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1.
mSphere ; 9(6): e0074823, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38771057

RESUMO

The placenta is a critical barrier against viral, bacterial, and eukaryotic pathogens. For most teratogenic pathogens, the precise molecular mechanisms of placental resistance are still being unraveled. Given the importance of understanding these mechanisms and challenges in replicating trophoblast-pathogen interactions using in vitro models, we tested an existing stem-cell-derived model of trophoblast development for its relevance to infection with Toxoplasma gondii. We grew human trophoblast stem cells (TSCT) under conditions leading to either syncytiotrophoblast (TSSYN) or cytotrophoblast (TSCYT) and infected them with T. gondii. We evaluated T. gondii proliferation and invasion, cell ultrastructure, as well as for transcriptome changes after infection. TSSYNs cells showed similar ultrastructure compared to primary cells and villous explants when analyzed by transmission electron microscopy and scanning electron microscopy (SEM), a resistance to T. gondii adhesion could be visualized on the SEM level. Furthermore, TSSYNs were highly refractory to parasite adhesion and replication, while TSCYTs were not. RNA-seq data on mock-treated and infected cells identified differences between cell types as well as how they responded to T. gondii infection. We also evaluated if TSSC-derived SYNs and CYTs had distinct resistance profiles to another vertically transmitted facultative intracellular pathogen, Listeria monocytogenes. We demonstrate that TSSYNs are highly resistant to L. monocytogenes, while TSCYTs are not. Like T. gondii, TSSYN resistance to L. monocytogenes was at the level of bacterial adhesion. Altogether, our data indicate that stem-cell-derived trophoblasts recapitulate resistance profiles of primary cells to T. gondii and highlight the critical importance of the placental surface in cell-autonomous resistance to teratogens.IMPORTANCECongenital toxoplasmosis can cause a devastating consequence to the fetus. To reach the fetus's tissues, Toxoplasma gondii must cross the placenta barrier. However, how this parasite crosses the placenta and the precise molecular mechanisms of placental resistance to this parasite are still unknown. In this study, we aimed to characterize a new cellular model of human trophoblast stem cells to determine their resistance, susceptibility, and response to T. gondii. Syncytiotrophoblast derived from trophoblast stem cells recapitulate the resistance profile similarly to placenta cells. We also showed that these cells are highly resistant to Listeria monocytogenes, at the level of bacterial adhesion. Our results suggest that resisting pathogen adhesion/attachment may be a generalized mechanism of syncytiotrophoblast resistance, and trophoblast stem cells represent a promising model to investigate cell-intrinsic mechanisms of resistance to pathogen adhesion and replication.


Assuntos
Listeria monocytogenes , Toxoplasma , Trofoblastos , Trofoblastos/microbiologia , Trofoblastos/parasitologia , Toxoplasma/genética , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/fisiologia , Toxoplasma/ultraestrutura , Humanos , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/genética , Listeria monocytogenes/fisiologia , Feminino , Gravidez , Adesão Celular , Placenta/microbiologia , Placenta/parasitologia , Toxoplasmose/parasitologia , Células-Tronco
2.
mBio ; 15(7): e0074324, 2024 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-38809022

