Lactoferrin impairs pathogen virulence through its proteolytic activity.

Antibiotics, often hailed as 'miracle drugs' in the 20th century, have revolutionised medicine by saving millions of lives in human and veterinary medicine, effectively combatting bacterial infections. However, the escalating global challenge of antimicrobial resistance and the appearance and spread of multidrug-resistant pathogens necessitates research into alternatives. One such alternative could be lactoferrin. Lactoferrin, an iron-binding multifunctional protein, is abundantly present in mammalian secretions and exhibits antimicrobial and immunomodulatory activities. An often overlooked aspect of lactoferrin is its proteolytic activity, which could contribute to its antibacterial activity. The proteolytic activity of lactoferrin has been linked to the degradation of virulence factors from several bacterial pathogens, impeding their colonisation and potentially limiting their pathogenicity. Despite numerous studies, the exact proteolytically active site of lactoferrin, the specific bacterial virulence factors it degrades and the underlying mechanism remain incompletely understood. This review gives an overview of the current knowledge concerning the proteolytic activity of lactoferrins and summarises the bacterial virulence factors degraded by lactoferrins. We further detail how a deeper understanding of the proteolytic activity of lactoferrin might position it as a viable alternative for antibiotics, being crucial to halt the spread of multi-drug resistant bacteria.

The Epistemology of Bacterial Virulence Factor Characterization.

The field of microbial pathogenesis seeks to identify the agents and mechanisms responsible for disease causation. Since Robert Koch introduced postulates that were used to guide the characterization of microbial pathogens, technological advances have substantially increased the capacity to rapidly identify a causative infectious agent. Research efforts currently focus on causation at the molecular level with a search for virulence factors (VFs) that contribute to different stages of the infectious process. We note that the quest to identify and characterize VFs sometimes lacks scientific rigor, and this suggests a need to examine the epistemology of VF characterization. We took this premise as an opportunity to explore the epistemology of VF characterization. In this perspective, we discuss how the characterization of various gene products that evolved to facilitate bacterial survival in the broader environment have potentially been prematurely mischaracterized as VFs that contribute to pathogenesis in the context of human biology. Examples of the reasoning that can affect misinterpretation, or at least a premature assignment of mechanistic causation, are provided. Our aim is to refine the categorization of VFs by emphasizing a broader biological view of their origin.

The Hidden World Within: Microbial Dynamics in Root Canal Systems.

Several hundred different microbial taxa have made the oral cavity their home because of their evolution in multiple species communities within the special ecosystem. On the other hand, the dental pulp or internal tissue of the tooth is a connective tissue that is physiologically sterile and where any microbial infiltration is a harmful indication. It causes the pulp tissue to become inflamed, which leads to the death of the pulp and diffuses infection with inflammation to the peri-radicular tissues. Comprehending the biology of biofilms, the microbial makeup, and the host's reaction to infections in the pathobiology of root canal infections has received a lot of attention throughout the last few decades. Such comprehensive knowledge is required to design preventive medicines as well as clinically effective treatment regimens. Surprisingly, clinical approaches have concentrated more on radiographically perfecting channel preparation than on debridement of these intricate root canal systems, despite the clear realization that root canal infections are biofilm mediated. Since the present comprehension of the microbial etiopathogenesis of apical periodontitis highlights the significance of focusing on procedures such as "canal cleaning" and chemo-mechanical disinfection, the exclusive purpose of endodontic therapy is mainly missed while discussing "canal shaping." We thoroughly examine the state of our knowledge of the composition and functional traits of the root canal microbiome in this review. We also go into the difficulties with root canal disinfection and the cutting-edge approaches that try to solve these difficulties. In conclusion, we present essential guidance for prospective research areas, underscoring their significance as crucial considerations in the field of frontiers in oral health.

Citric Acid Controls the Activity of YopH Bacterial Tyrosine Phosphatase.

