Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS.

Immunoglobulins M (IgM) are key natural antibodies produced initially in humoral immune response. Due to their large molecular weights and extensive glycosylation loads, IgMs represent a challenging target for conventional mass analysis. Charge detection mass spectrometry (CDMS) may provide a unique approach to tackle heterogeneous IgM assemblies, although this technique can be quite laborious and technically challenging. Here, we describe the use of online size exclusion chromatography (SEC) to automate buffer exchange and sample introduction, and demonstrate its adaptability with Orbitrap-based CDMS. We discuss optimal experimental parameters for online SEC-CDMS experiments, including ion activation, choice of column, and resolution. Using this approach, CDMS histograms containing hundreds of individual ion signals can be obtained in as little as 5 min from single injections of <1 µg of sample. To demonstrate the unique utility of online SEC-CDMS, we performed real-time kinetic monitoring of pentameric IgM digestion by the protease IgMBRAZOR, which cleaves specifically in the hinge region of IgM. Several digestion intermediates corresponding to processive losses of F(ab')2 subunits could be mass-resolved and identified by SEC-CDMS. Interestingly, we find that for the J-chain linked IgM pentamer, cleavage of one of the F(ab')2 subunits is much slower than the other four F(ab')2 subunits, which we attribute to the symmetry-breaking interactions of the J-chain within the pentameric IgM structure. The online SEC-CDMS methodologies described here open new avenues into the higher throughput automated analysis of heterogeneous, high-mass protein assemblies by CDMS.

Characterization of MdpS: an in-depth analysis of a MUC5B-degrading protease from Streptococcus oralis.

Oral biofilms, comprising hundreds of bacteria and other microorganisms on oral mucosal and dental surfaces, play a central role in oral health and disease dynamics. Streptococcus oralis, a key constituent of these biofilms, contributes significantly to the formation of which, serving as an early colonizer and microcolony scaffold. The interaction between S. oralis and the orally predominant mucin, MUC5B, is pivotal in biofilm development, yet the mechanism underlying MUC5B degradation remains poorly understood. This study introduces MdpS (Mucin Degrading Protease from Streptococcus oralis), a protease that extensively hydrolyses MUC5B and offers an insight into its evolutionary conservation, physicochemical properties, and substrate- and amino acid specificity. MdpS exhibits high sequence conservation within the species and also explicitly among early biofilm colonizing streptococci. It is a calcium or magnesium dependent serine protease with strict physicochemical preferences, including narrow pH and temperature tolerance, and high sensitivity to increasing concentrations of sodium chloride and reducing agents. Furthermore, MdpS primarily hydrolyzes proteins with O-glycans, but also shows activity toward immunoglobulins IgA1/2 and IgM, suggesting potential immunomodulatory effects. Significantly, MdpS extensively degrades MUC5B in the N- and C-terminal domains, emphasizing its role in mucin degradation, with implications for carbon and nitrogen sequestration for S. oralis or oral biofilm cross-feeding. Moreover, depending on substrate glycosylation, the amino acids serine, threonine or cysteine triggers the enzymatic action. Understanding the interplay between S. oralis and MUC5B, facilitated by MdpS, has significant implications for the management of a healthy eubiotic oral microenvironment, offering potential targets for interventions aimed at modulating oral biofilm composition and succession. Additionally, since MdpS does not rely on O-glycan removal prior to extensive peptide backbone hydrolysis, the MdpS data challenges the current model of MUC5B degradation. These findings emphasize the necessity for further research in this field.

Antibiotic resistance monitoring in wastewater in the Nordic countries: A systematic review.

