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Listeria monocytogenes is a Gram-positive, intracellular pathogen responsible for the highly fatal foodborne illness listeriosis. Establishing intracellular infections requires the coordinated expressions of a variety of virulence factors, such as the pore-forming toxin listeriolysin O (LLO), in response to various intra- and extracellular signals. For example, we previously reported that L. monocytogenes differentially modulated LLO production in response to exogenous propionate, a short chain fatty acid either used in salt form as a human food ingredient or produced endogenously by gut microbial fermentation. Therefore, propionate is likely a continuously present signal throughout the L. monocytogenes transmission and infection process. However, little is known about the role of propionate in modulating L. monocytogenes-host interactions. Here we investigated the impact of propionate treatment on L. monocytogenes intracellular infections using cell culture infection models. Propionate treatment was performed separately on L. monocytogenes or host cells before or during infections to better distinguish pathogen-versus-host responses to propionate. Intracellular CFU in RAW264.7 macrophages and plaque diameters in L-fibroblasts were measured as proxy for intracellular infection outcomes. Nitrite levels and cellular morphology were also measured to assess host responses to propionate. We found that propionate pretreatment of anaerobic, but not aerobic, L. monocytogenes significantly enhanced subsequent intracellular infections in both cell types and nitrite production by infected macrophages. Propionate treatment of uninfected macrophages significantly altered cell morphology, seen by longer cells and greater migration, and reduced nitrite concentration in activated macrophages. Treatment of macrophages with propionate prior to or during infections significantly inhibited intracellular growth of L. monocytogenes, including those pre-treated with propionate. These results showcased an opposing effect of propionate on L. monocytogenes intracellular infections and strongly support propionate as an important signaling molecule for both the pathogen and the host cell that can potentially alter the outcome of L. monocytogenes-host interactions.

Listeria monocytogenes is a foodborne pathogen capable of secreting listeriolysin O (LLO), a pore-forming toxin encoded by the hly gene. While the functions of LLO have been studied extensively, how the production of LLO is modulated by the intestinal environment, devoid of oxygen and enriched in short chain fatty acids (SCFAs), is not completely understood. Using L. monocytogenes strain 10403s, we found that hly transcription was moderately decreased by aerobic SCFA exposures but significantly increased by anaerobic SCFA exposures. Moreover, aerobic, but not anaerobic, exposure to low levels of SCFAs resulted in a significantly higher LLO activity. These results demonstrated that transcriptional and post-transcriptional regulations of LLO production were separately modulated by SCFAs and were responsive to oxygen levels. Examining isogenic mutants revealed that PrfA and SigB play a role in regulating LLO production in response to SCFAs. Effects of SCFAs were also present in the cardiotropic strain 07PF0776 but distinctly different from those in strain 10403s. For both strains, prior exposures to SCFAs altered intracellular infections in Caco-2 and RAW264.7 cells and the plaque sizes in L fibroblasts, a result confirming the ability of L. monocytogenes to adapt to SCFAs in ways that impact its subsequent infection outcomes.

Listeria monocytogenes is a human pathogen and a facultative anaerobe. To better understand how anaerobic growth affects L. monocytogenes pathogenesis, we first showed that anaerobic growth led to decreased growth and changes in surface morphology. Moreover, compared to aerobically grown bacteria, anaerobically grown L. monocytogenes established higher level of invasion but decreased intracellular growth and actin polymerization in cultured cells. The production of listeriolysin O (LLO) was significantly lower in anaerobic cultures-a phenotype observed in wild type and isogenic mutants lacking transcriptional regulators SigB or CodY or harboring a constitutively active PrfA. To explore potential regulatory mechanisms, we established that the addition of central carbon metabolism intermediates, such as acetate, citrate, fumarate, pyruvate, lactate, and succinate, led to an increase in LLO activity in the anaerobic culture supernatant. These results highlight the regulatory role of central carbon metabolism in L. monocytogenes pathogenesis under anaerobic conditions.

