RESUMO
Gastric Helicobacter pylori infection is diagnosed based on histopathological evaluation of gastric mucosal biopsies, urease test, urea breath test, H. pylori culturing, or direct detection using polymerase chain reaction (PCR). This study aimed to evaluate the efficacy of immunohistochemical (IHC) staining in detecting H. pylori in gastric biopsies from patients with chronic gastritis and minimal or atypical infection. Gastric biopsies from 50 patients with chronic gastritis were subjected to routine haematoxylin and eosin (H-E), modified Giemsa, and IHC staining. The results of staining were compared with those of quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis identified 32 (64%) H. pylori-positive cases, whereas IHC, H-E, and modified Giemsa staining identified 29 (58%), 27 (54%), and 21 (42%) positive cases. The sensitivity of IHC staining (87.50%) was higher than that of H-E (59.38%) and modified Giemsa (43.75%) staining. The specificity of H-E, modified Giemsa, and IHC staining was 55.56%, 61.11%, and 94.44%, respectively. IHC staining exhibited the highest diagnostic accuracy (90%), followed by H-E (58%) and modified Giemsa (50%) staining. Active gastritis, intestinal metaplasia, and lymphoid follicles were detected in 32 (64%), 4 (8%), and 22 (44%) cases, respectively, and all of these cases were H. pylori positive. In contrast to routine H-E and modified Giemsa staining, IHC allows for the accurate H. pylori detection in cases with minimal or atypical infection. Moreover, IHC can be an alternative diagnostic method to qRT-PCR for detection of H. pylori in such cases.
Assuntos
Gastrite/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/citologia , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Gastrite/patologia , Infecções por Helicobacter/patologia , Imuno-Histoquímica , Arábia SauditaRESUMO
Helicobacter pylori is associated with chronic gastritis, gastric or duodenal ulcers, and gastric cancer. Since the oral cavity is the entry port and the first component of the gastrointestinal system, the oral cavity has been discussed as a potential reservoir of H. pylori. Accordingly, a potential oral-oral transmission route of H. pylori raises the question concerning whether close contact such as kissing or sharing a meal can cause the transmission of H. pylori. Therefore, this topic has been investigated in many studies, applying different techniques for detection of H. pylori from oral samples, i.e. molecular techniques, immunological or biochemical methods and traditional culture techniques. While molecular, immunological or biochemical methods usually yield high detection rates, there is no definitive evidence that H. pylori has ever been isolated from the oral cavity. The specificity of those methods may be limited due to potential cross-reactivity, especially with H. pylori-like microorganisms such as Campylobacter spp. Furthermore, the influence of gastroesophageal reflux has not been investigated so far. This review aims to summarize and critically discuss previous studies investigating the potential colonization of H. pylori in the oral cavity and suggest novel research directions for targeting this critical research question.
Assuntos
Infecções por Helicobacter/microbiologia , Helicobacter pylori/crescimento & desenvolvimento , Boca/microbiologia , Animais , Infecções Assintomáticas , Técnicas Bacteriológicas , Placa Dentária/microbiologia , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/citologia , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Humanos , Técnicas Imunológicas , Técnicas de Diagnóstico Molecular , Saliva/microbiologiaRESUMO
Toxin-antitoxin systems are found in many bacterial chromosomes and plasmids with roles ranging from plasmid stabilization to biofilm formation and persistence. In these systems, the expression/activity of the toxin is counteracted by an antitoxin, which, in type I systems, is an antisense RNA. While the regulatory mechanisms of these systems are mostly well defined, the toxins' biological activity and expression conditions are less understood. Here, these questions were investigated for a type I toxin-antitoxin system (AapA1-IsoA1) expressed from the chromosome of the human pathogen Helicobacter pylori We show that expression of the AapA1 toxin in H. pylori causes growth arrest associated with rapid morphological transformation from spiral-shaped bacteria to round coccoid cells. Coccoids are observed in patients and during in vitro growth as a response to different stress conditions. The AapA1 toxin, first molecular effector of coccoids to be identified, targets H. pylori inner membrane without disrupting it, as visualized by cryoelectron microscopy. The peptidoglycan composition of coccoids is modified with respect to spiral bacteria. No major changes in membrane potential or adenosine 5'-triphosphate (ATP) concentration result from AapA1 expression, suggesting coccoid viability. Single-cell live microscopy tracking the shape conversion suggests a possible association of this process with cell elongation/division interference. Oxidative stress induces coccoid formation and is associated with repression of the antitoxin promoter and enhanced processing of its transcript, leading to an imbalance in favor of AapA1 toxin expression. Our data support the hypothesis of viable coccoids with characteristics of dormant bacteria that might be important in H. pylori infections refractory to treatment.
