Role of Proteus mirabilis flagella in biofilm formation / Papel de los flagelos de Proteus mirabilis en la formación de biofilms

Abstract Proteus mirabilis (P. mirabilis) is a common etiological agent of urinary tract infec-tions, particularly those associated with catheterization. P. mirabilis efficiently forms biofilms on different surfaces and shows a multicellular behavior called 'swarming', mediated by flagella. To date, the role of flagella in P. mirabilis biofilm formation has been under debate. In this study, we assessed the role of P. mirabilis flagella in biofilm formation using an isogenic allelic replacement mutant unable to express flagellin. Different approaches were used, such as the evaluation of cell surface hydrophobicity, bacterial motility and migration across catheter sections, measurements of biofilm biomass and biofilm dynamics by immunofluorescence and confocal microscopy in static and flow models. Our findings indicate that P. mirabilis flagella play a role in biofilm formation, although their lack does not completely avoid biofilm genera-tion. Our data suggest that impairment of flagellar function can contribute to biofilm prevention in the context of strategies focused on particular bacterial targets.

Resumen Proteus mirabilis (P mirabilis) es un agente etiológico común de infecciones del tracto urinario, en particular de aquellas asociadas con cateterización. P. mirabilis forma biofilms eficientemente en diferentes superficies y muestra un comportamiento multicelular llamado swarming, mediado por flagelos. Hasta el momento, el papel de los flagelos en la formación de biofilms de P. mirabilis ha estado en discusión. En este estudio, se evaluó el papel de los flagelos de P. mirabilis en la formación de biofilms, utilizando una mutante isogénica generada por reemplazo alélico, incapaz de expresar flagelina. Se utilizaron diferentes enfoques, como la evaluación de la hidrofobicidad de la superficie celular, de la movilidad y la migración bacteriana sobre secciones de catéteres y medidas de biomasa y de la dinámica del biofilm mediante inmunofluorescencia y microscopia confocal, tanto en modelos estáticos como de flujo. Nuestros hallazgos indican que los flagelos de P. mirabilis desempeñan un papel en la formación de biofilms, aunque su falta no suprime por completo su generación. Asimismo, evidencian que la interferencia de la función flagelar puede contribuir a evitar la formación de biofilms en el contexto de estrategias centradas en blancos bacterianos particulares.

A novel homozygous frameshift variant in DNAH8 causes multiple morphological abnormalities of the sperm flagella in a consanguineous Pakistani family / 亚洲男科学杂志(英文版)

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenozoospermia categorized by immotile spermatozoa with abnormal flagella in ejaculate. Whole-exome sequencing (WES) is used to detect pathogenic variants in patients with MMAF. In this study, a novel homozygous frameshift variant (c.6158_6159insT) in dynein axonemal heavy chain 8 (DNAH8) from two infertile brothers with MMAF in a consanguineous Pakistani family was identified by WES. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed DNAH8 mRNA decay in these patients with the DNAH8 mutation. Hematoxylin-eosin staining and transmission electron microscopy revealed highly divergent morphology and ultrastructure of sperm flagella in these patients. Furthermore, an immunofluorescence assay showed the absence of DNAH8 and a reduction in its associated protein DNAH17 in the patients' spermatozoa. Collectively, our study expands the phenotypic spectrum of patients with DNAH8-related MMAF worldwide.

Tactic movement of microalgae and its application in targeted transport: a review / 生物工程学报

Microalgae are a group of photosynthetic microorganisms, which have the general characteristics of plants such as photosynthesis, and some species have the ability of movement which resembles animals. Recently, it was reported that microalgae cells can be engineered to precisely deliver medicine-particles and other goods in microfluidic chips. These studies showed great application potential in biomedical treatment and pharmacodynamic analysis, which have become one of the current research hotspots. However, these developments have been rarely reviewed. Here, we summarized the advances in manageable movement exemplified by a model microalgae Chlamydomonas reinhardtii based on its characteristics of chemotaxis, phototaxis, and magnetotaxis. The bottlenecks and prospects in the application of microalgae-based tactic movement were also discussed. This review might be useful for rational design and modification of microalgal manageable movement to achieve targeted transport in medical and other fields.

