Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL)

Tetrahydrolipstatin (THL) is bactericidal but its precise target spectrum is poorly characterized. Here, we used a THL analog and activity-based protein profiling to identify target proteins after enrichment from whole cell lysates of Mycobacterium bovis Bacillus Calmette-Guérin cultured under replicating and non-replicating conditions. THL targets α/β-hydrolases, including many lipid esterases (LipD, G, H, I, M, N, O, V, W, and TesA). Target protein concentrations and total esterase activity correlated inversely with cellular triacylglycerol upon entry into and exit from non-replicating conditions. Cellular overexpression of lipH and tesA led to decreased THL susceptibility thus providing functional validation. Our results define the target spectrum of THL in a biological species with particularly diverse lipid metabolic pathways. We furthermore derive a conceptual approach that demonstrates the use of such THL probes for the characterization of substrate recognition by lipases and related enzymes.Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is responsible for nearly 2 million deaths each year. The host immune response toward aerosol infection is to quarantine tubercle bacilli in a granulomatous structure (1, 2). However, granuloma-associated mycobacteria can switch to a non-replicative, “dormant” state and successfully evade immune response for decades after infection (3, 4). The metabolic events that permit tubercle bacilli to enter host cells and revive from states of persistence suggest that lipids are utilized as a carbon source (5–7). During times of oxygen deprivation and in the absence of host cells, cultivated mycobacteria store fatty acids (FAs) in the form of triacylglycerol (TAG)¹-enriched lipid droplets (8–10). Upon resuscitation (by the re-introduction of oxygen), these lipid droplets vanish and TAGs are hydrolyzed (11). Unfortunately, the molecular mechanisms for TAG build-up and breakdown are far less well understood in bacteria when compared with those processes in eukaryotes.Comparative sequence analysis of the Mtb genome has revealed that it contains 250 genes encoding enzymes involved in lipid metabolism compared with only 50 enzymes in Escherichia coli, which has a genome of comparable size. Among these genes, 150 are predicted to encode proteins involved in lipid catabolism (12, 13). A family of 24 carboxyl ester hydrolases called “lip” genes (lipC to Z, except K and S) has been predicted to play a role in lipid catabolism (14). Among these, only a few have been functionally characterized and related to mycobacterial dormancy and resuscitation (15–18).Tetrahydrolipstatin, a serine esterase inhibitor, covalently binds to and inhibits mammalian lipases and fatty acid synthase (FAS) and is marketed as “Orlistat” for the treatment of severe forms of obesity (19). THL was previously shown to inhibit both active and latent forms of mycobacteria (11, 20–22) but the bacterial target spectrum remains poorly characterized. Therefore, to (1) define the THL target spectrum in a mycobacterial species and (2) to obtain biochemical insights into regulation of lipases and esterases in different metabolic states, we employed a chemical-proteomics approach using activity-based protein profiling (ABPP) with a bait that has been described to bind to lipolytic enzymes (23–25). We identified several known lipases (as anticipated), putative lipase and esterases, and hypothetical proteins of unknown functions, thereby providing a comprehensive resource of experimentally determined THL targets in mycobacteria. Importantly, we systematically compared readouts of fluorescently tagged THL-proteins (7 bands on one-dimensional SDS-PAGE) with those of mass spectrometry-based peptide identification of enriched protein fractions (247 in growing cells). This comparison led to the identification of 14 THL targets, two of which were further validated experimentally. We furthermore provide a conceptual framework for the evaluation of this target list using both experimental as well as bioinformatics approaches in two examples, lipH and tesA. Overall, our data indicate that THL is an anti-mycobacterial drug because of its potential to (1) bind to a relatively wide range of lipolytic enzymes and (2) prevent bacilli from resuscitating from a nonreplicating persistent (NRP) state when lipid metabolism is particularly important.     

