Enhanced Thermostability of D-Psicose 3-Epimerase from Clostridium bolteae through Rational Design and Engineering of New Disulfide Bridges

D-psicose 3-epimerase (DPEase) catalyzes the isomerization of D-fructose to D-psicose (aka D-allulose, a low-calorie sweetener), but its industrial application has been restricted by the poor thermostability of the naturally available enzymes. Computational rational design of disulfide bridges was used to select potential sites in the protein structure of DPEase from Clostridium bolteae to engineer new disulfide bridges. Three mutants were engineered successfully with new disulfide bridges in different locations, increasing their optimum catalytic temperature from 55 to 65 °C, greatly improving their thermal stability and extending their half-lives (t1/2) at 55 °C from 0.37 h to 4–4.5 h, thereby greatly enhancing their potential for industrial application. Molecular dynamics simulation and spatial configuration analysis revealed that introduction of a disulfide bridge modified the protein hydrogen–bond network, rigidified both the local and overall structures of the mutants and decreased the entropy of unfolded protein, thereby enhancing the thermostability of DPEase.

Mega- and meta-analyses of fecal metagenomic studies in predicting response to immune checkpoint inhibitors.

2570 Background: A number of studies have demonstrated that the gut microbiome of responders to immune checkpoint inhibitors (ICI) is compositionally different compared to that of non-responders. However, differences in study design, patient cohorts and bioinformatic analyses make it challenging to identify bacterial species consistently associated with response to ICI across different cohorts and cancer types. Methods: We leveraged the statistical power of mega- and meta-analyses to identify bacterial species consistently associated with response to ICI using data from three published fecal metagenomic studies (Gopalakrishnan et al., Science 2018; Matson et al., Science 2018; Routy et al., Science 2018). Metagenomic data was uniformly processed and analyzed using Metaphlan v2.0. We conducted a two-part modelling approach of bacterial species present in at least 20% of samples to account for both prevalence and relative abundance differences between responders/non-responders. Results: A total of 190 patients (n = 103 responders; n = 87 non-responders) were included from the three studies. Data from Routy et al., was analyzed as subsets based on tumor type for a total of 4 analyzed cohorts. We identified five species including Bacteroides thetaiotaomicron, Clostridium bolteae, Holdemania filiformis, Clostridiaceae bacterium JC118 and Escherichia coli that were concordantly significantly different between responders and non-responders using both meta- and mega-analyses. B. thetaiotaomicron and Clostridium bolteae relative abundance (RA) were independently predictive of non-response to immunotherapy when data sets were combined and analyzed using mega-analyses (AUC 0.59 95% CI 0.51-0.68 and AUC 0.61 95% CI 0.52-0.69, respectively). Conclusions: Despite inter-cohort heterogeneity in tumor type, treatment regimens, and sequencing modalities, meta- and mega analysis of published metagenomic studies identified generalizable bacterial species associated with ICI response or lack thereof. B. thetaiotaomicron and C. bolteae were predictors of non-response to ICI suggesting the clinical potential of narrow spectrum anti-biotics targeting non-response associated bacterial species to improve outcomes in ICI recipients.

Mega- and meta-analyses of fecal metagenomic studies assessing response to immune checkpoint inhibitors

PurposeGut microbiota have been associated with response to immune checkpoint inhibitors (ICI) including anti-PD-1 and anti-CTLA-4 antibodies. However, inter-study difference in design, patient cohorts and data analysis pose challenges to identifying species consistently associated with response to ICI or lack thereof.Experimental DesignWe uniformly processed and analyzed data from three studies of microbial metagenomes in cancer immunotherapy response (four distinct data sets) to identify species consistently associated with response or non-response (n=190 patient samples). Metagenomic data were processed and analyzed using Metaphlan v2.0. Meta- and mega-analyses were performed using a two-part modelling approach of species present in at least 20% of samples to account for both prevalence and relative abundance differences between responders/non-responders.ResultsMeta- and mega-analyses identified five species that were concordantly significantly different between responders and non-responders. Amongst them, Bacteroides thetaiotaomicron and Clostridium bolteae relative abundance (RA) were independently predictive of non-response to immunotherapy when data sets were combined and analyzed using mega-analyses (AUC 0.59 95% CI 0.51-0.68 and AUC 0.61 95% CI 0.52-0.69, respectively).ConclusionsMeta- and mega-analysis of published metagenomic studies identified bacterial species both positively and negatively associated with immunotherapy responsiveness across four published cohorts.

