Isolation, characterization, and fibroblast uptake of bacterial extracellular vesicles from Porphyromonas gingivalis strains.

Periodontitis is an inflammatory condition caused by bacteria and represents a serious health problem worldwide as the inflammation damages the supporting tissues of the teeth and may predispose to systemic diseases. Porphyromonas gingivalis is considered a keystone periodontal pathogen that releases bacterial extracellular vesicles (bEVs) containing virulence factors, such as gingipains, that may contribute to the pathogenesis of periodontitis. This study aimed to isolate and characterize bEVs from three strains of P. gingivalis, investigate putative bEV uptake into human oral fibroblasts, and determine the gingipain activity of the bEVs. bEVs from three bacterial strains, ATCC 33277, A7A1-28, and W83, were isolated through ultrafiltration and size-exclusion chromatography. Vesicle size distribution was measured by nano-tracking analysis (NTA). Transmission electron microscopy was used for bEV visualization. Flow cytometry was used to detect bEVs and gingipain activity was measured with an enzyme assay using a substrate specific for arg-gingipain. The uptake of bEVs into oral fibroblasts was visualized using confocal microscopy. NTA showed bEV concentrations from 108 to 1011 particles/mL and bEV diameters from 42 to 356 nm. TEM pictures demonstrated vesicle-like structures. bEV-gingipains were detected both by flow cytometry and enzyme assay. Fibroblasts incubated with bEVs labeled with fluorescent dye displayed intracellular localization consistent with bEV internalization. In conclusion, bEVs from P. gingivalis were successfully isolated and characterized, and their uptake into human oral fibroblasts was documented. The bEVs displayed active gingipains demonstrating their origin from P. gingivalis and the potential role of bEVs in periodontitis.

Cell culture media dependent in vitro dynamics and culture characteristics of adult caprine dermal fibroblast cells.

The enhanced availability of functional fibroblasts from precious tissue samples requires an ideal cell-culture system. Therefore, this study was designed to investigate the performance of caprine adult fibroblast cells (cadFibroblast) when cultivated in different culture media. The cadFibroblast cell lines from adult Barbari (Capra hircus) bucks were established and the effect of different media viz. DMEM/F-12 [with low-glucose (5.5 mM; DL) and high-glucose (30 mM; DH)], α-MEM [with low-glucose (5.5 mM; ML) and with high-glucose (30 mM; MH)], and fibroblast growth medium (FGM) were evaluated. Cells were then compared for growth characteristics and in-vitro dynamics through cellular morphology, proliferation, population-doubling time, double-immunocytochemistry, colony-forming units, wound healing, transwell migration, and differential expression of fibroblast-specific markers (FSP-1 and vimentin). The results of immunocytochemistry, transwell migration/invasion, and wound healing assays showed the superiority of DH over DL and other media tested. Whereas, similar effects of glucose supplementation and expression of FSP-1 were not observed in α-MEM. Transwell migration was significantly (p < 0.05) lower in FGM compared with other media tested. Overall, our results illustrate the media-dependent deviation in in-vitro dynamics and culture characteristics of cadFibroblasts that may be useful to develop strategies to cultivate these cells efficiently for research and downstream applications.

The Role of Gingival Fibroblasts in the Pathogenesis of Periodontitis.

Gingival fibroblasts (GFs) are essential components of the periodontium, which are responsible for the maintenance of tissue structure and integrity. However, the physiological role of GFs is not restricted to the production and remodeling of the extracellular matrix. GFs also act as sentinel cells that modulate the immune response to oral pathogens invading the gingival tissue. As an important "nonclassical" component of the innate immune system, GFs respond to bacteria and damage-related signals by producing cytokines, chemokines, and other inflammatory mediators. Although the activation of GFs supports the elimination of invading bacteria and the resolution of inflammation, their uncontrolled or excessive activation may promote inflammation and bone destruction. This occurs in periodontitis, a chronic inflammatory disease of the periodontium initiated and sustained by dysbiosis. In the inflamed gingival tissue, GFs acquire imprinted proinflammatory phenotypes that promote the growth of inflammophilic pathogens, stimulate osteoclastogenesis, and contribute to the chronicity of inflammation. In this review, we discuss the biological functions of GFs in healthy and inflamed gingival tissue, highlighting recent studies that provide insight into their role in the pathogenesis of periodontal diseases. We also draw parallels with the recently discovered fibroblast populations identified in other tissues and their roles in health and disease. This knowledge should be used in future studies to discover more about the role of GFs in periodontal diseases, especially chronic periodontitis, and to identify therapeutic strategies targeting their pathological interactions with oral pathogens and the immune system.

