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The translocation of stimulator of interferon genes (STING) from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC) enables its activation. However, the mechanism underlying the regulation of STING exit from the ER remains elusive. Here, we found that STING induces the activation of transforming growth factor beta-activated kinase 1 (TAK1) prior to STING trafficking in a TAK1 binding protein 1 (TAB1)-dependent manner. Intriguingly, activated TAK1 directly mediates STING phosphorylation on serine 355, which facilitates its interaction with STING ER exit protein (STEEP) and thereby promotes its oligomerization and translocation to the ERGIC for subsequent activation. Importantly, activation of TAK1 by monophosphoryl lipid A, a TLR4 agonist, boosts cGAMP-induced antitumor immunity dependent on STING phosphorylation in a mouse allograft tumor model. Taken together, TAK1 was identified as a checkpoint for STING activation by promoting its trafficking, providing a basis for combinatory tumor immunotherapy and intervention in STING-related diseases.
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Neoplasias , Animais , Camundongos , FosforilaçãoRESUMO
This article introduces the Lipids and Extracellular Materials theme of the Annual Review of Biochemistry, Volume 83.
Assuntos
Lipídeos/química , Animais , Bactérias/metabolismo , Heparitina Sulfato/química , Humanos , Bicamadas Lipídicas/química , Lipopolissacarídeos , Ligação ProteicaRESUMO
Lipopolysaccharide molecules represent a unique family of glycolipids based on a highly conserved lipid moiety known as lipid A. These molecules are produced by most gram-negative bacteria, in which they play important roles in the integrity of the outer-membrane permeability barrier and participate extensively in host-pathogen interplay. Few bacteria contain lipopolysaccharide molecules composed only of lipid A. In most forms, lipid A is glycosylated by addition of the core oligosaccharide that, in some bacteria, provides an attachment site for a long-chain O-antigenic polysaccharide. The complexity of lipopolysaccharide structures is reflected in the processes used for their biosynthesis and export. Rapid growth and cell division depend on the bacterial cell's capacity to synthesize and export lipopolysaccharide efficiently and in large amounts. We review recent advances in those processes, emphasizing the reactions that are essential for viability.
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Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Bactérias , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Glicolipídeos/metabolismo , Glicosilação , Bactérias Gram-Negativas/metabolismo , Antígenos O/metabolismo , Permeabilidade , Polissacarídeos/metabolismoRESUMO
We previously demonstrated that Alcaligenes-derived lipid A (ALA), which is produced from an intestinal lymphoid tissue-resident commensal bacterium, is an effective adjuvant for inducing antigen-specific immune responses. To understand the immunologic characteristics of ALA as a vaccine adjuvant, we here compared the adjuvant activity of ALA with that of a licensed adjuvant (monophosphoryl lipid A, MPLA) in mice. Although the adjuvant activity of ALA was only slightly greater than that of MPLA for subcutaneous immunization, ALA induced significantly greater IgA antibody production than did MPLA during nasal immunization. Regarding the underlying mechanism, ALA increased and activated CD11b+ CD103- CD11c+ dendritic cells in the nasal tissue by stimulating chemokine responses. These findings revealed the superiority of ALA as a mucosal adjuvant due to the unique immunologic functions of ALA in nasal tissue.
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Alcaligenes , Lipídeo A , Animais , Camundongos , Lipídeo A/farmacologia , Adjuvantes Imunológicos/farmacologia , Células DendríticasRESUMO
SignificanceYersinia pestis, the etiologic agent of plague, has been responsible for high mortality in several epidemics throughout human history. This plague bacillus has been used as a biological weapon during human history and is currently one of the deadliest biological threats. Currently, no licensed plague vaccines are available in the Western world. Since an array of immunogens are enclosed in outer membrane vesicles (OMVs), immune responses elicited by OMVs against a diverse range of antigens may reduce the likelihood of antigen circumvention. Therefore, self-adjuvanting OMVs from a remodeled Yersinia pseudotuberculosis strain as a type of plague vaccine could diversify prophylactic choices and solve current vaccine limitations.
