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1.
Nat Microbiol ; 4(10): 1716-1726, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31285586

RESUMO

The peptidoglycan cell wall is an essential structure for the growth of most bacteria. However, many are capable of switching into a wall-deficient L-form state in which they are resistant to antibiotics that target cell wall synthesis under osmoprotective conditions, including host environments. L-form cells may have an important role in chronic or recurrent infections. The cellular pathways involved in switching to and from the L-form state remain poorly understood. This work shows that the lack of a cell wall, or blocking its synthesis with ß-lactam antibiotics, results in an increased flux through glycolysis. This leads to the production of reactive oxygen species from the respiratory chain, which prevents L-form growth. Compensating for the metabolic imbalance by slowing down glycolysis, activating gluconeogenesis or depleting oxygen enables L-form growth in Bacillus subtilis, Listeria monocytogenes and Staphylococcus aureus. These effects do not occur in Enterococcus faecium, which lacks the respiratory chain pathway. Our results collectively show that when cell wall synthesis is blocked under aerobic and glycolytic conditions, perturbation of cellular metabolism causes cell death. We provide a mechanistic framework for many anecdotal descriptions of the optimal conditions for L-form growth and non-lytic killing by ß-lactam antibiotics.


Assuntos
Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Carbono/metabolismo , Formas L/efeitos dos fármacos , Formas L/metabolismo , beta-Lactamas/farmacologia , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Transporte de Elétrons/genética , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/crescimento & desenvolvimento , Enterococcus faecium/metabolismo , Gluconeogênese , Formas L/genética , Formas L/crescimento & desenvolvimento , Muramidase/farmacologia , Mutação , Penicilina G/farmacologia , Peptidoglicano/efeitos dos fármacos , Peptidoglicano/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/toxicidade
2.
Nat Commun ; 9(1): 5164, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30514921

RESUMO

The cell wall is a shape-defining structure that envelopes almost all bacteria and protects them from environmental stresses. Bacteria can be forced to grow without a cell wall under certain conditions that interfere with cell wall synthesis, but the relevance of these wall-less cells (known as L-forms) is unclear. Here, we show that several species of filamentous actinomycetes have a natural ability to generate wall-deficient cells in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the normal mycelial mode of growth. However, prolonged exposure of S-cells to hyperosmotic stress yields variants that are able to proliferate indefinitely without their cell wall, similarly to L-forms. We propose that formation of wall-deficient cells in actinomycetes may serve as an adaptation to osmotic stress.


Assuntos
Actinobacteria/citologia , Actinobacteria/fisiologia , Parede Celular/fisiologia , Pressão Osmótica , Actinobacteria/efeitos dos fármacos , Actinobacteria/genética , Adaptação Biológica , Fenômenos Fisiológicos Bacterianos/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Deleção de Genes , Formas L/citologia , Formas L/crescimento & desenvolvimento , Formas L/fisiologia , Viabilidade Microbiana , Penicilinas/farmacologia , Filogenia , RNA Ribossômico 16S , Alinhamento de Sequência , Esferoplastos/citologia , Esferoplastos/crescimento & desenvolvimento , Esferoplastos/fisiologia , Sacarose/metabolismo , Sequenciamento Completo do Genoma
3.
Bull Exp Biol Med ; 165(2): 239-242, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29923000

RESUMO

The dynamics of LPO marker malondialdehyde formation and peroxidase-destroying activity was studied in homogenized organs of guinea pigs, immunized with thermoextracts from S and L forms Brucella abortus I-206. The L form brucella thermoextract exhibited a lower reactogenicity and adequately activated the antioxidant system, due to which the destructive effects of ROS could be partially neutralized during the vaccinal process.


