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1.
World J Microbiol Biotechnol ; 40(11): 367, 2024 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-39455472

RESUMEN

Flagella is a well-known bacterial structure crucial for motility, which also plays pivotal roles in pathogenesis. Arcobacter butzleri, an enteropathogen, possesses a distinctive polar flagellum whose functional aspects remain largely unexplored. Upon investigating the factors influencing A. butzleri motility, we uncovered that environmental conditions like temperature, oxygen levels, and nutrient availability play a significant role. Furthermore, compounds that are found in human gut, such as short-chain fatty acids, mucins and bile salts, have a role in modulating the motility, and in turn, the pathogenicity of A. butzleri. Further investigation demonstrated that A. butzleri ΔflaA mutant showed a reduction in motility with a close to null average velocity, as well as a reduction on biofilm formation. In addition, compared with the wild-type, the ΔflaA mutant showed a decreased ability to invade Caco-2 cells and to adhere to mucins. Taken together, our findings support the role of environmental conditions and gut host associated compounds influencing key physiological aspects of the gastrointestinal pathogen A. butzleri, such as motility, and support the role of the flagellum on bacterial virulence.


Asunto(s)
Arcobacter , Adhesión Bacteriana , Biopelículas , Flagelos , Mucinas , Flagelos/genética , Flagelos/fisiología , Flagelos/metabolismo , Arcobacter/genética , Arcobacter/patogenicidad , Arcobacter/metabolismo , Humanos , Virulencia , Células CACO-2 , Biopelículas/crecimiento & desarrollo , Mucinas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ácidos y Sales Biliares/metabolismo , Ácidos y Sales Biliares/farmacología , Ácidos Grasos Volátiles/metabolismo
2.
Proc Natl Acad Sci U S A ; 121(41): e2412541121, 2024 Oct 08.
Artículo en Inglés | MEDLINE | ID: mdl-39352926

RESUMEN

Flagella are highly complex rotary molecular machines that enable bacteria to not only migrate to optimal environments but also to promote range expansion, competitiveness, virulence, and antibiotic survival. Flagellar motility is an energy-demanding process, where the sum of its production (biosynthesis) and operation (rotation) costs has been estimated to total ~10% of the entire energy budget of an Escherichia coli cell. The acquisition of such a costly adaptation process is expected to secure short-term benefits by increasing competitiveness and survival, as well as long-term evolutionary fitness gains. While the role of flagellar motility in bacterial survival has been widely reported, its direct influence on the rate of evolution remains unclear. We show here that both production and operation costs contribute to elevated mutation rates. Our findings suggest that flagellar movement may be an important player in tuning the rate of bacterial evolution.


Asunto(s)
Escherichia coli , Flagelos , Flagelos/metabolismo , Flagelos/fisiología , Flagelos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Mutación , Tasa de Mutación , Movimiento , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Evolución Biológica
3.
Int J Mol Sci ; 25(17)2024 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-39273173

RESUMEN

Escherichia coli O157:H7 (E. coli O157) is known for causing severe foodborne illnesses such as hemorrhagic colitis and hemolytic uremic syndrome. Although E. coli O157 is typically regarded as an extracellular pathogen and a weak biofilm producer, some E. coli O157 strains, including a clinical strain ATCC 43895, exhibit a notable ability to invade bovine crypt cells and other epithelial cells, as well as to form robust biofilm. This invasive strain persists in the bovine host significantly longer than non-invasive strains. Various surface-associated factors, including lipopolysaccharides (LPS), flagella, and other adhesins, likely contribute to this enhanced invasiveness and biofilm formation. In this study, we constructed a series of LPS-core deletion mutations (waaI, waaG, waaF, and waaC) in E. coli O157 ATCC 43895, resulting in stepwise truncations of the LPS. This approach enabled us to investigate the effects on the biosynthesis of key surface factors, such as flagella and curli, and the ability of this invasive strain to invade host cells. We confirmed the LPS structure and found that all LPS-core mutants failed to form biofilms, highlighting the crucial role of core oligosaccharides in biofilm formation. Additionally, the LPS inner-core mutants ΔwaaF and ΔwaaC lost the ability to produce flagella and curli. Furthermore, these inner-core mutants exhibited a dramatic reduction in adherence to and invasion of epithelial cells (MAC-T), showing an approximately 100-fold decrease in cell invasion compared with the outer-core mutants (waaI and waaG) and the wild type. These findings underscore the critical role of LPS-core truncation in impairing flagella and curli biosynthesis, thereby reducing the invasion capability of E. coli O157 ATCC 43895.