Novel biallelic loss-of-function mutations in / 亚洲男科学杂志(英文版)

Multiple morphological abnormalities of the sperm flagella (MMAF) is a specific type of asthenoteratozoospermia, presenting with multiple morphological anomalies in spermatozoa, such as absent, bent, coiled, short, or irregular caliber flagella. Previous genetic studies revealed pathogenic mutations in genes encoding cilia and flagella-associated proteins (CFAPs; e.g., CFAP43, CFAP44, CFAP65, CFAP69, CFAP70, and CFAP251) responsible for the MMAF phenotype in infertile men from different ethnic groups. However, none of them have been identified in infertile Pakistani males with MMAF. In the current study, two Pakistani families with MMAF patients were recruited. Whole-exome sequencing (WES) of patients and their parents was performed. WES analysis reflected novel biallelic loss-of-function mutations in CFAP43 in both families (Family 1: ENST00000357060.3, p.Arg300Lysfs*22 and p.Thr526Serfs*43 in a compound heterozygous state; Family 2: ENST00000357060.3, p.Thr526Serfs*43 in a homozygous state). Sanger sequencing further confirmed that these mutations were segregated recessively in the families with the MMAF phenotype. Semiquantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was carried out to detect the effect of the mutation on mRNA of the affected gene. Previous research demonstrated that biallelic loss-of-function mutations in CFAP43 accounted for the majority of all CFAP43-mutant MMAF patients. To the best of our knowledge, this is the first study to report CFAP43 biallelic loss-of-function mutations in a Pakistani population with the MMAF phenotype. This study will help researchers and clinicians to understand the genetic etiology of MMAF better.

Caracterización molecular y fenotípica de aislamientos clínicos de Salmonella Typhimurium variante monofásica (1, 4, [5],12: i: -) recuperados en Colombia / Molecular and phenotypic characterization of Salmonella Typhimurium monophasic variant (1, 4, [5],12: i: -) from Colombian clinical isolates

Resumen: Introducción. La variante monofásica (1,4,[5],12:i:-) de Salmonella Typhimurium ocupa los primeros lugares en los programas de vigilancia de Salmonella a nivel mundial. En Colombia, Salmonella enterica variante monofásica alcanza el cuarto lugar en cuanto a los aislamientos clínicos recuperados por medio de la vigilancia por laboratorio del Grupo de Microbiología del Instituto Nacional de Salud, pero se desconoce si dichos aislamientos están relacionados con la variante monofásica de Typhimurium que circula a nivel global, y con sus características genéticas y fenotípicas. Objetivo. Caracterizar los aislamientos de Salmonella monofásica recuperados en Colombia entre el 2015 y el 2018 por el Grupo de Microbiología del Instituto Nacional de Salud. Materiales y métodos. Se analizaron 286 aislamientos clínicos de Salmonella enterica variante monofásica mediante PCR o secuenciación del genoma completo (Whole Genome Sequencing, WGS) para confirmar si correspondían a Salmonella Typhimurium variante monofásica, en tanto que, en 54 aislamientos, se determinó la estructura genética del operón que codifica la segunda fase flagelar y, en 23, se evaluó la motilidad, el crecimiento y la expresión de las proteínas de membrana externa. Resultados. El 61 % (n=174) de los aislamientos de Salmonella monofásica correspondió a Salmonella Typhimurium serovar monofásico. El 64,8 % (n=35/54) se relacionó con el clon europeo-español y, el 13 % (n=7/54), con el estadounidense. En dos aislamientos de orina se encontró una diferencia significativa en la motilidad y el crecimiento, así como ausencia de la porina OmpD en medio mínimo M9. Conclusiones. En el periodo de estudio, circuló en Colombia la variante monofásica de Salmonella Typhimurium relacionada con el clon europeo-español, y se registró ausencia total del operón fljAB. Los resultados evidenciaron cambios fenotípicos en los aislamientos provenientes de muestras de orina que sugieren adaptación en procesos invasivos.