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Antígenos de Bactérias , Lipídeo A , Vacina contra a Peste , Peste , Proteínas Citotóxicas Formadoras de Poros , Yersinia pseudotuberculosis , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Dose Letal Mediana , Lipídeo A/genética , Lipídeo A/imunologia , Camundongos , Peste/prevenção & controle , Vacina contra a Peste/administração & dosagem , Vacina contra a Peste/genética , Vacina contra a Peste/imunologia , Plasmídeos/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/imunologia , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/imunologiaRESUMO
Salmonella enterica serovar Typhimurium (S. Typhimurium) controls lipopolysaccharide (LPS) biosynthesis by regulating proteolysis of LpxC, the rate-limiting enzyme and target of preclinical antibiotics. PbgA/YejM/LapC regulates LpxC levels and controls outer membrane (OM) LPS composition at the log-to-stationary phase transition. Suppressor substitutions in LPS assembly protein B (LapB/YciM) rescue the LPS and OM integrity defects of pbgA-mutant S. Typhimurium. We hypothesized that PbgA regulates LpxC proteolysis by controlling LapB's ability to bind LpxC as a function of the growth phase. According to existing models, when nutrients are abundant, PbgA binds and restricts LapB from interacting with LpxC and FtsH, which limits LpxC proteolysis. However, when nutrients are limited, there is debate whether LapB dissociates from PbgA to bind LpxC and FtsH to enhance degradation. We sought to examine these models and investigate how the structure of LapB enables salmonellae to control LpxC proteolysis and LPS biosynthesis. Salmonellae increase LapB levels during the stationary phase to promote LpxC degradation, which limits lipid A-core production and increases their survival. The deletion of lapB, resulting in unregulated lipid A-core production and LpxC overabundance, leads to bacterial growth retardation. Tetratricopeptide repeats near the cytosol-inner membrane interface are sufficient for LapB to bind LpxC, and remarkably, LapB and PbgA interact in both growth phases, yet LpxC only associates with LapB in the stationary phase. Our findings support that PbgA-LapB exists as a constitutive complex in S. Typhimurium, which differentially binds LpxC to control LpxC proteolysis and limit lipid A-core biosynthesis in response to changes in the environment.IMPORTANCEAntimicrobial resistance has been a costly setback for human health and agriculture. Continued pursuit of new antibiotics and targets is imperative, and an improved understanding of existing ones is necessary. LpxC is an essential target of preclinical trial antibiotics that can eliminate multidrug-resistant Gram-negative bacterial infections. LapB is a natural LpxC inhibitor that targets LpxC for degradation and limits lipopolysaccharide production in Enterobacteriaceae. Contrary to some studies, findings herein support that LapB remains in complex instead of dissociating from its presumed negative regulator, PbgA/YejM/LapC, under conditions where LpxC proteolysis is enhanced. Advanced comprehension of this critical protein-lipid signaling network will lead to future development and refinement of small molecules that can specifically interfere.
Assuntos
Proteínas de Escherichia coli , Lipopolissacarídeos , Humanos , Lipopolissacarídeos/metabolismo , Lipídeo A , Escherichia coli/metabolismo , Proteólise , Salmonella typhimurium/metabolismo , Antibacterianos/metabolismo , Amidoidrolases/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Alcohol binge drinking allows the translocation of bacterial lipopolysaccharide (LPS) from the gut to the blood, which activates the peripheral immune system with consequences in neuroinflammation. A possible access/direct signaling of LPS to/in the brain has not yet been described under alcohol abuse conditions. Apolipoproteins are compounds altered by alcohol with high affinity to LPS which may be involved in its transport to the brain or in its elimination. Here, we explored the expression of small components of LPS, in its free form or bound to apolipoproteins, in the brain of female and male rats exposed to alcohol binges. Animals received ethanol oral gavages (3 g/kg every 8 h) for 4 days. LPS or its components (Lipid A and core), LPS-binding protein, corticosterone, lipoproteins (HDL, LDL), apolipoproteins (ApoAI, ApoB, and ApoE), and their receptors were measured in plasma and/or in nonperfused prefrontal cortex (PFC) and cerebellum. Brain LipidA-apolipoprotein aggregates were determined by Western blotting and confirmed by co-immunoprecipitation. In animals exposed to alcohol binges: 1) plasma LPS-binding protein was elevated in both sexes; 2) females showed elevations in plasma ApoAI and corticosterone levels; 3) Lipid A formed aggregates with ApoAI in the female PFC and with ApoB in males, the latter showing Toll-like receptor 4 upregulation in PFC but not females. These results suggest that small bacterial components are present within the brain, forming aggregates with different apolipoproteins, depending on the sex, after alcohol binge intoxications. Results may have implications for the crosstalk between alcohol, LPS, and neuroinflammation.
