Armed to the Teeth—The Oral Mucosa Immunity System and Microbiota

The oral cavity is inhabited by a wide spectrum of microbial species, and their colonization is mostly based on commensalism. These microbes are part of the normal oral flora, but there are also opportunistic species that can cause oral and systemic diseases. Although there is a strong exposure to various microorganisms, the oral mucosa reduces the colonization of microorganisms with high rotation and secretion of various types of cytokines and antimicrobial proteins such as defensins. In some circumstances, the imbalance between normal oral flora and pathogenic flora may lead to a change in the ratio of commensalism to parasitism. Healthy oral mucosa has many important functions. Thanks to its integrity, it is impermeable to most microorganisms and constitutes a mechanical barrier against their penetration into tissues. Our study aims to present the role and composition of the oral cavity microbiota as well as defense mechanisms within the oral mucosa which allow for maintaining a balance between such numerous species of microorganisms. We highlight the specific aspects of the oral mucosa protecting barrier and discuss up-to-date information on the immune cell system that ensures microbiota balance. This study presents the latest data on specific tissue stimuli in the regulation of the immune system with particular emphasis on the resistance of the gingival barrier. Despite advances in understanding the mechanisms regulating the balance on the microorganism/host axis, more research is still needed on how the combination of these diverse signals is involved in the regulation of immunity at the oral mucosa barrier.

Eradication of Klebsiella Quasipneumoniae By Eudragit-Formulated Klebicin Kvaria In The Intestinal Tract of Mice

Background: Klebsiella quasipneumoniae is an opportunistic pathogen causing antibiotic-resistant infections of the gastrointestinal tract in many clinical cases. Orally delivered bioactive Klebsiella-specific antimicrobial proteins, klebicins, could be a promising method to eradicate Klebsiella species infecting the gut.Methods: Mouse infection model wasestablished based on infection of streptomycin treated BALB/C mice with K. quasipneumoniae strain DSM28212. Four study groups were used (3 animals/group) to test the antimicrobial efficacy of orally delivered klebicin KvarIa: vehicle-only group (control, phosphate-buffered saline), and other three groups with bacteria, antibiotic therapy and 100 µg of uncoated Kvarla, 100 µg coated KvarIa, 1000 µg coated-KvarIa. Because of the general sensitivity of bacteriocins to gastroduodenal proteases, Kvarla doses were coated with Eudragit®, a GMP-certified formulation agent that releases the protein at certain pH. The coating treatment was selected based on measurements of mouse GI tract pH. The quantity of Klebsiella haemolysin gene (khe) in faecal samples of the study animals was used to quantify the presence of Klebsiella.Results: GI colonization of K. quasipneumoniae was achieved only in the antibiotic-treated mice groups. Significant changes in khe marker quantification were found after the use of Eudragit® S100 formulated klebicin KvarIa, at both doses, with a significant reduction of K. quasipneumoniae colonization compared to the vehicle-only control group.Conclusions: Mouse GI tract colonization with K. quasipneumoniae can be achieved if natural gut microbiota is suppressed by prior antibiotic treatment. The study demonstrates that GI infection caused by K. quasipneumoniae can be significantly reduced using Eudragit®-protected klebicin KvarIa.

Antifungal Mechanism of Vip3Aa, a Vegetative Insecticidal Protein, against Pathogenic Fungal Strains

Discovering new antifungal agents is difficult, since, unlike bacteria, mammalian and fungal cells are both eukaryotes. An efficient strategy is to consider new antimicrobial proteins that have variety of action mechanisms. In this study, a cDNA encoding Bacillus thuringiensis Vip3Aa protein, a vegetative insecticidal protein, was obtained at the vegetative growth stage; its antifungal activity and mechanism were evaluated using a bacterially expressed recombinant Vip3Aa protein. The Vip3Aa protein demonstrated various concentration- and time-dependent antifungal activities, with inhibitory concentrations against yeast and filamentous fungi ranging from 62.5 to 125 µg/mL and 250 to 500 µg/mL, respectively. The uptake of propidium iodide and cellular distributions of rhodamine-labeled Vip3Aa into fungal cells indicate that its growth inhibition mechanism involves its penetration within cells and subsequent intracellular damage. Furthermore, we discovered that the death of Candida albicans cells was caused by the induction of apoptosis via the generation of mitochondrial reactive oxygen species and binding to nucleic acids. The presence of significantly enlarged Vip3Aa-treated fungal cells indicates that this protein causes intracellular damage. Our findings suggest that Vip3Aa protein has potential applications in the development of natural antimicrobial agents.

