Sex-dependent effects of carbohydrate source and quantity on caspase-1 activity in the mouse central nervous system.

BACKGROUND: Mounting evidence links glucose intolerance and diabetes as aspects of metabolic dysregulation that are associated with an increased risk of developing dementia. Inflammation and inflammasome activation have emerged as a potential link between these disparate pathologies. As diet is a key factor in both the development of metabolic disorders and inflammation, we hypothesize that long term changes in dietary factors can influence nervous system function by regulating inflammasome activity and that this phenotype would be sex-dependent, as sex hormones are known to regulate metabolism and immune processes. METHODS: 5-week-old male and female transgenic mice expressing a caspase-1 bioluminescent reporter underwent cranial window surgeries and were fed control (65% complex carbohydrates, 15% fat), high glycemic index (65% carbohydrates from sucrose, 15% fat), or ketogenic (1% complex carbohydrates, 79% fat) diet from 6 to 26 weeks of age. Glucose regulation was assessed with a glucose tolerance test following a 4-h morning fast. Bioluminescence in the brain was quantified using IVIS in vivo imaging. Blood cytokine levels were measured using cytokine bead array. 16S ribosomal RNA gene amplicon sequencing of mouse feces was performed to assess alterations in the gut microbiome. Behavior associated with these dietary changes was also evaluated. RESULTS: The ketogenic diet caused weight gain and glucose intolerance in both male and female mice. In male mice, the high glycemic diet led to increased caspase-1 biosensor activation over the course of the study, while in females the ketogenic diet drove an increase in biosensor activation compared to their respective controls. These changes correlated with an increase in inflammatory cytokines present in the serum of test mice and the emergence of anxiety-like behavior. The microbiome composition differed significantly between diets; however no significant link between diet, glucose tolerance, or caspase-1 signal was established. CONCLUSIONS: Our findings suggest that diet composition, specifically the source and quantity of carbohydrates, has sex-specific effects on inflammasome activation in the central nervous system and behavior. This phenotype manifested as increased anxiety in male mice, and future studies are needed to determine if this phenotype is linked to alterations in microbiome composition.

Metabolic syndrome and the urinary microbiome of patients undergoing percutaneous nephrolithotomy.

Objective: To identify possible stone-promoting microbes, we compared the profiles of microbes grown from stones of patients with and without metabolic syndrome (MetS). The association between MetS and urinary stone disease is well established, but the exact pathophysiologic relationship remains unknown. Recent evidence suggests urinary tract dysbiosis may lead to increased nephrolithiasis risk. Methods: At the time of percutaneous nephrolithotomy, bladder urine and stone fragments were collected from patients with and without MetS. Both sample types were subjected to expanded quantitative urine culture (EQUC) and 16 S ribosomal RNA gene sequencing. Results: Fifty-seven patients included 12 controls (21.1%) and 45 MetS patients (78.9%). Both cohorts were similar with respect to demographics and non-MetS comorbidities. No controls had uric acid stone composition. By EQUC, bacteria were detected more frequently in MetS stones (42.2%) compared to controls (8.3%) (p=0.041). Bacteria also were more abundant in stones of MetS patients compared to controls. To validate our EQUC results, we performed 16 S ribosomal RNA gene sequencing. In 12/16 (75.0%) sequence-positive stones, EQUC reliably isolated at least one species of the sequenced genera. Bacteria were detected in both "infectious" and "non-infectious" stone compositions. Conclusion: Bacteria are more common and more abundant in MetS stones than control stones. Our findings support a role for bacteria in urinary stone disease for patients with MetS regardless of stone composition.

Cataloging the phylogenetic diversity of human bladder bacterial isolates.

