scholarly journals Role of HrpA in Biofilm Formation of Neisseria meningitidis and Regulation of the hrpBAS Transcripts

2009 ◽  
Vol 77 (6) ◽  
pp. 2285-2293 ◽  
Author(s):  
R. Brock Neil ◽  
Michael A. Apicella
Keyword(s):  
Neisseria Meningitidis ◽  
Biofilm Formation ◽  
Cell Contact ◽  
Wild Type ◽  
Secretion Systems ◽  
Biofilm Growth ◽  
Bronchial Epithelial ◽  
Gram Negative Organisms ◽  
Biofilm Structure

ABSTRACT Two-partner secretion systems of gram-negative organisms are utilized in adherence, invasion, and biofilm formation. The HrpAB proteins of Neisseria meningitidis are members of a two-partner secretion system, and HrpA is established as being important to adherence and intracellular escape. This study set out to determine the expression pattern of members of the hrpBAS putative operon and to find a functional role for the HrpA protein. The upregulation of these genes was found in situations of anaerobiosis and cell contact. These observations prompted the study of the function of HrpA in biofilms on human bronchial epithelial cells. HrpA mutants in encapsulated and unencapsulated NMB strains demonstrated biofilm growth equivalent to that of the wild-type strain at 6 h but a decreased ability to form biofilms at 48 h. Biofilms formed by hrpA mutants for 48 h on collagen-coated coverslips demonstrated significant reductions compared to those of wild-type strains. Taken together, these observations imply a role for HrpA in the biofilm structure. Further analysis demonstrated the presence of HrpA on the surface of the bacterium.

scholarly journals Deletion of the rpoZ gene, encoding the ω subunit of RNA polymerase, results in pleiotropic surface-related phenotypes in Mycobacterium smegmatis

Microbiology ◽  
2006 ◽  
Vol 152 (6) ◽  
pp. 1741-1750 ◽  
Author(s):  
Renjith Mathew ◽  
Raju Mukherjee ◽  
Radhakrishnan Balachandar ◽  
Dipankar Chatterji
Keyword(s):  
Rna Polymerase ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Physiological Role ◽  
Wild Type ◽  
Gene Encoding ◽  
Biofilm Growth ◽  
Mutant Bacterium ◽  
Sliding Motility

The ω subunit, the smallest subunit of bacterial RNA polymerase, is known to be involved in maintaining the conformation of the β′ subunit and aiding its recruitment to the rest of the core enzyme assembly in Escherichia coli. It has recently been shown in Mycobacterium smegmatis, by creating a deletion mutation of the rpoZ gene encoding ω, that the physiological role of the ω subunit also includes providing physical protection to β′. Interestingly, the mutant had altered colony morphology. This paper demonstrates that the mutant mycobacterium has pleiotropic phenotypes including reduced sliding motility and defective biofilm formation. Analysis of the spatial arrangement of biofilms by electron microscopy suggests that the altered phenotype of the mutant arises from a deficiency in generation of extracellular matrix. Complementation of the mutant strain with a copy of the wild-type rpoZ gene integrated in the bacterial chromosome restored both sliding motility and biofilm formation to the wild-type state, unequivocally proving the role of ω in the characteristics observed for the mutant bacterium. Analysis of the cell wall composition demonstrated that the mutant bacterium had an identical glycopeptidolipid profile to the wild-type, but failed to synthesize the short-chain mycolic acids characteristic of biofilm growth in M. smegmatis.

scholarly journals The CpAL Quorum Sensing System Regulates Production of Hemolysins CPA and PFO To Build Clostridium perfringens Biofilms

2015 ◽  
Vol 83 (6) ◽  
pp. 2430-2442 ◽  
Author(s):  
Jorge E. Vidal ◽  
Joshua R. Shak ◽  
Adrian Canizalez-Roman
Keyword(s):  
Quorum Sensing ◽  
Clostridium Perfringens ◽  
Biofilm Formation ◽  
Extracellular Dna ◽  
Wild Type ◽  
Content Type ◽  
Type C ◽  
Enteritis Necroticans ◽  
Biofilm Structure

