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1.
PLoS Pathog ; 18(6): e1010561, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35714158

RESUMEN

The Neisseria gonorrhoeae Type IV pilus is a multifunctional, dynamic fiber involved in host cell attachment, DNA transformation, and twitching motility. We previously reported that the N. gonorrhoeae pilus is also required for resistance against hydrogen peroxide-, antimicrobial peptide LL-37-, and non-oxidative, neutrophil-mediated killing. We tested whether the hydrogen peroxide, LL-37, and neutrophil hypersensitivity phenotypes in non-piliated N. gonorrhoeae could be due to elevated iron levels. Iron chelation in the growth medium rescued a nonpiliated pilE mutant from both hydrogen peroxide- and antimicrobial peptide LL-37-mediated killing, suggesting these phenotypes are related to iron availability. We used the antibiotic streptonigrin, which depends on free cytoplasmic iron and oxidation to kill bacteria, to determine whether piliation affected intracellular iron levels. Several non-piliated, loss-of-function mutants were more sensitive to streptonigrin killing than the piliated parental strain. Consistent with the idea that higher available iron levels in the under- and non-piliated strains were responsible for the higher streptonigrin sensitivity, iron limitation by desferal chelation restored resistance to streptonigrin in these strains and the addition of iron restored the sensitivity to streptonigrin killing. The antioxidants tiron and dimethylthiourea rescued the pilE mutant from streptonigrin-mediated killing, suggesting that the elevated labile iron pool in non-piliated bacteria leads to streptonigrin-dependent reactive oxygen species production. These antioxidants did not affect LL-37-mediated killing. We confirmed that the pilE mutant is not more sensitive to other antibiotics showing that the streptonigrin phenotypes are not due to general bacterial envelope disruption. The total iron content of the cell was unaltered by piliation when measured using ICP-MS suggesting that only the labile iron pool is affected by piliation. These results support the hypothesis that piliation state affects N. gonorrhoeae iron homeostasis and influences sensitivity to various host-derived antimicrobial agents.


Asunto(s)
Peróxido de Hidrógeno , Neisseria gonorrhoeae , Proteínas Bacterianas/genética , Fimbrias Bacterianas , Peróxido de Hidrógeno/farmacología , Hierro , Neisseria gonorrhoeae/genética , Estreptonigrina
2.
Annu Rev Microbiol ; 72: 49-69, 2018 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-29852085

RESUMEN

Advances in understanding mechanisms of nucleic acids have revolutionized molecular biology and medicine, but understanding of nontraditional nucleic acid conformations is less developed. The guanine quadruplex (G4) alternative DNA structure was first described in the 1960s, but the existence of G4 structures (G4-S) and their participation in myriads of biological functions are still underappreciated. Despite many tools to study G4s and many examples of roles for G4s in eukaryotic molecular processes and issues with uncontrolled G4-S formation, there is relatively little knowledge about the roles of G4-S in viral or prokaryotic systems. This review summarizes the state of the art with regard to G4-S in eukaryotes and their potential roles in human disease before discussing the evidence that G4-S have equivalent importance in affecting viral and bacterial life.


Asunto(s)
Bacterias/genética , ADN/química , ADN/genética , Eucariontes/genética , G-Cuádruplex , Virus/genética , Regulación de la Expresión Génica
3.
Mol Cell ; 60(2): 242-55, 2015 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-26474066

RESUMEN

Type II CRISPR systems defend against invasive DNA by using Cas9 as an RNA-guided nuclease that creates double-stranded DNA breaks. Dual RNAs (CRISPR RNA [crRNA] and tracrRNA) are required for Cas9's targeting activities observed to date. Targeting requires a protospacer adjacent motif (PAM) and crRNA-DNA complementarity. Cas9 orthologs (including Neisseria meningitidis Cas9 [NmeCas9]) have also been adopted for genome engineering. Here we examine the DNA cleavage activities and substrate requirements of NmeCas9, including a set of unusually complex PAM recognition patterns. Unexpectedly, NmeCas9 cleaves single-stranded DNAs in a manner that is RNA guided but PAM and tracrRNA independent. Beyond the need for guide-target pairing, this "DNase H" activity has no apparent sequence requirements, and the cleavage sites are measured from the 5' end of the DNA substrate's RNA-paired region. These results indicate that tracrRNA is not strictly required for NmeCas9 enzymatic activation, and expand the list of targeting activities of Cas9 endonucleases.