Assuntos
Etanol , Lipopolissacarídeos , Ratos , Masculino , Feminino , Animais , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Doenças Neuroinflamatórias , Lipídeo A/metabolismo , Corticosterona/metabolismo , Apolipoproteínas/metabolismo , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Apolipoproteínas B/metabolismoRESUMO
Cyclooxygenase-2 converts arachidonic acid to prostaglandins (PGs) and the endocannabinoid, 2-arachidonoylglycerol (2-AG), to PG glyceryl esters (PG-Gs). The physiological function of PG biosynthesis has been extensively studied, but the importance of the more recently discovered PG-G synthetic pathway remains incompletely defined. This disparity is due in part to a lack of knowledge of the physiological conditions under which PG-G biosynthesis occurs. We have discovered that RAW264.7 macrophages stimulated with Kdo2-lipid A (KLA) produce primarily PGs within the first 12 h followed by robust PG-G synthesis between 12 h and 24 h. We suggest that the amount of PG-Gs quantified is less than actually synthesized, because PG-Gs are subject to a significant level of hydrolysis during the time course of synthesis. Inhibition of cytosolic phospholipase A2 by giripladib does not accelerate PG-G synthesis, suggesting the differential time course of PG and PG-G synthesis is not due to the competition between arachidonic acid and 2-AG. The late-phase PG-G formation is accompanied by an increase in the level of 2-AG and a concomitant decrease in 18:0-20:4 diacylglycerol (DAG). Inhibition of DAG lipases by KT-172 decreases the levels of 2-AG and PG-Gs, indicating that the DAG-lipase pathway is involved in delayed 2-AG metabolism/PG-G synthesis. These results demonstrate that physiologically significant levels of PG-Gs are produced by activated RAW264.7 macrophages well after the production of PGs plateaus.
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Polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae bacteria is associated with high morbidity and mortality in vulnerable populations throughout the world. Ineffective antimicrobial activity by these last resort therapeutics can occur by transfer of mcr-1, a plasmid-mediated resistance gene, causing modification of the lipid A portion of lipopolysaccharide (LPS) and disruption of the interactions between polymyxins and lipid A. Whether this modification alters the innate host immune response or carries a high fitness cost in the bacteria is not well established. To investigate this, we studied infection with K. pneumoniae (KP) ATCC 13883 harboring either the mcr-1 plasmid (pmcr-1) or the vector control (pBCSK) ATCC 13883. Bacterial fitness characteristics of mcr-1 acquisition were evaluated. Differentiated human monocytes (THP-1s) were stimulated with KP bacterial strains or purified LPS from both parent isolates and isolates harboring mcr-1. Cell culture supernatants were analyzed for cytokine production. A bacterial pneumonia model in WT C57/BL6J mice was used to monitor immune cell recruitment, cytokine induction, and bacterial clearance in the bronchoalveolar lavage fluid (BALF). Isolates harboring mcr-1 had increased colistin MIC compared to the parent isolates but did not alter bacterial fitness. Few differences in cytokines were observed with purified LPS from mcr-1 expressing bacteria in vitro. However, in a mouse pneumonia model, no bacterial clearance defect was observed between pmcr-1-harboring KP and parent isolates. Consistently, no differences in cytokine production or immune cell recruitment in the BALF were observed, suggesting that other mechanisms outweigh the effect of these lipid A mutations in LPS.
