Anaerobic Metabolism Occurs in the Substratum of Gonococcal Biofilms and May Be Sustained in Part by Nitric Oxide

ABSTRACT Neisseria gonorrhoeae is the etiologic agent of gonorrhea, which has been among the most frequently reported communicable diseases in the United States since 1960. Women frequently do not exhibit symptoms, which can lead to chronic infection. N. gonorrhoeae readily forms biofilms over abiotic surfaces, over primary and transformed cervical epithelial cells, and over cervical tissues in vivo. Biofilms are often associated with chronic infection, which suggests a link between biofilm formation and asymptomatic gonorrhea in women. Proteins involved in anaerobic metabolism and oxidative-stress tolerance are critical for normal biofilm formation of N. gonorrhoeae. Therefore, we examined the spatial profiles of anaerobic respiration in N. gonorrhoeae, using an aniA′-′gfp transcriptional fusion. Nitric oxide (NO) can elicit biofilm dispersal when present at sublethal concentrations in the surrounding medium. Some reports indicate that NO may also encourage biofilm formation at higher, potentially lethal concentrations. NO is produced by polymorphonuclear lymphocytes (PMNs) and cervical endothelial and epithelial cells. Thus, we also examined the effect of NO on N. gonorrhoeae biofilms. We found that anaerobic respiration occurs predominantly in the substratum of gonococcal biofilms and that expression of aniA is induced over time in biofilms. Treatment with high concentrations of a rapid-release NO donor prevents biofilm formation when supplied early in biofilm development but can also enhance biofilm formation once anaerobic respiration is initiated. NO treatment partially restores biofilm formation in an aniA::kan insertion mutant, which suggests that N. gonorrhoeae in biofilms may use NO as a substrate for anaerobic growth but prefer nitrite.

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Environmental and Genetic Determinants of Biofilm Formation inParacoccus denitrificans

mSphere ◽

10.1128/mspheredirect.00350-17 ◽

2017 ◽

Vol 2 (5) ◽

Cited By ~ 10

Author(s):

Santosh Kumar ◽

Stephen Spiro

Keyword(s):

Nitric Oxide ◽

Biofilm Formation ◽

Paracoccus Denitrificans ◽

Binding Protein ◽

Anaerobic Growth ◽

Type I ◽

Content Type ◽

Attached Growth ◽

Polystyrene Surface ◽

Biofilm Development

ABSTRACTThe genome of the denitrifying bacteriumParacoccus denitrificanspredicts the expression of a small heme-containing nitric oxide (NO) binding protein, H-NOX. The genome organization and prior work in other bacteria suggest that H-NOX interacts with a diguanylate cyclase that cyclizes GTP to make cyclic di-GMP (cdGMP). Since cdGMP frequently regulates attached growth as a biofilm, we first established conditions for biofilm development byP. denitrificans. We found that adhesion to a polystyrene surface is strongly stimulated by the addition of 10 mM Ca2+to rich media. The genome encodes at least 11 repeats-in-toxin family proteins that are predicted to be secreted by the type I secretion system (TISS). We deleted the genes encoding the TISS and found that the mutant is almost completely deficient for attached growth. Adjacent to the TISS genes there is a potential open reading frame encoding a 2,211-residue protein with 891 Asp-Ala repeats. This protein is also predicted to bind calcium and to be a TISS substrate, and a mutant specifically lacking this protein is deficient in biofilm formation. By analysis of mutants and promoter reporter fusions, we show that biofilm formation is stimulated by NO generated endogenously by the respiratory reduction of nitrite. A mutant lacking both predicted diguanylate cyclases encoded in the genome overproduces biofilm, implying that cdGMP is a negative regulator of attached growth. Our data are consistent with a model in which there are H-NOX-dependent and -independent pathways by which NO stimulates biofilm formation.IMPORTANCEThe bacteriumParacoccus denitrificansis a model for the process of denitrification, by which nitrate is reduced to dinitrogen during anaerobic growth. Denitrification is important for soil fertility and greenhouse gas emission and in waste and water treatment processes. The ability of bacteria to grow as a biofilm attached to a solid surface is important in many different contexts. In this paper, we report that attached growth ofP. denitrificansis stimulated by nitric oxide, an intermediate in the denitrification pathway. We also show that calcium ions stimulate attached growth, and we identify a large calcium binding protein that is required for growth on a polystyrene surface. We identify components of a signaling pathway through which nitric oxide may regulate biofilm formation. Our results point to an intimate link between metabolic processes and the ability ofP. denitrificansto grow attached to a surface.