Assuntos
Helicobacter pylori/citologia , Helicobacter pylori/efeitos dos fármacos , Peptídeos/farmacologia , Sistemas Toxina-Antitoxina , Trifosfato de Adenosina/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Helicobacter pylori/ultraestrutura , Peróxido de Hidrogênio/toxicidade , Espaço Intracelular/metabolismo , Cinética , Potenciais da Membrana/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peptidoglicano/metabolismoRESUMO
Here we present an easy flow cytometry protocol to study the viability of Helicobacter pylori which also enables the detection of even low live bacteria densities. This protocol has potential utility for a fast and accurate assessment of experimental eradication methods against H. pylori.
Assuntos
Técnicas Bacteriológicas/métodos , Citometria de Fluxo/métodos , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/isolamento & purificação , Antibacterianos , Infecções por Helicobacter/microbiologia , Helicobacter pylori/citologia , Humanos , Viabilidade Microbiana , Compostos Orgânicos , PropídioRESUMO
Background: Identification of novel Ure inhibitors with high potency has received considerable attention. Methodology & results: Ure inhibition was determined using the indophenol method, the affinities to Ure were estimated via surface plasmon resonance. Seventeen new plus ten known N-monosubstituted thiosemicarbazides were synthesized and identified as novel Ure inhibitors. Out of these compounds, compound b5 shows excellent activity against both crude Ure from Helicobacter pylori (IC50 = 0.04 µM) and Ure in living cell (IC50 = 0.27 µM), with the potency being over 600-fold higher than clinical used drug acetohyroxamic acid, respectively. Surface plasmon resonance demonstrated the high affinity (Kd.#x00A0;= 6.32 nM) of b5 to Ure. Conclusion: This work provides a class of novel and promising Ure inhibitors.
Assuntos
Antibacterianos/farmacologia , Helicobacter pylori/efeitos dos fármacos , Semicarbazidas/farmacologia , Fatores de Virulência/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Linhagem Celular , Helicobacter pylori/citologia , Helicobacter pylori/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Semicarbazidas/síntese química , Semicarbazidas/química , Fatores de Virulência/metabolismoRESUMO
A genetic screen for colonization factors of the human stomach pathogen Helicobacter pylori took a surprising turn with the discovery that some colonization mutants had lost helical cell morphology. Further pursuit of direct morphology screens revealed a large H. pylori 'shapesome' complex consisting of peptidoglycan modification and precursor synthesis enzymes, a cytoskeletal element and putative scaffold or regulatory proteins that promote enhanced asymmetric cell wall growth. Functional characterization of H. pylori shape mutants indicates multiple roles for cell shape during colonization of mucosal surfaces. Conservation of both the molecular constituents of the H. pylori cell shape program and a newly appreciated enrichment of this morphotype at mucosal surface suggests that helical organisms may be particularly well poised to exploit host perturbations to become pathogens.