Gut microbiota modulates lung fibrosis severity following acute lung injury in mice.

Independent studies demonstrate the significance of gut microbiota on the pathogenesis of chronic lung diseases; yet little is known regarding the role of the gut microbiota in lung fibrosis progression. Here we show, using the bleomycin murine model to quantify lung fibrosis in C57BL/6 J mice housed in germ-free, animal biosafety level 1 (ABSL-1), or animal biosafety level 2 (ABSL-2) environments, that germ-free mice are protected from lung fibrosis, while ABSL-1 and ABSL-2 mice develop mild and severe lung fibrosis, respectively. Metagenomic analysis reveals no notable distinctions between ABSL-1 and ABSL-2 lung microbiota, whereas greater microbial diversity, with increased Bifidobacterium and Lactobacilli, is present in ABSL-1 compared to ABSL-2 gut microbiota. Flow cytometric analysis reveals enhanced IL-6/STAT3/IL-17A signaling in pulmonary CD4 + T cells of ABSL-2 mice. Fecal transplantation of ABSL-2 stool into germ-free mice recapitulated more severe fibrosis than transplantation of ABSL-1 stool. Lactobacilli supernatant reduces collagen 1 A production in IL-17A- and TGFß1-stimulated human lung fibroblasts. These findings support a functional role of the gut microbiota in augmenting lung fibrosis severity.

Inflammasome dysregulation in human gingival fibroblasts in response to periodontal pathogens.

OBJECTIVE: Uncontrolled production of Interleukin-1ß (IL-1ß), a major proinflammatory cytokine, is associated with tissue destruction in periodontal disease. IL-1ß production is controlled by inflammasomes which are multiprotein regulatory complexes. The current study aimed to elucidate potential regulatory pathways by monitoring the effects of periodontal pathogens Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg) on inflammasomes and their regulators in human gingival fibroblasts (HGFs) in vitro. METHODS: HGFs were exposed to Fn and Pg alone or in combination for 24 hr at a multiplicity of infection of 100, ±30 min exposure with 5 mM adenosine triphosphate (ATP) incubation. Gene expression of NLRP3 and AIM2, inflammasome regulatory proteins POP1, CARD16 and TRIM16, and inflammasome components ASC and CASPASE 1, and IL-1ß, were evaluated by RT-PCR. Pro- and mature IL-1ß levels were monitored intracellularly by immunocytochemistry and extracellularly by ELISA. RESULTS: Fn + ATP significantly upregulated NLRP3, AIM2, IL-1ß, ASC, and CASPASE 1; however, it downregulated POP1 and TRIM16. Pg + ATP downregulated NLRP3, ASC, POP1, but upregulated IL-1ß and CARD16. Pg + Fn+ATP significantly upregulated AIM2, IL-1ß and CARD16, and downregulated POP1, TRIM16, and CASPASE 1. Pg + ATP exposure significantly increased pro- and mature IL-1ß production. CONCLUSION: Bacterial exposure with ATP may deregulate IL-1ß by dysregulating inflammasomes and their regulators in HGFs.

Cytotoxicity Comparison of a Calcium Silicate-Based Resin Cement versus Conventional Self-Adhesive Resin Cement and a Resin-Modified Glass Ionomer: Cell Viability Analysis

Abstract Objective: To compare the cytotoxicity level of a new calcium silicate-based resin cement (TheraCem) with two commonly used cements, including a conventional self-adhesive resin cement (Panavia SA) and a resinmodified glass ionomer cement (FujiCem2), on the human gingival fibroblast cells after 24 and 48 hours. Material and Methods: Twelve discs of each cement type were fabricated. The extract of cement disks was made by incubating them in the cell medium. Human gingival fibroblast cells were cultured and exposed to cement extracts for 24 h and 48 h. MTT assay was performed on extracts and optical density and cell viability rates were calculated by the spectrophotometer device at 570 nm. Data were analyzed using ANOVA and Tukey HSD tests. Results: The cell viability rates after 24 hours and 48 hours were as follows: TheraCem: 89.24% and 85.46%, Panavia SA: 49.51% and 46.57% and FujiCem2: 50.63% and 47.36%. TheraCem represented the highest cell viability rate. However, no significant difference was noted between Panavia SA and FujiCem2. Time had no significant effect on cell viability. Conclusion: TheraCem exhibited the best results among three tested cements and was considered non-toxic. Panavia SA and FujiCem2 were not significantly different regarding the cell viability rate. Time had no significant effect on the cytotoxicity level of cements (AU).