Assuntos
Estruturas Animais/efeitos dos fármacos , Antioxidantes/metabolismo , Vacina contra Brucelose/farmacologia , Brucella abortus/química , Peroxidação de Lipídeos/efeitos dos fármacos , Vacinas Atenuadas/farmacologia , Estruturas Animais/metabolismo , Animais , Animais de Laboratório , Vacina contra Brucelose/química , Brucella abortus/imunologia , Brucella abortus/patogenicidade , Feminino , Cobaias , Formas L/fisiologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Esferoplastos/fisiologia , Temperatura , Vacinas Atenuadas/química
4.
Cell ; 172(5): 1038-1049.e10, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29456081

RESUMO

ß-lactam antibiotics inhibit bacterial cell wall assembly and, under classical microbiological culture conditions that are generally hypotonic, induce explosive cell death. Here, we show that under more physiological, osmoprotective conditions, for various Gram-positive bacteria, lysis is delayed or abolished, apparently because inhibition of class A penicillin-binding protein leads to a block in autolytic activity. Although these cells still then die by other mechanisms, exogenous lytic enzymes, such as lysozyme, can rescue viability by enabling the escape of cell wall-deficient "L-form" bacteria. This protective L-form conversion was also observed in macrophages and in an animal model, presumably due to the production of host lytic activities, including lysozyme. Our results demonstrate the potential for L-form switching in the host environment and highlight the unexpected effects of innate immune effectors, such as lysozyme, on antibiotic activity. Unlike previously described dormant persisters, L-forms can continue to proliferate in the presence of antibiotic.


Assuntos
Antibacterianos/farmacologia , Formas L/efeitos dos fármacos , Muramidase/metabolismo , beta-Lactamas/farmacologia , Animais , Bacillus subtilis/efeitos dos fármacos , Bacteriólise/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Hidrolases/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Osmorregulação/efeitos dos fármacos , Penicilina G/farmacologia , Proteínas de Ligação às Penicilinas , Peptidoglicano/metabolismo , Prófagos/efeitos dos fármacos , Células RAW 264.7
5.
Sci Rep ; 7(1): 17366, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29234108

RESUMO

The ability of bacteria to exist as a population of self-replicating forms with defective or entirely missing cell wall (L-forms) is an adaptive mechanism for their survival and reproduction under unfavorable conditions. Bacterial mother-to-fetus transfer is a universal phenomenon in the animal kingdom. However, data about vertical transfer of L bacterial forms are extremely scarce. Bacille Calmette-Guérin is an attenuated strain of M. bovis and the only licensed vaccine used for tuberculosis prevention. We already have shown that filterable L-forms of BCG exist freely in the vaccine and are able to reproduce and to form colonies. The present study was focused on the placental microbiome in the context of mother's BCG vaccination. Here we report an isolation of filterable mycobacterial L-form cultures from gestational tissues and blood of healthy newborns delivered by healthy BCG-vaccinated mothers after normal pregnancy. Of note, vertically transmitted mycobacterial L-forms as a part of placentobiome of the pregnant women didn't influence the number of resident pathogen-reactive Vδ2 cells. Placenta colonization with mycobacterial L-forms occurs by maternal blood-to-decidua transfer very early in gestation. Together, these data showed that BCG L-forms have the capacity to pass trans-placental barrier and that maternal BCG vaccination affects the placentobiome.


Assuntos
Vacina BCG/imunologia , Transmissão Vertical de Doença Infecciosa , Linfócitos Intraepiteliais/imunologia , Formas L/isolamento & purificação , Microbiota/imunologia , Mycobacterium bovis/isolamento & purificação , Placenta/microbiologia , Vacina BCG/administração & dosagem , Feminino , Humanos , Recém-Nascido , Formas L/imunologia , Mães , Mycobacterium bovis/imunologia , Placenta/citologia , Gravidez , Simbiose/imunologia , Linfócitos T , Tuberculose/prevenção & controle , Vacinação/efeitos adversos
6.
Discov Med ; 23(128): 305-313, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28715646