Clostridium perfringensstrains produce severe diseases, including myonecrosis and enteritis necroticans, in humans and animals. Diseases are mediated by the production of potent toxins that often damage the site of infection, e.g., skin epithelium during myonecrosis. In planktonic cultures, the regulation of important toxins, such as CPA, CPB, and PFO, is controlled by theC. perfringensAgr-like (CpAL) quorum sensing (QS) system. Strains also encode a functional LuxS/AI-2 system. AlthoughC. perfringensstrains form biofilm-like structures, the regulation of biofilm formation is poorly understood. Therefore, our studies investigated the role of CpAL and LuxS/AI-2 QS systems and of QS-regulated factors in controlling the formation of biofilms. We first demonstrate that biofilm production by reference strains differs depending on the culture medium. Increased biomass correlated with the presence of extracellular DNA in the supernatant, which was released by lysis of a fraction of the biofilm population and planktonic cells. Whereas ΔagrBmutant strains were not able to produce biofilms, a ΔluxSmutant produced wild-type levels. The transcript levels of CpAL-regulatedcpaandpfoAgenes, but notcpb, were upregulated in biofilms compared to planktonic cultures. Accordingly, Δcpaand ΔpfoAmutants, in type A (S13) or type C (CN3685) backgrounds, were unable to produce biofilms, whereas CN3685Δcpbmade wild-type levels. Biofilm formation was restored in complemented Δcpa/cpaand ΔpfoA/pfoAstrains. Confocal microscopy studies further detected CPA partially colocalizing with eDNA on the biofilm structure. Thus, CpAL regulates biofilm formation inC. perfringensby increasing levels of certain toxins required to build biofilms.

scholarly journals An inside look at a biofilm: Pseudomonas aeruginosa flagella bio-tracking

2020 ◽  
Author(s):  
Eden Ozer ◽  
Karin Yaniv ◽  
Einat Chetrit ◽  
Anastasya Boyarski ◽  
Michael M. Meijler ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Opportunistic Pathogen ◽  
Unnatural Amino Acid ◽  
Model Organisms ◽  
Wild Type ◽  
Bacterial Flagella ◽  
Knockout Strain ◽  
Biofilm Structure

AbstractThe opportunistic pathogen, Pseudomonas aeruginosa, a flagellated bacterium, is one of the top model organisms for studying biofilm formation. In order to elucidate the role of the bacteria flagella in biofilm formation, we developed a new tool for flagella bio-tracking. We have site-specifically labeled the bacterial flagella by incorporating an unnatural amino acid into the flagella monomer via genetic code expansion. This enabled us to label and track the bacterial flagella during biofilm maturation. Direct, live imaging revealed for the first-time presence and synthesis of flagella throughout the biofilm lifecycle. To ascertain the possible role of the flagella in the strength of a biofilm we produced a “flagella knockout” strain and compared its biofilm to that of the wild type strain. Results showed a one order of magnitude stronger biofilm structure in the wild type in comparison to the flagella knockout strain. This suggests a newly discovered structural role for bacterial flagella in biofilm structure, possibly acting as a scaffold. Based on our findings we suggest a new model for biofilm maturation dynamic and underscore the importance of direct evidence from within the biofilm.

scholarly journals Disruption of c-di-GMP signaling networks unlocks cryptic expression of secondary metabolites during biofilm growth in Burkholderia pseudomallei

2021 ◽  
Author(s):  
Grace I Borlee ◽  
Mihnea R. Mangalea ◽  
Kevin H. Martin ◽  
Brooke A. Plumley ◽  
Samuel J. Golon ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Biofilm Formation ◽  
Burkholderia Pseudomallei ◽  
Gene Clusters ◽  
Etiologic Agent ◽  
Regulatory Control ◽  
Colony Morphology ◽  
Secretion Systems ◽  
Biofilm Growth ◽  
Conditional Expression

