Effect of xylitol and other carbon sources on Streptococcus pneumoniae biofilm formation and gene expression in vitro

Apmis ◽  
2010 ◽  
Vol 119 (2) ◽  
pp. 135-142 ◽  
Author(s):  
PAULA KUROLA ◽  
TERHI TAPIAINEN ◽  
JENNY SEVANDER ◽  
TARJA KAIJALAINEN ◽  
MAIJA LEINONEN ◽  
...  
Keyword(s):  
Gene Expression ◽  
Streptococcus Pneumoniae ◽  
Biofilm Formation ◽  
Carbon Sources

scholarly journals Synthetic gene regulatory networks in the opportunistic human pathogen Streptococcus pneumoniae

2019 ◽  
Author(s):  
Robin A. Sorg ◽  
Clement Gallay ◽  
Jan-Willem Veening
Keyword(s):  
Gene Expression ◽  
Streptococcus Pneumoniae ◽  
Regulatory Networks ◽  
Expression Patterns ◽  
Combinatorial Libraries ◽  
Regulatory Elements ◽  
Expression Control ◽  
Gene Expression Control

AbstractStreptococcus pneumoniae can cause disease in various human tissues and organs, including the ear, the brain, the blood and the lung, and thus in highly diverse and dynamic environments. It is challenging to study how pneumococci control virulence factor expression, because cues of natural environments and the presence of an immune system are difficult to simulate in vitro. Here, we apply synthetic biology methods to reverse-engineer gene expression control in S. pneumoniae. A selection platform is described that allows for straightforward identification of transcriptional regulatory elements out of combinatorial libraries. We present TetR- and LacI-regulated promoters that show expression ranges of four orders of magnitude. Based on these promoters, regulatory networks of higher complexity are assembled, such as logic AND and IMPLY gates. Finally, we demonstrate single-copy genome-integrated toggle switches that give rise to bimodal population distributions. The tools described here can be used to mimic complex expression patterns, such as the ones found for pneumococcal virulence factors, paving the way for in vivo investigations of the importance of gene expression control on the pathogenicity of S. pneumoniae.

scholarly journals Expression of biofilm-associated genes of Streptococcus mutans in response to glucose and sucrose

2007 ◽  
Vol 56 (11) ◽  
pp. 1528-1535 ◽  
Author(s):  
Moshe Shemesh ◽  
Avshalom Tam ◽  
Doron Steinberg
Keyword(s):  
Gene Expression ◽  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Fold Increase ◽  
Extracellular Polysaccharide ◽  
Tooth Surface ◽  
Differential Analysis ◽  
Nutrient Contents ◽  
Genes Encoding

Streptococcus mutans is known as a primary pathogen of dental caries, one of the most common human infectious diseases. Exopolysaccharide synthesis, adherence to tooth surface and biofilm formation are important physiological and virulence factors of S. mutans. In vitro comparative gene expression analysis was carried out to differentiate 10 selected genes known to be mostly involved in S. mutans biofilm formation by comparing the expression under biofilm and planktonic environments. Real-time RT-PCR analyses indicated that all of the genes tested were upregulated in the biofilm compared to cells grown in planktonic conditions. The influence of simple dietary carbohydrates on gene expression in S. mutans biofilm was tested also. Among the tested genes, in the biofilm phase, the greatest induction was observed for gtf and ftf, which are genes encoding the extracellular polysaccharide-producing enzymes. Biofilm formation was accompanied by a 22-fold induction in the abundance of mRNA encoding glucosyltransferase B (GTFB) and a 14.8 -fold increase in mRNA encoding GTFC. Levels of mRNA encoding fructosyltransferase were induced approximately 11.8-fold in biofilm-derived cells. Another notable finding of this study suggests that glucose affects the expression of S. mutans GS5 biofilm genes. In spite of a significant upregulation in biofilm-associated gene expression in the presence of sucrose, the presence of glucose with sucrose reduced expression of most tested genes. Differential analysis of the transcripts from S. mutans, grown in media with various nutrient contents, revealed significant shifts in the expression of the genes involved in biofilm formation. The results presented here provide new insights at the molecular level regarding gene expression in this bacterium when grown under biofilm conditions, allowing a better understanding of the mechanism of biofilm formation by S. mutans.

scholarly journals Reversible Gene Expression Control inYersinia pestisby Using an Optimized CRISPR Interference System

