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1.
J Med Microbiol ; 68(12): 1723-1731, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31746726

RESUMO

Introduction. Carbapenems are often described as the most effective weapon against infections caused by multidrug-resistant bacteria especially those belonging to the group of non-fermenting bacteria such as Pseudomonas. The main mechanisms leading to resistance are the hyperexpression of certain efflux pumps belonging to the resisto-nodular division and the lower expression of the transmembrane porin OprD, sometimes in combination with excessive production of the intrinsic AmpC. Carbapenemases are assumed to play a secondary role.Aim. The aim of this study was to determine the exact mechanisms of carbapenem resistance in Pseudomonas aeruginosa isolates from the largest Bulgarian University hospital 'St. George'- Plovdiv.Methodology. A total of 32 clinical isolates collected from different patients' samples resistant to imipenem and/or meropenem were examined via phenotypic and molecular-genetic tests.Results. No metallo-enzyme production was detected. Three isolates were positive for OXA-50-encoding genes in two of them in combination with other oxacillinases or the bla VEB-1 gene. For the first time, OXA-50-producing P. aeruginosa have been reported in Bulgaria. The increased expression or hyperexpression of MexXY-OprM efflux pump was observed as the main mechanism of resistance. In most cases, it was combined with lower expression or lack of OprD with or without MexAB-OprM hyperexpression. No excessive production of AmpC was detected in comparison to the reference ATCC 27853 P. aeruginosa strain.Conclusion. The increased expression or overexpression of MexXY-OprM efflux pumps is the leading cause of carbapenem resistance in our isolates Pseudomonas, detected in 94 % of the bacteria investigated.


Assuntos
Proteínas de Bactérias/análise , Proteínas de Bactérias/fisiologia , Carbapenêmicos/farmacologia , Porinas/fisiologia , Pseudomonas aeruginosa/isolamento & purificação , beta-Lactamases/análise , beta-Lactamases/fisiologia , Farmacorresistência Bacteriana , Humanos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia
2.
DNA Cell Biol ; 38(11): 1178-1187, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31580738

RESUMO

Mycobacterium tuberculosis proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family proteins, with >160 members, are crucial for virulence, cell wall, host cell fate, host Th1/Th2 balance, and CD8+ T cell recognition. Ca2+ signaling is involved in PE/PPE protein-mediated host-pathogen interaction. PE/PPE proteins also function in heme utilization and nitric oxide production. PE/PPE family proteins are intensively pursued as diagnosis biomarkers and vaccine components.


Assuntos
Proteínas de Bactérias/fisiologia , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune/genética , Mycobacterium tuberculosis/imunologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ácido Glutâmico/química , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Família Multigênica/fisiologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Prolina/química , Domínios Proteicos Ricos em Prolina/genética , Domínios Proteicos Ricos em Prolina/imunologia , Virulência/genética , Virulência/imunologia
3.
Nat Commun ; 10(1): 4653, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31604936

RESUMO

In mycobacteria, transcriptional activator PafBC is responsible for upregulating the majority of genes induced by DNA damage. Understanding the mechanism of PafBC activation is impeded by a lack of structural information on this transcription factor that contains a widespread, but poorly understood WYL domain frequently encountered in bacterial transcription factors. Here, we determine the crystal structure of Arthrobacter aurescens PafBC. The protein consists of two modules, each harboring an N-terminal helix-turn-helix DNA-binding domain followed by a central WYL and a C-terminal extension (WCX) domain. The WYL domains exhibit Sm-folds, while the WCX domains adopt ferredoxin-like folds, both characteristic for RNA-binding proteins. Our results suggest a mechanism of regulation in which WYL domain-containing transcription factors may be activated by binding RNA or other nucleic acid molecules. Using an in vivo mutational screen in Mycobacterium smegmatis, we identify potential co-activator binding sites on PafBC.


