Search

Helicobacter pyloriinfects the stomachs of half of all humans. It has a relatively benign relationship with most hosts but produces severe pathology, including gastric cancer, in others. Identifying the relative contributions of host, microbial, and environmental factors to the outcome of infection has been challenging. Here we describe one approach for identifying microbial genes that affect the magnitude of host responses to infection. Single colony purified H. pyloriisolates were obtained from 25 cases and 71 controls in a Swedish case-control study of gastric cancer. Strains were first phenotyped based on their ability to produce adhesins that recognize two classes of human gastric epithelial receptors. Thirteen binding strains and two non-binding controls were then subjected to whole genome genotyping using H. pyloriDNA microarrays. A cohort of “variable” genes was identified based on a microarray-determined call of “absent” in at least one member of the strain panel. Each strain was subsequently introduced into two types of germ-free transgenic mice, each programmed to express a different host factor postulated to pose increased risk for development of severe pathology. Expression of biomarkers of host defense was quantitated 4 weeks after inoculation, and the magnitude of the response correlated with bacterial genotype. The proportion of genes encoding HsdS homologs (specificity subunit of hetero-oligomeric type I restriction-modification systems) was significantly higher in the pool of 18 variable genes whose presence directly correlated with a robust host response than their proportion in the remaining 352 members of the variable gene pool. This suggests that the functions of these HsdS homologs may include control of expression of microbial determinants that affect the extent of gastric responses to this potentially virulent pathogen.

Yeast deprived of nutrients exhibit a marked life span extension that requires the activity of the NAD+-dependent histone deacetylase, Sir2p. Here we show that increased dosage of NPT1, encoding a nicotinate phosphoribosyltransferase critical for the NAD+salvage pathway, increases Sir2-dependent silencing, stabilizes the rDNA locus, and extends yeast replicative life span by up to 60%. Both NPT1and SIR2provide resistance against heat shock, demonstrating that these genes act in a more general manner to promote cell survival. We show that Npt1 and a previously uncharacterized salvage pathway enzyme, Nma2, are both concentrated in the nucleus, indicating that a significant amount of NAD+is regenerated in this organelle. Additional copies of the salvage pathway genes, PNC1, NMA1, and NMA2, increase telomeric and rDNA silencing, implying that multiple steps affect the rate of the pathway. Although SIR2-dependent processes are enhanced by additional NPT1, steady-state NAD+levels and NAD+/NADH ratios remain unaltered. This finding suggests that yeast life span extension may be facilitated by an increase in the availability of NAD+to Sir2, although not through a simple increase in steady-state levels. We propose a model in which increased flux through the NAD+salvage pathway is responsible for the Sir2-dependent extension of life span.

Saccharomyces cerevisiaecontains at least five acyl-CoA synthetases (fatty acid activation proteins, or Faaps). Four FAAgenes have been recovered to date. Recent genetic studies indicate that Faa1p and Faa4p are involved in the activation of imported fatty acids, while Faa2p activates endogenous pools of fatty acids. We have now purified Faa4p from S. cerevisiaeand compared its fatty acid substrate specificity in vitrowith the specificities of purified Faa1p, Faa2p, and Faa3p. Among C8-C18 saturated fatty acids, Faa4p and Faa1p both prefer C14:0. Surveys of C14 fatty acids with single cis-double bonds at C2-C12 indicated that Faa4p and Faa1p prefer Z9-tetradecenoic acid, although Faa4p's preference is much greater and also evident in C16 and C18 fatty acids. Faa4p's selectivity for fatty acids with a C9-C10 cis-double bond is a feature it shares with Faa3p and is notable since in yeast Ole1p, a microsomal cis-Δ9desaturase, accounts for de novoproduction of monoenoic acyl-CoAs from saturated acyl-CoA substrates. Faa4p has no detectable acyl-CoA synthetase activity when incubated with tetradecenoic acids having a trans-double bond at C2-3, C4-5, C5-6, C6-7, C7-8, or C9-10. Faa3p can only use E9-tetradecenoic acid as a substrate, while E4-, E6- and E9-tetradecenoic acids can be used by Faa1p and Faa2p. E2-tetradecenoic acid is an Faap inhibitor, with Faa2p exhibiting the greatest sensitivity (IC50= 2.6 ± 0.2 μM). Triacsin C (1-hydroxy3-(E,E,E,2′,4′,7′-undecatrienylidine)-1,2,3-triazene) has trans-double bonds at positions that correspond to those in E2-, E5-, and E7-tetradecenoic acids. This compound is a potent inhibitor of Faa2p (Ki= 15 ± 1 nM; competitive with fatty acid), less potent against Faa4p (Ki= 2 μM), and not active against Faa1p or Faa3p (IC50> 500 μM). Analysis of an n-tetradecanal plus a series of oximes (tridecanal oxime, 1-azadeca-1,3,5-trienol, and 1-azaundeca-1,3,5-trienol) indicated that the combination of an azenol moiety (R-CH⋕N-OH) plus adjacent unsaturation are critical for triacsin C's selective inhibition of Faa2p. Triacsin C and oxime derivatives appear to be very useful for defining differences in molecular recognition among S. cerevisiaeacyl-CoA synthetases. The >25,000-fold range in the inhibitory effects of triacsin C on these four Faaps suggests that it may be possible to develop other selective inhibitors of eukaryotic acyl-CoA synthetases.

