Neisseria meningitidis Polynucleotide Phosphorylase Affects Aggregation, Adhesion, and Virulence

Neisseria meningitidisautoaggregation is an important step during attachment to human cells. Aggregation is mediated by type IV pili and can be modulated by accessory pilus proteins, such as PilX, and posttranslational modifications of the major pilus subunit PilE. The mechanisms underlying the regulation of aggregation remain poorly characterized. Polynucleotide phosphorylase (PNPase) is a 3′–5′ exonuclease that is involved in RNA turnover and the regulation of small RNAs. In this study, we biochemically confirm that NMC0710 is theN. meningitidisPNPase, and we characterize its role inN. meningitidispathogenesis. We show that deletion of the gene encoding PNPase leads to hyperaggregation and increased adhesion to epithelial cells. The aggregation induced was found to be dependent on pili and to be mediated by excessive pilus bundling. PNPase expression was induced following bacterial attachment to human cells. Deletion of PNPase led to global transcriptional changes and the differential regulation of 469 genes. We also demonstrate that PNPase is required for full virulence in anin vivomodel ofN. meningitidisinfection. The present study shows that PNPase negatively affects aggregation, adhesion, and virulence inN. meningitidis.

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Release of collagen type IV degrading activity from C6 astrocytoma cells and cell density

Journal of Neurosurgery ◽

10.3171/jns.1996.84.6.1013 ◽

1996 ◽

Vol 84 (6) ◽

pp. 1013-1019 ◽

Cited By ~ 8

Author(s):

Masashi Tamaki ◽

Warren McDonald ◽

Rolando F. Del Maestro

Keyword(s):

Cell Density ◽

Collagen Type ◽

Cell Communication ◽

Collagen Type Iv ◽

Major Protein ◽

Content Type ◽

Type Iv ◽

Astrocytoma Cells ◽

C6 Astrocytoma

✓ Type IV collagen is a major protein component of the vascular basement membrane and its degradation is crucial to the initiation of tumor-associated angiogenesis. The authors have investigated the influence of cell density on the release of collagen type IV degrading activity by C6 astrocytoma cells in monolayer culture. The release of collagen type IV degrading activity was assessed biochemically, immunocytochemically, and by Western blot analysis. The results demonstrate that increasing plating density and increasing cell density are associated with decreased collagen type IV degrading activity released per tumor cell. These findings indicate the existence of regulatory mechanisms dependent on cell—cell communication, which modulate release of collagen type IV degrading activity. The extrapolation of these results to the in vivo tumor microenvironment would suggest that individual and/or small groups of invading tumor cells, distant from the main tumor mass, would release substantial collagen type IV degrading activity, which may be crucial to their continued invasion and to angiogenesis.

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Lantibiotic Reductase LtnJ Substrate Selectivity Assessed with a Collection of Nisin Derivatives as Substrates

Applied and Environmental Microbiology ◽

10.1128/aem.00475-15 ◽

2015 ◽

Vol 81 (11) ◽

pp. 3679-3687 ◽

Cited By ~ 11

Author(s):

Dongdong Mu ◽

Manuel Montalbán-López ◽

Jingjing Deng ◽

Oscar P. Kuipers

Keyword(s):

