effect analysis. A CI of 1 indicates additive effects, whereas a CI < 1 indicates synergy. Synergy was demonstrated with QBI-139 in combination with cisplatin against solid tumors of non-small cell lung cancer (A549 and H1975) and an additive effect in ovarian (Ovcar-3) cancer. 5-FU and docetaxel also were synergistic with QBI-139 against solid tumors of colon (HCT-116) and ovarian (SK-OV-3) cancer, respectively.The goal of this project is development of an innovative, differentiated therapy, QBI-139, to provide clinical benefit to patients with colon, non-small cell lung and ovarian cancer, which are among the seven deadliest forms of cancer in the United States. In addition to being serious diseases, effective treatment of these cancers also represent unmet medical needs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2573. doi:10.1158/1538-7445.AM2011-2573