Human Renal Fibroblasts, but Not Renal Epithelial Cells, Induce IL-1ß Release during a Uropathogenic Escherichia coli Infection In Vitro.

Understanding how uropathogenic Escherichia coli (UPEC) modulates the immune response in the kidney is essential to prevent UPEC from reaching the bloodstream and causing urosepsis. The purpose of this study was to elucidate if renal fibroblasts can release IL-1ß during a UPEC infection and to investigate the mechanism behind the IL-1ß release. We found that the UPEC strain CFT073 induced an increased IL-1ß and LDH release from renal fibroblasts, but not from renal epithelial cells. The UPEC-induced IL-1ß release was found to be NLRP3, caspase-1, caspase-4, ERK 1/2, cathepsin B and serine protease dependent in renal fibroblasts. We also found that the UPEC virulence factor α-hemolysin was necessary for IL-1ß release. Conditioned medium from caspase-1, caspase-4 and NLRP3-deficient renal fibroblasts mediated an increased reactive oxygen species production from neutrophils, but reduced UPEC phagocytosis. Taken together, our study demonstrates that renal fibroblasts, but not renal epithelial cells, release IL-1ß during a UPEC infection. This suggest that renal fibroblasts are vital immunoreactive cells and not only structural cells that produce and regulate the extracellular matrix.

Antimicrobial and cytotoxic activity of electrosprayed chitosan nanoparticles against endodontic pathogens and Balb/c 3T3 fibroblast cells.

The aims of this study were to synthesize highly positively charged chitosan nanoparticles (Ch-Np) using the electrospraying technique, and to test their antimicrobial activity against endodontic pathogens, and cytotoxicity against fibroblast cells. Ch-Np were synthesized from low molecular weight chitosan (LMW-Ch) using the electrospraying technique, and characterized. The antimicrobial activity was evaluated against Streptococcus mutans, Enterococcus faecalis, and Candida albicans in their planktonic state using a Time-Kill Test performed by using broth micro-dilution technique, and against biofilm biomass using a microtiter plate biofilm assay. The cytotoxicity was evaluated using Balb/c 3T3 fibroblast cells with the standard MTT assay. Electrospraying of LMW-Ch produced Ch-Np with an average size of 200 nm, and a surface charge of 51.7 mV. Ch-Np completely eradicated S. mutans and E. faecalis in the planktonic state and showed fungistatic activity against C. albicans. Furthermore, it significantly reduced the biofilm biomass for all the tested microbial species [S. mutans (p = 0.006), E. faecalis (p < 0.0001), and C. albicans (p = 0.004)]. When tested for cytotoxicity using 3T3 cells, Ch-Np showed no cytotoxicity. In conclusion, the highly positively charged, colloidal dispersion of Ch-Np are effective as a biocompatible endodontic antimicrobial agent.

HATMSC Secreted Factors in the Hydrogel as a Potential Treatment for Chronic Wounds-In Vitro Study.

Mesenchymal stem cells (MSCs) can improve chronic wound healing; however, recent studies suggest that the therapeutic effect of MSCs is mediated mainly through the growth factors and cytokines secreted by these cells, referred to as the MSC secretome. To overcome difficulties related to the translation of cell therapy into clinical use such as efficacy, safety and cost, we propose a hydrogel loaded with a secretome from the recently established human adipose tissue mesenchymal stem cell line (HATMSC2) as a potential treatment for chronic wounds. Biocompatibility and biological activity of hydrogel-released HATMSC2 supernatant were investigated in vitro by assessing the proliferation and metabolic activity of human fibroblast, endothelial cells and keratinocytes. Hydrogel degradation was measured using hydroxyproline assay while protein released from the hydrogel was assessed by interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) ELISAs. Pro-angiogenic activity of the developed treatment was assessed by tube formation assay while the presence of pro-angiogenic miRNAs in the HATMSC2 supernatant was investigated using real-time RT-PCR. The results demonstrated that the therapeutic effect of the HATMSC2-produced factors is maintained following incorporation into collagen hydrogel as confirmed by increased proliferation of skin-origin cells and improved angiogenic properties of endothelial cells. In addition, HATMSC2 supernatant revealed antimicrobial activity, and which therefore, in combination with the hydrogel has a potential to be used as advanced wound-healing dressing.