The Nordic countries (Denmark, Finland, Iceland, Norway, and Sweden) have effectively kept lower antibiotic-resistant bacterial (ARB) pathogen rates than many other countries. However, in recent years, these five countries have encountered a rise in ARB cases and challenges in treating infections due to the growing prevalence of ARB pathogens. Wastewater-based surveillance (WBS) is a valuable supplement to clinical methods for ARB surveillance, but there is a lack of comprehensive understanding of WBS application for ARB in the Nordic countries. This review aims to compile the latest state-of-the-art developments in WBS for ARB monitoring in the Nordic countries and compare them with clinical surveillance practices. After reviewing 1480 papers from the primary search, 54 were found relevant, and 15 additional WBS-related papers were included. Among 69 studies analyzed, 42 dedicated clinical epidemiology, while 27 focused on wastewater monitoring. The PRISMA review of the literature revealed that Nordic countries focus on four major WBS objectives of ARB: assessing ARB in the human population, identifying ARB evading wastewater treatment, quantifying removal rates, and evaluating potential ARB evolution during the treatment process. In both clinical and wastewater contexts, the most studied targets were pathogens producing carbapenemase and extended-spectrum beta-lactamase (ESBL), primarily Escherichia coli and Klebsiella spp. However, vancomycin-resistant Enterococcus (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) have received more attention in clinical epidemiology than in wastewater studies, probably due to their lower detection rates in wastewater. Clinical surveillance has mostly used culturing, antibiotic susceptibility testing, and genotyping, but WBS employed PCR-based and metagenomics alongside culture-based techniques. Imported cases resulting from international travel and hospitalization abroad appear to have frequently contributed to the rise in ARB pathogen cases in these countries. The many similarities between the Nordic countries (e.g., knowledge exchange practices, antibiotic usage patterns, and the current ARB landscape) could facilitate collaborative efforts in developing and implementing WBS for ARB in population-level screening.

Delivery of a sebum modulator by an engineered skin microbe in mice.

Microorganisms can be equipped with synthetic genetic programs for the production of targeted therapeutic molecules. Cutibacterium acnes is the most abundant commensal of the human skin, making it an attractive chassis to create skin-delivered therapeutics. Here, we report the engineering of this bacterium to produce and secrete the therapeutic molecule neutrophil gelatinase-associated lipocalin, in vivo, for the modulation of cutaneous sebum production.

Direct Comparison of the Hinge-Cleaving Proteases IgdE and BdpK for LC-MS-Based IgG1 Clonal Profiling.

Human antibodies are heterogeneous molecules primarily due to clonal sequence variations. Analytical techniques to assess antibody levels quantitatively, such as ELISA, lack the power to resolve abundances at the clonal level. Recently, we introduced an LC-MS-based approach that can distinguish and quantify antibody clones using the mass and retention time of their corresponding Fab-fragments. We used specific hinge-cleaving protease IgdE (FabALACTICA) to release the Fab-fragments from the constant Fc region of the antibody. Here, we explore an alternative IgG1 hinge-cleaving protease, BdpK (FabDELLO), and compare it directly to IgdE for use in IgG1 repertoire profiling. We used IgdE and BdpK in parallel to digest all IgG1s from the same set of plasma samples. Both proteases cleave IgG1 specifically in the hinge, albeit via different mechanisms and at two distinct cleavage sites. Notwithstanding these differences, the Fab fragments generated by IgdE or BdpK produced highly similar clonal repertoires. However, IgdE required ∼16 h of incubation to digest plasma IgG1s, while BdpK required ∼2 h. We authenticated the similarity of the clones by top-down proteomics using electron transfer dissociation. We conclude that BdpK performs very well in digesting polyclonal plasma IgG1s and that neither BdpK nor IgdE displays detectable biases in cleaving IgG1s. We anticipate that BdpK may emerge as the preferred protease for IgG1 hinge-digestion because it offers a shorter digestion time compared to IgdE, an equally specific digestion site, and no bias against any IgG1 present in plasma.

Unveiling Rare Pathogens and Antibiotic Resistance in Tanzanian Cholera Outbreak Waters.

