Protective Capsule Research Articles

Inorganic Polyphosphate Affects Biofilm Assembly, Capsule Formation, and Virulence of Hypervirulent ST23 Klebsiella pneumoniae.

The emergence of hypervirulent Klebsiella pneumoniae (hvKP) strains poses a significant threat to public health due to high mortality rates and propensity to cause severe community-acquired infections in healthy individuals. The ability to form biofilms and produce a protective capsule contributes to its enhanced virulence and is a significant challenge to effective antibiotic treatment. Polyphosphate kinase 1 (PPK1) is an enzyme responsible for inorganic polyphosphate synthesis and plays a vital role in regulating various physiological processes in bacteria. In this study, we investigated the impact of polyP metabolism on the biofilm and capsule formation and virulence traits in hvKP using Dictyostelium discoideum amoeba as a model host. We found that the PPK1 null mutant was impaired in biofilm and capsule formation and showed attenuated virulence in D. discoideum compared to the wild-type strain. We performed a proteomic analysis to gain further insights into the underlying molecular mechanism. The results revealed that the PPK1 mutant had a differential expression of proteins involved in capsule synthesis (Wzi-Ugd), biofilm formation (MrkC-D-H), synthesis of the colibactin genotoxin precursor (ClbB), as well as proteins associated with the synthesis and modification of lipid A (ArnB-LpxC-PagP). These proteomic findings corroborate the phenotypic observations and indicate that the PPK1 mutation is associated with impaired biofilm and capsule formation and attenuated virulence in hvKP. Overall, our study highlights the importance of polyP synthesis in regulating extracellular biomolecules and virulence in K. pneumoniae and provides insights into potential therapeutic targets for treating K. pneumoniae infections.

Neutron Scattering Analysis of Cryptococcus neoformans Polysaccharide Reveals Solution Rigidity and Repeating Fractal-like Structural Patterns.

Cryptococcus neoformans is a fungal pathogen that can cause life-threatening brain infections in immunocompromised individuals. Unlike other fungal pathogens, it possesses a protective polysaccharide capsule that is crucial for its virulence. During infections, Cryptococcus cells release copious amounts of extracellular polysaccharides (exo-PS) that interfere with host immune responses. Both exo-PS and capsular-PS play pivotal roles in Cryptococcus infections and serve as essential targets for disease diagnosis and vaccine development strategies. However, understanding their structure is complicated by their polydispersity, complexity, sensitivity to sample isolation and processing, and scarcity of methods capable of isolating and analyzing them while preserving their native structure. In this study, we employ small-angle neutron scattering (SANS) and ultra-small-angle neutron scattering (USANS) for the first time to investigate both fungal cell suspensions and extracellular polysaccharides in solution. Our data suggests that exo-PS in solution exhibits collapsed chain-like behavior and demonstrates mass fractal properties that indicate a relatively condensed pore structure in aqueous environments. This observation is also supported by scanning electron microscopy (SEM). The local structure of the polysaccharide is characterized as a rigid rod, with a length scale corresponding to 3-4 repeating units. This research not only unveils insights into exo-PS and capsular-PS structures but also demonstrates the potential of USANS for studying changes in cell dimensions and the promise of contrast variation in future neutron scattering studies.

Immunomodulating multivitamin complex and its role in antioxidant protection of the body

The article presents the results of experimental data on the development of an immunomodulatory multivitamin complex and the establishment of its role in the antioxidant defense of the body. The research objects were recipe ingredients, laboratory and industrial samples of the developed product. We used generally accepted and special methods for assessing the quality and safety of the developed product. The technology includes: preparation of raw materials, preparation of a mixture for encapsulation, encapsulation and sealing of capsules with gelatin, assessment of the quality of the resulting product. The composition of a new multivitamin composition with targeted antioxidant properties is given, mg / 1 capsule: red palm oil - 432; ascorbic acid - 70; ascorbate palmitate - 10; tocopherol acetate, 98% - 15.3 (vitamin E - 15); beta-carotene, 98% - 5.1 (beta - carotene - 0.5); silicon dioxide (carrier) - 17.6; gelatin capsule - 110. Total weight 660 mg. The functional properties of the developed product were assessed. The components of the developed product in the form of a multivitamin complex had a positive effect on the analyzed parameters of the SF-36 test. According to the test results, the general state of health in the group of volunteers after taking the multivitamin complex increased by 17.2%, while in the control group by only 9.5%, in terms of viability by 7.2 and 3.6% in the experimental and control groups respectively. The innovativeness of the new drug lies in the rejection of inert materials and the use as a carrier of a unique composition of red palm oil, rich, along with fat-soluble vitamins, highly active antioxidants - lycopene and coenzyme Q10. The proposed method of encapsulation in a protective gelatin capsule. This provides a fast action - a water-soluble and prolonged effect - of the fat-soluble fraction.