The regulation and production of secondary metabolites during biofilm growth of Burkholderia spp. is not well understood. To learn more about the crucial role and regulatory control of cryptic molecules produced during biofilm growth, we disrupted c-di-GMP signaling in Burkholderia pseudomallei, a soil-borne bacterial saprophyte and the etiologic agent of melioidosis. Our approach to these studies combined transcriptional profiling with genetic deletions that targeted key c-di-GMP regulatory components to characterize responses to changes in temperature. Mutational analyses and conditional expression studies of c-di-GMP genes demonstrates their contribution to phenotypes such as biofilm formation, colony morphology, motility, and expression of secondary metabolite biosynthesis when grown as a biofilm at different temperatures. RNA-seq analysis was performed at varying temperatures in a ΔII2523 mutant background that is responsive to temperature alterations resulting in hypo- and hyper- biofilm forming phenotypes. Differential regulation of genes was observed for polysaccharide biosynthesis, secretion systems, and nonribosomal peptide and polyketide synthase (NRPS/PKS) clusters in response to temperature changes. Deletion mutations of biosynthetic gene clusters (BGCs) clusters 2, 11, 14 (syrbactin), and 15 (malleipeptin) in wild-type and ΔII2523 backgrounds also reveals the contribution of these BGCs to biofilm formation and colony morphology in addition to inhibition of Bacillus subtilis and Rhizoctonia solani. Our findings suggest that II2523 impacts the regulation of genes that contribute to biofilm formation and competition. Characterization of cryptic BGCs under differing environmental conditions will allow for a better understanding of the role of secondary metabolites in the context of biofilm formation and microbe-microbe interactions.

LuxS modulates motility and secretion of extracellular protease in fish pathogen Vibrio harveyi

2021 ◽  
Author(s):  
yaqiu Zhang ◽  
Yiqing Deng ◽  
Juan Feng ◽  
Jianmei Hu ◽  
Haoxiang Chen ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Stationary Growth Phase ◽  
Extracellular Protease ◽  
Fish Pathogen ◽  
Wild Type ◽  
Stationary Growth ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Luxs Mutant

In this study, an in-frame deletion of the luxS gene was constructed to reveal the role of LuxS in the physiology and virulence of V. harveyi. The statistical analysis showed no significant differences in the growth ability, biofilm formation, antibiotic susceptibility, virulence by intraperitoneal injection, and the ability of V. harveyi to colonize the spleen and liver of the pearl gentian grouper between the wild-type (WT) and the luxS mutant. However, the deletion of luxS decreased the secretion of extracellular protease, while increased the ability of swimming and swarming. Simultaneously, a luxS-deleted mutant showed overproduction of lateral flagella, and an intact luxS complemented the defect. Since motility is flagella dependent, 16 of V. harveyi flagella biogenesis related genes were selected for further analysis. Based on quantitative real-time reverse transcription-PCR (qRT-PCR), the expression levels of these genes, including the polar flagella genes flaB, flhA, flhF, flhB, flhF, fliS, and flrA and the lateral flagella genes flgA, flgB, fliE, fliF, lafA, lafK, and motY, were significantly up-regulated in the ΔluxS: pMMB207 (ΔluxS+) strain as compared with the V. harveyi 345: pMMB207 (WT+) and C-ΔluxS strains during the early, mid-exponential, and stationary growth phase.

scholarly journals Motility-Independent Formation of Antibiotic-TolerantPseudomonas aeruginosaAggregates

2019 ◽  
Vol 85 (14) ◽  
Author(s):  
Sally Demirdjian ◽  
Hector Sanchez ◽  
Daniel Hopkins ◽  
Brent Berwin
Keyword(s):  
Biofilm Formation ◽  
Bacterial Pathogen ◽  
Aggregate Formation ◽  
Flagellar Motility ◽  
Wild Type ◽  
Chronic Infections ◽  
Content Type ◽  
Temporal Adaptation ◽  
Bacterial Aggregates

