Bacterial Surface Proteins Research Articles

Antigen 43 associated with Escherichia coli membrane vesicles contributes to bacterial cell association and biofilm formation.

Bacterial membrane vesicles (MVs) are produced by all bacteria and contribute to numerous bacterial functions due to their ability to package and transfer bacterial cargo. In doing so, MVs have been shown to facilitate horizontal gene transfer, mediate antimicrobial activity, and promote biofilm formation. Uropathogenic Escherichia coli is a pathogenic Gram-negative organism that persists in the urinary tract of its host due to its ability to form persistent, antibiotic-resistant biofilms. The formation of these biofilms is dependent upon proteins such as Antigen 43 (Ag43), which belongs to the widespread Autotransporter group of bacterial surface proteins. In E. coli, the autotransporter Ag43 has been shown to contribute to bacterial cell aggregation and biofilm formation via self-association of Ag43 between neighboring Ag43-expressing bacteria. As MVs package bacterial proteins, we investigated whether MVs produced by E. coli contained Ag43, and the ability of Ag43-expressing MVs to facilitate cell aggregation and biofilm formation. We showed that Ag43 expressing E. coli produced MVs that contained Ag43 on their surface and had an enhanced ability to bind to E. coli bacteria. Furthermore, we demonstrated that the addition of Ag43-containing MVs to Ag43-expressing E. coli significantly enhanced biofilm formation. These findings reveal the contribution of MVs harboring autotransporters in promoting bacterial aggregation and enhancing biofilm formation, highlighting the impact of MVs and their specific composition to bacterial adaptation and pathogenesis.IMPORTANCEAutotransporter proteins are the largest family of outer membrane and secreted proteins in Gram-negative bacteria which contribute to pathogenesis by promoting aggregation, biofilm formation, persistence, and cytotoxicity. Although the roles of bacterial autotransporters are well known, the ability of bacterial membrane vesicles (MVs) naturally released from the surface of bacteria to contain autotransporters and their role in promoting virulence remains less investigated. Our findings reveal that MVs produced by E. coli contain the autotransporter protein Ag43. Furthermore, we show that Ag43-containing MVs function to enhance bacterial cell interactions and biofilm formation. By demonstrating the ability of MVs to carry functional autotransporter adhesins, this work highlights the importance of MVs in disseminating autotransporters beyond the bacterial cell membrane to ultimately promote cellular interactions and enhance biofilm development. Overall, these findings have significant implications in furthering our understanding of the numerous ways in which MVs can facilitate bacterial persistence and pathogenesis.

A conserved bacterial genetic basis for commensal-host specificity.

Animals selectively acquire specific symbiotic gut bacteria from their environments that aid host fitness. To colonize, a symbiont must locate its niche and sustain growth within the gut. Adhesins are bacterial cell surface proteins that facilitate attachment to host tissues and are often virulence factors for opportunistic pathogens. However, the attachments are often transient and nonspecific, and additional mechanisms are required to sustain infection. In this work, we use live imaging of individual symbiotic bacterial cells colonizing the gut of living Drosophila melanogaster to show that Lactiplantibacillus plantarum specifically recognizes the fruit fly foregut as a distinct physical niche. L. plantarum establishes stably within its niche through host-specific adhesins encoded by genes carried on a colonization island. The adhesin binding domains are conserved throughout the Lactobacillales, and the island also encodes a secretion system widely conserved among commensal and pathogenic bacteria.

How nanoscale plastics facilitate the evolution of antibiotic resistance?