Clostridium bolteae is elevated in neuromyelitis optica spectrum disorder in India and shares sequence similarity with AQP4

ObjectiveTo understand the role of gut microbiome in influencing the pathogenesis of neuromyelitis optica spectrum disorders (NMOSDs) among patients of south Indian origin.MethodsIn this case-control study, stool and blood samples were collected from 39 patients with NMOSD, including 17 with aquaporin 4 IgG antibodies (AQP4+) and 36 matched controls. 16S ribosomal RNA (rRNA) sequencing was used to investigate the gut microbiome. Peripheral CD4+ T cells were sorted in 12 healthy controls, and in 12 patients with AQP4+ NMOSD, RNA was extracted and immune gene expression was analyzed using the NanoString nCounter human immunology kit code set.ResultsMicrobiota community structure (beta diversity) differed between patients with AQP4+ NMOSD and healthy controls (p < 0.001, pairwise PERMANOVA test). Linear discriminatory analysis effect size identified several members of the microbiota that were altered in patients with NMOSD, including an increase in Clostridium bolteae (effect size 4.23, p 0.00007). C bolteae was significantly more prevalent (p = 0.02) among patients with AQP4-IgG+ NMOSD (n = 8/17 subjects) compared with seronegative patients (n = 3/22) and was absent among healthy stool samples. C bolteae has a highly conserved glycerol uptake facilitator and related aquaporin protein (p59-71) that shares sequence homology with AQP4 peptide (p92-104), positioned within an immunodominant (AQP4 specific) T-cell epitope (p91-110). Presence of C bolteae correlated with expression of inflammatory genes associated with both innate and adaptive immunities and particularly involved in plasma cell differentiation, B cell chemotaxis, and Th17 activation.ConclusionOur study described elevated levels of C bolteae associated with AQP4+ NMOSD among Indian patients. It is possible that this organism may be causally related to the immunopathogenesis of this disease in susceptible individuals.

The composition of the bacterial microbiome in the sputum of patients with lung cancer and the assessment of its effect on clastogenic effects in blood lymphocytes

Впервые дана оценка влияния качественных и количественных изменений состава бактериального микробиома в мокроте 17 пациентов, страдающих раком легкого, и 17 контрольных доноров на показатели цитогенетического гомеостаза соматических клеток. Доноры с высокими значениями (свыше 3,5 %) частот хромосомных аберраций (ХА) имели в микробиоме достоверное снижение представителей родов Prevotella, Selenomonas, Veillonella, а также увеличение представителей рода Kocuria по сравнению с донорами с низким уровнем ХА в лимфоцитах. На видовом уровне были обнаружены различия в содержании Anaerosinus glycerini, Selenomonas bovis, Actinomyces hyovaginalis, Granulicatella balaenopterae и Clostridium bolteae. При этом только последний вид был представлен чаще в мокроте доноров с высоким уровнем ХА, остальные 4 вида бактерий были достоверно чаще представлены в мокроте доноров с низким уровнем мутаций в лимфоцитах. The impact of qualitative and quantitative changes in the composition of the bacterial microbiome in the sputum of 17 patients with lung cancer and 17 control donors on the parameters of somatic cell cytogenetic homeostasis was first assessed. Donors with high frequencies of chromosomal aberrations (CA) over 3.5 % had a significant decrease in the microbiome of the genera Prevotella, Selenomonas, Veillonella, as well as an increase in representatives of the genus Kocuria compared to donors with a low level of CA in lymphocytes. At the species level, differences were found in the contents of Anaerosinus glycerini, Selenomonas bovis, Actinomyces hyovaginalis, Granulicatella balaenopterae and Clostridium bolteae. Moreover, only the latter species was represented more often in the sputum of donors with high levels of CA, the remaining 4 types of bacteria were significantly more likely to be present in the sputum of donors with a low level of mutations in lymphocytes.

Inhibition of Autism Spectrum Disorder Associated Bacteria and C. difficile by Polyols

Objectives: To determine the effectiveness of erythritol and xylitol in the inhibition of gut bacteria possibly associated with Autism Spectrum Disorder (ASD) and Clostridium difficile Infection (CDI). Methods: Seven bacterial strains associated with ASD, or with CDI and a control probiotic were tested for polyol inhibitory activity: Clostridium histolyticum, Bacteroides vulgatus, Bifidobacterium longham, and two strains each of Clostridium bolteae and difficile. Each strain was grown in brain heart infusion/sucrose media with polyol concentrations varying from 0% to 15% for erythritol and 0% -30 % for xylitol. Growth of Clostridium histolyticum and Bifidobacterium longham was measured after 24 hours while all other strains were evaluated at 48 hours to permit additional growth. Optical density was measured using a spectrophotometer and the plates were read at 620 nm. Results: All strains had results indicating polyol inhibition of growth. Clostridium histolyticum (Chis), Bifidobacterium longham (Blof), and both Clostridium bolteae (Cbol) strains showed reduced growth with increasing polyol concentration with an inflection point of about 4% for both xylitol and erythritol (complete or near complete inhibition relative to control wells). Bacteroides vulgatus (Bvul) grew very lightly in the BHI/sucrose. This strain has visible growth but very low OD values. Inhibition of growth with increasing polyol concentrations was observed but assessing the polyol inhibition break point was difficult with this strain. Conclusions: Xylitol and erythritol at sufficient concentrations were able to inhibit the growth of bacterial strains that have been associated with the development of Autism Spectrum Disorder in recently published studies.