RESUMO

From a historical perspective, intriguing assumptions about unknown "live units" in human blood have attracted the attention of researchers, reflecting their desire to define a new class of microorganisms. Thus, the concept of "blood microbiota" brings about many questions about the nature, origin, and biological significance of the "unusual microbial cohabitants" in human blood. In contrast to current views that bloodstream in healthy humans is sterile, the hypothesis about the existence of microbes as L-forms (cell wall deficient bacteria) in human blood has evolved on the basis of known facts about their unique biology, as observed in our studies and those of other authors. Recently, we reported that bacterial L-forms persist in the human blood and that filterable, self-replicating bodies with a virus-like size of 100 nm are able to cross the maternal-fetal barrier by vertically transmitted pathway, then enter fetus blood circulation and colonize newborns. Subjects discussed here include the following: Is the existence of L-form bacteria in human blood a natural phenomenon? Are L-form bacteria commensal cohabitants in the human body? Since blood is an unfavorable compartment for the classical bacteria and their propagation, how do L-forms survive in blood circulation? How does L-form microbiota in blood influence the host immune system and contribute to systemic inflammatory, autoimmune, and tumor diseases? The current commentary presents the topic of "human microbiota and L-form bacteria" in its microcosm. It contains details of the hypothesis, supporting evidence and important implications.


Assuntos
Bactérias/citologia , Sangue/microbiologia , Doença , Saúde , Formas L/citologia , Bactérias/ultraestrutura , Humanos , Sistema Imunitário/fisiologia , Formas L/ultraestrutura , Microbiota
7.
Biochem Soc Trans ; 45(2): 287-295, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28408469

RESUMO

The peptidoglycan (PG) cell wall is a defining feature of the bacteria. It emerged very early in evolution and must have contributed significantly to the success of these organisms. The wall features prominently in our thinking about bacterial cell function, and its synthesis involves the action of several dozen proteins that are normally essential for viability. Surprisingly, it turns out to be relatively simple to generate bacterial genetic variants called L-forms that completely lack PG. They grow robustly provided that lack of the cell wall is compensated for by an osmoprotective growth medium. Although their existence has been noted and studied on and off for many decades, it is only recently that modern molecular and cellular methods have been applied to L-forms. We used Bacillus subtilis as an experimental model to understand the molecular basis for the L-form switch. Key findings included the discovery that L-forms use an unusual blebbing, or tubulation and scission mechanism to proliferate. This mechanism is completely independent of the normal FtsZ-based division machinery and seems to require only an increased rate of membrane synthesis, leading to an increased surface area-to-volume ratio. Antibiotics that block cell wall precursor synthesis, such as phosphomycin, efficiently induce the L-form switch without the need for genetic change. The same antibiotics turned out to induce a similar L-form switch in a wide range of bacteria, including Escherichia coli, in which we showed that proliferation was again FtsZ-independent. Aside from further basic science, future work on L-forms is likely to focus on their possible role in chronic or recurrent infections, their use as a model in studies of the origins of life, and possibly, biotechnological applications.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fosfomicina/farmacologia , Formas L/crescimento & desenvolvimento , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Formas L/metabolismo , Peptidoglicano/metabolismo
8.
Int J Mycobacteriol ; 5(4): 454-459, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27931687

RESUMO

OBJECTIVE/BACKGROUND: Cell wall-deficient bacterial forms (L-forms) may occur along with resistance to factors that trigger their appearance. It is of interest to study the relationship between the L-form transformation of Mycobacterium tuberculosis and the exhibition of drug tolerance to ethambutol (EMB), an inhibitor of cell wall synthesis. METHODS: L-form variant was produced from a sensitive EMB strain of M. tuberculosis through a cryogenic stress treatment protocol and was subsequently cultivated in Middlebrook 7H9 semisolid medium, containing EMB in a minimal inhibitory concentration of 2mg/L. Susceptibility to EMB of the parental strain and its L-form variant was evaluated phenotypically and using polymerase chain reaction-restriction fragment length polymorphism assay targeting a mutation in the embB306 gene fragment. RESULTS: In contrast to the sensitivity to EMB of the parental strain, its L-form variant showed phenotypic resistance to high concentrations of EMB (16mg/L), but the mutation in embB306 was not found. Electron microscopy observation of the L-form variant showed a heterogenic population of bacteria, with different degrees of cell wall deficiency, as well as cells of protoplastic type without cell walls. Of special interest were the observed capsule-like structures around the L-form cells and the biofilm-like matrix produced by the L-form population. CONCLUSION: We suggest that the expression of phenotypic resistance to EMB in M. tuberculosis can be associated with alterations or loss of cell walls in L-form bacteria, respectively, which results in a lack of a specific target for EMB action. In addition, production of capsule-like structures and biofilm matrix by L-forms could contribute to their resistance and survival in the presence of antibacterial agents.