The plastic can enhance the proliferation of antibiotic resistance genes (ARGs), however, the effect of nanoplastics (NPLs) on bacterial antibiotic resistance has not been clearly explained. Herein, we explored the effects and mechanisms of NPLs of different sizes (200 and 600nm) on the evolution of antibiotic resistance in Serratia marcescens. The results indicated that the evolution of bacterial antibiotic resistance could be promoted under NPLs exposure, which the median of relative abundance of ARGs was 1.11-1.46 times compared to the treatment without NPLs. Transcriptomic analysis showed that the larger size of NPLs mainly increased the permeability of bacterial cell membranes to efflux antibiotics, thus potentiating antibiotic resistance. While, the smaller NPLs is more than that, its enhanced the expression of antibiotic resistance by modulating bacterial metabolic processes. The genome SNP analysis found that the NPLs could cause the genetic mutation occurrence to alter the membrane transport and metabolism processes, and it increased at a size of 200nm more than at 600nm NPLs. Importantly, we demonstrated that the horizontal transfer of ARGs was augmented due to the NPLs could dock to bacterial surface proteins and pull their movement to contact with other bacteria (binding energy of membrane proteins: -8.54kcal/mol), especially the smaller size. It suggests that NPLs will also contribute to the proliferation of ARGs in the environment. This study provides data for understanding the risk of bacterial resistance.

In silico mechanistic insights of ecofriendly synthesized AgNPs, SeNPs, rGO and Ag&SeNPs@rGONM's for biological applications and its toxicity evaluation using Artemia salina

The present study focused on Rosmarinus officinalis Linn. leaves extract (ROE) mediated synthesis of silver nanoparticles (AgNPs), selenium nanoparticles (SeNPs), reduced graphene oxide (rGO) and silver and selenium nanoparticles decorated on rGO nanomaterials (Ag&SeNPs@rGONM's) for its antibacterial and antifungal in silico mechanistic insight applications. In addition, the toxicity of the synthesized nanomaterials was evaluated using Artemia salina. The formation of AgNPs, SeNPs, rGO and Ag&SeNPs@rGONM's was completed within 1.0, 140, 120 and 144h, respectively. Various optical and microscopic examinations were evident in the nanomaterial's synthesis. Further, the average size and stability of the synthesized nanomaterials were conformed through dynamic light scattering (DLS) and zeta potential analyzer, respectively. The synthesized Ag&SeNPs@rGONM's were pronounced promising results against Gram-negative bacteria of Escherichia coli and the results achieved from the route of entry and action, reactive oxygen species (ROS), and antioxidant nature of nanoparticles were evidence of its properties. Computational studies further supported these findings, indicating much of the phytochemicals present in ROE well interact with the bacterial surface proteins. Similarly, the synthesized Ag&SeNPs@rGONM's was effective against Fusarium graminearum and Alternaria alternata in a dose dependent manner than its original nanomaterials. In addition, the docking study also confirmed that rosmarinic acid and caffeic acid prominently interacted with the fungal proteins. Interestingly, Ag&SeNPs@rGONM's pronounced less toxic effect compared to AgNPs and SeNPs against Artemia salina, which shows its biocompatibility.

Surface proteins of Bifidobacterium bifidum DNG6 growing in 2′-fucosyllactose alleviating lipopolysaccharide-induced intestinal barrier injury in vitro

Human milk oligosaccharides promote the growth and adhesion of Bifidobacteria, thus exerting multiple biological functions on intestinal epithelial cells. Bacterial surface proteins play an important role in bacterial-host intestinal epithelial interactions. In this study, we aimed to investigate the effects of surface proteins extracted from Bifidobacterium bifidum DNG6 (B. bifidum DNG6) consuming 2'-fucosyllactose (2'-FL) on Caco-2 cells monolayer barrier injury induced by lipopolysaccharide, compared with lactose and galacto-oligosaccharides. Our results indicated that 2'-FL may promote the surface proteins of B. bifidum DNG6 to improve intestinal barrier injury by positively regulating the NF-κB signaling pathway, reducing inflammation (TNF-α reduced by 50.34%, IL-6 reduced by 22.83%, IL-1β reduced by 37.91%, and IL-10 increased by 63.47%) and strengthening tight junction proteins (ZO,1 2.39×; claudin,1 2.79×; and occluding, 4.70×). The findings of this study indicate that 2'-FL can further regulate intestinal barrier damage by promoting the alteration of B. bifidum DNG6 surface proteins. The findings of this research will also provide theoretical support for the development of synbiotic formulations.