Asunto(s)
Biopelículas , Escherichia coli O157 , Flagelos , Lipopolisacáridos , Flagelos/metabolismo , Flagelos/genética , Lipopolisacáridos/biosíntesis , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Escherichia coli O157/fisiología , Biopelículas/crecimiento & desarrollo , Animales , Bovinos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Adhesión Bacteriana , Células Epiteliales/microbiología , Células Epiteliales/metabolismo
4.
Microbiol Spectr ; 12(10): e0132524, 2024 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-39248473

RESUMEN

Loss of the flagellum marks the pathoadaptation of Pseudomonas aeruginosa to the cystic fibrosis (CF) airway environment during lung disease. Losing the flagellum is advantageous to the bacterium as the flagellum can be recognized by immune cells. The primary purpose of the flagellum is, however, to provide motility to the bacterium. Our goal was to determine whether the loss of flagellar motility or the loss of flagellum expression contributes to P. aeruginosa lung infection in CF. To address this, wild-type and gut-corrected FABP-human cystic fibrosis transmembrane conductance regulator (hCFTR) mice deficient in the murine Cftr gene were infected intratracheally with lethal doses of wild-type or flagellum-deficient P. aeruginosa. While there was no significant difference in the survival of wild-type mice after infection with either of the bacterial strains, a significantly higher mortality was observed in FABP-hCFTR mice infected with flagellum-deficient P. aeruginosa, compared to mice infected with their flagellated counterparts. When FABP-hCFTR mice were infected with isogenic, motility-deficient flagellated mutants, animal survival and lung bacterial titers were similar to those observed in mice infected with the wild-type bacterium. Airway levels of neutrophils and the amount neutrophil elastase were similar in mice infected with either the wild-type bacteria or the flagellum-deficient P. aeruginosa. Our results show that FABP-hCFTR mice have a different response to flagellum loss in P. aeruginosa compared to wild-type animals. The loss of flagellum expression, rather than the loss of motility, is the main driver behind the increased virulence of flagellum-deficient P. aeruginosa in CF. These observations provide new insight into P. aeruginosa virulence in CF.IMPORTANCEPseudomonas aeruginosa, a major respiratory pathogen in cystic fibrosis, is known to lose its flagellum during the course of infection in the airways. Here, we show that the loss of flagellum leads to a more enhanced virulence in Cftr-deficient cystic fibrosis mice than in control animals. Loss of flagellum expression, rather than the loss of flagellar swimming motility, represents the main driver behind this increased virulence suggesting that this appendage plays a specific role in P. aeruginosa virulence in cystic fibrosis airways.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Modelos Animales de Enfermedad , Flagelos , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Animales , Fibrosis Quística/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Ratones , Infecciones por Pseudomonas/microbiología , Flagelos/genética , Virulencia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/deficiencia , Humanos , Pulmón/microbiología , Pulmón/patología , Mutación , Femenino
5.
J Bacteriol ; 206(10): e0019924, 2024 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-39248522

RESUMEN

Motility allows microbes to explore and maximize success in their environment; however, many laboratory bacterial strains have a reduced or altered capacity for motility. Swimming motility in Bacillus subtilis depends on peritrichous flagella and is carried out individually as cells move by biased random walks toward attractants. Previously, we adapted Bacillus subtilis strain 3610 to the laboratory for 300 generations in lysogeny broth (LB) batch culture and isolated lab-adapted strains. Strain SH2 is motility-defective and in broth culture forms large, frequently spherical aggregates of cells. A single point mutation in the flagellin gene hag that causes amino acid 259 to switch from A to T is necessary and sufficient to cause these social cell aggregates, and aggregation occurs between flagellated cells bearing this point mutation regardless of the strain background. Cells associate when bearing this mutation, but flagellar rotation is needed to pull associating cells into spherical aggregates. Using electron microscopy, we are able to show that the SH2 flagellar filament has limited polymorphism when compared to other flagellar structures. This limited polymorphism hinders the flagellum's ability to function as a motility apparatus but appears to alter its function to that of cell aggregation/adhesion. We speculate that the genotype-specific aggregation of cells producing HagA259T flagella could have increased representation in a batch-culture experiment by allowing similar cells to go through a transfer together and also that this mutation could serve as an early step to evolve sociality in the natural world.IMPORTANCEThe first life forms on this planet were prokaryotic, and the earliest environments were aquatic, and from these relatively simple starting conditions, complex communities of microbes and ultimately multicellular organisms were able to evolve. Usually, motile cells in aqueous environments swim as individuals but become social by giving up motility and secreting extracellular substances to become a biofilm. Here, we identify a single point mutation in the flagellum that is sufficient to allow cells containing this mutation to specifically form large, suspended groups of cells. The specific change in the flagellar filament protein subunits causes a unique change in the flagellar structure. This could represent a distinct way for closely related cells to associate as an early precursor to sociality.


