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1.
Infect Immun ; 87(5)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30858343

RESUMO

The human protozoan Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection, which is accompanied by a species-diversified vaginal microbiota named community state type IV (CST-IV). Coincidently, CST-IV includes species associated with bacterial vaginosis (e.g. Gardnerella vaginalis, Atopobium vaginae, and Prevotella bivia). Both diseases are linked to the transmission of human immunodeficiency virus (HIV) and preterm birth, which complications are likely to result from the disruption of the cervicovaginal epithelial barrier. Here, we show that paracellular permeability of fluorescein isothiocyanate (FITC)-dextran through a monolayer of human ectocervical cells (hECs) is increased as a consequence of the activity of T. vaginalis and the aforementioned species of CST-IV bacteria cooperatively. T. vaginalis enhances paracellular permeability of hECs two times more than the individual bacterial species, by up to ∼10% versus ∼5%, respectively. However, any two or all three bacterial species are capable of synergizing this effect. T. vaginalis and the bacteria together increase the paracellular permeability of hECs by ∼50%, which is 5 to 10 times more than the results seen with the protozoan or bacteria alone. This effect is accompanied by enhancement of phosphatase activity, while phosphatase inhibition results in preservation of the integrity of the ectocervical cell monolayer. In addition, these microorganisms induce changes in the expression of tight junction proteins, particularly occludin, and of proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α). Together, our findings establish that cooperative interactions between CST-IV bacteria and T. vaginalis enhance the paracellular permeability of the cervicovaginal epithelium by disturbing the integrity of the tight junction complex. Our study results highlight the importance of understanding the contribution of the vaginal microbiota to trichomoniasis.


Assuntos
Células Epiteliais/fisiologia , Interações Microbianas , Junções Íntimas/fisiologia , Trichomonas vaginalis/fisiologia , Trichomonas vaginalis/patogenicidade , Vagina/fisiologia , Vaginose Bacteriana/fisiopatologia , Feminino , Humanos , Permeabilidade
2.
FEMS Immunol Med Microbiol ; 49(2): 235-42, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17328757

RESUMO

Free fatty acids and monoglycerides released from milkfat by partial pregastric lipase-catalysed hydrolysis are bactericidal towards Helicobacter pylori. Two milkfat preparations were investigated: a normal bovine milkfat, and a fractionated milkfat preparation, termed ModFat, enriched in triglycerides containing short- and medium-chain fatty acids. The released products were tested for bactericidal potency against H. pylori. The potencies of the respective preparations were consistent with expected potencies calculated from individual free fatty acid and monoglyceride concentrations and their lauric acid equivalence factors (Ki). ModFat products were more bactericidal, in accordance with release of free fatty acid types of high potency, and addition of the surfactant Tween 80 to the hydrolysed lipid increased potency eight times more than did addition of lecithin. Tween 80 micelles have smaller aggregation numbers, and the mixed micelles of Tween 80/free fatty acids would be more likely to expose the bacteria to higher apparent free fatty acid concentrations.


Assuntos
Antibacterianos/farmacologia , Helicobacter pylori/efeitos dos fármacos , Lipase/metabolismo , Lipídeos/farmacologia , Leite/química , Animais , Antibacterianos/isolamento & purificação , Bovinos , Contagem de Colônia Microbiana , Ácidos Graxos/análise , Hidrólise , Metabolismo dos Lipídeos , Lipídeos/química , Testes de Sensibilidade Microbiana , Viabilidade Microbiana , Leite/metabolismo , Modelos Biológicos
3.
J Clin Microbiol ; 43(11): 5504-8, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16272477

RESUMO

The modifications to the vaginal habitat accompanying a change to vaginal flora in bacterial vaginosis (BV) are poorly understood. In this study enzymes involved in mucin degradation were measured, including a novel glycosulfatase assay. Women attending an emergency walk-in sexually transmitted disease clinic were studied. One high vaginal swab (HVS) was used to prepare a gram-stained smear to determine BV status, using Ison and Hay's criteria, and a separate swab was used for the purposes of the assays. The median glycosulfatase activity was 8.5 (range, -1.2 to 31.9) nmol h(-1) 1.5 ml(-1) of HVS suspension in patients with BV compared to 0.5 (range, -0.7 to 9.4) nmol h(-1) 1.5 ml(-1) of HVS suspension in patients without BV (P = <0.001). The median glycoprotein sialidase activity was 29.2 (range, -17 to 190) nmol h(-1) 1.5 ml(-1) of HVS suspension in patients with BV compared to -1.1 (range, -41 to 48) nmol h(-1) 1.5 ml(-1) of HVS suspension in patients without BV (P < 0.001). A rapid spot test for sialidase was positive in 22/24 patients with BV (sensitivity, 91.7%; 95% confidence interval [CI], 73 to 99%) and negative in 32/35 patients without BV (specificity, 91.4%; 95% CI, 76.9 to 98.2%) (P < 0.001). Glycosulfatase activity significantly correlated with both glycoprotein sialidase activity and the sialidase spot test (P = 0.006 and P < 0.001, respectively). The results are consistent with the hypothesis that the consortium of bacteria present in BV requires the ability to break down mucins in order to colonize the vagina and replace the normal lactobacilli.