Shelf-life enhancement of bio-inoculants through synergistic effects of encapsulation technology and osmotic protectants

A novel protective capsule for encapsulating microorganism inoculants was developed based on a core/shell structured hydrogel doped with osmotic regulators, with the aim of protecting against salts stress. The resulting capsules showed favorable mechanical toughness, high encapsulated efficiency, low cell leakage and increased resistance against salt attack. Different types of sugar alcohol molecules were selected as the osmoprotectants and their effectiveness in retaining the microbial count of trichoderma harziana under adverse conditions was examined. It was found that by using trehalose-doped core/shell structured hydrogels as the microbial carrier, the survival rate and mycelial growth rate of trichoderma harziana increased during incubation in sodium chloride solution and on sodium chloride-containing solid medium. Furthermore, the shelf-life of the microorganisms demonstrated a significant increase from log 5.72 cfu/g to log 7.30 cfu/g after an incubation period of 180 days in compound fertilizers with 15 % nitrogen‐phosphorus‐potassium (NPK) content. This novel microbial encapsulated system showed remarkable preservation of microorganism inoculants under salt stress from fertilizer nutrients, suggesting a noticeable practical potential in agriculture.

Pectin-Coated Baclofen-Layered Zinc Hydroxide Nanohybrid as a Bio-Based Nanocomposite Carrier for Oral Delivery.

pH-sensitive pectin beads were proposed as a protective capsule for layered zinc hydroxide-drug (LZH-Drug) nanohybrids in the gastrointestinal tract in this paper. Baclofen was intercalated between LZH layers using the co-precipitation method as a model drug. By combining LZH-baclofen with pectin, the resulting nanohybrid (LZH-baclofen) was used to make bio-nanocomposite hydrogel beads. FTIR, XRD, and SEM analyses were used to characterize the produced products. Baclofen anions are vertical to the LZH layers in the shape of a monolayer, according to the interlayer space of 19.6Å. The presence of nanocomposites is demonstrated by FTIR, which exhibits a peak at 3489 cm-1 for the OH group, 1564 and 1384 cm-1 for the-COO- vibration mode, indicating that baclofen is intercalated between the layered structures. After intercalation, baclofen's thermal stability is greatly improved. The nanohybrid is more compact, with agglomerates and flat surfaces of the intercalated substance, shown by SEM. In vitro release behaviors of baclofen from LZH and bio-nanocomposites in buffer solution were examined under pH values (pH = 1.2, 6.8, 7.4) chosen from a model of the passing materials through the gastrointestinal tract. For pectin encapsulated LZH-baclofen nanohybrid, drug release studies indicated superior protection against stomach pH and regulated release under intestinal tract conditions. Furthermore, nanohybrid and nanocomposite treatment of a normal fibroblast cell line resulted in cell survival up to 12.5 g/mL for a 24-h period, with inhibition reducing dose-dependently at higher concentrations. A novel intercalation molecule with a sustained release mode and improved toxicity against normal fibroblast cell lines has been produced as a result of the strong host-guest contacts between the LZH lattice and the baclofen anion. Further study into the utilization of brucite-like host materials in drug delivery systems should be based on these findings.

Formation of Unique Placental Seed Capsules in the Maturation Process of the Tomato Fruit.