RESUMO

Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted protein activation and regulation post-membrane translocation remain largely unknown. Using proteomics, we identified several proteins that are dependent on the secreted chaperone PrsA2. We followed with phenotypic, biochemical, and biophysical assays and computational analyses to examine the regulation of a detected key secreted virulence factor, listeriolysin O (LLO), and its interaction with PrsA2 from the bacterial pathogen Listeria monocytogenes (Lm). Critical to Lm virulence is internalization by host cells and the subsequent action of the cholesterol-dependent pore-forming toxin, LLO, which enables bacterial escape from the host cell phagosome. Since Lm is a Gram-positive organism, the space between the cell membrane and wall is solvent exposed. Therefore, we hypothesized that the drop from neutral to acidic pH as the pathogen is internalized into a phagosome is critical to regulating the interaction of PrsA2 with LLO. Here, we demonstrate that PrsA2 directly interacts with LLO in a pH-dependent manner. We show that PrsA2 protects and sequesters LLO under neutral pH conditions where LLO can be observed to aggregate. In addition, we identify molecular features of PrsA2 that are required for interaction and ultimately the folding and activity of LLO. Moreover, protein-complex modeling suggests that PrsA2 interacts with LLO via its cholesterol-binding domain. These findings highlight a mechanism by which a Gram-positive secretion chaperone regulates the secretion, stability, and folding of a pore-forming toxin under conditions relevant to host cell infection. IMPORTANCE: Lm is a ubiquitous food-borne pathogen that can cause severe disease to vulnerable populations. During infection, Lm relies on a wide repertoire of secreted virulence factors including the LLO that enables the bacterium to invade the host and spread from cell to cell. After membrane translocation, secreted factors must become active in the challenging bacterial cell membrane-wall interface. However, the mechanisms required for secreted protein folding and function are largely unknown. Lm encodes a chaperone, PrsA2, that is critical for the activity of secreted factors. Here, we show that PrsA2 directly associates and protects the major Lm virulence factor, LLO, under conditions corresponding to the host cytosol, where LLO undergoes irreversible denaturation. Additionally, we identify molecular features of PrsA2 that enable its interaction with LLO. Together, our results suggest that Lm and perhaps other Gram-positive bacteria utilize secreted chaperones to regulate the activity of pore-forming toxins during infection.


Assuntos
Toxinas Bacterianas , Proteínas de Choque Térmico , Proteínas Hemolisinas , Listeria monocytogenes , Listeriose , Dobramento de Proteína , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/química , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Listeria monocytogenes/química , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/química , Listeriose/microbiologia , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/química , Peptidilprolil Isomerase/metabolismo , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/química , Concentração de Íons de Hidrogênio , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Estabilidade Proteica , Humanos
3.
Infect Immun ; 92(2): e0049023, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38226817

RESUMO

Streptococcus pneumoniae is a Gram-positive bacterium and a significant health threat with the populations most at risk being children, the elderly, and the immuno-compromised. To colonize and transition into an invasive infectious organism, S. pneumoniae secretes virulence factors that are translocated across the bacterial membrane and destined for surface exposure, attachment to the cell wall, or secretion into the host. The surface exposed protein chaperones PrsA, SlrA, and HtrA facilitate S. pneumoniae protein secretion; however, the distinct roles contributed by each of these secretion chaperones have not been well defined. Tandem Mass-Tagged Mass Spectrometry and virulence, adhesion, competence, and cell wall integrity assays were used to interrogate the individual and collective contributions of PrsA, SlrA, and HtrA to multiple aspects of S. pneumoniae physiology and virulence. PrsA, SlrA, and HtrA were found to play critical roles in S. pneumoniae host cell infection and competence, and the absence of each of these secretion chaperones significantly altered the S. pneumoniae secretome in distinct ways. PrsA and SlrA were additionally found to contribute to cell wall assembly and resistance to cell wall-active antimicrobials and were important for enabling S. pneumoniae host cell adhesion during colonization and invasive infection. These findings serve to further illustrate the pivotal contributions of PrsA, SlrA, and HtrA to S. pneumoniae protein secretion and virulence.


