The MprF homolog LysX synthesizes lysyl-diacylglycerol contributing to antibiotic resistance and virulence.

Lysyl-diacylglycerol (Lys-DAG) was identified three decades ago in Mycobacterium phlei, but the biosynthetic pathway and function of this aminoacylated lipid have since remained uncharacterized. Combining genetic methods, mass spectrometry, and biochemical approaches, we show that the multiple peptide resistance factor (MprF) homolog LysX from Corynebacterium pseudotuberculosis and two mycobacterial species is responsible for Lys-DAG synthesis. LysX is conserved in most Actinobacteria and was previously implicated in the synthesis of another modified lipid, lysyl-phosphatidylglycerol (Lys-PG), in Mycobacterium tuberculosis. Although we detected low levels of Lys-PG in the membrane of C. pseudotuberculosis, our data suggest that Lys-PG is not directly synthesized by LysX and may require an additional downstream pathway, which is as yet undefined. Our results show that LysX in C. pseudotuberculosis is a major factor of resistance against a variety of positively charged antibacterial agents, including cationic antimicrobial peptides (e.g., human peptide LL-37 and polymyxin B) and aminoglycosides (e.g., gentamycin and apramycin). Deletion of lysX caused an increase in cellular membrane permeability without dissipation of the membrane potential, suggesting that loss of the protein does not result in mechanical damage to the cell membrane. Furthermore, lysX-deficient cells exhibited an attenuated virulence phenotype in a Galleria mellonella infection model, supporting a role for LysX during infection. Altogether, Lys-DAG represents a novel molecular determinant for antimicrobial resistance and virulence that may be widespread in Actinobacteria and points to a richer landscape than previously realized of lipid components contributing to overall membrane physiology in this important bacterial phylum. IMPORTANCE In the past two decades, tRNA-dependent modification of membrane phosphatidylglycerol has been implicated in altering the biochemical properties of the cell surface, thereby enhancing the antimicrobial resistance and virulence of various bacterial pathogens. Here, we show that in several Actinobacteria, the multifunctional protein LysX attaches lysine to diacylglycerol instead of phosphatidylglycerol. We found that lysyl-diacylglycerol (Lys-DAG) confers high levels of resistance against various cationic antimicrobial peptides and aminoglycosides and also enhances virulence. Our data show that Lys-DAG is a lipid commonly found in important actinobacterial pathogens, including Mycobacterium and Corynebacterium species.

Provider perspectives on nutrition interventions in primary care: the role of organizational structure and community partnerships.

BACKGROUND: Poor nutrition remains a significant public health concern that is often managed within primary care settings. Meanwhile, to our knowledge, there have been few studies that evaluate the intent of primary care providers to offer nutrition services, nor what type of exchanges they engage in to ensure those programs can be implemented. METHODS: Semi-structured interviews were conducted with 16 primary care providers and support staff. Grounded theory analysis was utilized to identify themes and to develop a theoretical model of primary care nutrition program implementation. RESULTS: Three themes were identified. Patients approached primary care organizations with complex health beliefs, health severity, and barriers to care (theme 1). Providers and support staff responded by providing services that fit into existing organizational constraints, especially constraints related to workflow/time with patient, space and billing (theme 2). Providers see community as a major cue to action among patients but are unsure of the role of primary care (theme 3). CONCLUSIONS: Provider respondents found that implementing nutrition programs in primary care settings is difficult and that effective interventions for nutrition within health settings are limited without community-based partnerships and programming. Additional research is needed to measure existing community ties and how such ties could improve patient nutrition.

Contribution of the cyclic nucleotide gated channel subunit, CNG-3, to olfactory plasticity in Caenorhabditis elegans.

In Caenorhabditis elegans, the AWC neurons are thought to deploy a cGMP signaling cascade in the detection of and response to AWC sensed odors. Prolonged exposure to an AWC sensed odor in the absence of food leads to reversible decreases in the animal's attraction to that odor. This adaptation exhibits two stages referred to as short-term and long-term adaptation. Previously, the protein kinase G (PKG), EGL-4/PKG-1, was shown necessary for both stages of adaptation and phosphorylation of its target, the beta-type cyclic nucleotide gated (CNG) channel subunit, TAX-2, was implicated in the short term stage. Here we uncover a novel role for the CNG channel subunit, CNG-3, in short term adaptation. We demonstrate that CNG-3 is required in the AWC for adaptation to short (thirty minute) exposures of odor, and contains a candidate PKG phosphorylation site required to tune odor sensitivity. We also provide in vivo data suggesting that CNG-3 forms a complex with both TAX-2 and TAX-4 CNG channel subunits in AWC. Finally, we examine the physiology of different CNG channel subunit combinations.