ABSTRACTPseudomonas aeruginosais a bacterial pathogen that causes severe chronic infections in immunocompromised individuals. This bacterium is highly adaptable to its environments, which frequently select for traits that promote bacterial persistence. A clinically significant temporal adaptation is the formation of surface- or cell-adhered bacterial biofilms that are associated with increased resistance to immune and antibiotic clearance. Extensive research has shown that bacterial flagellar motility promotes formation of such biofilms, whereupon the bacteria subsequently become nonmotile. However, recent evidence shows that antibiotic-tolerant nonattached bacterial aggregates, distinct from surface-adhered biofilms, can form, and these have been reported in the context of lung infections, otitis media, nonhealing wounds, and soft tissue fillers. It is unclear whether the same bacterial traits are required for aggregate formation as for biofilm formation. In this report, using isogenic mutants, we demonstrate thatP. aeruginosaaggregates in liquid cultures are spontaneously formed independent of bacterial flagellar motility and independent of an exogenous scaffold. This contrasts with the role of the flagellum to initiate surface-adhered biofilms. Similarly to surface-attached biofilms, these aggregates exhibit increased antibiotic tolerance compared to planktonic cultures. These findings provide key insights into the requirements for aggregate formation that contrast with those for biofilm formation and that may have relevance for the persistence and dissemination of nonmotile bacteria found within chronic clinical infections.IMPORTANCEIn this work, we have investigated the role of bacterial motility with regard to antibiotic-tolerant bacterial aggregate formation. Previous work has convincingly demonstrated thatP. aeruginosaflagellar motility promotes the formation of surface-adhered biofilms in many systems. In contrast, aggregate formation byP. aeruginosawas observed for nonmotile but not for motile cells in the presence of an exogenous scaffold. Here, we demonstrate that both wild-typeP. aeruginosaand mutants that genetically lack motility spontaneously form antibiotic-tolerant aggregates in the absence of an exogenously added scaffold. Additionally, we also demonstrate that wild-type (WT) and nonmotileP. aeruginosabacteria can coaggregate, shedding light on potential physiological interactions and heterogeneity of aggregates.

scholarly journals Role of Flagellin-Homologous Proteins in Biofilm Formation by Pathogenic Vibrio Species

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
You-Chul Jung ◽  
Mi-Ae Lee ◽  
Kyu-Ho Lee
Keyword(s):  
Biofilm Formation ◽  
Specific Binding ◽  
Biological Significance ◽  
Wild Type ◽  
Forming Process ◽  
Homologous Proteins ◽  
Content Type ◽  
Pathogenic Vibrio ◽  
Biofilm Matrix

ABSTRACT The pathogenic bacterium Vibrio vulnificus exhibits the ability to form biofilm, for which initiation is dependent upon swimming motility by virtue of a polar flagellum. The filament of its flagellum is composed of multiple flagellin subunits, FlaA, -B, -C, and -D. In V. vulnificus genomes, however, open reading frames (ORFs) annotated by FlaE and -F are also present. Although neither FlaE nor FlaF is involved in filament formation and cellular motility, they are well expressed and secreted to the extracellular milieu through the secretion apparatus for flagellar assembly. In the extrapolymeric matrix of V. vulnificus biofilm, significant levels of FlaEF were detected. Mutants defective in both flaE and flaF formed significantly decreased biofilms compared to the wild-type biofilm. Thus, the potential role of FlaEF during the biofilm-forming process was investigated by exogenous addition of recombinant FlaEF (rFlaEF) to the biofilm assays. The added rFlaE and rFlaF were predominantly incorporated into the biofilm matrix formed by the wild type. However, biofilms formed by a mutant defective in exopolysaccharide (EPS) biosynthesis were not affected by added FlaEF. These results raised a possibility that FlaEF specifically interact with EPS within the biofilm matrix. In vitro pulldown assays using His-tagged rFlaEF or rFlaC revealed the specific binding of EPS to rFlaEF but not to rFlaC. Taken together, our results demonstrate that V. vulnificus FlaEF, flagellin-homologous proteins (FHPs), are crucial for biofilm formation by directly interacting with the essential determinant for biofilm maturation, EPS. Further analyses performed with other pathogenic Vibrio species demonstrated both the presence of FHPs and their important role in biofilm formation. IMPORTANCE Flagellar filaments of the pathogenic Vibrio species, including V. vulnificus, V. parahaemolyticus, and V. cholerae, are composed of multiple flagellin subunits. In their genomes, however, there are higher numbers of the ORFs encoding flagellin-like proteins than the numbers of flagellin subunits required for filament assembly. Since these flagellin-homologous proteins (FHPs) are well expressed and excreted to environments via a flagellin transport channel, their extracellular role in the pathogenic Vibrio has been enigmatic. Their biological significance, which is not related with flagellar functions, has been revealed to be in maturation of biofilm structures. Among various components of the extracellular polymeric matrix produced in the V. vulnificus biofilms, the exopolysaccharides (EPS) are dominant constituents and crucial in maturation of biofilms. The enhancing role of the V. vulnificus FHPs in biofilm formation requires the presence of EPS, as indicated by highly specific interactions among two FHPs and three EPS.

