Aeromonas Flagella (Polar and Lateral) Are Enterocyte Adhesins That Contribute to Biofilm Formation on Surfaces

ABSTRACT Aeromonas spp. (gram-negative, aquatic bacteria which include enteropathogenic strains) have two distinct flagellar systems, namely a polar flagellum for swimming in liquid and multiple lateral flagella for swarming over surfaces. Only ∼60% of mesophilic strains can produce lateral flagella. To evaluate flagellar contributions to Aeromonas intestinal colonization, we compared polar and lateral flagellar mutant strains of a diarrheal isolate of Aeromonas caviae for the ability to adhere to the intestinal cell lines Henle 407 and Caco-2, which have the characteristic features of human intestinal enterocytes. Strains lacking polar flagella were virtually nonadherent to these cell lines, while loss of the lateral flagellum decreased adherence by ∼60% in comparison to the wild-type level. Motility mutants (unable to swim or swarm in agar assays) had adhesion levels of ∼50% of wild-type values, irrespective of their flagellar expression. Flagellar mutant strains were also evaluated for the ability to form biofilms in a borosilicate glass tube model which was optimized for Aeromonas spp. (broth inoculum, with a 16- to 20-h incubation at 37°C). All flagellar mutants showed a decreased ability to form biofilms (at least 30% lower than the wild type). For the chemotactic motility mutant cheA, biofilm formation decreased >80% from the wild-type level. The complementation of flagellar phenotypes (polar flagellar mutants) restored biofilms to wild-type levels. We concluded that both flagellar types are enterocyte adhesins and need to be fully functional for optimal biofilm formation.

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vpaH, a Gene Encoding a Novel Histone-Like Nucleoid Structure-Like Protein That Was Possibly Horizontally Acquired, Regulates the Biogenesis of Lateral Flagella in trh-Positive Vibrio parahaemolyticus TH3996

Infection and Immunity ◽

10.1128/iai.73.9.5754-5761.2005 ◽

2005 ◽

Vol 73 (9) ◽

pp. 5754-5761 ◽

Cited By ~ 19

Author(s):

Kwon-Sam Park ◽

Michiko Arita ◽

Tetsuya Iida ◽

Takeshi Honda

Keyword(s):

Biofilm Formation ◽

Vibrio Parahaemolyticus ◽

Type Strain ◽

Wild Type Strain ◽

Enteric Bacteria ◽

Wild Type ◽

Gene Encoding ◽

Lateral Flagellum ◽

Global Gene Regulation ◽

Nucleoid Structure

ABSTRACT A histone-like nucleoid structure (H-NS) is a major component of the bacterial nucleoid and plays a crucial role in the global gene regulation of enteric bacteria. Here, we cloned and characterized the gene for the H-NS-like protein VpaH in Vibrio parahaemolyticus. vpaH encodes a protein of 134 amino acids that shows approximately 55%, 54%, and 41% identities with VicH in Vibrio cholerae, H-NS in V. parahaemolyticus, and H-NS in Escherichia coli, respectively. The vpaH gene was found in only trh-positive V. parahaemolyticus strains and not in Kanagawa-positive or in trh-negative environmental strains. Moreover, the G+C content of the vpaH gene was 38.6%, which is lower than the average G+C content of the whole genome of this bacterium (45.4%). These data suggest that vpaH was transmitted to trh-possessing V. parahaemolyticus strains by lateral transfer. The vpaH gene was located about 2.6 kb downstream of the trh gene, in the convergent direction of the trh transcription. An in-frame deletion mutant of vpaH lacked motility on semisolid motility assay plates. Western blot analysis and electron microscopy observations revealed that the mutant was deficient in lateral flagella biogenesis, whereas there was no defect in the expression of polar flagella. Additionally, the vpaH mutant showed a decreased adherence to HeLa cells and a decrease in biofilm formation compared with the wild-type strain. Introduction of the vpaH gene in the vpaH-negative strain increased the expression of lateral flagella compared with the wild-type strain. In conclusion, our findings suggest that VpaH affects lateral flagellum biogenesis in trh-positive V. parahaemolyticus strain TH3996.