Assuntos
Chaperonas Moleculares , Streptococcus pneumoniae , Criança , Humanos , Idoso , Chaperonas Moleculares/metabolismo , Fatores de Virulência/metabolismo , Virulência , Proteínas de Membrana/metabolismo , Resistência Microbiana a Medicamentos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
4.
bioRxiv ; 2023 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-37986837

RESUMO

The placenta is a critical barrier against viral, bacterial, and eukaryotic pathogens. For most teratogenic pathogens, the precise molecular mechanisms of placental resistance are still being unraveled. Given the importance to understand these mechanisms and challenges in replicating trophoblast- pathogen interactions using in vitro models, we tested an existing stem-cell derived model of trophoblast development for its relevance to infection with Toxoplasma gondii . We grew human trophoblast stem cells (TS CT ) under conditions leading to either syncytiotrophoblast (TS SYN ) or cytotrophoblast (TS CYT ) and infected them with T. gondii . We evaluated T. gondii proliferation and invasion, cell ultrastructure, as well as for transcriptome changes after infection. TS SYNs cells showed similar ultrastructure compared to primary cells and villous explants when analyzed by TEM and SEM, a resistance to T. gondii adhesion could be visualized on the SEM level. Furthermore, TS SYNs were highly refractory to parasite adhesion and replication, while TS CYT were not. RNA-seq data on mock-treated and infected cells identified differences between cell types as well as how they responded to T. gondii infection. We also evaluated if TS SC -derived SYNs and CYTs had distinct resistance profiles to another vertically transmitted facultative intracellular pathogen, Listeria monocytogenes . We demonstrate that TS SYNs are highly resistant to L. monocytogenes , while TS CYTs are not. Like T. gondii , TS SYN resistance to L. monocytogenes was at the level of bacterial adhesion. Altogether, our data indicate that stem-cell derived trophoblasts recapitulate resistance profiles of primary cells to T. gondii and highlight the critical importance of the placental surface in cell-autonomous resistance to teratogens.

5.
Mol Microbiol ; 118(3): 278-293, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35943959

RESUMO

Listeria monocytogenes (Lm) is a widespread environmental Gram-positive bacterium that can transition into a pathogen following ingestion by a susceptible host. To cross host barriers and establish infection, Lm is dependent upon the regulated secretion and activity of many proteins including PrsA2, a peptidyl-prolyl cis-trans isomerase with foldase activity. PrsA2 contributes to the stability and activity of a number of secreted virulence factors that are required for Lm invasion, replication, and cell-to-cell spread within the infected host. In contrast, a second related secretion chaperone, PrsA1, has thus far no identified contributions to Lm pathogenesis. Here we describe the characterization of a two-component signal transduction system PieRS that regulates the expression of a regulon that includes the secretion chaperones PrsA1 and PrsA2. PieRS regulated gene products are required for bacterial resistance to ethanol exposure and are important for bacterial survival during transit through the gastrointestinal tract. PrsA1 was also found to make a unique contribution to Lm survival in the GI tract, revealing for the first time a non-overlapping requirement for both secretion chaperones PrsA1 and PrsA2 during the process of intra-gastric infection.


Assuntos
Listeria monocytogenes , Listeriose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Translocação Bacteriana , Humanos , Intestinos , Listeria monocytogenes/genética , Listeriose/microbiologia , Chaperonas Moleculares/metabolismo , Fatores de Virulência/metabolismo
6.
Biochemistry ; 59(10): 1104-1112, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32078293

RESUMO

Neisseria gonorrhoeae possesses a programmed recombination system that allows the bacteria to alter the major subunit of the type IV pilus, pilin or PilE. An alternate DNA structure known as a guanine quadruplex (G4) is required for pilin antigenic variation (pilin Av). The G-C base pairs within the G4 motif are required for pilin Av, but simple mutation of the loop bases does not affect pilin Av. We show that more substantial changes to the loops, in both size and nucleotide composition, with the core guanines unchanged, decrease or abrogate pilin Av. We investigated why these loop changes might influence the efficiency of pilin Av. RecA is a recombinase required for pilin Av that can bind the pilE G4 in vitro. RecA binds different G4 structures with altered loops with varied affinities. However, changes in RecA binding affinities to the loop mutants do not absolutely correlate with the pilin Av phenotypes. Interestingly, the yeast RecA ortholog, Rad51, also binds the pilE G4 structure with a higher affinity than it binds single-stranded DNA, suggesting that RecA G4 binding is conserved in eukaryotic orthologs. The thermal stability the pilE G4 structure and its loop mutants showed that the parental G4 structure had the highest melting temperature, and the melting temperature of the loop mutants correlated with pilin Av phenotype. These results suggest that the folding kinetics and stability of G4 structures are important for the efficiency of pilin Av.