Asunto(s)
Bacillus subtilis , Flagelos , Flagelina , Mutación Puntual , Bacillus subtilis/genética , Bacillus subtilis/fisiología , Flagelos/genética , Flagelos/fisiología , Flagelina/genética , Flagelina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica
6.
Braz J Microbiol ; 55(3): 2727-2738, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39222218

RESUMEN

Due to the increasing occurrence of drug resistant urinary tract infections (UTI) among children, there is a need to investigate alternative effective treatment protocols such as nanoparticles. Flagella and fimbriae are primary factors contributing the virulence of urinary tract infecting bacteria. The aim of this study was to assess the antibacterial effects of zinc oxide nanoparticles which have been synthesized using both chemical and green methods on multi-drug resistant (MDR) uropathogenic bacteria encoding fli and fim genes and investigating their binding ability to bacterial appendage proteins. A total of 30 urine culture samples were collected from children under 2 years old diagnosed with urinary tract infection. The isolates underwent antibiotic suseptibility assessment and the isolates demonstrating MDR were subjected to molecular amplification of fimG (fimbrial) and fliD and fliT (flagellal) genes. The confirmation of cellular appendages was achieved through silver nitrate staining. The antibacterial efficacy of the synthetized nanoparticles was assessed using the micro and macrodilution methods. The successful binding of nanoparticles to bacterial appendage proteins was confirmed through mobility shift and membrane filter assays. The dimensions of chemically synthesized ZnO nanoparticles and green nanoparticles were measured at 30 nm and 85 nm, respectively, with the exhibition of hexagonal geometries. The nanoparticles synthesized through chemical and green methods exhibited minimum inhibitory concentrations (MIC) of 0.0062-0.025 g/L and 0.3 g/L, respectively. The ability of ZnO nanoparticles to bind bacterial appendage proteins and to combat MDR uropathogenic bacteria are promising for new treatment protocols against UTI in children in future.


Asunto(s)
Antibacterianos , Farmacorresistencia Bacteriana Múltiple , Flagelos , Infecciones Urinarias , Óxido de Zinc , Óxido de Zinc/farmacología , Óxido de Zinc/química , Óxido de Zinc/metabolismo , Antibacterianos/farmacología , Humanos , Infecciones Urinarias/microbiología , Infecciones Urinarias/tratamiento farmacológico , Flagelos/efectos de los fármacos , Flagelos/genética , Flagelos/metabolismo , Pruebas de Sensibilidad Microbiana , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/efectos de los fármacos , Nanopartículas/química , Lactante , Nanopartículas del Metal/química
7.
mBio ; 15(10): e0126124, 2024 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-39240115

RESUMEN

The marine bacterium Vibrio alginolyticus possesses a polar flagellum driven by a sodium ion flow. The main components of the flagellar motor are the stator and rotor. The C-ring and MS-ring, which are composed of FliG and FliF, respectively, are parts of the rotor. Here, we purified an MS-ring composed of FliF-FliG fusion proteins and solved the near-atomic resolution structure of the S-ring-the upper part of the MS-ring-using cryo-electron microscopy. This is the first report of an S-ring structure from Vibrio, whereas, previously, only those from Salmonella have been reported. The Vibrio S-ring structure reveals novel features compared with that of Salmonella, such as tilt angle differences of the RBM3 domain and the ß-collar region, which contribute to the vertical arrangement of the upper part of the ß-collar region despite the diversity in the RBM3 domain angles. Additionally, there is a decrease of the inter-subunit interaction between RBM3 domains, which influences the efficiency of the MS-ring formation in different bacterial species. Furthermore, although the inner-surface electrostatic properties of Vibrio and Salmonella S-rings are altered, the residues potentially interacting with other flagellar components, such as FliE and FlgB, are well structurally conserved in the Vibrio S-ring. These comparisons clarified the conserved and non-conserved structural features of the MS-ring across different species.IMPORTANCEUnderstanding the structure and function of the flagellar motor in bacterial species is essential for uncovering the mechanisms underlying bacterial motility and pathogenesis. Our study revealed the structure of the Vibrio S-ring, a part of its polar flagellar motor, and highlighted its unique features compared with the well-studied Salmonella S-ring. The observed differences in the inter-subunit interactions and in the tilt angles between the Vibrio and Salmonella S-rings highlighted the species-specific variations and the mechanism for the optimization of MS-ring formation in the flagellar assembly. By concentrating on the region where the S-ring and the rod proteins interact, we uncovered conserved residues essential for the interaction. Our research contributes to the advancement of bacterial flagellar biology.


