The potential interplay between the glandular microbiome and scent marking behavior in owl monkeys (Aotus nancymaae).

In mammals, scent marking behavior is a pervasive form of chemical communication that regulates social interactions within and between groups. Glandular microbiota consist of bacterial communities capable of producing chemical cues used in olfactory communication. Despite countless studies on scent marking in primates, few have examined the microbiota associated with glandular secretions. Nancy Ma's owl monkeys (Aotus nancymaae) are nocturnal, socially monogamous primates that frequently scent mark using their subcaudal glands. Previous analyses revealed that unique chemical signatures of Aotus may convey information about sex and age. We used positive reinforcement to sample the subcaudal glands of 23 captive owl monkeys to describe their glandular microbiomes and examine how patterns in these bacterial communities vary with age, sex, rearing environment and/or social group (pair identity). We coupled these analyses with behavioral observations to examine patterns in their scent marking behavior. We isolated 31 bacterial species from Phyla Firmicutes, Proteobacteria, and Actinobacteria, consistent with the dermal and glandular microbiomes of other primates. Several bacterial taxa we identified produce volatile organic compounds, which may contribute to olfactory communication. These bacterial communities are best predicted by an interaction between sex, rearing environment and pair identity rather than any of these variables alone. Within mated pairs of A. nancymaae, males and females scent mark their nest boxes at similar frequencies. In some pairs, rates of scent marking by males and females fluctuated over time in a similar manner. Pairs that had been together longer tended to exhibit the greatest similarities in their rates of scent marking. Together, these findings suggest that scent marking behavior and close social interactions with pair mates in Aotus may influence bacterial transmission and their glandular microbiomes. Chemical communication, including coordinated scent marking, may play a role in strengthening pair bonds, signaling pair status and/or in mate guarding in this socially monogamous primate.

Candida albicans Czf1 and Efg1 coordinate the response to farnesol during quorum sensing, white-opaque thermal dimorphism, and cell death.

Quorum sensing by farnesol in Candida albicans inhibits filamentation and may be directly related to its ability to cause both mucosal and systemic diseases. The Ras1-cyclic AMP signaling pathway is a target for farnesol inhibition. However, a clear understanding of the downstream effectors of the morphological farnesol response has yet to be unraveled. To address this issue, we screened a library for mutants that fail to respond to farnesol. Six mutants were identified, and the czf1Δ/czf1Δ mutant was selected for further characterization. Czf1 is a transcription factor that regulates filamentation in embedded agar and also white-to-opaque switching. We found that Czf1 is required for filament inhibition by farnesol under at least three distinct environmental conditions: on agar surfaces, in liquid medium, and when embedded in a semisolid agar matrix. Since Efg1 is a transcription factor of the Ras1-cyclic AMP signaling pathway that interacts with and regulates Czf1, an efg1Δ/efg1Δ czf1Δ/czf1Δ mutant was tested for filament inhibition by farnesol. It exhibited an opaque-cell-like temperature-dependent morphology, and it was killed by low farnesol levels that are sublethal to wild-type cells and both efg1Δ/efg1Δ and czf1Δ/czf1Δ single mutants. These results highlight a new role for Czf1 as a downstream effector of the morphological response to farnesol, and along with Efg1, Czf1 is involved in the control of farnesol-mediated cell death in C. albicans.

Helicobacter pylori arginase mutant colonizes arginase II knockout mice.