The Balance between Protealysin and Its Substrate, the Outer Membrane Protein OmpX, Regulates Serratia proteamaculans Invasion.

Serratia are opportunistic bacteria, causing infections in plants, insects, animals and humans under certain conditions. The development of bacterial infection in the human body involves several stages of host-pathogen interaction, including entry into non-phagocytic cells to evade host immune cells. The facultative pathogen Serratia proteamaculans is capable of penetrating eukaryotic cells. These bacteria synthesize an actin-specific metalloprotease named protealysin. After transformation with a plasmid carrying the protealysin gene, noninvasive E. coli penetrate eukaryotic cells. This suggests that protealysin may play a key role in S. proteamaculans invasion. This review addresses the mechanisms underlying protealysin's involvement in bacterial invasion, highlighting the main findings as follows. Protealysin can be delivered into the eukaryotic cell by the type VI secretion system and/or by bacterial outer membrane vesicles. By cleaving actin in the host cell, protealysin can mediate the reversible actin rearrangements required for bacterial invasion. However, inactivation of the protealysin gene leads to an increase, rather than decrease, in the intensity of S. proteamaculans invasion. This indicates the presence of virulence factors among bacterial protealysin substrates. Indeed, protealysin cleaves the virulence factors, including the bacterial surface protein OmpX. OmpX increases the expression of the EGFR and β1 integrin, which are involved in S. proteamaculans invasion. It has been shown that an increase in the invasion of genetically modified S. proteamaculans may be the result of the accumulation of full-length OmpX on the bacterial surface, which is not cleaved by protealysin. Thus, the intensity of the S. proteamaculans invasion is determined by the balance between the active protealysin and its substrate OmpX.

Exploring Residue-Level Interactions between the Biofilm-Driving R2ab Protein and Polystyrene Nanoparticles.

In biological systems, proteins can bind to nanoparticles to form a "corona" of adsorbed molecules. The nanoparticle corona is of significant interest because it impacts an organism's response to a nanomaterial. Understanding the corona requires knowledge of protein structure, orientation, and dynamics at the surface. A residue-level mapping of protein behavior on nanoparticle surfaces is needed, but this mapping is difficult to obtain with traditional approaches. Here, we have investigated the interaction between R2ab and polystyrene nanoparticles (PSNPs) at the level of individual residues. R2ab is a bacterial surface protein from Staphylococcus epidermidis and is known to interact strongly with polystyrene, leading to biofilm formation. We have used mass spectrometry after lysine methylation and hydrogen-deuterium exchange (HDX) NMR spectroscopy to understand how the R2ab protein interacts with PSNPs of different sizes. Lysine methylation experiments reveal subtle but statistically significant changes in methylation patterns in the presence of PSNPs, indicating altered protein surface accessibility. HDX rates become slower overall in the presence of PSNPs. However, some regions of the R2ab protein exhibit faster than average exchange rates in the presence of PSNPs, while others are slower than the average behavior, suggesting conformational changes upon binding. HDX rates and methylation ratios support a recently proposed "adsorbotope" model for PSNPs, wherein adsorbed proteins consist of unfolded anchor points interspersed with partially structured regions. Our data also highlight the challenges of characterizing complex protein-nanoparticle interactions using these techniques, such as fast exchange rates. While providing insights into how R2ab adsorbs onto PSNP surfaces, this research emphasizes the need for advanced methods to comprehend residue-level interactions in the nanoparticle corona.

Introduction of Spontaneous Mutations Using Streptomycin as a Method for Lactic Acid Bacteria Breeding.