On the history of the study of endogenous antibiotics

The review presents data on the history of the discovery of the first endogenous antibiotic compounds, on the contribution of Russian scientists, in particular, researchers from the Institute of Experimental Medicine (St. Petersburg), in the study of the structural and functional properties of antimicrobial proteins and peptides important molecular factors of innate immunity that can act as an alternative to conventional antibiotics in the fight to control pathogenic microorganisms.

Sequence edition of single domains modulates the final immune and antimicrobial potential of a new generation of multidomain recombinant proteins

Combining several innate immune peptides into a single recombinant antimicrobial and immunomodulatory polypeptide has been recently demonstrated. However, the versatility of the multidomain design, the role that each domain plays and how the sequence edition of the different domains affects their final protein activity is unknown. Parental multidomain antimicrobial and immunomodulatory protein JAMF1 and several protein variants (JAMF1.2, JAMF2 and AM2) have been designed and recombinantly produced to explore how the tuning of domain sequences affects their immunomodulatory potential in epithelial cells and their antimicrobial capacity against Gram-positive and Gram-negative bacteria. The replacement of the sequence of defensin HD5 and phospholipase sPLA2 by shorter active fragments of both peptides improves the final immunomodulatory (IL-8 secretion) and antimicrobial function of the multidomain protein against antimicrobial-resistant Klebsiella pneumoniae and Enterococcus spp. Further, the presence of Jun and Fos leucine zippers in multidomain proteins is crucial in preventing toxic effects on producer cells. The generation of antimicrobial proteins based on multidomain polypeptides allows specific immunomodulatory and antimicrobial functions, which can be easily edited by modifying of each domain sequence.

An ancient antimicrobial protein co-opted by a fungal plant pathogen for in planta mycobiome manipulation

Microbes typically secrete a plethora of molecules to promote niche colonization. Soil-dwelling microbes are well-known producers of antimicrobials that are exploited to outcompete microbial coinhabitants. Also, plant pathogenic microbes secrete a diversity of molecules into their environment for niche establishment. Upon plant colonization, microbial pathogens secrete so-called effector proteins that promote disease development. While such effectors are typically considered to exclusively act through direct host manipulation, we recently reported that the soil-borne, fungal, xylem-colonizing vascular wilt pathogen Verticillium dahliae exploits effector proteins with antibacterial properties to promote host colonization through the manipulation of beneficial host microbiota. Since fungal evolution preceded land plant evolution, we now speculate that a subset of the pathogen effectors involved in host microbiota manipulation evolved from ancient antimicrobial proteins of terrestrial fungal ancestors that served in microbial competition prior to the evolution of plant pathogenicity. Here, we show that V. dahliae has co-opted an ancient antimicrobial protein as effector, named VdAMP3, for mycobiome manipulation in planta. We show that VdAMP3 is specifically expressed to ward off fungal niche competitors during resting structure formation in senescing mesophyll tissues. Our findings indicate that effector-mediated microbiome manipulation by plant pathogenic microbes extends beyond bacteria and also concerns eukaryotic members of the plant microbiome. Finally, we demonstrate that fungal pathogens can exploit plant microbiome-manipulating effectors in a life stage–specific manner and that a subset of these effectors has evolved from ancient antimicrobial proteins of fungal ancestors that likely originally functioned in manipulation of terrestrial biota.

Lasso Proteins—Unifying Cysteine Knots and Miniproteins

Complex lasso proteins are a recently identified class of biological compounds that are present in considerable fraction of proteins with disulfide bridges. In this work, we look at complex lasso proteins as a generalization of well-known cysteine knots and miniproteins (lasso peptides). In particular, we show that complex lasso proteins with the same crucial topological features—cysteine knots and lasso peptides—are antimicrobial proteins, which suggests that they act as a molecular plug. Based on an analysis of the stability of the lasso piercing residue, we also introduce a method to determine which lasso motif is potentially functional. Using this method, we show that the lasso motif in antimicrobial proteins, as well in that in cytokines, is functionally relevant. We also study the evolution of lasso motifs, their conservation, and the usefulness of the lasso fingerprint, which extracts all topologically non-triviality concerning covalent loops. The work is completed by the presentation of extensive statistics on complex lasso proteins to analyze, in particular, the strange propensity for “negative” piercings. We also identify 21 previously unknown complex lasso proteins with an ester and a thioester bridge.

