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OBJECTIVES: Vitamin K (VK) is a family of structurally-related quinones, phylloquinone (PK) and menaquinones (MKn, n = prenyl units in side chain), that share a common napthoquinone ring (menadione, MD). VK quinones function as an essential dietary nutrient for humans. MD is considered a pro-vitamin form of VK. Plants and bacteria that produce VK quinones (PK and MKn, respectively) use them as an electron carrier in energy production. Little is known about the interaction of dietary VK quinones with gut bacteria, which may be bi-directional. The objective of this study was to investigate the influence of VK quinones and MD on human gut bacteria composition and MKn production. METHODS: Stool from 5 healthy male donors was pooled and inoculated in bioreactors under conditions mimicking the colon (anaerobic, pH 6.8, 37°C) for 48 h. Bioreactors were treated with deuterium ((2)H)-labeled quinones ((2)H-PK, (2)H-MK4, (2)H-MK9 or (2)H-MD); no quinones (cell controls); or (2)H-quinone treatment with no stool (cell-free controls). Culture aliquots were collected at 0, 5, 10, 24, and 48 h, and separated into pellet and supernatant fractions. Experiments were conducted in triplicate. All fractions were analyzed for VK quinone content using LC-MS. DNA from 0 and 24 h pellet fractions was extracted and amplified for paired-end 16S sequencing on an Illumina MiSeq 2500. Differences in bacterial composition were assessed using PERMANOVA. RESULTS: Supplemented (2)H-quinones accumulated in the pellet fraction over time. This was not observed in cell-free controls and was thus not a function of culture media solubility. Endogenous (unlabeled) production of MKn was unaffected by supplementation of (2)H-quinones. Generated (2)H-MKn ((2)H-MK4, (2)H-MK9, (2)H-MK10, and (2)H-MK11) were only detected in (2)H-MD supplemented vessels. Community-wide bacterial composition significantly differed between 0 h and 24 h (r(2 )= 0.85, P = 0.001), but not by quinone treatment. CONCLUSIONS: PK and MKn, dietary viamin K quinones, were not transformed by gut microbes to MKn in vitro, whereas the pro-vitamin quinone MD was transformed to MKn of multiple side chain lengths. Although no quinone induced community-wide changes in bacteria composition, additional analyses are needed to assess species-specific growth promotion. FUNDING SOURCES: USDA ARS and DOD Health Program.

Interactions between gut microbes and dietary components modulate intestinal permeability (IP) and inflammation. Recent studies have reported altered fecal microbiota composition together with increased IP and inflammation in individuals consuming military food rations in austere environments, but could not isolate effects of the diet from environmental factors. To determine how the U.S. Meal, Ready-to-Eat food ration affects fecal microbiota composition, IP and inflammation, 60 adults (95% male,18-61 years) were randomized to consume their usual ad libitum diet for 31 days (CON) or a strictly controlled Meal, Ready-to-Eat-only diet for 21 days followed by their usual diet for 10 days (MRE). In both groups, fecal microbiota composition was measured before, during (INT, days 1-21) and after the intervention period. IP and inflammation [high-sensitivity C-reactive protein (hsCRP)] were measured on days 0, 10, 21 and 31. Longitudinal changes in fecal microbiota composition differed between groups (P=.005), and fecal samples collected from MRE during INT were identified with 88% accuracy using random forest models. The genera making the strongest contribution to that prediction accuracy included multiple lactic acid bacteria (Lactobacillus, Lactococcus, Leuconostoc), which demonstrated lower relative abundance in MRE, and several genera known to dominate the ileal microbiota (Streptococcus, Veillonella, Clostridium), the latter two demonstrating higher relative abundance in MRE. IP and hsCRP were both lower (34% and 41%, respectively) in MRE relative to CON on day 21 (P

The Tri-Service Microbiome Consortium (TSMC) was recently established to enhance collaboration, coordination, and communication of microbiome research among Department of Defense (DoD) organizations. The TSMC aims to serve as a forum for sharing information related to DoD microbiome research, policy, and applications, to monitor global advances relevant to human health and performance, to identify priority objectives, and to facilitate Tri-Service (Army, Navy, and Air Force) collaborative research., The Tri-Service Microbiome Consortium (TSMC) was recently established to enhance collaboration, coordination, and communication of microbiome research among Department of Defense (DoD) organizations. The TSMC aims to serve as a forum for sharing information related to DoD microbiome research, policy, and applications, to monitor global advances relevant to human health and performance, to identify priority objectives, and to facilitate Tri-Service (Army, Navy, and Air Force) collaborative research. The inaugural TSMC workshop held on 10 to 11 May 2017 brought together almost 100 attendees from across the DoD and several key DoD partners. The meeting outcomes informed attendees of the scope of current DoD microbiome research efforts and identified knowledge gaps, collaborative/leveraging opportunities, research barriers/challenges, and future directions. This report details meeting presentations and discussions with special emphasis on Tri-Service labs’ current research activities.