The emergence of antibiotic resistance is a global health concern. Therefore, understanding the mechanisms of its spread is crucial for implementing evidence-based strategies to tackle resistance in the context of the One Health approach. In developing countries where sanitation systems and access to clean and safe water are still major challenges, contamination may introduce bacteria and bacteriophages harboring antibiotic resistance genes (ARGs) into the environment. This contamination can increase the risk of exposure and community transmission of ARGs and infectious pathogens. However, there is a paucity of information on the mechanisms of bacteriophage-mediated spread of ARGs and patterns through the environment. Here, we deploy Droplet Digital PCR (ddPCR) and metagenomics approaches to analyze the abundance of ARGs and bacterial pathogens disseminated through clean and wastewater systems. We detected a relatively less-studied and rare human zoonotic pathogen, Vibrio metschnikovii, known to spread through fecal--oral contamination, similarly to V. cholerae. Several antibiotic resistance genes were identified in both bacterial and bacteriophage fractions from water sources. Using metagenomics, we detected several resistance genes related to tetracyclines and beta-lactams in all the samples. Environmental samples from outlet wastewater had a high diversity of ARGs and contained high levels of blaOXA-48. Other identified resistance profiles included tetA, tetM, and blaCTX-M9. Specifically, we demonstrated that blaCTX-M1 is enriched in the bacteriophage fraction from wastewater. In general, however, the bacterial community has a significantly higher abundance of resistance genes compared to the bacteriophage population. In conclusion, the study highlights the need to implement environmental monitoring of clean and wastewater to inform the risk of infectious disease outbreaks and the spread of antibiotic resistance in the context of One Health.

Oxygen Exposure and Tolerance Shapes the Cell Wall-Associated Lipids of the Skin Commensal Cutibacterium acnes.

Cutibacterium acnes is one of the most abundant bacteria on the skin. Being exposed to oxygen and oxic stress, the secretion of the bacterial antioxidant protein RoxP ensures an endogenous antioxidant system for the preservation of skin health. To investigate the impact of the antioxidant RoxP on oxidation of the bacteria, wildtype and an isogenic roxp mutant were cultured in anaerobic and oxic conditions. The carbonylated status of proteins were recorded, as were the most significant modifications in a relative intensity of free fatty acids (FFA) and lipids containing fatty acids (FA), such as di- (DG) and triglycerides (TG), di- (DGDG) and sulfoquinozyldiacylglycerol (SQDG) and ceramides. Concerning the fatty acid types, it was observed that the free fatty acids contained mainly C12:0-C26:0 in hydroxy and acylated forms, the DG contained mainly C29:0-C37:0, the TG contained mainly C19:0-C33:0, and the DGDG/SQDGs contained very long fatty acids (C29:0-C37:0) demonstrating the interdependence of de novo synthesis of lipids and RoxP. The area of DGDG peaks (924.52, 929.56 and 930.58) were affected by bacterial growth conditions, with the exception of m/z 910.61. Moreover, the FFA unsaturation is wider in the SQDG species (C30:0 to C36:6) than in DG, TG or free FFA species. It could be concluded that both environmental oxidative statuses, as well as the prevalence of bacterial antioxidant systems, significantly shape the lipidome of C. acnes.

Expression of the Bacterial Enzyme IdeS Using a GFP Fusion in the Yeast Saccharomyces cerevisiae.

Bacterial proteases are important enzymes used in several technical applications where controlled cleavage of proteins is needed. They are challenging enzymes to express recombinantly as parts of the proteome can be hydrolyzed by their activity. The eukaryotic model organism Saccharomyces cerevisiae is potentially a good expression host as it tolerates several stress conditions and is known to better express insoluble proteins compared to bacterial systems. In this chapter we describe how the protease IdeS from Streptococcus pyogenes can be expressed in S. cerevisiae. The expression of IdeS was followed by constructing a fused protein with GFP and measuring the fluorescence with flow cytometry. The protease presence was confirmed with a Western blot assay and activity was measured with an in vitro assay. To reduce potentially toxic effect on the host cell, the growth and production phases were separated by using the inducible promoter GAL1p to control recombinant gene expression. The protocol provided may be adopted for other bacterial proteases through minor modifications of the fused protein.

Characterization of a highly conserved MUC5B-degrading protease, MdpL, from Limosilactobacillus fermentum.

