Human-to-bovine jump of Staphylococcus aureus CC8 is associated with the loss of a β-hemolysin converting prophage and the acquisition of a new staphylococcal cassette chromosome.

Staphylococcus aureus can colonize and infect both humans and animals, but isolates from both hosts tend to belong to different lineages. Our recent finding of bovine-adapted S. aureus showing close genetic relationship to the human S. aureus clonal complex 8 (CC8) allowed us to examine the genetic basis of host adaptation in this particular CC. Using total chromosome microarrays, we compared the genetic makeup of 14 CC8 isolates obtained from cows suffering subclinical mastitis, with nine CC8 isolates from colonized or infected human patients, and nine S. aureus isolates belonging to typical bovine CCs. CC8 isolates were found to segregate in a unique group, different from the typical bovine CCs. Within this CC8 group, human and bovine isolates further segregated into three subgroups, among which two contained a mix of human and bovine isolates, and one contained only bovine isolates. This distribution into specific clusters and subclusters reflected major differences in the S. aureus content of mobile genetic elements (MGEs). Indeed, while the mixed human-bovine clusters carried commonly human-associated β-hemolysin converting prophages, the bovine-only isolates were devoid of such prophages but harbored an additional new non-mec staphylococcal cassette chromosome (SCC) unique to bovine CC8 isolates. This composite cassette carried a gene coding for a new LPXTG-surface protein sharing homologies with a protein found in the environmental bacterium Geobacillus thermoglucosidans. Thus, in contrast to human CC8 isolates, the bovine-only CC8 group was associated with the combined loss of β-hemolysin converting prophages and gain of a new SCC probably acquired in the animal environment. Remaining questions are whether the new LPXTG-protein plays a role in bovine colonization or infection, and whether the new SCC could further acquire antibiotic-resistance genes and carry them back to human.

Azole resistance by loss of function of the sterol Δ⁵,⁶-desaturase gene (ERG3) in Candida albicans does not necessarily decrease virulence.

The inactivation of ERG3, a gene encoding sterol Δ⁵,⁶-desaturase (essential for ergosterol biosynthesis), is a known mechanism of in vitro resistance to azole antifungal drugs in the human pathogen Candida albicans. ERG3 inactivation typically results in loss of filamentation and attenuated virulence in animal models of disseminated candidiasis. In this work, we identified a C. albicans clinical isolate (VSY2) with high-level resistance to azole drugs in vitro and an absence of ergosterol but normal filamentation. Sequencing of ERG3 in VSY2 revealed a double base deletion leading to a premature stop codon and thus a nonfunctional enzyme. The reversion of the double base deletion in the mutant allele (erg3-1) restored ergosterol biosynthesis and full fluconazole susceptibility in VSY2, confirming that ERG3 inactivation was the mechanism of azole resistance. Additionally, the replacement of both ERG3 alleles by erg3-1 in the wild-type strain SC5314 led to the absence of ergosterol and to fluconazole resistance without affecting filamentation. In a mouse model of disseminated candidiasis, the clinical ERG3 mutant VSY2 produced kidney fungal burdens and mouse survival comparable to those obtained with the wild-type control. Interestingly, while VSY2 was resistant to fluconazole both in vitro and in vivo, the ERG3-derived mutant of SC5314 was resistant only in vitro and was less virulent than the wild type. This suggests that VSY2 compensated for the in vivo fitness defect of ERG3 inactivation by a still unknown mechanism(s). Taken together, our results provide evidence that contrary to previous reports inactivation of ERG3 does not necessarily affect filamentation and virulence.

Decreased glucose-induced thermogenesis after weight loss in obese subjects: a predisposing factor for relapse of obesity?