Assuntos
Mucosa Gástrica/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/citologia , Helicobacter pylori/patogenicidade , Animais , Helicobacter pylori/genética , Helicobacter pylori/crescimento & desenvolvimento , Humanos , Movimento , Mutação , Estômago/microbiologia , VirulênciaRESUMO
BACKGROUND: Proton pump inhibitors (PPIs) with lipophilic nature may interact with lipid components of H. pylori cell membrane, disrupting cell structure and viability. In this study, the effect of PPIs on fatty acid and cholesterol components of H. pylori cell membrane was assessed. METHODS: One H. pylori isolate was treated with 1X and 2X MICs (µg/mL) of lansoprazole (LPZ: 8 and 16) and pantoprazole (PAN: 128 and 256) in brain heart infusion broth plus serum. Treated H. pylori was cultured on brucella blood agar (BBA) and tetrazolium egg yolk agar (TEYA). Bacterial cells stained with Live/Dead kit were examined by fluorescent microscopy. Fatty acid and cholesterol contents of treated H. pylori were measured by gas chromatography. RESULTS: PPI-treated H. pylori did not grow on BBA but grew on TEYA. Fluorescent microscopy showed H. pylori stained red. Analyses showed high frequency of saturated fatty acids, C14:0, C16:0 and C18:0. Among unsaturated fatty acids, C18:1 and C18:2c were increased, while five were eliminated and five were synthesized de novo. Cholesteryl-6-O-tetradecanoyl-α-D- glucopyranoside was detected as the only glycosylated cholesterol in treated H. pylori. Growth of PPI-treated H. pylori on cholesterol-rich TEYA showed that occurrence of cholesterol can reverse the growth inhibition by PPIs. Red- bacilli form of H. pylori showed dye entry through damaged cell membrane without lysis. CONCLUSION: Incorporation of lipophilic PPI into H. pylori cell membrane disrupted lipids and inhibited growth. However, H. pylori adjusted the defected membrane by replacing the lipid components and resisted lysis.
Assuntos
Colesterol/análise , Ácidos Graxos/análise , Helicobacter pylori/efeitos dos fármacos , Inibidores da Bomba de Prótons/farmacologia , Membrana Celular/química , Helicobacter pylori/citologia , Lansoprazol/farmacologia , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Pantoprazol/farmacologiaRESUMO
Helical cell shape is necessary for efficient stomach colonization by Helicobacter pylori, but the molecular mechanisms for generating helical shape remain unclear. The helical centerline pitch and radius of wild-type H. pylori cells dictate surface curvatures of considerably higher positive and negative Gaussian curvatures than those present in straight- or curved-rod H. pylori. Quantitative 3D microscopy analysis of short pulses with either N-acetylmuramic acid or D-alanine metabolic probes showed that cell wall growth is enhanced at both sidewall curvature extremes. Immunofluorescence revealed MreB is most abundant at negative Gaussian curvature, while the bactofilin CcmA is most abundant at positive Gaussian curvature. Strains expressing CcmA variants with altered polymerization properties lose helical shape and associated positive Gaussian curvatures. We thus propose a model where CcmA and MreB promote PG synthesis at positive and negative Gaussian curvatures, respectively, and that this patterning is one mechanism necessary for maintaining helical shape.
Round spheres, straight rods, and twisting corkscrews, bacteria come in many different shapes. The shape of bacteria is dictated by their cell wall, the strong outer barrier of the cell. As bacteria grow and multiply, they must add to their cell wall while keeping the same basic shape. The cells walls are made from long chain-like molecules via processes that are guided by protein scaffolds within the cell. Many common antibiotics, including penicillin, stop bacterial infections by interrupting the growth of cell walls. Helicobacter pylori is a common bacterium that lives in the gut and, after many years, can cause stomach ulcers and stomach cancer. H. pylori are shaped in a twisting helix, much like a corkscrew. This shape helps H. pylori to take hold and colonize the stomach. It remains unclear how H. pylori creates and maintains its helical shape. The helix is much more curved than other bacteria, and H. pylori does not have the same helpful proteins that other curved bacteria do. If H. pylori grows asymmetrically, adding more material to the cell wall on its long outer side to create