Abstract: Introduction. The Salmonella Typhimurium monophasic variant (1,4,[5],12:i:-) is currently the most commonly detected variant in Salmonella surveillance programs worldwide. In Colombia, the Salmonella enterica monophasic variant is the fourth most common clinical isolate recovered through the laboratory surveillance of the Grupo de Microbiología from the Instituto Nacional de Salud; however, it is unknown whether these isolates are closely related to the monophasic Typhimurium variant, which circulates globally, and their genetic and phenotypic characteristics have not been reported. Objective. To characterize monophasic Salmonella enterica isolates identified in Colombia from 2015 to 2018 by the Instituto Nacional de Salud. Materials and methods. Two hundred eighty-six clinical isolates of the monophasic Salmonella enterica variant were analyzed by PCR or whole-genome sequencing to confirm whether they corresponded to the Salmonella Typhimurium monophasic variant while the genetic structure of the operon encoding the second flagellar phase was determined in 54 isolates. Motility, growth, and expression of the outer membrane proteins were evaluated in 23 isolates. Results. During the study period in Colombia, 61% (n=174) of Salmonella monophasic isolates belonged to Salmonella Typhimurium serovar monophasic (1,4,[5],12:i-). Of these, 64.8% (n=35/54) were related to the European/Spanish clone and 13% (n=7/54) to the U.S. clone. Two isolates recovered from urine samples showed differences in motility, growth, and the absence of the OmpD porin in M9 minimal medium. Conclusions. Most of the monophasic Salmonella Typhimurium variants that have circulated in Colombia since 2015 lacked the second phase of operon fljAB, which is related to the European/Spanish clone. The results evidenced phenotypic changes in urine samples suggesting bacterial adaptation in the case of these invasive samples.

Insight on multiple morphological abnormalities of sperm flagella in male infertility: What is new? / 亚洲男科学杂志(英文版)

The syndrome of multiple morphological abnormalities of the sperm flagella (MMAF) is a specific kind of asthenoteratozoospermia with a mosaic of flagellar morphological abnormalities (absent, short, bent, coiled, and irregular flagella). MMAF was proposed in 2014 and has attracted increasing attention; however, it has not been clearly understood. In this review, we elucidate the definition of MMAF from a systematical view, the difference between MMAF and other conditions with asthenoteratozoospermia or asthenozoospermia (such as primary mitochondrial sheath defects and primary ciliary dyskinesia), the knowledge regarding its etiological mechanism and related genetic findings, and the clinical significance of MMAF for intracytoplasmic sperm injection and genetic counseling. This review provides the basic knowledge for MMAF and puts forward some suggestions for further investigations.

FlaA gene deletion of LM90 SB2 inducing sheep encephalitis and the influence on virulence / 中国人兽共患病学报

The flagellin of Listeria monocytogenes is encoded by flaA gene;there is no detail report about the influence of flaA gene on the virulence of LM90 SB2.FlaA gene-deleted strain was constructed successfully with recombination technology.The influence of FlaA on LM90 SB2 virulence was evaluated by motility,BF formation,LD50,etc.Results showed that the colony morphology did not change,gene-deleted strain still had good genetic stability,but its growth was slow and the motility was decreased in the environment at 25 ℃,the morphological structure of the mutant BF was looser and incomplete,LD50 was increased from 4.27 × 106 cfu to 1.62 × 107 cfu.Results indicated that the flaA gene affected the flagellar formation of LM90 SB2 significantly,the ability of the BF formation and the virulence of the flaA deleted strain were decreased obviously.