Assuntos
Proteínas de Fímbrias/metabolismo , Neisseria gonorrhoeae/genética , Variação Antigênica/genética , Pareamento de Bases/genética , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Quadruplex G , Guanina/metabolismo , Mutação/genética , Neisseria gonorrhoeae/imunologia , Ligação Proteica/genética , Recombinação Genética/genética
7.
Mol Microbiol ; 112(4): 1219-1234, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31338863

RESUMO

Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the Type IV pilus. Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis-acting sRNA (G4-sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av). We show that the reduced sRNA transcription lowers pilin Av frequencies. Extended transcriptional elongation is not required for Av, since limiting the transcript to 32 nt allows for normal Av frequencies. Using chromatin immunoprecipitation (ChIP) assays, we show that cellular G4s are less abundant when sRNA transcription is lower. In addition, using ChIP, we demonstrate that the G4-sRNA forms a stable RNA:DNA hybrid (R-loop) with its template strand. However, modulating R-loop levels by controlling RNase HI expression does not alter G4 abundance quantified through ChIP. Since pilin Av frequencies were not altered when modulating R-loop levels by controlling RNase HI expression, we conclude that transcription of the sRNA is necessary, but stable R-loops are not required to promote pilin Av.


Assuntos
Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Neisseria gonorrhoeae/genética , Variação Antigênica/genética , Fímbrias Bacterianas/metabolismo , Conversão Gênica/genética , Gonorreia/genética , Neisseria gonorrhoeae/metabolismo , Estruturas R-Loop/genética , RNA/metabolismo , Estabilidade de RNA/genética , Recombinação Genética/genética
8.
Nature ; 562(7725): 43-44, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30275549
9.
Structure ; 25(2): 295-304, 2017 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-28089449

RESUMO

Active in the aqueous cellular environment where a massive excess of water is perpetually present, enzymes that catalyze the transfer of an electrophile to a non-water nucleophile (transferases) require specific strategies to inhibit mechanistically related hydrolysis reactions. To identify principles that confer transferase versus hydrolase reaction specificity, we exploited two enzymes that use highly similar catalytic apparatuses to catalyze the transglycosylation (a transferase reaction) or hydrolysis of α-1,3-glucan linkages in the cyclic tetrasaccharide cycloalternan (CA). We show that substrate binding to non-catalytic domains and a conformationally stable active site promote CA transglycosylation, whereas a distinct pattern of active site conformational change is associated with CA hydrolysis. These findings defy the classic view of induced-fit conformational change and illustrate a mechanism by which a stable hydrophobic binding site can favor transferase activity and disfavor hydrolysis. Application of these principles could facilitate the rational reengineering of transferases with desired catalytic properties.


Assuntos
Actinomycetales/enzimologia , Glucosidases/química , Glicosídeo Hidrolases/química , Listeria monocytogenes/enzimologia , Oligossacarídeos/química , Água/química , Actinomycetales/genética , Motivos de Aminoácidos , Sítios de Ligação , Biocatálise , Configuração de Carboidratos , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glucosidases/genética , Glucosidases/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Glicosilação , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Cinética , Listeria monocytogenes/genética , Modelos Moleculares , Oligossacarídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Água/metabolismo
10.
Nat Microbiol ; 2: 16202, 2016 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-27819654

RESUMO

Here we employ a 'systems structural biology' approach to functionally characterize an unconventional α-glucan metabolic pathway from the food-borne pathogen Listeria monocytogenes (Lm). Crystal structure determination coupled with basic biochemical and biophysical assays allowed for the identification of anabolic, transport, catabolic and regulatory portions of the cycloalternan pathway. These findings provide numerous insights into cycloalternan pathway function and reveal the mechanism of repressor, open reading frame, kinase (ROK) transcription regulators. Moreover, by developing a structural overview we were able to anticipate the cycloalternan pathway's role in the metabolism of partially hydrolysed starch derivatives and demonstrate its involvement in Lm pathogenesis. These findings suggest that the cycloalternan pathway plays a role in interspecies resource competition-potentially within the host gastrointestinal tract-and establish the methodological framework for characterizing bacterial systems of unknown function.