BACKGROUND: Although the human bladder is reported to harbor unique microbiota, our understanding of how these microbial communities interact with their human hosts is limited, mostly owing to the lack of isolates to test mechanistic hypotheses. Niche-specific bacterial collections and associated reference genome databases have been instrumental in expanding knowledge of the microbiota of other anatomical sites, such as the gut and oral cavity. RESULTS: To facilitate genomic, functional, and experimental analyses of the human bladder microbiota, we present a bladder-specific bacterial isolate reference collection comprising 1134 genomes, primarily from adult females. These genomes were culled from bacterial isolates obtained by a metaculturomic method from bladder urine collected by transurethral catheterization. This bladder-specific bacterial isolate reference collection includes 196 different species, including representatives of major aerobes and facultative anaerobes, as well as some anaerobes. It captures 72.2% of the genera found when re-examining previously published 16S rRNA gene sequencing of 392 adult female bladder urine samples. Comparative genomic analysis finds that the taxonomies and functions of the bladder microbiota share more similarities with the vaginal microbiota than the gut microbiota. Whole-genome phylogenetic and functional analyses of 186 bladder Escherichia coli isolates and 387 gut Escherichia coli isolates support the hypothesis that phylogroup distribution and functions of Escherichia coli strains differ dramatically between these two very different niches. CONCLUSIONS: This bladder-specific bacterial isolate reference collection is a unique resource that will enable bladder microbiota research and comparison to isolates from other anatomical sites.

One Health Assessment of Bacillus anthracis Incidence and Detection in Anthrax-Endemic Areas of Pakistan.

Anthrax, a severe zoonotic disease, is infrequently reported in anthrax-endemic regions of Pakistan. Despite clinical reports indicating its presence, particularly cutaneous anthrax, there is insufficient laboratory evidence regarding disease occurrence and environmental persistence. The present study aimed to confirm Bacillus anthracis presence, accountable for animal mortality and human infection, while exploring environmental transmission factors. Between March 2019 and July 2021, a total of 19 outbreaks were documented. Of these, 11 affected sheep/goats in Zhob district and 8 affected cattle/sheep in Bajour Agency. Clinical signs suggestive of Bacillus anthracis outbreak were observed in 11 animals. Blood and swab samples were collected for confirmation. The study followed a One Health approach, analyzing animal, environmental (soil/plant), and human samples. Of the 19 outbreaks, 11 were confirmed positive for anthrax based on growth characteristics, colony morphology, and PCR. Soil and plant root samples from the outbreak areas were collected and analyzed microscopically and molecularly. Cutaneous anthrax was observed in six humans, and swab samples were taken from the lesions. Human serum samples (n = 156) were tested for IgG antibodies against PA toxin and quantitative analysis of anthrax toxin receptor 1 (ANTXR1). Bacillus anthracis was detected in 65 out of 570 (11.40%) soil samples and 19 out of 190 (10%) plant root samples from the outbreak areas. Four out of six human samples from cutaneous anthrax lesions tested positive for Bacillus anthracis. Human anthrax seroprevalence was found to be 11% and 9% in two districts, with the highest rates among butchers and meat consumers. The highest ANTXR1 levels were observed in butchers, followed by meat consumers, farm employees, meat vendors, veterinarians, and farm owners. These findings highlight the persistence of anthrax in the region and emphasize the potential public health risks.

Cataloging the Phylogenetic Diversity of Human Bladder Bacterial Isolates.

Although the human bladder is reported to harbor unique microbiota, our understanding of how these microbial communities interact with their human hosts is limited, mostly owing to the lack of isolates to test mechanistic hypotheses. Niche-specific bacterial collections and associated reference genome databases have been instrumental in expanding knowledge of the microbiota of other anatomical sites, e.g., the gut and oral cavity. To facilitate genomic, functional, and experimental analyses of the human bladder microbiota, here we present a bladder-specific bacterial reference collection comprised of 1134 genomes. These genomes were culled from bacterial isolates obtained by a metaculturomic method from bladder urine collected by transurethral catheterization. This bladder-specific bacterial reference collection includes 196 different species, including representatives of major aerobes and facultative anaerobes, as well as some anaerobes. It captures 72.2 % of the genera found when we reexamined previously published 16S rRNA gene sequencing of 392 adult female bladder urine samples. Comparative genomic analysis found that the taxonomies and functions of the bladder microbiota shared more similarities with the vaginal microbiota than the gut microbiota. Whole-genome phylogenetic and functional analyses of 186 bladder E. coli isolates and 387 gut E. coli isolates supports the hypothesis that phylogroup distribution and functions of E. coli strains differ dramatically between these two very different niches. This bladder-specific bacterial reference collection is a unique resource that will enable hypothesis-driven bladder microbiota research and comparison to isolates from other anatomical sites.