Asunto(s)
Proteínas Bacterianas , Microscopía por Crioelectrón , Flagelos , Vibrio alginolyticus , Flagelos/metabolismo , Flagelos/química , Flagelos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Vibrio alginolyticus/genética , Vibrio alginolyticus/metabolismo , Vibrio alginolyticus/química , Salmonella/genética , Salmonella/metabolismo , Salmonella/química , Conformación Proteica , Proteínas de la Membrana
8.
mBio ; 15(10): e0100224, 2024 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-39230277

RESUMEN

During its cell cycle, the bacterium Caulobacter crescentus switches from a motile, free-living state, to a sessile surface-attached cell. During this coordinated process, cells undergo irreversible morphological changes, such as shedding of their polar flagellum and synthesis of an adhesive holdfast at the same pole. In this work, we used genetic screens to identify genes involved in the regulation of the transition from the motile to the sessile lifestyle. We identified a predicted hybrid histidine kinase that inhibits biofilm formation and promotes the motile lifestyle: HmrA (holdfast and motility regulator A). Genetic screens and genomic localization led to the identification of additional genes that form a putative phosphorelay pathway with HmrA. We postulate that the Hmr pathway acts as a rheostat to control the proportion of cells harboring a flagellum or a holdfast in the population. Further genetic analysis suggests that the Hmr pathway impacts c-di-GMP synthesis through the diguanylate cyclase DgcB pathway. Our results also indicate that the Hmr pathway is involved in the regulation of motile to sessile lifestyle transition as a function of various environmental factors: biofilm formation is repressed when excess copper is present and derepressed under non-optimal temperatures. Finally, we provide evidence that the Hmr pathway regulates motility and adhesion without modulating the transcription of the holdfast synthesis regulator HfiA. IMPORTANCE: Complex communities attached to a surface, or biofilms, represent the major lifestyle of bacteria in the environment. Such a sessile state enables the inhabitants to be more resistant to adverse environmental conditions. Thus, having a deeper understanding of the underlying mechanisms that regulate the transition between the motile and the sessile states could help design strategies to improve biofilms when they are beneficial or impede them when they are detrimental. For Caulobacter crescentus motile cells, the transition to the sessile lifestyle is irreversible, and this decision is regulated at several levels. In this work, we describe a putative phosphorelay that promotes the motile lifestyle and inhibits biofilm formation, providing new insights into the control of adhesin production that leads to the formation of biofilms.


Asunto(s)
Proteínas Bacterianas , Biopelículas , Caulobacter crescentus , Regulación Bacteriana de la Expresión Génica , Caulobacter crescentus/genética , Caulobacter crescentus/fisiología , Caulobacter crescentus/metabolismo , Biopelículas/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Histidina Quinasa/metabolismo , Histidina Quinasa/genética , GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , Flagelos/genética , Flagelos/metabolismo , Flagelos/fisiología , Transcripción Genética , Adhesión Bacteriana , Locomoción
9.
ISME J ; 18(1)2024 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-39113613

RESUMEN

Biofilms aid bacterial adhesion to surfaces via direct and indirect mechanisms, and formation of biofilms is considered as an important strategy for adaptation and survival in suboptimal environmental conditions. However, the molecular underpinnings of biofilm formation in subsurface sediment/groundwater ecosystems where microorganisms often experience fluctuations in nutrient input, pH, and nitrate or metal concentrations are underexplored. We examined biofilm formation under different nutrient, pH, metal, and nitrate regimens of 16 Rhodanobacter strains isolated from subsurface groundwater wells spanning diverse levels of pH (3.5 to 5) and nitrates (13.7 to 146 mM). Eight Rhodanobacter strains demonstrated significant biofilm growth under low pH, suggesting adaptations for survival and growth at low pH. Biofilms were intensified under aluminum stress, particularly in strains possessing fewer genetic traits associated with biofilm formation, findings warranting further investigation. Through random barcode transposon-site sequencing (RB-TnSeq), proteomics, use of specific mutants, and transmission electron microscopy analysis, we discovered flagellar loss under aluminum stress, indicating a potential relationship between motility, metal tolerance, and biofilm growth. Comparative genomic analyses revealed the absence of flagella and chemotaxis genes and the presence of a putative type VI secretion system in the highly biofilm-forming strain FW021-MT20. In this study we identified genetic determinants associated with biofilm growth under metal stress in a predominant environmental genus, Rhodanobacter, and identified traits aiding survival and adaptation to contaminated subsurface environments.


