Increased metabolism is a requirement for tumor cell proliferation. To understand the dependence of tumor cells on fatty acid metabolism, we evaluated various nodes of the fatty acid synthesis pathway. Using RNAi we have demonstrated that depletion of fatty-acid synthesis pathway enzymes SCD1, FASN, or ACC1 in HCT116 colon cancer cells results in cytotoxicity that is reversible by addition of exogenous fatty acids. This conditional phenotype is most pronounced when SCD1 is depleted. We used this fatty-acid rescue strategy to characterize several small-molecule inhibitors of fatty acid synthesis, including identification of TOFA as a potent SCD1 inhibitor, representing a previously undescribed activity for this compound. Reference FASN and ACC inhibitors show cytotoxicity that is less pronounced than that of TOFA, and fatty-acid rescue profiles consistent with their proposed enzyme targets. Two reference SCD1 inhibitors show low-nanomolar cytotoxicity that is offset by at least two orders of magnitude by exogenous oleate. One of these inhibitors slows growth of HCT116 xenograft tumors. Our data outline an effective strategy for interrogation of on-mechanism potency and pathway-node-specificity of fatty acid synthesis inhibitors, establish an unambiguous link between fatty acid synthesis and cancer cell survival, and point toward SCD1 as a key target in this pathway.

SUMMARY Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM2 isoform, effectively constraining lower glycolysis. Here, we report the discovery of PKM2 activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM2 activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the nonessential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM2 activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM2 expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress.

Regulation of bacterial gene expression by small RNA (sRNA) molecules is an increasingly recognized phenomenon but one that is not yet fully understood. We show that the sRNA RyhB suppresses several virulence-associated phenotypes of Shigella dysenteriae, a causative agent of bacillary dysentery in humans. The virulence genes repressed by S. dysenteriae RyhB include those encoding the type III secretion apparatus, its secreted effectors, and specific chaperones. Suppression of Shigella virulence occurs via RyhB-dependent repression of the transcriptional activator VirB, leading to reduced expression of genes within the VirB regulon. Efficient repression of virB is mediated by a single-stranded region of RyhB that is distinct from the region required for repression of Shigella sodB. Regulation of virB by RyhB implicates iron as an environmental factor contributing to the complex regulation of Shigella virulence determinants.Shigella species, which are closely related to Escherichia coli, cause bacillary dysentery, a disease associated with invasion of the colonic epithelium and provocation of an intense inflammatory response (12). Using cultured epithelial cells to measure bacterial invasion (invasion assay) (8) and intercellular spread (plaque assay) (22), investigators have identified a number of the genes required for Shigella pathogenesis. Many Shigella virulence-associated genes, including those encoding the type III secretion apparatus (mxi and spa genes), its secreted effectors (ipaA-D, ipgD, icsB, and virA), and specific chaperones (ipgC, ipgA, ipgE, and spa15), map to a 220-kbp virulence plasmid. Expression of these Shigella virulence determinants is highly regulated in response to environmental signals, such as temperature, osmolarity, and pH (12). This complex regulation is accomplished primarily by the transcriptional activators VirF and VirB (1). Transcription of many Shigella virulence-associated genes, including those encoding the type III secretion system (TTSS), is positively regulated by direct binding of VirB to the regulated promoters. Transcription of virB, in turn, is controlled primarily by the opposing activities of VirF, a transcriptional activator, and HNS, a transcriptional repressor. (For a review of the regulation of Shigella virulence gene expression, see reference 6.) VirF and VirB are both required to induce expression of virulence determinants that allow efficient invasion of epithelial cells by Shigella, an essential step in disease initiation. While the regulation of Shigella virulence has been the focus of intense investigation, the identification and characterization of all contributing environmental factors and regulatory elements have not yet been achieved.Noncoding RNA molecules (ncRNAs) control diverse cellular functions in organisms ranging from bacteria to humans. Although it is known that ncRNAs employ a variety of mechanisms, including methylation of rRNA, inhibition of translation or transcription, and sequestration of regulatory proteins (11, 13, 21), the full spectr...

The initiation, progression and transmission of most bacterial infections is dependent upon the ability of the invading pathogen to acquire iron from each of the varied environments encountered during the course of a natural infection. In total, 95% of iron within the human body is complexed within heme, making heme a potentially rich source of host-associated nutrient iron for invading bacteria. As heme is encountered only within the host, pathogenic bacteria often regulate synthesis of heme utilization factors such that production is maximal under host-associated environmental conditions. This study examines the regulated production of ShuA, an outer-membrane receptor required for the utilization of heme as a source of nutrient iron by Shigella dysenteriae, a pathogenic bacterium that causes severe diarrheal diseases in humans. Specifically, the impact of the distinct environmental temperatures encountered during infection within a host (37°C) and transmission between hosts (25°C) on shuA expression is investigated. We show that shuA expression is subject to temperature-dependent post-transcriptional regulation resulting in increased ShuA production at 37°C. The observed thermoregulation is mediated by nucleic acid sequences within the 5′ untranslated region. In addition, we have identified similar nucleotide sequences within the 5′ untranslated region of the orthologous chuA transcript of enteropathogenic E. coli and have demonstrated that it also functions to confer temperature-dependent post-transcriptional regulation. In both function and predicted structure, the regulatory element within the shuA and chuA 5′ untranslated regions closely resembles a FourU RNA thermometer, a zipper-like RNA structure that occludes the Shine-Dalgarno sequence at low temperatures. Increased production of ShuA and ChuA in response to the host body temperature allows for maximal production of these heme acquisition factors within the environment where S. dysenteriae and pathogenic E. coli strains would encounter heme, a host-specific iron source.