AIM: To investigate the role of host and bacterial arginases in the colonization of mice by Helicobacter pylori (H. pylori). METHODS: H. pylori produces a very powerful urease that hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Urease is absolutely essential to H. pylori pathogenesis; therefore, the urea substrate must be in ample supply for urease to work efficiently. The urea substrate is most likely provided by arginase activity, which hydrolyzes L-arginine to L-ornithine and urea. Previous work has demonstrated that H. pylori arginase is surprisingly not required for colonization of wild-type mice. Hence, another in vivo source of the critical urea substrate must exist. We hypothesized that the urea source was provided by host arginase II, since this enzyme is expressed in the stomach, and H. pylori has previously been shown to induce the expression of murine gastric arginase II. To test this hypothesis, wild-type and arginase (rocF) mutant H. pylori strain SS1 were inoculated into arginase II knockout mice. RESULTS: Surprisingly, both the wild-type and rocF mutant bacteria still colonized arginase II knockout mice. Moreover, feeding arginase II knockout mice the host arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC), while inhibiting > 50% of the host arginase I activity in several tissues, did not block the ability of the rocF mutant H. pylori to colonize. In contrast, BEC poorly inhibited H. pylori arginase activity. CONCLUSION: The in vivo source for the essential urea utilized by H. pylori urease is neither bacterial arginase nor host arginase II; instead, either residual host arginase I or agmatinase is probably responsible.

Activity and toxicity of farnesol towards Candida albicans are dependent on growth conditions.

Farnesol interacts with Candida albicans as both a quorum-sensing molecule and toxic agent, but confusion abounds regarding which conditions promote these distinct responses. Farnesol sensitivity was measured when inoculum cell history and size, temperature, and growth media were altered. Parameters for farnesol tolerance/sensitivity were defined, validating previous studies and identifying new variables, such as energy availability. This study clearly defines what farnesol concentrations are lethal to C. albicans, based on environmental conditions.

Cellular interactions of farnesol, a quorum-sensing molecule produced by Candida albicans.

Farnesol is a quorum-sensing molecule produced by Candida albicans that has many effects, including filament inhibition of this polymorphic fungus. In the past 9 years, the effect of farnesol on C. albicans has been reported in nearly 160 publications, with early work examining its influence on morphology. This article presents an update on the literature published since 2006, focusing on points that still need to be resolved as well as identifying possible artifacts that might interfere with this goal. In addition, the regulation of C. albicans farnesol production, C. albicans' resistance/sensitivity to farnesol and the influence of farnesol on other species as well as the host are discussed. It is intriguing that we still do not know precisely how farnesol works, but interference with the Ras1-cAMP pathway is part of the story.

Candida albicans Tup1 is involved in farnesol-mediated inhibition of filamentous-growth induction.

Candida albicans is a dimorphic fungus that can interconvert between yeast and filamentous forms. Its ability to regulate morphogenesis is strongly correlated with virulence. Tup1, a transcriptional repressor, and the signaling molecule farnesol are both capable of negatively regulating the yeast to filamentous conversion. Based on this overlap in function, we tested the hypothesis that the cellular response to farnesol involves, in part, the activation of Tup1. Tup1 functions with the DNA binding proteins Nrg1 and Rfg1 as a transcription regulator to repress the expression of hypha-specific genes. The tup1/tup1 and nrg1/nrg1 mutants, but not the rfg1/rfg1 mutant, failed to respond to farnesol. Treatment of C. albicans cells with farnesol caused a small but consistent increase in both TUP1 mRNA and protein levels. Importantly, this increase corresponds with the commitment point, beyond which added farnesol no longer blocks germ tube formation, and it correlates with a strong decrease in the expression of two Tup1-regulated hypha-specific genes, HWP1 and RBT1. Tup1 probably plays a direct role in the response to farnesol because farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 heterozygote. Farnesol did not affect EFG1 (a transcription regulator of filament development), NRG1, or RFG1 mRNA levels, demonstrating specific gene regulation in response to farnesol. Furthermore, the tup1/tup1 and nrg1/nrg1 mutants produced 17- and 19-fold more farnesol, respectively, than the parental strain. These levels of excess farnesol are sufficient to block filamentation in a wild-type strain. Our data are consistent with the role of Tup1 as a crucial component of the response to farnesol in C. albicans.

Genetic microheterogeneity and phenotypic variation of Helicobacter pylori arginase in clinical isolates.