Asunto(s)
Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , ADN Bacteriano/genética , Endodesoxirribonucleasas/genética , Neisseria meningitidis/genética , Proteínas Bacterianas/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN Bacteriano/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Endodesoxirribonucleasas/metabolismo , Datos de Secuencia Molecular , Neisseria meningitidis/enzimología , Motivos de Nucleótidos , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo
4.
Annu Rev Genet ; 48: 405-31, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25251852

RESUMEN

Neisseria gonorrhoeae and Neisseria meningitidis are closely related organisms that cause the sexually transmitted infection gonorrhea and serious bacterial meningitis and septicemia, respectively. Both species possess multiple mechanisms to alter the expression of surface-exposed proteins through the processes of phase and antigenic variation. This potential for wide variability in surface-exposed structures allows the organisms to always have subpopulations of divergent antigenic types to avoid immune surveillance and to contribute to functional variation. Additionally, the Neisseria are naturally competent for DNA transformation, which is their main means of genetic exchange. Although bacteriophages and plasmids are present in this genus, they are not as effective as DNA transformation for horizontal genetic exchange. There are barriers to genetic transfer, such as restriction-modification systems and CRISPR loci, that limit particular types of exchange. These host-restricted pathogens illustrate the rich complexity of genetics that can help define the similarities and differences of closely related organisms.


Asunto(s)
Genoma Bacteriano , Neisseria gonorrhoeae/genética , Neisseria meningitidis/genética , Recombinación Genética , Bacteriófagos/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Transferencia de Gen Horizontal/genética , Gonorrea/genética , Gonorrea/microbiología , Meningitis Bacterianas/genética , Meningitis Bacterianas/microbiología , Neisseria gonorrhoeae/patogenicidad , Neisseria meningitidis/patogenicidad , Sepsis/genética , Sepsis/microbiología
5.
Antimicrob Agents Chemother ; 65(8): e0057021, 2021 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-34097477

RESUMEN

The Acinetobacter baumannii RND efflux pump AdeABC is regulated by the 2-component regulator AdeRS. In this study, we compared the regulation and expression of AdeABC of the reference strains ATCC 17978 and ATCC 19606. A clearly stronger efflux activity was demonstrated for ATCC 19606. An amino acid substitution at residue 172 of adeS was identified as a potential cause for differential expression of the pump. Therefore, we recommend caution with exclusively using single reference strains for research.


Asunto(s)
Acinetobacter baumannii , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana/genética , Farmacorresistencia Bacteriana Múltiple/genética , Proteínas de Transporte de Membrana/genética , Pruebas de Sensibilidad Microbiana
6.
Mol Cell ; 50(4): 488-503, 2013 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-23706818

RESUMEN

CRISPR interference confers adaptive, sequence-based immunity against viruses and plasmids and is specified by CRISPR RNAs (crRNAs) that are transcribed and processed from spacer-repeat units. Pre-crRNA processing is essential for CRISPR interference in all systems studied thus far. Here, our studies of crRNA biogenesis and CRISPR interference in naturally competent Neisseria spp. reveal a unique crRNA maturation pathway in which crRNAs are transcribed from promoters that are embedded within each repeat, yielding crRNA 5' ends formed by transcription and not by processing. Although crRNA 3' end formation involves RNase III and trans-encoded tracrRNA, as in other type II CRISPR systems, this processing is dispensable for interference. The meningococcal pathway is the most streamlined CRISPR/Cas system characterized to date. Endogenous CRISPR spacers limit natural transformation, which is the primary source of genetic variation that contributes to immune evasion, antibiotic resistance, and virulence in the human pathogen N. meningitidis.


Asunto(s)
Secuencias Invertidas Repetidas/genética , Neisseria meningitidis/genética , ARN Bacteriano/genética , Transformación Bacteriana , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Genes Bacterianos/genética , Interacciones Huésped-Patógeno , Humanos , Infecciones Meningocócicas/microbiología , Modelos Genéticos , Neisseria meningitidis/patogenicidad , Neisseria meningitidis/fisiología , Regiones Promotoras Genéticas/genética , Procesamiento Postranscripcional del ARN , ARN Bacteriano/metabolismo , Ribonucleasa III/metabolismo , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Virulencia/genética
7.
Biochemistry ; 59(10): 1104-1112, 2020 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-32078293

RESUMEN

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.


