Megan A. Inglis

Production of the Lantibiotic Salivaricin A and Its Variants by Oral Streptococci and Use of a Specific Induction Assay To Detect Their Presence in Human Saliva

ABSTRACT Salivaricin A (SalA), the first Streptococcus salivarius lantibiotic to be characterized, appears to be inhibitory to most Streptococcus pyogenes strains. A variant of the SalA structural gene (salA1) is present in more than 90% of S. pyogenes strains, but only strains of M serotype 4 and T pattern 4 produce the biologically active peptide. The present study identifies four additional variants (salA2 to salA5) of the SalA structural gene and demonstrates that each of the corresponding inhibitory peptides (SalA2 to SalA5) is produced in vitro. These variants appear to be similar to SalA and SalA1 in their inhibitory activity against Micrococcus luteus and in their ability to act as inducers of SalA production. It had previously been shown that S. pyogenes strain SF370 had a deletion (of approximately 2.5 kb) in the salM and salT genes of the salA1 locus. In the present study, several additional characteristic deletions within the salA1 loci were identified. S. pyogenes strains of the same M serotype all share the same salA1 locus structure. Since S. salivarius is a predominant member of the normal oral flora of healthy humans, strains producing anti-S. pyogenes lantibiotics, such as SalA, may have excellent potential for use as oral probiotics. In the present study, we have used a highly specific SalA induction system to directly detect the presence of SalA in the saliva of humans who either naturally harbor populations of SalA-producing S. salivarius or who have been colonized with the SalA2-producing probiotic S. salivarius K12.

Neither Signal Transducer and Activator of Transcription 3 (STAT3) or STAT5 Signaling Pathways Are Required for Leptin's Effects on Fertility in Mice

The hormone leptin is critical for the regulation of energy balance and fertility. The long-form leptin receptor (LepR) regulates multiple intracellular signaling cascades, including the classic Janus kinase-signal transducer and activator of transcription (STAT) pathways. Previous studies have shown that deletion of STAT3 or the closely related STAT5 from the brain results in an obese phenotype, but their roles in fertility regulation are not clear. This study tested whether STAT3 and STAT5 pathways of leptin signaling are required for fertility, and whether absence of one pathway might be compensated for by the other in a redundant manner. A Cre-loxP approach was used to generate 3 models of male and female transgenic mice with LepR-specific deletion of STAT3, STAT5, or both STAT3 and STAT5. Body weight, puberty onset, estrous cyclicity, and fertility were measured in all knockout (KO) mice and their control littermates. Knocking out STAT3 or both STAT3 and 5 from LepR expressing cells, but not STAT5 alone, led to significant increase in body weight. All STAT3 and STAT5 single KO mice exhibited normal puberty onset and subsequent fertility compared to their control littermates. Surprisingly, all STAT3 and STAT5 double KO mice also exhibited normal puberty onset, estrous cyclicity, and fertility, although they had severely disrupted body weight regulation. These results suggest that, although STAT3 signaling is crucial for body weight regulation, neither STAT3 nor STAT5 is required for the regulation of fertility by leptin. It remains to be determined what other signaling molecules mediate this effect of leptin, and whether they interact in a redundant manner.

Evidence that hypothalamic RFamide related peptide-3 neurones are not leptin-responsive in mice and rats.

Leptin, a permissive hormonal regulator of fertility, provides information about the bodys energy reserves to the hypothalamic gonadotrophin‐releasing hormone (GnRH) neuronal system that drives reproduction. Leptin does not directly act on GnRH neurones, and the neuronal pathways that it uses remain unclear. RFamide‐related peptide‐3 (RFRP‐3) neurones project to GnRH neurones and primarily inhibit their activity. We tested whether leptin could act via RFRP‐3 neurones to potentially modulate GnRH activity. First, the effects of leptin deficiency or high‐fat diet‐induced obesity on RFRP‐3 cell numbers and gene expression were assessed in male and female mice. There was no significant difference in Rfrp mRNA levels or RFRP‐3‐immunoreactive cell counts in wild‐type versus leptin‐deficient ob/ob animals, or in low‐fat versus high‐fat diet fed wild‐type mice. Second, the presence of leptin‐induced signalling in RFRP‐3 neurones was examined in male and female wild‐type mice and rats. Dual label immunohistochemistry revealed leptin‐induced phosphorylated signal transducer and activator of transcription‐3 in close proximity to RFRP‐3 neurones, although there was very little (2–13%) colocalisation and no significant differences between vehicle and leptin‐treated animals. Furthermore, we were unable to detect leptin receptor mRNA in a semi‐purified RFRP‐3 cell preparation. Because GABA neurones form critical leptin‐responsive GnRH inputs, we also determined whether RFRP‐3 and GABA cells were colocalised. No such colocalisation was detected. These results support the concept that leptin has little or no effects on RFRP‐3 neurones, and that these neurones are unlikely to be an important neuronal pathway for the metabolic regulation of fertility by leptin.