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Role of FAD-I in Fusobacterial Interspecies Interaction and Biofilm Formation

Microorganisms ◽

10.3390/microorganisms8010070 ◽

2020 ◽

Vol 8 (1) ◽

pp. 70 ◽

Cited By ~ 1

Author(s):

Bhumika Shokeen ◽

Jane Park ◽

Emily Duong ◽

Sonam Rambhia ◽

Manash Paul ◽

...

Keyword(s):

Biofilm Formation ◽

Fusobacterium Nucleatum ◽

Interspecies Interaction ◽

Wild Type ◽

Oral Epithelial Cells ◽

Small Proteins ◽

Mutant Strains ◽

Oral Epithelial ◽

Wild Type Parent

RadD, a major adhesin of oral fusobacteria, is part of a four-gene operon encoding the small lipoprotein FAD-I and two currently uncharacterized small proteins encoded by the rapA and rapB genes. Previously, we described a role for FAD-I in the induction of human B-defensin 2 (hBD2) upon contact with oral epithelial cells. Here, we investigated potential roles for fad-I, rapA, and rapB in interspecies interaction and biofilm formation. Gene inactivation mutants were generated for each of these genes in the nucleatum and polymorphum subspecies of Fusobacterium nucleatum and characterized for their adherence to partner species, biofilm formation, and operon transcription. Binding to Streptococcus gordonii was increased in all mutant strains with Δfad-I having the most significant effect. This increased adherence was directly proportional to elevated radD transcript levels and resulted in significantly different architecture and height of the biofilms formed by Δfad-I and S. gordonii compared to the wild-type parent. In conclusion, FAD-I is important for fusobacterial interspecies interaction as its lack leads to increased production of the RadD adhesin suggesting a role of FAD-I in its regulation. This regulatory effect does not require the presence of functional RadD.

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Role of Streptococcus gordoniiAmylase-Binding Protein A in Adhesion to Hydroxyapatite, Starch Metabolism, and Biofilm Formation

Infection and Immunity ◽

10.1128/iai.69.11.7046-7056.2001 ◽

2001 ◽

Vol 69 (11) ◽

pp. 7046-7056 ◽

Cited By ~ 61

Author(s):

Jeffrey D. Rogers ◽

Robert J. Palmer ◽

Paul E. Kolenbrander ◽

Frank A. Scannapieco

Keyword(s):

Biofilm Formation ◽

Binding Protein ◽

Protein A ◽

Scatchard Analysis ◽

Wild Type ◽

Salivary Amylase ◽

Carbohydrate Source ◽

Whole Saliva ◽

Mutant Strains ◽

Type Strains

ABSTRACT Interactions between bacteria and salivary components are thought to be important in the establishment and ecology of the oral microflora. α-Amylase, the predominant salivary enzyme in humans, binds to Streptococcus gordonii, a primary colonizer of the tooth. Previous studies have implicated this interaction in adhesion of the bacteria to salivary pellicles, catabolism of dietary starches, and biofilm formation. Amylase binding is mediated at least in part by the amylase-binding protein A (AbpA). To study the function of this protein, an erythromycin resistance determinant [erm(AM)] was inserted within the abpAgene of S. gordonii strains Challis and FAS4 by allelic exchange, resulting in abpA mutant strains Challis-E1 and FAS4-E1. Comparison of the wild-type and mutant strains did not reveal any significant differences in colony morphology, biochemical metabolic profiles, growth in complex or defined media, surface hydrophobicity, or coaggregation properties. Scatchard analysis of adhesion isotherms demonstrated that the wild-type strains adhered better to human parotid-saliva- and amylase-coated hydroxyapatite than did the AbpA mutants. In contrast, the mutant strains bound to whole-saliva-coated hydroxyapatite to a greater extent than did the wild-type strains. While the wild-type strains preincubated with purified salivary amylase grew well in defined medium with potato starch as the sole carbohydrate source, the AbpA mutants did not grow under the same conditions even after preincubation with amylase. In addition, the wild-type strain produced large microcolonies in a flow cell biofilm model, while the abpA mutant strains grew much more poorly and produced relatively small microcolonies. Taken together, these results suggest that AbpA ofS. gordonii functions as an adhesin to amylase-coated hydroxyapatite, in salivary-amylase-mediated catabolism of dietary starches and in human saliva-supported biofilm formation by S. gordonii.