Purpose: Citric acid (CA) is a tricarboxylic acid with antioxidant and antimicrobial properties. Based on previous studies, the small compound with its three carboxylic groups can be considered a protein tyrosine phosphatase inhibitor. YopH, a protein tyrosine phosphatase, is an essential virulence factor in Yersinia bacteria. Materials and Methods: We performed enzymatic activity assays of YopH phosphatase after treatment with citric acid in comparison with the inhibitory compound trimesic acid, which has a similar structure. We also measured the cytotoxicity of these compounds in Jurkat T E6.1 and macrophage J774.2 cell lines. We performed molecular docking analysis of the binding of citric acid molecules to YopH phosphatase. Results: Citric acid and trimesic acid reversibly reduced the activity of YopH enzyme and decreased the viability of Jurkat and macrophage cell lines. Importantly, these two compounds showed greater inhibitory properties against bacterial YopH activity than against human CD45 phosphatase activity. Molecular docking simulations confirmed that citric acid could bind to YopH phosphatase. Conclusion: Citric acid, a known antioxidant, can be considered an inhibitor of bacterial phosphatases.

Staphylococcus aureus and Pseudomonas aeruginosa infectious keratitis: key bacterial mechanisms that mediate pathogenesis and emerging therapeutics.

Bacterial keratitis (bacterial infection of the cornea) is a major cause of vision loss worldwide. Given the rapid and aggressive nature of the disease, immediate broad-spectrum antibiotics are essential to adequately treat this disease. However, rising antibiotic resistance continues to accelerate, rendering many commonly used therapeutics increasingly ineffective. As such, there is a significant effort to understand the basic pathogenesis of common causative organisms implicated in keratitis in part, to fuel the development of novel therapies to treat this blinding disease. This review explores two common causes of bacterial keratitis, Staphylococcus aureus and Pseudomonas aeruginosa, with regards to the bacterial mediators of virulence as well as novel therapies on the horizon.

Insights into oral microbiome and colorectal cancer - on the way of searching new perspectives.

Microbiome is a keystone polymicrobial community that coexist with human body in a beneficial relationship. These microorganisms enable the human body to maintain homeostasis and take part in mechanisms of defense against infection and in the absorption of nutrients. Even though microbiome is involved in physiologic processes that are beneficial to host health, it may also cause serious detrimental issues. Additionally, it has been proven that bacteria can migrate to other human body compartments and colonize them even although significant structural differences with the area of origin exist. Such migrations have been clearly observed when the causes of genesis and progression of colorectal cancer (CRC) have been investigated. It has been demonstrated that the oral microbiome is capable of penetrating into the large intestine and cause impairments leading to dysbiosis and stimulation of cancerogenic processes. The main actors of such events seem to be oral pathogenic bacteria belonging to the red and orange complex (regarding classification of bacteria in the context of periodontal diseases), such as Porphyromonas gingivalis and Fusobacterium nucleatum respectively, which are characterized by significant amount of cancerogenic virulence factors. Further examination of oral microbiome and its impact on CRC may be crucial on early detection of this disease and would allow its use as a precise non-invasive biomarker.

Exploiting Natural Maltol for Synthesis of Novel Hydroxypyridone Derivatives as Promising Anti-Virulence Agents in Bactericides Discovery.

Anti-infection strategies based on suppression of bacterial virulence factors represent a crucial direction for the development of new antibacterial agents to address the resistance triggered by traditional drugs'/pesticides' bactericidal activity. To identify and obtain more effective and diverse molecules targeting virulence, we prepared a series of 3-hydroxy-2-methyl-1-pyridin-4-(1H)-one derivatives and evaluated their antibacterial behaviors. Compound B6 exhibited the highest bioactivity, with half-maximal effective concentration (EC50) values ranging fro9m 10.03 to 30.16 µg mL-1 against three plant pathogenic bacteria. The antibacterial mechanism showed that it could considerably reduce various virulence factors (such as extracellular enzymes, biofilm, and T3SS effectors) and inhibit the expression of virulence factor-related genes. In addition, the control efficiency of compound B6 against rice bacterial leaf blight at 200 µg mL-1 was 46.15-49.15%, and their control efficiency was improved by approximately 12% after the addition of pesticide additives. Thus, a new class of bactericidal candidates targeting bacterial virulence factors was developed for controlling plant bacterial diseases.