a twisting helix, what controls the process? To find out, Taylor et al. grew H. pylori cells and watched how the cell walls took shape. First, a fluorescent dye was attached to the building blocks of the cell wall or to underlying proteins that were thought to help direct its growth. The cells were then imaged in 3D, and images from hundreds of cells were reconstructed to analyze the growth patterns of the bacteria's cell wall. A protein called CcmA was found most often on the long side of the twisting H. pylori. When the CcmA protein was isolated in a dish, it spontaneously formed sheets and helical bundles, confirming its role as a structural scaffold for the cell wall. When CcmA was absent from the cell of H. pylori, Taylor et al. observed that the pattern of cell growth changed substantially. This work identifies a key component directing the growth of the cell wall of H. pylori and therefore, a new target for antibiotics. Its helical shape is essential for H. pylori to infect the gut, so blocking the action of the CcmA protein may interrupt cell wall growth and prevent stomach infections.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Helicobacter pylori/metabolismo , Alanina/metabolismo , Helicobacter pylori/citologia , Ácidos Murâmicos/metabolismo , Peptidoglicano/biossínteseRESUMO
BACKGROUND: In this study, one Helicobacter pylori isolate, from gastric biopsy of a dyspeptic patient that turned into mucoid-coccoid (MC) form upon consecutive subcultures, was identified. The culturability, antibiotic resistance, and lipid contents of MC were compared with those of non-mucoid (NM) spiral H pylori. MATERIALS AND METHODS: Mucoid-coccoid and NM H pylori were subcultured on Brucella blood agar (BBA) and incubated under aerobic and microaerobic atmospheres at 37°C. Cultures were examined for colony characteristics and bacterial morphology after 1-3 days. The isolates were identified by biochemical tests and detection of H pylori-16S rDNA. Antibiogram was performed with currently used antibiotics for H pylori eradication. Cellular lipid contents were extracted and analyzed by gas chromatography. RESULTS: Compared with pin-pointed and glistening colonies of NM H pylori that appeared under microaerobic conditions, MC H pylori grew well in consecutive subcultures under aerobic and microaerobic atmospheres and produced white patches of mucoid colonies. MC exhibited coccoid and NM spiral morphology. Both isolates were catalase, oxidase, and urease positive and contained 16S rDNA. Compared with NM that was susceptible to almost all the antibiotics, MC was resistant to all the antibiotics. Lipid analyses showed high frequency of unsaturated fatty acids and cholesterol in MC. CONCLUSIONS: Coccoid forms with high fatty acid and cholesterol contents that show resistance to antibiotics might resist against other stressful conditions such as gastric acidity and immune response. Moreover, mucoid property may enhance resistance of coccoids to stresses. With mucoid-coccoid lifestyle, H pylori may establish a chronic infection refractory to antimicrobial therapy.
Assuntos
Helicobacter pylori/citologia , Helicobacter pylori/crescimento & desenvolvimento , Helicobacter pylori/isolamento & purificação , Colesterol/química , Resistência Microbiana a Medicamentos , Ácidos Graxos/química , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/metabolismo , Humanos , Testes de Sensibilidade MicrobianaRESUMO
A urease inhibitor with good in vivo profile is considered as an alternative agent for treating infections caused by urease-producing bacteria such as Helicobacter pylori. Here, we report a series of N-monosubstituted thioureas, which act as effective urease inhibitors with very low cytotoxicity. One compound (b19) was evaluated in detail and shows promising features for further development as an agent to treat H. pylori caused diseases. Excellent values for the inhibition of b19 against both extracted urease and urease in intact cell were observed, which shows IC50 values of 0.16 ± 0.05 and 3.86 ± 0.10 µM, being 170- and 44-fold more potent than the clinically used drug AHA, respectively. Docking simulations suggested that the monosubstituted thiourea moiety penetrates urea binding site. In addition, b19 is a rapid and reversible urease inhibitor, and displays nM affinity to urease with very slow dissociation (koff=1.60 × 10-3 s-1) from the catalytic domain.
