scholarly journals Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Emilie Verplaetse ◽  
Leyla Slamti ◽  
Michel Gohar ◽  
Didier Lereclus
Keyword(s):  
Cell Differentiation ◽  
Bacillus Thuringiensis ◽  
Biofilm Formation ◽  
Regulatory Networks ◽  
Infection Process ◽  
Insect Host ◽  
Insect Larvae ◽  
Content Type ◽  
Insect Cadaver ◽  
Quorum Sensor

ABSTRACT Bacillus thuringiensis (Bt) is armed to complete a full cycle in its insect host. During infection, virulence factors are expressed under the control of the quorum sensor PlcR to kill the host. After the host's death, the quorum sensor NprR controls a necrotrophic lifestyle, allowing the vegetative cells to use the insect cadaver as a bioincubator and to survive. Only a part of the Bt population sporulates in the insect cadaver, and the precise composition of the whole population and its evolution over time are unknown. Using fluorescent reporters to record gene expression at the single-cell level, we have determined the differentiation course of a Bt population and explored the lineage existing among virulent, necrotrophic, and sporulating cells. The dynamics of cell differentiation were monitored during growth in homogenized medium, biofilm formation, and colonization of insect larvae. We demonstrated that in the insect host and in planktonic culture in rich medium, the virulence, necrotrophism, and sporulation regulators are successively activated in the same cell. In contrast, in biofilms, activation of PlcR is dispensable for NprR activation and we observed a greater heterogeneity than under the other two growth conditions. We also showed that sporulating cells arise almost exclusively from necrotrophic cells. In biofilm and in the insect cadaver, we identified an as-yet-uncharacterized category of cells that do not express any of the reporters used. Overall, we showed that PlcR, NprR, and Spo0A act as interconnected integrators to allow finely tuned adaptation of the pathogen to its environment. IMPORTANCE Bt is an entomopathogen found ubiquitously in the environment and is a widely used biopesticide. Studies performed at the population level suggest that the infection process of Bt includes three successive steps (virulence, necrotrophism, and sporulation) controlled by different regulators. This study aimed to determine how these phenotypes are activated at the cellular level and if they are switched on in all cells. We used an insect model of infection and biofilms to decipher the cellular differentiation of this bacterium under naturalistic conditions. Our study reveals the connection and lineage existing among virulent, necrotrophic, and sporulating cells. It also shows that the complex conditions encountered in biofilms and during infection generate great heterogeneity inside the population, which might reflect a bet-hedging strategy to ameliorate survival. These data generate new insights into the role of regulatory networks in the adaptation of a pathogen to its host.

scholarly journals Metabolic Remodeling during Biofilm Development ofBacillus subtilis

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Tippapha Pisithkul ◽  
Jeremy W. Schroeder ◽  
Edna A. Trujillo ◽  
Ponlkrit Yeesin ◽  
David M. Stevenson ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Regulatory Networks ◽  
Developmental Process ◽  
Central Carbon Metabolism ◽  
Bacterial Biofilm ◽  
Biofilm Growth ◽  
Time Resolved ◽  
Content Type ◽  
Metabolic Remodeling ◽  
Biofilm Development

ABSTRACTBiofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such asBacillus subtilishas been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development inB. subtilisby combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring duringB. subtilisbiofilm development that will serve as a useful road map for future studies on biofilm physiology.IMPORTANCEBacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such asBacillus subtilis, very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development inB. subtilisby combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism.

scholarly journals Rap-Phr Systems from Plasmids pAW63 and pHT8-1 Act Together To Regulate Sporulation in the Bacillus thuringiensis Serovar kurstaki HD73 Strain

2020 ◽  
Vol 86 (18) ◽  
Author(s):  
Priscilla Cardoso ◽  
Fernanda Fazion ◽  
Stéphane Perchat ◽  
Christophe Buisson ◽  
Gislayne Vilas-Bôas ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Quorum Sensing ◽  
Effective Control ◽  
Moderate Activity ◽  
Insect Larvae ◽  
Content Type ◽  
Sensing Systems ◽  
Insecticidal Toxins ◽  
Negative Effect ◽  
Plasmid Genes