MUC5B is the predominant glycoprotein in saliva and is instrumental in the establishment and maintenance of multi-species eubiotic biofilms in the oral cavity. Investigations of the aciduric Lactobacillaceae family, and its role in biofilms emphasizes the diversity across different genera of the proteolytic systems involved in the nutritional utilization of mucins. We have characterized a protease from Limosilactobacillus fermentum, MdpL (Mucin degrading protease from Limosilactobacillus) with a high protein backbone similarity with commensals that exploit mucins for attachment and nutrition. MdpL was shown to be associated with the bacterial cell surface, in close proximity to MUC5B, which was sequentially degraded into low molecular weight fragments. Mapping the substrate preference revealed multiple hydrolytic sites of proteins with a high O-glycan occurrence, although hydrolysis was not dependent on the presence of O-glycans. However, since proteolysis of immunoglobulins was absent, and general protease activity was low, a preference for glycoproteins similar to MUC5B in terms of glycosylation and structure is suggested. MdpL preferentially hydrolyzed C-terminally located hydrophobic residues in peptides larger than 20 amino acids, which hinted at a limited sequence preference. To secure proper enzyme folding and optimal conditions for activity, L. fermentum incorporates a complex system that establishes a reducing environment. The importance of overall reducing conditions was confirmed by the activity boosting effect of the added reducing agents L-cysteine and DTT. High activity was retained in low to neutral pH 5.5-7.0, but the enzyme was completely inhibited in the presence of Zn2+. Here we have characterized a highly conserved mucin degrading protease from L. fermentum. MdpL, that together with the recently discovered O-glycanase and O-glycoprotease enzyme groups, increases our understanding of mucin degradation and complex biofilm dynamics.

Enrichment of antibiotic resistance genes within bacteriophage populations in saliva samples from individuals undergoing oral antibiotic treatments.

Spread of antibiotic resistance is a significant challenge for our modern health care system, and even more so in developing countries with higher prevalence of both infections and resistant bacteria. Faulty usage of antibiotics has been pinpointed as a driving factor in spread of resistant bacteria through selective pressure. However, horizontal gene transfer mediated through bacteriophages may also play an important role in this spread. In a cohort of Tanzanian patients suffering from bacterial infections, we demonstrate significant differences in the oral microbial diversity between infected and non-infected individuals, as well as before and after oral antibiotics treatment. Further, the resistome carried both by bacteria and bacteriophages vary significantly, with bla CTX-M1 resistance genes being mobilized and enriched within phage populations. This may impact how we consider spread of resistance in a biological context, as well in terms of treatment regimes.

Biogeographical variation in antimicrobial resistance in rivers is influenced by agriculture and is spread through bacteriophages.

Antibiotic resistance is currently an extensive medical challenge worldwide, with global numbers increasing steadily. Recent data have highlighted wastewater treatment plants as a reservoir of resistance genes. The impact of these findings for human health can best be summarized using a One Health concept. However, the molecular mechanisms impacting resistance spread have not been carefully evaluated. Bacterial viruses, that is bacteriophages, have recently been shown to be important mediators of bacterial resistance genes in environmental milieus and are transferrable to human pathogens. Herein, we investigated the biogeographical impact on resistance spread through river-borne bacteriophages using amplicon deep sequencing of the microbiota, absolute quantification of resistance genes using ddPCR, and phage induction capacity within wastewater. Microbial biodiversity of the rivers is significantly affected by river site, surrounding milieu and time of sampling. Furthermore, areas of land associated with agriculture had a significantly higher ability to induce bacteriophages carrying antibiotic resistance genes, indicating their impact on resistance spread. It is imperative that we continue to analyse global antibiotic resistance problem from a One Health perspective to gain novel insights into mechanisms of resistance spread.

Engineering selectivity of Cutibacterium acnes phages by epigenetic imprinting.