The morphological and anatomical study of the seed formation features in a juicy tomato fruit was carried out. The ovules, which form on the placenta, have been shown to be gradually enveloped by the protrusions of placental tissue that arises simultaneously with them. As a result of this process, each seed is enclosed in an individual capsule. These seed capsules have been shown in vivo to be airtight and air-filled. Tomato seeds, as has been shown in this study, develop inside these capsules until the full maturity of the fruit and do not come into contact with the detached and moldered cells of the placenta protrusions, which convert into a gel (pulp). Using scanning electron microscopy, it was possible to reveal the details of a ribbon-like “pubescence” formation of the tomato seed, as well as to understand the mechanism of cracking of the outer layer cells in the seed coat, associated with the detection of calcium oxalate crystals in these cells. The unique outer layer of the tomato seed coat seems to play the role of a scaffold that maintains a constant volume of the protective capsule.

Enterococcus faecalis V583 cell membrane protein expression to alkaline stress.

Enterococcus faecalis is able to adapt to alkaline conditions and is commonly recovered from teeth in which endodontic treatment has failed. The role that E. faecalis membrane proteins play in survival strategies to extreme alkaline conditions is unclear. We grew E. faecalis V583 in a chemostat at pH 8 and 11 at one-tenth the organism’s relative maximum growth rate. Following membrane shaving, isotope-coding protein labels were added at the peptide level to samples and then combined. The relative proportion of membrane proteins were identified using LC-ESI mass spectrometry and MaxQuant analysis. Ratios of membrane proteins were log2 transformed, with proteins deviating by more than 1 SD of the mean considered to be up- or down-regulated. A total of six proteins were up-regulated in pH 11 including: EF0669 (polysaccharide biosynthesis family); EF1927 (glycerol uptake facilitator), and EF0114 (glycosyl hydrolase). A total of five proteins were down-regulated including: EF0108 (C4-dicarboxylate transporter); EF1838 (PTS system IIC component); EF0456 (PTS system IID component); and EF0022 (PTS mannose-specific IID component). In extreme alkaline conditions, the membrane proteins of E. faecalis seem to be involved in a shift of carbohydrate metabolism from the PTS system to glycerol, which supports the formation of a protective capsule protecting the cell.

Neisseria-Meningitis Diseases

Meningococcal disease is caused by the Gram-negative bacterium Neisseria meningitidis (the meningococcus). It remains a significant public health issue globally causing both endemic and epidemic disease in developed and developing countries. Approximately 10% of humans harmlessly carry N. meningitidis in the oronasopharynx. On very rare occasions the bacteria may cross the epithelium and enter the blood stream causing sudden onset of sepsis and or meningitis with high complication and case fatality rates, even with appropriate antibiotic treatment. A limited number of strains cause the majority of invasive disease and, in normally healthy individuals, these practically always express a protective polysaccharide capsule on their cell surface. There are 12 capsular serogroups, of which A, B, C, W, X and Y cause the vast majority of invasive meningococcal disease worldwide. Polysaccharide-based vaccines target the capsule and so are serogroup-specific. Plain (unconjugated) polysaccharide vaccines were developed first and have been used in control of serogroup A epidemics in sub-Saharan Africa and for controlling serogroup C disease in the military and college students. Associated limitations include poor immunogenicity in young children, hyporesponsiveness with repeat doses, inability to induce immune memory and lack of an effect on carriage. Conjugated polysaccharide vaccines have none of these limitations and, most importantly, significantly reduce carriage. Therefore, large scale vaccination of cohorts with high carriage (catch-up campaigns) are highly effective in inducing herd protection. Serogroup C conjugate vaccines have been hugely successful in dramatically reducing disease in the countries that have instigated immunization programs together with appropriate catch-up campaigns. Meningococcal quadrivalent conjugate vaccines are now being implemented into schedules. With the development and introduction of a meningococcal serogroup A conjugate vaccine, serogroup A disease has disappeared from those sub-Saharan countries who have implemented campaigns. The serogroup B polysaccharide is poorly immunogenic and so broad coverage protein-based serogroup B vaccines have been developed.

