Identification of Functional Spo0A Residues Critical for Sporulation in Clostridioides difficile.

Clostridioides difficile is an anaerobic, Gram-positive pathogen that is responsible for C. difficile infection (CDI). To survive in the environment and spread to new hosts, C. difficile must form metabolically dormant spores. The formation of spores requires activation of the transcription factor Spo0A, which is the master regulator of sporulation in all endospore-forming bacteria. Though the sporulation initiation pathway has been delineated in the Bacilli, including the model spore-former Bacillus subtilis, the direct regulators of Spo0A in C. difficile remain undefined. C. difficile Spo0A shares highly conserved protein interaction regions with the B. subtilis sporulation proteins Spo0F and Spo0A, although many of the interacting factors present in B. subtilis are not encoded in C. difficile. To determine if comparable Spo0A residues are important for C. difficile sporulation initiation, site-directed mutagenesis was performed at conserved receiver domain residues and the effects on sporulation were examined. Mutation of residues important for homodimerization and interaction with positive and negative regulators of B. subtilis Spo0A and Spo0F impacted C. difficile Spo0A function. The data also demonstrated that mutation of many additional conserved residues altered C. difficile Spo0A activity, even when the corresponding Bacillus interacting proteins are not apparent in the C. difficile genome. Finally, the conserved aspartate residue at position 56 of C. difficile Spo0A was determined to be the phosphorylation site that is necessary for Spo0A activation. The finding that Spo0A interacting motifs maintain functionality suggests that C. difficile Spo0A interacts with yet unidentified proteins that regulate its activity and control spore formation.

Targeted Delivery of DNA Topoisomerase Inhibitor SN38 to Intracranial Tumors of Glioblastoma Using Sub-5 Ultrafine Iron Oxide Nanoparticles.

Effectively delivering therapeutics for treating brain tumors is hindered by the physical and biological barriers in the brain. Even with the compromised blood-brain barrier and highly angiogenic blood-tumor barrier seen in glioblastoma (GBM), most drugs, including nanomaterial-based formulations, hardly reach intracranial tumors. This work investigates sub-5 nm ultrafine iron oxide nanoparticles (uIONP) with 3.5 nm core diameter as a carrier for delivering DNA topoisomerase inhibitor 7-ethyl-10-hydroxyl camptothecin (SN38) to treat GBM. Given a higher surface-to-volume ratio, uIONP shows one- or three-folds higher SN38 loading efficiency (48.3 ± 6.1%, mg/mg Fe) than those with core sizes of 10 or 20 nm. SN38 encapsulated in the coating polymer exhibits pH sensitive release with <10% over 48 h at pH 7.4, but 86% at pH 5, thus being protected from converting to inactive glucuronide by UDP-glucuronosyltransferase 1A1. Conjugating αv ß3 -integrin-targeted cyclo(Arg-Gly-Asp-D-Phe-Cys) (RGD) as ligands, RGD-uIONP/SN38 demonstrates targeted cytotoxicity to αv ß3 -integrin-overexpressed U87MG GBM cells with a half-maximal inhibitory concentration (IC50 ) of 30.9 ± 2.2 nm. The efficacy study using an orthotopic mouse model of GBM reveals tumor-specific delivery of 11.5% injected RGD-uIONP/SN38 (10 mg Fe kg-1 ), significantly prolonging the survival in mice by 41%, comparing to those treated with SN38 alone (p < 0.001).

Cationic Homopolymers Inhibit Spore and Vegetative Cell Growth of Clostridioides difficile.

A wide range of synthetic polymers have been explored for antimicrobial activity. These materials usually contain both cationic and hydrophobic subunits because these two characteristics are prominent among host-defense peptides. Here, we describe a series of nylon-3 polymers containing only cationic subunits and their evaluation against the gastrointestinal, spore-forming pathogen Clostridioides difficile. Despite their highly hydrophilic nature, these homopolymers showed efficacy against both the vegetative and spore forms of the bacterium, including an impact on C. difficile spore germination. The polymer designated P34 demonstrated the greatest efficacy against C. difficile strains, along with low propensities to lyse human red blood cells or intestinal epithelial cells. To gain insight into the mechanism of P34 action, we evaluated several cell-surface mutant strains of C. difficile to determine the impacts on growth, viability, and cell morphology. The results suggest that P34 interacts with the cell wall, resulting in severe cell bending and death in a concentration-dependent manner. The unexpected finding that nylon-3 polymers composed entirely of cationic subunits display significant activities toward C. difficile should expand the range of other polymers considered for antibacterial applications.

Phase variation of a signal transduction system controls Clostridioides difficile colony morphology, motility, and virulence.