2019 ◽  
Vol 85 (12) ◽  
Author(s):  
Tong Wang ◽  
Min Wang ◽  
Qingwen Zhang ◽  
Shiyang Cao ◽  
Xiang Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Biofilm Formation ◽  
Prevention And Control ◽  
Target Gene ◽  
Gene Knockdown ◽  
Content Type ◽  
Short Palindromic Repeat ◽  
And Control

ABSTRACTMany genes in the bacterial pathogenYersinia pestis, the causative agent of three plague pandemics, remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been shown to be an effective tool for gene knockdown in model bacteria. In this system, a catalytically dead Cas9 (dCas9) and a small guide RNA (sgRNA) form a complex, binding to the specific DNA target through base pairing, thereby impeding RNA polymerase binding and causing target gene repression. Here, we introduce an optimized CRISPRi system usingStreptococcus pyogenesCas9-derived dCas9 for gene knockdown inY. pestis. Multiple genes harbored on either the chromosome or plasmids ofY. pestiswere efficiently knocked down (up to 380-fold) in a strictly anhydrotetracycline-inducible manner using this CRISPRi approach. Knockdown ofhmsH(responsible for biofilm formation) orcspB(encoding a cold shock protein) resulted in greatly decreased biofilm formation or impaired cold tolerance inin vitrophenotypic assays. Furthermore, silencing of the virulence-associated genesyscBorailusing this CRISPRi system resulted in attenuation of virulence in HeLa cells and mice similar to that previously reported foryscBandailnull mutants. Taken together, our results confirm that this optimized CRISPRi system can reversibly and efficiently repress the expression of target genes inY. pestis, providing an alternative to conventional gene knockdown techniques, as well as a strategy for high-throughput phenotypic screening ofY. pestisgenes with unknown functions.IMPORTANCEYersiniapestisis a lethal pathogen responsible for millions of human deaths in history. It has also attracted much attention for potential uses as a bioweapon or bioterrorism agent, against which new vaccines are desperately needed. However, manyY. pestisgenes remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been successfully used in a variety of bacteria in functional genomic studies, but no such genetic tool has been reported inY. pestis. Here, we systematically optimized the CRISPRi approach for use inY. pestis, which ultimately repressed target gene expression with high efficiency in a reversible manner. Knockdown of functional genes using this method produced phenotypes that were readily detected byin vitroassays, cell infection assays, and mouse infection experiments. This is a report of a CRISPRi approach inY. pestisand highlights the potential use of this approach in high-throughput functional genomics studies of this pathogen.

scholarly journals The N-Acetylglucosaminidase LytB of Streptococcus pneumoniae Is Involved in the Structure and Formation of Biofilms

2020 ◽  
Vol 86 (10) ◽  
Author(s):  
Mirian Domenech ◽  
Ernesto García
Keyword(s):  
Streptococcus Pneumoniae ◽  
Biofilm Formation ◽  
Single Gene ◽  
Extracellular Polysaccharide ◽  
Extracellular Dna ◽  
Nasopharyngeal Colonization ◽  
Content Type ◽  
Daughter Cells ◽  
Biofilm Matrix

ABSTRACT The N-acetylglucosaminidase LytB of Streptococcus pneumoniae is involved in nasopharyngeal colonization and is responsible for cell separation at the end of cell division; thus, ΔlytB mutants form long chains of cells. This paper reports the construction and properties of a defective pneumococcal mutant producing an inactive LytB protein (LytBE585A). It is shown that an enzymatically active LytB is required for in vitro biofilm formation, as lytB mutants (either ΔlytB or producing the inactive LytBE585A) are incapable of forming substantial biofilms, despite that extracellular DNA is present in the biofilm matrix. Adding small amounts (0.5 to 2.0 μg/ml) of exogenous LytB or some LytB constructs restored the biofilm-forming capacity of lytB mutants to wild-type levels. The LytBE585A mutant formed biofilm more rapidly than ΔlytB mutants in the presence of LytB. This suggests that the mutant protein acted in a structural role, likely through the formation of complexes with extracellular DNA. The chain-dispersing capacity of LytB allowed the separation of daughter cells, presumably facilitating the formation of microcolonies and, finally, of biofilms. A role for the possible involvement of LytB in the synthesis of the extracellular polysaccharide component of the biofilm matrix is also discussed. IMPORTANCE It has been previously accepted that biofilm formation in S. pneumoniae must be a multigenic trait because the mutation of a single gene has led to only to partial inhibition of biofilm production. In the present study, however, evidence that the N-acetylglucosaminidase LytB is crucial in biofilm formation is provided. Despite the presence of extracellular DNA, strains either deficient in LytB or producing a defective LytB enzyme formed only shallow biofilms.