Clinical and microbiological features of host-bacterial interplay in chronic venous ulcers versus other types of chronic skin ulcers.

Introduction: Chronic venous ulcers of the lower limbs develop in the context of advanced venous disease and have a significant impact on the patient's quality of life, being associated with depression and worrisome suicide rates, as well as with an economic burden caused by increased medical care costs and high epidemiological risks of healthcare associated infections and emergence of strains resistant to multiple classes of antibiotics and/ or antiseptics. Although numerous studies have investigated the composition of the chronic wounds microbiome, either by culture-dependent or independent methods, there are no data on the association between virulence and resistance profiles of strains isolated from venous ulcers and the clinical picture of this pathology. The elucidation of pathogenic mechanisms, at both phenotypic and molecular level, is crucial in the fight against these important human microbial agents, in order to develop novel biomarkers and discover new therapeutic targets. Methods: In this study we aimed to characterize the phenotypic virulence profiles (including the ability to develop biofilms) of microorganisms isolated from chronic skin wounds and to correlate them with the clinical symptomatology. Considering the high incidence of Staphylococcus aureus infections in chronic ulcers, but also the ability of this species to develop multi-drug resistance, we performed an more in-depth study of the phenotypic and genotypic virulence profiles of methicillin-resistant Staphylococcus. Results: The study revealed important differences regarding the clinical evolution and virulence profiles of microorganisms isolated from lower limb wounds, as well as between patients diagnosed with chronic venous ulcers and those with lesions of different etiology.

ExlA: A New Contributor to Pseudomonas aeruginosa Virulence.

ExlA (also called exolysin) is a recently discovered virulence factor secreted by a subset of Pseudomonas aeruginosa strains in which a type 3 secretion system is lacking. exlA-positive strains were identified worldwide in the clinic, causing several types of infectious diseases, and were detected in various locations in the environment. ExlA possesses pore-forming activity and is cytolytic for most human cell types. It belongs to a class of poorly characterized bacterial toxins, sharing a similar protein domain organization and a common secretion pathway. This review summarizes the recent findings regarding ExlA synthesis, its secretion pathway, and its toxic behavior for host cells.

Antimicrobial resistance and virulence profiles of Enterobacterales isolated from two-finger and three-finger sloths (Choloepus hoffmanni and Bradypus variegatus) of Costa Rica.

Background: Wildlife has been recently recognized as an environmental reservoir for antimicrobial resistance (AMR). However, less information on this topic is available in animals released back into the wild after rehabilitation in wildlife facilities, compared with studies performed exclusively in captive or free-ranging wildlife. This study aimed to evaluate the potential influence of captivity and/or treatment while in captivity of wild sloths on the AMR and virulence profiles of sloths' Enterobacterales. Methods: Oral and rectal swab samples were collected from 39 two-finger (Choloepus hoffmanni) and three-finger sloths (Bradypus variegatus) of Costa Rica (n = 78) and analyzed using conventional bacteriological techniques. A generalized linear mixed model was applied to estimate the isolates' multiple antimicrobial resistance and virulence indices as a function of animal status. Results: A considerable level of resistance was detected, especially for Citrobacter youngae and Escherichia coli, with 17.5% of isolates classified as multidrug-resistant. Virulence indices of isolates from rehabilitated sloths were significantly higher than the ones from sloths being hand-reared for shorter periods. Conclusions: To our knowledge, this is the first description of sloths' antimicrobial resistant Enterobacterales, suggesting that sloths' rehabilitation and consequent exposure to humans, may promote the selection of bacteria with higher virulence. Ultimately, these bacteria may represent a threat to human and animal health due to their zoonotic potential and AMR and virulence profiles.

Phages from Genus Bruynoghevirus and Phage Therapy: Pseudomonas Phage Delta Case.