Recent work has revealed that Clostridioides difficile, a major cause of nosocomial diarrheal disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase variation. Many C. difficile strains representing multiple ribotypes develop two colony morphotypes, termed rough and smooth, but the biological implications of this phenomenon have not been explored. Here, we examine the molecular basis and physiological relevance of the distinct colony morphotypes produced by this bacterium. We show that C. difficile reversibly differentiates into rough and smooth colony morphologies and that bacteria derived from the isolates display discrete motility behaviors. We identified an atypical phase-variable signal transduction system consisting of a histidine kinase and two response regulators, named herein colony morphology regulators RST (CmrRST), which mediates the switch in colony morphology and motility behaviors. The CmrRST-regulated surface motility is independent of flagella and type IV pili, suggesting a novel mechanism of cell migration in C. difficile. Microscopic analysis of cell and colony structure indicates that CmrRST promotes the formation of elongated bacteria arranged in bundled chains, which may contribute to bacterial migration on surfaces. In a hamster model of acute C. difficile disease, the CmrRST system is required for disease development. Furthermore, we provide evidence that CmrRST phase varies during infection, suggesting that the intestinal environment impacts the proportion of CmrRST-expressing C. difficile. Our findings indicate that C. difficile employs phase variation of the CmrRST signal transduction system to generate phenotypic heterogeneity during infection, with concomitant effects on bacterial physiology and pathogenesis.

The C. difficile clnRAB operon initiates adaptations to the host environment in response to LL-37.

To cause disease, Clostridioides (Clostridium) difficile must resist killing by innate immune effectors in the intestine, including the host antimicrobial peptide, cathelicidin (LL-37). The mechanisms that enable C. difficile to adapt to the intestine in the presence of antimicrobial peptides are unknown. Expression analyses revealed an operon, CD630_16170-CD630_16190 (clnRAB), which is highly induced by LL-37 and is not expressed in response to other cell-surface active antimicrobials. This operon encodes a predicted transcriptional regulator (ClnR) and an ABC transporter system (ClnAB), all of which are required for function. Analyses of a clnR mutant indicate that ClnR is a pleiotropic regulator that directly binds to LL-37 and controls expression of numerous genes, including many involved in metabolism, cellular transport, signaling, gene regulation, and pathogenesis. The data suggest that ClnRAB is a novel regulatory mechanism that senses LL-37 as a host signal and regulates gene expression to adapt to the host intestinal environment during infection.

Ethanolamine is a valuable nutrient source that impacts Clostridium difficile pathogenesis.

Clostridium (Clostridioides) difficile is a gastrointestinal pathogen that colonizes the intestinal tract of mammals and can cause severe diarrheal disease. Although C. difficile growth is confined to the intestinal tract, our understanding of the specific metabolites and host factors that are important for the growth of the bacterium is limited. In other enteric pathogens, the membrane-derived metabolite, ethanolamine (EA), is utilized as a nutrient source and can function as a signal to initiate the production of virulence factors. In this study, we investigated the effects of ethanolamine and the role of the predicted ethanolamine gene cluster (CD1907-CD1925) on C. difficile growth. Using targeted mutagenesis, we disrupted genes within the eut cluster and assessed their roles in ethanolamine utilization, and the impact of eut disruption on the outcome of infection in a hamster model of disease. Our results indicate that the eut gene cluster is required for the growth of C. difficile on ethanolamine as a primary nutrient source. Further, the inability to utilize ethanolamine resulted in greater virulence and a shorter time to morbidity in the animal model. Overall, these data suggest that ethanolamine is an important nutrient source within the host and that, in contrast to other intestinal pathogens, the metabolism of ethanolamine by C. difficile can delay the onset of disease.

The Effects of Increased Protein Intake on Fullness: A Meta-Analysis and Its Limitations.

BACKGROUND: Higher protein intake has been implicated in weight management because of its appetitive properties. However, the effects of protein intake on appetitive sensations such as fullness have not been systematically assessed. Meta-analysis is a useful technique to evaluate evidence of an intervention's effect on testable outcomes, but it also has important limitations. OBJECTIVE: The primary aim of this study was to synthesize the available evidence on the effect of protein intake on fullness using a quantitative meta-analysis and a secondary directional analysis using the vote-counting procedure. A tertiary aim was to address limitations of meta-analyses as they pertain to findings from this meta-analysis. DESIGN: We searched multiple databases for interventional studies that evaluated the effect of increased protein intake on fullness ratings. Inclusion criteria for both analyses were as follows: healthy human participants, preload studies that utilized intact dietary protein, delivery of protein load orally, and studies reporting fullness as an outcome. For the meta-analysis, an additional criterion was that the studies also needed to report 2- to 4-hour area under the curve value for fullness. RESULTS: Five studies met all criteria for the meta-analysis. Twenty-eight studies met all criteria for the directional analysis. The meta-analysis indicated higher protein preloads have a greater effect on fullness than lower protein preloads (overall effect estimate: 2,435.74 mm.240 min, (95% CI 1,375.18 to 3,496.31 mm.240 min; P<0.0001). The directional analysis also revealed a positive effect on fullness with higher protein preloads (P<0.01). Many related scientifically rigorous studies were excluded from the analysis because analytical criteria required a narrowly focused research question. CONCLUSIONS: The present analyses show that higher protein preloads increase fullness ratings more than lower protein preloads under tightly defined conditions. Extrapolation of findings to common conditions outside the specified criteria of this analysis must be made cautiously, as must speculation about the influence of fullness sensations on ingestive behavior, body weight, and various health outcomes.