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Nitric oxide production duringVibrio choleraeinfection

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1997.273.5.g1160 ◽

1997 ◽

Vol 273 (5) ◽

pp. G1160-G1167 ◽

Cited By ~ 10

Author(s):

Edward N. Janoff ◽

Hiroshi Hayakawa ◽

David N. Taylor ◽

Claudine E. Fasching ◽

Julie R. Kenner ◽

...

Keyword(s):

Nitric Oxide ◽

Blood Flow ◽

Epithelial Cells ◽

Regional Blood Flow ◽

Fluid Secretion ◽

The United States ◽

No Production ◽

Loop Model ◽

Ileal Loop

Vibrio cholerae induces massive intestinal fluid secretion that continues for the life of the stimulated epithelial cells. Enhanced regional blood flow and peristalsis are required to adapt to this obligatory intestinal secretory challenge. Nitric oxide (NO) is a multifunctional molecule that modulates blood flow and peristalsis and possesses both cytotoxic and antibacterial activity. We demonstrate that, compared with those in asymptomatic control subjects, levels of stable NO metabolites ([Formula: see text]/[Formula: see text]) are significantly increased in sera from acutely ill Peruvian patients with natural cholera infection as well as from symptomatic volunteers from the United States infected experimentally with V. cholerae. In a rabbit ileal loop model in vivo, cholera toxin (CT) elicited fluid secretion and dose-dependent increases in levels of[Formula: see text]/[Formula: see text]in the fluid ( P < 0.01). In contrast, lipopolysaccharide (LPS) elicited no such effects when applied to the intact mucosa. NO synthase (NOS) catalytic activity also increased in toxin-exposed tissues ( P< 0.05), predominantly in epithelial cells. The CT-induced NOS activity was Ca2+dependent and was not suppressed by dexamethasone. In conclusion, symptomatic V. cholerae infection induces NO production in humans. In the related animal model, CT, but not LPS, stimulated significant production of NO in association with increases in local Ca2+-dependent NOS activity in the tissues.

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Enhanced Biofilm Formation and Loss of Capsule Synthesis: Deletion of a Putative Glycosyltransferase in Porphyromonas gingivalis

Journal of Bacteriology ◽

10.1128/jb.01685-05 ◽

2006 ◽

Vol 188 (15) ◽

pp. 5510-5523 ◽

Cited By ~ 76

Author(s):

Mary E. Davey ◽

Margaret J. Duncan

Keyword(s):

Porphyromonas Gingivalis ◽

Biofilm Formation ◽

Capsular Polysaccharide ◽

Sequence Similarity ◽

Opportunistic Pathogen ◽

Insertion Mutant ◽

Wild Type ◽

High Sequence Similarity ◽

Biofilm Development

ABSTRACT Periodontitis is a biofilm-mediated disease. Porphyromonas gingivalis is an obligate anaerobe consistently associated with severe manifestations of this disease. As an opportunistic pathogen, the ability to proliferate within and disseminate from subgingival biofilm (plaque) is central to its virulence. Here, we report the isolation of a P. gingivalis transposon insertion mutant altered in biofilm development and the reconstruction and characterization of this mutation in three different wild-type strains. The mutation responsible for the altered biofilm phenotype was in a gene with high sequence similarity (∼61%) to a glycosyltransferase gene. The gene is located in a region of the chromosome that includes up to 16 genes predicted to be involved in the synthesis and transport of capsular polysaccharide. The phenotype of the reconstructed mutation in all three wild-type backgrounds is that of enhanced biofilm formation. In addition, in strain W83, a strain that is encapsulated, the glycosyltransferase mutation resulted in a loss of capsule. Further experiments showed that the W83 mutant strain was more hydrophobic and exhibited increased autoaggregation. Our results indicate that we have identified a gene involved in capsular-polysaccharide synthesis in P. gingivalis and that the production of capsule prevented attachment and the initiation of in vitro biofilm formation on polystyrene microtiter plates.

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Role of Type IV Pilins in Persistence of Vibrio vulnificus in Crassostrea virginica Oysters

Applied and Environmental Microbiology ◽

10.1128/aem.00641-07 ◽

2007 ◽

Vol 73 (15) ◽

pp. 5041-5044 ◽

Cited By ~ 37

Author(s):

Rohinee N. Paranjpye ◽

Asta B. Johnson ◽

Anne E. Baxter ◽

Mark S. Strom

Keyword(s):