THYROID ABSCESS: A CASE SERIES

Thyroid abscess is a rare surgical pathology, accounting for less than 0.7% of surgical pathologies involving the thyroid 1,2 gland. This is due to its rich blood supply and lymphatic drainage, high concentration oodine that inhibits bacterial growth, protective brous capsule, and fascial planes 2–4 separating it from other neck structures. It may lead to complications like septicaemia, paralysis of the vocal cords, retropharyngeal abscess and suppurative media stinitis and 2 may occasionally lead to osteomyelitis or septic thrombophlebitis. It 5 may also lead to tracheal and oesophageal perforation. The 6 left lobe is commonly affected. If left untreated, thyroid abscess can be life threatening resulting in a mortality of 12% 6 or more

Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage.

ABSTRACTThe production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen.IMPORTANCE Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse “capsule depolymerases” which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.

Capsular polysaccharide correlates with immune response to the human gut microbe Ruminococcus gnavus.

Active inflammatory bowel disease (IBD) often coincides with increases of Ruminococcus gnavus, a gut microbe found in nearly everyone. It was not known how, or if, this correlation contributed to disease. We investigated clinical isolates of R. gnavus to identify molecular mechanisms that would link R. gnavus to inflammation. Here, we show that only some isolates of R. gnavus produce a capsular polysaccharide that promotes a tolerogenic immune response, whereas isolates lacking functional capsule biosynthetic genes elicit robust proinflammatory responses in vitro. Germ-free mice colonized with an isolate of R. gnavus lacking a capsule show increased measures of gut inflammation compared to those colonized with an encapsulated isolate in vivo. These observations in the context of our earlier identification of an inflammatory cell-wall polysaccharide reveal how some strains of R. gnavus could drive the inflammatory responses that characterize IBD.

Essential Gene Analysis in Acinetobacter baumannii by High-Density Transposon Mutagenesis and CRISPR Interference.

Acinetobacter baumannii is a poorly understood bacterium capable of life-threatening infections in hospitals. Few antibiotics remain effective against this highly resistant pathogen. Development of rationally designed antimicrobials that can target A. baumannii requires improved knowledge of the proteins that carry out essential processes allowing growth of the organism. Unfortunately, studying essential genes has been challenging using traditional techniques, which usually require time-consuming recombination-based genetic manipulations. Here, we performed saturating mutagenesis with dual transposon systems to identify essential genes in A. baumannii, and we developed a CRISPR interference (CRISPRi) system for facile analysis of these genes. We show that the CRISPRi system enables efficient transcriptional silencing in A. baumannii. Using these tools, we confirmed the essentiality of the novel cell division protein AdvA and discovered a previously uncharacterized AraC family transcription factor (ACX60_RS03245) that is necessary for growth. In addition, we show that capsule biosynthesis is a conditionally essential process, with mutations in late-acting steps causing toxicity in strain ATCC 17978 that can be bypassed by blocking early-acting steps or activating the BfmRS stress response. These results open new avenues for analysis of essential pathways in A. baumannii. IMPORTANCE New approaches are urgently needed to control A. baumannii, one of the most drug-resistant pathogens known. To facilitate the development of novel targets that allow inhibition of the pathogen, we performed a large-scale identification of genes whose products the bacterium needs for growth. We also developed a CRISPR-based gene knockdown tool that operates efficiently in A. baumannii, allowing rapid analysis of these essential genes. We used these methods to define multiple processes vital to the bacterium, including a previously uncharacterized gene regulatory factor and export of a protective polymeric capsule. These tools will enhance our ability to investigate processes critical for the essential biology of this challenging hospital-acquired pathogen.

Isolation and Characterization of a Novel Lytic Bacteriophage against the K2 Capsule-Expressing Hypervirulent Klebsiella pneumoniae Strain 52145, and Identification of Its Functional Depolymerase.