The chapter presents a technique for inducing spontaneous mutations using antibiotics that target microbial ribosomes and/or RNA polymerase, employed in bacterial breeding. In contrast to UV-based mutagenesis, this method allows control of the mutation sites, specifically targeting the rpsL gene. The outlined methodology introduces spontaneous mutations using streptomycin in Lacticaseibacillus rhamnosus GG ATCC 53103 (LGG), a widely studied lactic acid bacterium. Streptomycin has been shown to induce mutations in the rpsL gene, particularly altering lysine residues at position 56 or 101. It has also been reported to affect bacterial morphology and surface protein composition, thereby enhancing adhesion to human mucin.

The Application of Bacteriophage and Photoacoustic Flow Cytometry in Bacterial Identification.

Infection with resistant bacteria has become an ever-increasing problem in modern medical practice. Bacteremia is a serious and potentially lethal condition that can lead to sepsis without early intervention. Currently, broad-spectrum antibiotics are prescribed until bacteria can be identified through blood cultures, a process that can take 2-3 days and is unable to provide quantitative information. Staphylococcus aureus (S. aureus) is a leading cause of bacteremia, and methicillin-resistant S. aureus (MRSA) accounts for more than a third of the cases. Other bacteria such as Clostridium difficile, Acinetobacter baumannii, and Carbapenem-resistant Enterobacteriaceae are becoming more prevalent and antibiotic-resistant. Rapid diagnostics for each of these superbugs has been a priority for health organizations around the world. Bacteriophages have evolved for millions of years to develop exquisite specificity in target binding using their host attachment proteins. Bacteriophages are viruses that infect bacteria. Bacteriophages use tail spikes, specialized attachment proteins, to bind specifically to their target bacterial cell surface proteins. We use bacteriophages and parts of bacteriophages as specific tags coupled with photoacoustic flow cytometry for the detection and quantification of bacteria. In photoacoustic flow cytometry, laser light is absorbed by particles under flow, and the ultrasound waves generated on the release of the energy are detected. Photoacoustics involves the detection of ultrasound waves resulting from laser irradiation. In photoacoustic flow cytometry, pulsed laser light is delivered to a sample flowing past a focused transducer, and particles that absorb laser light create a photoacoustic response. Bacteria can be tagged with dyed bacteriophage and processed through a photoacoustic flow cytometer where they are detected by the acoustic response. In this chapter, we describe the procedure and methods used to accomplish this. Often the limiting factor for the treatment of patients is the time spent waiting for results. It is our hope that the work presented in this chapter can be a foundation for future work and provide an ability to detect bacterial pathogens in blood cultures. Bacterial plate cultures and Gram staining are nineteenth-century technologies that have been the gold standards for decades, but current trends in resistant bacteria have necessitated a move toward more rapid and quantifiable diagnostic tools.

Amyloid Fibrils Produced by Streptococcus sanguinis Contribute to Biofilm Formation and Immune Evasion.

Bacterial surface proteins assembled into amyloids contribute to biofilm formation and host immune evasion. Streptococcus sanguinis, a pioneer colonizer of teeth commonly involved in cardiovascular infections, expresses about thirty-three proteins anchored to the cell wall by sortase A. Here, we characterized the production of amyloid in S. sanguinis strains differing in biofilm and immune evasion phenotypes and investigated the role of sortase A in amyloidogenesis. Amyloid was identified in biofilms formed by nine strains, using Congo red (CR) staining and cross-polarized light microscopy. Additionally, EGCG, an amyloid inhibitor, impaired biofilm maturation in a strain-specific fashion. The amounts of amyloid-like components quantified in culture fluids of nine strains using thioflavin T and fluorimetry negatively correlated with bacterial binding to complement-activating proteins (SAP, C1q), C3b deposition and rates of opsonophagocytosis in PMNs, implying amyloid production in immune evasion. The deletion of the sortase A gene (srtA) in strain SK36 compromised amyloid production and sucrose-independent biofilm maturation. The srtA mutant further showed increased susceptibility to C3b deposition and altered interactions with PMNs as well as reduced persistence in human blood. These findings highlight the contribution of amyloids to biofilm formation and host immune evasion in S. sanguinis strains, further indicating the participation of sortase A substrates in amyloidogenesis.