Assuntos
Proteínas de Bactérias/química , Dano ao DNA , Micrococcaceae/genética , Fatores de Transcrição/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Regulação para Cima
4.
PLoS Pathog ; 15(9): e1008044, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31518377

RESUMO

ß-lactam antibiotics interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Serendipitously we found that sub-lethal doses of ß-lactams rescue growth and prevent spontaneous lysis of Staphylococcus aureus mutants lacking the widely conserved chaperone ClpX, and we reasoned that a better understanding of the clpX phenotypes could provide novel insights into the downstream effects of ß-lactam binding to the PBP targets. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30°C, but not at 37°C, demonstrating that ClpX becomes critical for coordinating the S. aureus cell cycle as the temperature decreases. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, oxacillin antagonized the septum progression defects of clpX cells and prevented lysis of prematurely splitting clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with ß-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, as did genetic inactivation of the gene encoding the septal autolysin, Sle1. Taken together, our data support a model in which Sle1 causes premature splitting and lysis of clpX daughter cells unless Sle1-dependent lysis is antagonized by ß-lactams or by inhibiting an early step in WTA biosynthesis. The finding that ß-lactams and inhibitors of WTA biosynthesis specifically prevent lysis of a mutant with dysregulated autolytic activity lends support to the idea that PBPs and WTA biosynthesis play an important role in coordinating cell division with autolytic splitting of daughter cells, and that ß-lactams do not kill S. aureus simply by weakening the cell wall.


Assuntos
Proteínas de Bactérias/fisiologia , Endopeptidase Clp/fisiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriólise/efeitos dos fármacos , Bacteriólise/fisiologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Endopeptidase Clp/genética , Humanos , Modelos Biológicos , Mutação , Oxacilina/farmacologia , Staphylococcus aureus/genética , Ácidos Teicoicos/biossíntese , Tunicamicina/farmacologia , beta-Lactamas/farmacologia
5.
PLoS Pathog ; 15(9): e1007651, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31513674

RESUMO

Bacterial type IV secretion systems (T4SS) are a highly diversified but evolutionarily related family of macromolecule transporters that can secrete proteins and DNA into the extracellular medium or into target cells. It was recently shown that a subtype of T4SS harboured by the plant pathogen Xanthomonas citri transfers toxins into target cells. Here, we show that a similar T4SS from the multi-drug-resistant opportunistic pathogen Stenotrophomonas maltophilia is proficient in killing competitor bacterial species. T4SS-dependent duelling between S. maltophilia and X. citri was observed by time-lapse fluorescence microscopy. A bioinformatic search of the S. maltophilia K279a genome for proteins containing a C-terminal domain conserved in X. citri T4SS effectors (XVIPCD) identified twelve putative effectors and their cognate immunity proteins. We selected a putative S. maltophilia effector with unknown function (Smlt3024) for further characterization and confirmed that it is indeed secreted in a T4SS-dependent manner. Expression of Smlt3024 in the periplasm of E. coli or its contact-dependent delivery via T4SS into E. coli by X. citri resulted in reduced growth rates, which could be counteracted by expression of its cognate inhibitor Smlt3025 in the target cell. Furthermore, expression of the VirD4 coupling protein of X. citri can restore the function of S. maltophilia ΔvirD4, demonstrating that effectors from one species can be recognized for transfer by T4SSs from another species. Interestingly, Smlt3024 is homologous to the N-terminal domain of large Ca2+-binding RTX proteins and the crystal structure of Smlt3025 revealed a topology similar to the iron-regulated protein FrpD from Neisseria meningitidis which has been shown to interact with the RTX protein FrpC. This work expands our current knowledge about the function of bacteria-killing T4SSs and increases the panel of effectors known to be involved in T4SS-mediated interbacterial competition, which possibly contribute to the establishment of S. maltophilia in clinical and environmental settings.


Assuntos
Proteínas de Bactérias/fisiologia , Stenotrophomonas maltophilia/fisiologia , Stenotrophomonas maltophilia/patogenicidade , Sistemas de Secreção Tipo IV/fisiologia , Sequência de Aminoácidos , Antibiose/genética , Antibiose/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência Conservada , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Genes Bacterianos , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Proteínas Reguladoras do Ferro/química , Proteínas Reguladoras do Ferro/genética , Proteínas Reguladoras do Ferro/fisiologia , Modelos Moleculares , Infecções Oportunistas/microbiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Stenotrophomonas maltophilia/genética , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/genética , Xanthomonas/genética , Xanthomonas/crescimento & desenvolvimento
6.
Biochimie ; 167: 34-41, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31491441