Leptin Signaling in AgRP Neurons Modulates Puberty Onset and Adult Fertility in Mice

The hormone leptin indirectly communicates metabolic information to brain neurons that control reproduction, using GABAergic circuitry. Agouti-related peptide (AgRP) neurons in the arcuate nucleus are GABAergic, express leptin receptors (LepR), and are known to influence reproduction. This study tested whether leptin actions on AgRP neurons are required and sufficient for puberty onset and subsequent fertility. First, Agrp-Cre and Lepr-flox mice were used to target deletion of LepR to AgRP neurons. AgRP-LepR knock-out female mice exhibited mild obesity and adiposity as described previously, as well as a significant delay in the pubertal onset of estrous cycles compared with control animals. No significant differences in male puberty onset or adult fecundity in either sex were observed. Next, mice with a floxed polyadenylation signal causing premature transcriptional termination of the Lepr gene were crossed with AgRP-Cre mice to generate mice with AgRP neuron-specific rescue of LepR. Lepr-null control males and females were morbidly obese and exhibited delayed puberty onset, no evidence of estrous cycles, and minimal fecundity. Remarkably, AgRP-LepR rescue partially or fully restored all of these reproductive attributes to levels similar to those of LepR-intact controls despite minimal rescue of metabolic function. These results indicate that leptin signaling in AgRP neurons is sufficient for puberty onset and normal adult fecundity in both sexes when leptin signaling is absent in all other cells and that in females, the absence of AgRP neuron leptin signaling delays puberty. These actions appear to be independent of leptins metabolic effects. SIGNIFICANCE STATEMENT Sexual maturation and fertility are dispensable at the individual level but critical for species survival. Conditions such as nutritional imbalance may therefore suppress puberty onset and fertility in an individual. In societies characterized by widespread obesity, the sensitivity of reproduction to metabolic imbalance has significant public health implications. Deficient leptin signaling attributable to diet-induced leptin resistance is associated with infertility in humans and rodents, and treatments for human infertility show a decreased success rate with increasing body mass index. Here we show that the transmission of metabolic information to the hypothalamo-pituitary-gonadal axis is mediated by leptin receptors on AgRP neurons. These results provide conclusive new insights into the mechanisms that cause infertility attributable to malnourishment.

Salivaricin G32, a Homolog of the Prototype Streptococcus pyogenes Nisin-Like Lantibiotic SA-FF22, Produced by the Commensal Species Streptococcus salivarius

Salivaricin G32, a 2667 Da novel member of the SA-FF22 cluster of lantibiotics, has been purified and characterized from Streptococcus salivarius strain G32. The inhibitory peptide differs from the Streptococcus pyogenes—produced SA-FF22 in the absence of lysine in position 2. The salivaricin G32 locus was widely distributed in BLIS-producing S. salivarius, with 6 (23%) of 26 strains PCR-positive for the structural gene, slnA. As for most other lantibiotics produced by S. salivarius, the salivaricin G32 locus can be megaplasmid encoded. Another member of the SA-FF22 family was detected in two Streptococcus dysgalactiae of bovine origin, an observation supportive of widespread distribution of this lantibiotic within the genus Streptococcus. Since the inhibitory spectrum of salivaricin G32 includes Streptococcus pyogenes, its production by S. salivarius, either as a member of the normal oral microflora or as a commercial probiotic, could serve to enhance protection of the human host against S. pyogenes infection.

Deletion of Suppressor of Cytokine Signaling 3 from Forebrain Neurons Delays Infertility and Onset of Hypothalamic Leptin Resistance in Response to a High Caloric Diet.