Saccharomyces cerevisiaemyristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential 455-residue, monomeric enzyme that catalyzes the transfer of myristate from myristoyl-CoA to the NH2-terminal Gly residue of cellular proteins. Nmt1p has an ordered Bi Bi reaction mechanism with binding of myristoyl-CoA occurring before binding of peptide substrates. To define residues important for function, the polymerase chain reaction was used to generate random mutations in the NMT1gene. A colony color sectoring assay was used to screen a library of 52,000 transformants for nmt1alleles encoding enzymes with reduced activity. nmt1alleles were identified that produced temperature-sensitive (ts) growth arrest due to substitutions affecting eight residues conserved in orthologous Nmts: Asn102, Ala202, Cys217, Ser328, Val395, Asn404, Leu420, and Asn426. Ala202→ Thr, Cys217→ Arg, Ser328→ Pro, Asn404→ Tyr, and Asn426→ Ile produced the most severe ts phenotype. Their effects on the functional properties of the enzyme's myristoyl-CoA and peptide binding sites were defined by purifying each mutant from Escherichia coliand conducting in vitrokinetic analyses with acyl-CoA and peptide substrates and with two competitive inhibitors: S-(2-oxo)pentadecyl-CoA, a nonhydrolyzable myristoyl-CoA analog, and SC-58272, a peptidomimetic derived from the NH2-terminal sequence of an Nmt1p substrate (ADP-ribosylation factor-2, Arf2p). None of the substitutions affect the enzyme's acyl chain length selectivity. When compared with wild type Nmt1p, Cys217→ Arg produces 3- and 6-fold increases in Kifor SC-58272 at 24 and 37°C but no change in Kifor S-(2-oxo)pentadecyl-CoA, indicating that the substitution selectively affects Nmt1p's peptide binding site. Asn426→ Ile selectively perturbs the myristoyl-CoA binding site, resulting in the most pronounced reduction in affinity for S-(2-oxo)pentadecyl-CoA (12- and 20-fold). Ala202→ Thr, which confers the most severe ts phenotype, provides an example of a substitution that affects both sites, producing 3- and 6-fold increases in the Kifor S-(2-oxo)pentadecyl-CoA and 6- and 9-fold increases in the Kifor SC-58272 at 24 and 37°C. An N-myristoylation-dependent change in the electrophoretic mobility of Arf1p was used to assay the effects of the mutants on cellular levels of protein N-myristoylation under a variety of growth conditions. The ts growth arrest produced by nmt1alleles correlates with a reduction in myristoyl-Arf1p to ≤50% of total cellular Arf1p.

Cryptococcus neoformansis a fungal pathogen that causes chronic meningitis in 10% of patients with AIDS. Genetic and biochemical studies were conducted to determine whether myristoyl-CoA:protein N-myristoyltransferase (Nmt) is a target for development of a new class of fungicidal drugs. A single copy of a conditional lethal C. neoformans NMTallele was introduced into the fungal genome by homologous recombination. The allele (nmt487D) produces temperature-sensitive myristic acid auxotrophy. This phenotype is due, in part, to under-myristoylation of a cellular ADP ribosylation factor (Arf) and can be rescued by forced expression of human Nmt. Two isogenic strains with identical growth kinetics at 35 °C were used to test the biological effects of an Nmt inhibitor. CPA8 contained a single copy of wild type C. neoformans NMT. HMC1 containednmt487Dplus 10 copies of human NMT. Since a single copy of nmt487Dwill not support growth at 35 °C, survival of HMC1 depends upon its human Nmt. ALYASKLS-NH2, an inhibitor derived from an Arf, was fully depeptidized:p-[(2-methyl-1-imidazol-1-yl)butyl]phenyl-acetyl was used to represent the GLYA tetrapeptide, whereas SKLS was replaced with a chiral tyrosinol scaffold. Kinetic studies revealedKi(app)values of 1.8 ± 1 and 9 ± 2.4 μmfor purified fungal and human Nmts, respectively. The minimal inhibitory concentration of the compound was 2-fold lower for CPA8 compared with HMC1. A single dose of 100 μmproduced a 5-fold greater inhibition of protein synthesis in CPA8 versusHMC1. The strain specificity of these responses indicates that the fungicidal effect was Nmt-dependent. These two strains may be useful for screening chemical libraries for Nmt-based fungicidal compounds with relatively little activity against the human enzyme.

