Feature-based 3D+t descriptors of hyperactivated human sperm beat patterns.

The flagellar movement of the mammalian sperm plays a crucial role in fertilization. In the female reproductive tract, human spermatozoa undergo a process called capacitation which promotes changes in their motility. Only capacitated spermatozoa may be hyperactivated and only those that transition to hyperactivated motility are capable of fertilizing the egg. Hyperactivated motility is characterized by asymmetric flagellar bends of greater amplitude and lower frequency. Historically, clinical fertilization studies have used two-dimensional analysis to classify sperm motility, despite the inherently three-dimensional (3D) nature of sperm motion. Recent research has described several 3D beating features of sperm flagella. However, the 3D motility pattern of hyperactivated spermatozoa has not yet been characterized. One of the main challenges in classifying these patterns in 3D is the lack of a ground-truth reference, as it can be difficult to visually assess differences in flagellar beat patterns. Additionally, it is worth noting that only a relatively small proportion, approximately 10-20% of sperm incubated under capacitating conditions exhibit hyperactivated motility. In this work, we used a multifocal image acquisition system that can acquire, segment, and track sperm flagella in 3D+t. We developed a feature-based vector that describes the spatio-temporal flagellar sperm motility patterns by an envelope of ellipses. The classification results obtained using our 3D feature-based descriptors can serve as potential label for future work involving deep neural networks. By using the classification results as labels, it will be possible to train a deep neural network to automatically classify spermatozoa based on their 3D flagellar beating patterns. We demonstrated the effectiveness of the descriptors by applying them to a dataset of human sperm cells and showing that they can accurately differentiate between non-hyperactivated and hyperactivated 3D motility patterns of the sperm cells. This work contributes to the understanding of 3D flagellar hyperactive motility patterns and provides a framework for research in the fields of human and animal fertility.

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.

Role of Proteus mirabilis flagella in biofilm formation.

Proteus mirabilis(P. mirabilis) is a common etiological agent of urinary tract infections, 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 generation. Our data suggest that impairment of flagellar function can contribute to biofilm prevention in the context of strategies focused on particular bacterial targets.

The Assembly of Flagella in Enteropathogenic Escherichia coli Requires the Presence of a Functional Type III Secretion System.

In enteropathogenic Escherichia coli (EPEC), the production of flagella and the type III secretion system (T3SS) is activated in the presence of host cultured epithelial cells. The goal of this study was to investigate the relationship between expression of flagella and the T3SS. Mutants deficient in assembling T3SS basal and translocon components (ΔespA, ΔespB, ΔespD, ΔescC, ΔescN, and ΔescV), and in secreting effector molecules (ΔsepD and ΔsepL) were tested for flagella production under several growth conditions. The ΔespA mutant did not produce flagella in any condition tested, although fliC was transcribed. The remaining mutants produced different levels of flagella upon growth in LB or in the presence of cells but were significantly diminished in flagella production after growth in Dulbecco's minimal essential medium. We also investigated the role of virulence and global regulator genes in expression of flagella. The ΔqseB and ΔqseC mutants produced abundant flagella only when growing in LB and in the presence of HeLa cells, indicating that QseB and QseC act as negative regulators of fliC transcription. The ΔgrlR, ΔperA, Δler, Δhns, and Δfis mutants produced low levels of flagella, suggesting these regulators are activators of fliC expression. These data suggest that the presence of an intact T3SS is required for assembly of flagella highlighting the existence in EPEC of a cross-talk between these two virulence-associated T3SSs.

The Transcription of Flagella of Enteropathogenic Escherichia coli O127:H6 Is Activated in Response to Environmental and Nutritional Signals.

The flagella of enteropathogenic Escherichia coli (EPEC) O127:H6 E2348/69 mediate adherence to host proteins and epithelial cells. What environmental and nutritional signals trigger or down-regulate flagella expression in EPEC are largely unknown. In this study, we analyzed the influence of pH, oxygen tension, cationic and anionic salts (including bile salt), carbon and nitrogen sources, and catecholamines on the expression of the flagellin gene (fliC) of E2348/69. We found that sodium bicarbonate, which has been shown to induce the expression of type III secretion effectors, down-regulated flagella expression, explaining why E2348/69 shows reduced motility and flagellation when growing in Dulbecco's Minimal Essential Medium (DMEM). Further, growth under a 5% carbon dioxide atmosphere, in DMEM adjusted to pH 8.2, in M9 minimal medium supplemented with 80 mM glucose or sucrose, and in DMEM containing 150 mM sodium chloride, 0.1% sodium deoxycholate, or 30 µM epinephrine significantly enhanced fliC transcription to different levels in comparison to growth in DMEM alone. When EPEC was grown in the presence of HeLa cells or in supernatants of cultured HeLa cells, high levels (4-fold increase) of fliC transcription were detected in comparison to growth in DMEM alone. Our data suggest that nutritional and host signals that EPEC may encounter in the intestinal niche activate fliC expression in order to favor motility and host colonization.

Methods to Evaluate Bacterial Motility and Its Role in Bacterial-Host Interactions.

Bacterial motility is a widespread characteristic that can provide several advantages for the cell, allowing it to move towards more favorable conditions and enabling host-associated processes such as colonization. There are different bacterial motility types, and their expression is highly regulated by the environmental conditions. Because of this, methods for studying motility under realistic experimental conditions are required. A wide variety of approaches have been developed to study bacterial motility. Here, we present the most common techniques and recent advances and discuss their strengths as well as their limitations. We classify them as macroscopic or microscopic and highlight the advantages of three-dimensional imaging in microscopic approaches. Lastly, we discuss methods suited for studying motility in bacterial-host interactions, including the use of the zebrafish model.