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Consequences of Reduction of Klebsiella pneumoniae Capsule Expression on Interactions of This Bacterium with Epithelial Cells

Infection and Immunity ◽

10.1128/iai.67.2.554-561.1999 ◽

1999 ◽

Vol 67 (2) ◽

pp. 554-561 ◽

Cited By ~ 101

Author(s):

Sabine Favre-Bonte ◽

Bernard Joly ◽

Christiane Forestier

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Klebsiella Pneumoniae ◽

Cell Lines ◽

Type Strain ◽

Wild Type Strain ◽

Conjugative Plasmid ◽

Intestinal Cell ◽

Wild Type ◽

Defective Mutant

ABSTRACT Most Klebsiella pneumoniae clinical isolates are fully encapsulated and adhere in vitro to intestinal cell lines with an aggregative pattern. In this study, the influence of the capsule on interactions with epithelial cells was investigated by creating an isogenic mutant defective in the synthesis of the capsule. Determination of the uronic acid content of bacterial extracts confirmed that the mutant did not produce capsular polysaccharides whereas, with the wild-type strain, the level of encapsulation was growth phase dependent and reached a maximum during the lag and early log phases. Assays performed with different epithelial cell lines, Int-407, A-549, and HEp-2, showed that the capsule-defective mutant demonstrated greater adhesion than did the wild-type strain and that the aggregative pattern was maintained, indicating that the capsule was not related to the adhesion phenotype. In contrast, when the mucus-producing HT-29-MTX cells were used, the encapsulated wild-type strain adhered more strongly than did the capsule-defective mutant. No invasion properties were observed with any of the capsular phenotypes or cell lines used. The K. pneumoniae adhesin CF29K was detected by Western blot analysis and enzyme-linked immunosorbent assay on the surface of transconjugants obtained after transfer of a conjugative plasmid harboring the CF29K-encoding genes into both the wild-type and the capsule-defective strains. The amounts of adhesin detected were greater in the capsule-defective background strain than in the wild-type encapsulated strain and were associated with an increase in the level of adhesion to Caco-2 cells. Moreover, RNA slot blot experiments showed that transcription of the adhesin-encoding gene was markedly increased in the capsule-defective mutant compared to the wild-type encapsulated background. These results suggest (i) that the capsule plays an active role during the initial steps of the pathogenesis by interacting with mucus-producing cells but is subsequently not required for the adhesin-related interaction with the epithelial cell surface and (ii) that the expression of the adhesin is modulated by the presence of a capsule at a transcriptional level.

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Mutagenic strategies against luxS gene affect the early stage of biofilm formation of Campylobacter jejuni

10.21203/rs.3.rs-204944/v1 ◽

2021 ◽

Author(s):

Martin Tereň ◽

Ekaterina Shagieva ◽

Lucie Vondrakova ◽

Jitka Viktorova ◽

Viviana Svarcova ◽

...

Keyword(s):