Amino Acids ◽

Posttranslational Modifications ◽

Substrate Selectivity ◽

Biosynthesis Gene Cluster ◽

Content Type ◽

Hydrophobic Residues ◽

Lacticin 3147 ◽

Biosynthesis Gene ◽

Biosynthetic Machinery

ABSTRACTLantibiotics are potent antimicrobial peptides characterized by the presence of dehydrated amino acids, dehydroalanine and dehydrobutyrine, and (methyl)lanthionine rings. In addition to these posttranslational modifications, some lantibiotics exhibit additional modifications that usually confer increased biological activity or stability on the peptide. LtnJ is a reductase responsible for the introduction ofd-alanine in the lantibiotic lacticin 3147. The conversion ofl-serine intod-alanine requires dehydroalanine as the substrate, which is producedin vivoby the dehydration of serine by a lantibiotic dehydratase, i.e., LanB or LanM. In this work, we probe the substrate specificity of LtnJ using a system that combines the nisin modification machinery (dehydratase, cyclase, and transporter) and the stereospecific reductase LtnJ inLactococcus lactis. We also describe an improvement in the production yield of this system by inserting a putative attenuator from the nisin biosynthesis gene cluster in front of theltnJgene. In order to clarify the sequence selectivity of LtnJ, peptides composed of truncated nisin and different mutated C-terminal tails were designed and coexpressed with LtnJ and the nisin biosynthetic machinery. In these tails, serine was flanked by diverse amino acids to determine the influence of the surrounding residues in the reaction. LtnJ successfully hydrogenated peptides when hydrophobic residues (Leu, Ile, Phe, and Ala) were flanking the intermediate dehydroalanine, while those in which dehydroalanine was flanked by one or two polar residues (Ser, Thr, Glu, Lys, and Asn) or Gly were either less prone to be modified by LtnJ or not modified at all. Moreover, our results showed that dehydrobutyrine cannot serve as a substrate for LtnJ.

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The Catabolite Repressor/Activator Cra Is a Bridge Connecting Carbon Metabolism and Host Colonization in the Plant Drought Resistance-Promoting Bacterium Pantoea alhagi LTYR-11Z

Applied and Environmental Microbiology ◽

10.1128/aem.00054-18 ◽

2018 ◽

Vol 84 (13) ◽

Cited By ~ 3

Author(s):

Lei Zhang ◽

Muhang Li ◽

Qiqi Li ◽

Chaoqiong Chen ◽

Meng Qu ◽

...

Keyword(s):

Carbon Source ◽

Carbon Metabolism ◽

Carbon Sources ◽

Endophytic Bacterium ◽

Bacterial Attachment ◽

Gene Encoding ◽

Wheat Roots ◽

Host Colonization ◽

Content Type

ABSTRACT Efficient root colonization is a prerequisite for application of plant growth-promoting (PGP) bacteria in improving health and yield of agricultural crops. We have recently identified an endophytic bacterium, Pantoea alhagi LTYR-11Z, with multiple PGP properties that effectively colonizes the root system of wheat and improves its growth and drought tolerance. To identify novel regulatory genes required for wheat colonization, we screened an LTYR-11Z transposon (Tn) insertion library and found cra to be a colonization-related gene. By using transcriptome (RNA-seq) analysis, we found that transcriptional levels of an eps operon, the ydiV gene encoding an anti-FlhD 4 C 2 factor, and the yedQ gene encoding an enzyme for synthesis of cyclic dimeric GMP (c-di-GMP) were significantly downregulated in the Δ cra mutant. Further studies demonstrated that Cra directly binds to the promoters of the eps operon, ydiV , and yedQ and activates their expression, thus inhibiting motility and promoting exopolysaccharide (EPS) production and biofilm formation. Consistent with previous findings that Cra plays a role in transcriptional regulation in response to carbon source availability, the activating effects of Cra were much more pronounced when LTYR-11Z was grown within a gluconeogenic environment than when it was grown within a glycolytic environment. We further demonstrate that the ability of LTYR-11Z to colonize wheat roots is modulated by the availability of carbon sources. Altogether, these results uncover a novel strategy utilized by LTYR-11Z to achieve host colonization in response to carbon nutrition in the environment, in which Cra bridges a connection between carbon metabolism and colonization capacity of LTYR-11Z. IMPORTANCE Rapid and appropriate response to environmental signals is crucial for bacteria to adapt to competitive environments and to establish interactions with their hosts. Efficient colonization and persistence within the host are controlled by various regulatory factors that respond to specific environmental cues. The most common is nutrient availability. In this work, we unraveled the pivotal role of Cra in regulation of colonization ability of Pantoea alhagi LTYR-11Z in response to carbon source availability. Moreover, we identified three novel members of the Cra regulon involved in EPS synthesis, regulation of flagellar biosynthesis, and synthesis of c-di-GMP and propose a working model to explain the Cra-mediated regulatory mechanism that links carbon metabolism to host colonization. This study elucidates the regulatory role of Cra in bacterial attachment and colonization of plants, which raises the possibility of extending our studies to other bacteria associated with plant and human health.