RESUMO

ArgR, a transcriptional regulator belonging to the AraC/XylS family, plays a key role in arginine metabolism regulation. ArgR has also been found to repress the transcription of a lipase gene, but its molecular mechanism is still unknown. In this study, we investigated the molecular mechanism acting on the expression of intracellular lipase gene lipA regulated by ArgR in Pseudomonas protegens Pf-5 through knockout and overexpression of argR, detection of DNA-protein interaction in vivo, determining whole-cell lipase activities of various strains derived from Pf-5, and examining ß-galactosidase activities of various lacZ fusions. The results demonstrated that ArgR inhibits lipA expression at the transcriptional level. Further results showed that the inhibition of lipA transcription by ArgR is mediated by binding to the ArgR binding site of lipA promoter to produce steric hindrance, in which the common sequence, TGTCGC is crucial for the ArgR binding. Besides, arginine inhibits lipA expression in both wild-type and argR mutant, and shows a synergistic inhibition on lipA expression when combined with ArgR. To the best of our knowledge, this is the first report on ArgR directly repressing the transcription of a lipase gene.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica , Pseudomonas/genética , Proteínas Repressoras/fisiologia , Arginina/metabolismo , Sítios de Ligação , Regiões Promotoras Genéticas , Proteínas Repressoras/genética
7.
Invest Ophthalmol Vis Sci ; 60(12): 3727-3739, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31479113

RESUMO

Purpose: Bacillus causes a sight-threating infection of the posterior segment of the eye. The robust intraocular inflammatory response in this disease is likely activated via host innate receptor interactions with components of the Bacillus cell envelope. S-layer proteins (SLPs) of some Gram-positive pathogens contribute to the pathogenesis of certain infections. The potential contributions of SLPs in eye infection pathogenesis have not been considered. Here, we explored the role of a Bacillus SLP (SlpA) in endophthalmitis pathogenesis. Methods: The phenotypes and infectivity of wild-type (WT) and S-layer deficient (ΔslpA) Bacillus thuringiensis were compared. Experimental endophthalmitis was induced in C57BL/6J mice by intravitreally injecting 100-CFU WT or ΔslpA B. thuringiensis. Infected eyes were analyzed by bacterial counts, retinal function analysis, histology, and inflammatory cell influx. SLP-induced inflammation was also analyzed in vitro. Muller cells (MIO-M1) were treated with purified SLP. Nuclear factor-κB (NF-κB) DNA binding was measured by ELISA and expression of proinflammatory mediators from Muller cells was measured by RT-qPCR. Results: Tested phenotypes of WT and ΔslpA B. thuringiensis were similar, with the exception of absence of the S-layer in the ΔslpA mutant. Intraocular growth of WT and ΔslpA B. thuringiensis was also similar. However, eyes infected with the ΔslpA mutant had significantly reduced inflammatory cell influx, less inflammatory damage to the eyes, and significant retention of retinal function compared with WT-infected eyes. SLP was also a potent stimulator of the NF-κB pathway and induced the expression of proinflammatory mediators (IL6, TNFα, CCL2, and CXCL-1) in human retinal Muller cells. Conclusions: Taken together, our results suggest that SlpA contributes to the pathogenesis of Bacillus endophthalmitis, potentially by triggering innate inflammatory pathways in the retina.


Assuntos
Bacillus thuringiensis/patogenicidade , Proteínas de Bactérias/fisiologia , Endoftalmite/microbiologia , Infecções Oculares Bacterianas/microbiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Glicoproteínas de Membrana/fisiologia , Animais , Contagem de Colônia Microbiana , Citocinas/metabolismo , Eletrorretinografia , Endoftalmite/metabolismo , Endoftalmite/patologia , Ensaio de Imunoadsorção Enzimática , Células Ependimogliais/microbiologia , Infecções Oculares Bacterianas/metabolismo , Infecções Oculares Bacterianas/patologia , Infecções por Bactérias Gram-Positivas/metabolismo , Infecções por Bactérias Gram-Positivas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Modelos Animais , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Retina/microbiologia , Retina/fisiopatologia , Virulência/fisiologia
8.
Shanghai Kou Qiang Yi Xue ; 28(2): 113-117, 2019.
Artigo em Chinês | MEDLINE | ID: mdl-31384892