Campylobacter Jejuni ◽

Biofilm Formation ◽

Early Stage ◽

Wild Type ◽

Genetic Determinants ◽

Autoinducer 2 ◽

Naturally Occurring ◽

Mutant Strains ◽

The Impact

Abstract Currently, it is clear that the luxS gene has an impact on the process of biofilm formation in Campylobacter jejuni. However, even within the species naturally occurring strains of Campylobacter lacking the luxS gene exist, which can form biofilms. In order to better understand the genetic determinants and the role of quorum sensing through the LuxS/AI-2 pathway in biofilm formation, a set of mutant/complemented strains of C. jejuni 81–176 were prepared. Additionally, the impact of the mutagenic strategy used against the luxS gene was investigated. Biofilm formation was affected by both the presence and absence of the luxS gene, and by the mutagenic strategy used. Analysis by CLSM showed that all mutant strains formed significantly less biofilm mass when compared to the wild-type. Interestingly, the deletion mutant (∆luxS) showed a larger decrease in biofilm mass than the substitution (∙luxS) and insertional inactivated (⸬luxS) mutants, even though all the mutant strains lost the ability to produce autoinducer-2 molecules. Moreover, the biofilm of the ∆luxS mutant lacked the characteristic microcolonies observed in all other strains. The complementation of all mutant strains resulted in restored ability to produce AI-2, to form a complex biofilm, and to develop microcolonies at the level of the wild-type.

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Comparative genomics of wild-type and laboratory-evolved biofilm-overproducing Deinococcus metallilatus strains

Microbial Genomics ◽

10.1099/mgen.0.000464 ◽

2020 ◽

Vol 6 (12) ◽

Author(s):

Chulwoo Park ◽

Bora Shin ◽

Wonjae Kim ◽

Hoon Cheong ◽

Soyoon Park ◽

...

Keyword(s):

Cell Death ◽

Biofilm Formation ◽

Type Species ◽

High Rate ◽

Wild Type ◽

Content Type ◽

Link Type ◽

Extracellular Matrix Components ◽

Mutant Strains ◽

Mutant Cells

Deinococcus metallilatus MA1002 was exposed to ultraviolet radiation to generate mutants with enhanced biofilm production. Two strains (nos 5 and 6) were then selected based on their high biofilm formation, as well as their possession of higher concentrations of extracellular matrix components (eDNA, protein and saccharides) than the wild-type (WT). Genomic sequencing revealed the presence of large genome deletions in a secondary chromosome in the mutants. Expression analyses of the WT and mutant strains indicated the upregulation of genes associated with exopolysaccharide synthesis and stress response. The mutant strains showed high mortality in glucose-supplemented (TYG) medium; however, cell death and biofilm formation were not increased in mutant cells grown under acetate- or glyoxylate-added media, suggesting that metabolic toxicity during glucose metabolism induced a high rate of cell death but improved biofilm formation in mutant strains. In damaged cells, eDNAs contributed to the enhanced biofilm formation of D. metallilatus .

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Glycoproteomics of Haloferax volcanii reveals an extensive glycoproteome and concurrence of different N-glycosylation pathways

10.1101/2021.01.21.427637 ◽

2021 ◽

Author(s):

Stefan Schulze ◽

Friedhelm Pfeiffer ◽

Benjamin A. Garcia ◽

Mechthild Pohlschroder

Keyword(s):

Biofilm Formation ◽

Cell Biology ◽

Culture Conditions ◽

Haloferax Volcanii ◽

Spectrometric Analysis ◽

Wild Type ◽

Cellular Processes ◽

Phenotypic Characterisation ◽

Mutant Strains ◽

Glycosylation Pathway

AbstractGlycosylation is one of the most complex post-translational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here we extend the phenotypic characterisation of N-glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project, we identify the largest archaeal glycoproteome described so far. We further show that different N-glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx. volcanii N-glycoproteome revealed here provides new insights into the roles of N-glycosylation in archaeal cell biology.

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TatD DNases Contribute to Biofilm Formation and Virulence in Trueperella pyogenes

Frontiers in Microbiology ◽

10.3389/fmicb.2021.758465 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zehui Zhang ◽

Yinfeng Liang ◽

Lihui Yu ◽

Menghan Chen ◽

Yuru Guo ◽

...

