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1.
Cell ; 139(4): 770-9, 2009 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-19914169

RESUMO

Riboswitches are RNA elements acting in cis, controlling expression of their downstream genes through a metabolite-induced alteration of their secondary structure. Here, we demonstrate that two S-adenosylmethionine (SAM) riboswitches, SreA and SreB, can also function in trans and act as noncoding RNAs in Listeria monocytogenes. SreA and SreB control expression of the virulence regulator PrfA by binding to the 5'-untranslated region of its mRNA. Absence of the SAM riboswitches SreA and SreB increases the level of PrfA and virulence gene expression in L. monocytogenes. Thus, the impact of the SAM riboswitches on PrfA expression highlights a link between bacterial virulence and nutrient availability. Together, our results uncover an unexpected role for riboswitches and a distinct class of regulatory noncoding RNAs in bacteria.


Assuntos
Proteínas de Bactérias/genética , Listeria monocytogenes/genética , Fatores de Terminação de Peptídeos/genética , Sequências Reguladoras de Ácido Ribonucleico , Regiões 5' não Traduzidas , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Dados de Sequência Molecular , Temperatura , Virulência
2.
Nucleic Acids Res ; 50(19): 11285-11300, 2022 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-36300626

RESUMO

HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than in HflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes.


Assuntos
Listeria monocytogenes , Listeria monocytogenes/genética , Proteínas de Ligação ao GTP/genética , Resistência Microbiana a Medicamentos , Ribossomos/genética , Ribossomos/metabolismo , Lincosamidas/farmacologia , Antibacterianos/farmacologia , Antibacterianos/metabolismo
3.
Infect Immun ; 84(1): 67-76, 2016 01.
Artigo em Inglês | MEDLINE | ID: mdl-26483402

RESUMO

RNA helicases have been shown to be important for the function of RNA molecules at several levels, although their putative involvement in microbial pathogenesis has remained elusive. We have previously shown that Listeria monocytogenes DExD-box RNA helicases are important for bacterial growth, motility, ribosomal maturation, and rRNA processing. We assessed the importance of the RNA helicase Lmo0866 (here named CshA) for expression of virulence traits. We observed a reduction in hemolytic activity in a strain lacking CshA compared to the wild type. This phenomenon was less evident in strains lacking other RNA helicases. The reduced hemolysis was accompanied by lower expression of major listerial virulence factors in the ΔcshA strain, mainly listeriolysin O, but also to some degree the actin polymerizing factor ActA. Reduced expression of these virulence factors in the strain lacking CshA did not, however, correlate with a decreased level of the virulence regulator PrfA. When combining the ΔcshA knockout with a mutation creating a constitutively active PrfA protein (PrfA*), the effect of the ΔcshA knockout on LLO expression was negated. These data suggest a role for the RNA helicase CshA in posttranslational activation of PrfA. Surprisingly, although the expression of several virulence factors was reduced, the ΔcshA strain did not demonstrate any reduced ability to infect nonphagocytic cells compared to the wild-type strain.


Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Hemólise/genética , Listeria monocytogenes/patogenicidade , RNA Helicases/genética , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Células CACO-2 , Linhagem Celular , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/biossíntese , Proteínas Hemolisinas/genética , Humanos , Listeria monocytogenes/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/genética , Fatores de Virulência/biossíntese
4.
Nature ; 459(7249): 950-6, 2009 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-19448609

RESUMO

The bacterium Listeria monocytogenes is ubiquitous in the environment and can lead to severe food-borne infections. It has recently emerged as a multifaceted model in pathogenesis. However, how this bacterium switches from a saprophyte to a pathogen is largely unknown. Here, using tiling arrays and RNAs from wild-type and mutant bacteria grown in vitro, ex vivo and in vivo, we have analysed the transcription of its entire genome. We provide the complete Listeria operon map and have uncovered far more diverse types of RNAs than expected: in addition to 50 small RNAs (<500 nucleotides), at least two of which are involved in virulence in mice, we have identified antisense RNAs covering several open-reading frames and long overlapping 5' and 3' untranslated regions. We discovered that riboswitches can act as terminators for upstream genes. When Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs with a SigB-mediated activation of virulence genes. In contrast, in the blood, PrfA controls transcription of virulence genes. Remarkably, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same expression patterns as the virulence genes. Together, our data unravel successive and coordinated global transcriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.