Asunto(s)
Adaptación Fisiológica , Aluminio , Biopelículas , Flagelos , Estrés Fisiológico , Biopelículas/crecimiento & desarrollo , Flagelos/genética , Flagelos/fisiología , Aluminio/toxicidad , Concentración de Iones de Hidrógeno , Nitratos/metabolismo , Agua Subterránea/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo
10.
Microbiol Spectr ; 12(10): e0030424, 2024 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-39189752

RESUMEN

Atypical enteropathogenic Escherichia coli (aEPEC) is a significant cause of diarrhea in low- and middle-income countries. Certain aEPEC strains, including the Brazilian representative strain of serotype O51:H40 called aEPEC 1711-4, can use flagella to attach to, invade, and persist in T84 and Caco-2 intestinal cells. It can also translocate from the gut to extraintestinal sites in a rat model. Although various aspects of the virulence of this strain were studied and the requirement of a type III secretion system for the efficiency of the invasion process was demonstrated, the expression of the locus of enterocyte effacement (LEE) genes during the invasion and intracellular persistence remains unclear. To address this question, the expression of flagella and the different LEE operons was evaluated during kinetic experiments of the interaction of aEPEC 1711-4 with enterocytes in vitro. The genome of the strain was also sequenced. The results showed that flagella expression remained unchanged, but the expression of eae and escJ increased during the early interaction and invasion of aEPEC 1711-4 into Caco-2 cells, and there was no change 24 h post-infection during the persistence period. The number of actin accumulation foci formed on HeLa cells also increased during the 6-h analysis. No known gene related to the invasion process was identified in the genome of aEPEC 1711-4, which was shown to belong to the global EPEC lineage 10. These findings suggest that the LEE components and the intimate adherence promoted by intimin are necessary for the invasion and persistence of aEPEC 1711-4, but the detailed mechanism needs further study.IMPORTANCEAtypical enteropathogenic Escherichia coli (aEPEC) is a major cause of diarrhea, especially in low- and middle-income countries, like Brazil. However, due to the genome heterogeneity of each clonal group, it is difficult to comprehend the pathogenicity of this strain fully. Among aEPEC strains, 1711-4 can invade eukaryotic cells in vitro, cross the gut barrier, and reach extraintestinal sites in animal models. By studying how different known aEPEC virulence factors are expressed during the invasion process, we can gain insight into the commonalities of this phenotype among other aEPEC strains. This will help in developing preventive measures to control infections caused by invasive strains. No known virulence-encoding genes linked to the invasion process were found. Nevertheless, additional studies are still necessary to evaluate the role of other factors in this phenotype.


Asunto(s)
Enterocitos , Escherichia coli Enteropatógena , Infecciones por Escherichia coli , Proteínas de Escherichia coli , Flagelos , Serogrupo , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/patogenicidad , Escherichia coli Enteropatógena/metabolismo , Humanos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Enterocitos/microbiología , Células CACO-2 , Infecciones por Escherichia coli/microbiología , Flagelos/genética , Flagelos/metabolismo , Virulencia/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regulación Bacteriana de la Expresión Génica , Adhesión Bacteriana/genética , Animales , Brasil , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Operón/genética , Ratas
11.
Mol Microbiol ; 122(4): 447-454, 2024 10.
Artículo en Inglés | MEDLINE | ID: mdl-39096095

RESUMEN

FliL is a bacterial flagellar protein demonstrated to associate with, and regulate ion flow through, the stator complex in a diverse array of bacterial species. FliL is also implicated in additional functions such as stabilizing the flagellar rod, modulating rotor bias, sensing the surface, and regulating gene expression. How can one protein do so many things? Its location is paramount to understanding its numerous functions. This review will look at the evidence, attempt to resolve some conflicting findings, and offer new thoughts on FliL.


Asunto(s)
Proteínas Bacterianas , Flagelos , Flagelos/metabolismo , Flagelos/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Bacterias/metabolismo , Bacterias/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de la Membrana
12.
Mol Microbiol ; 122(2): 255-270, 2024 08.
Artículo en Inglés | MEDLINE | ID: mdl-39030901

RESUMEN

The flagellar MS-ring, uniquely constituted by FliF, is essential for flagellar biogenesis and functionality in several bacteria. The aim of this study was to dissect the role of FliF in the Gram-positive and peritrichously flagellated Bacillus cereus. We demonstrate that fliF forms an operon with the upstream gene fliE. In silico analysis of B. cereus ATCC 14579 FliF identifies functional domains and amino acid residues that are essential for protein functioning. The analysis of a ΔfliF mutant of B. cereus, constructed in this study using an in frame markerless gene replacement method, reveals that the mutant is unexpectedly able to assemble flagella, although in reduced amounts compared to the parental strain. Nevertheless, motility is completely abolished by fliF deletion. FliF deprivation causes the production of submerged biofilms and affects the ability of B. cereus to adhere to gastrointestinal mucins. We additionally show that the fliF deletion does not compromise the secretion of the three components of hemolysin BL, a toxin secreted through the flagellar type III secretion system. Overall, our findings highlight the important role of B. cereus FliF in flagella-related functions, being the protein required for complete flagellation, motility, mucin adhesion, and pellicle biofilms.