Assuntos
Antibacterianos , Colistina , Modelos Animais de Doenças , Imunidade Inata , Infecções por Klebsiella , Klebsiella pneumoniae , Lipídeo A , Animais , Klebsiella pneumoniae/imunologia , Klebsiella pneumoniae/efeitos dos fármacos , Colistina/farmacologia , Lipídeo A/imunologia , Camundongos , Infecções por Klebsiella/imunologia , Infecções por Klebsiella/microbiologia , Humanos , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Camundongos Endogâmicos C57BL , Citocinas/metabolismo , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/microbiologia , FemininoRESUMO
The protection afforded by acellular pertussis vaccines wanes over time, and there is a need to develop improved vaccine formulations. Options to improve the vaccines involve the utilization of different adjuvants and administration via different routes. While intramuscular (IM) vaccination provides a robust systemic immune response, intranasal (IN) vaccination theoretically induces a localized immune response within the nasal cavity. In the case of a Bordetella pertussis infection, IN vaccination results in an immune response that is similar to natural infection, which provides the longest duration of protection. Current acellular formulations utilize an alum adjuvant, and antibody levels wane over time. To overcome the current limitations with the acellular vaccine, we incorporated a novel TLR4 agonist, BECC438b, into both IM and IN acellular formulations to determine its ability to protect against infection in a murine airway challenge model. Following immunization and challenge, we observed that DTaP + BECC438b reduced bacterial burden within the lung and trachea for both administration routes when compared with mock-vaccinated and challenged (MVC) mice. Interestingly, IN administration of DTaP + BECC438b induced a Th1-polarized immune response, while IM vaccination polarized toward a Th2 immune response. RNA sequencing analysis of the lung demonstrated that DTaP + BECC438b activates biological pathways similar to natural infection. Additionally, IN administration of DTaP + BECC438b activated the expression of genes involved in a multitude of pathways associated with the immune system. Overall, these data suggest that BECC438b adjuvant and the IN vaccination route can impact efficacy and responses of pertussis vaccines in pre-clinical mouse models.
Assuntos
Vacinas contra Difteria, Tétano e Coqueluche Acelular , Coqueluche , Animais , Camundongos , Coqueluche/prevenção & controle , Receptor 4 Toll-Like , Vacina contra Coqueluche , Vacina contra Difteria, Tétano e Coqueluche , Bordetella pertussis , Adjuvantes Imunológicos , Imunidade , Anticorpos AntibacterianosRESUMO
Lipopolysaccharide (LPS) is a fundamental tripartite glycolipid found on the surface of nearly all Gram-negative bacteria. It acts as a protective shield for the bacterial cell and is a potent agonist of the innate immune system. This primer serves to introduce the basic properties of LPS, its function in bacterial physiology and pathogenicity, and its use as a therapeutic target.
Assuntos
Bactérias Gram-Negativas , Lipopolissacarídeos , Bactérias Gram-Negativas/genéticaRESUMO
A new Yersinia pseudotuberculosis mutant strain, YptbS46, carrying the lpxE insertion and pmrF-J deletion is constructed and shown to exclusively produce monophosphoryl lipid A (MPLA) having adjuvant properties. Outer membrane vesicles (OMVs) isolated from YptbS46 harboring an lcrV expression plasmid, pSMV13, are designated OMV46-LcrV, which contained MPLA and high amounts of LcrV (Low Calcium response V) and displayed low activation of Toll-like receptor 4 (TLR4). Intramuscular prime-boost immunization with 30 µg of of OMV46-LcrV exhibited substantially reduced reactogenicity than the parent OMV44-LcrV and conferred complete protection to mice against a high-dose of respiratory Y. pestis challenge. OMV46-LcrV immunization induced robust adaptive responses in both lung mucosal and systemic compartments and orchestrated innate immunity in the lung, which are correlated with rapid bacterial clearance and unremarkable lung damage during Y. pestis challenge. Additionally, OMV46-LcrV immunization conferred long-term protection. Moreover, immunization with reduced doses of OMV46-LcrV exhibited further lower reactogenicity and still provided great protection against pneumonic plague. The studies strongly demonstrate the feasibility of OMV46-LcrV as a new type of plague vaccine candidate.