Asunto(s)
Proteínas Fimbrias/metabolismo , Neisseria gonorrhoeae/genética , Variación Antigénica/genética , Emparejamiento Base/genética , Proteínas Fimbrias/química , Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , G-Cuádruplex , Guanina/metabolismo , Mutación/genética , Neisseria gonorrhoeae/inmunología , Unión Proteica/genética , Recombinación Genética/genética
8.
J Bacteriol ; 201(13)2019 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-30988037

RESUMEN

The major subunit of the type IV pilus (T4p) of Neisseria gonorrhoeae undergoes antigenic variation (AV) dependent on a guanine quadruplex (G4) DNA structure located upstream of the pilin gene. Since the presence of G4 DNA induces genome instability in both eukaryotic and prokaryotic chromosomes, we tested whether a double-strand break (DSB) at the site of the pilE G4 sequence could substitute for G4-directed pilin AV. The G4 motif was replaced by an I-SceI cut site, and the cut site was also introduced to locations near the origin of replication and the terminus. Expression of the I-SceI endonuclease from an irrelevant chromosomal site confirmed that the endonuclease functions to induce double-strand breaks at all three locations. No antigenic variants were detected when the G4 was replaced with the I-SceI cut site, but there was a growth defect from having a DSB in the chromosome, and suppressor mutations that were mainly deletions of the cut site and/or the entire pilE gene accumulated. Thus, the pilE G4 does not act to promote pilin AV by generating a DSB but requires either a different type of break, a nick, or more complex interactions with other factors to stimulate this programmed recombination system.IMPORTANCENeisseria gonorrhoeae, the causative agent of gonorrhea, possesses a DNA recombination system to change one of its surface-exposed antigens. This recombination system, known as antigenic variation, uses an alternate DNA structure to initiate variation. The guanine quadruplex DNA structure is known to cause nicks or breaks in DNA; however, much remains unknown about how this structure functions in cells. We show that inducing a break by different means does not allow antigenic variation, indicating that the DNA structure may have a more complicated role.


Asunto(s)
Variación Antigénica , Roturas del ADN de Doble Cadena , Proteínas Fimbrias/inmunología , Neisseria gonorrhoeae/genética , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , ADN Bacteriano/genética , Proteínas Fimbrias/genética , Fimbrias Bacterianas/inmunología , G-Cuádruplex , Recombinación Genética
9.
Mol Microbiol ; 109(2): 135-149, 2018 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-29573486

RESUMEN

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea and is adapted to survive in humans, its only host. The N. gonorrhoeae cell wall is critical for maintaining envelope integrity, resisting immune cell killing and production of cytotoxic peptidoglycan (PG) fragments. Deletion of the N. gonorrhoeae strain FA1090 genes encoding two predicted low-molecular-mass, penicillin-binding proteins (LMM PBPs), DacB and DacC, substantially altered the PG cross-linking. Loss of the DacB peptidase resulted in global alterations to the PG composition, while loss of the DacC protein affected a much narrower subset of PG peptide components. A double ΔdacB/ΔdacC mutant resembled the ΔdacB single mutant, but had an even greater level of cross-linked PG. While single ΔdacB or ΔdacC mutants did not show any major phenotypes, the ΔdacB/ΔdacC mutant displayed an altered cellular morphology, decreased resistance to antibiotics and increased sensitivity to detergent-mediated death. Loss of the two proteins also drastically reduced the number of Type IV pili (Tfp), a critical virulence factor. The decreased piliation reduced transformation efficiency and correlated with increased growth rate. While these two LMM PBPs differentially alter the PG composition, their overlapping effects are essential to proper envelope function and expression of factors critical for pathogenesis.

10.
PLoS Genet ; 12(5): e1006069, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-27213957

RESUMEN

The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3' third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants and suggest there are alternate forms of the Tfp assembly apparatus that mediate various functions including transformation.