Asunto(s)
Bacillus cereus , Proteínas Bacterianas , Biopelículas , Flagelos , Operón , Bacillus cereus/metabolismo , Bacillus cereus/genética , Flagelos/metabolismo , Flagelos/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/genética , Adhesión Bacteriana , Regulación Bacteriana de la Expresión Génica , Eliminación de Gen , Proteínas de la Membrana
13.
BMC Microbiol ; 24(1): 234, 2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38951769

RESUMEN

BACKGROUND: Klebsiella aerogenes is an opportunistic pathogen that causes a wide variety of infections. Due to the rising problem of antibiotic resistance, novel antibiotics and strategies to combat bacterial infections are needed. Host-specific bacteriophages are natural enemies of bacteria and can be used in phage therapy as an alternative form of treatment against bacterial infections. Jumbo phages are defined as phages with genomes larger than 200 kb. Relatively few studies have been done on jumbo phages compared to smaller phages. RESULTS: A novel phage, fENko-Kae01, was isolated from a commercial phage cocktail. Genomic analysis revealed that fENko-Kae01 is a lytic jumbo phage with a 360 kb genome encoding 578 predicted genes. No highly similar phage genomes were identified and fENko-Kae01 may be a completely new genus representative. No known genes associated with lysogenic life cycle, bacterial virulence, or antibiotic resistance were identified. The phage had myovirus morphology and a narrow host range. Phage resistant bacterial mutants emerged under phage selection. Whole genome sequencing revealed that the biogenesis of the flagellum was affected in four mutants and the lack of functional flagellum was confirmed in motility assays. Furthermore, phage fENKo-Kae01 failed to adsorb on the non-motile mutants indicating that the bacterial flagellum is the phage-binding receptor. CONCLUSIONS: fENko-Kae01 is a novel jumbo bacteriophage that is considered safe for phage therapy. fENko-Kae01 uses the flagellum as the phage-binding receptor and may represent a completely novel genus.


Asunto(s)
Bacteriófagos , Enterobacter aerogenes , Flagelos , Genoma Viral , Especificidad del Huésped , Bacteriófagos/genética , Bacteriófagos/clasificación , Bacteriófagos/aislamiento & purificación , Bacteriófagos/fisiología , Flagelos/virología , Flagelos/genética , Enterobacter aerogenes/virología , Enterobacter aerogenes/genética , Secuenciación Completa del Genoma , Myoviridae/genética , Myoviridae/aislamiento & purificación , Myoviridae/clasificación , Myoviridae/fisiología
14.
Nat Commun ; 15(1): 5921, 2024 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-39004688

RESUMEN

The bacterial flagellum, which facilitates motility, is composed of ~20 structural proteins organized into a long extracellular filament connected to a cytoplasmic rotor-stator complex via a periplasmic rod. Flagellum assembly is regulated by multiple checkpoints that ensure an ordered gene expression pattern coupled to the assembly of the various building blocks. Here, we use epifluorescence, super-resolution, and transmission electron microscopy to show that the absence of a periplasmic protein (FlhE) prevents proper flagellar morphogenesis and results in the formation of periplasmic flagella in Salmonella enterica. The periplasmic flagella disrupt cell wall synthesis, leading to a loss of normal cell morphology resulting in cell lysis. We propose that FlhE functions as a periplasmic chaperone to control assembly of the periplasmic rod, thus preventing formation of periplasmic flagella.


Asunto(s)
Proteínas Bacterianas , Flagelos , Chaperonas Moleculares , Periplasma , Flagelos/metabolismo , Flagelos/ultraestructura , Flagelos/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Periplasma/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Salmonella enterica/metabolismo , Salmonella enterica/genética , Microscopía Electrónica de Transmisión , Proteínas Periplasmáticas/metabolismo , Proteínas Periplasmáticas/genética , Regulación Bacteriana de la Expresión Génica
15.
mBio ; 15(8): e0071524, 2024 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-39037271