Cutibacterium acnes (C. acnes) is a gram-positive bacterium and a member of the human skin microbiome. Despite being the most abundant skin commensal, certain members have been associated with common inflammatory disorders such as acne vulgaris. The availability of the complete genome sequences from various C. acnes clades have enabled the identification of putative methyltransferases, some of them potentially belonging to restriction-modification (R-M) systems which protect the host of invading DNA. However, little is known on whether these systems are functional in the different C. acnes strains. To investigate the activity of these putative R-M and their relevance in host protective mechanisms, we analyzed the methylome of six representative C. acnes strains by Oxford Nanopore Technologies (ONT) sequencing. We detected the presence of a 6-methyladenine modification at a defined DNA consensus sequence in strain KPA171202 and recombinant expression of this R-M system confirmed its methylation activity. Additionally, a R-M knockout mutant verified the loss of methylation properties of the strain. We studied the potential of one C. acnes bacteriophage (PAD20) in killing various C. acnes strains and linked an increase in its specificity to phage DNA methylation acquired upon infection of a methylation competent strain. We demonstrate a therapeutic application of this mechanism where phages propagated in R-M deficient strains selectively kill R-M deficient acne-prone clades while probiotic ones remain resistant to phage infection.

A review of green solvent extraction techniques and their use in antibiotic residue analysis.

Antibiotic residues are being continuously recognized in the aquatic environment and in food. Though the concentration of antibiotic residues is typically low, adverse effects on the environment and human health have been observed. Hence, an efficient method to determine numerous antibiotic residues should be simple, inexpensive, selective, with high throughput and with low detection limits. Liquid-based extractions have been exceedingly used for clean-up and preconcentration of antibiotics prior to chromatographic analysis. In order to make methods more green and environmentally sustainable, conventional hazardous organic solvents can be replaced with green solvents. This review presents sampling strategies as well as comprehensive and up-to-date methods for chemical analysis of antibiotic residues in different sample matrices. Particularly, solvent-based sample preparation techniques using green solvents are discussed along with applications in antibiotic residue analysis.

Streptococcus pyogenes Forms Serotype- and Local Environment-Dependent Interspecies Protein Complexes.

Streptococcus pyogenes is known to cause both mucosal and systemic infections in humans. In this study, we used a combination of quantitative and structural mass spectrometry techniques to determine the composition and structure of the interaction network formed between human plasma proteins and the surfaces of different S. pyogenes serotypes. Quantitative network analysis revealed that S. pyogenes forms serotype-specific interaction networks that are highly dependent on the domain arrangement of the surface-attached M protein. Subsequent structural mass spectrometry analysis and computational modeling of one of the M proteins, M28, revealed that the network structure changes across different host microenvironments. We report that M28 binds secretory IgA via two separate binding sites with high affinity in saliva. During vascular leakage mimicked by increasing plasma concentrations in saliva, the binding of secretory IgA was replaced by the binding of monomeric IgA and C4b-binding protein (C4BP). This indicates that an upsurge of C4BP in the local microenvironment due to damage to the mucosal membrane drives the binding of C4BP and monomeric IgA to M28. These results suggest that S. pyogenes has evolved to form microenvironment-dependent host-pathogen protein complexes to combat human immune surveillance during both mucosal and systemic infections. IMPORTANCE Streptococcus pyogenes (group A Streptococcus [GAS]), is a human-specific Gram-positive bacterium. Each year, the bacterium affects 700 million people globally, leading to 160,000 deaths. The clinical manifestations of S. pyogenes are diverse, ranging from mild and common infections like tonsillitis and impetigo to life-threatening systemic conditions such as sepsis and necrotizing fasciitis. S. pyogenes expresses multiple virulence factors on its surface to localize and initiate infections in humans. Among all these expressed virulence factors, the M protein is the most important antigen. In this study, we perform an in-depth characterization of the human protein interactions formed around one of the foremost human pathogens. This strategy allowed us to decipher the protein interaction networks around different S. pyogenes strains on a global scale and to compare and visualize how such interactions are mediated by M proteins.

A Janus-Faced Bacterium: Host-Beneficial and -Detrimental Roles of Cutibacterium acnes.