Propionate is a common food preservative and one of the major fermentation acids in the intestines. Therefore, exposure to propionate is frequent for foodborne pathogens and likely takes place under suboxic conditions. However, it is not clear whether the absence of oxygen affects how pathogens respond to propionate. Here, we investigated how propionate exposure affects Listeria monocytogenes growth and virulence factor production under aerobic or anaerobic conditions and showed that oxygen indeed plays a key role in modulating L. monocytogenes response to propionate. Under aerobic conditions, propionate supplementations had no effect on planktonic growth but resulted in decreased adherent growth. Under anaerobic conditions, propionate supplementations resulted in a pH-dependent inhibition of planktonic growth and increased adherent growth. Cultures grown with propionate accumulated higher levels of acetoin under aerobic conditions but lower levels of ethanol under both aerobic and anaerobic conditions. Metabolic perturbations by propionate were also evident by the increase in straight chain fatty acids. Finally, propionate supplementations resulted in increased listeriolyin O (LLO) production under anaerobic conditions but decreased LLO production under aerobic conditions. These results demonstrate for the first time that the presence or absence of oxygen plays a critical role in shaping L. monocytogenes responses to propionate.

Bacterial enteric pathogens are responsible for a tremendous amount of foodborne illnesses every year through the consumption of contaminated food products. During their transit from contaminated food sources to the host gastrointestinal tract, these pathogens are exposed and must adapt to fluctuating oxygen levels to successfully colonize the host and cause diseases. However, the majority of enteric infection research has been conducted under aerobic conditions. To raise awareness of the importance in understanding the impact of oxygen, or lack of oxygen, on enteric pathogenesis, we describe in this review the metabolic and physiological responses of nine bacterial enteric pathogens exposed to environments with different oxygen levels. We further discuss the effects of oxygen levels on virulence regulation to establish potential connections between metabolic adaptations and bacterial pathogenesis. While not providing an exhaustive list of all bacterial pathogens, we highlight key differences and similarities among nine facultative anaerobic and microaerobic pathogens in this review to argue for a more in-depth understanding of the diverse impact oxygen levels have on enteric pathogenesis.

Fatty acids (FAs) are the major structural component of cellular membranes, which provide a physical and chemical barrier that insulates intracellular reactions from environmental fluctuations. The native composition of membrane FAs establishes the topological and chemical parameters for membrane-associated functions and is therefore modulated diligently by microorganisms especially in response to environmental stresses. However, the consequences of altered FA composition during host-pathogen interactions are poorly understood. The food-borne pathogen Listeria monocytogenes contains mostly saturated branched-chain FAs (BCFAs), which support growth at low pH and low temperature. In this study, we show that anteiso-BCFAs enhance bacterial resistance against phagosomal killing in macrophages. Specifically, BCFAs protect against antimicrobial peptides and peptidoglycan hydrolases, two classes of phagosome antimicrobial defense mechanisms. In addition, the production of the critical virulence factor, listeriolysin O, was compromised by FA modulation, suggesting that FAs play a key role in virulence regulation. In summary, our results emphasize the significance of FA metabolism, not only in bacterial virulence regulation but also in membrane barrier function by providing resistance against host antimicrobial stress.

Anteiso-branched-chain fatty acids (BCFA) represent the dominant group of membrane fatty acids and have been established as crucial determinants in resistance against environmental stresses in Listeria monocytogenes , a facultative intracellular pathogen. Here, we investigate the role of anteiso-BCFA in L. monocytogenes virulence by using mutants deficient in branched-chain alpha-keto acid dehydrogenase (BKD), an enzyme complex involved in the synthesis of BCFA. In tissue culture models of infection, anteiso-BCFA contributed to intracellular growth and survival in macrophages and significantly enhanced plaque formation upon prolonged infection in L2 fibroblasts. The intracellular defects observed could be attributed partially to insufficient listeriolysin O (LLO) production, indicating a requirement for anteiso-BCFA in regulating virulence factor production. In a murine model of infection, the BKD-deficient mutant was highly attenuated, further emphasizing the importance of BKD-mediated metabolism in L. monocytogenes virulence. This study demonstrates an underappreciated role for BCFA in bacterial pathogenesis, which may provide insight into the development and application of antimicrobial agents.