ABSTRACT Bacillus thuringiensis is a Gram-positive spore-forming bacterium pathogenic to various insect species. This property is due to the Cry toxins encoded by plasmid genes and mostly produced during sporulation. B. thuringiensis contains a remarkable number of extrachromosomal DNA molecules and a great number of plasmid rap-phr genes. Rap-Phr quorum-sensing systems regulate different bacterial processes, notably the commitment to sporulation in Bacillus species. Rap proteins are quorum sensors acting as phosphatases on Spo0F, an intermediate of the sporulation phosphorelay, and are inhibited by Phr peptides that function as signaling molecules. In this study, we characterize the Rap63-Phr63 system encoded by the pAW63 plasmid from the B. thuringiensis serovar kurstaki HD73 strain. Rap63 has moderate activity on sporulation and is inhibited by the Phr63 peptide. The rap63-phr63 genes are cotranscribed, and the phr63 gene is also transcribed from a σH-specific promoter. We show that Rap63-Phr63 regulates sporulation together with the Rap8-Phr8 system harbored by plasmid pHT8_1 of the HD73 strain. Interestingly, the deletion of both phr63 and phr8 genes in the same strain has a greater negative effect on sporulation than the sum of the loss of each phr gene. Despite the similarities in the Phr8 and Phr63 sequences, there is no cross talk between the two systems. Our results suggest a synergism of these two Rap-Phr systems in the regulation of the sporulation of B. thuringiensis at the end of the infectious cycle in insects, thus pointing out the roles of the plasmids in the fitness of the bacterium. IMPORTANCE The life cycle of Bacillus thuringiensis in insect larvae is regulated by quorum-sensing systems of the RNPP family. After the toxemia caused by Cry insecticidal toxins, the sequential activation of these systems allows the bacterium to trigger first a state of virulence (regulated by PlcR-PapR) and then a necrotrophic lifestyle (regulated by NprR-NprX); ultimately, sporulation is controlled by the Rap-Phr systems. Our study describes a new rap-phr operon carried by a B. thuringiensis plasmid and shows that the Rap protein has a moderate effect on sporulation. However, this system, in combination with another plasmidic rap-phr operon, provides effective control of sporulation when the bacteria develop in the cadavers of infected insect larvae. Overall, this study highlights the important adaptive role of the plasmid Rap-Phr systems in the developmental fate of B. thuringiensis and its survival within its ecological niche.

scholarly journals Death Becomes Them: Bacterial Community Dynamics and Stilbene Antibiotic Production in Cadavers of Galleria mellonella Killed by Heterorhabditis and Photorhabdus spp.

2016 ◽  
Vol 82 (19) ◽  
pp. 5824-5837 ◽  
Author(s):  
Amanda C. Wollenberg ◽  
Tanush Jagdish ◽  
Greg Slough ◽  
Megan E. Hoinville ◽  
Michael S. Wollenberg
Keyword(s):  
Entomopathogenic Nematodes ◽  
Community Dynamics ◽  
Resource Competition ◽  
Galleria Mellonella ◽  
Insect Larvae ◽  
Content Type ◽  
Bacterial Community Dynamics ◽  
Insect Cadaver ◽  
Generation Sequencing

ABSTRACTInsect larvae killed by entomopathogenic nematodes are thought to contain bacterial communities dominated by a single bacterial genus, that of the nematode's bacterial symbiont. In this study, we used next-generation sequencing to profile bacterial community dynamics in greater wax moth (Galleria mellonella) larvae cadavers killed byHeterorhabditisnematodes and theirPhotorhabdussymbionts. We found that, althoughPhotorhabdusstrains did initially displace anEnterococcus-dominated community present in uninfectedG. mellonellainsect larvae, the cadaver community was not static. Twelve days postinfection,Photorhabdusshared the cadaver withStenotrophomonasspecies. Consistent with this result,Stenotrophomonasstrains isolated from infected cadavers were resistant toPhotorhabdus-mediated toxicity in solid coculture assays. We isolated and characterized aPhotorhabdus-produced antibiotic fromG. mellonellacadavers, produced it synthetically, and demonstrated that both the natural and synthetic compounds decreasedG. mellonella-associatedEnterococcusgrowth, but notStenotrophomonasgrowth,in vitro. Finally, we showed that theStenotrophomonasstrains described here negatively affectedPhotorhabdusgrowthin vitro. Our results add an important dimension to a broader understanding ofHeterorhabditis-Photorhabdusbiology and also demonstrate that interspecific bacterial competition likely characterizes even a theoretically monoxenic environment, such as aHeterorhabditis-Photorhabdus-parasitized insect cadaver.IMPORTANCEUnderstanding, and eventually manipulating, both human and environmental health depends on a complete accounting of the forces that act on and shape microbial communities. One of these underlying forces is hypothesized to be resource competition. A resource that has received little attention in the general microbiological literature, but likely has ecological and evolutionary importance, is dead/decaying multicellular organisms. Metazoan cadavers, including those of insects, are ephemeral and nutrient-rich environments, where resource competition might shape interspecific macrobiotic and microbiotic interactions. This study is the first to use a next-generation sequencing approach to study the community dynamics of bacteria within a model insect cadaver system: insect larvae parasitized by entomopathogenic nematodes and their bacterial symbionts. By integrating bioinformatic, biochemical, and classicin vitromicrobiological approaches, we have provided mechanistic insight into how antibiotic-mediated bacterial interactions may shape community dynamics within insect cadavers.