Asunto(s)
Proteínas Fimbrias/genética , Gonorrea/genética , Neisseria gonorrhoeae/genética , Codón sin Sentido , Cisteína/genética , Proteínas Fimbrias/biosíntesis , Fimbrias Bacterianas , Regulación Bacteriana de la Expresión Génica , Gonorrea/microbiología , Humanos , Mutagénesis , Neisseria gonorrhoeae/patogenicidad , Dominios Proteicos , Transformación Bacteriana/genética
11.
J Bacteriol ; 200(22)2018 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-30181126

RESUMEN

Many pathogenic microbes evade host immune surveillance by varying the surface antigens, a process termed antigenic variation. While the process of pilin antigenic variation has been extensively studied in the human pathogen Neisseria gonorrhoeae (gonococcus [Gc]), relatively few studies of pilin antigenic variation have been conducted with Neisseria meningitidis (meningococcus [Mc]). Mc is usually a commensal organism that colonizes the human nasopharynx, but when it translocates to the bloodstream or meninges, it results in the severe and often deadly meningococcal disease. The type IV pili of Mc isolates play a critical role in host surface adherence, and its major pilin component (PilE) can undergo antigenic variation. In this study, Roche 454 pyrosequencing was used to examine the pilin antigenic variation of Mc strain 8013, as well as 8013 recA, recX, recQ, rep, and recJ mutants, Gc orthologues which have been shown to play a role in pilin antigenic variation. This study confirms that the Mc recA, rep, and recJ genes are essential for pilin antigenic variation. While the Mc recQ and recX gene products contribute to normal frequencies of antigenic variation, the loss of these factors does not alter the types of pilin variants produced. Overall, this study shows that the mechanisms of pilin antigenic variation are conserved between Gc and Mc.IMPORTANCE Antigenic variation is a strategy used by many pathogens to escape host immune surveillance and establish persistent infections. This study successfully applies next-generation sequencing to study pilin antigenic variation in the human pathogen Neisseria meningitidis This assay provides an affordable and efficient solution for quantifying antigenic variation frequency in mutant strains and for defining the recombination products of the process. We determined that there is a nonuniformity of silent donor copies used during meningococcus antigenic variation, and by the analysis of selected mutants deficient for specific recombination pathways, we show for the first time that the processes are conserved between N. meningitidis and Neisseria gonorrhoeae.


Asunto(s)
Variación Antigénica , Proteínas Fimbrias/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Neisseria gonorrhoeae/genética , Neisseria meningitidis/genética , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Mutación , Neisseria gonorrhoeae/patogenicidad , Neisseria meningitidis/patogenicidad , Fenotipo
12.
J Clin Microbiol ; 56(7)2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29720433

RESUMEN

For the diagnosis of Clostridium difficile infection (CDI), microbiological testing is almost always accomplished through the analysis of stool specimens. We evaluated the performances of rectal swabs with liquid transport medium (FS) and nylon flocked dry swabs for the detection of C. difficile Additionally, the impact on the diagnostic yield of storing swabs at -80°C for up to 3 months was evaluated. Sixty clinical stool samples positive for C. difficile by PCR were used for simulating rectal swabbing. FS and dry swabs were dipped into the stool and tested by PCR directly after swabbing at 1 and 3 months after storage at -80°C. Stool and the liquid medium of FS were additionally tested by a combination of glutamate dehydrogenase antigen (GDH) testing and toxin A/B enzyme immunoassay (EIA), as well as by toxigenic culture (TC). Using dry swabs, the PCR-based detection rate of C. difficile was equal to the rate using stool samples (30/30 [100%]), whereas the detection rate in FS was significantly lower (25/30 [83.2%]; P = 0.019). The sensitivities of FS for detecting C. difficile by PCR, TC, GDH testing, and toxin A/B EIA were 83.3%, 85.7%, 88%, and 68.9%, respectively. Storage of swabs at -80°C had no impact on the detection rate. FS cannot replace stool samples in the two-step laboratory diagnosis of CDI, as the sensitivities were too low, probably due to diluting effects of the fecal sample in the liquid medium. For simple PCR-based detection of C. difficile, dry swabs proved to be a suitable alternative to the use of stool samples.