RESUMEN

The bacterial predator Bdellovibrio bacteriovorus is considered to be obligatorily prey (host)-dependent (H-D), and thus unable to form biofilms. However, spontaneous host-independent (H-I) variants grow axenically and can form robust biofilms. A screen of 350 H-I mutants revealed that single mutations in stator genes fliL or motA were sufficient to generate flagellar motility-defective H-I strains able to adhere to surfaces but unable to develop biofilms. The variants showed large transcriptional shifts in genes related to flagella, prey-invasion, and cyclic-di-GMP (CdG), as well as large changes in CdG cellular concentration relative to the H-D parent. The introduction of the parental fliL allele resulted in a full reversion to the H-D phenotype, but we propose that specific interactions between stator proteins prevented functional complementation by fliL paralogs. In contrast, specific mutations in a pilus-associated protein (Bd0108) mutant background were necessary for biofilm formation, including secretion of extracellular DNA (eDNA), proteins, and polysaccharides matrix components. Remarkably, fliL disruption strongly reduced biofilm development. All H-I variants grew similarly without prey, showed a strain-specific reduction in predatory ability in prey suspensions, but maintained similar high efficiency in prey biofilms. Population-wide allele sequencing suggested additional routes to host independence. Thus, stator and invasion pole-dependent signaling control the H-D and the H-I biofilm-forming phenotypes, with single mutations overriding prey requirements, and enabling shifts from obligate to facultative predation, with potential consequences on community dynamics. Our findings on the facility and variety of changes leading to facultative predation also challenge the concept of Bdellovibrio and like organisms being obligate predators. IMPORTANCE: The ability of bacteria to form biofilms is a central research theme in biology, medicine, and the environment. We show that cultures of the obligate (host-dependent) "solitary" predatory bacterium Bdellovibrio bacteriovorus, which cannot replicate without prey, can use various genetic routes to spontaneously yield host-independent (H-I) variants that grow axenically (as a single species, in the absence of prey) and exhibit various surface attachment phenotypes, including biofilm formation. These routes include single mutations in flagellar stator genes that affect biofilm formation, provoke motor instability and large motility defects, and disrupt cyclic-di-GMP intracellular signaling. H-I strains also exhibit reduced predatory efficiency in suspension but high efficiency in prey biofilms. These changes override the requirements for prey, enabling a shift from obligate to facultative predation, with potential consequences on community dynamics.


Asunto(s)
Proteínas Bacterianas , Bdellovibrio bacteriovorus , Biopelículas , Flagelos , Biopelículas/crecimiento & desarrollo , Flagelos/genética , Flagelos/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bdellovibrio bacteriovorus/genética , Bdellovibrio bacteriovorus/fisiología , Mutación , Regulación Bacteriana de la Expresión Génica , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo
16.
Fish Shellfish Immunol ; 151: 109752, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38977112

RESUMEN

Pseudomonas plecoglossicida is a vital pathogen that poses a substantial risk to aquaculture. Small RNAs (sRNAs) are non-coding regulatory molecules capable of sensing environmental changes and modulating virulence-associated signaling pathways, such as the assembly of flagella. However, the relevant researches on P. plecoglossicida are an urgent need. Here, we report a novel sRNA, sRNA562, which has potential to regulate the post-transcriptional of fliP, a key component of the lateral flagellar type III secretion system. In this study, the effects of sRNA562 on the virulence of P. plecoglossicida and its role in regulating the pathogenic process were investigated through the use of a constructed sRNA562 deletion strain. The deletion of sRNA562 resulted in an up-regulation of fliP in P. plecoglossicida, and leading to increased swarming motility and enhanced the ability of biofilm formation, adhesion and chemotaxis. Subsequent artificial infection experiment demonstrated that the deletion of sRNA562 increased the virulence of P. plecoglossicida towards hybrid grouper, as evidenced by a reduction in survival rate, elevation of tissue bacterial load, and the exacerbation of histopathological damage. Further studies have found that the deletion of sRNA562 lead to an up-regulation of fliP expression during hybrid grouper infection, thereby enhancing bacterial swarming ability and ultimately heightening pathogenicity, leading to a dysregulated host response to infection, tissue damage and eventually death. Our work revealed a sRNA that exerts negative regulation on the expression of lateral flagella in P. plecoglossicida, thereby impacting its virulence. These findings provide a new perspective on the virulence regulation mechanism of P. plecoglossicida, contributing to a more comprehensive understanding in the field of pathogenicity research.


Asunto(s)
Enfermedades de los Peces , Flagelos , Regulación Bacteriana de la Expresión Génica , Pseudomonas , ARN Pequeño no Traducido , Pseudomonas/patogenicidad , Pseudomonas/genética , Pseudomonas/fisiología , Virulencia/genética , Animales , Enfermedades de los Peces/microbiología , ARN Pequeño no Traducido/genética , Flagelos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ARN Bacteriano/genética , Sistemas de Secreción Tipo III/genética , Lubina , Infecciones por Pseudomonas/inmunología
17.
Soft Matter ; 20(37): 7397-7404, 2024 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-39021099

RESUMEN

Escherichia coli expresses surface appendages including fimbriae, flagella, and curli, at various levels in response to environmental conditions and external stimuli. Previous studies have revealed an interplay between expression of fimbriae and flagella in several E. coli strains, but how this regulation between fimbrial and flagellar expression affects adhesion to interfaces is incompletely understood. Here, we investigate how the concurrent expression of fimbriae and flagella by engineered strains of E. coli MG1655 affects their adhesion at liquid-solid and liquid-liquid interfaces. We tune fimbrial and flagellar expression on the cell surface through plasmid-based inducible expression of the fim operon and fliC-flhDC genes. We show that increased fimbrial expression increases interfacial adhesion as well as bacteria-driven actuation of micron-sized objects. Co-expression of flagella in fimbriated bacteria, however, does not greatly affect either of these properties. Together, these results suggest that interfacial adhesion as well as motion actuated by adherent bacteria can be altered by controlling the expression of surface appendages.