RESUMO

PURPOSE: To evaluate the effect of S.mutans luxS gene on mixed-species biofilms communities. METHODS: Biofilms were formed by S. mutans (wild type strain, its luxS overexpression strain and luxS knockout strain) and Lactobacillus acidophilus (ATCC4356) with a ratio of 1:1 at 37℃ for 4 h, 14 h and 24 h. MTT assay was used to detect the quantification of the biofilms formed. The structures of biofilms were observed under confocal laser scanning microscopy after 24 h, and expression of biofilm-related genes (ftf, smu630, brpA, gbpB, gtfB, vicR, comDE and relA) was investigated by real-time PCR. Statistical analysis was performed with SPSS17.0 software package. RESULTS: The results showed that biofilm formed by S. mutans(wild type strain, its luxS overexpression strain and luxS knockout strain) and L.acidophilus after 14 h were 0.481±0.024, 0.591±0.023 and 0.279±0.019, respectively. The same findings were present after 24 h, the biofilm formed by S.mutans overexpression strain with L.acidophilus was higher than wild type strain, and the biofilm formed by knockout strain significantly decreased; but there was no significant difference at 4 h time points. CLSM images revealed that both S.mutans overexpression strain and its wild type strain tended to aggregate into distinct clusters and dense structures, whereas the luxS knockout strain appeared relatively sparse. Compared with wild type strain, all of the genes examined were upregulated in the biofilms formed by the overexpression strain, and were downregulated in the biofilms formed by the luxS mutant strain in mixed-species biofilm. CONCLUSIONS: S.mutans luxS gene can affect mixed-species biofilm formation with L.acidophilus, which provides evidences for further study.


Assuntos
Proteínas de Bactérias , Biofilmes , Liases de Carbono-Enxofre , Streptococcus mutans , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/fisiologia , Lactobacillus acidophilus , Reação em Cadeia da Polimerase em Tempo Real , Streptococcus mutans/genética
9.
Biochimie ; 165: 156-160, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377193

RESUMO

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase (PtpA) has so far been known to control intracellular survival of mycobacteria; whereas the ATP synthase which is essential for mycobacterial growth has recently been contemplated in developing a breakthrough anti-TB drug, diarylquinoline. Since both of these enzymes have been established as validated drug targets; we report a robust and functional relationship between these two enzymes through a series of experiments using Mtb H37Ra. In the present study we report that the mycobacterial ATP synthase alpha subunit is regulated by PtpA. We generated gene knock-out for the enzyme PtpA and subjected to determine the mycobacterial replication and the proteome profile of wild type, mutant (ΔptpA) and complemented (ΔptpA:ptpA) strains of Mtb H37Ra. A substantial amount of decrease in the protein level of ATP synthase alpha subunit (AtpA) in case of mutant H37Ra was observed, while the levels of the enzyme were either increased or remained unchanged, in wild type and in the complemented strains.


Assuntos
Proteínas de Bactérias/fisiologia , ATPases Bacterianas Próton-Translocadoras/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Proteínas Tirosina Fosfatases/fisiologia , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Diarilquinolinas/farmacologia , Técnicas de Inativação de Genes , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Proteínas Tirosina Fosfatases/genética
10.
PLoS Genet ; 15(8): e1008086, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31412020

RESUMO

DNA methyltransferases are ubiquitous enzymes conserved in bacteria, plants and opisthokonta. These enzymes, which methylate cytosines, are involved in numerous biological processes, notably development. In mammals and higher plants, methylation patterns established and maintained by the cytosine DNA methyltransferases (DMTs) are essential to zygotic development. In fungi, some members of an extensively conserved fungal-specific DNA methyltransferase class are both mediators of the Repeat Induced Point mutation (RIP) genome defense system and key players of sexual reproduction. Yet, no DNA methyltransferase activity of these purified RID (RIP deficient) proteins could be detected in vitro. These observations led us to explore how RID-like DNA methyltransferase encoding genes would play a role during sexual development of fungi showing very little genomic DNA methylation, if any. To do so, we used the model ascomycete fungus Podospora anserina. We identified the PaRid gene, encoding a RID-like DNA methyltransferase and constructed knocked-out ΔPaRid defective mutants. Crosses involving P. anserina ΔPaRid mutants are sterile. Our results show that, although gametes are readily formed and fertilization occurs in a ΔPaRid background, sexual development is blocked just before the individualization of the dikaryotic cells leading to meiocytes. Complementation of ΔPaRid mutants with ectopic alleles of PaRid, including GFP-tagged, point-mutated and chimeric alleles, demonstrated that the catalytic motif of the putative PaRid methyltransferase is essential to ensure proper sexual development and that the expression of PaRid is spatially and temporally restricted. A transcriptomic analysis performed on mutant crosses revealed an overlap of the PaRid-controlled genetic network with the well-known mating-types gene developmental pathway common to an important group of fungi, the Pezizomycotina.