Four unlinked fatty acid activation (FAA) genes encoding acyl-CoA synthetases have been identified in Saccharomyces cerevisiaeand characterized by noting the phenotypes of isogenic strains containing all possible combinations of faanull alleles. None of these genes is required for vegetative growth when acyl-CoA production by the fatty acid synthetase (Fas) complex is active. When Fas is inhibited by cerulenin, exponentially growing cells are not viable on media containing a fermentable carbon source unless supplemented with fatty acids such as myristate, palmitate, or oleate. The functionally interchangeable FAA1and FAA4genes are responsible for activation of these imported fatty acids. Analysis of lysates prepared from isogenic FAA1FAA4and faa1Δfaa4Δ strains indicated that Faa1p and Faa4p together account for 99% of total cellular myristoyl-CoA and palmitoyl-CoA synthetase activities. Genetic complementation studies revealed that rat liver acyl-CoA synthetase (RLACS) rescues the viability of faa1Δfaa4Δ cells in media containing a fermentable carbon source, myristate or palmitate, plus cerulenin. Rescue is greater at 37°C compared with 24°C, paralleling the temperature-dependent changes in RLACS activity in vitroas well as the enzyme's ability to direct incorporation of tritiated myristate and palmitate into cellular phospholipids in vivo. Complementation by RLACS is blocked by treatment of cells with triacsin C (1-hydroxy-3-(E, E, E,2’,4’,7’-undecatrienylidine)triazene). Even though Faa1p, Faa4p, and RLACS are all able to activate imported myristate and palmitate in S. cerevisiae, the sensitivity of Faa4p and RLACS, but not Faa1p, to inhibition by triacsin C suggests that the rat liver enzyme is functionally more analogous to Faa4p than to Faa1p. Finally, an assessment of myristate and palmitate import into FAA1FAA4and faa1Δfaa4Δ strains, with or without episomes that direct overexpression of Faa1p, Faa4p or RLACS, indicated that fatty acid uptake is not coupled to activation in S. cerevisiae.

Saccharomyces cerevisiaecontains four known acyl-CoA synthetases (fatty acid activation proteins, Faaps). Faa1p and Faa4p activate exogenously derived fatty acids. Acyl-CoA metabolism plays a critical role in regulating protein N-myristoylation by the essential enzyme, myristoyl-CoA:protein N-myristoyltransferase (Nmt1p). In this report, we have examined whether Faa1p and Faa4p have distinct roles in affecting protein N-myristoylation as cells transition from growth in rich media to a growth-arrested state during nutrient deprivation (stationary phase). The colony-forming potential of 10 isogenic strains was defined as a function of time spent in stationary phase. These strains contained either a wild type or mutant NMT1allele, and wild type or null alleles of each FAA. Only the combination of the Nmt mutant (nmt451Dp; reduced affinity for myristoyl-CoA) and loss of Faa4p produced a dramatic loss of colony-forming units (CFU). The progressive millionfold reduction in CFU was associated with a deficiency in protein N-myristoylation that first appeared during logarithmic growth, worsened through the post-diauxic phase, and became extreme in stationary phase. Northern and Western blot analyses plusN-myristoyltransferase assays showed that Nmt is normally present only during the log and diauxic/post-diauxic periods, indicating that N-myristoylproteins present in stationary phase are “inherited” from these earlier phases. Moreover,FAA4is the only FAAinduced during the critical diauxic/early post-diauxic transition. Although substitution of nmt1–451Dfor NMT1results in deficiencies in protein N-myristoylation, these deficiencies are modest and limited by compensatory responses that include augmented expression of nmt1–451Dand precocious induction of FAA4in log phase. Loss of Faa4p from nmt1–451Dcells severely compromises their capacity to adequately myristoylate Nmt substrates prior to entry into stationary phase since none of the other Faaps are able to functionally compensate for its absence. To identify Nmt1p substrates that may affect maintenance of proliferative potential during stationary phase, we searched the yeast genome for known and putative N-myristoylproteins. Of the 64 genes found, 48 were successfully deleted in NMT1cells. Removal of any one of the following nine substrates produced a loss of CFU similar to that observed in nmt1–451Dfaa4Δcells: Arf1p, Arf2p, Sip2p, Van1p, Ptc2p, YBL049W (homology to Snf7p), YJR114W, YKR007W, and YMR077C. These proteins provide opportunities to further define the molecular mechanisms that regulate survival during stationary phase.