Urogenital urobiome of healthy children does not differ from that of children with bladder and bowel dysfunction.

INTRODUCTION: The pediatric urinary microbiome (urobiome) has been studied in the context of healthy children and children with genitourinary pathologies including neuropathic bladder, urinary tract infection (UTI) and nephrolithiasis. Little is known about the urobiome of children with bladder and bowel dysfunction (BBD), a condition that is an established risk factor of UTI. We hypothesized that the symptoms of a child with BBD may be related to urobiome composition. OBJECTIVE: To evaluate the urogenital urobiome's role in BBD, we compared the urogenital urobiomes of children with and without BBD. STUDY DESIGN: We performed a prospective case-control pilot study at a single large, academic children's hospital. Cases included toilet trained prepubertal females over 2 years of age with BBD established through a validated scoring system and controls included asymptomatic, presumably healthy, children. Children were excluded if they had symptoms or lab work consistent with a concurrent UTI or antibiotic course for any reason within the prior 14 days. We performed 16 S ribosomal RNA gene sequencing and expanded quantitative urine culture on clean catch urine samples. To compare within sample (alpha) diversity, we used the Kruskal-Wallis test. To compare between sample (beta) diversity, we calculated the Bray-Curtis distance and performed the PERMANOVA test. RESULTS: Data from 25 children with BBD and 8 asymptomatic controls were analyzed. The demographic and clinical characteristics of the two comparison groups were similar, though a higher proportion of Black children were included in the asymptomatic control group. Neither alpha diversity nor beta diversity was significantly different between the two groups. The core microbiome of the BBD group included all the genera in the core urogenital urobiome of the controls, plus additional genera associated with opportunistic infection and/or UTI, including Escherichia, Campylobacter and Streptococcus. DISCUSSION: The results of both the 16 S sequencing and expanded quantitative urine culture in this small study suggest that the urogenital urobiomes of children with BBD do not differ significantly from those of asymptomatic children. However, the core urogenital urobiome of children with BBD included genera associated with opportunistic infection and/or UTI. This study was limited by the sample collection method ("clean catch" midstream voided urine samples, which introduce the possibility of vulvovaginal contamination), small sample size, and unequal balance of patient characteristics between the two study groups. CONCLUSION: The urogenital urobiomes of children with and without BBD do not appear to significantly differ. Larger studies are needed to confirm these findings.

Role of novel polysaccharide layers in assembly of the exosporium, the outermost protein layer of the Bacillus anthracis spore.

A fundamental question in cell biology is how cells assemble their outer layers. The bacterial endospore is a well-established model for cell layer assembly. However, the assembly of the exosporium, a complex protein shell comprising the outermost layer in the pathogen Bacillus anthracis, remains poorly understood. Exosporium assembly begins with the deposition of proteins at one side of the spore surface, followed by the progressive encirclement of the spore. We seek to resolve a major open question: the mechanism directing exosporium assembly to the spore, and then into a closed shell. We hypothesized that material directly underneath the exosporium (the interspace) directs exosporium assembly to the spore and drives encirclement. In support of this, we show that the interspace possesses at least two distinct layers of polysaccharide. Secondly, we show that putative polysaccharide biosynthetic genes are required for exosporium encirclement, suggesting a direct role for the interspace. These results not only significantly clarify the mechanism of assembly of the exosporium, an especially widespread bacterial outer layer, but also suggest a novel mechanism in which polysaccharide layers drive the assembly of a protein shell.

Characterization of Bacillus anthracis Spore Proteins Using a Nanoscaffold Vaccine Platform.