Assuntos
Sulfatases/análise , Vagina/enzimologia , Vaginose Bacteriana/metabolismo , Adolescente , Adulto , Bactérias/isolamento & purificação , Feminino , Humanos , Pessoa de Meia-Idade , Mucinas/metabolismo , Neuraminidase/análise , Vagina/microbiologia , Vaginose Bacteriana/microbiologia
4.
J Bacteriol ; 187(5): 1543-51, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15716424

RESUMO

A novel enzyme which may be important in mucin degradation has been discovered in the mucin-utilizing anaerobe Prevotella strain RS2. This enzyme cleaves terminal 2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sulfate (6-SO3-GlcNAc) residues from sulfomucin and from the model substrate 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sodium sulfate. The existence of this mucin-desulfating glycosidase (sulfoglycosidase) suggests an alternative mechanism by which this bacterium may desulfate sulfomucins, by glycosidic removal of a sulfated sugar from mucin oligosaccharide chains. Previously, mucin desulfation was thought to take place by the action of a specific desulfating enzyme, which then allowed glycosidases to remove desulfated sugar. Sulfate removal from sulfomucins is thought to be a rate-limiting step in mucin degradation by bacteria in the regions of the digestive tract with a significant bacterial flora. The sulfoglycosidase was induced by growth of the Prevotella strain on mucin and was purified 284-fold from periplasmic extracts. Tryptic digestion and sequencing of peptides from the 100-kDa protein enabled the sulfoglycosidase gene to be cloned and sequenced. Active recombinant enzyme was made in an Escherichia coli expression system. The sulfoglycosidase shows sequence similarity to hexosaminidases. The only other enzyme that has been shown to remove 6-SO3-GlcNAc from glycoside substrates is the human lysosomal enzyme beta-N-acetylhexosaminidase A, point mutations in which cause the inheritable, lysosomal storage disorder Tay-Sachs disease. The human enzyme removes GlcNAc from glycoside substrates also, in contrast to the Prevotella enzyme, which acts on a nonsulfated substrate at a rate that is only 1% of the rate observed with a sulfated substrate.


Assuntos
Glicosídeo Hidrolases/metabolismo , Mucinas/metabolismo , Prevotella/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Expressão Gênica , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Dados de Sequência Molecular , Proteínas Recombinantes , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
5.
J Biol Chem ; 278(49): 49600-9, 2003 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-12958310

RESUMO

Human intestinal mucin genes MUC3A and MUC3B are members of a membrane mucin gene family residing at chromosome 7q22. In this paper, we utilized genomic and cDNA cloning to elucidate the sequence of the 5'-region of the MUC3A gene including the gene promoter and the amino terminus coding sequence. Following its 21-residue signal peptide, the amino terminus of the mucin consists of a 233-residue Thr-, Ser-, and Pro-rich nonrepetitive sequence that is contiguous with its hypervariable domain of 375-residue repeats. RNase protection analysis and 5'-GeneRacer PCR indicated that MUC3A gene transcripts initiate from multiple start sites along a region spanning approximately 180 bases. The 5'-flanking region of the gene had promoter activity when fused to a luciferase reporter gene in all of the tested cell lines. This region contained binding sites for several transcription factors, including those implicated in the regulation of intestinal genes, but lacked a cognate TATA box. These features of the gene promoter may enable the gene to be expressed at variable levels in several cell types with different repertoires of transcription factors. We also utilized 5'-GeneRacer PCR to determine the sequence of the 5'-terminus of the MUC3B message. The amino termini of the MUC3A and MUC3B mucins are 91% conserved at the amino acid level. Thus, MUC3A and MUC3B have highly conserved amino and carboxyl termini, suggesting a recent duplication of the entire ancestral gene. It remains to be determined whether other members of the 7q22 membrane mucin gene family have amino-terminal domains similar to MUC3A and MUC3B.


Assuntos
Mucosa Intestinal/metabolismo , Mucinas/genética , Regiões Promotoras Genéticas , TATA Box , Transcrição Genética , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar , Humanos , Dados de Sequência Molecular , Mucina-3 , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos
6.
FEMS Immunol Med Microbiol ; 36(1-2): 9-17, 2003 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-12727360