Assuntos
Antituberculosos/farmacologia , Tolerância a Medicamentos , Etambutol/farmacologia , Formas L/citologia , Formas L/efeitos dos fármacos , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/efeitos dos fármacos , Técnicas Bacteriológicas , Parede Celular/efeitos dos fármacos , Meios de Cultura/química , Humanos
9.
Artigo em Inglês | MEDLINE | ID: mdl-27672147

RESUMO

The peptidoglycan cell wall is widely conserved across the bacterial domain, suggesting that it appeared early in the evolution of bacteria. It is normally essential but under certain conditions wall-deficient or 'L-form' bacteria can be isolated. In Bacillus subtilis this normally requires two genetic changes. The first, exemplified by mutations shutting down wall precursor synthesis, works by increasing membrane synthesis. This promotes the unusual form of proliferation used by L-forms, involving a range of relatively disorganized membrane blebbing or vesiculation events. The secondary class of mutations probably work by relieving oxidative stress that L-forms may incur due to their unbalanced metabolism. Repression or inhibition of cell wall precursor synthesis can stimulate the L-form transition in a wide range of bacteria, of both Gram-positive and -negative lineages. L-forms are completely resistant to most antibiotics working specifically on cell wall synthesis, such as penicillins and cephalosporins, consistent with the many reports of their involvement in various chronic diseases. They are potentially important in biotechnology, because lack of a wall can be advantageous in a range of production or strain improvement applications. Finally, L-forms provide an interesting model system for studying early steps in the evolution of cellular life.This article is part of the themed issue 'The new bacteriology'.


Assuntos
Bacillus subtilis/fisiologia , Parede Celular/metabolismo , Doença Crônica , Formas L/fisiologia , Antibacterianos/metabolismo , Bacillus subtilis/genética , Formas L/genética , Mutação , Peptidoglicano/metabolismo
10.
Hum Vaccin Immunother ; 12(10): 2565-2571, 2016 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-27294392

RESUMO

Our previous studies showed that mycobacterial L-forms persist in the blood of BCG vaccinated people and that BCG vaccine is able to produce, under appropriate conditions, filterable, self-replicating L-bodies with virus-like size. Because filterability is one of the characteristics of L-forms, considerable interest has been shown in their capacity to cross the maternal-fetal barrier. The current study demonstrated isolation of mycobacterial L-form cultures from umbilical cord blood of 5 healthy newborns of healthy mothers vaccinated previously with BCG. The isolated cultures showed distinctive growth characteristics of cell wall deficient L-form bacteria. Transmission electron microscopy demonstrated presence of L-bodies with extremely small size of 100 nm and revealed morphological transformations, typical for L-forms. IS6110 Real Time PCR assay confirmed that all L-form isolates were of mycobacterial origin and belonged to Mycobacterium tuberculosis complex which includes vaccinal BCG substrains. In conclusion, we could suggest that reproductive filterable L-bodies of BCG origin are able to fall in blood circulation of the fetus by vertical transmitted pathway and colonize newborns.