The impacts of a fliD mutation on the biofilm formation of Helicobacter pylori

Objective To investigate the impact of the fliD gene on the biofilm formation of Helicobacter pylori (H. pylori). Methods H. pylori fliD mutant was constructed using inverse PCR mutagenesis. The mobility of the bacteria and its adhesion ability to human epithelial cells were assessed using a motility assay and a fluorescein isothiocyanate staining adhesion assay, respectively. The formation of biofilm was evaluated using a pellicle assay and a crystal violet staining assay. The cyto-architecture of the biofilm was documented with scanning electron microscopy. Results It was found that there was no significant difference in the levels of bacterial adhesion and the biofilm formation between the wild-type ATCC 43504 and the fliD mutant. Apart from a poor motility, the fliD mutant had a slightly delayed formation of its biofilm and an incomplete cyto-architecture of its biofilm. The bacterial cells residing in the biofilm of the fliD mutant showed a loose accumulation with less apparent cross-linking fibrils. Most of the mutant cells had truncated flagella. Conclusions This study provides the preliminary evidences that fliD potentially regulates biofilm formation and is required for the motility of H. pylori. Further studies need to be performed in order to develop fliD as a novel target for vaccine or antimicrobial agent in future.

Mechanical characteristics of the spirochete flagella in 2D micro-periplasmic space / 医用生物力学

Objective To investigate mechanical characteristics of the spirochete flagella with tight-fitting ribbon configuration in micro-periplasmic space. Methods The 2D model of two parallel plates was used to simplify the periplasmic space, and the effects of flagellum spacing and eccentricity on force and torque acted on the spirochete flagella, and wall shear stress acted on the spirochete protoplasmic cylinder were studied by using numerical simulation method. Results (1) The relationship between the flagellum horizontal force and eccentricity was presented as a parabolic curve, and the peak value of the flagellum horizontal force was mainly caused by the gradual increase of pressure difference at two sides of the cylinder and the resistance viscous force as well. Flagellum spacing had no significant influence on flagellum horizontal force. (2) The relationship between the flagellum torque and eccentricity was presented as an exponential curve, and smaller flagella spacing would cause bigger flagella torque. (3) Flagellum spacing had no significant effect on wall shear stress of the protoplasmic cylinder, but it would be increased with the number of flagella and the eccentricity increasing. Conclusions Numerical simulation results in this study can qualitatively reflect mechanical characteristics of the spirochete flagella, and also provide references for further understanding the morphology of spirochete as well as its kinematic mechanism and pathogenic characteristics.

Transcriptional Profiling of an Attenuated Salmonella Typhimurium ptsI Mutant Strain Under Low-oxygen Conditions using Microarray Analysis

Salmonella causes a wide variety of diseases ranging from mild diarrhea to severe systemic infections, such as like typhoid fever, in multiple organisms, ranging from mice to humans. A lack of ptsI, which encodes the first component of phosphoenolpyruvate (PEP) : carbohydrate phosphotransferase system (PTS), is known to cause Salmonella Typhimurium attenuation; however, the mechanisms behind this have not yet been elucidated. In this study, a DNA microarray was performed to determine why the virulence of ptsI mutants is attenuated under low-oxygen conditions in which the ptsI expression is enhanced. Of 106 down-regulated genes, the most repressed were pdu and tdc genes, which are required for propanediol utilization and threonine and serine metabolism, respectively. In addition, half the flagellar genes were down-regulated in the ptsI mutant strain. Because pdu genes are induced during infection and Tdc products and flagella-mediated motility are necessary for the invasion of S. Typhimurium, the invasive ability of ptsI mutants was examined. We found that ptsI mutation reduced the ability of S. Typhimurium to invade into host cells, suggesting that reduced expression of the pdu, tdc, and flagellar genes is involved in the attenuation of ptsI mutants.