Asunto(s)
Técnicas de Laboratorio Clínico/métodos , Infecciones por Clostridium/diagnóstico , Medios de Cultivo , Heces/microbiología , Toxinas Bacterianas/análisis , Clostridioides difficile , Humanos , Sensibilidad y Especificidad
13.
Pneumologie ; 72(1): 15-63, 2018 01.
Artículo en Alemán | MEDLINE | ID: mdl-29341032

RESUMEN

Nosocomial pneumonia (HAP) is a frequent complication of hospital care. Most data are available on ventilator-associated pneumonia. However, infections on general wards are increasing. A central issue are infections with multidrug resistant (MDR) pathogens which are difficult to treat in the empirical setting potentially leading to inappropriate use of antimicrobial therapy.This guideline update was compiled by an interdisciplinary group on the basis of a systematic literature review. Recommendations are made according to GRADE giving guidance for the diagnosis and treatment of HAP on the basis of quality of evidence and benefit/risk ratio.This guideline has two parts. First an update on epidemiology, spectrum of pathogens and antimicrobials is provided. In the second part recommendations for the management of diagnosis and treatment are given. New recommendations with respect to imaging, diagnosis of nosocomial viral pneumonia and prolonged infusion of antibacterial drugs have been added. The statements to risk factors for infections with MDR pathogens and recommendations for monotherapy vs combination therapy have been actualised. The importance of structured deescalation concepts and limitation of treatment duration is emphasized.


Asunto(s)
Neumonía Asociada a la Atención Médica/diagnóstico , Neumonía Asociada a la Atención Médica/terapia , Adulto , Estudios Transversales , Alemania , Neumonía Asociada a la Atención Médica/epidemiología , Humanos
14.
J Biol Chem ; 291(20): 10916-33, 2016 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-26984407

RESUMEN

The human-restricted pathogen Neisseria gonorrhoeae encodes a single N-acetylmuramyl-l-alanine amidase involved in cell separation (AmiC), as compared with three largely redundant cell separation amidases found in Escherichia coli (AmiA, AmiB, and AmiC). Deletion of amiC from N. gonorrhoeae results in severely impaired cell separation and altered peptidoglycan (PG) fragment release, but little else is known about how AmiC functions in gonococci. Here, we demonstrated that gonococcal AmiC can act on macromolecular PG to liberate cross-linked and non-cross-linked peptides indicative of amidase activity, and we provided the first evidence that a cell separation amidase can utilize a small synthetic PG fragment as substrate (GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc(pentapeptide)). An investigation of two residues in the active site of AmiC revealed that Glu-229 is critical for both normal cell separation and the release of PG fragments by gonococci during growth. In contrast, Gln-316 has an autoinhibitory role, and its mutation to lysine resulted in an AmiC with increased enzymatic activity on macromolecular PG and on the synthetic PG derivative. Curiously, the same Q316K mutation that increased AmiC activity also resulted in cell separation and PG fragment release defects, indicating that activation state is not the only factor determining normal AmiC activity. In addition to displaying high basal activity on PG, gonococcal AmiC can utilize metal ions other than the zinc cofactor typically used by cell separation amidases, potentially protecting its ability to function in zinc-limiting environments. Thus gonococcal AmiC has distinct differences from related enzymes, and these studies revealed parameters for how AmiC functions in cell separation and PG fragment release.


Asunto(s)
Proteínas Bacterianas/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Neisseria gonorrhoeae/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dominio Catalítico/genética , Cationes Bivalentes/metabolismo , Activación Enzimática , Humanos , N-Acetil Muramoil-L-Alanina Amidasa/química , N-Acetil Muramoil-L-Alanina Amidasa/genética , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/patogenicidad , Peptidoglicano/química , Peptidoglicano/metabolismo , Dominios y Motivos de Interacción de Proteínas , Especificidad por Sustrato
15.
J Antimicrob Chemother ; 72(12): 3277-3282, 2017 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-28961773