Tumor suppressor p53 plays a central role in tumor prevention. p53 protein levels and activity are under a tight and complex regulation in cells to maintain the proper function of p53. MicroRNAs play a key role in the regulation of gene expression. Here we report the regulation of p53 through miR-504. miR-504 acts as a negative regulator of human p53 through its direct binding to two sites in the p53 3' untranslated region. Overexpression of miR-504 decreases p53 protein levels and functions in cells, including p53 transcriptional activity, p53-mediated apoptosis, and cell-cycle arrest in response to stress, and furthermore promotes tumorigenecity of cells in vivo. These results demonstrate the direct negative regulation of p53 by miR-504 as a mechanism for p53 regulation in cells, which highlights the importance of microRNAs in tumorigenesis.

Whereas cell cycle arrest, apoptosis, and senescence are traditionally thought of as the major functions of the tumor suppressor p53, recent studies revealed two unique functions for this protein: p53 regulates cellular energy metabolism and antioxidant defense mechanisms. Here, we identify glutaminase 2 (GLS2) as a previously uncharacterized p53 target gene to mediate these two functions of the p53 protein. GLS2 encodes a mitochondrial glutaminase catalyzing the hydrolysis of glutamine to glutamate. p53 increases the GLS2 expression under both nonstressed and stressed conditions. GLS2 regulates cellular energy metabolism by increasing production of glutamate and α-ketoglutarate, which in turn results in enhanced mitochondrial respiration and ATP generation. Furthermore, GLS2 regulates antioxidant defense function in cells by increasing reduced glutathione (GSH) levels and decreasing ROS levels, which in turn protects cells from oxidative stress (e.g., H 2 O 2 )-induced apoptosis. Consistent with these functions of GLS2, the activation of p53 increases the levels of glutamate and α-ketoglutarate, mitochondrial respiration rate, and GSH levels and decreases reactive oxygen species (ROS) levels in cells. Furthermore, GLS2 expression is lost or greatly decreased in hepatocellular carcinomas and the overexpression of GLS2 greatly reduced tumor cell colony formation. These results demonstrated that as a unique p53 target gene, GLS2 is a mediator of p53’s role in energy metabolism and antioxidant defense, which can contribute to its role in tumor suppression.

Germline deletion of the p53 gene in mice gives rise to spontaneous thymic (T-cell) lymphomas. In this study, the p53 knockout mouse was employed as a model to study the mutational evolution of tumorigenesis. The clonality of the T-cell repertoire from p53 knockout and wild-type thymic cells was analyzed at various ages employing TCRβ sequencing. These data demonstrate that p53 knockout thymic lymphomas arose in an oligoclonal fashion, with tumors evolving dominant clones over time. Exon sequencing of tumor DNA revealed that all of the independently derived oligoclonal mouse tumors had a deletion in the Pten gene prior to the formation of the TCRβ rearrangement, produced early in development. This was followed in each independent clone of the thymic lymphoma by the amplification or overexpression of cyclin Ds and Cdk6. Alterations in the expression of Ikaros were common and blocked further development of CD-4/CD-8 T cells. While the frequency of point mutations in the genome of these lymphomas was one per megabase, there were a tremendous number of copy number variations producing the tumors’ driver mutations. The initial inherited loss of p53 functions appeared to delineate an order of genetic alterations selected for during the evolution of these thymic lymphomas.

The p53 protein ensures cellular fidelity by suppressing or killing cells under stresses that enhance the mutation rate. Evidence suggests that the p53 protein may also ensure the fidelity of the epigenome. In this study a group of drugs that alter the deoxycytosine methylation patterns in cellular DNA are shown to preferentially kill human and mouse cells that contain p53 mutations or deficiencies. These observations are extended to mice that contain p53 deficiencies or missense mutations in their genome, which are preferentially killed when compared to mice with a wild type p53 gene. This is also the case for human cancer cell xenografts containing p53 mutations, which preferentially are killed by these drugs when compared to similar tumors with wild type p53. The loss of p53 function enhances a synthetic lethality with drugs that block or alter the patterns of deoxycytidine methylation in the genome.