Assuntos
Vacina BCG/administração & dosagem , Sangue Fetal/microbiologia , Formas L/isolamento & purificação , Mycobacterium bovis/isolamento & purificação , Feminino , Voluntários Saudáveis , Humanos , Recém-Nascido , Formas L/genética , Formas L/ultraestrutura , Microscopia Eletrônica de Transmissão , Mycobacterium bovis/genética , Mycobacterium bovis/ultraestrutura , Reação em Cadeia da Polimerase em Tempo Real
11.
PLoS One ; 11(5): e0154925, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27149671

RESUMO

L-forms are cell wall-deficient variants of otherwise walled bacteria that maintain the ability to survive and proliferate in absence of the surrounding peptidoglycan sacculus. While transient or unstable L-forms can revert to the walled state and may still rely on residual peptidoglycan synthesis for multiplication, stable L-forms cannot revert to the walled form and are believed to propagate in the complete absence of peptidoglycan. L-forms are increasingly studied as a fundamental biological model system for cell wall synthesis. Here, we show that a stable L-form of the intracellular pathogen Listeria monocytogenes features a surprisingly intact peptidoglycan synthesis pathway including glycosyl transfer, in spite of the accumulation of multiple mutations during prolonged passage in the cell wall-deficient state. Microscopic and biochemical analysis revealed the presence of peptidoglycan precursors and functional glycosyl transferases, resulting in the formation of peptidoglycan polymers but without the synthesis of a mature cell wall sacculus. In conclusion, we found that stable, non-reverting L-forms, which do not require active PG synthesis for proliferation, may still continue to produce aberrant peptidoglycan.


Assuntos
Parede Celular/metabolismo , Formas L/metabolismo , Listeria monocytogenes/metabolismo , Peptidoglicano/metabolismo , Transferases/metabolismo
12.
Curr Biol ; 25(12): 1613-8, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-26051891

RESUMO

The peptidoglycan (PG) cell wall is a defining feature of the bacterial lineage and an important target for antibiotics, such as ß-lactams and glycopeptides. Nevertheless, many bacteria are capable of switching into a cell-wall-deficient state, called the "L-form" [1-3]. These variants have been classically identified as antibiotic-resistant forms in association with a wide range of infectious diseases [4]. L-forms become completely independent of the normally essential FtsZ cell division machinery [3, 5]. Instead, L-form proliferation is driven by a simple biophysical process based on an increased ratio of surface area to cell volume synthesis [6, 7]. We recently showed that only two genetic changes are needed for the L-form transition in Bacillus subtilis [7]. Class 1 mutations work to generate excess membrane synthesis [7]. Until now, the function of the class 2 mutations was unclear. We now show that these mutations work by counteracting an increase in the cellular levels of reactive oxygen species (ROS) originating from the electron transport pathway, which occurs in wall-deficient cells. Consistent with this, addition of a ROS scavenger or anaerobic culture conditions also worked to promote L-form growth without the class 2 mutations in both Gram-positive B. subtilis and Gram-negative Escherichia coli. Our results suggest that physiological compensation for the metabolic imbalance that occurs when cell wall synthesis is blocked is crucial for L-form proliferation in a wide range of bacteria and also provide new insights into the mode of action of antibiotics that target the bacterial cell wall.


Assuntos
Bacillus subtilis/citologia , Parede Celular , Escherichia coli/citologia , Formas L/citologia , Estresse Oxidativo , Bacillus subtilis/genética , Transporte de Elétrons , Mutação , Oxirredução
13.
Hum Vaccin Immunother ; 11(5): 1192-200, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25874947