Assuntos
Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , RNA Bacteriano/genética , Transcrição Gênica/genética , Animais , Genes Bacterianos/genética , Genoma Bacteriano/genética , Intestinos/microbiologia , Camundongos , Fases de Leitura Aberta/genética , Óperon/genética , RNA Bacteriano/análise , Sequências Reguladoras de Ácido Ribonucleico/genética , Regiões não Traduzidas/genética , Virulência/genética
5.
RNA Biol ; 11(11): 1457-66, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25590644

RESUMO

RNA-helicases are proteins required for the unwinding of occluding secondary RNA structures, especially at low temperatures. In this work, we have deleted all 4 DExD-box RNA helicases in various combinations in the Gram-positive pathogen Listeria monocytogenes. Our results show that 3 out of 4 RNA-helicases were important for growth at low temperatures, whereas the effect was less prominent at 37°C. Over-expression of one RNA-helicase, Lmo1450, was able to overcome the reduced growth of the quadruple mutant strain at temperatures above 26°C, but not at lower temperatures. The maturation of ribosomes was affected in different degrees in the various strains at 20°C, whereas the effect was marginal at 37°C. This was accompanied by an increased level of immature 23S rRNA precursors in some of the RNA-helicase mutants at low temperatures. Although the expression of the PrfA regulated virulence factors ActA and LLO decreased in the quadruple mutant strain, this strain showed a slightly increased infection ability. Interestingly, even though the level of the virulence factor LLO was decreased in the quadruple mutant strain as compared with the wild-type strain, the hly-transcript (encoding LLO) was increased. Hence, our results could suggest a role for the RNA-helicases during translation. In this work, we show that DExD-box RNA-helicases are involved in bacterial virulence gene-expression and infection of eukaryotic cells.


Assuntos
Proteínas de Bactérias/genética , Listeria monocytogenes/genética , RNA Helicases/genética , RNA Ribossômico/genética , Ribossomos/genética , Fatores de Virulência/genética , Proteínas de Bactérias/metabolismo , Células CACO-2 , Regulação Bacteriana da Expressão Gênica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Viabilidade Microbiana/genética , Mutação , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , RNA Helicases/metabolismo , RNA Ribossômico/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Ribossomos/metabolismo , Temperatura , Virulência/genética
6.
Nucleic Acids Res ; 40(4): 1818-27, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22053088

RESUMO

Expression of virulence factors in the human bacterial pathogen Listeria monocytogenes is almost exclusively regulated by the transcriptional activator PrfA. The translation of prfA is controlled by a thermosensor located in the 5'-untranslated RNA (UTR), and is high at 37°C and low at temperatures <30°C. In order to develop a thermoregulated translational expression system, the 5'-UTR and different lengths of the prfA-coding sequences were placed in front of lacZ. When expressed in Escherichia coli, the ß-galactosidase expression was directly correlated to the length of the prfA-coding mRNA lying in front of lacZ. A similar effect was detected with gfp as a reporter gene in both L. monocytogenes and E. coli, emphasizing the requirement of the prfA-coding RNA for maximal expression. In vitro transcription/translation and mutational analysis suggests a role for the first 20 codons of the native prfA-mRNA for maximal expression. By toe-print and RNA-probing analysis, a flexible hairpin-loop located immediately downstream of the start-codon was shown to be important for ribosomal binding. The present work determines the importance of an unstructured part of the 5'-coding region of the prfA-mRNA for efficient translation.


Assuntos
Proteínas de Bactérias/genética , Códon , Fatores de Terminação de Peptídeos/genética , Biossíntese de Proteínas , Proteínas de Bactérias/biossíntese , Genes Reporter , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Mutação , Conformação de Ácido Nucleico , Fatores de Terminação de Peptídeos/biossíntese , Estabilidade Proteica , Estabilidade de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ribossomos/metabolismo
7.
mBio ; 14(3): e0044923, 2023 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-37120759