A naturally occurring antimicrobial peptide, SMAP-29, was synthesized with an n-terminal or c-terminal cysteine, termed c_SMAP and SMAP_c, respectively, for site-directed immobilization to superparamagnetic beads. Immobilized SMAP orientation-dependent activity was probed against multiple bacteria of clinical interest including Acinetobacter baumannii, Pseudomonas aeruginosa, Bacillus anthracis sterne and Staphylococcus aureus. A kinetic microplate assay was employed to reveal both concentration and time-dependent activity for elucidation of minimum bactericidal concentration (MBC) and sub-lethal effects. Immobilized SMAP activity was equivalent or reduced compared with soluble SMAP_c and c_SMAP regardless of immobilization orientation, with only one exception. A comparison of immobilized SMAP_c and c_SMAP activity revealed a bacteria-specific potency dependent on immobilization orientation, which was contrary to that seen in solution, wherein SMAP_c was more potent against all bacteria than c_SMAP. Sub-MBC kinetic studies displayed the influence of peptide exposure to the cells with multiple bacteria exhibiting increased susceptibility and efficacy at lower concentrations upon extended exposure (i.e. MBC enhancement). For instances in which complete killing was not achieved, two predominant effects were evident: retardation of growth rate and an increased lag phase. Both effects, seen independently and concomitantly, indicate some degree of induced cellular damage that can serve as a predictor toward eventual cell death. SMAP_c immobilized on glass through standard silanization chemistry was also investigated to ascertain the influence of substrate on activity against select bacteria.

The interaction of cecropin P1 (CP1) with Escherichiacoli was investigated to gain insight into the time-dependent antimicrobial action. Biophysical characterizations of CP1 with whole bacterial cells were performed using both fluorescent and colorimetric assays to investigate the role of membrane permeability and lipopolysaccharide (LPS) binding in lytic behavior. The kinetics of CP1 growth inhibition assays indicated a minimal inhibitory concentration (MIC) of 3 microM. Bactericidal kinetics at the MIC indicated rapid killing of E.coli (

The studies presented here explore antimicrobial peptide preferential binding behavior for a target pathogen, Escherichia coli O157:H7. A modified immunoassay and surface plasmon resonance were employed to evaluate immobilized peptide binding of whole bacterial cells. The knowledge gained may guide the rational design of peptides with enhanced species binding selectivity.

This report documents research conducted by Natick Soldier Research, Development and Engineering Center (NSRDEC),between 2002 and 2010, to discover, design, and assess the differential bacterial binding ability of short antimicrobial peptides (AMPs) as part of an effort to provide rapid, accurate, and highly sensitive detection of bacterial contamination in Soldier wounds, food, and water sources. This detection capability is pivotal to maximize Warfighter survivability and quality of life. Current biosensor platforms incorporate recognition elements, such as antibodies, that are highly selective but have limited stability and sensitivity. The current drawbacks of the biosensor have limited its usefulness for rapid, real-time detection in an operational environment. Toward development of more robust and sensitive recognition elements, a series of truncated AMPs (7-15 amino acid residues) was designed using three methods: 1) fragmentation of naturally-occurring AMP sequences, 2) scanning alanine mutagenesis (applied to three fragments), wherein each residue in the sequence is systematically replaced with alanine to produce a set of well-defined mutations, and 3) sequence generation via a Markov chain algorithm utilizing a database of naturally-occurring AMPs. The fragmentation approach yielded multiple sequences possessing binding to all three organisms; additionally, several fragments exhibited selectivity for E. coli O157:H7 relative to S. aureus. Several of the fragments screened also displayed discriminatory binding to pathogenic E. coli O157:H7 relative to non-pathogenic E. coli ML35. The three fragments that were further engineered via scanning alanine mutagenesis had elevated binding responses and differential binding characteristics. Sequence generation via Markov chain also yielded peptides capable of selectivity between E. coli and S. aureus.

Antimicrobial peptide immobilization onto surfaces is of great interest, although characterization of activity can be problematic. The kinetic microplate method described here determines the minimum bactericidal concentration (MBC) of immobilized antimicrobial peptides through a combination and modification of traditional solution assays, overcoming the difficulties of working with a solid substrate. The technique enables rapid, accurate evaluation of immobilized peptide lytic behavior, elucidating both dose- and time-dependent activity at multiple concentrations. Furthermore, the method yields information regarding sublethal concentrations not realized in the traditional assays.

A procedure has been developed for the isolation of recombinant spider silk proteins based upon their unique stability and solubilization characteristics. Three recombinant silk proteins, (SpI)7, NcDS, and [(SpI)4/(SpII)1]4, were purified by extraction with organic acids followed by affinity or ion exchange chromatography resulting in 90-95% pure silk solutions. The protein yield of NcDS (15 mg/L culture) and (SpI)7 (35 mg/L) increased 4- and 5-fold, respectively, from previously reported values presumably due to a more complete solubilization of the expressed recombinant protein. [(SpI)4/(SpII)1]4, a hybrid protein based on the repeat sequences of spidroin I and spidroin II, had a yield of 12.4 mg/L. This method is an effective, reproducible technique that has broad applicability for a variety of silk proteins as well as other acid stable biopolymers.

Antimicrobial peptides (AMPs) have been discovered in insects, mammals, reptiles, and plants to protect against microbial infection. Many of these peptides have been isolated and studied exhaustively to decipher the molecular mechanisms that impart protection against infectious bacteria, fungi, and viruses. Unfortunately, the molecular mechanisms are still being debated within the scientific community but valuable clues have been obtained through structure/function relationship studies1. Biophysical studies have revealed that cecropins, isolated from insects and pigs, exhibit random structure in solution but undergo a conformational change to an amphipathic α-helix upon interaction with a membrane surface2. The lack of secondary structure in solution results in an extremely durable peptide able to survive exposure to high temperatures, organic solvents and incorporation into fibers and films without compromising antibacterial activity. Studies to better understand the antimicrobial action of cecropins and other AMPs have provided insight into the importance of peptide sequence and structure in antimicrobial activities. Therefore, enhancing our knowledge of how peptide structure imparts function may result in customized peptide sequences tailored for specific applications such as targeted cell delivery systems, novel antibiotics and food preservation additives. This review will summarize the current state of knowledge with respect to cell binding and antimicrobial activity of AMPs focusing primarily upon cecropins.