Assuntos
Helicobacter pylori/efeitos dos fármacos , Ureia/farmacologia , Urease/antagonistas & inibidores , Antibacterianos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos , Helicobacter pylori/citologia , Helicobacter pylori/enzimologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Ureia/análogos & derivados , Ureia/química , Urease/metabolismoRESUMO
Evident in its name, the gastric pathogen Helicobacter pylori has a helical cell morphology which facilitates efficient colonization of the human stomach. An improved light-focusing strategy allowed us to robustly distinguish even subtle perturbations of H. pylori cell morphology by deviations in light-scattering properties measured by flow cytometry. Profiling of an arrayed genome-wide deletion library identified 28 genes that influence different aspects of cell shape, including properties of the helix, cell length or width, cell filament formation, cell shape heterogeneity, and cell branching. Included in this mutant collection were two that failed to form any helical cells, a soluble lytic transglycosylase and a previously uncharacterized putative multipass inner membrane protein HPG27_0728, renamed Csd7. A combination of cell fractionation, mutational, and immunoprecipitation experiments show that Csd7 and Csd2 collaborate to stabilize the Csd1 peptidoglycan (PG) endopeptidase. Thus, both csd2 and csd7 mutants show the same enhancement of PG tetra-pentapeptide cross-linking as csd1 mutants. Csd7 also links Csd1 with the bactofilin CcmA via protein-protein interactions. Although Csd1 is stable in ccmA mutants, these mutants show altered PG tetra-pentapeptide cross-linking, suggesting that Csd7 may directly or indirectly activate as well as stabilize Csd1. These data begin to illuminate a highly orchestrated program to regulate PG modifications that promote helical shape, which includes nine nonessential nonredundant genes required for helical shape and 26 additional genes that further modify H. pylori's cell morphology.IMPORTANCE The stomach ulcer and cancer-causing pathogen Helicobacter pylori has a helical cell shape which facilitates stomach infection. Using light scattering to measure perturbations of cell morphology, we identified 28 genes that influence different aspects of cell shape. A mutant in a previously uncharacterized protein renamed Csd7 failed to form any helical cells. Biochemical analyses showed that Csd7 collaborates with other proteins to stabilize the cell wall-degrading enzyme Csd1. Csd7 also links Csd1 with a putative filament-forming protein via protein-protein interactions. These data suggest that helical cell shape arises from a highly orchestrated program to regulate cell wall modifications. Targeting of this helical cell shape-promoting program could offer new ways to block infectivity of this important human pathogen.
Assuntos
Membrana Externa Bacteriana/química , Proteínas de Bactérias/química , Endopeptidases/química , Genoma Bacteriano , Helicobacter pylori/citologia , Helicobacter pylori/genética , Proteínas de Bactérias/genética , Parede Celular , Citoesqueleto/química , Endopeptidases/genética , MutaçãoRESUMO
Half of all humans harbor Helicobacter pylori in their stomachs. Helical cell shape is thought to facilitate H. pylori's ability to bore into the protective mucus layer in a corkscrew-like motion, thereby enhancing colonization of the stomach. H. pylori cell shape mutants show impaired colonization of the mouse stomach, highlighting the importance of cell shape in infection. To gain a deeper understanding of how helical cell morphology promotes host colonization by H. pylori, we used three-dimensional confocal microscopy to visualize the clinical isolate PMSS1 and an isogenic straight-rod mutant (Δcsd6) within thick longitudinal mouse stomach sections. We also performed volumetric image analysis to quantify the number of bacteria residing within corpus and antral glands in addition to measuring total CFU. We found that straight rods show attenuation during acute colonization of the stomach (1 day or 1 week postinfection) as measured by total CFU. Our quantitative imaging revealed that wild-type bacteria extensively colonized antral glands at 1 week postinfection, while csd6 mutants showed variable colonization of the antrum at this time point. During chronic infection (1 or 3 months postinfection), total CFU were highly variable but similar for wild-type and straight rods. Both wild-type and straight rods persisted and expanded in corpus glands during chronic infection. However, the straight rods showed reduced inflammation and disease progression. Thus, helical cell shape contributes to tissue interactions that promote inflammation during chronic infection, in addition to facilitating niche acquisition during acute infection.
Assuntos
Infecções por Helicobacter/microbiologia , Helicobacter pylori/citologia , Helicobacter pylori/crescimento & desenvolvimento , Estômago/patologia , Animais , Aderência Bacteriana , Doença Crônica , Feminino , Infecções por Helicobacter/patologia , Helicobacter pylori/genética , Humanos , Camundongos Endogâmicos C57BL , Antro Pilórico/microbiologia , Antro Pilórico/patologia , Estômago/microbiologiaRESUMO
Helicobacter pylori (Hp) infection is related to the pathogenesis of chronic gastric disorders and extragastric diseases. Here, we examined the anorexigenic and anxiogenic effects of Hp vacuolating cytotoxin A (VacA) through activation of hypothalamic urocortin1 (Ucn1). VacA was detected in the hypothalamus after peripheral administration and increased Ucn1 mRNA expression and c-Fos-positive cells in the hypothalamus but not in the nucleus tractus solitarius. c-Fos and Ucn1-double positive cells were detected. CRF1 and CRF2 receptor antagonists suppressed VacA-induced anxiety and anorexia, respectively. VacA activated single paraventricular nucleus neurons and A7r5 cells; this activation was inhibited by phospholipase C (PLC) and protein kinase C (PKC) inhibitors. VacA causes anorexia and anxiety through the intracellular PLC-PKC pathway, migrates across the blood-brain barrier, and activates the Ucn1-CRF receptor axis.