RESUMO

Possible persistence of bacteria in human blood as cell wall deficient forms (L-forms) represents a top research priority for microbiologists. Application of live BCG vaccine and L-form transformation of vaccine strain may display a new intriguing aspect concerning the opportunity for occurrence of unpredictable colonization inside the human body by unusual microbial life forms. L-form cultures were isolated from 141 blood samples of people previously vaccinated with BCG, none with a history of exposure to tuberculosis. Innovative methodology to access the unusual L-form elements derived from human blood was developed. The methodology outlines the path of transformation of non- cultivable L-form element to cultivable bacteria and their adaptation for growth in vitro. All isolates showed typical L-forms growth features ("fried eggs" colonies and biofilm). Electron microscopy revealed morphology evidencing peculiar characteristics of bacterial L-form population (cell wall deficient polymorphic elements of variable shape and size). Regular detection of acid fast bacteria in smears of isolated blood L-form cultures, led us to start their identification by using specific Mycobactrium spp. genetic tests. Forty five of 97 genetically tested blood cultures provided specific positive signals for mycobacteria, confirmed by at least one of the 3 specific assays (16S rRNA PCR; IS6110 Real Time PCR and spoligotyping). In conclusion, the obtained genetic evidence suggests that these L-forms are of mycobacterial origin. As the investigated people had been vaccinated with BCG, we can assume that the identified mycobacterial L-forms may be produced by persisting live BCG vaccine.


Assuntos
Vacina BCG/administração & dosagem , Sangue/microbiologia , Formas L/isolamento & purificação , Mycobacterium/isolamento & purificação , Adolescente , Adulto , Biofilmes/crescimento & desenvolvimento , Criança , Pré-Escolar , Elementos de DNA Transponíveis , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Feminino , Humanos , Lactente , Formas L/genética , Formas L/fisiologia , Formas L/ultraestrutura , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Tipagem Molecular , Mycobacterium/genética , Mycobacterium/fisiologia , Mycobacterium/ultraestrutura , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
14.
Clin Microbiol Infect ; 21(5): 470.e9-16, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25703211

RESUMO

Bacterial L-forms have always been considered as osmotic-pressure-sensitive cell-wall-deficient bacteria and isolation culture of L-forms must use media with high osmotic pressure. However, isolation culture of stable L-forms formed in humans and animals is very difficult because they have adapted to the physiological osmotic pressure condition of the host. We use a non-high osmotic isolation technique to isolate stable L-forms of Salmonella Typhi and Salmonella Paratyphi A from bile-inducer cultures in vitro and from patients' gallbladder specimens. Multiplex PCR assay for Salmonella-specific genes and nucleotide sequencing are used to identify the Salmonella L-forms in stable L-form isolates. Using this method, we confirmed that Salmonella Paratyphi A and Salmonella Typhi cannot be isolated from bile-inducer cultures cultured for 6 h or 48 h, but the L-forms can be isolated from 1 h to 45 days. In the 524 gallbladder samples, the positive rate for bacterial forms was 19.7% and the positive rate for Salmonella spp. was 0.6% by routine bacteriological methods. The positive rate for bacterial L-forms was 75.4% using non-high osmotic isolation culture. In the L-form isolates, the positive rate of Salmonella invA gene was 3.1%. In these invA-positive L-form isolates, four were positive for the invA and flic-d genes of Salmonella Typhi, and ten were positive for the invA and flic-a genes of Salmonella Paratyphi A.


Assuntos
Técnicas Bacteriológicas/métodos , Bile/microbiologia , Vesícula Biliar/microbiologia , Formas L/isolamento & purificação , Salmonella paratyphi A/isolamento & purificação , Salmonella typhi/isolamento & purificação , Proteínas de Bactérias/genética , Meios de Cultura/química , Humanos , Febre Paratifoide/microbiologia , Reação em Cadeia da Polimerase , Salmonella paratyphi A/genética , Salmonella typhi/genética , Análise de Sequência de DNA , Febre Tifoide/microbiologia , Fatores de Virulência/genética
15.
Helicobacter ; 20(2): 98-105, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25381932