scholarly journals Dissection of the Role of Pili and Type 2 and 3 Secretion Systems in Adherence and Biofilm Formation of an Atypical Enteropathogenic Escherichia coli Strain

2013 ◽  
Vol 81 (10) ◽  
pp. 3793-3802 ◽  
Author(s):  
Rodrigo T. Hernandes ◽  
Miguel A. De la Cruz ◽  
Denise Yamamoto ◽  
Jorge A. Girón ◽  
Tânia A. T. Gomes
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Coli Strain ◽  
Mass Spectrometry Analysis ◽  
Secretion Systems ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Rigid Rod

ABSTRACTAtypical enteropathogenicEscherichia coli(aEPEC) strains are diarrheal pathogens that lack bundle-forming pilus production but possess the virulence-associated locus of enterocyte effacement. aEPEC strain 1551-2 produces localized adherence (LA) on HeLa cells; however, its isogenic intimin (eae) mutant produces a diffuse-adherence (DA) pattern. In this study, we aimed to identify the DA-associated adhesin of the 1551-2eaemutant. Electron microscopy of 1551-2 identified rigid rod-like pili composed of an 18-kDa protein, which was identified as the major pilin subunit of type 1 pilus (T1P) by mass spectrometry analysis. Deletion offimAin 1551-2 affected biofilm formation but had no effect on adherence properties. Analysis of secreted proteins in supernatants of this strain identified a 150-kDa protein corresponding to SslE, a type 2 secreted protein that was recently reported to be involved in biofilm formation of rabbit and human EPEC strains. However, neither adherence nor biofilm formation was affected in a 1551-2sslEmutant. We then investigated the role of the EspA filament associated with the type 3 secretion system (T3SS) in DA by generating a doubleeae espAmutant. This strain was no longer adherent, strongly suggesting that the T3SS translocon is the DA adhesin. In agreement with these results, specific anti-EspA antibodies blocked adherence of the 1551-2eaemutant. Our data support a role for intimin in LA, for the T3SS translocon in DA, and for T1P in biofilm formation, all of which may act in concert to facilitate host intestinal colonization by aEPEC strains.

scholarly journals Characterization of Biofilm Formation and the Role of BCR1 in Clinical Isolates of Candida parapsilosis

2013 ◽  
Vol 13 (4) ◽  
pp. 438-451 ◽  
Author(s):  
Srisuda Pannanusorn ◽  
Bernardo Ramírez-Zavala ◽  
Heinrich Lünsdorf ◽  
Birgitta Agerberth ◽  
Joachim Morschhäuser ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Candida Parapsilosis ◽  
Clinical Isolates ◽  
Wild Type ◽  
Content Type ◽  
Initial Adhesion ◽  
High Level ◽  
Increased Susceptibility

ABSTRACT In Candida parapsilosis , biofilm formation is considered to be a major virulence factor. Previously, we determined the ability of 33 clinical isolates causing bloodstream infection to form biofilms and identified three distinct groups of biofilm-forming strains (negative, low, and high). Here, we establish two different biofilm structures among strains forming large amounts of biofilm in which strains with complex spider-like structures formed robust biofilms on different surface materials with increased resistance to fluconazole. Surprisingly, the transcription factor Bcr1, required for biofilm formation in Candida albicans and C. parapsilosis , has an essential role only in strains with low capacity for biofilm formation. Although BCR1 leads to the formation of more and longer pseudohyphae, it was not required for initial adhesion and formation of mature biofilms in strains with a high level of biofilm formation. Furthermore, an additional phenotype affected by BCR1 was the switch in colony morphology from rough to crepe, but only in strains forming high levels of biofilm. All bcr1 Δ/Δ mutants showed increased proteolytic activity and increased susceptibility to the antimicrobial peptides protamine and RP-1 compared to corresponding wild-type and complemented strains. Taken together, our results demonstrate that biofilm formation in clinical isolates of C. parapsilosis is both dependent and independent of BCR1 , but even in strains which showed a BCR1 -independent biofilm phenotype, BCR1 has alternative physiological functions.

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

2020 ◽  
Vol 8 (1) ◽  
pp. 70 ◽  
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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