Bile induces pleiotropic responses that affect production of virulence factors, motility, and other phenotypes in the enteric pathogen Vibrio cholerae. Since bile is a heterogeneous mixture, crude bile was fractionated, and the components that mediate virulence gene repression and enhancement of motility were identified by nuclear magnetic resonance, gas chromatography (GC), and GC-mass spectrometry analyses. The unsaturated fatty acids detected in bile, arachidonic, linoleic, and oleic acids, drastically repressed expression of the ctxAB and tcpA genes, which encode cholera toxin and the major subunit of the toxin-coregulated pilus, respectively. The unsaturated fatty acid-dependent repression was due to silencing of ctxAB and tcpA expression by the histonelike nucleoid-structuring protein H-NS, even in the presence of the transcriptional activator ToxT. Unsaturated fatty acids also enhanced motility of V. cholerae due to increased expression of flrA, the first gene of a regulatory cascade that controls motility. H-NS had no role in the fatty acid-mediated enhancement of motility. It is likely that the ToxR/ToxT system that negatively regulates motility is rendered nonfunctional in the presence of unsaturated fatty acids, leading to an increase in motility. Motility and flrA expression were also increased in the presence of cholesterol, another component of bile, in an H-NS-and ToxR/ToxT-independent manner.Vibrio cholerae, a noninvasive enteric bacterium, is the causative agent of the diarrheal disease cholera. Cholera continues to cause devastating outbreaks, particularly in the developing world, resulting in more than 100,000 deaths every year, and the case fatality ratio may exceed 20% in affected populations (29). The pathogenicity of V. cholerae is largely due to the production of cholera toxin (CT) and a toxin-coregulated pilus (TCP), thought to be essential for colonization of the intestinal epithelium by the bacterium (13). Expression of CT, TCP, and several other virulence factors is coordinately controlled by the hierarchical expression of regulatory proteins comprising the ToxR regulon (14), in which the inner membrane DNA binding proteins ToxR and TcpP (7, 18) activate expression of ToxT, a transcriptional regulator that is required for the expression of ctxAB and tcpA as well as several other virulence genes (3). The ToxR regulon is strongly influenced by physicochemical parameters/factors such as temperature, osmolarity, pH, amino acids, and bile, which exert their effects at different levels of the regulatory cascade (16, 25).Bile, a heterogeneous mixture of conjugated and unconjugated bile acids, bile pigments, inorganic salts, cholesterol, phospholipids, and probably other unidentified components, is secreted into the lumen of the duodenum from the gall bladder through the bile duct and is inevitably encountered by all enteric bacteria in their human hosts (9). The role of bile in normal gastrointestinal physiology is to aid the emulsification of lipids and also to protect the host from bacteri...

Esteban Rossi-Hansberg, and trade workshop participants at Princeton University for many helpful comments and suggestions. Many thanks to João De Negri and IPEA-Brasília for granting access to Secex and RAIS. Comments and questions are welcome. Please contact

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3’-P and 5’-OH, are processed by mammalian polynucleotide kinase 3’-phosphatase (PNKP), a bifunctional enzyme with 3’-phosphatase and 5’-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14–41 to 55–82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP’s 3’ phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3’-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients’ brain. Finally, long amplicon quantitative PCR analysis of human MJD patients’ brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3’-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

The repair of reactive oxygen species-induced base lesions and single strand breaks (SSBs) in the nuclear genome via the base excision (BER) and SSB repair (SSBR) pathways, respectively, is well characterize, and important for maintaining genomic integrity. However, the role of mitochondrial (mt) BER and SSBR proteins in mt genome maintenance is not completely clear. Here we show the presence of the oxidized base-specific DNA glycosylase Nei-like 2 (NEIL2) and the DNA end-processing enzyme polynucleotide kinase 3 -phosphatase (PNKP) in purified human mitochondrial extracts (MEs). Confocal microscopy revealed co-localization of PNKP and NEIL2 with the mitochondrion-specific protein cytochrome c oxidase subunit 2 (MT-CO2). Further, chromatin immunoprecipitation analysis showed association of NEIL2 and PNKP with the mitochondrial genes MT-CO2 and MT-CO3 (cytochrome c oxidase subunit 3); importantly, both enzymes also associated with the mitochondrion-specific DNA polymerase ␥. In cell association of NEIL2 and PNKP with polymerase ␥ was further confirmed by proximity ligation assays. PNKP-depleted ME showed a significant decrease in both BER and SSBR activities, and PNKP was found to be the major 3 -phosphatase in human ME. Furthermore, individual depletion of NEIL2 and PNKP in human HEK293 cells caused increased levels of oxidized bases and SSBs in the mt genome, respectively. Taken together, these studies demonstrate the critical role of NEIL2 and PNKP in maintenance of the mammalian mitochondrial genome.Reactive oxygen species-induced genomic damage includes a plethora of oxidized bases, apurinic/apyrimidinic (AP) 3 sites, and DNA single strand breaks (SSBs) that are often mutagenic and are etiologically linked to various pathophysiologies, including sporadic cancer, and a multitude of age-related degenerative diseases (1, 2). All of these DNA base lesions are primarily repaired by the evolutionarily conserved base excision repair (BER) pathway in both the nucleus and mitochondria (3). BER for oxidized bases is initiated by the recognition and cleavage of the base lesion from DNA by a DNA glycosylase; the resulting AP site is then cleaved by the intrinsic lyase activity of the glycosylase. In mammalian cells, five oxidized base-specific DNA glycosylases have been identified; they belong to two families based on their reaction mechanisms and structural conservation of their catalytic motifs. OGG1 and NTH1 of the Nth family remove base lesions only from duplex DNA and have ␤-elimination activity, generating 3Ј-deoxyribose phosphate and 5Ј-phosphate (5Ј-P) groups at the resulting SSB (4). In contrast, the recently discovered NEILs 1-3 of the Nei family are active with duplex as well as single-stranded DNA (occurring transiently during DNA replication and transcription). Our recent studies now show that NEIL2 preferentially repairs oxidative damage from the transcribed genes, and NEIL1 primarily associates with the replication-associated proteins, thus indicating its involvement in repair during DNA replication (5, 6...