scholarly journals Evaluation of antimicrobial and antibiofilm activities of stingless bee Trigona honey (Malaysia) against Streptococcus pneumoniae

10.3823/856 ◽  
2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Mohammad A. Alkafaween ◽  
Hamid A. Nagi Al-Jamal ◽  
Abu Bakar Mohmd Hilmi
Keyword(s):  
Antibacterial Activity ◽  
Streptococcus Pneumoniae ◽  
Biofilm Formation ◽  
Stingless Bee ◽  
Expression Of Genes ◽  
Zones Of Inhibition

Background: The study aims to evaluate the antibacterial activity of Trigona honey against S. pneumonia. Methods: The effect of Trigona honey on S. pneumonia investigated using agar well diffusion, MIC, MBC, biofilm formation and RT-qPCR. Results: Trigona honey samples showed the larger zones of inhibition against S. pneumonia, 22.2±0.4 at 100% concentration. Trigona honey possessed the lowest MIC, MBC, MIC50 and MIC90 against S. pneumoniae, 25%, 30%, 12.5% and 25% (w/v) respectively. Trigona honey permeated established biofilms of S. pneumonia, resulting in significant decreased the cells from the biofilm. RT-qPCR revealed that the expression of genes amiF, ftsY, mvaS, pnpA, argG, mvd1, purN, miaA and pbp2a were upregulated, glcK, marR, prmA and ccpA­­­ were downregulated after exposure to honey. Conclusion: Trigona honey demonstrated the highest antibacterial activity against S. pneumoniae. By limiting study in vitro on Trigona honey, we infer that Trigona honey impacts on S. pneumoniae.

Different carbon sources and their concentrations change alkaloid production and gene expression in Catharanthus roseus shoots in vitro

2021 ◽  
Vol 48 (1) ◽  
pp. 40
Author(s):  
Elham Khataee ◽  
Farah Karimi ◽  
Khadijeh Razavi
Keyword(s):  
Gene Expression ◽  
Regulatory Gene ◽  
Carbon Sources ◽  
Basal Medium ◽  
Alkaloid Production ◽  
Defensive Responses ◽  
Mitogen Activated Protein ◽  
Catalase Peroxidase ◽  
Plant Alkaloids

To compare the effects of different carbon sources on physiological aspects, especially medicinal alkaloid biosynthesis and related gene expression in Catharantus roseus (L.) G.Don, we employed sucrose and sorbitol with two concentrations (87.64 mM, the equimolar concentration of sucrose in MS basal medium, and 150 mM) on the plant’s shoots in vitro in presence of 100 μM methyl jasmonate. The production of plant alkaloids including vincristine, vinblastine, ajmalicine, vindoline and catharantine and their biosynthetic and regulatory gene expression was measured. Both treatments had incremental effects on alkaloid production, upregulated the mitogen-activated protein kinase3 (MAPK3) and a downstream responsive transcription factor, ORCA3, which resulted in elevated transcript contents of the important genes in terpenoid indol alkaloids biosynthetic pathway including peroxidase1 (PRX1), geissoschizine synthase (GS), strictosidine synthase (STR) and deacetylvindoline acetyltransferase (DAT). Defensive responses such as antioxidant enzymes (catalase, peroxidase and superoxide dismutase) activities and non-enzymatic metabolites (total phenolics, flavonoids and carotenoids) contents increased under both treatments but the effects of sorbitol were stronger. Reduced fresh weight and chlorophylls contents, increased malondialdehyde (MDA) and carotenoid contents were shown after a week under all employed treatments. It seems that replacement of sucrose with sorbitol and also, increased concentrations of both carbon sources via increasing osmotic pressure make stressful conditions for the plant especially in longer times.

Gene expression profile of the plant pathogen Xylella fastidiosa during biofilm formation in vitro

2004 ◽  
Vol 237 (2) ◽  
pp. 341-353 ◽  
Author(s):  
Alessandra A. Souza ◽  
Marco A. Takita ◽  
Helvécio D. Coletta-Filho ◽  
Camila Caldana ◽  
Giane M. Yanai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Plant Pathogen ◽  
Biofilm Formation ◽  
Xylella Fastidiosa

scholarly journals Streptococcus pneumoniae Biofilm Formation Is Strain Dependent, Multifactorial, and Associated with Reduced Invasiveness and Immunoreactivity during Colonization