The cellular processes that cause high caloric diet (HCD)-induced infertility are poorly understood but may involve upregulation of suppressor of cytokine signaling (SOCS-3) proteins that are associated with hypothalamic leptin resistance. Deletion of SOCS-3 from brain cells is known to protect mice from diet-induced obesity, but the effects on HCD-induced infertility are unknown. We used neuron-specific SOCS3 knock-out mice to elucidate this and the effects on regional hypothalamic leptin resistance. As expected, male and female neuron-specific SOCS3 knock-out mice were protected from HCD-induced obesity. While female wild-type mice became infertile after 4 months of HCD feeding, infertility onset in knock-out females was delayed by 4 weeks. Similarly, knock-out mice had delayed leptin resistance development in the medial preoptic area and anteroventral periventricular nucleus, regions important for generation of the surge of GnRH and LH that induces ovulation. We therefore tested whether the suppressive effects of HCD on the estradiol-induced GnRH/LH surge were overcome by neuron-specific SOCS3 knock-out. Although only 20% of control HCD-mice experienced a preovulatory-like LH surge, LH surges could be induced in almost all neuron-specific SOCS3 knock-out mice on this diet. In contrast to females, HCD-fed male mice did not exhibit any fertility decline compared with low caloric diet-fed males despite their resistance to the satiety effects of leptin. These data show that deletion of SOCS3 delays the onset of leptin resistance and infertility in HCD-fed female mice, but given continued HCD feeding this state does eventually occur, presumably in response to other mechanisms inhibiting leptin signal transduction. SIGNIFICANCE STATEMENT Obesity is commonly associated with infertility in humans and other animals. Treatments for human infertility show a decreased success rate with increasing body mass index. A hallmark of obesity is an increase in circulating leptin levels; despite this, the brain responds as if there were low levels of leptin, leading to increased appetite and suppressed fertility. Here we show that leptin resistant infertility is caused in part by the leptin signaling molecule SOCS3. Deletion of SOCS3 from brain neurons delays the onset of diet-induced infertility.

Central RFRP-3 Stimulates LH Secretion in Male Mice and Has Cycle Stage–Dependent Inhibitory Effects in Females

&NA; RFamide‐related peptide (RFRP)‐3 is a neuropeptide thought to play an inhibitory role in the regulation of reproduction in various mammalian species, although some stimulatory effects have been reported. To date, the effects of RFRP‐3 on gonadotropin secretion have been scarcely studied in mice. The aim of the current study was to characterize the effect of RFRP‐3 administration on gonadotropin secretion in male and female mice. Adult intact and castrated male mice received acute central injections of 0.5 to 5 nmol of RFRP‐3, and luteinizing hormone (LH) concentration was assayed in tail‐tip blood samples. RFRP‐3 had a dose‐dependent stimulatory effect on LH secretion when administered centrally to both intact and castrated mice, and this effect was diminished when RFRP‐3 was administered to kisspeptin receptor knockout mice. In female mice, central RFRP‐3 had an inhibitory effect on LH secretion when administered at the time of the preovulatory LH surge in intact mice, or of an estradiol‐induced LH surge in ovariectomized mice. Conversely, central RFRP‐3 administration had no effect on LH levels in intact diestrus or ovariectomized, low‐dose estradiol‐implanted mice. Finally, peripheral administration of RFRP‐3 to intact males and to females at the time of the preovulatory LH surge or during diestrus had no effect on LH secretion. Taken together, these results provide a detailed description of sex‐ and cycle stage‐dependent effects of RFRP‐3 on gonadotrophin secretion in mice. Moreover, it appears that the stimulatory effects are mediated in part by the receptor for kisspeptin, a potent stimulator of the reproductive axis.

Leptin and insulin do not exert redundant control of metabolic or emotive function via dopamine neurons

Abstract Leptin and insulins hunger‐suppressing and activity‐promoting actions on hypothalamic neurons are well characterized, yet the mechanisms by which they modulate the midbrain dopamine system to influence energy balance remain less clear. A subset of midbrain dopamine neurons express receptors for leptin (Lepr) and insulin (Insr). Leptin‐dopamine signaling reduces running reward and homecage activity. However, dopamine‐specific deletion of Lepr does not affect body weight or food intake in mice. We hypothesized insulin‐dopamine signaling might compensate for disrupted leptin‐dopamine signaling. To investigate the degree to which insulin and leptin exert overlapping (i.e. redundant) versus discrete control over dopamine neurons, we generated transgenic male and female mice exhibiting dopamine‐specific deletion of either Lepr (Lepr KO), Insr (Insr KO) or both Lepr and Insr (Dbl KO) and assessed their feeding behavior, voluntary activity, and energy expenditure compared to control mice. No differences in body weight, daily food intake, energy expenditure or hyperphagic feeding of palatable chow were observed between Lepr, Insr or Dbl KO mice and control mice. However, consistent with previous findings, Lepr KO (but not Insr or Dbl KO) male mice exhibited significantly increased running wheel activity compared to controls. These data demonstrate that insulin and leptin do not exert redundant control of dopamine neuron‐mediated modulation of energy balance. Furthermore, our results indicate neither leptin nor insulin plays a critical role in the modulation of dopamine neurons regarding hedonic feeding behavior or anxiety‐related behavior. HighlightsLeptin and insulin do not exert redundant control over dopamine neurons.Direct leptin‐dopamine signaling suppresses voluntary running behavior in mice.Leptin signaling via dopamine neurons is not required for normal feeding behavior.Dopamine‐specific insulin receptor knockout mice do not exhibit a phenotype.