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TIN2 Functions with TPP1/POT1 To Stimulate Telomerase Processivity

Molecular and Cellular Biology ◽

10.1128/mcb.00593-18 ◽

2019 ◽

Vol 39 (21) ◽

Cited By ~ 6

Author(s):

Alexandra M. Pike ◽

Margaret A. Strong ◽

John Paul T. Ouyang ◽

Carol W. Greider

Keyword(s):

Telomere Length ◽

Telomerase Activity ◽

Human Cells ◽

Short Telomere ◽

Gene Encoding ◽

Important Regulator

ABSTRACT TIN2 is an important regulator of telomere length, and mutations in TINF2, the gene encoding TIN2, cause short-telomere syndromes. While the genetics underscore the importance of TIN2, the mechanism through which TIN2 regulates telomere length remains unclear. Here, we tested the effects of human TIN2 on telomerase activity. We identified a new isoform in human cells, TIN2M, that is expressed at levels similar to those of previously studied TIN2 isoforms. All three TIN2 isoforms localized to and maintained telomere integrity in vivo, and localization was not disrupted by telomere syndrome mutations. Using direct telomerase activity assays, we discovered that TIN2 stimulated telomerase processivity in vitro. All of the TIN2 isoforms stimulated telomerase to similar extents. Mutations in the TPP1 TEL patch abrogated this stimulation, suggesting that TIN2 functions with TPP1/POT1 to stimulate telomerase processivity. We conclude from our data and previously published work that TIN2/TPP1/POT1 is a functional shelterin subcomplex.

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Cyclic Di-GMP Binding by an Assembly ATPase (PilB2) and Control of Type IV Pilin Polymerization in the Gram-Positive Pathogen Clostridium perfringens

Journal of Bacteriology ◽

10.1128/jb.00034-17 ◽

2017 ◽

Vol 199 (10) ◽

Cited By ~ 17

Author(s):

William A. Hendrick ◽

Mona W. Orr ◽

Samantha R. Murray ◽

Vincent T. Lee ◽

Stephen B. Melville

Keyword(s):

Clostridium Perfringens ◽

Biochemical Characterization ◽

Core Protein ◽

Type Iv Pili ◽

Host Cells ◽

Dependent Manner ◽

Gram Positive ◽

Content Type ◽

Type Iv

ABSTRACT The Gram-positive pathogen Clostridium perfringens possesses type IV pili (TFP), which are extracellular fibers that are polymerized from a pool of pilin monomers in the cytoplasmic membrane. Two proteins that are essential for pilus functions are an assembly ATPase (PilB) and an inner membrane core protein (PilC). Two homologues each of PilB and PilC are present in C. perfringens, called PilB1/PilB2 and PilC1/PilC2, respectively, along with four pilin proteins, PilA1 to PilA4. The gene encoding PilA2, which is considered the major pilin based on previous studies, is immediately downstream of the pilB2 and pilC2 genes. Purified PilB2 had ATPase activity, bound zinc, formed hexamers even in the absence of ATP, and bound the second messenger molecule cyclic di-GMP (c-di-GMP). Circular dichroism spectroscopy of purified PilC2 indicated that it retained its predicted degree of alpha-helical secondary structure. Even though no direct interactions between PilB2 and PilC2 could be detected in vivo or in vitro even in the presence of c-di-GMP, high levels of expression of a diguanylate cyclase from C. perfringens (CPE1788) stimulated polymerization of PilA2 in a PilB2- and PilC2-dependent manner. These results suggest that PilB2 activity is controlled by c-di-GMP levels in vivo but that PilB2-PilC2 interactions are either transitory or of low affinity, in contrast to results reported previously from in vivo studies of the PilB1/PilC1 pair in which PilC1 was needed for polar localization of PilB1. This is the first biochemical characterization of a c-di-GMP-dependent assembly ATPase from a Gram-positive bacterium. IMPORTANCE Type IV pili (TFP) are protein fibers involved in important bacterial functions, including motility, adherence to surfaces and host cells, and natural transformation. All clostridia whose genomes have been sequenced show evidence of the presence of TFP. The genetically tractable species Clostridium perfringens was used to study proteins involved in polymerizing the pilin, PilA2, into a pilus. The assembly ATPase PilB2 and its cognate membrane protein partner, PilC2, were purified. PilB2 bound the intracellular signal molecule c-di-GMP. Increased levels of intracellular c-di-GMP led to increased polymerization of PilA2, indicating that Gram-positive bacteria use this molecule to regulate pilus synthesis. These findings provide valuable information for understanding how pathogenic clostridia regulate TFP to cause human diseases.