Assuntos
Enzimas/química , Enzimas/metabolismo , Listeria monocytogenes/enzimologia , Listeria monocytogenes/metabolismo , Redes e Vias Metabólicas , Oligossacarídeos/metabolismo , Fenômenos Biofísicos , Cristalografia por Raios X , Conformação Proteica
11.
Mol Microbiol ; 101(1): 42-61, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27007641

RESUMO

Listeria monocytogenes is a Gram-positive environmental bacterium that lives within soil but transitions into a pathogen upon contact with a mammalian host. The transition of L. monocytogenes from soil dweller to cytosolic pathogen is dependent upon secreted virulence factors that mediate cell invasion and intracellular growth. PrsA1 and PrsA2 are secreted bacterial lipoprotein chaperones that contribute to the folding of proteins translocated across the bacterial membrane; PrsA2 is required for L. monocytogenes virulence, whereas the function of PrsA1 remains to be determined. We have solved an X-ray crystal structure of PrsA1 and have used this model to guide comparison structure-based mutagenesis studies with PrsA2. Targeted mutagenesis of PrsA2 demonstrates that oligomerization of PrsA2 as well as molecular features of the foldase domain are required for protein secretion and virulence, whereas a functional role was uncovered for PrsA1 in bacterial resistance to alcohol. Interestingly, PrsA2 membrane localization is not required for all PrsA2-dependent activities, suggesting that the lipoprotein retains function when released from the bacterial cell. PrsA chaperones are thus multifaceted proteins with distinct domains adapted to accommodate the functional needs of a diverse array of secreted substrates.


Assuntos
Listeria monocytogenes/metabolismo , Peptidilprolil Isomerase/metabolismo , Bacillus subtilis/enzimologia , Cristalografia por Raios X , Citosol/enzimologia , Citosol/metabolismo , Isoenzimas , Lipoproteínas/metabolismo , Listeria monocytogenes/enzimologia , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/química , Relação Estrutura-Atividade , Virulência , Fatores de Virulência/metabolismo
12.
Infect Immun ; 83(10): 4028-41, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26216425

RESUMO

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen that relies on the regulated secretion and activity of a variety of proteins that sustain life within diverse environments. PrsA2 has recently been identified as a secreted peptidyl-prolyl cis/trans isomerase and chaperone that is dispensable for bacterial growth in broth culture but essential for L. monocytogenes virulence. Following host infection, PrsA2 contributes to the proper folding and activity of secreted proteins that are required for bacterial replication within the host cytosol and for bacterial spread to adjacent cells. PrsA2 is one member of a family of Gram-positive secretion chaperones that appear to play important roles in bacterial physiology; however, it is not known how these proteins recognize their substrate proteins or the degree to which their function is conserved across diverse Gram-positive species. We therefore examined PrsA proteins encoded by a variety of Gram-positive bacteria for functional complementation of L. monocytogenes mutants lacking prsA2. PrsA homologues encoded by Bacillus subtilis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus mutans, Staphylococcus aureus, and Lactococcus lactis were examined for functional complementation of a variety of L. monocytogenes PrsA2-associated phenotypes central to L. monocytogenes pathogenesis and bacterial cell physiology. Our results indicate that while selected aspects of PrsA2 function are broadly conserved among diverse Gram-positive bacteria, PrsA2 exhibits unique specificity for L. monocytogenes target proteins required for pathogenesis. The L. monocytogenes PrsA2 chaperone thus appears evolutionarily optimized for virulence factor secretion within the host cell cytosol while still maintaining aspects of activity relevant to more general features of Gram-positive protein translocation.