scholarly journals Negative Interplay between Biofilm Formation and Competence in the Environmental Strains of Bacillus subtilis

mSystems ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Qianxuan She ◽  
Evan Hunter ◽  
Yuxuan Qin ◽  
Samantha Nicolau ◽  
Eliza A. Zalis ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Differentiation ◽  
Biofilm Formation ◽  
Competence Development ◽  
Bacterial Biofilm ◽  
Soil Bacterium ◽  
Natural Competence ◽  
Content Type ◽  
Pathway Regulation

The soil bacterium Bacillus subtilis can form robust biofilms, which are important for its survival in the environment. B. subtilis also exhibits natural competence. By investigating competence development in B. subtilis in situ during biofilm formation, we reveal that robust biofilm formation often greatly reduces the frequency of competent cells within the biofilm. We then characterize a cross-pathway regulation that allows cells in these two developmental events to undergo mutually exclusive cell differentiation during biofilm formation. Finally, we discuss potential biological implications of limiting competence in a bacterial biofilm.

scholarly journals SuhB Is a Regulator of Multiple Virulence Genes and Essential for Pathogenesis of Pseudomonas aeruginosa

mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Kewei Li ◽  
Chang Xu ◽  
Yongxin Jin ◽  
Ziyu Sun ◽  
Chang Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Virulence Factors ◽  
Biofilm Formation ◽  
Regulatory Networks ◽  
Virulence Genes ◽  
Chronic Infections ◽  
Content Type ◽  
Factors Associated ◽  
Acute Infections

ABSTRACTDuring initial colonization and chronic infection, pathogenic bacteria encounter distinct host environments. Adjusting gene expression accordingly is essential for the pathogenesis.Pseudomonas aeruginosahas evolved complicated regulatory networks to regulate different sets of virulence factors to facilitate colonization and persistence. The type III secretion system (T3SS) and motility are associated with acute infections, while biofilm formation and the type VI secretion system (T6SS) are associated with chronic persistence. To identify novel regulatory genes required for pathogenesis, we screened aP. aeruginosatransposon (Tn) insertion library and foundsuhBto be an essential gene for the T3SS gene expression. The expression ofsuhBwas upregulated in a mouse acute lung infection model, and loss ofsuhBresulted in avirulence. Suppression of T3SS gene expression in thesuhBmutant is linked to a defective translation of the T3SS master regulator, ExsA. Further studies demonstrated thatsuhBmutation led to the upregulation of GacA and its downstream small RNAs, RsmY and RsmZ, triggering T6SS expression and biofilm formation while inhibiting the T3SS. Our results demonstrate that anin vivo-inducible gene,suhB, reciprocally regulates genes associated with acute and chronic infections and plays an essential role in the pathogenesis ofP. aeruginosa.IMPORTANCEA variety of bacterial pathogens, such asPseudomonas aeruginosa, cause acute and chronic infections in humans. During infections, pathogens produce different sets of virulence genes for colonization, tissue damage, and dissemination and for countering host immune responses. Complex regulatory networks control the delicate tuning of gene expression in response to host environments to enable the survival and growth of invading pathogens. Here we identifiedsuhBas a critical gene for the regulation of virulence factors inP. aeruginosa. The expression ofsuhBwas upregulated during acute infection in an animal model, and mutation ofsuhBrenderedP. aeruginosaavirulent. Moreover, we demonstrate that SuhB is required for the activation of virulence factors associated with acute infections while suppressing virulence factors associated with chronic infections. Our report provides new insights into the multilayered regulatory network of virulence genes inP. aeruginosa.

scholarly journals Role of Intracellular Proteases in the Antibiotic Resistance, Motility, and Biofilm Formation of Pseudomonas aeruginosa

2011 ◽  
Vol 56 (2) ◽  
pp. 1128-1132 ◽  
Author(s):  
Lucía Fernández ◽  
Elena B. M. Breidenstein ◽  
Diana Song ◽  
Robert E. W. Hancock
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Biofilm Formation ◽  
Regulatory Networks ◽  
Swarming Motility ◽  
Content Type ◽  
Adverse Conditions ◽  
Related Proteins ◽  
Intracellular Proteases