Assuntos
Proteínas de Bactérias/fisiologia , Metilases de Modificação do DNA/fisiologia , Redes Reguladoras de Genes/genética , Podospora/fisiologia , Citosina/metabolismo , Metilação de DNA/fisiologia , Epigênese Genética/fisiologia , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Fúngicos Tipo Acasalamento/genética , Genoma Bacteriano
11.
Microbiol Res ; 228: 126304, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31422235

RESUMO

Streptococcus suis (S. suis) is an important zoonotic pathogen that causes major economic losses in the pig industry worldwide. The S. suis cell division process is an integral part of its growth and reproduction, which is controlled by a complex regulatory network. Pyruvate dehydrogenase (PDH), which catalyzes the oxidative decarboxylation of pyruvate to form acetyl-CoA, while reducing NAD + to NADH, plays an important role in energy metabolism. Recently, we reported that pdh regulates virulence by reducing stress tolerance and biofilm formation in S. suis serotype 2. In this study, we found that deletion of the pdh gene in S. suis resulted in abnormal cell chains, plump morphology and abnormal localization of the Z rings, indicating that the knockout mutant is impaired in its ability to divide. In addition, the interaction between FtsZ and PDH in vitro was confirmed by ELISA, and qRT-PCR analysis revealed that the deletion of the pdh gene results in differential expression of the division-related genes ftsZ, ftsK, ftsl, zapA, divIC, pbp1a, rodA, mreD, and sepF. These results indicate that pdh is involved in the normal formation of Z rings and cell morphology during S. suis cell division.


Assuntos
Divisão Celular/genética , Divisão Celular/fisiologia , Complexo Piruvato Desidrogenase/genética , Streptococcus suis/citologia , Streptococcus suis/genética , Streptococcus suis/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Proteínas do Citoesqueleto/genética , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/veterinária , Streptococcus suis/patogenicidade , Suínos , Virulência , Fatores de Virulência/genética
12.
Appl Microbiol Biotechnol ; 103(17): 7055-7070, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31273395

RESUMO

Thermostability plays an important role in the application of L-asparaginase in the pharmaceutical and food industries. Understanding the key residues and structures that influence thermostability in L-asparaginase is necessary to obtain suitable L-asparaginase candidates. In this study, special residues and structures that altered thermostability in thermophilic L-asparaginase and non-thermophilic L-asparaginase II were identified. Interchanging these special residues and structures of L-asparaginases from the four strains, that is, Pyrococcus yayanosii CH1 (PYA), Thermococcus gammatolerans (TGA), Bacillus subtilis (BSA II), and Escherichia coli (ECA II), revealed the 51st and 298th residues of PYA (corresponding to 57th, 305th residues of ECA II) as the key residues responsible for thermal stability of thermophilic L-asparaginase and non-thermophilic L-asparaginase II. Moreover, the C terminal tightness, loop rigidity, and low surface charge around activity sites were of great significance to the thermostability of L-asparaginase. This study therefore revealed the crucial amino acid residues and structures responsible for the difference in thermostability of the thermophilic and non-thermophilic L-asparaginase and provides a reference for engineering thermostability in L-asparaginase II.