Proliferation and bystander suppression induced by membrane and flagellar antigens of Trypanosoma cruzi

We have studied, in vitro, proliferation induced by flagella (FE) and membrane (ME) antigenic fractions of T. cruzi epimastigotes, as well as their regulatory effect on the proliferative response to PPD (Protein Purified Derivative). Crude flagella as well as bands from Western blots of flagella and membrane of epimastigotes were tested. Crude flagella elicited higher proliferation in mononuclear cells from patients with Chagasic cardiomyopathy (CDM) than in patients with no evidence of cardiac pathology (INF). Fractionated antigens induced a lower proliferative response, in intensity as well as in frequency, than the crude extracts. With FE, bands between 150 and 24.3 kDa (B3 to B18 with the exception of B4 and B13) induced higher CPM (Counts Per Minute) in CDM. In INF only bands B7 (87.3 to 80.1 kDa), 9 (69.8 to 64.6 kDa) and 13 (45.4 to 41.5 kDa) had high CPM. ME bands also elicited higher proliferation in CDM. However, only 5 out of 14 bands gave CPM higher than 1000 in CDM and none in INF. The mean down regulation (DR) of most bands was similar in both groups. But the frequency of relevant DR elicited by FE was significantly higher in CDM. In contrast the frequency of up regulation (UR) was higher in INF. Bands 13 and 14 of ME did not induce DR in most INF. The discordance between the frequency of relevant DR in CDM and INF was more evident with ME than with FE. The frequency of (UR) was 50% or higher with all ME bands in INF, but, lower than 12% in CDM. The higher UR in INF and of DR in CDM, suggest the presence of some balance or interaction in INF that is lost in CDM. In ME there might be antigens that could be relevant for the immunoprofilaxis of Chagas' disease. The difference in the clinical status of the two groups seems to be associated with the recognition of different groups of antigens together with variations in the nature of the regulation of the response of mononuclear cells to these antigens.

Hemos estudiado, in vitro, la respuesta proliferativa de células mononucleares a antígenos de Flagelo (FE) y Membrana (ME) de epimastigotes de T. cruzi, así como su efecto regulador en la respuesta proliferativa a PPD (Protein Purified derivative). Fue evaluada tanto la respuesta a Flagelo crudo como a las bandas de Western blots de FE y ME. El flagelo crudo indujo una proliferación de las células mononucleares más intensa en los pacientes con cardiomiopatía (CDM) que en los pacientes sin evidencia de patología cardiaca (INF). Los antígenos fraccionados causaron una menor respuesta proliferativa, tanto en intensidad como en frecuencia, que los antígenos crudos. Las bandas de FE, entre 150 y 24,3 kDa (B3 a B18 con la excepción de B4 y B13), indujeron CPM mas altas en CDM. En INF solo las bandas B7 (87,3 a 80,1 kDa), 9 (69,8 a 64,6 kDa) y 13 (45,4 a 41,5 kDa) causaron CPM (Cuentas Por Minuto) altas. Las bandas de ME también indujeron una proliferación mayor en CDM. Sin embargo, solo 5 de 14 bandas tuvieron CPM promedio mayores de 1000 en INF. La mayor parte de las bandas causaron una baja regulación (BR) que fue similar en ambos grupos. Sin embargo, la frecuencia de BR relevante producida por FE fue significativamente mayor en CDM. Por el contrario la sobre regulación (SB) fue mayor en INF. Las bandas 13 y 14 de ME no indujeron BR en la mayor parte de los INF. La discordancia entre la frecuencia de BR relevante en CDM e INF fue mas evidente con ME que con FE. La frecuencia de SB fue 50% o mas con todas las bandas de ME en INF, pero menor que 12% en los CDM. El predominio de SB en INF y de BR en CDM sugiere la presencia, en los INF, de algún tipo de equilibrio o interacción que esta ausente en los CDM. En ME pudieran estar presente antígenos relevantes para la inmunoprofilaxis de la enfermedad de Chagas. En conjunto nuestros resultados sugieren que las diferencias en los cuadros clínicos pudieran tener relación con la respuesta observada a los diferentes...