ABSTRACTPseudomonas aeruginosapossesses complex regulatory networks controlling virulence and survival under adverse conditions, including antibiotic pressure, which are interconnected and share common regulatory proteins. Here, we screen a panel of 13 mutants defective in intracellular proteases and demonstrate that, in addition to the known alterations in Lon and AsrA mutants, mutation of three protease-related proteins PfpI, ClpS, and ClpP differentially affected antibiotic resistance, swarming motility, and biofilm formation.

scholarly journals AraC-Type Regulator Rsp Adapts Staphylococcus aureus Gene Expression to Acute Infection

2015 ◽  
Vol 84 (3) ◽  
pp. 723-734 ◽  
Author(s):  
Tianming Li ◽  
Lei He ◽  
Yan Song ◽  
Amer E. Villaruz ◽  
Hwang-Soo Joo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Regulatory Networks ◽  
Acute Infection ◽  
Toxin Production ◽  
Transcriptional Analysis ◽  
Chronic Infections ◽  
Accessory Gene ◽  
Content Type ◽  
Regulatory Impact

Staphylococcus aureusis an important human pathogen that can cause two categories of severe infections. Acute infections are characterized by pronounced toxin production, while chronic infections often involve biofilm formation. However, it is poorly understood howS. aureuscontrols the expression of genes associated with acute versus biofilm-associated virulence. We here identified an AraC-type transcriptional regulator, Rsp, that promotes the production of key toxins while repressing major biofilm-associated genes and biofilm formation. Genome-wide transcriptional analysis and modeling of regulatory networks indicated that upregulation of the accessory gene regulator (Agr) and downregulation of theicaoperon coding for the biofilm exopolysaccharide polysaccharide intercellular adhesin (PIA) were central to the regulatory impact of Rsp on virulence. Notably, the Rsp protein directly bound to theagrP2andicaADBCpromoters, resulting in strongly increased levels of the Agr-controlled toxins phenol-soluble modulins (PSMs) and alpha-toxin and reduced production of PIA. Accordingly, Rsp was essential for the development of bacteremia and skin infection, representing major types of acuteS. aureusinfection. Our findings give important insight into howS. aureusadapts the expression of its broad arsenal of virulence genes to promote different types of disease manifestations and identify the Rsp regulator as a potential target for strategies to control acuteS. aureusinfection.

scholarly journals Influence of Lysogeny of Tectiviruses GIL01 and GIL16 on Bacillus thuringiensis Growth, Biofilm Formation, and Swarming Motility

2014 ◽  
Vol 80 (24) ◽  
pp. 7620-7630 ◽  
Author(s):  
Annika Gillis ◽  
Jacques Mahillon
Keyword(s):  
Life Cycle ◽  
Bacillus Thuringiensis ◽  
Biofilm Formation ◽  
Metabolic Profiles ◽  
Swarming Motility ◽  
Content Type ◽  
Life Traits ◽  
Antibiotics Susceptibility ◽  
Species Specific ◽  
Sporulation Rate

ABSTRACTBacillus thuringiensisis an entomopathogenic bacterium that has been used as an efficient biopesticide worldwide. Despite the fact that this bacterium is usually described as an insect pathogen, its life cycle in the environment is still largely unknown.B. thuringiensisbelongs to theBacillus cereusgroup of bacteria, which has been associated with many mobile genetic elements, such as species-specific temperate or virulent bacteriophages (phages). Temperate (lysogenic) phages are able to establish a long-term relationship with their host, providing, in some cases, novel ecological traits to the bacterial lysogens. Therefore, this work focuses on evaluating the potential influence of temperate tectiviruses GIL01 and GIL16 on the development of different life traits ofB. thuringiensis. For this purpose, aB. thuringiensisserovar israelensis plasmid-cured (nonlysogenic) strain was used to establish bacterial lysogens for phages GIL01 and GIL16, and, subsequently, the following life traits were compared among the strains: kinetics of growth, metabolic profiles, antibiotics susceptibility, biofilm formation, swarming motility, and sporulation. The results revealed that GIL01 and GIL16 lysogeny has a significant influence on the bacterial growth, sporulation rate, biofilm formation, and swarming motility ofB. thuringiensis. No changes in metabolic profiles or antibiotic susceptibilities were detected. These findings provide evidence that tectiviruses have a putative role in theB. thuringiensislife cycle as adapters of life traits with ecological advantages.

scholarly journals Pseudomonas aeruginosa Expresses a Functional Human Natriuretic Peptide Receptor Ortholog: Involvement in Biofilm Formation