In vitro antibiofilm and bacteriostatic activity of diacerein against Enterococcus faecalis

Enterococcus faecalis is one of the main pathogens that causes hospital-acquired infections because it is intrinsically resistant to some antibiotics and often is capable of biofilm formation, which plays a critical role in resisting the external environment. Therefore, attacking biofilms is a potential therapeutic strategy for infections caused by E. faecalis. Current research indicates that diacerein used in the treatment of osteoarthritis showed antimicrobial activity on strains of gram-positive cocci in vitro. In this study, we tested the MICs of diacerein using the broth microdilution method, and successive susceptibility testing verified that E. faecalis is unlikely to develop resistance to diacerein. In addition, we obtained a strain of E. faecalis HE01 with strong biofilm-forming ability from an eye hospital environment and demonstrated that diacerein affected the biofilm development of HE01 in a dose-dependent manner. Then, we explored the mechanism by which diacerein inhibits biofilm formation through qRT-PCR, extracellular protein assays, hydrophobicity assays and transcriptomic analysis. The results showed that biofilm formation was inhibited at the initial adhesion stage by inhibition of the expression of the esp gene, synthesis of bacterial surface proteins and reduction in cell hydrophobicity. In addition, transcriptome analysis showed that diacerein not only inhibited bacterial growth by affecting the oxidative phosphorylation process and substance transport but also inhibited biofilm formation by affecting secondary metabolism, biosynthesis, the ribosome pathway and luxS expression. Thus, our findings provide compelling evidence for the substantial therapeutic potential of diacerein against E. faecalis biofilms.

Analysis of Bacterial Phosphorylcholine-Related Genes Reveals an Association between Type-Specific Biosynthesis Pathways and Biomolecules Targeted for Phosphorylcholine Modification.

Many bacterial surface proteins and carbohydrates are modified with phosphorylcholine (ChoP), which contributes to host mimicry and can also promote colonization and survival in the host. However, the ChoP biosynthetic pathways that are used in bacterial species that express ChoP have not been systematically studied. For example, the well-studied Lic-1 pathway is absent in some ChoP-expressing bacteria, such as Neisseria meningitidis and Neisseria gonorrhoeae. This raises a question as to the origin of the ChoP used for macromolecule biosynthesis in these species. In the current study, we used in silico analyses to identify the potential pathways involved in ChoP biosynthesis in genomes of the 26 bacterial species reported to express a ChoP-modified biomolecule. We used the four known ChoP biosynthetic pathways and a ChoP transferase as search terms to probe for their presence in these genomes. We found that the Lic-1 pathway is primarily associated with organisms producing ChoP-modified carbohydrates, such as lipooligosaccharide. Pilin phosphorylcholine transferase A (PptA) homologs were detected in all bacteria that express ChoP-modified proteins. Additionally, ChoP biosynthesis pathways, such as phospholipid N-methyltransferase (PmtA), phosphatidylcholine synthase (Pcs), or the acylation-dependent phosphatidylcholine biosynthesis pathway, which generate phosphatidylcholine, were also identified in species that produce ChoP-modified proteins. Thus, a major finding of this study is the association of a particular ChoP biosynthetic pathway with a cognate, target ChoP-modified surface factor; i.e., protein versus carbohydrate. This survey failed to identify a known biosynthetic pathway for some species that express ChoP, indicating that a novel ChoP biosynthetic pathway(s) may remain to be identified. IMPORTANCE The modification of bacterial surface virulence factors with phosphorylcholine (ChoP) plays an important role in bacterial virulence and pathogenesis. However, the ChoP biosynthetic pathways in bacteria have not been fully understood. In this study, we used in silico analysis to identify potential ChoP biosynthetic pathways in bacteria that express ChoP-modified biomolecules and found the association between a specific ChoP biosynthesis pathway and the cognate target ChoP-modified surface factor.