Assuntos
Lipídeo A/análogos & derivados , Vacina contra a Peste , Peste , Yersinia pestis , Camundongos , Animais , Yersinia , Peste/prevenção & controle , Antígenos de BactériasRESUMO
Colistin (polymyxin E) is a group of cationic antimicrobial cyclic peptides and is recognized as a last-resort defense against lethal infections with carbapenem-resistant pathogens. In addition to the plasmid-borne mobilized phosphoethanolamine (PEA) transferases, the functional expression of lipid A-modifying enzymes encoded on chromosomes has been attributed to intrinsic bacterial colistin resistance. However, the mechanisms of colistin resistance in Riemerella anatipestifer remain unknown. Herein, the GE296_RS09715 gene-encoded Lipid A PEA transferases (RaEptA) was identified in R. anatipestifer. Genetic and structural analyses revealed that the amino acid sequence of RaEptA shared 26.6%-33.1% similarities with the family of Lipid A PEA transferases (EptA) and MCR-like proteins and have defined 12 residues that contribute to the formation of phosphatidylethanolamine (PE)-recognizable cavities. Comparative analyses of colistin resistance in RA-LZ01 and RA-LZ01ΔRaEptA showed the level of colistin has fallen from 96 µg mL-1 down to 24 ~ 32 µg mL-1 . Site-directed mutagenesis assay of the PE-binding cavity and expression of the mutants reveals that K309-rRaEptA can remodel the surface of Escherichia coli and rendering it resistant to colistin, suggesting this point-mutation of P309K is necessary for EptA-mediated lipid A modification. Moreover, the virulence of RA-LZ01ΔRaEptA was attenuated compared with RA-LZ01 both in vivo and vitro. Taken together, the results represent the RaEptA involved in the colistin resistance and pathogenicity, and the P309K mutation might alter bacterial adaptation and increase the spread of colistin resistance from R. anatipestifer to other gram-negative bacteria. The findings of this study suggest another scenario for the spread of colistin resistance genes and should be considered by a wide audience.
Assuntos
Antibacterianos , Colistina , Colistina/farmacologia , Colistina/química , Antibacterianos/farmacologia , Virulência/genética , Lipídeo A/química , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fenótipo , TransferasesRESUMO
Gram-negative bacteria living in marine waters have evolved peculiar adaptation strategies to deal with the numerous stress conditions that characterize aquatic environments. Among the multiple mechanisms for efficient adaptation, these bacteria typically exhibit chemical modifications in the structure of the lipopolysaccharide (LPS), which is a fundamental component of their outer membrane. In particular, the glycolipid anchor to the membrane of marine bacteria LPSs, i.e. the lipid A, frequently shows unusual chemical structures, which are reflected in equally singular immunological properties with potential applications as immune adjuvants or anti-sepsis drugs. In this work, we determined the chemical structure of the lipid A from Cellulophaga pacifica KMM 3664T isolated from the Sea of Japan. This bacterium showed to produce a heterogeneous mixture of lipid A molecules that mainly display five acyl chains and carry a single phosphate and a D-mannose disaccharide on the glucosamine backbone. Furthermore, we proved that C. pacifica KMM 3664T LPS acts as a weaker activator of Toll-like receptor 4 (TLR4) compared to the prototypical enterobacterial Salmonella typhimurium LPS. Our results are relevant to the future development of novel vaccine adjuvants and immunomodulators inspired by marine LPS chemistry.
Assuntos
Lipídeo A , Lipídeo A/química , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/química , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/química , Animais , Lipopolissacarídeos/química , CamundongosRESUMO
During the COVID-19 pandemic, expedient vaccine production has been slowed by the shortage of safe and effective raw materials, such as adjuvants, essential components to enhance the efficacy of vaccines. Monophosphoryl lipid A (MPLA) is a potent and safe adjuvant used in human vaccines, including the Shingles vaccine, Shingrix. 3-O-desacyl-4'-monophosphoryl lipid A (MPL), a representative MPLA adjuvant commercialized by GSK, was prepared via chemical conversion of precursors isolated from Salmonella typhimurium R595. However, the high price of these materials limits their use in premium vaccines. To combat the scarcity and high cost of safe raw materials for vaccines, we need to develop a feasible MPLA production method that is easily scaled up to meet industrial requirements. In this study, we engineered peptidoglycan and outer membrane biosynthetic pathways in Escherichia coli and developed a Escherichia coli strain, KHSC0055, that constitutively produces EcML (E. coli-produced monophosphoryl lipid A) without additives such as antibiotics or overexpression inducers. EcML production was optimized on an industrial scale via high-density fed-batch fermentation, and obtained 2.7 g of EcML (about 135,000 doses of vaccine) from a 30-L-scale fermentation. Using KHSC0055, we simplified the production process and decreased the production costs of MPLA. Then, we applied EcML purified from KHSC0055 as an adjuvant for a COVID-19 vaccine candidate (EuCorVac-19) currently in clinical trial stage III in the Philippines. By probing the efficacy and safety of EcML in humans, we established KHSC0055 as an efficient cell factory for MPLA adjuvant production.