Mitochondrial DNA Haplogroup Related to the Prevalence of Helicobacter pylori.

Mitochondria are essential organelles that are not only responsible for energy production but are also involved in cell metabolism, calcium homeostasis, and apoptosis. Targeting mitochondria is a key strategy for bacteria to subvert host cells' physiology and promote infection. Helicobacter (H.) pylori targets mitochondria directly. However, mitochondrial genome (mtDNA) polymorphism (haplogroup) is not yet considered an important factor for H. pylori infection. Here, we clarified the association of mitochondrial haplogroups with H. pylori prevalence and the ability to perform damage. Seven mtDNA haplogroups were identified among 28 H. pylori-positive subjects. Haplogroup B was present at a higher frequency and haplotype D at a lower one in the H. pylori population than in that of the H. pylori-negative one. The fibroblasts carrying high-frequency haplogroup displayed a higher apoptotic rate and diminished mitochondrial respiration following H. pylori infection. mtDNA mutations were accumulated more in the H. pylori-positive population than in that of the H. pylori-negative one in old age. Among the mutations, 57% were located in RNA genes or nonsynonymous protein-coding regions in the H. pylori-positive population, while 35% were in the H. pylori-negative one. We concluded that gastric disease caused by Helicobacter virulence could be associated with haplogroups and mtDNA mutations.

The Bigger Picture: Why Oral Mucosa Heals Better Than Skin.

Wound healing is an essential process to restore tissue integrity after trauma. Large skin wounds such as burns often heal with hypertrophic scarring and contractures, resulting in disfigurements and reduced joint mobility. Such adverse healing outcomes are less common in the oral mucosa, which generally heals faster compared to skin. Several studies have identified differences between oral and skin wound healing. Most of these studies however focus only on a single stage of wound healing or a single cell type. The aim of this review is to provide an extensive overview of wound healing in skin versus oral mucosa during all stages of wound healing and including all cell types and molecules involved in the process and also taking into account environmental specific factors such as exposure to saliva and the microbiome. Next to intrinsic properties of resident cells and differential expression of cytokines and growth factors, multiple external factors have been identified that contribute to oral wound healing. It can be concluded that faster wound closure, the presence of saliva, a more rapid immune response, and increased extracellular matrix remodeling all contribute to the superior wound healing and reduced scar formation in oral mucosa, compared to skin.

Human cytomegalovirus blocks canonical TGFß signaling during lytic infection to limit induction of type I interferons.

Human cytomegalovirus (HCMV) microRNAs (miRNAs) significantly rewire host signaling pathways to support the viral lifecycle and regulate host cell responses. Here we show that SMAD3 expression is regulated by HCMV miR-UL22A and contributes to the IRF7-mediated induction of type I IFNs and IFN-stimulated genes (ISGs) in human fibroblasts. Addition of exogenous TGFß interferes with the replication of a miR-UL22A mutant virus in a SMAD3-dependent manner in wild type fibroblasts, but not in cells lacking IRF7, indicating that downregulation of SMAD3 expression to limit IFN induction is important for efficient lytic replication. These findings uncover a novel interplay between SMAD3 and innate immunity during HCMV infection and highlight the role of viral miRNAs in modulating these responses.