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Disease-Associated Neisseria meningitidis Isolates Inhibit Wound Repair in Respiratory Epithelial Cells in a Type IV Pilus-Independent Manner

Infection and Immunity ◽

10.1128/iai.02001-14 ◽

2014 ◽

Vol 82 (12) ◽

pp. 5023-5034 ◽

Cited By ~ 2

Author(s):

Xiaoyun Ren ◽

Joanna K. MacKichan

Keyword(s):

Cell Migration ◽

Neisseria Meningitidis ◽

Meningococcal Disease ◽

Wound Repair ◽

Disease Onset ◽

Independent Manner ◽

Content Type ◽

Type Iv ◽

Respiratory Epithelial

ABSTRACTNeisseria meningitidisis the causative agent of meningococcal disease. Onset of meningococcal disease can be extremely rapid and can kill within a matter of hours. However, although a much-feared pathogen,Neisseria meningitidisis frequently found in the nasopharyngeal mucosae of healthy carriers. The bacterial factors that distinguish disease- from carriage-associated meningococci are incompletely understood. Evidence suggesting that disruptions to the nasopharynx may increase the risk of acquiring meningococcal disease led us to evaluate the ability of disease- and carriage-associated meningococcal isolates to inhibit cell migration, using anin vitroassay for wound repair. We found that disease-associated isolates in our collection inhibited wound closure, while carriage-associated isolates were more variable, with many isolates not inhibiting wound repair at all. For isolates selected for further study, we found that actin morphology, such as presence of lamellipodia, correlated with cell migration. We demonstrated that multiple meningococcal virulence factors, including the type IV pili, are dispensable for inhibition of wound repair. Inhibition of wound repair was also shown to be an active process, i.e., requiring live bacteria undergoing active protein synthesis.

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Use of the mCherry Fluorescent Protein To Study Intestinal Colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in Mice

Applied and Environmental Microbiology ◽

10.1128/aem.01247-15 ◽

2015 ◽

Vol 81 (17) ◽

pp. 5993-6002 ◽

Cited By ~ 17

Author(s):

Winschau F. van Zyl ◽

Shelly M. Deane ◽

Leon M. T. Dicks

Keyword(s):

Lactobacillus Plantarum ◽

Fluorescent Protein ◽

Intestinal Colonization ◽

Red Fluorescent Protein ◽

Gene Encoding ◽

Content Type ◽

Enterococcus Mundtii ◽

Probiotic Strains ◽

Reporter Systems

ABSTRACTLactic acid bacteria (LAB) are natural inhabitants of the gastrointestinal tract (GIT) of humans and animals, and some LAB species receive considerable attention due to their health benefits. Although many papers have been published on probiotic LAB, only a few reports have been published on the migration and colonization of the cells in the GIT. This is due mostly to the lack of efficient reporter systems. In this study, we report on the application of the fluorescent mCherry protein in thein vivotagging of the probiotic strainsEnterococcus mundtiiST4SA andLactobacillus plantarum423. ThemCherrygene, encoding a red fluorescent protein (RFP), was integrated into a nonfunctional region on the genome ofL. plantarum423 by homologous recombination. In the case ofE. mundtiiST4SA, themCherrygene was cloned into the pGKV223D LAB/Escherichia coliexpression vector. Expression of themCherrygene did not alter the growth rate of the two strains and had no effect on bacteriocin production. Both strains colonized the cecum and colon of mice.