Roles of Flagellar Hook-Associated Proteins in Vibrio vulnificus Motility and Virulence

The bacterial flagellar structure can be divided into the basal body, the hook and the filament. Three minor components called hook associated proteins (HAP1, HAP2 and HAP3) form a junction between the hook and the filament (HAP1 and HAP3) and a capping structure at the distal end of flagellar filament (HAP2). Vibrio vulnificus is a halophilic pathogenic bacterium that is locomotive by means of a polar flagellum. From a V. vulnificus genome sequencing project, we obtained sequences of V. vulnificus flgK (Vv-flgK), flgL (Vv-flgL), and flaH (Vv-flaH) genes that encode HAP1, HAP3, and HAP2, respectively. To investigate roles of the HAP proteins, deletion mutants of the Vv-flgK, Vv-flgL and Vv-flaH were constructed. Electron microscopic analysis showed that the Vv-flgK or Vv-flgL mutant did not produce an intact polar flagellum while the Vv-flaH mutant produced a fragile flagellar structure. Western blot analysis against a major polar flagellin proposed that the null HAP1 and HAP3 mutations resulted in a failure of normal flagellar assembly since flagellins produced by the mutants were secreted out in the culture supernatants without long flagellar filaments. Motility was completely abolished by a single mutation in HAP1 or HAP3, and the HAP2 mutant showed a decreased motility. Also each of the mutants showed an impaired cytotoxicity and adherence to HeLa cell compared with the isogenic wild type strain. LD(50) increased by 10- and 11-fold in the V. vulnificus HAP3 and HAP2 mutant, respectively. These results suggest that the HAP proteins play important roles in polar flagellation and the virulence of V. vulnificus.

Some adaptation ways of creature in liquid environment / 国际生物医学工程杂志

Creatures living in liquid environment can form special exterior structure, internal framework, movement and substance distribution according to their respective living condition. This paper focused on the analysis via examples of macroscopic and microscopic organism. Quantitative analysis is used to make the description more exact and internal features of the organism easier to be examed.

Development of a sandwich ELISA for the detection of Listeria spp. using specific flagella antibodies

Five monoclonal antibodies (MAbs) and chicken immunoglobulin (IgY) were developed by immunizing with flagella purified from Listeria monocytogenes 4b and the five MAbs have been confirmed to be specific against three different epitopes of flagellin. The antibodies showed specific reaction to Listeria genus and no cross-reactivity with other bacteria tested in this experiment including E.coli O157:H7 and Salmonella enteritidis. Sandwich enzyme-linked immunosorbent assays (ELISA) using the MAbs and IgY were developed to detect Listeria species and the sensitivity and specificity of the developed ELISA have been analyzed. The detection limit of ELISA using MAb 2B1 and HRP labeled IgY was 1 x105cells/0.1 ml at 22degrees C and 1x106 cells/0.1 ml at 30degrees C. ELISA using the pair of MAbs (MAbs 2B1 and HRP labeled MAbs 7A3) detected up to 104cells/0.1 ml at 22degrees C and 30degrees C. Detection limit of sandwich ELISA using IgY was 10 times lower than MAb pair. Using the developed ELISA, we could detect several Listeria contaminated in food samples after 48 h-culturing. In conclusion, both MAbs and IgY have been proved to be highly specific to detect Listeria flagella and the developed sandwich ELISA using these antibodies would be useful tool for screening Listeria spp. in food.

The Research Advances on the Bacterial Flagella / 微生物学通报

Flagella is an unique structure that presents in appropriate half of bacillus,few coccus,and all of spirilla and vibrio.It is associated with bacterial motility and plays an important role in bacterial infection and immunity as well as classification.This review focused on the research progress on bacterial flagella,including staining methods,the structure and function of flagella gene,as well as immunegenicity.of flagellin.

Antiserum directed against cell surface antigens is lethal to Leishmania donovani promastigotes.

The purified flagellar fraction of Leishmania donovani promastigotes consists of 30-35 polypeptides. Antiserum raised against this fraction reacts with both flagella and pellicular membrane antigens as evident from immunoblot and immunofluorescence studies. Only 3 of these immunoreactive polypeptides are flagellum-specific. The antiserum agglutinates the cells and inhibits their growth in liquid culture medium. Moreover, glucose uptake and glucose-stimulated oxygen uptake of the promastigotes are significantly inhibited by the antiserum. The results indicate that the antiserum has a profound lethal effect on the in vitro propagation of the parasite.