The applicability and safety of bacteriophage Delta as a potential anti-Pseudomonas aeruginosa agent belonging to genus Bruynoghevirus (family Podoviridae) was characterised. Phage Delta belongs to the species Pseudomonas virus PaP3, which has been described as a temperate, with cos sites at the end of the genome. The phage Delta possesses a genome of 45,970 bp that encodes tRNA for proline (Pro), aspartic acid (Asp) and asparagine (Asn) and does not encode any known protein involved in lysogeny formation or persistence. Analysis showed that phage Delta has 182 bp direct terminal repeats at the end of genome and lysogeny was confirmed, neither upon infection at low nor at high multiplicity of infection (MOI). The turbid plaques that appear on certain host lawns can result from bacteriophage insensitive mutants that occur with higher frequency (10-4). In silico analysis showed that the genome of Delta phage does not encode any known bacterial toxin or virulence factor, determinants of antibiotic resistance and known human allergens. Based on the broad host range and high lytic activity against planktonic and biofilm cells, phage Delta represents a promising candidate for phage therapy.

Antibiotic Resistance and Virulence Profiles of Gram-Negative Bacteria Isolated from Loggerhead Sea Turtles (Caretta caretta) of the Island of Maio, Cape Verde.

Previous studies revealed high levels of antimicrobial resistance (AMR) in loggerhead sea turtles (Caretta caretta), describing this species as prime reservoir of antimicrobial-resistant bacteria. This study aimed to characterise, for the first time, the AMR and virulence profiles of Gram-negative bacteria isolated from 33 nesting loggerhead turtles of the island of Maio, Cape Verde. Cloacal, oral, and egg content swab samples (n = 99) were collected and analysed using conventional bacteriological techniques. Shewanella putrefaciens, Morganella morganii, and Vibrio alginolyticus were isolated from the samples under study. The isolates obtained from this loggerhead subpopulation (North-East Atlantic) revealed lower levels of AMR, compared with the results of studies performed in other subpopulations (e.g., Mediterranean). However, the detection of resistance to carbapenems and multiple antimicrobial resistance indices higher than 0.20, raises concern about the potential association of these animals to points of high antimicrobial exposure. Furthermore, virulence phenotypic characterisation revealed that the isolates presented complex virulence profiles, including the ability to produce biofilms. Finally, due to their pathogenic potential, and considering the evidence of illegal consumption of turtle-related products on the island of Maio, the identified bacteria may represent a significant threat to public health.

Inhibition of bacterial and human zinc-metalloproteases by bisphosphonate- and catechol-containing compounds.

Compounds containg catechol or bisphosphonate were tested as inhibitors of the zinc metalloproteases, thermolysin (TLN), pseudolysin (PLN) and aureolysin (ALN) which are bacterial virulence factors, and the human matrix metalloproteases MMP-9 and -14. Inhibition of virulence is a putative strategy in the development of antibacterial drugs, but the inhibitors should not interfere with human enzymes. Docking indicated that the inhibitors bound MMP-9 and MMP-14 with the phenyl, biphenyl, chlorophenyl, nitrophenyl or methoxyphenyl ringsystem in the S1'-subpocket, while these ringsystems entered the S2'- or S1 -subpockets or a region involving amino acids in the S1'- and S2'-subpockets of the bacterial enzymes. An arginine conserved among the bacterial enzymes seemed to hinder entrance deeply into the S1'-subpocket. Only the bisphosphonate containing compound RC2 bound stronger to PLN and TLN than to MMP-9 and MMP-14. Docking indicated that the reason was that the conserved arginine (R203 in TLN and R198 in PLN) interacts with phosphate groups of RC2.

Cadherin-mediated host-pathogen interactions.

Cell adhesion molecules mediate cell-to-cell and cell-to-matrix adhesions and play an immense role in a myriad of physiological processes during the growth and development of a multicellular organism. Cadherins belong to a major group of membrane-bound cell surface proteins that, in coordination with nectins, drive the formation and maintenance of adherens junctions for mediating cell to cell adhesion, cellular communication and signalling. Alongside adhesive function, the involvement of cadherins in mediating host-pathogen interactions has been extensively explored in recent years. In this review, we provide an in-depth understanding of microbial pathogens and their virulence factors that exploit cadherins for their strategical invasion into the host cell. Furthermore, macromolecular interactions involving cadherins and various microbial factors such as secretory toxins and adhesins lead to the disintegration of host cell junctions followed by the entry of the pathogen or triggering downstream signalling pathways responsible for successful invasion of the pathogenic microbes are discussed. Besides providing a comprehensive insight into some of the structural complexes involving cadherins and microbial factors to offer the mechanistic details of host-pathogen interactions, the current review also highlights novel constituents of various cell signalling events such as endocytosis machinery elicited upon microbial infections.