RESUMEN

OBJECTIVES: To investigate the molecular epidemiology, antimicrobial susceptibility and carbapenem resistance determinants of Acinetobacter baumannii isolates from respiratory tract samples of patients diagnosed with ventilator-associated pneumonia (VAP) who were enrolled in the MagicBullet clinical trial. METHODS: A. baumannii isolates were prospectively cultured from respiratory tract samples from 65 patients from 15 hospitals in Greece, Italy and Spain. Susceptibility testing was performed by broth microdilution. Carbapenem resistance determinants were identified by PCR and sequencing. Molecular epidemiology was investigated using rep-PCR (DiversiLab) and international clones (IC) were identified using our in-house database. RESULTS: Of 65 isolates, all but two isolates (97%) were resistant to imipenem and these were always associated with an acquired carbapenemase, OXA-23 (80%), OXA-40 (4.6%), OXA-58 (1.5%) or OXA-23/58 (1.5%). Resistance to colistin was 47.7%. Twenty-two isolates were XDR, and 20 isolates were pandrug-resistant (PDR). The majority of isolates clustered with IC2 (n = 54) with one major subtype comprising isolates from 12 hospitals in the three countries, which included 19 XDR and 16 PDR isolates. CONCLUSIONS: Carbapenem resistance rates were very high in A. baumannii recovered from patients with VAP. Almost half of the isolates were colistin resistant, and 42 (64.6%) isolates were XDR or PDR. Rep-PCR confirmed IC2 is the predominant clonal lineage in Europe and suggests the presence of an epidemic XDR/PDR A. baumannii clone that has spread in Greece, Italy and Spain. These data highlight the difficulty in empirical treatment of patients with A. baumannii VAP in centres with a high prevalence of carbapenem-resistant A. baumannii.


Asunto(s)
Infecciones por Acinetobacter/epidemiología , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/aislamiento & purificación , Farmacorresistencia Bacteriana Múltiple , Neumonía Asociada al Ventilador/epidemiología , Neumonía Asociada al Ventilador/microbiología , Genotipo , Grecia/epidemiología , Humanos , Incidencia , Italia/epidemiología , Pruebas de Sensibilidad Microbiana , Epidemiología Molecular , Tipificación Molecular , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , Análisis de Secuencia de ADN , España/epidemiología
16.
J Antimicrob Chemother ; 72(5): 1359-1363, 2017 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-28108677

RESUMEN

Objectives: As part of the multicentre Antibiotic Therapy Optimisation Study, MIC values of 19 non-ß-lactam agents were determined for third-generation cephalosporin-resistant Escherichia coli , Klebsiella species and Enterobacter species (3GCREB) isolates collected in German hospitals. Methods: A total of 328 E. coli , 35 Klebsiella spp. (1 Klebsiella oxytoca and 34 Klebsiella pneumoniae ) and 16 Enterobacter spp. (1 Enterobacter aerogenes and 15 Enterobacter cloacae ) isolates were submitted to broth microdilution antimicrobial susceptibility testing with the MICRONAUT system. MICs of fluoroquinolones (levofloxacin and moxifloxacin), aminoglycosides (gentamicin, tobramycin, amikacin, streptomycin, neomycin and paromomycin), tetracyclines (tetracycline, minocycline and tigecycline), macrolides (erythromycin, clarithromycin and azithromycin) and miscellaneous agents [trimethoprim/sulfamethoxazole, chloramphenicol, nitrofurantoin, colistin and fosfomycin intravenous (iv)] were determined and reviewed against 2016 EUCAST breakpoints. Results: The MIC of levofloxacin was >2 mg/L for 128 of 328 E. coli and 8 of 35 Klebsiella spp., but only 1 of 16 Enterobacter spp. Rates of resistance to trimethoprim/sulfamethoxazole were high (>70%), except for Enterobacter spp. Rates of resistance to colistin and fosfomycin iv were still low. About 20% of the tested isolates were resistant to chloramphenicol. Only 1 (of 328) E. coli isolate had an MIC of amikacin >16 mg/L and only 33 of 328 E. coli and 1 of 35 Klebsiella spp. had an MIC of tobramycin >4 mg/L, whereas average gentamicin MICs were in general more elevated. A tigecycline MIC >2 mg/L was only found for 1 of 16 Enterobacter spp., but in none of the E. coli or Klebsiella spp. isolates. Conclusions: Our study gives insight into previously unreported non-ß-lactam MIC distributions of 3GCREB isolates.