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Adjuvantes de Vacinas , Lipídeo A/análogos & derivados , Vacinas , Humanos , Escherichia coli/genética , Vacinas contra COVID-19 , Pandemias , Adjuvantes ImunológicosRESUMO
BACKGROUND: A cost-effective Escherichia coli expression system has gained popularity for producing virus-like particle (VLP) vaccines. However, the challenge lies in balancing the endotoxin residue and removal costs, as residual endotoxins can cause inflammatory reactions in the body. RESULTS: In this study, porcine parvovirus virus-like particles (PPV-VLPs) were successfully assembled from Decreased Endotoxic BL21 (BL21-DeE), and the effect of structural changes in the lipid A of BL21 on endotoxin activity, immunogenicity, and safety was investigated. The lipopolysaccharide purified from BL21-DeE produced lower IL-6 and TNF-α than that from wild-type BL21 (BL21-W) in both RAW264.7 cells and BALB/c mice. Additionally, mice immunized with PPV-VLP derived form BL21-DeE (BL21-DeE-VLP) showed significantly lower production of inflammatory factors and a smaller increase in body temperature within 3 h than those immunized with VLP from BL21-W (BL21-W-VLP) and endotoxin-removed VLP (ReE-VLP). Moreover, mice in the BL21-DeE-VLP immunized group had similar levels of serum antibodies as those in the BL21-W-VLP group but significantly higher levels than those in the ReE-VLP group. Furthermore, the liver, lungs, and kidneys showed no pathological damage compared with the BL21-W-VLP group. CONCLUSION: Overall, this study proposes a method for producing VLP with high immunogenicity and minimal endotoxin activity without chemical or physical endotoxin removal methods. This method could address the issue of endotoxin residues in the VLP and provide production benefits.
Assuntos
Endotoxinas , Escherichia coli , Lipídeo A , Camundongos Endogâmicos BALB C , Parvovirus Suíno , Vacinas de Partículas Semelhantes a Vírus , Animais , Camundongos , Escherichia coli/genética , Escherichia coli/metabolismo , Parvovirus Suíno/imunologia , Parvovirus Suíno/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Endotoxinas/imunologia , Células RAW 264.7 , Lipídeo A/imunologia , Lipídeo A/análogos & derivados , Interleucina-6/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Feminino , Suínos , Lipopolissacarídeos/imunologiaRESUMO
BACKGROUND: Bioscaffolds and cells are two main components in the regeneration of damaged tissues via cell therapy. Umbilical cord stem cells are among the most well-known cell types for this purpose. The main objective of the present study was to evaluate the effect of the pretreatment of the foreskin acellular matrix (FAM) by monophosphoryl lipid A (MPLA) and Lactobacillus casei supernatant (LCS) on the attraction of human umbilical cord mesenchymal stem cells (hucMSC). METHODS AND RESULTS: The expression of certain cell migration genes was studied using qRT-PCR. In addition to cell migration, transdifferentiation of these cells to the epidermal-like cells was evaluated via immunohistochemistry (IHC) and immunocytochemistry (ICC) of cytokeratin 19 (CK19). The hucMSC showed more tissue tropism in the presence of MPLA and LCS pretreated FAM compared to the untreated control group. We confirmed this result by scanning electron microscopy (SEM) analysis, glycosaminoglycan (GAG), collagen, and DNA content. Furthermore, IHC and ICC data demonstrated that both treatments increase the protein expression level of CK19. CONCLUSION: Pretreatment of acellular bioscaffolds by MPLA or LCS can increase the migration rate of cells and also transdifferentiation of hucMSC to epidermal-like cells without growth factors. This strategy suggests a new approach in regenerative medicine.