Zinc-Chelating Compounds as Inhibitors of Human and Bacterial Zinc Metalloproteases.

Inhibition of bacterial virulence is believed to be a new treatment option for bacterial infections. In the present study, we tested dipicolylamine (DPA), tripicolylamine (TPA), tris pyridine ethylene diamine (TPED), pyridine and thiophene derivatives as putative inhibitors of the bacterial virulence factors thermolysin (TLN), pseudolysin (PLN) and aureolysin (ALN) and the human zinc metalloproteases, matrix metalloprotease-9 (MMP-9) and matrix metalloprotease-14 (MMP-14). These compounds have nitrogen or sulfur as putative donor atoms for zinc chelation. In general, the compounds showed stronger inhibition of MMP-14 and PLN than of the other enzymes, with Ki values in the lower µM range. Except for DPA, none of the compounds showed significantly stronger inhibition of the virulence factors than of the human zinc metalloproteases. TPA and Zn230 were the only compounds that inhibited all five zinc metalloproteinases with a Ki value in the lower µM range. The thiophene compounds gave weak or no inhibition. Docking indicated that some of the compounds coordinated zinc by one oxygen atom from a hydroxyl or carbonyl group, or by oxygen atoms both from a hydroxyl group and a carbonyl group, and not by pyridine nitrogen as in DPA and TPA.

Arming the troops: Post-translational modification of extracellular bacterial proteins.

Protein secretion is almost universally employed by bacteria. Some proteins are retained on the cell surface, whereas others are released into the extracellular milieu, often playing a key role in virulence. In this review, we discuss the diverse types and potential functions of post-translational modifications (PTMs) occurring to extracellular bacterial proteins.

Bacterial intracellularly active toxins: Membrane localisation of the active domain.

Numerous bacterial toxins exert their activity by inactivating or modulating a specific intracellular host target. For this purpose, these toxins have developed efficient strategies to overcome the different host cell defences including specific binding to cell surface, internalisation, passage through the endosome or plasma membrane, exploiting intracellular trafficking and addressing to intracellular targets. Several intracellularly active toxins deliver an active domain into the cytosol that interacts with a target localised to the inner face of the plasma membrane. Thus, the large clostridial glucosylating toxins (LCGTs) target Rho/Ras-GTPases, certain virulence factors of Gram negative bacteria, Rho-GTPases, while Pasteurella multocida toxin (PMT) targets trimeric G-proteins. Others such as botulinum neurotoxins and tetanus neurotoxin have their substrate on synaptic vesicle membrane. LCGTs, PMT, and certain virulence factors from Vibrio sp. show a particular structure constituted of a four-helix bundle membrane (4HBM) protruding from the catalytic site that specifically binds to the membrane phospholipids and then trap the catalytic domain at the proximity of the membrane anchored substrate. Structural and functional analysis indicate that the 4HBM tip of the Clostridium sordellii lethal toxin (TcsL) from the LCGT family contain two loops forming a cavity that mediates the binding to phospholipids and more specifically to phosphatidylserine.

Factors affecting the secretion of vascular endothelial growth factor from dental pulp stem cells / 中国组织工程研究