Klebsiella pneumoniae is among the leading bacteria that cause nosocomial infections. The capsule of this Gram-negative bacterium is a dominant virulence factor, with a prominent role in defense and biofilm formation. Bacteriophages, which are specific for one bacterial strain and its capsule type, can evoke the lysis of bacterial cells, aided by polysaccharide depolymerase enzymes. In this study, we isolated and characterized a bacteriophage against the nosocomial K. pneumoniae 52145 strain with K2 capsular serotype. The phage showed a narrow host range and stable lytic activity, even when exposed to different temperatures or detergents. Preventive effect of the phage in a nasal colonization model was investigated in vivo. Phlyogenetic analysis showed that the newly isolated Klebsiella phage B1 belongs to the Webervirus genus in Drexlerviridae family. We identified the location of the capsule depolymerase gene of the new phage, which was amplified, cloned, expressed, and purified. The efficacy of the recombinant B1dep depolymerase was tested by spotting on K. pneumoniae strains and it was confirmed that the extract lowers the thickness of the bacterium lawn as it degrades the protective capsule on bacterial cells. As K. pneumoniae strains possessing the K2 serotype have epidemiological importance, the B1 phage and its depolymerase are promising candidates for use as possible antimicrobial agents.

Recombinant Human Secretory IgA Induces Salmonella Typhimurium Agglutination and Limits Bacterial Invasion into Gut-Associated Lymphoid Tissues.

As the predominant antibody type in mucosal secretions, human colostrum, and breast milk, secretory IgA (SIgA) plays a central role in safeguarding the intestinal epithelium of newborns from invasive enteric pathogens like the Gram-negative bacterium Salmonella enterica serovar Typhimurium (STm). SIgA is a complex molecule, consisting of an assemblage of two or more IgA monomers, joining (J)-chain, and secretory component (SC), whose exact functions in neutralizing pathogens are only beginning to be elucidated. In this study, we produced and characterized a recombinant human SIgA variant of Sal4, a well-characterized monoclonal antibody (mAb) specific for the O5-antigen of STm lipopolysaccharide (LPS). We demonstrate by flow cytometry, light microscopy, and fluorescence microscopy that Sal4 SIgA promotes the formation of large, densely packed bacterial aggregates in vitro. In a mouse model, passive oral administration of Sal4 SIgA was sufficient to entrap STm within the intestinal lumen and reduce bacterial invasion into gut-associated lymphoid tissues by several orders of magnitude. Bacterial aggregates induced by Sal4 SIgA treatment in the intestinal lumen were recalcitrant to immunohistochemical staining, suggesting the bacteria were encased in a protective capsule. Indeed, a crystal violet staining assay demonstrated that STm secretes an extracellular matrix enriched in cellulose following even short exposures to Sal4 SIgA. Collectively, these results demonstrate that recombinant human SIgA recapitulates key biological activities associated with mucosal immunity and raises the prospect of oral passive immunization to combat enteric diseases.

Effect of Simulated Gastrointestinal Tract Conditions on Survivability of Probiotic Bacteria Present in Commercial Preparations.

Probiotics are recommended, among others, in the diet of children who are under antibiotic therapy, or that suffer from food allergies or travel diarrhea, etc. In the case of toddlers taking probiotic preparations, it is highly recommended to first remove the special capsule, which normally protects probiotic strains against hard conditions in the gastrointestinal tract. Otherwise, the toddler may choke. This removal can impair probiotic survival and reduce its efficacy in a toddler’s organism. The aim of this study was to evaluate the survivability of five strains of lactic acid bacteria from the commercial probiotics available on the Polish market under simulated conditions of the gastrointestinal tract. Five probiotics (each including one of these strains: Bifidobacterium BB-12, Lactobacillus (Lb.) rhamnosus GG, Lb. casei, Lb. acidophilus, Lb. plantarum) were protective capsule deprived, added in a food matrix (chicken–vegetable soup) and subjected under simulated conditions of the gastric and gastrointestinal passage. Strain survivability and possibility to growth were evaluated. Obtained results showed that, among all analyzed commercial probiotic strains, the Lb. plantarum was the most resistant to the applied conditions of the culture medium. They showed a noticeable growth under both in vitro gastric conditions at pH 4.0 and 5.0, as well as in vitro intestinal conditions at all tested concentrations of bile salts.

Confining Iron Oxide Nanocubes inside Submicrometric Cavities as a Key Strategy To Preserve Magnetic Heat Losses in an Intracellular Environment.