The bacterial species Cutibacterium acnes (formerly known as Propionibacterium acnes) is tightly associated with humans. It is the dominant bacterium in sebaceous regions of the human skin, where it preferentially colonizes the pilosebaceous unit. Multiple strains of C. acnes that belong to phylogenetically distinct types can co-exist. In this review we summarize and discuss the current knowledge of C. acnes regarding bacterial properties and traits that allow host colonization and play major roles in host-bacterium interactions and also regarding the host responses that C. acnes can trigger. These responses can have beneficial or detrimental consequences for the host. In the first part of the review, we highlight and critically review disease associations of C. acnes, in particular acne vulgaris, implant-associated infections and native infections. Here, we also analyse the current evidence for a direct or indirect role of a C. acnes-related dysbiosis in disease development or progression, i.e., reduced C. acnes strain diversity and/or the predominance of a certain phylotype. In the second part of the review, we highlight historical and recent findings demonstrating beneficial aspects of colonization by C. acnes such as colonization resistance, immune system interactions, and oxidant protection, and discuss the molecular mechanisms behind these effects. This new insight led to efforts in skin microbiota manipulation, such as the use of C. acnes strains as probiotic options to treat skin disorders.

Entamoeba gingivalis: epidemiology, genetic diversity and association with oral microbiota signatures in North Eastern Tanzania.

BACKGROUND: Entamoeba gingivalis has been associated with periodontal diseases. Baseline data from the background population, which could help delimit the role of the parasite in health and disease, remain limited. OBJECTIVE: To describe epidemiological features, genetic diversity, and associations with oral microbiome signatures of E. gingivalis colonisation in Tanzanians with non-oral/non-dental diseases. METHODS: DNAs from 92 oral washings from 52 participants were subject to metabarcoding of ribosomal genes. DNA sequences were identified to genus level and submitted to oral microbiota diversity analyses. RESULTS: Sixteen (31%) of the 52 study participants were E. gingivalis-positive, with no difference in positivity rate according to gender or age. Only one subtype (ST1) was found. Individuals testing positive for E. gingivalis had higher oral microbiota alpha diversity than those testing negative (P = 0.03). Eight of the top-ten most common bacterial genera were shared between the two groups (Alloprevotella, Fusobacterium, Gemella, Haemophilus, Neisseria, Porphyromonas, Prevotella, Streptococcus, and Veillonella). Meanwhile, E. gingivalis carriers and non-carriers were more likely to have Aggregatibacter and Rothia, respectively, among the top-ten most common genera. CONCLUSION: About one third of the cohort carried E. gingivalis ST1, and carriers had higher oral microbiome diversity and were more predominantly colonized by Aggregatibacter.

Lack of Opsonic Antibody Responses to Invasive Infections With Streptococcus dysgalactiae.

INTRODUCTION: Streptococcus dysgalactiae can cause severe recurrent infections. This study aimed to investigate antibody responses following S. dysgalactiae bacteraemia and possible development of protective immunity. MATERIALS AND METHODS: Patients with S. dysgalactiae bacteraemia in the county of Skåne between 2017 and 2018 were prospectively included. Acute and convalescent sera were obtained. All isolates were emm typed and enzyme-linked immunosorbent assay (ELISA) was utilised to analyse specific antibody responses to bacteria and antigens. Bactericidal- and phagocytosis assays were applied to further establish antibody function. RESULTS: Sixteen patients with S. dysgalactiae bacteraemia were included of whom one had recurrent episodes of bacteraemia. Using ELISA with S. dysgalactiae isolates and mutants, development of IgG antibodies was demonstrated in few patients. Type-specific antibodies were demonstrated in one patient when recombinant M proteins as antigens, were applied. The type-specific serum mediated a small increase in phagocytosis but did not facilitate increased killing of the S. dysgalactiae isolate, carrying that M protein, in blood or by phagocytic cells. CONCLUSION: S. dysgalactiae bacteraemia sometimes results in increased levels of antibodies to the infecting pathogen. We did not find evidence that these antibodies are effectively opsonising. Apparent failure to produce opsonising antibodies might partially explain why S. dysgalactiae can cause recurrent invasive infections in the same host.