BACKGROUND: How to regulate the secretion of vascular endothelial growth factor from dental pulp stem cells is of great significance for promoting dental pulp regeneration by dental pulp stem cells, especially promoting dentinogenesis, dental pulp angiogenesis and neurogenesis. OBJECTIVE: To review the factors affecting the secretion of vascular endothelial growth factor from dental pulp stem cells, providing ideas for pulp regeneration and other clinical applications. METHODS: We searched the articles in PubMed, CNKI, WanFang databases with the keywords of “vascular endothelial growth factor; dental pulp stem cell; dental pulp regeneration; hypoxia; inflammatory mediator; bacterial virulence factor; growth factor; material” in Chinese and English, respectively. Finally, 56 articles met the criteria for review. RESULTS AND CONCLUSION: Vascular endothelial growth factor is the most important cytokine in angiogenesis and neovasculization, which promotes the proliferation and differentiation of stem cells as well as protecting nerves and promoting neurogenesis. Dental pulp stem cells are the most important stem cells in dental pulp tissues. They are also important seed cells in pulp regeneration. Dental pulp stem cells have biological characteristics such as high proliferation, self-renewal and multi-lineage differentiation, and have certain secretory activities, which can be used as an alternative source of exogenous vascular endothelial growth factor. A variety of factors, such as hypoxia, bacterial virulence factors, inflammatory factors, growth factors and materials, are associated with the cytokine secretion activity of dental pulp stem cells, which can affect the expression and secretion of vascular endothelial growth factor in dental pulp stem cells. Therefore, increasing concern has been emphasized on the regulation of vascular endothelial growth factor ecpression and secretion in dental pulp stem cells and the better use in pulp regeneration.

The effect of smoking on caries-related microorganisms.

INTRODUCTION: Epidemiological studies have shown a close relationship between smoking and dental caries. Bacteria are one of the essential factors of caries formation. The imbalance of cariogenic bacteria and commensal bacteria in dental plaque results in higher production of acid that can corrode dental hard tissue. The aim of our review is to summarize the effect of smoking on caries-related bacteria. METHODS: English articles available in Pubmed and ScienceDirect databases and published before December 2018 were searched. A variety of evidence was collected including not only the influence of cigarette products on bacteria strains in vitro but also their effect on bacterial composition in saliva and dental plaque in vivo. We particularly emphasize the mechanisms by which nicotine acts on oral bacteria. RESULTS: The components of cigarettes promote the growth of cariogenic microorganisms. The mechanisms of how nicotine enhances Streptococcus mutans, Lactobacilli, Streptococcus gordonii, Actinomyces and Candida albicans are described separately in detail. The commensal bacteria, Streptococcus sanguinis, show less competitive capability in the presence of nicotine. Smoking influences saliva by lowering the buffer capability, altering its chemical agent and bacterial components, and therefore promotes the formation of a caries-susceptible environment. CONCLUSIONS: Cigarette smoking and nicotine exposure promote the cariogenic activity of oral microorganisms and the formation of a caries-susceptible environment. This suggests that smokers should quit smoking, amongst other health reasons, also for their oral health.

Phylogenetically Defined Isoforms of Listeria monocytogenes Invasion Factor InlB Differently Activate Intracellular Signaling Pathways and Interact with the Receptor gC1q-R.

The pathogenic Gram-positive bacterium Listeria monocytogenes has been evolving into a few phylogenetic lineages. Phylogenetically defined substitutions were described in the L. monocytogenes virulence factor InlB, which mediates active invasion into mammalian cells via interactions with surface receptors c-Met and gC1q-R. InlB internalin domain (idInlB) is central to interactions with c-Met. Here we compared activity of purified recombinant idInlB isoforms characteristic for L. monocytogenes phylogenetic lineage I and II. Size exclusion chromatography and intrinsic fluorescence were used to characterize idInlBs. Western blotting was used to study activation of c-Met-dependent MAPK- and PI3K/Akt-pathways. Solid-phase microplate binding and competition assay was used to quantify interactions with gCq1-R. Isogenic recombinant L. monocytogenes strains were used to elucidate the input of idInlB isoforms in HEp-2 cell invasion. Physicochemical parameters of idInlB isoforms were similar but not identical. Kinetics of Erk1/2 and Akt phosphorylation in response to purified idInlBs was lineage specific. Lineage I but not lineage II idInlB specifically bound gC1q-R. Antibody against gC1q-R amino acids 221-249 inhibited invasion of L. monocytogenes carrying lineage I but not lineage II idInlB. Taken together, obtained results suggested that phylogenetically defined substitutions in idInlB provide functional distinctions and might be involved in phylogenetically determined differences in virulence potential.