Los glicosaminoglicanos se encuentran implicados en la adherencia de Candida albicans y Malassezia spp. a queratinocitos, pero no a fibroblastos dérmicos / Glycosaminoglycans Are Involved in the Adhesion of Candida albicans and Malassezia Species to Keratinocytes But Not to Dermal Fibroblasts

Antecedentes y objetivo Las micosis superficiales son algunas de las enfermedades más comunes en todo el mundo, siendo los agentes causales más frecuentes las levaduras de los géneros Malassezia y Candida, comensales habituales de la piel que pueden actuar como patógenos oportunistas. El objetivo de este trabajo es investigar si los glicosaminoglicanos (GAG) de las células epiteliales son utilizados por estos microrganismos como receptores de adhesión a las mismas. Materiales y métodos Se utilizaron cultivos de queratinocitos y fibroblastos dérmicos. La participación de los GAG en la adhesión de Candida albicans (C. albicans) y Malassezia spp. se estudió mediante inhibición específica de la síntesis de estas moléculas empleando rodamina B o genisteína. También se analizó mediante digestión enzimática in situ empleando liasas específicas. Resultados El tratamiento con rodamina B produjo una inhibición parcial de la adherencia de ambas especies fúngicas a queratinocitos, pero no a fibroblastos. La digestión selectiva del heparán sulfato produjo un aumento de la unión de Malassezia a los queratinocitos y de ambas especies a los fibroblastos. La digestión del condroitín sulfato redujo la unión de C. albicans en los queratinocitos, pero favoreció la unión de la forma filamentada de esta levadura en los fibroblastos. Conclusiones Los GAG de superficie celular de queratinocitos parecen estar implicados en la adherencia de Candida y Malasezzia a la superficie celular. En los fibroblastos, por el contrario, su eliminación favorece la adherencia, sugiriendo la implicación de otro tipo de receptores (AU)

Background and objective Superficial mycoses are some of the most common diseases worldwide. The usual culprits — yeasts belonging to the genera Malassezia and Candida — are commensal species in the skin that can cause opportunistic infections. We aimed to determine whether these yeasts use glycosaminoglycans (GAGs) as adhesion receptors to mediate binding to epithelial cells. Material and methods In keratinocyte and dermal fibroblast cultures, we used rhodamine B and genistein to inhibit GAG synthesis to study the role these molecules play in the adhesion of Candida albicans (C. albicans) and Malassezia species to cells. We also analyzed GAG involvement by means of enzyme digestion, using specific lyases. Results Rhodamine B partially inhibited the adhesion of both fungi to keratinocytes but not to fibroblasts. Selective digestion of heparan sulfate enhanced the binding of Malassezia species to keratinocytes and of both fungi to fibroblasts. Chondroitin sulfate digestion decreased C. albicans adhesion to keratinocytes, but increased the adhesion of the filamentous forms of this species to fibroblasts. Conclusions Cell surface GAGs appear to play a role in the adhesion of C albicans and Malasezzia species to keratinocytes. In contrast, their adhesion to fibroblasts appears to be enhanced by GAG inhibition, suggesting that some other type of receptor is the mediator (AU)

The Co-Culture of Staphylococcal Biofilm and Fibroblast Cell Line: The Correlation of Biological Phenomena with Metabolic NMR1 Footprint.

Staphylococcus aureus is one of the most prevalent pathogens associated with several types of biofilm-based infections, including infections of chronic wounds. Mature staphylococcal biofilm is extremely hard to eradicate from a wound and displays a high tendency to induce recurring infections. Therefore, in the present study, we aimed to investigate in vitro the interaction between S. aureus biofilm and fibroblast cells searching for metabolites that could be considered as potential biomarkers of critical colonization and infection. Utilizing advanced microscopy and microbiological methods to examine biofilm formation and the staphylococcal infection process, we were able to distinguish 4 phases of biofilm development. The analysis of staphylococcal biofilm influence on the viability of fibroblasts allowed us to pinpoint the moment of critical colonization-12 h post contamination. Based on the obtained model we performed a metabolomics analysis by 1H NMR spectroscopy to provide new insights into the pathophysiology of infection. We identified a set of metabolites related to the switch to anaerobic metabolism that was characteristic for staphylococcal biofilm co-cultured with fibroblast cells. The data presented in this study may be thus considered a noteworthy but preliminary step in the direction of developing a new, NMR-based tool for rapid diagnosing of infection in a chronic wound.

Staphylococcus aureus impairs dermal fibroblast functions with deleterious effects on wound healing.