Among patients with neuroendocrine tumors, males are more likely than females to develop liver metastatic disease. We show that this male bias toward liver metastasis is also observed in a mouse model for pancreatic neuroendocrine tumorigenesis (PanNETs). Interestingly, primary tumors from mice with metastatic disease display hepatomimicry, expressing genes normally expressed in liver. Both male and female primary tumors from mice with metastatic disease show hepatomimicry, although one of the liver genes, complement C5, is only induced in males. In mice knocked out for the complement C5 gene, the frequency of liver metastasis dropped significantly; moreover, males and females lacking a gene for complement C5 developed liver metastasis at the same frequency. Liver metastasis was also prevented by PMX53, a small molecule antagonist of C5aR1/CD88, which is the receptor for complement C5a. Thus complement C5 is epistatic to metastasis and promotes gender bias of liver metastatic disease. Citation Format: Tanupriya Contractor, Chang Chan, Shinta Kobayashi, Richard Clausen, Yvonne Sun, Edaise da Silva, Evan Vosburgh, Arnold J. Levine, Laura Tang, Chris R. Harris. Complement C5 promotes male bias of pancreatic neuroendocrine tumor metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3185. doi:10.1158/1538-7445.AM2015-3185

Loss of the tumor suppressor, p53, occurs in over 50% of cancers. The ability for p53 to protect the cell from tumorigenesis is based on its ability to induce cell cycle arrest, apoptosis, and senescence following genotoxic stress. Furthermore, genetic deletion of p53 in mice gives rise to spontaneous thymic (T-cell) lymphomas and sarcomas. In this study, we used the p53-knockout (KO) mouse as a model for progression of tumorigenesis. We first analyzed genomic DNA of p53-KO and WT normal thymus at various ages and p53-KO thymic lymphomas using TCRβ sequencing, allowing us to determine the clonality of the T cell repertoire. These data suggest that p53-KO thymic lymphomas were mostly oligoclonal, with some tumors comprising of a dominant clone. We further analyzed tumors with dominant clones for genomic instability by exome sequencing. Sequencing analysis revealed that there were many non-synonymous mutations occurring in each tumor, but with no similarities between tumors. On the other hand, all of the murine tumors had deletion of Pten and amplification of Cdk6. This is in stark contrast to what is seen in most human T cell lymphoblastic leukemias, which have activating mutations in Notch1 and loss of Arf. Interestingly, loss of Pten not only occurred in all tumors, but also in most, if not all, clones in the tumor. This suggests that Pten loss is an early event driving T cell transformation in a p53-KO background. Using this data, we found the somatic mutation rate in the tumors to be 1000-fold greater than the germline mutation rate. Loss of p53 also caused chromosomal abnormalities as seen with increased copy number variations. Taken together, our work suggests that, with initial loss of p53, the path to T cell tumorigenesis is vastly different from that of human tumors. Without p53, the somatic genome increases in mutation rate and copy number variations, thus solidifying p53's role as guardian of the genome. Citation Format: Crissy Dudgeon, Chang Chan, Yvonne Sun, Arnold J. Levine. Exome sequencing of murine p53-knockout T-cell lymphomas reveals Pten loss and Cdk6 amplification as key steps toward cellular transformation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-80. doi:10.1158/1538-7445.AM2014-LB-80

The tumor suppressor p53 is nonfunctional in over 50% of cancers. The ability for p53 to protect the cell from tumorigenesis is rooted in its ability to induce cell cycle arrest, apoptosis, and senescence following genotoxic stress. Furthermore, loss of p53 in mice gives rise to spontaneous thymic lymphomas and sarcomas. In this study, we used the p53-KO mouse as a model for progression of tumorigenesis. We first analyzed genomic DNA of p53-KO and WT normal thymus at various ages using TCRβ sequencing. This procedure allowed us to determine the clonality of the T cell repertoire. We then analyzed the clonality of p53-KO thymic lymphomas. Our results indicated that while the highest T cell clone was 0.08% for normal thymus, the highest T cell clone for the p53-KO tumor was 70%, indicating that the p53-KO thymic lymphomas were monoclonal. Building on this data, we analyzed the mutations occurring in the thymic lymphomas by mouse exome sequencing. We believe that combining TCRβ sequencing with mouse exome sequencing will allow for a better understanding of the clonality and genetic mutations required for cellular transformation. Citation Format: Crissy Dudgeon, Chang Chan, Yvonne Sun, Arnold J. Levine. Utilizing TCRβ and whole exome sequencing to determine the clonality and mutational spectrum of p53-null thymic lymphomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 796. doi:10.1158/1538-7445.AM2013-796