RESUMO

The bactericidal potencies of saturated and unsaturated fatty acids (FAs) and monoglycerides (MGs) against Helicobacter pylori were determined following short incubations with freshly harvested cells over a range of pHs. FAs and their derivatives with an equivalent-carbon number of 12 were the most potent: lauric acid had a minimum bactericidal concentration (MBC) at pH 7.4 of 1 mM, myristoleic and linolenic acid were the most potent unsaturated FAs (MBCs of 0.5 mM, pH 7.4), and monolaurin was the most potent MG (MBC 0.5 mM). Potencies of saturated FAs were increased sharply by lowering pH, and a decrease of only 0.5 pH units can cause a change from non-lethal to lethal conditions. Conversely, the bactericidal action of monolaurin was not pH-dependent. The bactericidal potencies of unsaturated FAs increased with degree of unsaturation. When more than one FA or FA plus MGs were present, their combined action was additive. Urea and endogenous urease did not protect H. pylori from the bactericidal action of FAs. These results suggest that H. pylori present in the stomach contents (but not necessarily within the mucus barrier) should be rapidly killed by the millimolar concentrations of FAs and MGs that are produced by pre-intestinal lipase(s) acting on suitable triglycerides such as milk fat.


Assuntos
Antibacterianos/farmacologia , Ácidos Graxos não Esterificados/farmacologia , Ácidos Graxos Insaturados/farmacologia , Glicerídeos/farmacologia , Helicobacter pylori/efeitos dos fármacos , Ácidos Graxos não Esterificados/antagonistas & inibidores , Ácidos Graxos Insaturados/antagonistas & inibidores , Glicerídeos/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Ureia/farmacologia , Urease/antagonistas & inibidores , Urease/farmacologia
7.
Chem Biol Interact ; 140(2): 185-98, 2002 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-12076524

RESUMO

Studies on the kinetic characteristics of calf pregastric lipase (EC 3.1.1.3) have shown that it preferentially releases short chain fatty acids (SCFAs) from bovine milkfat. The released fatty acids form mixed micelle structures. The aim of this investigation has been to test whether hydrolysed milkfat is antimicrobial, and how the state of the emulsion alters the bactericidal or bacteriostatic effects. Partial hydrolysis of milkfat by pregastric lipase was carried out in two types of emulsion systems, containing either Triton X-100 or casein/lecithin, plus milkfat in citrate/phosphate buffer (pH 5.0-6.0). The concentrations and compositions of fatty acids were determined by gas chromatography. The minimum percentages of hydrolysed milkfat which affected growth and survival of selected Gram-positive and Gram-negative bacteria were measured. The bacterial experiments were repeated using pure fatty acids at similar concentrations. Lauric acid (C12:0) was found to be the most potent bactericidal fatty acid against Enterococcae (Gram-positive), and caprylic acid (C8:0) was the most potent against coliforms (Gram-negative). Use of Triton X-100 for milkfat emulsification provided a more compatible medium for studying bacterial growth in the hydrolysed milkfat than did use of casein/lecithin. The results also show that the antimicrobial effects of individual fatty acids released from hydrolysed milkfat were at least additive and suggest that hydrolysis of milkfat may be a significant factor in controlling growth of organisms imbibed with food in pre-weaned animals. The amount of pregastric catalyzed triglyceride hydrolysis in the digestive tract is sufficient to produce an antibacterial concentration of fatty acids and monoglycerides.


Assuntos
Antibacterianos/farmacologia , Antibacterianos/farmacocinética , Escherichia coli/efeitos dos fármacos , Glicolipídeos/farmacologia , Glicolipídeos/farmacocinética , Glicoproteínas/farmacologia , Glicoproteínas/farmacocinética , Klebsiella pneumoniae/efeitos dos fármacos , Lipase/metabolismo , Animais , Bovinos , Enterococcus/efeitos dos fármacos , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Hidrólise , Klebsiella pneumoniae/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana
8.
Carbohydr Res ; 337(12): 1095-111, 2002 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-12062525

RESUMO

A chromogenic substrate, 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sodium sulfate was synthesized and used in combination with beta-N-acetylhexosaminidase for detection of the sulfatase, MdsA, by release of 4-nitrophenol. MdsA was originally isolated from the bacterium Prevotella strain RS2 and is believed to be involved in desulfation of sulfomucins, major components of the mucus barrier protecting the human colon surface. The exo nature of the MdsA sulfatase was indicated by its inability to de-esterify the disaccharide 4-nitrophenyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sodium sulfate. This latter compound was prepared from monosaccharide precursors by two different methods, the shorter requiring just six steps from 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and giving an overall yield of 26.4%. The syntheses of 4-nitrophenyl beta-D-galactopyranoside 3-triethylammonium sulfate and 6-triethylammonium sulfate and their use in combination with beta-galactosidase as chromogenic substrates for detecting Bacteroides fragilis sulfatases with different specificities was also demonstrated.


Assuntos
Compostos Cromogênicos/síntese química , Mucinas/metabolismo , Oligossacarídeos/síntese química , Sulfatases/metabolismo , Sulfatos/síntese química , Aspergillus oryzae/enzimologia , Bacteroides fragilis/enzimologia , Compostos Cromogênicos/química , Modelos Químicos , Estrutura Molecular , Mucinas/química , Oligossacarídeos/química , Sulfatos/química
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