Asunto(s)
Antibacterianos/farmacología , Cefalosporinas/farmacología , Farmacorresistencia Bacteriana , Enterobacter/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Klebsiella/efectos de los fármacos , Resistencia a las Cefalosporinas , Colistina/farmacología , Enterobacter/aislamiento & purificación , Infecciones por Enterobacteriaceae/microbiología , Escherichia coli/aislamiento & purificación , Hospitalización , Humanos , Klebsiella/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Minociclina/análogos & derivados , Minociclina/farmacología , Centros de Atención Terciaria , Tetraciclina/farmacología , Tigeciclina , beta-Lactamasas/metabolismo , beta-Lactamas/farmacología
17.
PLoS Pathog ; 11(9): e1005162, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26367394

RESUMEN

The ability of pathogens to cause disease depends on their aptitude to escape the immune system. Type IV pili are extracellular filamentous virulence factors composed of pilin monomers and frequently expressed by bacterial pathogens. As such they are major targets for the host immune system. In the human pathogen Neisseria meningitidis, strains expressing class I pilins contain a genetic recombination system that promotes variation of the pilin sequence and is thought to aid immune escape. However, numerous hypervirulent clinical isolates express class II pilins that lack this property. This raises the question of how they evade immunity targeting type IV pili. As glycosylation is a possible source of antigenic variation it was investigated using top-down mass spectrometry to provide the highest molecular precision on the modified proteins. Unlike class I pilins that carry a single glycan, we found that class II pilins display up to 5 glycosylation sites per monomer on the pilus surface. Swapping of pilin class and genetic background shows that the pilin primary structure determines multisite glycosylation while the genetic background determines the nature of the glycans. Absence of glycosylation in class II pilins affects pilus biogenesis or enhances pilus-dependent aggregation in a strain specific fashion highlighting the extensive functional impact of multisite glycosylation. Finally, molecular modeling shows that glycans cover the surface of class II pilins and strongly decrease antibody access to the polypeptide chain. This strongly supports a model where strains expressing class II pilins evade the immune system by changing their sugar structure rather than pilin primary structure. Overall these results show that sequence invariable class II pilins are cloaked in glycans with extensive functional and immunological consequences.


Asunto(s)
Endotelio Vascular/microbiología , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Evasión Inmune , Modelos Moleculares , Neisseria meningitidis/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Adhesión Bacteriana , Línea Celular , Células Cultivadas , Secuencia Conservada , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Endotelio Vascular/patología , Proteínas Fimbrias/química , Proteínas Fimbrias/genética , Fimbrias Bacterianas/inmunología , Fimbrias Bacterianas/ultraestructura , Eliminación de Gen , Glicosilación , Interacciones Huésped-Patógeno , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/microbiología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Infecciones Meningocócicas/inmunología , Infecciones Meningocócicas/metabolismo , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/patología , Microscopía Electrónica de Transmisión , Neisseria meningitidis/inmunología , Neisseria meningitidis/ultraestructura , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Propiedades de Superficie
18.
Cell Microbiol ; 18(4): 546-60, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26426083

RESUMEN

The human-adapted organism Neisseria gonorrhoeae is the causative agent of gonorrhoea, a sexually transmitted infection. It readily colonizes the genital, rectal and nasalpharyngeal mucosa during infection. While it is well established that N. gonorrhoeae recruits and modulates the functions of polymorphonuclear leukocytes during infection, how N. gonorrhoeae interacts with macrophages present in infected tissue is not fully defined. We studied the interactions of N. gonorrhoeae with two human monocytic cell lines, THP-1 and U937, and primary monocytes, all differentiated into macrophages. Most engulfed bacteria were killed in the phagolysosome, but a subset of bacteria was able to survive and replicate inside the macrophages suggesting that those cells may be an unexplored cellular reservoir for N. gonorrhoeae during infection. N. gonorrhoeae was able to modulate macrophage apoptosis: N. gonorrhoeae induced apoptosis in THP-1 cells whereas it inhibited induced apoptosis in U937 cells and primary human macrophages. Furthermore, N. gonorrhoeae induced expression of inflammatory cytokines in macrophages, suggesting a role for macrophages in recruiting polymorphonuclear leukocytes to the site of infection. These results indicate macrophages may serve as a significant replicative niche for N. gonorrhoeae and play an important role in gonorrheal pathogenesis.