United States ◽

Epithelial Cells ◽

Crassostrea Virginica ◽

Biofilm Formation ◽

Vibrio Vulnificus ◽

The United States ◽

Type Iv ◽

Abiotic Surfaces ◽

Prepilin Peptidase

ABSTRACT Vibrio vulnificus is part of the natural estuarine microflora and accumulates in shellfish through filter feeding. It is responsible for the majority of seafood-associated fatalities in the United States mainly through consumption of raw oysters. Previously we have shown that a V. vulnificus mutant unable to express PilD, the type IV prepilin peptidase, does not express pili on the surface of the bacterium and is defective in adherence to human epithelial cells (R. N. Paranjpye, J. C. Lara, J. C. Pepe, C. M. Pepe, and M. S. Strom, Infect. Immun. 66:5659-5668, 1998). A mutant unable to express one of the type IV pilins, PilA, is also defective in adherence to epithelial cells as well as biofilm formation on abiotic surfaces (R. N. Paranjpye and M. S. Strom, Infect. Immun. 73:1411-1422, 2005). In this study we report that the loss of PilD or PilA significantly reduces the ability of V. vulnificus to persist in Crassostrea virginica over a 66-h interval, strongly suggesting that pili expressed by this bacterium play a role in colonization or persistence in oysters.

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Inactivations of rsbU and sarA by IS256 Represent Novel Mechanisms of Biofilm Phenotypic Variation in Staphylococcus epidermidis

Journal of Bacteriology ◽

10.1128/jb.186.18.6208-6219.2004 ◽

2004 ◽

Vol 186 (18) ◽

pp. 6208-6219 ◽

Cited By ~ 84

Author(s):

Kevin M. Conlon ◽

Hilary Humphreys ◽

James P. O'Gara

Keyword(s):

Phenotypic Variation ◽

Staphylococcus Epidermidis ◽

Biofilm Formation ◽

Insertion Mutant ◽

Regulation Of Transcription ◽

Phase Variable ◽

Rapid Rate ◽

Global Regulation ◽

Repeated Passage ◽

Biofilm Development

ABSTRACT Expression of ica operon-mediated biofilm formation in Staphylococcus epidermidis RP62A is subject to phase variable regulation. Reversible transposition of IS256 into icaADBC or downregulation of icaADBC expression are two important mechanisms of biofilm phenotypic variation. Interestingly, the presence of IS256 was generally associated with a more rapid rate of phenotypic variation, suggesting that IS256 insertions outside the ica locus may affect ica transcription. Consistent with this, we identified variants with diminished ica expression, which were associated with IS256 insertions in the σB activator rsbU or sarA. Biofilm development and ica expression were activated only by ethanol and not NaCl in rsbU::IS256 insertion variants, which were present in ∼11% of all variants. σB activity was impaired in rsbU::IS256 variants, as evidenced by reduced expression of the σB-regulated genes asp23, csb9, and rsbV. Moreover, expression of sarA, which is σB regulated, and SarA-regulated RNAIII were also suppressed. A biofilm-forming phenotype was restored to rsbU::IS256 variants only after repeated passage and was not associated with IS256 excision from rsbU. Only one sarA::IS256 insertion mutant was identified among 43 biofilm-negative variants. Both NaCl and ethanol-activated ica expression in this sarA::IS256 variant, but only ethanol increased biofilm development. Unlike rsbU::IS256 variants, reversion of the sarA::IS256 variant to a biofilm-positive phenotype was accompanied by precise excision of IS256 from sarA and restoration of normal ica expression. These data identify new roles for IS256 in ica and biofilm phenotypic variation and demonstrate the capacity of this element to influence the global regulation of transcription in S. epidermidis.

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Cost-Effectiveness of Inhaled Nitric Oxide in the Treatment of Neonatal Respiratory Failure in the United States

PEDIATRICS ◽

10.1542/peds.112.6.1351 ◽

2003 ◽

Vol 112 (6) ◽

pp. 1351-1360 ◽

Cited By ~ 27

Author(s):

D. C. Angus ◽

G. Clermont ◽

R. S. Watson ◽

W. T. Linde-Zwirble ◽

R. H. Clark ◽

...

Keyword(s):

Nitric Oxide ◽

United States ◽

Cost Effectiveness ◽

Respiratory Failure ◽

The United States ◽

Inhaled Nitric Oxide

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Phylogenomic analysis of Clostridioides difficile ribotype 106 strains reveals novel genetic islands and emergent phenotypes

Scientific Reports ◽

10.1038/s41598-020-79123-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Bryan Angelo P. Roxas ◽

Jennifer Lising Roxas ◽

Rachel Claus-Walker ◽

Anusha Harishankar ◽

Asad Mansoor ◽

...