Assuntos
Listeria monocytogenes/enzimologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Sequência de Aminoácidos , Sistemas de Secreção Bacterianos , Sequência Conservada , Evolução Molecular , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/enzimologia , Bactérias Gram-Positivas/genética , Listeria monocytogenes/química , Listeria monocytogenes/genética , Listeriose , Chaperonas Moleculares/química , Dados de Sequência Molecular , Peptidilprolil Isomerase/química , Filogenia , Alinhamento de Sequência , Especificidade da Espécie , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
13.
Artigo em Inglês | MEDLINE | ID: mdl-24575392

RESUMO

In Gram-positive bacteria, the secretion of proteins requires translocation of polypeptides across the bacterial membrane into the highly charged environment of the membrane-cell wall interface. Here, proteins must be folded and often further delivered across the matrix of the cell wall. While many aspects of protein secretion have been well studied in Gram-negative bacteria which possess both an inner and outer membrane, generally less attention has been given to the mechanics of protein secretion across the single cell membrane of Gram-positive bacteria. In this review, we focus on the role of a post-translocation secretion chaperone in Listeria monocytogenes known as PrsA2, and compare what is known regarding PrsA2 with PrsA homologs in other Gram-positive bacteria. PrsA2 is a member of a family of membrane-associated lipoproteins that contribute to the folding and stability of secreted proteins as they cross the bacterial membrane. PrsA2 contributes to the integrity of the L. monocytogenes cell wall as well as swimming motility and bacterial resistance to osmotic stress; however its most critical role may be its requirement for L. monocytogenes virulence and viability within host cells. A better understanding of the role of PrsA2 and PrsA-like homologs will provide insight into the dynamics of protein folding and stability in Gram-positive bacteria and may result in new strategies for optimizing protein secretion as well as inhibiting the production of virulence factors.


Assuntos
Listeria monocytogenes/enzimologia , Listeria monocytogenes/metabolismo , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/metabolismo , Dobramento de Proteína , Fatores de Virulência/metabolismo , Parede Celular/metabolismo , Humanos , Lipoproteínas/metabolismo , Locomoção , Viabilidade Microbiana , Estresse Fisiológico , Virulência
14.
J Bacteriol ; 195(10): 2255-61, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23475972

RESUMO

The strict human pathogen Neisseria gonorrhoeae utilizes homologous recombination to antigenically vary the pilus, thus evading the host immune response. High-frequency gene conversion reactions between many silent pilin loci and the expressed pilin locus (pilE) allow for numerous pilus variants per strain to be produced from a single strain. For pilin antigenic variation (Av) to occur, a guanine quartet (G4) structure must form upstream of pilE. The RecQ helicase is one of several recombination or repair enzymes required for efficient levels of pilin Av, and RecQ family members have been shown to bind to and unwind G4 structures. Additionally, the vast majority of RecQ helicase family members encode one "helicase and RNase D C-terminal" (HRDC) domain, whereas the N. gonorrhoeae RecQ helicase gene encodes three HRDC domains, which are critical for pilin Av. Here, we confirm that deletion of RecQ HRDC domains 2 and 3 causes a decrease in the frequency of pilin Av comparable to that obtained with a functional knockout. We demonstrate that the N. gonorrhoeae RecQ helicase can bind and unwind the pilE G4 structure. Deletion of the RecQ HRDC domains 2 and 3 resulted in a decrease in G4 structure binding and unwinding. These data suggest that the decrease in pilin Av observed in the RecQ HRDC domain 2 and 3 deletion mutant is a result of the enzyme's inability to efficiently bind and unwind the pilE G4 structure.