Glucose-induced thermogenesis (GIT) after a 100-g oral glucose load was measured by continuous indirect calorimetry in 32 nondiabetic and diabetic obese subjects and compared to 17 young and 13 middle aged control subjects. The obese subjects were divided into three groups: A (n = 12) normal glucose tolerance, B (n = 13) impaired glucose tolerance, and C (n = 7) diabetics, and were studied before and after a body weight loss ranging from 9.6 to 33.5 kg consecutive to a 4 to 6 months hypocaloric diet. GIT, measured over 3 h and expressed as percentage of the energy content of the load, was significantly reduced in obese groups A and C (6.2 +/- 0.6, and 3.8 +/- 0.7%, respectively) when compared to their age-matched control groups: 8.6 +/- 0.7 (young) and 5.8 +/- 0.3% (middle aged). Obese group B had a GIT of 6.1 +/- 0.6% which was lower than that of the young control group but not different from the middle-aged control group. After weight loss, GIT in the obese was further reduced in groups A and B than before weight loss: ie, 3.4 +/- 0.6 (p less than 0.001), 3.7 +/- 0.5 (p less than 0.01) respectively, whereas in group C, weight loss induced no further diminution in GIT (3.8 +/- 0.6%). These results support the concept of a thermogenic defect after glucose ingestion in obese individuals which is not the consequence of their excess body weight but may be one of the factors favoring the relapse of obesity after weight loss.

NR2A at CA1 synapses is obligatory for the susceptibility of hippocampal plasticity to sleep loss.

A loss in the necessary amount of sleep alters expression of genes and proteins implicated in brain plasticity, but key proteins that render neuronal circuits sensitive to sleep disturbance are unknown. We show that mild (4-6 h) sleep deprivation (SD) selectively augmented the number of NR2A subunits of NMDA receptors on postsynaptic densities of adult mouse CA1 synapses. The greater synaptic NR2A content facilitated induction of CA3-CA1 long-term depression in the theta frequency stimulation range and augmented the synaptic modification threshold. NR2A-knock-out mice maintained behavioral response to SD, including compensatory increase in post-deprivation resting time, but hippocampal synaptic plasticity was insensitive to sleep loss. After SD, the balance between synaptically activated and slowly recruited NMDA receptor pools during temporal summation was disrupted. Together, these results indicate that NR2A is obligatory for the consequences of sleep loss on hippocampal synaptic plasticity. These findings could advance pharmacological strategies aiming to sustain hippocampal function during sleep restriction.

Membrane lipid composition affects plant heat sensing and modulates Ca ( 2+) -dependent heat shock response.

Understanding how plants sense and respond to heat stress is central to improve crop tolerance and productivity. Recent findings in Physcomitrella patensdemonstrated that the controlled passage of calcium ions across the plasma membrane regulates the heat shock response (HSR). To investigate the effect of membrane lipid composition on the plant HSR, we acclimated P. patens to a slightly elevated yet physiological growth temperature and analysed the signature of calcium influx under a mild heat shock. Compared to tissues grown at 22°C, tissues grown at 32°C had significantly higher overall membrane lipid saturation level and, when submitted to a short heat shock at 35°C, displayed a noticeably reduced calcium influx and a consequent reduced heat shock gene expression. These results show that temperature differences, rather than the absolute temperature, determine the extent of the plant HSR and indicate that membrane lipid composition regulates the calcium-dependent heat-signaling pathway.

Loss of mating flight and shift in the pattern of carbohydrate storage in sexuals of ants (Hymenoptera; Formicidae)

In ants, energy for flying is derived from carbohydrates (glycogen and free sugars). The amount of these substrates was compared in sexuals participating or not participating in mating flights. Results show that in participating females (Lasius niger, L. flavus, Myrmica scabrinodis, Formica rufa, F. polyctena, F. lugubris), the amount of carbohydrates, especially glycogen, was higher than in non-participating females (Cataglyphis cursor, Iridomyrmex humilis). Similarly, male C. cursor and I. humilis which fly, exhibit a much higher carbohydrate content than do the non-flying females of these species. Furthermore, the quantity of carbohydrates stored was generally higher in males than in females for each species. These results are discussed with regard to the loss of the nuptial flight by some species of ants.

Loss of penicillin tolerance by inactivating the carbon catabolite repression determinant CcpA in Streptococcus gordonii.