Eradication of Klebsiella quasipneumoniae by Eudragit-formulated klebicin KvarIa in the intestinal tract of mice

Background Klebsiella quasipneumoniae is an opportunistic pathogen causing antibiotic-resistant infections of the gastrointestinal tract in many clinical cases. Orally delivered bioactive Klebsiella-specific antimicrobial proteins, klebicins, could be a promising method to eradicate Klebsiella species infecting the gut. Methods Mouse infection model wasestablished based on infection of streptomycin treated BALB/C mice with K. quasipneumoniae strain DSM28212. Four study groups were used (3 animals/group) to test the antimicrobial efficacy of orally delivered klebicin KvarIa: vehicle-only group (control, phosphate-buffered saline), and other three groups with bacteria, antibiotic therapy and 100 µg of uncoated Kvarla, 100 µg coated KvarIa, 1000 µg coated-KvarIa. Because of the general sensitivity of bacteriocins to gastroduodenal proteases, Karla doses were coated with Eudragit®, a GMP-certified formulation agent that releases the protein at certain pH. The coating treatment was selected based on measurements of mouse GI tract pH. The quantity of Klebsiella haemolysin gene (khe) in faecal samples of the study animals was used to quantify the presence of Klebsiella. Results GI colonization of K. quasipneumoniae was achieved only in the antibiotic-treated mice groups. Significant changes in khe marker quantification were found after the use of Eudragit® S100 formulated klebicin KvarIa, at both doses, with a significant reduction of K. quasipneumoniae colonization compared to the vehicle-only control group. Conclusions Mouse GI tract colonization with K. quasipneumoniae can be achieved if natural gut microbiota is suppressed by prior antibiotic treatment. The study demonstrates that GI infection caused by K. quasipneumoniae can be significantly reduced using Eudragit®-protected klebicin KvarIa.

Myxobacterial Genomics and Post-Genomics: A Review of Genome Biology, Genome Sequences and Related ‘Omics Studies

Myxobacteria are fascinating and complex microbes. They prey upon other members of the soil microbiome by secreting antimicrobial proteins and metabolites, and will undergo multicellular development if starved. The genome sequence of the model myxobacterium Myxococcus xanthus DK1622 was published in 2006 and 15 years later, 163 myxobacterial genome sequences have now been made public. This explosion in genomic data has enabled comparative genomics analyses to be performed across the taxon, providing important insights into myxobacterial gene conservation and evolution. The availability of myxobacterial genome sequences has allowed system-wide functional genomic investigations into entire classes of genes. It has also enabled post-genomic technologies to be applied to myxobacteria, including transcriptome analyses (microarrays and RNA-seq), proteome studies (gel-based and gel-free), investigations into protein–DNA interactions (ChIP-seq) and metabolism. Here, we review myxobacterial genome sequencing, and summarise the insights into myxobacterial biology that have emerged as a result. We also outline the application of functional genomics and post-genomic approaches in myxobacterial research, highlighting important findings to emerge from seminal studies. The review also provides a comprehensive guide to the genomic datasets available in mid-2021 for myxobacteria (including 24 genomes that we have sequenced and which are described here for the first time).

Local Defence System in Healthy Lungs

Background: The respiratory system is one of the main entrance gates for infection. The aim of this work was to compare the appearance of specific mucosal pro-inflammatory and common anti-microbial defence factors in healthy lung tissue, from an ontogenetic point of view. Materials and methods: Healthy lung tissues were collected from 15 patients (three females and 12 males) in the age range from 18 to 86. Immunohistochemistry to human β defensin 2 (HBD-2), human β defensin 3 (HBD-3), human β defensin 4 (HBD-4), cathelicidine (LL-37) and interleukine 17A (IL-17A) were performed. Results: The lung tissue material contained bronchial and lung parenchyma material in which no histological changes, connected with the inflammatory process, were detected. During the study, various statistically significant differences were detected in immunoreactive expression between different factors in all lung tissue structures. Conclusion: All healthy lung structures, but especially the cartilage, alveolar epithelium and the alveolar macrophages, are the main locations for the baseline synthesis of antimicrobial proteins and IL-17A. Cartilage shows high functional plasticity of this structure, including significant antimicrobial activity and participation in local lung protection response. Interrelated changes between antimicrobial proteins in different tissue confirm baseline synergistical cooperation of all these factors in healthy lung host defence.