Effects of daily consumption of one or varied peanut flavors on acceptance and intake.

To realize the health benefits associated with peanut consumption, it is important that they remain acceptable with regular intake. Peanuts are marketed with various flavorings so that consumers will not become fatigued by frequent consumption of any single flavor. This study sought to determine whether liking of peanuts or compliance to a peanut feeding intervention would differ based on receiving an individual flavor or a variety of flavors. Participants (n=151) were directed to consume 42 g peanuts/d for 12 weeks as 14-g servings of 3 different flavors (n=50) or a single flavor (n=25-26/group). The trial was randomized, with a parallel-group design. Neither the peanut flavor consumed nor the presence of variety had an impact on liking of the peanuts or compliance with the study protocol (p>0.05). Men had significantly greater compliance and liking ratings than women (p<0.05), but liking declines did not differ based on sex. Eating attitudes and dietary restraint did not correlate with liking or compliance. Peanuts were generally well-liked in the study, with a mean liking of 69 on a 100-mm visual analogue scale and a decrease of less than 15% over 12 weeks. Overall compliance to the protocol was 96.9%, suggesting participants were able to incorporate peanuts into their diet on a daily basis. These findings suggest a recommendation to regularly consume peanuts would be well-tolerated, facilitating their contribution to a healthy diet. The study was conducted between February 2010 and May 2012 at Purdue University, West Lafayette, IN, USA. This trial was registered at clinicaltrials.gov as NCT01886326.

Effects of learning and food form on energy intake and appetitive responses.

Energy-yielding beverages reportedly contribute to positive energy balance uniquely. They are highly consumed and evoke weaker satiety signaling and dietary energy compensation than solid foods of the same energy content. This study measured the contribution of learning to appetitive sensations and adjustments of energy intake for preloads varying in energy content and food form in lean and obese adults. One-hundred seven participants received four preload trials before and after a dietary intervention in this randomized cross-over trial with the stipulation that lean and obese individuals were evenly assigned to each intervention. The study entailed monitoring appetitive sensations and daily energy intake after consumption of low and high energy beverage and solid food loads on weekly visit days. Preload testing was conducted at baseline, followed by daily ingestion of one load for 14 days and then retesting responses to the four treatments. Lean individuals compensated precisely for the high energy beverage and solid loads from the onset of the study, whereas the obese did not alter eating patterns after consuming the higher energy beverage load. The learning intervention did not have an effect on the responses to the preloads, as responses in both lean and obese participants did not differ from baseline values. Responses to personality and eating behavior questionnaires revealed differences between the lean and obese groups and weakly, but significantly, predicted challenge meal and total daily energy intake. These data suggest that lean and obese individuals respond to energy in beverage form differently, and this is not altered by purposeful daily exposure to loads varying in physical form and energy content for two weeks.

A randomized trial on the effects of flavorings on the health benefits of daily peanut consumption.

BACKGROUND: Chronic peanut consumption is associated with health benefits. To encourage consumption, peanuts are marketed with various flavorings, but questions have been raised as to whether seasonings offset the benefits of peanuts alone. OBJECTIVE: This study sought to determine whether flavorings on peanuts affect health benefits over 12 wk. DESIGN: In a randomized, parallel-group trial, 151 participants received 42 g peanuts/d as 14-g servings of 3 different flavors (n = 50) or a single flavor (n = 25-26/group). Anthropometric indexes, blood pressure, and heart rate were measured biweekly. Cardiovascular disease risk factors (serum lipids, insulin, glucose, and cortisol) were assessed monthly. RESULTS: No single added flavor differentially altered body weight, body fat, body mass index, heart rate, or blood indexes in the total sample. Participants at greater risk of cardiovascular disease had significantly greater mean (± SE) reductions in diastolic blood pressure (-5.0 ± 1.7 mm Hg compared with -0.7 ± 0.6 mm Hg), cholesterol (-12.1 ± 8.5 mg/dL compared with +5.6 ± 2.0 mg/dL), and triglycerides (-31.7 ± 15.8 mg/dL compared with +2.3 ± 3.0 mg/dL) (n = 27, 24, and 15, respectively; P < 0.01) than did those at lower risk, who did not have significantly different cholesterol or triglyceride concentrations. Consumption of a variety of flavors led to greater weight gain (0.9 ± 0.3 kg) compared with individual flavors alone (0.5 ± 0.2 kg) (P < 0.05), but increases in fat and lean masses were not significant. CONCLUSIONS: Regardless of flavoring, peanut consumption offered significant benefits to participants with elevated serum lipids and blood pressure. Sensory variety led to a small, but significant, increase in body weight in comparison with ingestion of a single flavor but no change in fat mass.