The design of magnetic nanostructures whose magnetic heating efficiency remains unaffected at the tumor site is a fundamental requirement to further advance magnetic hyperthermia in the clinic. This work demonstrates that the confinement of magnetic nanoparticles (NPs) into a sub-micrometer cavity is a key strategy to enable a certain degree of nanoparticle motion and minimize aggregation effects, consequently preserving the magnetic heat loss of iron oxide nanocubes (IONCs) under different conditions, including intracellular environments. We fabricated magnetic layer-by-layer (LbL) self-assembled polyelectrolyte sub-micrometer capsules using three different approaches, and we studied their heating efficiency as obtained in aqueous dispersions and after internalization by tumor cells. First, IONCs were added to the hollow cavities of LbL submicrocapsules, allowing the IONCs to move to a certain extent in the capsule cavities. Second, IONCs were coencapsulated into solid calcium carbonate cores coated with LbL polymer shells. Third, IONCs were incorporated within the polymer layers of the LbL capsule walls. In aqueous solution, higher specific absorption rate (SAR) values were related to those of free IONCs, while lower SAR values were recorded for capsule/core assemblies. However, after uptake by cancer cell lines (SKOV-3 cells), the SAR values of the free IONCs were significantly lower than those observed for capsule/core assemblies, especially after prolonged incubation periods (24 and 48 h). These results show that IONCs packed into submicrocavities preserve the magnetic losses, as the SAR values remained almost invariable. Conversely, free IONCs without the protective capsule shell agglomerated and their magnetic losses were strongly reduced. Indeed, IONC-loaded capsules and free IONCs reside inside endosomal and lysosomal compartments after cellular uptake and show strongly reduced magnetic losses due to the immobilization and aggregation in centrosymmetrical structures in the intracellular vesicles. The confinement of IONCs into sub-micrometer cavities is a key strategy to provide a sustained and predictable heating dose inside biological matrices.

Streptococcus pyogenes Capsule Promotes Microcolony-Independent Biofilm Formation.

Biofilms play an important role in the pathogenesis of group A streptococcus (GAS), a Gram-positive pathogen responsible for a wide range of infections and with a significant public health impact. Although most GAS serotypes are able to form biofilms, there is a large amount of heterogeneity between individual strains in biofilm formation, as measured by standard crystal violet assays. It is generally accepted that biofilm formation includes the initial adhesion of bacterial cells to a surface followed by microcolony formation, biofilm maturation, and extensive production of extracellular matrix that links together proliferating cells and provides a scaffold for the three-dimensional (3D) biofilm structure. However, our studies show that for GAS strain JS95, microcolony formation is not an essential step in static biofilm formation, and instead, biofilm can be effectively formed from slow-growing or nonreplicating late-exponential- or early-stationary-phase planktonic cells via sedimentation and fixation of GAS chains. In addition, we show that the GAS capsule specifically contributes to the alternative sedimentation-initiated biofilms. Microcolony-independent sedimentation biofilms are similar in morphology and 3D structure to biofilms initiated by actively dividing planktonic bacteria. We conclude that GAS can form biofilms by an alternate noncanonical mechanism that does not require transition from microcolony formation to biofilm maturation and which may be obscured by biofilm phenotypes that arise via the classical biofilm maturation processes.IMPORTANCE The static biofilm assay is a common tool for easy biomass quantification of biofilm-forming bacteria. However, Streptococcus pyogenes biofilm formation as measured by the static assay is strain dependent and yields heterogeneous results for different strains of the same serotype. In this study, we show that two independent mechanisms, for which the protective capsule contributes opposing functions, may contribute to static biofilm formation. We propose that separation of these mechanisms for biofilm formation might uncover previously unappreciated biofilm phenotypes that may otherwise be masked in the classic static assay.