Chronic wounds are a major disease burden worldwide. The breach of the epithelial barrier facilitates transition of skin commensals to invasive facultative pathogens. Therefore, we investigated the potential effects of Staphylococcus aureus (SA) on dermal fibroblasts as key cells for tissue repair. In co-culture systems combining live or heat-killed SA with dermal fibroblasts derived from the BJ-5ta cell line, healthy individuals, and patients with systemic sclerosis, we assessed tissue repair including pro-inflammatory cytokines, matrix metalloproteases (MMPs), myofibroblast functions, and host defense responses. Only live SA induced the upregulation of IL-1ß/-6/-8 and MMP1/3 as co-factors of tissue degradation. Additionally, the increased cell death reduced collagen production, proliferation, migration, and contractility, prerequisite mechanisms for wound closure. Intracellular SA triggered inflammatory and type I IFN responses via intracellular dsDNA sensor molecules and MyD88 and STING signaling pathways. In conclusion, live SA affected various key tissue repair functions of dermal fibroblasts from different sources to a similar extent. Thus, SA infection of dermal fibroblasts should be taken into account for future wound management strategies.

The cellular architecture of the antimicrobial response network in human leprosy granulomas.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.

The biological synthesis of gold/perlite nanocomposite using Urtica dioica extract and its chitosan-capped derivative for healing wounds infected with methicillin-resistant Staphylococcus aureus.

The preparation of ointments from natural compounds is essential for accelerating infected wounds. This study investigated the effects of topical uses of gold nanoparticles (Au)/perlite (Au/Perl) nanocomposites (NCs) by the help of Urtica dioica extract and its chitosan-capped derivative (Chit) on methicillin-resistant Staphylococcus aureus (MRSA)-infected wound healing in a mouse model. Furthermore, Au/Perl/Chit nanocomposite was prepared using protonated chitosan solution. The physicochemical properties of the as-synthesized nanocomposites were also investigated. The effects of Au/Perl/Chit NC were assessed by antibacterial, histopathological parameters as well as molecular evaluations. Then, they were compared with synthetic agent of mupirocin. The results revealed that Au/Perl NC was mesoporous and spherical in a range of 13-15 nm. Topical administration of Au/Perl/Chit ointment accelerated wound healing by reducing bacteria colonization and wound rate enhancing collagen biosynthesis and re-epithelialization, the expressions of IL-10, PI3K, AKT, bFGF, and COL1A genes, which is in agreement with the obtained results for mupirocin. In conclusion, the results strongly demonstrated that administration of ointments prepared from Au/Perl and Au/Perl/Chit nanocomposites stimulates MRSA-infected wound healing by decreasing the length of healing time and regulating PI3K/AKT/bFGF signaling pathway and is a promising candidate in stimulating MRSA-infected wound regeneration.

Mycoplasma affects baseline gene expression and the response to glucocorticoids in vocal fold fibroblasts.

Introduction. In vitro experimentation is intentionally contrived to isolate specific phenomena in the context of profound biological complexity. Mycoplasmas in the upper airway likely contribute to this complexity and play a largely unknown role in both health and disease. Similarly, the presence and role of mycoplasma in in vitro investigation are largely unknown.Hypothesis. We hypothesize mycoplasma in human vocal fold fibroblasts (VFF) will affect both basal gene-expression patterns as well as the cell response to exogenous stimuli.Aim. We sought to determine mycoplasma presence across vocal fold fibroblast cultures, basal transcriptional changes as a function of mycoplasma, and responsiveness to exogenous glucocorticoids in mycoplasma-positive and -negative VFF.Methodology. PCR-based mycoplasma detection was performed in an immortalized human VFF line as well as rat and rabbit primary VFF cultures and extracted rat laryngeal tissue. RNA sequencing was performed in mycoplasma-positive and -negative human cells at baseline and in response to dexamethasone.Results. Mycoplasma was identified in the human cell line as well as primary culture from rabbits. Mycoplasma was not detected in tissue or primary culture from rat vocal folds. Basal mRNA expression in human VFF differed significantly following mycoplasma treatment. In addition, differential responses to dexamethasone were observed across multiple pathways as a function of mycoplasma presence in these cells. Pathways including apoptosis, DNA damage repair, and G1 to S cell cycle signalling were significantly enriched in mycoplasma-positive cells.Conclusion. Variability of mycoplasma presence across culture conditions and differential responses to exogenous stimuli as a function of mycoplasma presence are potentially problematic for the translation of in vitro experimentation in the upper aerodigestive tract. It remains unclear if these findings represent contamination or the baseline state of this specialized tissue.