Assuntos
Anorexia/induzido quimicamente , Ansiedade/induzido quimicamente , Citotoxinas/toxicidade , Helicobacter pylori/citologia , Hipotálamo/efeitos dos fármacos , Urocortinas/metabolismo , Vacúolos/metabolismo , Animais , Anorexia/genética , Ansiedade/genética , Peso Corporal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Gerbillinae , Helicobacter pylori/fisiologia , Hipotálamo/citologia , Hipotálamo/metabolismo , Camundongos , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Urocortinas/genéticaRESUMO
Infection of the human stomach caused by Helicobacter pylori is very common, as the pathogen colonizes more than half of the world's population. It is associated with varied outcomes of infection, such as peptic ulcer disease, gastric ulcers, and mucosa-associated lymphoid tissue lymphoma, and is generally considered a risk factor for the development of gastric adenocarcinoma. Cholesteryl glucosides (CGs) constitute a vital component of the cell wall of H. pylori and contribute to its pathogenicity and virulence. The hp0421 gene, which encodes cholesteryl-α-glucoside transferase (CGT), appears critical for the enzymatic function of integrating unique CGs into the cell wall of H. pylori, and deletion of this gene leads to depletion of CGs and their variants. Herein, we report that the deletion of hp0421 and consequent deficiency of cholesterol alter the morphology, shape, and cell wall composition of H. pylori cells, as demonstrated by high-resolution confocal microscopy and flow cytometry analyses of two different type strains of H. pylori, their isogenic knockouts as well as a reconstituted strain. Moreover, measurement of ethidium bromide (EtBr) influx by flow cytometry showed that lack of CGs increased cell wall permeability. Antimicrobial susceptibility testing revealed that the hp0421 isogenic knockout strains (Hp26695Δ421 and Hp76Δ421) were sensitive to antibiotics, such as fosfomycin, polymyxin B, colistin, tetracycline, and ciprofloxacin, in contrast to the wild-type strains that were resistant to the above antibiotics and tended to form denser biofilms. Lipid profile analysis of both Hp76 and Hp76Δ421 strains showed an aberrant profile of lipopolysaccharides (LPS) in the Hp76Δ421 strain. Taken together, we herein provide a set of mechanistic evidences to demonstrate that CGs play critical roles in the maintenance of the typical spiral morphology of H. pylori and its cell wall integrity, and any alteration in CG content affects the characteristic morphological features and renders the H. pylori susceptible to various antibiotics.IMPORTANCEHelicobacter pylori is an important cause of chronic gastritis leading to peptic ulcer and is a major risk factor for gastric malignancies. Failure in the eradication of H. pylori infection and increasing antibiotic resistance are two major problems in preventing H. pylori colonization. Hence, a deeper understanding of the bacterial survival strategies is needed to tackle the increasing burden of H. pylori infection by an appropriate intervention. Our study demonstrated that the lack of cholesteryl glucosides (CGs) remarkably altered the morphology of H. pylori and increased permeability of the bacterial cell wall. Further, this study highlighted the substantial role of CGs in maintaining the typical H. pylori morphology that is essential for retaining its pathogenic potential. We also demonstrated that the loss of CGs in H. pylori renders the bacterium susceptible to different antibiotics.