Subunit vaccines are theoretically safe and easy to manufacture but require effective adjuvants and delivery systems to yield protective immunity, particularly at critical mucosal sites such as the lung. We investigated nanolipoprotein particles (NLPs) containing the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) as a platform for intranasal vaccination against Bacillus anthracis. Modified lipids enabled attachment of disparate spore and toxin protein antigens. Intranasal vaccination of mice with B. anthracis antigen-MPLA-NLP constructs induced robust IgG and IgA responses in serum and in bronchoalveolar and nasal lavage. Typically, a single dose sufficed to induce sustained antibody titers over time. When multiple immunizations were required for sustained titers, specific antibodies were detected earlier in the boost schedule with MPLA-NLP-mediated delivery than with free MPLA. Administering combinations of constructs induced responses to multiple antigens, indicating potential for a multivalent vaccine preparation. No off-target responses to the NLP scaffold protein were detected. In summary, the NLP platform enhances humoral and mucosal responses to intranasal immunization, indicating promise for NLPs as a flexible, robust vaccine platform against B. anthracis and potentially other inhalational pathogens.

Expansion of the Spore Surface Polysaccharide Layer in Bacillus subtilis by Deletion of Genes Encoding Glycosyltransferases and Glucose Modification Enzymes.

Polysaccharides (PS) decorate the surface of dormant endospores (spores). In the model organism for sporulation, Bacillus subtilis, the composition of the spore PS is not known in detail. Here, we have assessed how PS synthesis enzymes produced during the late stages of sporulation affect spore surface properties. Using four methods, bacterial adhesion to hydrocarbons (BATH) assays, India ink staining, transmission electron microscopy (TEM) with ruthenium red staining, and scanning electron microscopy (SEM), we characterized the contributions of four sporulation gene clusters, spsABCDEFGHIJKL, yfnHGF-yfnED, ytdA-ytcABC, and cgeAB-cgeCDE, on the morphology and properties of the crust, the outermost spore layer. Our results show that all mutations in the sps operon result in the production of spores that are more hydrophobic and lack a visible crust, presumably because of reduced PS deposition, while mutations in cgeD and the yfnH-D cluster noticeably expand the PS layer. In addition, yfnH-D mutant spores exhibit a crust with an unusual weblike morphology. The hydrophobic phenotype from sps mutant spores was partially rescued by a second mutation inactivating any gene in the yfnHGF operon. While spsI, yfnH, and ytdA are paralogous genes, all encoding glucose-1-phosphate nucleotidyltransferases, each paralog appears to contribute in a distinct manner to the spore PS. Our data are consistent with the possibility that each gene cluster is responsible for the production of its own respective deoxyhexose. In summary, we found that disruptions to the PS layer modify spore surface hydrophobicity and that there are multiple saccharide synthesis pathways involved in spore surface properties.IMPORTANCE Many bacteria are characterized by their ability to form highly resistant spores. The dormant spore state allows these species to survive even the harshest treatments with antimicrobial agents. Spore surface properties are particularly relevant because they influence spore dispersal in various habitats from natural to human-made environments. The spore surface in Bacillus subtilis (crust) is composed of a combination of proteins and polysaccharides. By inactivating the enzymes responsible for the synthesis of spore polysaccharides, we can assess how spore surface properties such as hydrophobicity are modulated by the addition of specific carbohydrates. Our findings indicate that several sporulation gene clusters are responsible for the assembly and allocation of surface polysaccharides. Similar mechanisms could be modulating the dispersal of infectious spore-forming bacteria.

Contributions of crust proteins to spore surface properties in Bacillus subtilis.