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Thibaut Rosay ◽  
Alexis Bazire ◽  
Suraya Diaz ◽  
Thomas Clamens ◽  
Anne-Sophie Blier ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Natriuretic Peptide ◽  
Biofilm Formation ◽  
Receptor Agonist ◽  
Three Dimensional ◽  
Specific Protein ◽  
Infection Process ◽  
Content Type ◽  
Exciting Opportunity ◽  
Immunocompromised Hosts

ABSTRACTConsiderable evidence exists that bacteria detect eukaryotic communication molecules and modify their virulence accordingly. In previous studies, it has been demonstrated that the increasingly antibiotic-resistant pathogenPseudomonas aeruginosacan detect the human hormones brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) at micromolar concentrations. In response, the bacterium modifies its behavior to adapt to the host physiology, increasing its overall virulence. The possibility of identifying the bacterial sensor for these hormones and interfering with this sensing mechanism offers an exciting opportunity to directly affect the infection process. Here, we show that BNP and CNP strongly decreaseP. aeruginosabiofilm formation. Isatin, an antagonist of human natriuretic peptide receptors (NPR), prevents this effect. Furthermore, the human NPR-C receptor agonist cANF4-23mimics the effects of natriuretic peptides onP. aeruginosa, while sANP, the NPR-A receptor agonist, appears to be weakly active. We showin silicothat NPR-C, a preferential CNP receptor, and theP. aeruginosaprotein AmiC have similar three-dimensional (3D) structures and that both CNP and isatin bind to AmiC. We demonstrate that CNP acts as an AmiC agonist, enhancing the expression of theamioperon inP. aeruginosa. Binding of CNP and NPR-C agonists to AmiC was confirmed by microscale thermophoresis. Finally, using anamiCmutant strain, we demonstrated that AmiC is essential for CNP effects on biofilm formation. In conclusion, the AmiC bacterial sensor possesses structural and pharmacological profiles similar to those of the human NPR-C receptor and appears to be a bacterial receptor for human hormones that enablesP. aeruginosato modulate biofilm expression.IMPORTANCEThe bacteriumPseudomonas aeruginosais a highly dangerous opportunist pathogen for immunocompromised hosts, especially cystic fibrosis patients. The sites ofP. aeruginosainfection are varied, with predominance in the human lung, in which bacteria are in contact with host molecular messengers such as hormones. The C-type natriuretic peptide (CNP), a hormone produced by lung cells, has been described as a bacterial virulence enhancer. In this study, we showed that the CNP hormone counteractsP. aeruginosabiofilm formation and we identified the bacterial protein AmiC as the sensor involved in the CNP effects. We showed that AmiC could bind specifically CNP. These results show for the first time that a human hormone could be sensed by bacteria through a specific protein, which is an ortholog of the human receptor NPR-C. The bacterium would be able to modify its lifestyle by favoring virulence factor production while reducing biofilm formation.

scholarly journals Utilization of Variant and Fusion Proteins To Functionally Map the Aggregatibacter actinomycetemcomitans Trimeric Autotransporter Protein ApiA

2017 ◽  
Vol 86 (3) ◽  
Author(s):  
Carla Cugini ◽  
Yongyi Mei ◽  
David Furgang ◽  
Nisha George ◽  
Narayanan Ramasubbu ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Aggregatibacter Actinomycetemcomitans ◽  
Surface Expression ◽  
Aggressive Periodontitis ◽  
Infection Process ◽  
Extracellular Polysaccharides ◽  
Passenger Domain ◽  
Content Type ◽  
C Terminus ◽  
N Terminus

ABSTRACT The Gram-negative bacterium Aggregatibacter actinomycetemcomitans is a causative agent of localized aggressive periodontitis. Critical to its infection process is the first and essential step of attachment, which is related to the coordinated functions of surface components comprised of proteins and extracellular polysaccharides. One such protein is the outer membrane trimeric autotransporter protein ApiA, a versatile virulence factor with numerous functions, including cell binding, invasion, serum resistance, autoaggregation, and induction of cytokine release. Here we report on the use of Escherichia coli strains expressing protein variants to define the separate functions ascribed to the N terminus and those related to the C terminus. Importantly, a hybrid protein that comprised the N terminus of trimeric ApiA and the β-barrel domain of monomeric autotransporter Aae was constructed, which allowed the expression of a monomer surface-exposed domain of ApiA. Functional and phenotypic analyses demonstrated that the C terminus of ApiA forms an independent domain that is crucial for general stability and trimer formation, which appears to be associated with autoaggregation, biofilm formation, and surface expression. Importantly, the results show that the monomeric form of the N-terminal passenger domain of ApiA, while surface exposed, is sufficient for binding to buccal epithelial cells; however, it is not sufficient to allow aggregation and biofilm formation, strengthening the importance of the role of trimerization in these phenotypes.

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