Structural evolution of an immune evasion determinant shapes pathogen host tropism

Modern infectious disease outbreaks often involve changes in host tropism, the preferential adaptation of pathogens to specific hosts. The Lyme disease-causing bacterium Borrelia burgdorferi (Bb) is an ideal model to investigate the molecular mechanisms of host tropism, because different variants of these tick-transmitted bacteria are distinctly maintained in rodents or bird reservoir hosts. To survive in hosts and escape complement-mediated immune clearance, Bb produces the outer surface protein CspZ that binds the complement inhibitor factor H (FH) to facilitate bacterial dissemination in vertebrates. Despite high sequence conservation, CspZ variants differ in human FH-binding ability. Together with the FH polymorphisms between vertebrate hosts, these findings suggest that minor sequence variation in this bacterial outer surface protein may confer dramatic differences in host-specific, FH-binding-mediated infectivity. We tested this hypothesis by determining the crystal structure of the CspZ-human FH complex, and identifying minor variation localized in the FH-binding interface yielding bird and rodent FH-specific binding activity that impacts infectivity. Swapping the divergent region in the FH-binding interface between rodent- and bird-associated CspZ variants alters the ability to promote rodent- and bird-specific early-onset dissemination. We further linked these loops and respective host-specific, complement-dependent phenotypes with distinct CspZ phylogenetic lineages, elucidating evolutionary mechanisms driving host tropism emergence. Our multidisciplinary work provides a novel molecular basis for how a single, short protein motif could greatly modulate pathogen host tropism.

The characteristics of nano-micron calcite particles/common bacteria complex and its interfacial interaction.

Based on the composite pollution of atmospheric microbial aerosol, this paper selects the calcite/bacteria complex as the research object which was prepared by calcite particles and two common strains of bacteria (Escherichia coli, Staphylococcus aureus) in the solution system. The morphology, particle size, surface potential, and surface groups of the complex were explored by modern analysis and testing methods, with an emphasis on the interfacial interaction between calcite and bacteria. The SEM, TEM, and CLSM results showed that the morphology of the complex could be divided into three types: bacteria adhering to the surface or edge of micro-CaCO3, bacteria aggregating with nano-CaCO3, and single nano-CaCO3 wrapping bacteria. The complex's particle size was about 2.07 ~ 192.4 times larger than the original mineral particles, and the nano-CaCO3/bacteria complex's particle size variation was caused by the fact that nano-CaCO3 has agglomeration in solution. The surface potential of the micro-CaCO3/bacteria complex (isoelectric point pH = 3.0) lies between micro-CaCO3 and bacteria, while the surface potential of the nano-CaCO3/bacteria complex (isoelectric point pH = 2.0) approaches the nano-CaCO3. The complex's surface groups were based primarily on the infrared characteristics of calcite particles, accompanied by the infrared characteristics of bacteria, displaying the interfacial interaction from the protein, polysaccharides, and phosphodiester groups of bacteria. The interfacial action of the micro-CaCO3/bacteria complex is mainly driven by electrostatic attraction and hydrogen bonding force, while the nano-CaCO3/bacteria complex is guided by surface complexation and hydrogen bonding force. The increase in the β-fold/α-helix ratio of the calcite/S. aureus complex indicated that the secondary structure of bacterial surface proteins was more stable and the hydrogen bond effect was strong than the calcite/E. coli complex. The findings are expected to provide basic data for the mechanism research of atmospheric composite particles closer to the real environment.