Assuntos
Variação Antigênica/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Neisseria gonorrhoeae/enzimologia , Neisseria gonorrhoeae/metabolismo , RecQ Helicases/química , RecQ Helicases/metabolismo , Variação Antigênica/genética , Proteínas de Bactérias/genética , Dicroísmo Circular , Proteínas de Fímbrias/genética , Polarização de Fluorescência , Neisseria gonorrhoeae/genética , Ligação Proteica , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , RecQ Helicases/genética
15.
PLoS Pathog ; 9(1): e1003074, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23349628

RESUMO

The strict human pathogen Neisseria gonorrhoeae can utilize homologous recombination to generate antigenic variability in targets of immune surveillance. To evade the host immune response, N. gonorrhoeae promotes high frequency gene conversion events between many silent pilin copies and the expressed pilin locus (pilE), resulting in the production of variant pilin proteins. Previously, we identified a guanine quartet (G4) structure localized near pilE that is required for the homologous recombination reactions leading to pilin antigenic variation (Av). In this work, we demonstrate that inactivating the promoter of a small non-coding RNA (sRNA) that initiates within the G4 forming sequence blocks pilin Av. The sRNA promoter is conserved in all sequenced gonococcal strains, and mutations in the predicted transcript downstream of the G4 forming sequence do not alter pilin Av. A mutation that produces a stronger promoter or substitution of the pilE G4-associated sRNA promoter with a phage promoter (when the phage polymerase was expressed) produced wild-type levels of pilin Av. Altering the direction and orientation of the pilE G4-associated sRNA disrupted pilin Av. In addition, expression of the sRNA at a distal site on the gonococcal chromosome in the context of a promoter mutant did not support pilin Av. We conclude that the DNA containing the G-rich sequence can only form the G4 structure during transcription of this sRNA, thus providing a unique molecular step for the initiation of programmed recombination events.


Assuntos
Variação Antigênica/genética , Antígenos de Bactérias/genética , Proteínas de Fímbrias/imunologia , Neisseria gonorrhoeae/genética , Pequeno RNA não Traduzido/genética , Proteínas de Fímbrias/genética , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune/genética , Neisseria gonorrhoeae/imunologia , Transcrição Gênica
16.
Structure ; 20(12): 2090-102, 2012 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-23085077

RESUMO

Neisseria gonorrhoeae is an obligate human pathogen that can escape immune surveillance through antigenic variation of surface structures such as pili. A G-quadruplex-forming (G4) sequence (5'-G(3)TG(3)TTG(3)TG(3)) located upstream of the N. gonorrhoeae pilin expression locus (pilE) is necessary for initiation of pilin antigenic variation, a recombination-based, high-frequency, diversity-generation system. We have determined NMR-based structures of the all parallel-stranded monomeric and 5' end-stacked dimeric pilE G-quadruplexes in monovalent cation-containing solutions. We demonstrate that the three-layered all parallel-stranded monomeric pilE G-quadruplex containing single-residue double-chain reversal loops, which can be modeled without steric clashes into the 3 nt DNA-binding site of RecA, binds and promotes E. coli RecA-mediated strand exchange in vitro. We discuss how interactions between RecA and monomeric pilE G-quadruplex could facilitate the specialized recombination reactions leading to pilin diversification.


Assuntos
Antígenos de Bactérias/genética , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Proteínas de Fímbrias/genética , Quadruplex G , Neisseria gonorrhoeae/genética , Recombinases Rec A/química , Antígenos de Bactérias/química , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Medição da Troca de Deutério , Proteínas de Fímbrias/química , Sequência Rica em GC , Evasão da Resposta Imune , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica
17.
Mol Microbiol ; 81(5): 1136-43, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21812841

RESUMO

Some pathogenic microbes utilize homologous recombination to generate antigenic variability in targets of immune surveillance. These specialized systems rely on the cellular recombination machinery to catalyse dedicated, high-frequency reactions that provide extensive diversity in the genes encoding surface antigens. A description of the specific mechanisms that allow unusually high rates of recombination without deleterious effects on the genome in the well-characterized pilin antigenic variation systems of Neisseria gonorrhoeae and Neisseria meningitidis is presented. We will also draw parallels to selected bacterial and eukaryotic antigenic variation systems, and suggest the most pressing unanswered questions related to understanding these important processes.