Assuntos
Parede Celular/metabolismo , Colesterol/análogos & derivados , Glucosiltransferases/metabolismo , Helicobacter pylori/citologia , Helicobacter pylori/enzimologia , Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Colesterol/metabolismo , Citometria de Fluxo , Deleção de Genes , Teste de Complementação Genética , Glucosiltransferases/genética , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Testes de Sensibilidade Microbiana , Microscopia Confocal , PermeabilidadeRESUMO
There is an ongoing need to develop ultrasensitive nanomechanical instrumentation that has high spatial and force resolution, as well as an ability to operate in various biological environments. Here, we present a compact nanofiber optic force transducer (NOFT) with sub-piconewton force sensitivity and a nanoscale footprint that paves the way to the probing of complex mechanical phenomena inside biomolecular systems. The NOFT platform comprises a SnO2 nanofiber optic equipped with a thin, compressible polymer cladding layer studded with plasmonic nanoparticles (NPs). This combination allows angstrom-level movements of the NPs to be quantified by tracking the optical scattering of the NPs as they interact with the near-field of the fiber. The distance-dependent optical signals can be converted to force once the mechanical properties of the compressible cladding are fully characterized. In this protocol, the details of the synthesis, characterization, and calibration of the NOFT system are described. The overall protocol, from the synthesis of the nanofiber optic devices to acquisition of nanomechanical data, takes ~72 h.
Assuntos
Tecnologia de Fibra Óptica/métodos , Mecanotransdução Celular/fisiologia , Microscopia de Força Atômica/métodos , Nanofibras/química , Nanotecnologia/métodos , Animais , Animais Recém-Nascidos , Tecnologia de Fibra Óptica/instrumentação , Ouro/química , Helicobacter pylori/citologia , Helicobacter pylori/fisiologia , Camundongos , Microscopia de Força Atômica/instrumentação , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Nanofibras/ultraestrutura , Nanopartículas/química , Nanopartículas/ultraestrutura , Nanotecnologia/instrumentação , Polietilenoglicóis/química , Cultura Primária de Células , Som , Compostos de Estanho/química , VibraçãoRESUMO
Two series of ω-phenoxy contained acylhydroxamic acids as novel urease inhibitors were designed and synthesized. Biological activity evaluations revealed that ω-phenoxypropinoylhydroxamic acids were more active than phenoxyacetohydroxamic acids. Out of these compounds, 3-(3,4-dichlorophenoxy)propionylhydroxamic acid c24 showed significant potency against urease in both cell free extract (IC50â¯=â¯0.061⯱â¯0.003⯵M) and intact cell (IC50â¯=â¯0.89⯱â¯0.05⯵M), being over 450- and 120-fold more potent than the clinically prescribed urease inhibitor AHA, repectively. Non-linear fitting of experimental data (V-[S]) suggested a mixed-type inhibition mechanism and a dual site binding mode of these compounds.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Urease/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Infecções por Helicobacter/metabolismo , Helicobacter pylori/citologia , Helicobacter pylori/enzimologia , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Cinética , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Urease/isolamento & purificação , Urease/metabolismoRESUMO
Chronic infection with Helicobacter pylori can lead to the development of gastric ulcers and stomach cancers. The helical cell shape of H. pylori promotes stomach colonization. Screens for loss of helical shape have identified several periplasmic peptidoglycan (PG) hydrolases and non-enzymatic putative scaffolding proteins, including Csd5. Both over and under expression of the PG hydrolases perturb helical shape, but the mechanism used to coordinate and localize their enzymatic activities is not known. Using immunoprecipitation and mass spectrometry we identified Csd5 interactions with cytosolic proteins CcmA, a bactofilin required for helical shape, and MurF, a PG precursor synthase, as well as the inner membrane spanning ATP synthase. A combination of Csd5 domain deletions, point mutations, and transmembrane domain chimeras revealed that the N-terminal transmembrane domain promotes MurF, CcmA, and ATP synthase interactions, while the C-terminal SH3 domain mediates PG binding. We conclude that Csd5 promotes helical shape as part of a membrane associated, multi-protein shape complex that includes interactions with the periplasmic cell wall, a PG precursor synthesis enzyme, the bacterial cytoskeleton, and ATP synthase.