Involvement of host iron-withholding strategy on Streptococcus pyogenes strain KSU-1 growth and pathogenicity

Background and objectives Streptococcus pyogenes is a highly adaptable human pathogen that can cause a wide spectrum of infections ranging from mild to a life-threating systemic infection. This study discussed the effectiveness of iron-depriving strategy on growth, survival, and virulence of S. pyogenes. Materials and methods Some comparisons between different iron-saturated and iron-depleted forms of the main human iron reservoirs (hemoglobin, hemin, transferrin, lactoferrin, and human milk) were tested for their effect on growth and pathogenicity of S. pyogenes. Results and conclusion Although the iron-saturated forms enhanced the growth and survival, the iron-free forms had a bacteriostatic/bactericidal activity against the microbe, and these results were emphasized by the in vivo study. Finally, the bacterial surface proteins as virulence factors were secreted upon iron depletion as indicated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This study proved that iron-depletion environment increased the resistance and virulence factors of S. pyogenes. Moreover, these results can give an insight about the interaction between the host and pathogen, which can lead to designing of new antimicrobial agents or vaccine that may target these pathways.

Berberine disrupts staphylococcal proton motive force to cause potent anti-staphylococcal effects in vitro

The presence of antibiotic resistance has increased the urgency for more effective treatments of bacterial infections. Biofilm formation has complicated this issue as biofilm bacteria become tolerant to antibiotics due to environmental factors such as nutrient deprivation and adhesion. In septic arthritis, a disease with an 11% mortality rate, bacteria in synovial fluid organize into floating, protein-rich, bacterial aggregates (mm-cm) that display depressed metabolism and antibiotic tolerance. In this study, Staphylococcus aureus (S. aureus), which is the most common pathogen in septic arthritis, was tested against different inhibitors that modulate bacterial surface protein availability and that should decrease bacterial aggregation. One of these, berberine, a quaternary ammonium compound, was found to reduce bacterial counts by 3-7 logs in human synovial fluid (aggregating medium) with no effect in tryptic soy broth (TSB, non-aggregating). Unlike traditional antibiotics, the bactericidal activity of berberine appeared to be independent of bacterial metabolism. To elucidate the mechanism, we used synovial fluid fractionation, targeted MRSA transposon insertion mutants, dyes to assess changes in membrane potential (DiSC3(5)) and membrane permeability (propidium iodide (PI)), colony counting, and fluorescence spectroscopy. We showed that berberine's activity was dependent on an alkaline pH and berberine killed both methicillin-sensitive S. aureus and MRSA in alkaline media (pH 8.5-9.0; p<0.0001 vs. same pH controls). Under these alkaline conditions, berberine localized to S. aureus where berberine was isolated in cytoplasmic (∼95%) and DNA (∼5%) fractions. Importantly, berberine increased bacterial cell membrane permeability, and disrupted the proton motive force, suggesting a mechanism whereby it may be able to synergize with other antibacterial compounds under less harsh conditions. We suggest that berberine, which is cheap and readily available, can be made into an effective treatment.

The Antioxidant Activity and Protection of Probiotic Bacteria in the In Vitro Gastrointestinal Digestion of a Blueberry Juice and Whey Protein Fermentation System

Blueberries have received great attention due to the health effects of their bioactive compounds, such as antioxidant, antitumor, and anti-obesity properties. Probiotics also have these health-promoting benefits. However, these biological activities may be affected by the processs of gastrointestinal digestion, which decreases their functionality. This study aimed to use a more convenient method to improve the blueberries’ antioxidant activity and protective effects on probiotic cells by fermentation with whey protein, and to explore the possible mechanisms underlying these effects. This result showed that the total phenolic content, anthocyanin content, reducing power, DPPH radical scavenging capacity, and probiotic cells’ survival in a blueberry juice and whey protein fermentation system were enhanced in a model of in vitro gastrointestinal digestion. The bioactive compounds in blueberry juice interacted with whey protein, as shown through FTIR. The stability of phenolic compounds was enhanced, and the release of functional compounds in the mixture fermentation system was delayed through CLSM. Interactions between bioactive compounds in blueberries, whey protein, and bacterial surface proteins, glycoproteins or polysaccharides during fermentation were studied by SDS-PAGE. Thus, the stability of bioactive activities in the mixed system after fermentation was strengthened by the interaction. The mixed fermentation system has promising potential for improving antioxidant activity and protecting probiotic cells.

An innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins.

Sensing of pathogens by ubiquitination is a critical arm of cellular immunity. However, universal ubiquitination targets on microbes remain unidentified. Here, using in vitro, ex vivo, and in vivo studies, we identify the first protein-based ubiquitination substrates on phylogenetically diverse bacteria by unveiling a strategy that uses recognition of degron-like motifs. Such motifs form a new class of intra-cytosolic pathogen-associated molecular patterns (PAMPs). Their incorporation enabled recognition of nonubiquitin targets by host ubiquitin ligases. We find that SCFFBW7 E3 ligase, supported by the regulatory kinase, glycogen synthase kinase 3β, is crucial for effective pathogen detection and clearance. This provides a mechanistic explanation for enhanced risk of infections in patients with chronic lymphocytic leukemia bearing mutations in F-box and WD repeat domain containing 7 protein. We conclude that exploitation of this generic pathogen sensing strategy allows conservation of host resources and boosts antimicrobial immunity.

Staphylococcal Periscope proteins Aap, SasG, and Pls project noncanonical legume-like lectin adhesin domains from the bacterial surface

Staphylococcus aureus and Staphylococcus epidermidis are frequently associated with medical device infections that involve establishment of a bacterial biofilm on the device surface. Staphylococcal surface proteins Aap, SasG, and Pls are members of the Periscope Protein class and have been implicated in biofilm formation and host colonization; they comprise a repetitive region ("B region") and an N-terminal host colonization domain within the "A region," predicted to be a lectin domain. Repetitive E-G5 domains (as found in Aap, SasG, and Pls) form elongated "stalks" that would vary in length with repeat number, resulting in projection of the N-terminal A domain variable distances from the bacterial cell surface. Here, we present the structures of the lectin domains within A regions of SasG, Aap, and Pls and a structure of the Aap lectin domain attached to contiguous E-G5 repeats, suggesting the lectin domains will sit at the tip of the variable length rod. We demonstrate that these isolated domains (Aap, SasG) are sufficient to bind to human host desquamated nasal epithelial cells. Previously, proteolytic cleavage or a deletion within the A domain had been reported to induce biofilm formation; the structures suggest a potential link between these observations. Intriguingly, while the Aap, SasG, and Pls lectin domains bind a metal ion, they lack the nonproline cis peptide bond thought to be key for carbohydrate binding by the lectin fold. This suggestion of noncanonical ligand binding should be a key consideration when investigating the host cell interactions of these bacterial surface proteins.

Recombinant vaccine candidates with integrated adjuvants provide stimulation of an effective immune response against bacterial infections

The use of recombinant proteins as vaccine preparations is limited by their weak immunogenicity, which can be enhanced by the use of adjuvants, the development of which is an important and urgent problem of modern vaccinology. Significantly, adjuvants as additives to vaccine preparations are of concern to clinicians. From this point of view, the idea of including an internal adjuvant into the structure of a recombinant protein molecule is of undoubted interest. Previously, we synthesized and studied two recombinant vaccine preparations specific for S. agalactiae (Su4) and S. pneumoniae (PSPF). Each of them was a tandem of immunogenic bacterial surface proteins in combination with an additional adjuvant site. The amino acid sequence identical to flagellin acted as an internal adjuvant. In this work, we investigated the possibility of additional enhancement of the body’s immune response to immunization with recombinant Su4 and PSPF proteins due to the simultaneous administration of an external adjuvant, carboxymethylchitosan or Imject Alum.Studies have shown that the additional introduction of these adjuvants into the composition of the vaccine preparation did not affect the immunogenicity of the Su4 and PSPF proteins, which included the internal adjuvant flagellin. The protective efficacy of the immune response to all immunization options was comparable.Thus, the inclusion of a flagellin insert as an internal adjuvant into the composition of recombinant proteins ensures the development of the highest possible level of the immune response and its protective efficacy against the corresponding pathogens of a bacterial infection.