Assuntos
Lacticaseibacillus casei , Lipídeo A , Células-Tronco Mesenquimais , Humanos , Células-Tronco Mesenquimais/metabolismo , Lacticaseibacillus casei/metabolismo , Lipídeo A/metabolismo , Lipídeo A/análogos & derivados , Movimento Celular/efeitos dos fármacos , Pele/metabolismo , Alicerces Teciduais/química , Masculino , Cordão Umbilical/citologia , Cordão Umbilical/metabolismo , Prepúcio do Pênis/citologia , Transdiferenciação Celular/efeitos dos fármacos , Engenharia Tecidual/métodos , Matriz Extracelular/metabolismo , Queratina-19/metabolismo , Queratina-19/genéticaRESUMO
The rate of pandrug-resistant Acinetobacter baumannii strains is on the rise in all continents. This bacterium can acquire resistance to all antibiotics, even to colistin. Alterations in the lipid A or/and the two-component pmrAB were earlier detected in colistin resistance. We investigated and analyzed two strains of A. baumannii (ABRC1 and ABRC2) isolated from two patients admitted to intensive care unit with a septic shock. Both strains were resistant to all tested antibiotics including colistin with a MIC >256 mg L-1. Colistin resistance genes (pmrA, pmrB, lpxA, lpxC, lpxD, and lpsB) of two strains (ABRC1 and ABRC2) were investigated by PCR and sequencing. Obtained nucleic acid sequences were aligned with reference sequences of ATCC 19606 and 17987. In this study two amino acid mutations, N287D in the lpxC gene and E117K in the lpxD gene, were detected in both ABRC1 and ABRC2 strains. ABRC1 had an additional H200L mutation in the pmrA gene. Both colistin resistant strains harbored the same A138T mutation in the pmrB gene. The ABRC2 strain also had an alteration in the kinase domain, specifically an R263S substitution of the histidine kinase domain. Three identical mutations were found in the lpsB gene of both A. baumannii strains: Q216K + H218G + S219E. As a result, a newly deduced protein sequence in both ABRC1 and ABRC2 strains differed from those described in ATCC 17978 and 19606 strains was determined. Colistin resistance is multifactorial in A. baumannii. In our study we detected novel mutations in colistin resistant A. baumannii clinical isolates.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Proteínas de Bactérias , Lipídeo A , Testes de Sensibilidade Microbiana , Acinetobacter baumannii/genética , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/metabolismo , Humanos , Lipídeo A/genética , Lipídeo A/metabolismo , Lipídeo A/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antibacterianos/farmacologia , Infecções por Acinetobacter/microbiologia , Farmacorresistência Bacteriana/genética , Polimixinas/farmacologia , Colistina/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , MutaçãoRESUMO
Complex microbial communities have been reported to be involved in endodontic infections. The microorganisms invade the dental pulp leading to pulpitis and initiating pulp inflammation. Fusobacterium nucleatum is a dominant bacterium implicated in both primary and secondary endodontic infections. Drugs targeting the molecular machinery of F. nucleatum will minimize pulp infection. LpxA and LpxD are early acyltransferases involved in the formation of lipid A, a major component of bacterial membranes. The identification of leads which exhibit preference towards successive enzymes in a single pathway can also prevent the development of bacterial resistance. A stringent screening strategy utilizing physicochemical and pharmacokinetic parameters along with a virtual screening approach identified two compounds, Lomefloxacin and Enoxacin, with good binding affinity towards the early acyltransferases LpxA and LpxD. Lomefloxacin and Enoxacin, members of the fluoroquinolone antibiotic class, exhibit wide-ranging activity against diverse bacterial strains. Nevertheless, their effectiveness in the context of endodontic treatment requires further investigation. This study explored the potential of Lomefloxacin and Enoxacin to manage endodontic infections via computational analysis. Moreover, the compounds identified herein serve as a foundation for devising novel combinatorial libraries with enhanced efficacy for endodontic therapeutic strategies.
Assuntos
Antibacterianos , Fusobacterium nucleatum , Lipopolissacarídeos , Fusobacterium nucleatum/efeitos dos fármacos , Fusobacterium nucleatum/metabolismo , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Lipopolissacarídeos/metabolismo , Simulação de Acoplamento Molecular , Simulação por Computador , Infecções por Fusobacterium/tratamento farmacológico , Infecções por Fusobacterium/microbiologia , Enoxacino/farmacologia , Proteínas de Bactérias/metabolismo , Pulpite/tratamento farmacológico , Pulpite/metabolismo , Pulpite/microbiologiaRESUMO
Polymyxins are a group of detergent-like antimicrobial peptides that are the ultimate line of defense against carbapenem-resistant pathogens in clinical settings. Polymyxin resistance primarily originates from structural remodeling of lipid A anchored on bacterial surfaces. We integrate genetic, structural, and biochemical aspects of three major types of lipid A modifiers that have been shown to confer intrinsic colistin resistance. Namely, we highlight ArnT, a glycosyltransferase, EptA, a phosphoethanolamine transferase, and the AlmEFG tripartite system, which is restricted to EI Tor biotype of Vibrio cholerae O1. We also discuss the growing family of mobile colistin resistance (MCR) enzymes, each of which is analogous to EptA, and which pose great challenges to global public health.