Structure and Specificity of the Bacterial Cysteine Methyltransferase Effector NleE Suggests a Novel Substrate in Human DNA Repair Pathway

Enteropathogenic E. coli (EPEC) and related enterobacteria rely on a type III secretion system (T3SS) effector NleE to block host NF-κB signaling. NleE is a first in class, novel S-adenosyl-L-methionine (SAM)-dependent methyltransferase that methylates a zinc-coordinating cysteine in the Npl4-like Zinc Finger (NZF) domains in TAB2/3 adaptors in the NF-κB pathway, but its mechanism of action and other human substrates are unknown. Here we solve crystal structure of NleE-SAM complex, which reveals a methyltransferase fold different from those of known ones. The SAM, cradled snugly at the bottom of a deep and narrow cavity, adopts a unique conformation ready for nucleophilic attack by the methyl acceptor. The substrate NZF domain can be well docked into the cavity, and molecular dynamic simulation indicates that Cys673 in TAB2-NZF is spatially and energetically favorable for attacking the SAM. We further identify a new NleE substrate, ZRANB3, that functions in PCNA binding and remodeling of stalled replication forks at the DNA damage sites. Specific inactivation of the NZF domain in ZRANB3 by NleE offers a unique opportunity to suggest that ZRANB3-NZF domain functions in DNA repair processes other than ZRANB3 recruitment to DNA damage sites. Our analyses suggest a novel and unexpected link between EPEC infection, virulence proteins and genome integrity.     

Metabolomic profiling of three brain regions from a postnatal infected Borna disease virus Hu-H1 rat model

Neonatal rat infection with Borna disease virus (BDV), termed neonatal Borna disease, is an established model for investigating the BDV-associated pathogenesis of neurodevelopmental abnormalities. BDV produces a persistent noncytolytic infection in all culture cell systems assayed to date, while persistent infection in neonatal rats results in a progressive loss of hippocampal granule cells, cerebellar Purkinje cells, and cortical GABA-ergic neurons. Persistent infection also results in behavioral deficits including hyperactivity, cognitive impairment, and abnormal social behavior. However, the molecular mechanisms underlying the neuronal degeneration and behavioral abnormalities remain unclear. Using a metabolomic approach based on gas chromatography coupled with mass spectrometry in conjunction with statistical pattern recognition, the metabolic changes in response to BDV Hu-H1 infection were characterized in the rat hippocampus, cerebellum, and cortex. Metabonomic profiling revealed significant perturbations in nucleotide (e.g., adenosine, uracil, inosine, adenosine-5′-monophosphate, uridine-5′-monophosphate, d-ribose 5-phosphate, and sedoheptulose 7-phosphate), amino acid (e.g., lysine, glycine, phenylalanine, tyrosine, proline, serine, cysteine, aspartic acid, pyroglutamic acid, and γ-aminobutyric acid), lipid (e.g., cholesterol, myristic acid, stearic acid, palmitic acid, 1-monopalmitoylglycerol, and arachidonic acid), and energy (e.g., glucose, lactose, 3-phosphoglyceric acid, and pyruvic acid) metabolites. These metabolites participate in pathways crucial to viral proliferation and neurotransmitter homeostasis. This metabolomic profiling study provides insight into the pathogenic mechanisms of BDV and new directions with which to investigate the in vivo effects of persistent BDV infection.     

Establishment of a proteomic profile associated with gonocyte and spermatogonial stem cell maturation and differentiation in neonatal mice

Initiation of the first wave of spermatogenesis in the neonatal mouse testis is characterized by differentiation of a transient population of germ cells called gonocytes in the center of the seminiferous tubules. After resuming mitotic activity, gonocytes relocate on the basement membrane, giving rise to spermatogonial stem cells (SSCs). These processes begin from birth in mice, and differentiated type A spermatogonia first appear by day 6 postpartum. During these processes, Sertoli cells within the seminiferous tubules and Leydig cells in the interstitial tissue form the stem cell “niche,” and influence SSC fate decisions. Thus, we collected whole mouse testis tissues during the first wave of spermatogenesis at specific time points (days 0.5, 1.5, 2.5, 3.5, 4.5, and 5.5 postpartum) and constructed a comparative proteomic profile. We identified 252 differentially expressed proteins classified into three clusters based on expression, and bioinformatics analysis correlated each protein pattern to specific cell processes. Expression patterns of nine selected proteins were verified via Western blot, and cellular localizations of three proteins with little known information in testes were further investigated during spermatogenesis. Taken together, the results provide an important reference profile of a functional proteome during neonatal mouse gonocyte and SSC maturation and differentiation.     