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Krystle Blanchette-Cain ◽  
Cecilia A. Hinojosa ◽  
Ramya Akula Suresh Babu ◽  
Anel Lizcano ◽  
Norberto Gonzalez-Juarbe ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Biofilm Formation ◽  
Lavage Fluid ◽  
Cytokine Response ◽  
Host Cells ◽  
Nasal Lavage ◽  
Content Type ◽  
Nasal Lavage Fluid

ABSTRACT Biofilms are thought to play an important role during colonization of the nasopharynx by Streptococcus pneumoniae, yet how they form in vivo and the determinants responsible remain unknown. Using scanning electron microscopy, we show that biofilm aggregates of increasing complexity form on murine nasal septa following intranasal inoculation. These biofilms were highly distinct from in vitro biofilms, as they were discontiguous and appeared to incorporate nonbacterial components such as intact host cells. Biofilms initially formed on the surface of ciliated epithelial cells and, as cells were sloughed off, were found on the basement membrane. The size and number of biofilm aggregates within nasal lavage fluid were digitally quantitated and revealed strain-specific capabilities that loosely correlated with the ability to form robust in vitro biofilms. We tested the ability of isogenic mutants deficient in CbpA, pneumolysin, hydrogen peroxide, LytA, LuxS, CiaR/H, and PsrP to form biofilms within the nasopharynx. This analysis revealed that CiaR/H was absolutely required for colonization, that PsrP and SpxB strongly impacted aggregate formation, and that other determinants affected aggregate morphology in a modest fashion. We determined that mice colonized with ΔpsrP mutants had greater levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-1β, and KC in nasal lavage fluid than did mice colonized with wild-type controls. This phenotype correlated with a diminished capacity of biofilm pneumococci to invade host cells in vitro despite enhanced attachment. Our results show that biofilms form during colonization and suggest that they may contribute to persistence through a hyperadhesive, noninvasive state that elicits a dampened cytokine response. IMPORTANCE This work demonstrates the first temporal characterization of Streptococcus pneumoniae biofilm formation in vivo. Our results show that the morphology of biofilms formed by both invasive and noninvasive clinical isolates in vivo is distinct from that of formed biofilms in vitro, yet propensity to form biofilms in vivo loosely correlates with the degree of in vitro biofilm formation on a microtiter plate. We show that host components, including intact host cells, influence the formation of in vivo structures. We also found that efficient biofilm formation in vivo requires multiple bacterial determinants. While some factors are essential for in vivo biofilm formation (CiaRH, PsrP, and SpxB), other factors are less critical (CbpA, LytA, LuxS, and pneumolysin). In comparison to their planktonic counterparts, biofilm pneumococci are hyperadhesive but less invasive and elicit a weaker proinflammatory cytokine response. These findings give insight into the requirements for and potential role of biofilms during prolonged asymptomatic colonization.

scholarly journals Shin’iseihaito (Xinyiqingfeitang) Suppresses the Biofilm Formation of Streptococcus pneumoniae In Vitro

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Masaaki Minami ◽  
Toru Konishi ◽  
Hiroshi Takase ◽  
Toshiaki Makino
Keyword(s):  
Streptococcus Pneumoniae ◽  
Cell Surface ◽  
Bacterial Cell ◽  
Biofilm Formation ◽  
Antimicrobial Agents ◽  
Time Dependent ◽  
Antibiofilm Activity ◽  
Inhibitory Effects ◽  
Crude Drugs

Streptococcus pneumoniae (S. pneumoniae) is the important pathogen that causes otolaryngeal diseases such as sinusitis. S. pneumoniae frequently forms the biofilm to prevent severe circumstances such as antimicrobial agents. Shin’iseihaito (xinyiqingfeitang) is a formula of Japanese traditional Kampo medicine that has 9 crude drugs and provides the medicinal usage for sinusitis. The objective of the present study is to reveal the mechanism of antibiofilm activity by Shin’iseihaito extract (SSHT). SSHT significantly inhibited the formation of biofilm from S. pneumoniae ATCC 49619 in dose- and time-dependent manners. SSHT also significantly suppressed the biofilm formation by other five different cps types of S. pneumoniae clinical isolates. We found that the extracts of 8 out of 9 components in Shin’iseihaito had the inhibitory effects of biofilm formation, and the extract of the root of Scutellaria baicalensis had the strongest effect among the ingredients of Shin’iseihaito. We found that the capsule of SSHT-treated S. pneumoniae was significantly thinner than that of the untreated group and that SSHT reduced the hydrophobicity of bacterial cell surface. Our results suggest that Shin’iseihaito may be a useful agent for the treatment of S. pneumoniae-induced sinusitis because of the inhibition of biofilm formation of S. pneumoniae.

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