Assuntos
Variação Antigênica , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/imunologia , Recombinação Homóloga/genética , Neisseria gonorrhoeae/imunologia , Neisseria meningitidis/imunologia , Animais , Humanos , Neisseria gonorrhoeae/genética , Neisseria meningitidis/genética
18.
Mol Microbiol ; 79(3): 729-42, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21255115

RESUMO

Neisseria gonorrhoeae (Gc) is an obligate human pathogen and the causative agent of the sexually transmitted infection, gonorrhoea. Despite the fact that the gonococcus is not normally exposed to UV irradiation or visible light, the bacterium expresses a phrB orthologue, which in other organisms encodes a DNA photolyase that repairs UV-induced pyrimidine dimers with energy provided by visible light. We show that a Gc phrB mutant is not more sensitive to UV irradiation, independent of visible light exposure, and that the Gc phrB cannot complement an Escherichia coli phrB mutant strain. The Gc phrB mutant had a reduced colony size that was not a result of a growth defect and the mutant cells exhibited an altered morphology. Although the phrB mutant exhibited increased sensitivity to oxidative killing; it showed increased survival on media containing nalidixic acid or rifampicin, but did not have an increased mutation rate to these antibiotics or spectinomycin and kasugamycin. The Gc phrB mutant showed increased negative DNA supercoiling, but while the protein bound double-stranded DNA, it did not express topoisomerase activity. We conclude that the Gc PhrB has a previously unrecognized role in maintaining DNA supercoiling that is important for normal cell physiology.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Super-Helicoidal/metabolismo , Desoxirribodipirimidina Fotoliase/metabolismo , Luz , Neisseria gonorrhoeae/enzimologia , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Contagem de Colônia Microbiana , DNA Bacteriano/metabolismo , Desoxirribodipirimidina Fotoliase/química , Desoxirribodipirimidina Fotoliase/genética , Ativação Enzimática/efeitos da radiação , Escherichia coli/efeitos da radiação , Evolução Molecular , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Humanos , Viabilidade Microbiana/efeitos da radiação , Dados de Sequência Molecular , Ácido Nalidíxico/farmacologia , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/crescimento & desenvolvimento , Neisseria gonorrhoeae/efeitos da radiação , Conformação de Ácido Nucleico , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Fenótipo , Filogenia , Espectinomicina/farmacologia
19.
Science ; 325(5941): 764-7, 2009 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-19661435

RESUMO

Pathogens can use DNA recombination to promote antigenic variation (Av) of surface structures to avoid immune detection. We identified a cis-acting DNA sequence near the antigenically variable pilin locus of the human pathogen, Neisseria gonorrhoeae. This 16-base pair guanine (G)-rich sequence was required for pilin Av and formed a guanine quartet (G4) structure in vitro. Individual mutations that disrupted the structure also blocked pilin Av and prevented nicks required for recombination from occurring within the G4 region. A compound that binds and stabilizes G4 structures also inhibited pilin Av and prevented nicks from occurring on the G-rich strand. This site constitutes a recombination initiation sequence/structure that directs gene conversion to a specific chromosomal locus.


Assuntos
Variação Antigênica , DNA Bacteriano/genética , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/imunologia , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/imunologia , Pareamento de Bases , Sequência de Bases , Dano ao DNA , DNA Bacteriano/química , Proteínas de Fímbrias/química , Fímbrias Bacterianas , Conversão Gênica , Guanina/química , Mesoporfirinas/metabolismo , Mesoporfirinas/farmacologia , Neisseria gonorrhoeae/crescimento & desenvolvimento , Oligodesoxirribonucleotídeos/química , Recombinação Genética
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