Surface properties, such as adhesion and hydrophobicity, constrain dispersal of bacterial spores in the environment. In Bacillus subtilis, these properties are influenced by the outermost layer of the spore, the crust. Previous work has shown that two clusters, cotVWXYZ and cgeAB, encode the protein components of the crust. Here, we characterize the respective roles of these genes in surface properties using Bacterial Adherence to Hydrocarbons assays, negative staining of polysaccharides by India ink and Transmission Electron Microscopy. We showed that inactivation of crust genes caused increases in spore relative hydrophobicity, disrupted the spore polysaccharide layer, and impaired crust structure and attachment to the rest of the coat. We also found that cotO, previously identified for its role in outer coat formation, is necessary for proper encasement of the spore by the crust. In parallel, we conducted fluorescence microscopy experiments to determine the full network of genetic dependencies for subcellular localization of crust proteins. We determined that CotZ is required for the localization of most crust proteins, while CgeA is at the bottom of the genetic interaction hierarchy.

Coordinated Assembly of the Bacillus anthracis Coat and Exosporium during Bacterial Spore Outer Layer Formation.

Bacterial spores produced by the Bacillales are composed of concentric shells, each of which contributes to spore function. Spores from all species possess a cortex and coat, but spores from many species possess additional outer layers. The outermost layer of Bacillus anthracis spores, the exosporium, is separated from the coat by a gap known as the interspace. Exosporium and interspace assembly remains largely mysterious. As a result, we have a poor understanding of the overarching mechanisms driving the assembly of one of the most ubiquitous cell types in nature. To elucidate the mechanisms directing exosporium assembly, we generated strains bearing mutations in candidate exosporium-controlling genes and analyzed the effect on exosporium formation. Biochemical and cell biological analyses argue that CotE directs the assembly of CotO into the spore and that CotO might be located at or close to the interior side of the cap. Taken together with data showing that CotE and CotO interact directly in vitro, we propose a model in which CotE and CotO are important components of a protein interaction network that connects the exosporium to the forespore during cap formation and exosporium elongation. Our data also suggest that the cap interferes with coat assembly at one pole of the spore, altering the pattern of coat deposition compared to the model organism Bacillus subtilis We propose that the difference in coat assembly patterns between these two species is due to an inherent flexibility in coat assembly, which may facilitate the evolution of spore outer layer complexity.IMPORTANCE This work dramatically improves our understanding of the assembly of the outermost layer of the B. anthracis spore, the exosporium, a layer that encases spores from many bacterial species and likely plays important roles in the spore's interactions with the environment, including host tissues. Nonetheless, the mechanisms directing exosporium assembly into a shell surrounding the spore are still very poorly understood. In this study, we clarify these mechanisms by the identification of a novel protein interaction network that directs assembly to initiate at a specific subcellular location in the developing cell. Our results further suggest that the presence or absence of an exosporium has a major impact on the assembly of other more interior spore layers, thereby potentially explaining long-noted differences in spore assembly between B. anthracis and the model organism B. subtilis.

Ultrasound directs a transposase system for durable hepatic gene delivery in mice.

Our aim was to evaluate the delivery of transposase-based vectors by ultrasound targeted microbubble destruction (UTMD) in mice. DNA vectors were attached to cationic lipid microbubbles (1-3 µm in diameter), injected intravenously and delivered to the liver by destruction of the carrier bubbles with ultrasound in burst mode at 1.0 MHz, 20-µs pulse duration, 10-Hz pulse repetition frequency and ∼1.3-MPa acoustic peak negative pressure. We evaluated the expression and genomic integration of conventional (pcDNA3) and piggyBac transposase-based (pmGENIE) reporter vectors. In vivo, we observed UTMD-mediated liver-specific expression of pmGENIE for an average of 24 d, compared with 4 d with pcDNA3. Reporter expression was located predominately near blood vessels initially, whereas expression after 3 d was more evenly distributed through the parenchyma of the liver. We confirmed random genomic integration for pmGENIE in vitro; however, integration events for pmGENIE in vivo were targeted to specific areas of chromosome 14. Our results suggest that a combination of UTMD and non-viral DNA transposase vectors can mediate weeks of hepatic-specific gene transfer in vivo, and analyses performed by non-restrictive linear amplification-mediated (nrLAM) polymerase chain reaction, cloning and sequencing identify an unexpected tropism for integration within a specific sequence on chromosome 14 in mice. UTMD delivery of transgenes may be useful for the treatment of hepatic gene deficiency disorders.