RESUMO

Listeria monocytogenes is a facultative Gram-positive bacterium that causes listeriosis, a severe foodborne disease. We previously discovered that ring-fused 2-pyridone compounds can decrease virulence factor expression in Listeria by binding and inactivating the PrfA virulence activator. In this study, we tested PS900, a highly substituted 2-pyridone that was recently discovered to be bactericidal to other Gram-positive pathogenic bacteria, such as Staphylococcus aureus and Enterococcus faecalis. We show that PS900 can interact with PrfA and reduce the expression of virulence factors. Unlike previous ring-fused 2-pyridones shown to inactivate PrfA, PS900 had an additional antibacterial activity and was found to potentiate sensitivity toward cholic acid. Two PS900-tolerant mutants able to grow in the presence of PS900 carried mutations in the brtA gene, encoding the BrtA repressor. In wild-type (WT) bacteria, cholic acid binds and inactivates BrtA, thereby alleviating the expression of the multidrug transporter MdrT. Interestingly, we found that PS900 also binds to BrtA and that this interaction causes BrtA to dissociate from its binding site in front of the mdrT gene. In addition, we observed that PS900 potentiated the effect of different osmolytes. We suggest that the increased potency of cholic acid and osmolytes to kill bacteria in the presence of PS900 is due to the ability of the latter to inhibit general efflux, through a yet-unknown mechanism. Our data indicate that thiazolino 2-pyridones constitute an attractive scaffold when designing new types of antibacterial agents. IMPORTANCE Bacteria resistant to one or several antibiotics are a very large problem, threatening not only treatment of infections but also surgery and cancer treatments. Thus, new types of antibacterial drugs are desperately needed. In this work, we show that a new generation of substituted ring-fused 2-pyridones not only inhibit Listeria monocytogenes virulence gene expression, presumably by inactivating the PrfA virulence regulator, but also potentiate the bactericidal effects of cholic acid and different osmolytes. We identified a multidrug repressor as a second target of 2-pyridones. The repressor-2-pyridone interaction displaces the repressor from DNA, thus increasing the expression of a multidrug transporter. In addition, our data suggest that the new class of ring-fused 2-pyridones are efficient efflux inhibitors, possibly explaining why the simultaneous addition of 2-pyridones together with cholic acid or osmolytes is detrimental for the bacterium. This work proves conclusively that 2-pyridones constitute a promising scaffold to build on for future antibacterial drug design.


Assuntos
Listeria monocytogenes , Piridonas/farmacologia , Piridonas/metabolismo , Fatores de Virulência/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ácido Cólico/metabolismo , Ácido Cólico/farmacologia , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Regulação Bacteriana da Expressão Gênica
8.
J Bacteriol ; 194(16): 4377-85, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22707705

RESUMO

Listeria monocytogenes, a Gram-positive food-borne human pathogen, is able to grow at temperatures close to 0°C and is thus of great concern for the food industry. In this work, we investigated the physiological role of one DExD-box RNA helicase in Listeria monocytogenes. The RNA helicase Lmo1722 was required for optimal growth at low temperatures, whereas it was dispensable at 37°C. A Δlmo1722 strain was less motile due to downregulation of the major subunit of the flagellum, FlaA, caused by decreased flaA expression. By ribosomal fractionation experiments, it was observed that Lmo1722 was mainly associated with the 50S subunit of the ribosome. Absence of Lmo1722 decreased the fraction of 50S ribosomal subunits and mature 70S ribosomes and affected the processing of the 23S precursor rRNA. The ribosomal profile could be restored to wild-type levels in a Δlmo1722 strain expressing Lmo1722. Interestingly, the C-terminal part of Lmo1722 was redundant for low-temperature growth, motility, 23S rRNA processing, and appropriate ribosomal maturation. However, Lmo1722 lacking the C terminus showed a reduced affinity for the 50S and 70S fractions, suggesting that the C terminus is important for proper guidance of Lmo1722 to the 50S subunit. Taken together, our results show that the Listeria RNA helicase Lmo1722 is essential for growth at low temperatures, motility, and rRNA processing and is important for ribosomal maturation, being associated mainly with the 50S subunit of the ribosome.