Investigation of the invasion mechanism mediated by the outer membrane protein PagN of Salmonella Typhimurium.

BACKGROUND: Salmonella can invade host cells via a type three secretion system called T3SS-1 and its outer membrane proteins, PagN and Rck. However, the mechanism of PagN-dependent invasion pathway used by Salmonella enterica, subspecies enterica serovar Typhimurium remains unclear. RESULTS: Here, we report that PagN is well conserved and widely distributed among the different species and subspecies of Salmonella. We showed that PagN of S. Typhimurium was sufficient and necessary to enable non-invasive E. coli over-expressing PagN and PagN-coated beads to bind to and invade different non-phagocytic cells. According to the literature, PagN is likely to interact with heparan sulfate proteoglycan (HSPG) as PagN-mediated invasion could be inhibited by heparin treatment in a dose-dependent manner. This report shows that this interaction is not sufficient to allow the internalization mechanism. Investigation of the role of ß1 integrin as co-receptor showed that mouse embryo fibroblasts genetically deficient in ß1 integrin were less permissive to PagN-mediated internalization. Moreover, PagN-mediated internalization was fully inhibited in glycosylation-deficient pgsA-745 cells treated with anti-ß1 integrin antibody, supporting the hypothesis that ß1 integrin and HSPG cooperate to induce the PagN-mediated internalization mechanism. In addition, use of specific inhibitors and expression of dominant-negative derivatives demonstrated that tyrosine phosphorylation and class I phosphatidylinositol 3-kinase were crucial to trigger PagN-dependent internalization, as for the Rck internalization mechanism. Finally, scanning electron microscopy with infected cells showed microvillus-like extensions characteristic of Zipper-like structure, engulfing PagN-coated beads and E. coli expressing PagN, as observed during Rck-mediated internalization. CONCLUSIONS: Our results supply new comprehensions into T3SS-1-independent invasion mechanisms of S. Typhimurium and highly indicate that PagN induces a phosphatidylinositol 3-kinase signaling pathway, leading to a Zipper-like entry mechanism as the Salmonella outer membrane protein Rck.

Natural flavonoid morin showed anti-bacterial activity against Vibrio cholera after binding with cell division protein FtsA near ATP binding site.

BACKGROUND: Increasing antibiotic-resistance in bacterial strains has boosted the need to find new targets for drug delivery. FtsA, a major bacterial divisome protein can be a potent novel drug-target. METHODS AND RESULTS: This study finds, morin (3,5,7,2',4'-pentahydroxyflavone), a bio-available flavonoid, had anti-bacterial activities against Vibrio cholerae, IC50 (50 µM) and MIC (150 µM). Morin (2 mM) kills ~20% of human lung fibroblast (WI38) and human intestinal epithelial (HIEC-6) cells in 24 h in-vitro. Fluorescence studies showed morin binds to VcFtsA (FtsA of V. cholerae) with a Kd of 4.68 ± 0.4 µM, inhibiting the protein's polymerization by 72 ± 7% at 25 µM concentration. Morin also affected VcFtsA's ATPase activity, recording ~80% reduction at 20 µM concentration. The in-silico binding study indicated binding sites of morin and ATP on VcFtsA had overlapping amino acids. Mant-ATP, a fluorescent ATP-derivative, showed increased fluorescence on binding to VcFtsA in absence of morin, but in its presence, Mant-ATP fluorescence decreased. VcFtsA-S40A mutant protein did not bind to morin. CONCLUSIONS: VcFtsA-morin interaction inhibits the polymerization of the protein by affecting its ATPase activity. The destabilized VcFtsA assembly in-turn affected the cell division in V. cholerae, yielding an elongated morphology. GENERAL SIGNIFICANCE: Collectively, these findings explore the anti-bacterial effect of morin on V. cholerae cells targeting VcFtsA, encouraging it to become a potent anti-bacterial agent. Low cytotoxicity of morin against human cells (host) is therapeutically advantageous. This study will also help in synthesizing novel derivatives that can target VcFtsA more efficiently.