RESUMO

BACKGROUND: The Helicobacter pylori is considered the important causative agent causing biliary diseases, but the H. pylori can be isolated from very few gallbladder specimens with diseases. We studied the formation of H. pylori L-forms in bile in vitro and isolated the H. pylori L-forms from gallbladder of patients with biliary diseases. METHODS: We inoculated the H. pylori into the human bile to induce the L-form in vitro. The gallbladder specimens were collected from patients with biliary diseases to isolate the bacterial L-forms by the nonhigh osmotic isolation technique, and the H. pylori L-forms in the L-form isolates were identified by the gene assay for the H. pylori-specific genes 16S rRNA and UreA. RESULTS: The H. Pylori cannot be isolated from the bile-induced cultures, but the H. pylori L-form can be isolated from the H. pylori-negative bile-induced cultures. The L-form isolates of bile-induced cultures showed a positive reaction of the H. pylori-specific genes by PCR, and the coincidence ratio of the nucleotide sequences between the L-forms and the H. pylori is 99%. The isolation rate of bacteria L-form is 93.2% in the gallbladder specimens with bacteria-negative isolation culture by the nonhigh osmotic isolation technique, and the positive rate of the H. pylori-specific genes in the L-form isolates is 7.1% in the bacterial L-form-positive isolation cultures by the PCR. CONCLUSIONS: H. pylori can be rapidly induced into the L-form in the human bile; the L-form, as the latent bacteria, can live in the host gallbladder for a long times, and they made the host became a latent carrier of the H. pylori L-form. The H. pylori L-form can be isolated by the nonhigh osmotic isolation technique, and the variant can be identified by the gene assay for the H. pylori-specific genes 16S rRNA and reA.


Assuntos
Bile/microbiologia , Doenças Biliares/microbiologia , Vesícula Biliar/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/isolamento & purificação , Formas L/isolamento & purificação , Portador Sadio/microbiologia , Helicobacter pylori/classificação , Helicobacter pylori/genética , Humanos , Formas L/classificação , Formas L/genética , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Urease/genética
16.
Elife ; 32014 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-25427009
17.
Elife ; 32014 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-25358088

RESUMO

The peptidoglycan cell wall is a defining structural feature of the bacterial kingdom. Curiously, some bacteria have the ability to switch to a wall-free or 'L-form' state. Although known for decades, the general properties of L-forms are poorly understood, largely due to the lack of systematic analysis of L-forms in the molecular biology era. Here we show that inhibition of peptidoglycan precursor synthesis promotes the generation of L-forms from both Gram-positive and Gram-negative bacteria. We show that the L-forms generated have in common a mechanism of proliferation involving membrane blebbing and tubulation, which is dependent on an altered rate of membrane synthesis. Crucially, this mode of proliferation is independent of the essential FtsZ based division machinery. Our results suggest that the L-form mode of proliferation is conserved across the bacterial kingdom, reinforcing the idea that it could have been used in primitive cells, and opening up its use in the generation of synthetic cells.


Assuntos
Bactérias/citologia , Parede Celular/metabolismo , Formas L/citologia , Bactérias/crescimento & desenvolvimento , Divisão Celular , Proliferação de Células , Corynebacterium glutamicum/citologia , Escherichia coli/citologia , Ácidos Graxos/biossíntese , Peptidoglicano/metabolismo , Imagem com Lapso de Tempo
18.
Mol Microbiol ; 93(5): 883-96, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24995493

RESUMO

In virtually all bacteria, the cell wall is crucial for mechanical integrity and for determining cell shape. Escherichia coli's rod-like shape is maintained via the spatiotemporal patterning of cell-wall synthesis by the actin homologue MreB. Here, we transiently inhibited cell-wall synthesis in E. coli to generate cell-wall-deficient, spherical L-forms, and found that they robustly reverted to a rod-like shape within several generations after inhibition cessation. The chemical composition of the cell wall remained essentially unchanged during this process, as indicated by liquid chromatography. Throughout reversion, MreB localized to inwardly curved regions of the cell, and fluorescent cell wall labelling revealed that MreB targets synthesis to those regions. When exposed to the MreB inhibitor A22, reverting cells regrew a cell wall but failed to recover a rod-like shape. Our results suggest that MreB provides the geometric measure that allows E. coli to actively establish and regulate its morphology.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Formas L/crescimento & desenvolvimento , Formas L/genética , Parede Celular/genética , Parede Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Formas L/metabolismo
19.
Artigo em Inglês | MEDLINE | ID: mdl-24904838