Characterization of the most abundant Lactobacillus species in chicken gastrointestinal tract and potential use as probiotics for genetic engineering

The count and diffusion of Lactobacilli species in the differ ent gastrointestinal tract （GI） regions of broilers were investigated by quantitative realtime polymerase chain reaction, and the probiotic characteristics of six L. reuteri species isolated from broilers＇ GI tract were also investi gated to obtain the potential target for genetic engineering. Lactobacilli had the highest diversity in the crop and the lowest one in the cecum. Compared with the lower GI tract, more LactobaciUi were found in the upper GI tract. Lactobacillus reuteri, johnsonii, L. acidophilus, L. crispatus, L. salivarius, and L. aviarius were the predominant Lactobacillus species and present throughout the GI tract of chickens. Lactobacillus reuteri was the most abundant Lactobacillus species. Lactobacillus reuteri XC1 had good probiotic characteristics that would be a potential and desirable target for genetic engineering.     

A novel mitovirus from Buergenerula spartinae infecting the invasive species Spartina alterniflora

Spartina alterniflora,a coastal saltmarsh plant,has been classified as one of sixteen invasive alien species by the State Environmental Protection Administration of China.Nearly forty years of propagation and intru-sion of this plant has resulted in some harmful effects on the economic development of coastal areas,due to its proliferation.     

The β-alanyl-monoamine synthase ebony is regulated by schizophrenia susceptibility gene dysbindin in Drosophila

The Drosophila homolog of schizophrenia susceptibility gene dysbindin(Ddysb)affects a range of behaviors through regulation of multiple neurotransmitter signals,including dopamine activity.To gain insights into mechanisms underlying Ddysb-dependent regulation of dopamine signal,we investigated interaction between Ddysb and Ebony,the Drosophilaβ-alanyl-monoamine synthase involved in dopamine recycling.We found that Ddysb was capable of regulating expression of Ebony in a bi-directional manner and its subcellular distribution.Such regulation is confined to glial cells.The expression level of ebony and its accumulation in glial soma depend positively on Ddysb activity,whereas its distribution in glial processes is bound to be reduced in response to any alterations of Ddysb from the normal control level,either an increase or decrease.An optimal binding ratio between Dysb and Ebony might contribute to such non-linear effects.Thus,Ddysb-dependent regulation of Ebony could be one of the mechanisms that mediate dopamine signal.     

Effects of host-egg ages on host selection and suitability of four Chinese Trichogramma species,egg parasitoids of the rice striped stem borer,Chilo suppressalis

The striped stem borer, Chilo suppressalis (Walker), is one of the most economically important rice pests worldwide. However, biological control of this pest using natural-enemy insects has been rarely documented to date. With the objective of screening suitable candidate species for controlling the striped stem borer, we investigated the effect of the age of host eggs on the host selection and suitability by four indigenous Trichogramma species on their native host, C. suppressalis. The results indicated that the differently aged eggs of C. suppressalis were all accepted by T. japonicum, T. dendrolimi and T. chilonis, and there was a clear tendency to parasitize older eggs less under no-choice and choice conditions. The number of parasitized host eggs by T. ostriniae also decreased with the increasing host age in the no-choice test, but more 2-day-old host eggs were parasitized in the choice test. When 0-, 2-day-old eggs were offered, T. dendrolimi, T. japonicum and T. chilonis exhibited similar parasitism ability, whereas T. ostriniae appeared to have a stronger ability to attack the older host eggs (4-day-old). Trichogramma japonicum developed and emerged on parasitized C. suppressalis eggs of all ages tested, while showing a better adaptation to younger host eggs with significantly faster developmental time, higher survival and more female progeny on 0-day-old eggs. No adults for each of the other three Trichogramma species emerged from parasitized 4-day-old host eggs, and they had similar developmental time, survival and female progeny on parasitized 0-, 2-day-old host eggs with an exception of female progeny for T. chilonis. On 0-day-old host eggs, T. japonicum developed faster and T. ostriniae had lower progeny survival than the other three Trichogramma species evaluated, respectively. The current study provides useful information to select suitable Trichogramma species for controlling the striped stem borer, C. suppressalis.