Assuntos
Asparaginase/química , Asparaginase/fisiologia , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Domínio Catalítico , Biologia Computacional , Estabilidade Enzimática , Temperatura Alta , Modelos Moleculares , Mutação , Conformação Proteica , Relação Estrutura-Atividade
13.
Infect Immun ; 87(9)2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31262979

RESUMO

Helicobacter pylori is a pathogen that chronically colonizes the stomachs of approximately half of the world's population and contributes to the development of gastric inflammation. We demonstrated previously in vivo that H. pylori uses motility to preferentially colonize injury sites in the mouse stomach. However, the chemoreceptor responsible for sensing gastric injury has not yet been identified. In this study, we utilized murine gastric organoids (gastroids) and mutant H. pylori strains to investigate the components necessary for H. pylori chemotaxis. High-intensity 730-nm light (two-photon photodamage) was used to cause single-cell damage in gastroids, and repair of the damage was monitored over time; complete repair occurred within ∼10 min in uninfected gastroids. Wild-type H. pylori accumulated at the damage site after gastric damage induction. In contrast, mutants lacking motility (ΔmotB) or chemotaxis (ΔcheY) did not accumulate at the injury site. Using mutants lacking individual chemoreceptors, we found that only TlpB was required for H. pylori accumulation, while TlpA, TlpC, and TlpD were dispensable. All strains that were able to accumulate at the damage site limited repair. When urea (an identified chemoattractant sensed by TlpB) was microinjected into the gastroid lumen, it prevented the accumulation of H. pylori at damage sites. Overall, our findings demonstrate that H. pylori colonizes and limits repair at damage sites via chemotactic motility that requires the TlpB chemoreceptor to sense signals generated by gastric epithelial cells.


Assuntos
Proteínas de Bactérias/fisiologia , Fatores Quimiotáticos/farmacologia , Quimiotaxia/fisiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/efeitos dos fármacos , Gastropatias/microbiologia , Animais , Modelos Animais de Doenças , Mucosa Gástrica/microbiologia , Camundongos
14.
Elife ; 82019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31313986

RESUMO

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator-rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator-rotor interaction at an unprecedented detail. Importantly, the stator-rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.


Assuntos
Proteínas de Bactérias/química , Borrelia burgdorferi/química , Flagelos/química , Proteínas Motores Moleculares/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidade , Tomografia com Microscopia Eletrônica , Flagelos/genética , Flagelos/fisiologia , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/fisiologia , Mutação/genética , Rotação , Deleção de Sequência , Sódio/química , Torque
15.
PLoS Pathog ; 15(7): e1007987, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31356624

RESUMO

Streptococcus pneumoniae (pneumococci) is a leading cause of severe bacterial meningitis in many countries worldwide. To characterize the repertoire of fitness and virulence factors predominantly expressed during meningitis we performed niche-specific analysis of the in vivo proteome in a mouse meningitis model, in which bacteria are directly inoculated into the cerebrospinal fluid (CSF) cisterna magna. We generated a comprehensive mass spectrometry (MS) spectra library enabling bacterial proteome analysis even in the presence of eukaryotic proteins. We recovered 200,000 pneumococci from CSF obtained from meningitis mice and by MS we identified 685 pneumococci proteins in samples from in vitro filter controls and 249 in CSF isolates. Strikingly, the regulatory two-component system ComDE and substrate-binding protein AliB of the oligopeptide transporter system were exclusively detected in pneumococci recovered from the CSF. In the mouse meningitis model, AliB-, ComDE-, or AliB-ComDE-deficiency resulted in attenuated meningeal inflammation and disease severity when compared to wild-type pneumococci indicating the crucial role of ComDE and AliB in pneumococcal meningitis. In conclusion, we show here mechanisms of pneumococcal adaptation to a defined host compartment by a proteome-based approach. Further, this study provides the basis of a promising strategy for the identification of protein antigens critical for invasive disease caused by pneumococci and other meningeal pathogens.


Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Transporte/fisiologia , Lipoproteínas/fisiologia , Meningite Pneumocócica/microbiologia , Streptococcus pneumoniae/fisiologia , Streptococcus pneumoniae/patogenicidade , Fatores de Virulência/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Genes Bacterianos , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Lipoproteínas/deficiência , Lipoproteínas/genética , Masculino , Meningite Pneumocócica/líquido cefalorraquidiano , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteômica , Regulon , Streptococcus pneumoniae/genética , Virulência/genética , Virulência/fisiologia , Fatores de Virulência/genética
16.
PLoS Biol ; 17(6): e3000311, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31233493