SummaryShigella flexneri requires iron for survival, and the genes for iron uptake and homeostasis are regulated by the Fur protein. Microarrays were used to identify genes regulated by Fur and to study the physiological effects of iron availability in S. flexneri . These assays showed that the expression of genes involved in iron acquisition and acid response was induced by lowiron availability and by inactivation of fur . A fur null mutant was acid sensitive in media at pH 2.5, and acid sensitivity was also observed in the wild-type strain grown under iron-limiting conditions. Acid resistance of the fur mutant in minimal medium was restored by addition of glutamate during acid challenge, indicating that the glutamate-dependent acid resistance system was not defective. Inactivation of ryhB , a small regulatory RNA whose expression is repressed by Fur, restored acid resistance in the fur mutant, while overexpressing ryhB increased acid sensitivity in the wild-type strain. RyhB-regulated genes were identified by microarray analysis. The expression of one of the RyhB-repressed genes, ydeP , which encodes a putative oxidoreductase, suppressed acid sensitivity in the fur mutant. Furthermore, an S. flexneri ydeP mutant was defective for both glutamate-independent and glutamate-dependent acid resistance. The repression of ydeP by RyhB may be indirect, as real time polymerase chain reaction (PCR) experiments indicated that RyhB negatively regulates evgA, which encodes an activator of ydeP . These results demonstrate that the acid sensitivity defect of the S. flexneri fur mutant is due to repression of ydeP by RyhB, most likely via repression of evgA .

For most living organisms, iron is both essential and potentially toxic, making the precise maintenance of iron homeostasis necessary for survival. To manage this paradox, bacteria regulate the acquisition, utilization, and storage of iron in response to its availability. The iron-dependent ferric uptake repressor (Fur) often mediates this iron-responsive regulation, by both direct and indirect mechanisms. In 2002, Masse and Gottesman identified a novel target of Fur-mediated regulation in Escherichia coli: a gene encoding a small regulatory RNA (sRNA) termed RyhB. Under conditions of iron-limitation, RyhB is produced and functions to regulate the expression of several target genes encoding iron-utilizing enzymes, iron acquisition systems, and iron storage factors. This pivotal finding provided the missing link between environmental iron-limitation and previously observed decreases in certain iron-dependent metabolic pathways, a phenomenon now referred to as an “iron-sparing” response. The discovery of RyhB opened the door to the rapidly expanding field of bacterial iron-regulated sRNAs, which continue to be identified and described in numerous bacterial species. Most striking are findings that the impact of iron-responsive sRNA regulation often extends beyond iron homeostasis, particularly with regard to production of virulence-associated factors by pathogenic bacteria. This review discusses trends in the collective body of work on iron-regulated sRNAs, highlighting both the regulatory mechanisms they utilize to control target gene expression and the impact of this regulation on basic processes controlling bacterial physiology and virulence.

Efficient utilization of heme as an iron (Fe) source by Bordetella avium requires bhuR, an Fe-regulated gene which encodes an outer membrane heme receptor. Upstream of bhuR is a 507-bp open reading frame, hereby designated rhuI (for regulator of heme uptake), which codes for a 19-kDa polypeptide. Whereas the 19-kDa polypeptide had homology to a subfamily of alternative sigma factors known as the extracytoplasmic function (ECF) sigma factors, it was hypothesized that rhuI encoded a potential in-trans regulator of the heme receptor gene in trans. Support for the model was strengthened by the identification of nucleotide sequences common to ECF sigma-dependent promoters in the region immediately upstream of bhuR. Experimental evidence for the regulatory activities of rhuI was first revealed by recombinant experiments in which overproduction of rhuI was correlated with a dramatically increased expression of BhuR. A putative rhuI-dependent bhuR promoter was identified in the 199-bp region located proximal to bhuR. When a transcriptional fusion of the 199-bp region and a promoterless lacZ gene was introduced into Escherichia coli, promoter activity was evident, but only when rhuI was coexpressed in the cell. Sigma competition experiments in E. coli demonstrated that rhuI conferred biological properties on the cell that were consistent with RhuI having sigma factor activity. Heme, hemoglobin, and several other heme-containing proteins were shown to be the extracellular inducers of the rhuI-dependent regulatory system. Fur titration assays indicated that expression of rhuI was probably Fur dependent.

Shigella species are able to grow in a variety of environments, including intracellularly in host epithelial cells. Shigella have a number of different iron transport systems that contribute to their ability to grow in these diverse environments. Siderophore iron uptake systems, heme transporters, and ferric and ferrous iron transport systems are present in these bacteria, and the genes encoding some of these systems appear to have spread among the Shigella species by horizontal transmission. Iron is not only essential for growth of Shigella but also plays an important role in regulation of metabolic processes and virulence determinants in Shigella. This regulation is mediated by the repressor protein Fur and the small RNA RyhB.