Assuntos
Listeria monocytogenes/enzimologia , Listeria monocytogenes/crescimento & desenvolvimento , Mapeamento de Interação de Proteínas , RNA Helicases/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico 23S/metabolismo , Deleção de Genes , Teste de Complementação Genética , Listeria monocytogenes/metabolismo , Locomoção , Ligação Proteica , RNA Helicases/genética , Temperatura
9.
Nat Commun ; 12(1): 3577, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117249

RESUMO

Target protection proteins confer resistance to the host organism by directly binding to the antibiotic target. One class of such proteins are the antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F-subtype (ARE-ABCFs), which are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition from antibiotics that target the large ribosomal subunit. Here, we present single-particle cryo-EM structures of ARE-ABCF-ribosome complexes from three Gram-positive pathogens: Enterococcus faecalis LsaA, Staphylococcus haemolyticus VgaALC and Listeria monocytogenes VgaL. Supported by extensive mutagenesis analysis, these structures enable a general model for antibiotic resistance mediated by these ARE-ABCFs to be proposed. In this model, ABCF binding to the antibiotic-stalled ribosome mediates antibiotic release via mechanistically diverse long-range conformational relays that converge on a few conserved ribosomal RNA nucleotides located at the peptidyltransferase center. These insights are important for the future development of antibiotics that overcome such target protection resistance mechanisms.


Assuntos
Antibacterianos/farmacologia , Diterpenos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Lincosamidas/farmacologia , Compostos Policíclicos/farmacologia , Estreptograminas/farmacologia , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Farmacorresistência Bacteriana/genética , Bactérias Gram-Positivas/genética , Modelos Moleculares , Peptidil Transferases/metabolismo , Conformação Proteica , RNA Mensageiro , Ribossomos/metabolismo , Pleuromutilinas
10.
STAR Protoc ; 1(2): 100046, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-33111092

RESUMO

The structure of 5' untranslated regions (5' UTRs) of bacterial mRNAs often determines the fate of the transcripts. Using a dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) approach, we developed a protocol to generate sequence libraries to determine the base-pairing status of adenines and cytosines in the 5' UTRs of bacterial mRNAs. Our method increases the sequencing depth of the 5' UTRs and allows detection of changes in their structures by sequencing libraries of moderate sizes. For complete details on the use and execution of this protocol, please refer to Ignatov et al. (2020).


Assuntos
Regiões 5' não Traduzidas/genética , Biblioteca Gênica , RNA Bacteriano/genética , RNA-Seq/métodos
11.
Cell Rep ; 30(12): 4027-4040.e7, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32209466

RESUMO

Bacterial pathogens often employ RNA regulatory elements located in the 5' untranslated regions (UTRs) to control gene expression. Using a comparative structural analysis, we examine the structure of 5' UTRs at a global scale in the pathogenic bacterium Listeria monocytogenes under different conditions. In addition to discovering an RNA thermoswitch and detecting simultaneous interaction of ribosomes and small RNAs with mRNA, we identify structural changes in the 5' UTR of an mRNA encoding the post-translocation chaperone PrsA2 during infection conditions. We demonstrate that the 5' UTR of the prsA2 mRNA base pairs with the 3' UTR of the full-length hly mRNA encoding listeriolysin O, thus preventing RNase J1-mediated degradation of the prsA2 transcript. Mutants lacking the hly-prsA2 interaction exhibit reduced virulence properties. This work highlights an additional level of RNA regulation, where the mRNA encoding a chaperone is stabilized by the mRNA encoding its substrate.


Assuntos
Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Chaperonas Moleculares/metabolismo , Fatores de Virulência/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Sequência de Bases , Proteínas e Peptídeos de Choque Frio/metabolismo , Biblioteca Gênica , Modelos Biológicos , Peptidilprolil Isomerase/metabolismo , Estabilidade de RNA/genética , RNA Bacteriano/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Ribossomos/metabolismo , Temperatura , Virulência/genética , Fatores de Virulência/metabolismo
12.
Infect Immun ; 77(3): 935-42, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19103773

RESUMO

We identified the mutated gene locus in a pigment-overproducing Vibrio cholerae mutant of strain A1552. The deduced gene product is suggested to be an oxidoreductase based on partial homology to putative homogentisate 1,2-dioxygenase in Pseudomonas aeruginosa and Mesorhizobium loti, and we propose that the gene VC1345 in the V. cholerae genome be denoted hmgA in accordance with the nomenclature for other species. The hmgA::mini-Tn5 mutant showed a nonpigmented phenotype after complementation with a plasmid clone carrying the WT hmgA(+) locus. Microarray transcription analysis revealed that expression of hmgA and the neighboring genes encoding a postulated two-component sensor system was growth phase dependent. Results from quantitative reverse transcription-PCR analysis showed that hmgA operon expression was reduced in the rpoS mutant, but pigment production by the WT V. cholerae or the hmgA mutant was not detectably influenced by the stationary-phase regulator RpoS. The pigmented mutant showed increased UV resistance in comparison with the WT strain. Interestingly, the pigment-producing mutant expressed more toxin-coregulated pilus and cholera toxin than WT V. cholerae. Moreover, the hmgA mutant showed a fivefold increase in the ability to colonize the intestines of infant mice. A possible mechanism by which pigment production might cause induction of the ToxR regulon due to generation of hydrogen peroxide was supported by results from tests showing that externally supplied H(2)O(2) led to higher TcpA levels. Taken together, our findings suggest that melanin pigment formation may play a role in V. cholerae virulence factor expression.


Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Melaninas/metabolismo , Oxirredutases/genética , Vibrio cholerae/genética , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Western Blotting , Elementos de DNA Transponíveis , Melaninas/genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator sigma/genética , Vibrio cholerae/patogenicidade
13.
BMC Microbiol ; 9: 220, 2009 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-19835618

RESUMO

BACKGROUND: Background: Cytolethal distending toxin (CDT) is one of the well-characterized virulence factors of Campylobacter jejuni, but it is unknown how CDT becomes surface-exposed or is released from the bacterium to the surrounding environment. RESULTS: Our data suggest that CDT is secreted to the bacterial culture supernatant via outer membrane vesicles (OMVs) released from the bacteria. All three subunits (the CdtA, CdtB, and CdtC proteins) were detected by immunogold labeling and electron microscopy of OMVs. Subcellular fractionation of the bacteria indicated that, apart from the majority of CDT detected in the cytoplasmic compartment, appreciable amounts (20-50%) of the cellular pool of CDT proteins were present in the periplasmic compartment. In the bacterial culture supernatant, we found that a majority of the extracellular CDT was tightly associated with the OMVs. Isolated OMVs could exert the cell distending effects typical of CDT on a human intestinal cell line, indicating that CDT is present there in a biologically active form. CONCLUSION: Our results strongly suggest that the release of outer membrane vesicles is functioning as a route of C. jejuni to deliver all the subunits of CDT toxin (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.


Assuntos
Toxinas Bacterianas/metabolismo , Campylobacter jejuni/metabolismo , Vesículas Transportadoras/metabolismo , Linhagem Celular Tumoral , Humanos , Microscopia de Força Atômica , Microscopia Imunoeletrônica , Vesículas Transportadoras/microbiologia
14.
FEBS J ; 275(12): 3167-77, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18479458

RESUMO

The Vibrio metalloprotease PrtV was purified from the culture supernatant of a Vibrio cholerae derivative that is deficient in several other secreted peptidases, including the otherwise abundant hemagglutinin/protease HapA. The PrtV is synthesized as a 102 kDa protein, but undergoes several N- and C-terminal processing steps during V. cholerae envelope translocation and prolonged incubation. Purified V. cholerae PrtV protease forms of 81 or 73 kDa were stabilized by calcium ions. Removal of calcium resulted in further rapid autoproteolysis. The two major products of autoproteolysis of the PrtV protease were approximately 37 and 18 kDa and could not be separated under non-denaturing conditions, indicating they are interacting domains. In an assay using cultured cells of the human intestinal cell line HCT8, the PrtV protein showed a cytotoxic effect leading to cell death. Using human blood plasma as a source of potential substrates of mammalian origin for the PrtV protease, we found that the extracellular matrix components fibronectin and fibrinogen were degraded by the enzyme. Additional tests with individual protein substrates revealed that plasminogen was also a possible target for the PrtV protease.


Assuntos
Metaloproteases/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas Sanguíneas/metabolismo , Linhagem Celular Tumoral , Estabilidade Enzimática , Humanos , Metaloproteases/química , Metaloproteases/toxicidade , Peptídeo Hidrolases/química , Peptídeo Hidrolases/toxicidade , Inibidores de Proteases/farmacologia , Especificidade por Substrato
15.
Cell Chem Biol ; 23(3): 404-14, 2016 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-26991105

RESUMO

The transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes cellular uptake by reducing the expression of virulence genes. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA-binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds at two separate sites on the protein: one within a hydrophobic pocket or tunnel, located between the C- and N-terminal domains of PrfA, and the second in the vicinity of the DNA-binding helix-turn-helix motif. At both sites the compound interacts with residues important for PrfA activation and helix-turn-helix formation. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes.