RESUMO

Clostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2 C. difficile-secreted exotoxins-toxin A (TcdA) and toxin B (TcdB)-that disrupt the tight junctions between epithelial cells leading to the loss of colonic epithelial barrier function. Here, we report the engineering of a series of monomeric and dimeric designed ankyrin repeat proteins (DARPins) for the neutralization of TcdB. The best dimeric DARPin, DLD-4, inhibited TcdB with a half maximal effective concentration (EC50) of 4 pM in vitro, representing an approximately 330-fold higher potency than the Food and Drug Administration (FDA)-approved anti-TcdB monoclonal antibody bezlotoxumab in the same assay. DLD-4 also protected mice from a toxin challenge in vivo. Cryo-electron microscopy (cryo-EM) studies revealed that the 2 constituent DARPins of DLD-4-1.4E and U3-bind the central and C-terminal regions of the delivery domain of TcdB. Competitive enzyme-linked immunosorbent assay (ELISA) studies showed that the DARPins 1.4E and U3 interfere with the interaction between TcdB and its receptors chondroitin sulfate proteoglycan 4 (CSPG4) and frizzled class receptor 2 (FZD2), respectively. Our cryo-EM studies revealed a new conformation of TcdB (both apo- and DARPin-bound at pH 7.4) in which the combined repetitive oligopeptides (CROPS) domain points away from the delivery domain. This conformation of the CROPS domain is in stark contrast to that seen in the negative-stain electron microscopy (EM) structure of TcdA and TcdB at the same pH, in which the CROPS domain bends toward and "kisses" the delivery domain. The ultrapotent anti-TcdB molecules from this study serve as candidate starting points for CDI drug development and provide new biological tools for studying the pathogenicity of C. difficile. The structural insights regarding both the "native" conformation of TcdB and the putative sites of TcdB interaction with the FZD2 receptor, in particular, should help accelerate the development of next-generation anti-C. difficile toxin therapeutics.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/fisiologia , Toxinas Bacterianas/antagonistas & inibidores , Infecções por Clostridium/metabolismo , Animais , Repetição de Anquirina/genética , Anticorpos Monoclonais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Células CACO-2 , Clostridium difficile/metabolismo , Clostridium difficile/patogenicidade , Microscopia Crioeletrônica , Enterotoxinas/metabolismo , Humanos , Camundongos , Engenharia de Proteínas/métodos
17.
Int J Infect Dis ; 84: 143-150, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31204002

RESUMO

OBJECTIVES: Carbapenem resistance in Pseudomonas aeruginosa is growing and results from variable mechanisms. The objectives of the current study were to investigate mechanisms of carbapenem resistance and genetic relatedness of P. aeruginosa isolates recovered in Dubai hospitals. METHODS: From June 2015 through June 2016, carbapenem-nonsusceptible P. aeruginosa were collected from 4 hospitals in Dubai, and subjected to antimicrobial susceptibility testing, molecular investigation of carbapenemases by PCR-sequencing, analysis of outer membrane porin OprD2 and multidrug efflux channel MexAB-OprM levels by qPCR, and fingerprinting by ERIC-PCR. RESULTS: Out of 1969 P. aeruginosa isolated during the study period, 471 (23.9%) showed reduced carbapenem susceptibility. Of these, 37 were analyzed and 32% of them produced VIM-type metallo-ß-lactamases, including VIM-2, VIM-30, VIM-31, and VIM-42, while GES-5 and GES-9 co-existed with VIM in 5.4% of isolates. Outer membrane impermeability was observed in 73% of isolates and 75.6% displayed overproduced MexAB-OprM. ERIC-PCR revealed one large clone including most carbapenemase-producing isolates indicating clonal dissemination. CONCLUSION: This is the first study on carbapenem-nonsusceptible P. aeruginosa from Dubai, incriminating VIM production as well as outer membrane permeability and efflux systems as resistance mechanisms. Further studies on carbapenem-nonsusceptible P. aeruginosa in Dubai are warranted for containment of such health hazard.