Keyword(s):

Biofilm Formation ◽

Wild Type Strain ◽

Divalent Cations ◽

Bacterial Load ◽

Economic Losses ◽

Wild Type ◽

Trueperella Pyogenes ◽

Mutant Strains ◽

Sequence Characteristics

TatD DNases are conserved proteins in a variety of organisms and are considered potential virulence factors in Plasmodium falciparum and Streptococcus pneumoniae. However, the function of TatD DNases has not yet been determined in Trueperella pyogenes, which causes various infections in animals and leads to economic losses. In this study, we describe the roles of TatD DNases in T. pyogenes (TpTatDs). A bioinformatics analysis was performed to investigate the sequence characteristics of TpTatDs, and then the ability of recombinant TatD proteins to hydrolyze DNA was determined in the presence of divalent cations. Moreover, we constructed tatD-deficient mutants. The biofilms formed by the wild-type and mutant strains were observed under a microscope. The mortality and bacterial load in the spleen of mice infected with the wild-type strain and tatD-deficient mutants were determined to obtain insights into the role of TatDs in the virulence of T. pyogenes. Two TatD DNases were identified in T. pyogenes. They were Mg2+-dependent DNases and exhibited DNA endonuclease activity. Compared with those formed by the parental strain, biofilms formed by mutants showed a significantly reduced thickness and biomass. Moreover, mutants produced a lower bacterial load in the spleen of mice and compromised virulence. Our data indicated that TatD DNases in T. pyogenes are involved in biofilm formation and required for virulence during infections.

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Quorum-Sensing Regulation of the Biofilm Matrix Genes (pel) of Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00137-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 5383-5386 ◽

Cited By ~ 187

Author(s):

Yumiko Sakuragi ◽

Roberto Kolter

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Biofilm Formation ◽

Homoserine Lactone ◽

Wild Type ◽

Wild Type Level ◽

Dna Release ◽

Biofilm Development ◽

Development Regulation ◽

Biofilm Matrix

ABSTRACT Quorum sensing (QS) has been previously shown to play an important role in the development of Pseudomonas aeruginosa biofilms (D. G. Davies et al., Science 280:295-298, 1998). Although QS regulation of swarming and DNA release has been shown to play important roles in biofilm development, regulation of genes directly involved in biosynthesis of biofilm matrix has not been described. Here, transcription of the pel operon, essential for the production of a glucose-rich matrix exopolysaccharide, is shown to be greatly reduced in lasI and rhlI mutants. Chemical complementation of the lasI mutant with 3-oxo-dodecanoyl homoserine lactone restores pel transcription to the wild-type level and biofilm formation ability. These findings thus connect QS signaling and transcription of genes responsible for biofilm matrix biosynthesis.

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The Exopolysaccharide of Xylella fastidiosa Is Essential for Biofilm Formation, Plant Virulence, and Vector Transmission

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-09-12-0211-r ◽

2013 ◽

Vol 26 (9) ◽

pp. 1044-1053 ◽

Cited By ~ 42

Author(s):

N. Killiny ◽

R. Hernandez Martinez ◽

C. Korsi Dumenyo ◽

D. A. Cooksey ◽

R. P. P. Almeida

Keyword(s):

Biofilm Formation ◽

Pathogenic Bacteria ◽

Xylella Fastidiosa ◽

Growth Characteristics ◽

Gas Chromatography Mass Spectrometry ◽

Wild Type ◽

Mutant Strains ◽

Plant Movement

Exopolysaccharides (EPS) synthesized by plant-pathogenic bacteria are generally essential for virulence. The role of EPS produced by the vector-transmitted bacterium Xylella fastidiosa was investigated by knocking out two genes implicated in the EPS biosynthesis, gumD and gumH. Mutant strains were affected in growth characteristics in vitro, including adhesion to surfaces and biofilm formation. In addition, different assays were used to demonstrate that the mutant strains produced significantly less EPS compared with the wild type. Furthermore, gas chromatography–mass spectrometry showed that both mutant strains did not produce oligosaccharides. Biologically, the mutants were deficient in movement within plants, resulting in an avirulent phenotype. Additionally, mutant strains were affected in transmission by insects: they were very poorly transmitted by and retained within vectors. The gene expression profile indicated upregulation of genes implicated in cell-to-cell signaling and adhesins while downregulation in genes was required for within-plant movement in EPS-deficient strains. These results suggest an essential role for EPS in X. fastidiosa interactions with both plants and insects.

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