Asunto(s)
Apoptosis , Citocinas/metabolismo , Interacciones Huésped-Patógeno , Macrófagos/inmunología , Macrófagos/microbiología , Neisseria gonorrhoeae/inmunología , Neisseria gonorrhoeae/fisiología , Células Cultivadas , Humanos
19.
J Bacteriol ; 198(18): 2470-82, 2016 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-27381912

RESUMEN

UNLABELLED: Many pathogens use homologous recombination to vary surface antigens in order to avoid immune surveillance. Neisseria gonorrhoeae, the bacterium responsible for the sexually transmitted infection gonorrhea, achieves this in part by changing the sequence of the major subunit of the type IV pilus in a process termed pilin antigenic variation (Av). The N. gonorrhoeae chromosome contains one expression locus (pilE) and many promoterless, partial-coding silent copies (pilS) that act as reservoirs for variant pilin information. Pilin Av occurs by high-frequency gene conversion reactions, which transfer pilS sequences into the pilE locus. We have developed a 454 sequencing-based assay to analyze the frequency and characteristics of pilin Av that allows a more robust analysis of pilin Av than previous assays. We used this assay to analyze mutations and conditions previously shown to affect pilin Av, confirming many but not all of the previously reported phenotypes. We show that mutations or conditions that cause growth defects can result in Av phenotypes when analyzed by phase variation-based assays. Adapting the 454 sequencing to analyze pilin Av demonstrates the utility of this technology to analyze any diversity generation system that uses recombination to develop biological diversity. IMPORTANCE: Measuring and analyzing complex recombination-based systems constitute a major barrier to understanding the mechanisms used to generate diversity. We have analyzed the contributions of many gonococcal mutations or conditions to the process of pilin antigenic variation.


Asunto(s)
Variación Antigénica , Antígenos Bacterianos/metabolismo , Proteínas Fimbrias/metabolismo , Neisseria gonorrhoeae/metabolismo , Secuencia de Aminoácidos , Antígenos Bacterianos/genética , Proteínas Fimbrias/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Mutación
20.
J Bacteriol ; 198(16): 2228-35, 2016 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-27274027

RESUMEN

UNLABELLED: Neisseria gonorrhoeae causes the human-specific disease gonorrhea and is transmitted from person to person primarily via sexual contact. During transmission, N. gonorrhoeae is often exposed to seminal fluid and must adapt to this change in environment. Previous work demonstrated that seminal fluid facilitates N. gonorrhoeae motility and alters epithelial cell interactions. In this study, exposure to seminal fluid was found to decrease surface adherence of gonococci in a manner that was independent of Opa adhesin proteins or type IV pilus retraction. Semen was also shown to cause dispersal of bacteria that had previously established surface adherence. Although surface adherence decreased, interbacterial interactions were increased by seminal plasma both in long-term static culture and on a cell-to-cell basis over shorter time periods. The result of increased bacterium-bacterium interactions resulted in the formation of microcolonies, an important step in the N. gonorrhoeae infectious process. Seminal fluid also facilitated increased bacterial aggregation in the form of shear-resistant three-dimensional biofilms. These results emphasize the importance of the gonococcal response to the influx of seminal fluid within the genital niche. Further characterization of the N. gonorrhoeae response to semen will advance our understanding of the mechanisms behind the establishment of infection in naive hosts and the process of transmission. IMPORTANCE: N. gonorrhoeae is the causative agent of the globally prevalent sexually transmitted infection gonorrhea. An understudied aspect of this human-adapted pathogen is the change in bacterial physiology that occurs during sexual transmission. N. gonorrhoeae encounters semen when transmitted from host to host, and it is known that, when N. gonorrhoeae is exposed to seminal fluid, alterations in bacterial motility and type IV pilus arrangement occur. This work extends our previous observations on this modulation of gonococcal physiology by seminal fluid and demonstrates that seminal plasma decreases surface adherence, promotes interbacterial interactions, and enhances biofilm formation.


Asunto(s)
Adhesión Bacteriana , Biopelículas/crecimiento & desarrollo , Neisseria gonorrhoeae/fisiología , Semen , Humanos
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