OBJECTIVES: Antibiotic tolerance is a phenomenon allowing bacteria to withstand drug-induced killing. Here, we studied a penicillin-tolerant mutant of Streptococcus gordonii (Tol1), which was shown to be deregulated in the expression of the arginine deiminase operon (arc). arc was not directly responsible for tolerance, but is controlled by the global regulator CcpA. Therefore, we sought whether CcpA might be implicated in tolerance. METHODS: The ccpA gene was characterized and subsequently inactivated by PCR ligation mutagenesis in both the susceptible wild-type (WT) and Tol1. The minimal inhibitory concentration and time-kill curves for the strains were determined and the outcome of penicillin treatment in experimental endocarditis assessed. RESULTS: ccpA sequence and expression were similar between the WT and Tol1 strains. In killing assays, the WT lost 3.5 +/- 0.6 and 5.3 +/- 0.6 log(10) cfu/mL and Tol1 lost 0.4 +/- 0.2 and 1.4 +/- 0.9 log(10) cfu/mL after 24 and 48 h of penicillin exposure, respectively. Deletion of ccpA almost totally restored Tol1 kill susceptibility (loss of 2.5 +/- 0.7 and 4.9 +/- 0.7 log(10) cfu/mL at the same endpoints). In experimental endocarditis, penicillin treatment induced a significant reduction in vegetation bacterial densities between Tol1 (4.1 log(10) cfu/g) and Tol1DeltaccpA (2.4 log(10) cfu/g). Restitution of ccpA re-established the tolerant phenotype both in vitro and in vivo. CONCLUSIONS: CcpA, a global regulator of the carbon catabolite repression system, is implicated in penicillin tolerance both in vitro and in vivo. This links antibiotic survival to bacterial sugar metabolism. However, since ccpA sequence and expression were similar between the WT and Tol1 strains, other factors are probably involved in tolerance.

Loss of insulin synthesis and beta cell survival induced by oxidized LDL require activation of reticulum endoplasmic stress: a mechanism countered by HDL

Elevated circulating concentrations in modified LDL-cholesterol particles (e.g. oxidised LDL) and low levels in HDL increase not only the risk for diabetic patients to develop cardiovascular diseases but also may contribute to development and progression of diabetes by directly having adverse effects on β-cells. Chronic exposure of β-cells to 2 mM human oxidised LDL-cholesterol (oxLDL) increases the rate of apoptosis, reduce insulin biosynthesis and the secretory capacity of the cells in response to nutrients. In line with the protective role, HDL efficiently antagonised the harmful effects of ox- LDL, suggesting that low levels of HDL would be inefficient to protect β-cells against oxLDL attack in patients. Activation of endoplasmic reticulum (ER) stress is pointed out to contribute to β-cell dysfunction elicited by environmental stressors. In this study we investigated whether activation of ER stress is required for oxLDL to mediate detrimental effects on β-cells and we tested the potential antagonist properties of HDL: The mouse MIN6 insulin-secreting cells were cultured with 2 mM of LDL-cholesterol preparation (native or in vitro oxidized) in the presence or absence of 1 mM of HDL-cholesterol or the ER stress inhibitor 4-phenylbutyrate (4-PBA): Prolonged exposure of MIN6 cells to 2 mM oxLDL-cholesterol for 48 hours led to an increase in expression of ER stress markers such as ATF4, CHOP and p58 and stimulated the splicing of XBP-1 whereas, induction of these markers was not observable in the cells cultured with native LDL. Treatment of the cells with the 4-PBA chemical chaperone molecule efficiently blocked activation of the ER stress markers induced by oxLDL. The latter mediates β-cell dysfunction and apoptosis by diminishing the expression of islet brain 1 (IB1) and Bcl2. The levels of these two proteins were preserved in the cells that were co-treated with oxLDL and the 4-PBA. Consistent with this result we found that blockade of ER stress activation alleviated the loss of insulin synthesis and abolished apoptosis evoked by oxLDL. However incubation of the cells with 4-PBA did not prevent impairment of insulin secretion elicited by oxLDL, indicating that ER stress is not responsible for the oxLDL-mediated defect of insulin secretion. Co-incubation of the cells with HDL mimicked the effects of 4-PBA on the expression of IB1 and Blc2 and thereby counteracted oxLDL attacks on insulin synthesis and cell survivals. We found that HDL efficiently inhibited activation of the ER stress mediated by oxLDL: These data highlight the contribution of the ER stress in the defects of insulin synthesis and cell survivals induced by oxLDL and emphasize the potent role of HDL to counter activation of the oxLDL-mediated ER-stress activation:

Selective inhibition of JNK with a peptide inhibitor attenuates pain hypersensitivity and tumor growth in a mouse skin cancer pain model.