Assuntos
Parede Celular/metabolismo , Citoesqueleto/metabolismo , Helicobacter pylori/citologia , Helicobacter pylori/enzimologia , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptídeo Sintases/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Deleção de Genes , Helicobacter pylori/genética , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/genética , Peptídeo Sintases/química , Peptídeo Sintases/genética , Periplasma/metabolismo , Análise de Sequência de ProteínaRESUMO
DNA replication and cell division are two fundamental processes in the life cycle of a cell. The majority of prokaryotic cells undergo division by means of binary fission in coordination with replication of the genome. Both processes, but especially their coordination, are poorly understood in Helicobacter pylori. Here, we studied the cell divisome assembly and the subsequent processes of membrane and peptidoglycan synthesis in the bacterium. To our surprise, we found the cell divisome assembly to be polar, which was well-corroborated by the asymmetric membrane and peptidoglycan synthesis at the poles. The divisome components showed its assembly to be synchronous with that of the replisome and the two remained associated throughout the cell cycle, demonstrating a tight coordination among chromosome replication, segregation and cell division in H. pylori. To our knowledge, this is the first report where both DNA replication and cell division along with their possible association have been demonstrated for this pathogenic bacterium.
Assuntos
Proteínas de Bactérias/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Helicobacter pylori/genética , Complexos Multienzimáticos/genética , Proteínas de Bactérias/metabolismo , Divisão Celular/genética , Membrana Celular/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Helicobacter pylori/citologia , Helicobacter pylori/metabolismo , Microscopia de Fluorescência , Modelos Genéticos , Complexos Multienzimáticos/metabolismo , Peptidoglicano/metabolismoRESUMO
Helicobacter pylori (H. pylori), a gram-negative microaerophilic bacterial pathogen that colonizes the stomachs of more than half of all humans, is linked to chronic gastritis, peptic ulcers and gastric cancer. Spiral-shaped H. pylori undergo morphologic conversion to a viable but not culturable coccoid form when they transit from the microaerobic stomach into the anaerobic intestinal tract. However, little is known about the morphological and pathogenic characteristics of H. pylori under prolonged anaerobic conditions. In this study, scanning electron microscopy was used to document anaerobiosis-induced morphological changes of H. pylori, from helical to coccoid to a newly defined fragmented form. Western blot analysis indicated that all three forms express certain pathogenic proteins, including the bacterial cytotoxin-associated gene A (CagA), components of the cag-Type IV secretion system (TFSS), the blood group antigen-binding adhesin BabA, and UreA (an apoenzyme of urease), almost equally. Similar urease activities were also detected in all three forms of H. pylori. However, in contrast to the helical form, bacterial motility and TFSS activity were found to have been abrogated in the anaerobiosis-induced coccoid and fragmented forms of H. pylori. Notably, it was demonstrated that some of the anaerobiosis-induced fragmented state cells could be converted to proliferation-competent helical bacteria in vitro. These results indicate that prolonged exposure to the anaerobic intestine may not eliminate the potential for H. pylori to revert to the helical pathogenic state.
Assuntos
Proteínas de Bactérias/genética , Helicobacter pylori/citologia , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Anaerobiose , Antibacterianos , Antígenos de Bactérias/genética , Linhagem Celular , Proliferação de Células , Regulação Bacteriana da Expressão Gênica , Infecções por Helicobacter/microbiologia , Humanos , Microscopia Eletrônica de Varredura , Sistemas de Secreção Tipo IV/genética , Urease/genética , Fatores de Virulência/genéticaRESUMO
AIM: This study aims to investigate the morphological transition of Helicobacter pylori during adaptation to water. MATERIALS & METHODS: Different strains were adapted to water. Changes regarding cultivability and cellular morphology were recorded. Expression of 11 genes involved in H. pylori morphological changes was evaluated by real-time PCR. RESULTS: H. pylori presented increased cultivability in water after adaptation. The permanent loss of the spiral shape was observed, but no transition into coccoid form has occurred. Expression levels of genes involved in peptidoglycan assembly of H. pylori 26695 have shown significant changes between adapted and nonadapted strains. CONCLUSION: Adaption to water favors the culturable phenotype and the morphological transition to the rod shape, into a process that implicates the peptidoglycan turnover.