Keyword(s):

Biofilm Formation ◽

Diarrheal Disease ◽

The United States ◽

Genomic Islands ◽

Rapid Expansion ◽

Phylogenomic Analysis ◽

Community Settings ◽

Hamster Model ◽

Clostridioides Difficile ◽

Healthcare Associated

AbstractClostridioides difficile infection (CDI) is a major healthcare-associated diarrheal disease. Consistent with trends across the United States, C. difficile RT106 was the second-most prevalent molecular type in our surveillance in Arizona from 2015 to 2018. A representative RT106 strain displayed robust virulence and 100% lethality in the hamster model of acute CDI. We identified a unique 46 KB genomic island (GI1) in all RT106 strains sequenced to date, including those in public databases. GI1 was not found in its entirety in any other C. difficile clade, or indeed, in any other microbial genome; however, smaller segments were detected in Enterococcus faecium strains. Molecular clock analyses suggested that GI1 was horizontally acquired and sequentially assembled over time. GI1 encodes homologs of VanZ and a SrtB-anchored collagen-binding adhesin, and correspondingly, all tested RT106 strains had increased teicoplanin resistance, and a majority displayed collagen-dependent biofilm formation. Two additional genomic islands (GI2 and GI3) were also present in a subset of RT106 strains. All three islands are predicted to encode mobile genetic elements as well as virulence factors. Emergent phenotypes associated with these genetic islands may have contributed to the relatively rapid expansion of RT106 in US healthcare and community settings.

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Sodium Salicylate Influences the Pseudomonas aeruginosa Biofilm Structure and Susceptibility Towards Silver

International Journal of Molecular Sciences ◽

10.3390/ijms22031060 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1060

Author(s):

Erik Gerner ◽

Sofia Almqvist ◽

Peter Thomsen ◽

Maria Werthén ◽

Margarita Trobos

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Sodium Salicylate ◽

Treatment Strategies ◽

Cell Aggregation ◽

Wound Fluid ◽

Global Challenge ◽

3D Collagen ◽

Biofilm Development ◽

Biofilm Structure

Hard-to-heal wounds are typically infected with biofilm-producing microorganisms, such as Pseudomonas aeruginosa, which strongly contribute to delayed healing. Due to the global challenge of antimicrobial resistance, alternative treatment strategies are needed. Here, we investigated whether inhibition of quorum sensing (QS) by sodium salicylate in different P. aeruginosa strains (QS-competent, QS-mutant, and chronic wound strains) influences biofilm formation and tolerance to silver. Biofilm formation was evaluated in simulated serum-containing wound fluid in the presence or absence of sodium salicylate (NaSa). Biofilms were established using a 3D collagen-based biofilm model, collagen coated glass, and the Calgary biofilm device. Furthermore, the susceptibility of 48-h-old biofilms formed by laboratory and clinical strains in the presence or absence of NaSa towards silver was evaluated by assessing cell viability. Biofilms formed in the presence of NaSa were more susceptible to silver and contained reduced levels of virulence factors associated with biofilm development than those formed in the absence of NaSa. Biofilm aggregates formed by the wild-type but not the QS mutant strain, were smaller and less heterogenous in size when grown in cultures with NaSa compared to control. These data suggest that NaSa, via a reduction of cell aggregation in biofilms, allows the antiseptic to become more readily available to cells.

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The Role of Exopolysaccharides in Oral Biofilms

Journal of Dental Research ◽

10.1177/0022034519845001 ◽

2019 ◽

Vol 98 (7) ◽

pp. 739-745 ◽

Cited By ~ 7

Author(s):

C. Cugini ◽

M. Shanmugam ◽

N. Landge ◽

N. Ramasubbu

Keyword(s):

Biofilm Formation ◽

Bacterial Colonization ◽

Super Resolution ◽

Extracellular Proteins ◽

Extracellular Polysaccharides ◽

Oral Biofilms ◽

The Matrix ◽

Oral Microbes ◽

Biofilm Development

The oral cavity contains a rich consortium of exopolysaccharide-producing microbes. These extracellular polysaccharides comprise a major component of the oral biofilm. Together with extracellular proteins, DNA, and lipids, they form the biofilm matrix, which contributes to bacterial colonization, biofilm formation and maintenance, and pathogenesis. While a number of oral microbes have been studied in detail with regard to biofilm formation and pathogenesis, the exopolysaccharides have been well characterized for only select organisms, namely Streptococcus mutans and Aggregatibacter actinomycetemcomitans. Studies on the exopolysaccharides of other oral organisms, however, are in their infancy. In this review, we present the current research on exopolysaccharides of oral microbes regarding their biosynthesis, regulation, contributions to biofilm formation and stability of the matrix, and immune evasion. In addition, insight into the role of exopolysaccharides in biofilms is highlighted through the evaluation of emerging techniques such as pH probing of biofilm colonies, solid-state nuclear magnetic resonance for macromolecular interactions within biofilms, and super-resolution microscopy analysis of biofilm development. Finally, exopolysaccharide as a potential nutrient source for species within a biofilm is discussed.

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