Asunto(s)
Adhesión Bacteriana , Proteínas de Escherichia coli , Escherichia coli , Fimbrias Bacterianas , Flagelos , Flagelos/metabolismo , Flagelos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Operón , Regulación Bacteriana de la Expresión Génica , Flagelina , Transactivadores
18.
J Assist Reprod Genet ; 41(9): 2271-2278, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38963606

RESUMEN

PURPOSE: To identify novel variants in ACTL9 and new phenotypes responsible for male infertility. METHODS: Genomic DNA was extracted from peripheral blood samples for whole-exome sequencing (WES). Computer-assisted sperm analysis (CASA) was used to test the motility of spermatozoa. The ultrastructure of flagella and the mitochondrial sheath were assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Immunostaining was used to validate the localization and expression of ACTL9 and ACTL7A. An Actl9-mutated mouse model was used to validate the phenotypes by CASA and TEM. RESULTS: We identified novel homozygous variants in ACTL9 in two independent Chinese families. Spermatozoa with ACTL9 mutations showed decreased CASA parameters and a higher proportion of spermatozoa with abnormal morphology, exhibiting coiled flagella and a thickened midpiece. The spermatozoa were characterized by chaotic or irregular '9+2' structures and irregular mitochondrial sheath arrangements in the flagellum. Actl9 knock-in mice also showed abnormal CASA parameters and irregular '9+2' structures in flagella. CONCLUSIONS: Our study expands the mutation spectrum and phenotypic spectrum of ACTL9.


Asunto(s)
Flagelos , Homocigoto , Infertilidad Masculina , Mitocondrias , Mutación , Motilidad Espermática , Cola del Espermatozoide , Espermatozoides , Masculino , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Humanos , Ratones , Espermatozoides/patología , Espermatozoides/ultraestructura , Espermatozoides/metabolismo , Animales , Mitocondrias/genética , Mitocondrias/ultraestructura , Mitocondrias/patología , Mitocondrias/metabolismo , Mutación/genética , Cola del Espermatozoide/patología , Cola del Espermatozoide/metabolismo , Cola del Espermatozoide/ultraestructura , Flagelos/genética , Flagelos/ultraestructura , Flagelos/metabolismo , Motilidad Espermática/genética , Secuenciación del Exoma , Linaje , Adulto , Análisis de Semen
20.
Int J Mol Sci ; 25(13)2024 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-39000482

RESUMEN

Plesiomonas shigelloides, a Gram-negative bacillus, is the only member of the Enterobacteriaceae family able to produce polar and lateral flagella and cause gastrointestinal and extraintestinal illnesses in humans. The flagellar transcriptional hierarchy of P. shigelloides is currently unknown. In this study, we identified FlaK, FlaM, FliA, and FliAL as the four regulators responsible for polar and lateral flagellar regulation in P. shigelloides. To determine the flagellar transcription hierarchy of P. shigelloides, the transcriptomes of the WT and ΔflaK, ΔflaM, ΔfliA, and ΔfliAL were carried out for comparison in this study. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and luminescence screening assays were used to validate the RNA-seq results, and the Electrophoretic Mobility Shift Assay (EMSA) results revealed that FlaK can directly bind to the promoters of fliK, fliE, flhA, and cheY, while the FlaM protein can bind directly to the promoters of flgO, flgT, and flgA. Meanwhile, we also observed type VI secretion system (T6SS) and type II secretion system 2 (T2SS-2) genes downregulated in the transcriptome profiles, and the killing assay revealed lower killing abilities for ΔflaK, ΔflaM, ΔfliA, and ΔfliAL compared to the WT, indicating that there was a cross-talk between the flagellar hierarchy system and bacterial secretion system. Invasion assays also showed that ΔflaK, ΔflaM, ΔfliA, and ΔfliAL were less effective in infecting Caco-2 cells than the WT. Additionally, we also found that the loss of flagellar regulators causes the differential expression of some of the physiological metabolic genes of P. shigelloides. Overall, this study aims to reveal the transcriptional hierarchy that controls flagellar gene expression in P. shigelloides, as well as the cross-talk between motility, virulence, and physiological and metabolic activity, laying the groundwork for future research into P. shigelloides' coordinated survival in the natural environment and the mechanisms that infect the host.


Asunto(s)
Proteínas Bacterianas , Flagelos , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Plesiomonas , Flagelos/metabolismo , Flagelos/genética , Plesiomonas/genética , Plesiomonas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Transcriptoma , Regiones Promotoras Genéticas , Sistemas de Secreción Bacterianos/genética , Sistemas de Secreción Bacterianos/metabolismo , Transcripción Genética , Humanos
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