Assuntos
Proteínas de Bactérias/metabolismo , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/patogenicidade , Fatores de Terminação de Peptídeos/metabolismo , Piridonas/farmacologia , Proteínas de Bactérias/genética , Sítios de Ligação/efeitos dos fármacos , Células CACO-2 , Linhagem Celular , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Modelos Moleculares , Fatores de Terminação de Peptídeos/genética , Piridonas/química , Relação Estrutura-Atividade , Virulência/efeitos dos fármacos
16.
PLoS One ; 4(11): e7806, 2009 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-19907657

RESUMO

BACKGROUND: Vibrio cholerae is the causal intestinal pathogen of the diarrheal disease cholera. It secretes the protease PrtV, which protects the bacterium from invertebrate predators but reduces the ability of Vibrio-secreted factor(s) to induce interleukin-8 (IL-8) production by human intestinal epithelial cells. The aim was to identify the secreted component(s) of V. cholerae that induces an epithelial inflammatory response and to define whether it is a substrate for PrtV. METHODOLOGY/PRINCIPAL FINDINGS: Culture supernatants of wild type V. cholerae O1 strain C6706, its derivatives and pure V. cholerae cytolysin (VCC) were analyzed for the capacity to induce changes in cytokine mRNA expression levels, IL-8 and tumor necrosis factor-alpha (TNF-alpha) secretion, permeability and cell viability when added to the apical side of polarized tight monolayer T84 cells used as an in vitro model for human intestinal epithelium. Culture supernatants were also analyzed for hemolytic activity and for the presence of PrtV and VCC by immunoblot analysis. CONCLUSIONS/SIGNIFICANCE: We suggest that VCC is capable of causing an inflammatory response characterized by increased permeability and production of IL-8 and TNF-alpha in tight monolayers. Pure VCC at a concentration of 160 ng/ml caused an inflammatory response that reached the magnitude of that caused by Vibrio-secreted factors, while higher concentrations caused epithelial cell death. The inflammatory response was totally abolished by treatment with PrtV. The findings suggest that low doses of VCC initiate a local immune defense reaction while high doses lead to intestinal epithelial lesions. Furthermore, VCC is indeed a substrate for PrtV and PrtV seems to execute an environment-dependent modulation of the activity of VCC that may be the cause of V. cholerae reactogenicity.


Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Peptídeo Hidrolases/metabolismo , Perforina/fisiologia , Vibrio cholerae/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Hemólise , Humanos , Inflamação , Interleucina-8/metabolismo , Mutação , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
17.
Proc Natl Acad Sci U S A ; 103(24): 9280-5, 2006 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-16754867

RESUMO

Vibrio cholerae is the causal bacterium of the diarrheal disease cholera, and its growth and survival are thought to be curtailed by bacteriovorous predators, e.g., ciliates and flagellates. We explored Caenorhabditis elegans as a test organism after finding that V. cholerae can cause lethal infection of this nematode. By reverse genetics we identified an extracellular protease, the previously uncharacterized PrtV protein, as being necessary for killing. The killing effect is associated with the colonization of bacteria within the Caenorhabditis elegans intestine. We also show that PrtV is essential for V. cholerae in the bacterial survival from grazing by the flagellate Cafeteria roenbergensis and the ciliate Tetrahymena pyriformis. The PrtV protein appears to have an indirect role in the interaction of V. cholerae with mammalian host cells as judged from tests with tight monolayers of human intestinal epithelial cells. Our results demonstrate a key role for PrtV in V. cholerae interaction with grazing predators, and we establish Caenorhabditis elegans as a convenient organism for identification of V. cholerae factors involved in host interactions and environmental persistence.


Assuntos
Proteínas de Bactérias/metabolismo , Caenorhabditis elegans/microbiologia , Caenorhabditis elegans/fisiologia , Peptídeo Hidrolases/metabolismo , Comportamento Predatório , Vibrio cholerae/enzimologia , Vibrio cholerae/patogenicidade , Animais , Proteínas de Bactérias/genética , Biofilmes , Caenorhabditis elegans/citologia , Comunicação Celular , Linhagem Celular Tumoral , Toxina da Cólera/metabolismo , Comportamento Alimentar , Fímbrias Bacterianas/metabolismo , Humanos , Interleucina-8/metabolismo , Intestinos/citologia , Intestinos/microbiologia , Peptídeo Hidrolases/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Taxa de Sobrevida , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vibrio cholerae/genética
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