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CodY Deletion EnhancesIn VivoVirulence of Community-Associated Methicillin-Resistant Staphylococcus aureus Clone USA300

Infection and Immunity ◽

10.1128/iai.06172-11 ◽

2012 ◽

Vol 80 (7) ◽

pp. 2382-2389 ◽

Cited By ~ 36

Author(s):

Christopher P. Montgomery ◽

Susan Boyle-Vavra ◽

Agnès Roux ◽

Kazumi Ebine ◽

Abraham L. Sonenshein ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Virulence Genes ◽

Target Genes ◽

Gene Encoding ◽

Methicillin Resistant ◽

Content Type ◽

Global Regulators ◽

Alpha Hemolysin

ABSTRACTTheStaphylococcus aureusglobal regulator CodY responds to nutrient availability by controlling the expression of target genes.In vitro, CodY represses the transcription of virulence genes, but it is not known if CodY also represses virulencein vivo. The dominant community-associated methicillin-resistantS. aureus(CA-MRSA) clone, USA300, is hypervirulent and has increased transcription of global regulators and virulence genes; these features are reminiscent of a strain defective in CodY. Sequence analysis revealed, however, that thecodYgenes of USA300 and other sequencedS. aureusisolates are not significantly different from thecodYgenes in strains known to have active CodY.codYwas expressed in USA300, as well as in other pulsotypes assessed. Deletion ofcodYfrom a USA300 clinical isolate resulted in modestly increased expression of the global regulatorsagrandsaeRS, as well as the gene encoding the toxin alpha-hemolysin (hla). A substantial increase (>30-fold) in expression of thelukF-PVgene, encoding part of the Panton-Valentine leukocidin (PVL), was observed in thecodYmutant. All of these expression differences were reversed by complementation with a functionalcodYgene. Moreover, purified CodY protein bound upstream of thelukSF-PVoperon, indicating that CodY directly represses expression oflukSF-PV. Deletion ofcodYincreased the virulence of USA300 in necrotizing pneumonia and skin infection. Interestingly, deletion oflukSF-PVfrom thecodYmutant did not attenuate virulence, indicating that the hypervirulence of thecodYmutant was not explained by overexpression of PVL. These results demonstrate that CodY is active in USA300 and that CodY-mediated repression restrains the virulence of USA300.

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Strain-Specific Regulatory Role of Eukaryote-Like Serine/Threonine Phosphatase in Pneumococcal Adherence

Infection and Immunity ◽

10.1128/iai.06311-11 ◽

2012 ◽

Vol 80 (4) ◽

pp. 1361-1372 ◽

Cited By ~ 21

Author(s):

Shivangi Agarwal ◽

Shivani Agarwal ◽

Preeti Pancholi ◽

Vijay Pancholi

Keyword(s):

High Efficiency ◽

Regulatory System ◽

Gene Encoding ◽

Content Type ◽

Knockout Mutants ◽

Catalytically Active

ABSTRACTStreptococcus pneumoniaeexploits a battery of virulence factors to colonize the host. Although the eukaryote-like Ser/Thr kinase ofS. pneumoniae(StkP) has been implicated in physiology and virulence, the role of its cotranscribing phosphatase (PhpP) has remained elusive. The construction of nonpolar markerlessphpPknockout mutants (ΔphpP) in two pathogenic strains, D39 (type 2) and 6A-EF3114 (type 6A), indicated that PhpP is not indispensable for pneumococcal survival. Further, PhpP also participates in the regulation of cell wall biosynthesis/division, adherence, and biofilm formation in a strain-specific manner. Additionally, we provide hitherto-unknownin vitroandin vivoevidence of a physiologically relevant biochemical link between the StkP/PhpP-mediated cognate regulation and the two-component regulatory system TCS06 (RR06/HK06) that regulates the expression of the gene encoding an important pneumococcal surface adhesin, CbpA, which was found to be significantly upregulated in ΔphpPmutants. In particular, StkP (threonine)-phosphorylated RR06 bound to thecbpApromoter with high efficiency even in the absence of the HK06-responsive and catalytically active aspartate 51 residue. Together, our findings unravel the significant contributions of PhpP in pneumococcal physiology and adherence.

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The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response

Infection and Immunity ◽

10.1128/iai.00531-16 ◽

2016 ◽

Vol 84 (12) ◽

pp. 3458-3470 ◽

Cited By ~ 11

Author(s):

Mike Khan ◽

Jerome S. Harms ◽

Fernanda M. Marim ◽

Leah Armon ◽

Cherisse L. Hall ◽

...

Keyword(s):

Immune Response ◽

Second Messenger ◽

Host Immune Response ◽

Vital Role ◽

Host Cells ◽

Content Type ◽

Type Iv

Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host- Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a Δ bpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the Δ bpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase Δ cgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, Δ bpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection.

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