Cancer pain significantly affects the quality of cancer patients, and current treatments for this pain are limited. C-Jun N-terminal kinase (JNK) has been implicated in tumor growth and neuropathic pain sensitization. We investigated the role of JNK in cancer pain and tumor growth in a skin cancer pain model. Injection of luciferase-transfected B16-Fluc melanoma cells into a hindpaw of mouse induced robust tumor growth, as indicated by increase in paw volume and fluorescence intensity. Pain hypersensitivity in this model developed rapidly (<5 days) and reached a peak in 2 weeks, and was characterized by mechanical allodynia and heat hyperalgesia. Tumor growth was associated with JNK activation in tumor mass, dorsal root ganglion (DRG), and spinal cord and a peripheral neuropathy, such as loss of nerve fibers in the hindpaw skin and induction of ATF-3 expression in DRG neurons. Repeated systemic injections of D-JNKI-1 (6 mg/kg, i.p.), a selective and cell-permeable peptide inhibitor of JNK, produced an accumulative inhibition of mechanical allodynia and heat hyperalgesia. A bolus spinal injection of D-JNKI-1 also inhibited mechanical allodynia. Further, JNK inhibition suppressed tumor growth in vivo and melanoma cell proliferation in vitro. In contrast, repeated injections of morphine (5 mg/kg), a commonly used analgesic for terminal cancer, produced analgesic tolerance after 1 day and did not inhibit tumor growth. Our data reveal a marked peripheral neuropathy in this skin cancer model and important roles of the JNK pathway in cancer pain development and tumor growth. JNK inhibitors such as D-JNKI-1 may be used to treat cancer pain.

Chaperones and proteases: cellular fold-controlling factors of proteins in neurodegenerative diseases and aging.

The formation of toxic protein aggregates is a common denominator to many neurodegenerative diseases and aging. Accumulation of toxic, possibly infectious protein aggregates induces a cascade of events, such as excessive inflammation, the production of reactive oxygen species, apoptosis and neuronal loss. A network of highly conserved molecular chaperones and of chaperone-related proteases controls the fold-quality of proteins in the cell. Most molecular chaperones can passively prevent protein aggregation by binding misfolding intermediates. Some molecular chaperones and chaperone-related proteases, such as the proteasome, can also hydrolyse ATP to forcefully convert stable harmful protein aggregates into harmless natively refoldable, or protease-degradable, polypeptides. Molecular chaperones and chaperone-related proteases thus control the delicate balance between natively folded functional proteins and aggregation-prone misfolded proteins, which may form during the lifetime and lead to cell death. Abundant data now point at the molecular chaperones and the proteases as major clearance mechanisms to remove toxic protein aggregates from cells, delaying the onset and the outcome of protein-misfolding diseases. Therapeutic approaches include treatments and drugs that can specifically induce and sustain a strong chaperone and protease activity in cells and tissues prone to toxic protein aggregations.

Sequencing of Lp-PLA2-encoding PLA2G7 gene in 2000 Europeans reveals several rare loss-of-function mutations.

Elevated plasma levels of lipoprotein-associated phospholipase A(2) (Lp-PLA2) activity have been shown to be associated with increased risk of coronary heart disease and an inhibitor of this enzyme is under development for the treatment of that condition. A Val279Phe null allele in this gene, that may influence patient eligibility for treatment, is relatively common in East Asians but has not been observed in Europeans. We investigated the existence and functional effects of low frequency alleles in a Western European population by re-sequencing the exons of PLA2G7 in 2000 samples. In all, 19 non-synonymous single-nucleotide polymorphisms (nsSNPs) were found, 14 in fewer than four subjects (minor allele frequency <0.1%). Lp-PLA2 activity was significantly lower in rare nsSNP carriers compared with non-carriers (167.8±63.2 vs 204.6±41.8, P=0.01) and seven variants had enzyme activities consistent with a null allele. The cumulative frequency of these null alleles was 0.25%, so <1 in 10,000 Europeans would be expected to be homozygous, and thus not potentially benefit from treatment with an Lp-PLA2 inhibitor.