Commensal polysaccharide-induced T cell communication and peripheral immune regulation

Abstract The increasing prevalence of inflammatory and autoimmune diseases across the world has been attributed in part to a shift in microbial exposure both in the exogenous environment and the endogenous microbiome. Here we identify and adopt a strategy used by a common commensal gut microbe, Bacteroides fragilis, to suppress inappropriate immune activation. Bacteroides fragilis produces a protective capsule containing polysaccharide A (PSA), which has been shown to suppress a host of inflammatory diseases in an IL-10-dependent manner. We have discovered that PSA is endocytosed, processed, and presented by APCs through MHCII and αβTCR engagement, and clonally expands CD4+FoxP3−CD25−CD45Rblo cells of an effector memory subset (RbloTEM). RbloTEM are capable of suppressing peripheral inflammation in way dependent on IL-10, however, they themselves do not produce the IL-10 necessary for suppression of disease. We show here that RbloTEM cells communicate directly with FoxP3+ Regulatory T cells, resulting in enhanced IL-10 production. Using novel FoxP3RFP/IL-10GFP dual reporter mice, we further demonstrate that this communication induces synergistic IL-10 expression specifically in Tregs, enhances Treg proliferation, and drives Treg-mediated immune inhibition by preferentially favoring the expansion of IL-10+ Tregs. Through promoting a robust and rapid Treg response, we show that the cytokine signature of PSA-responding RbloTEM cells and coordinating FoxP3+ Tregs can suppress mouse models of inflammatory disease, including HDM-induced asthma and EAE.

Rethinking resting eggs decapsulating

Abstract Aim Temperature and light have been recognized as important factors for ephippia hatching in temperate and tropical freshwater systems. Oddly some authors suggested that decapsulation of resting eggs would be a pro when it comes to ex situ hatching studies, exposing those eggs to a greater amount of light. This study aimed to compare the difference in the hatching rate between resting eggs decapsulated and intact ephippia of Daphnia laevis, a zooplankton that occurs at lakes, in tropical freshwaters (Cladocera). Methods The ephippia used in this work were collected at the sediment of a reservoir, in Belo Horizonte city (Minas Gerais, Brazil). We set up the laboratory experiment with two distinct groups: intact ephippia and decapsulated resting eggs. For that, we manually decapsulated 120 ephippia and kept 120 others intact (six replicas with 20 ephippia each), then incubated them all with culture water at 22ºC (12h photoperiod) for 30 days with daily monitoring. Results The results showed that decapsulation influenced negatively the hatching success, as the intact ephippia had a hatching rate of 22%, while those decapsulated only had 6%. In addition, Daphnia hatchlings were observed for intact ephippia group up to the twenty-seventh day, while for the decapsulated the last hatching occurred on the tenth day. Decapsulated eggs are subject to high exposure to light and it may jeopardize the embryo development. Conclusions In this context, we suggest that removing the protective capsule from the eggs needs to be done with caution, since in some species this can damage the resting eggs, which alters the viability and compromise the accuracy of the hatching rates studies.

Descripción morfológica y biomecánica de la articulación de la rodilla del canino (Canis lupus familiaris)

Las articulaciones sinoviales o también conocidas como diartroideas se caracterizan por presentar gran movilidad, estar constituidas por una cápsula articular de protección, la que a su vez internamente permite la formación de una cavidad, donde es posible observar el líquido sinovial,responsable de proveer elementos nutricios; producir la depuración de los desechos celulares; realizar una amortiguación entre las caras articulares y lubricar las superficies articulares. La rodilla es una de las articulaciones sinoviales más grandes del cuerpo y una de las más complejas en su morfología y biomecánica. Su conformación anatómica consta de la unión de estructuras óseas, cartilaginosas, ligamentosas, vasculares y musculares que permiten otorgar una funcionalidad específica en su capacidad extensora, flexora y leve rotación. Por lo que el objetivo del siguiente estudio fue realizar una descripción morfológica de las estructuras anatómicas macroscópicas,que participan en la conformación de la articulación de la rodilla y como ellas pueden permitir una funcionalidad biomecánica particular. Para ello se realizaron disecciones de 10 rodillas de perros machos conservados en el laboratorio de anatomía veterinaria de la Universidad Santo Tomás, sede Puerto Montt. Los resultados indican características detalladas de la morfología de la rodilla del canino y como cada uno de ellos permiten entender y explicar la capacidad biomecánica de esta articulación.