Assuntos
Proteínas de Bactérias/fisiologia , Carbapenêmicos/farmacologia , Porinas/fisiologia , Pseudomonas aeruginosa/efeitos dos fármacos , beta-Lactamases/fisiologia , Permeabilidade da Membrana Celular , Estudos Transversais , Farmacorresistência Bacteriana , Humanos , Pseudomonas aeruginosa/enzimologia
18.
Photosynth Res ; 142(1): 105-125, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31187340

RESUMO

Biological water oxidation, performed by a single enzyme, photosystem II, is a central research topic not only in understanding the photosynthetic apparatus but also for the development of water splitting catalysts for technological applications. Great progress has been made in this endeavor following the report of a high-resolution X-ray crystallographic structure in 2011 resolving the cofactor site (Umena et al. in Nature 473:55-60, 2011), a tetra-manganese calcium complex. The electronic properties of the protein-bound water oxidizing Mn4OxCa complex are crucial to understand its catalytic activity. These properties include: its redox state(s) which are tuned by the protein matrix, the distribution of the manganese valence and spin states and the complex interactions that exist between the four manganese ions. In this short review we describe how magnetic resonance techniques, particularly EPR, complemented by quantum chemical calculations, have played an important role in understanding the electronic structure of the cofactor. Together with isotope labeling, these techniques have also been instrumental in deciphering the binding of the two substrate water molecules to the cluster. These results are briefly described in the context of the history of biological water oxidation with special emphasis on recent work using time resolved X-ray diffraction with free electron lasers. It is shown that these data are instrumental for developing a model of the biological water oxidation cycle.


Assuntos
Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/fisiologia , Água/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Cristalografia por Raios X , Cianobactérias , Cinética , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Oxirredução , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Estrutura Terciária de Proteína
19.
Infect Immun ; 87(9)2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31209148

RESUMO

The SaeRS two-component system in Staphylococcus aureus is critical for regulation of many virulence genes, including hla, which encodes alpha-toxin. However, the impact of regulation of alpha-toxin by Sae on S. aureus pathogenesis has not been directly addressed. Here, we mutated the SaeR-binding sequences in the hla regulatory region and determined the contribution of this mutation to hla expression and pathogenesis in strain USA300 JE2. Western blot analyses revealed drastic reduction of alpha-toxin levels in the culture supernatants of SaeR-binding mutant in contrast to the marked alpha-toxin production in the wild type. The SaeR-binding mutation had no significant effect on alpha-toxin regulation by Agr, MgrA, and CcpA. In animal studies, we found that the SaeR-binding mutation did not contribute to USA300 JE2 pathogenesis using a rat infective endocarditis model. However, in a rat skin and soft tissue infection model, the abscesses on rats infected with the mutant were significantly smaller than the abscesses on those infected with the wild type but similar to the abscesses on those infected with a saeR mutant. These studies indicated that there is a direct effect of hla regulation by SaeR on pathogenesis but that the effect depends on the animal model used.


Assuntos
Proteínas de Bactérias/fisiologia , Toxinas Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Quinases/fisiologia , Infecções Estafilocócicas , Staphylococcus aureus , Animais , Toxinas Bacterianas/genética , Ratos , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Virulência
20.
Cells ; 8(6)2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31216697

RESUMO

In preparation for division, bacteria replicate their DNA and segregate the newly formed chromosomes. A division septum then assembles between the chromosomes, and the mother cell splits into two identical daughters due to septum degradation. A major constituent of bacterial septa and of the whole cell wall is peptidoglycan (PGN), an essential cell wall polymer, formed by glycan chains of ß-(1-4)-linked-N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), cross-linked by short peptide stems. Depending on the amino acid located at the third position of the peptide stem, PGN is classified as either Lys-type or meso-diaminopimelic acid (DAP)-type. Hydrolytic enzymes play a crucial role in the degradation of bacterial septa to split the cell wall material shared by adjacent daughter cells to promote their separation. In mycobacteria, a key PGN hydrolase, belonging to the NlpC/P60 endopeptidase family and denoted as RipA, is responsible for the degradation of septa, as the deletion of the gene encoding for this enzyme generates abnormal bacteria with multiple septa. This review provides an update of structural and functional data highlighting the central role of RipA in mycobacterial cytokinesis and the fine regulation of its catalytic activity, which involves multiple molecular partners.


Assuntos
Citocinese/fisiologia , Endopeptidases/metabolismo , Mycobacterium/metabolismo , Acetilglucosamina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Divisão Celular , Parede Celular/metabolismo , Cristalografia por Raios X , Hidrólise , Lipoproteínas/metabolismo , Lipoproteínas/fisiologia , Modelos Moleculares , Ácidos Murâmicos/metabolismo , Mycobacterium/enzimologia , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/química , Peptidoglicano/genética , Conformação Proteica
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