BACKGROUND: Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid. RESULTS: The degree of variation in arginase was explored at the DNA sequence, enzyme activity and protein expression levels. To this end, arginase activity was measured from 73 minimally-passaged clinical isolates and six laboratory-adapted strains of H. pylori. The rocF gene from 21 of the strains was cloned into genetically stable E. coli and the enzyme activities measured. Arginase activity was found to substantially vary (>100-fold) in both different H. pylori strains and in the E. coli model. Western blot analysis revealed a positive correlation between activity and amount of protein expressed in most H. pylori strains. Several H. pylori strains featured altered arginase activity upon in vitro passage. Pairwise alignments of the 21 rocF genes plus strain J99 revealed extensive microheterogeneity in the promoter region and 3' end of the rocF coding region. Amino acid S232, which was I232 in the arginase-negative clinical strain A2, was critical for arginase activity. CONCLUSION: These studies demonstrated that H. pylori arginase exhibits extensive genotypic and phenotypic variation which may be used to understand mechanisms of microheterogeneity in H. pylori.

In vitro and in vivo complementation of the Helicobacter pylori arginase mutant using an intergenic chromosomal site.

BACKGROUND: Gene complementation strategies are important in validating the roles of genes in specific phenotypes. Complementation systems in Helicobacter pylori include shuttle vectors, which transform H. pylori at relatively low frequencies, and chromosomally based approaches. Chromosomal complementation strategies are susceptible to polar effects and disruption of other H. pylori genes, leading to unwanted pleiotropic effects. MATERIALS AND METHODS: A new complementation strategy was developed for H. pylori by utilizing a suicide plasmid vector that contains fragments of an H. pylori intergenic region (hp0203-hp0204), a chloramphenicol acetyltransferase cassette (cat), and a multiple-cloning site. Genes of interest could be cloned into the intergenic plasmid and the genes integrated into H. pylori by homologous recombination into the intergenic chromosomal region without disrupting any annotated H. pylori gene. The complementation system was validated using the gene encoding arginase (rocF). RESULTS: A rocF mutant unable to hydrolyze or consume l-arginine regained these functions by complementation with the wild-type rocF gene. Complemented strains also had restored arginase protein as determined by Western blot analysis. The complementation system could be successfully applied to multiple H. pylori strains. The intergenic region varied in length and sequence across 17 H. pylori strains, but the flanking-3' ends of the hp0203 and hp0204 coding regions were highly conserved. Inserting a cat cassette and wild-type rocF into the intergenic region did not alter the ability of strain SS1 to colonize mice. CONCLUSIONS: This complementation strategy should greatly facilitate genetic experiments in H. pylori.

Colonization and inflammation deficiencies in Mongolian gerbils infected by Helicobacter pylori chemotaxis mutants.

Helicobacter pylori causes disease in the human stomach and in mouse and gerbil stomach models. Previous results have shown that motility is critical for H. pylori to colonize mice, gerbils, and other animal models. The role of chemotaxis, however, in colonization and disease is less well understood. Two genes in the H. pylori chemotaxis pathway, cheY and tlpB, which encode the chemotaxis response regulator and a methyl-accepting chemoreceptor, respectively, were disrupted. The cheY mutation was complemented with a wild-type copy of cheY inserted into the chromosomal rdxA gene. The cheY mutant lost chemotaxis but retained motility, while all other strains were motile and chemotactic in vitro. These strains were inoculated into gerbils either alone or in combination with the wild-type strain, and colonization and inflammation were assessed. While the cheY mutant completely failed to colonize gerbil stomachs, the tlpB mutant colonized at levels similar to those of the wild type. With the tlpB mutant, there was a substantial decrease in inflammation in the gerbil stomach compared to that with the wild type. Furthermore, there were differences in the numbers of each immune cell in the tlpB-mutant-infected stomach: the ratio of lymphocytes to neutrophils was about 8 to 1 in the wild type but only about 1 to 1 in the mutant. These results suggest that the TlpB chemoreceptor plays an important role in the inflammatory response while the CheY chemotaxis regulator plays a critical role in initial colonization. Chemotaxis mutants may provide new insights into the steps involved in H. pylori pathogenesis.