Development of a Molecular Imprinting-Based Surface Plasmon Resonance Biosensor for Rapid and Sensitive Detection of Staphylococcus aureus Alpha Hemolysin From Human Serum.

Stapylococcus aureus is a common infectious agent in e.g. sepsis, associated with both high mortality rates and severe long-term effects. The cytolytic protein α-hemolysin has repeatedly been shown to enhance the virulence of S. aureus. Combined with an unhindered spread of multi drug-resistant strains, this has triggered research into novel anti virulence (i.e. anti α-hemolysin) drugs. Their functionality will depend on our ability to identify infections that might be alleviated by such. We therefore saw a need for detection methods that could identify individuals suffering from S. aureus infections where α-hemolysin was a major determinant. Molecular imprinted polymers were subsequently prepared on gold coated sensor chips. Used in combination with a surface plasmon resonance biosensor, α-hemolysin could therethrough be quantified from septic blood samples (n = 9), without pre-culturing of the infectious agent. The biosensor recognized α-hemolysin with high affinity (KD = 2.75 x 10-7 M) and demonstrated a statistically significant difference (p < 0.0001) between the α-hemolysin response and potential sample contaminants. The detection scheme proved equally good, or better, when compared to antibody-based detection methods. This novel detection scheme constitutes a more rapid, economical, and user-friendly alternative to many methods currently in use. Heightening both reproducibility and sensitivity, molecular imprinting in combination with surface plasmon resonance (SPR)-technology could be a versatile new tool in clinical- and research-settings alike.

Studies on Bd0934 and Bd3507, Two Secreted Nucleases from Bdellovibrio bacteriovorus, Reveal Sequential Release of Nucleases during the Predatory Cycle.

Bdellovibrio bacteriovorus is an obligate predatory bacterium that invades and kills a broad range of Gram-negative prey cells, including human pathogens. Its potential therapeutic application has been the subject of increased research interest in recent years. However, an improved understanding of the fundamental molecular aspects of the predatory life cycle is crucial for developing this bacterium as a "living antibiotic." During intracellular growth, B. bacteriovorus secretes an arsenal of hydrolases, which digest the content of the host cell to provide growth nutrients for the predator, e.g., prey DNA is completely degraded by the nucleases. Here, we have, on a genetic and molecular level, characterized two secreted DNases from B. bacteriovorus, Bd0934 and Bd3507, and determined the temporal expression profile of other putative secreted nucleases. We conclude that Bd0934 and Bd3507 are likely a part of the predatosome but are not essential for the predation, host-independent growth, prey biofilm degradation, and self-biofilm formation. The detailed temporal expression analysis of genes encoding secreted nucleases revealed that these enzymes are produced in a sequential orchestrated manner. This work contributes to our understanding of the sequential breakdown of the prey nucleic acid by the nucleases secreted during the predatory life cycle of B. bacteriovorusIMPORTANCE Antibiotic resistance is a major global concern with few available new means to combat it. From a therapeutic perspective, predatory bacteria constitute an interesting tool. They not only eliminate the pathogen but also reduce the overall pool of antibiotic resistance genes through secretion of nucleases and complete degradation of exogenous DNA. Molecular knowledge of how these secreted DNases act will give us further insight into how antibiotic resistance, and the spread thereof, can be limited through the action of predatory bacteria.

Proliferation and benevolence-A framework for dissecting the mechanisms of microbial virulence and health promotion.

Key topics in the study of host-microbe interactions-such as the prevention of drug resistance and the exploitation of beneficial effects of bacteria-would benefit from concerted efforts with both mechanistic and evolutionary approaches. But due to differences in intellectual traditions, insights gained in one field rarely benefit the other. Here, we develop a conceptual and analytical framework for the integrated study of host-microbe interactions. This framework partitions the health effects of microbes and the effector molecules they produce into components with different evolutionary implications. It thereby facilitates the prediction of evolutionary responses to inhibition and exploitation of specific molecular mechanisms.