RESUMO

Stable L-forms are cell wall-deficient bacteria which are able to multiply and propagate indefinitely, despite the absence of a rigid peptidoglycan cell wall. We investigated whether L-forms of the intracellular pathogen L. monocytogenes possibly retain pathogenicity, and if they could trigger an innate immune response. While phagocytosis of L. monocytogenes L-forms by non-activated macrophages sometimes resulted in an unexpected persistence of the bacteria in the phagocytes, they were effectively eliminated by IFN-γ preactivated or bone marrow-derived macrophages (BMM). These findings were in line with the observed down-regulation of virulence factors in the cell-wall deficient L. monocytogenes. Absence of Interferon-ß (IFN-ß) triggering indicated inability of L-forms to escape from the phagosome into the cytosol. Moreover, abrogated cytokine response in MyD88-deficient dendritic cells (DC) challenged with L. monocytogenes L-forms suggested an exclusive TLR-dependent host response. Taken together, our data demonstrate a strong attenuation of Listeria monocytogenes L-form pathogenicity, due to diminished expression of virulence factors and innate immunity recognition, eventually resulting in elimination of L-form bacteria from phagocytes.


Assuntos
Formas L/patogenicidade , Listeria monocytogenes/patogenicidade , Macrófagos/microbiologia , Animais , Células Cultivadas , Regulação para Baixo , Formas L/imunologia , Listeria monocytogenes/imunologia , Camundongos Endogâmicos C57BL , Fagossomos/imunologia , Fagossomos/microbiologia , Virulência , Fatores de Virulência/biossíntese
20.
Nan Fang Yi Ke Da Xue Xue Bao ; 34(2): 180-7, 2014 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-24589592

RESUMO

OBJECTIVE: To investigate the correlation between infection with L-form of Helicobacter pylori (Hp-L) and the expressions of macrophage migration inhibition factor (MIF), matrix metalloproteinase 9 (MMP9), and vascular endothelial growth factor (VEGF) in gastric cancer. METHODS: Hp-L was examined in 80 gastric carcinoma and 50 adjacent normal tissues by Gram staining and immunohistochemical staining, and the expressions of MIF, MMP9 and VEGF were detected by immunohistochemical staining; the expression of MIF mRNA was detected by RT-PCR and the expression of MIF, MMP9 and VEGF proteins were detected by Western blotting in 30 fresh gastric cancer tissues and the corresponding adjacent tissues. RESULTS: Of the 80 gastric carcinoma tissues, 57 (71.25%) showed Hp-L positivity detected by both Gram staining and immunohistochemical staining, as compared with a rate of only 14% in the adjacent normal tissues (P<0.05). The gastric carcinoma tissues showed higher expression levels of MIF, MMP9 and VEGF proteins than the corresponding adjacent normal mucosa; the positivity MIF, MMP-9 and VEGF proteins were significantly higher in Hp-L-positive gastric carcinoma than in Hp-L-negative cases (P<0.05). Positive correlations were found between Hp-L positivity and the expressions of MIF, MMP-9 and VEGF (r=0.598, 0.292, 0.341, respectively, P<0.05). The 30 fresh gastric cancer tissues showed also significantly higher MIF mRNA expression and MIF, MMP-9 and VEGF protein expressions than the adjacent tissues (t=3.729, P<0.01). The expressions of MIF and MMP-9 were also related to the clinicopathological factors including lymph node metastasis and depth of invasion (P<0.05). CONCLUSION: Infection with L-form of Hp-L can be an important factor that contributes to the invasion and metastasis of gastric carcinoma, the mechanism of which involves up-regulated expressions of MIF, MMP-9 and VEGF.


Assuntos
Infecções por Helicobacter/metabolismo , Helicobacter pylori , Formas L , Fatores Inibidores da Migração de Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias Gástricas/microbiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Infecções por Helicobacter/patologia , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia
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