Hibiscus Acid from Hibiscus sabdariffa L. Inhibits Flagellar Motility and Cell Invasion in Salmonella enterica.

Extracts of Hibiscus sabdariffa L. (commonly called Rosselle or "Jamaica flower" in Mexico) have been shown to have antibiotic and antivirulence properties in several bacteria. Here, an organic extract of H. sabdariffa L. is shown to inhibit motility in Salmonella enterica serovars Typhi and Typhimurium. The compound responsible for this effect was purified and found to be the hibiscus acid. When tested, this compound also inhibited motility and reduced the secretion of both flagellin and type III secretion effectors. Purified hibiscus acid was not toxic in tissue-cultured eukaryotic cells, and it was able to reduce the invasion of Salmonella Typhimurium in epithelial cells. Initial steps to understand its mode of action showed it might affect membrane proton balance.

Genome-wide analysis reveals a rhamnolipid-dependent modulation of flagellar genes in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an opportunistic pathogen and an important model organism for the study of bacterial group behaviors, including cell motility and biofilm formation. Rhamnolipids play a pivotal role in biofilm formation and motility phenotypes in P. aeruginosa, possibly acting as wetting agents and mediating chemotactic stimuli. However, no biochemical mechanism or gene regulatory network has been investigated in regard to rhamnolipids' modulation of those group behaviors. Using DNA microarrays, we investigated the transcriptomic profiles in the stationary phase of growth of wild-type P. aeruginosa PAO1 and a rhlA-mutant strain, unable to produce rhamnolipids. A total of 134 genes were differentially expressed, comprising different functional categories, indicating a significant physiological difference between the rhamnolipid-producing and -non-producing strains. Interestingly, several flagellar genes are repressed in the mutant strain, which directly relates to the inability of the rhlA-minus strain to develop a swarming-motility phenotype. Supplementation with exogenous rhamnolipids has partially restored flagellar gene expression in the mutant strain. Our results show significant evidence that rhamnolipids, the major biosynthetic products of rhlABC pathway, seem to modulate gene expression in P. aeruginosa.

Ultrastructural and Functional Analysis of a Novel Extra-Axonemal Structure in Parasitic Trichomonads.

Trichomonas vaginalis and Tritrichomonas foetus are extracellular flagellated parasites that inhabit humans and other mammals, respectively. In addition to motility, flagella act in a variety of biological processes in different cell types, and extra-axonemal structures (EASs) have been described as fibrillar structures that provide mechanical support and act as metabolic, homeostatic, and sensory platforms in many organisms. It has been assumed that T. vaginalis and T. foetus do not have EASs. However, here, we used complementary electron microscopy techniques to reveal the ultrastructure of EASs in both parasites. Such EASs are thin filaments (3-5 nm diameter) running longitudinally along the axonemes and surrounded by the flagellar membrane, forming prominent flagellar swellings. We observed that the formation of EAS increases after parasite adhesion on the host cells, fibronectin, and precationized surfaces. A high number of rosettes, clusters of intramembrane particles that have been proposed as sensorial structures, and microvesicles protruding from the membrane were observed in the EASs. Our observations demonstrate that T. vaginalis and T. foetus can connect to themselves by EASs present in flagella. The protein VPS32, a member of the ESCRT-III complex crucial for diverse membrane remodeling events, the pinching off and release of microvesicles, was found in the surface as well as in microvesicles protruding from EASs. Moreover, we demonstrated that the formation of EAS also increases in parasites overexpressing VPS32 and that T. vaginalis-VPS32 parasites showed greater motility in semisolid agar. These results provide valuable data about the role of the flagellar EASs in the cell-to-cell communication and pathogenesis of these extracellular parasites.

Difference of MreBCD Complex Interactome in Salmonella Typhimurium ST19 and ST213 Genotypes on Pathogenesis and Stress Response Pathways.

BACKGROUND: Salmonella enterica is the etiological agent of salmonellosis, with a high infection rate worldwide in Mexico, ST213 genotype of S. enterica ser. Typhimurium is displacing the ancestral ST19 genotype. Bacterial cytoskeleton protein complex MreBCD plays an important role in S. enterica pathogenesis, but underlying mechanisms are unknown. RESULTS: In this study, 106 interactions among MreBCD and 15 proteins from S. Typhimurium Pathogenicity Islands 1 (SP-I) and 2 (SP-2) involved in both bacterial virulence and stress response were predicted in ST213 and ST19 genotypes, of which 12 interactions were confirmed in vitro. In addition, gene cluster analysis in 100 S. Typhimurium genomes was performed for these genes. RESULTS AND CONCLUSION: The in silico and in vitro results showed a novel MreBCD interactome involved in regulating pathogenesis and stress response through interactions with virulence factors located at SPI-1 and SPI-2. Furthermore, both pseudogene presence and sequence variations in four tested proteins between genotypes resulted in differential interaction patterns involved in Salmonella motility and survival in eukaryotic cells, which could explain the replacement of ST19 by ST213 in Mexico.

Multiple CheY Proteins Control Surface-Associated Lifestyles of Azospirillum brasilense.

Bacterial chemotaxis is the directed movement of motile bacteria in gradients of chemoeffectors. This behavior is mediated by dedicated signal transduction pathways that couple environment sensing with changes in the direction of rotation of flagellar motors to ultimately affect the motility pattern. Azospirillum brasilense uses two distinct chemotaxis pathways, named Che1 and Che4, and four different response regulators (CheY1, CheY4, CheY6, and CheY7) to control the swimming pattern during chemotaxis. Each of the CheY homologs was shown to differentially affect the rotational bias of the polar flagellum and chemotaxis. The role, if any, of these CheY homologs in swarming, which depends on a distinct lateral flagella system or in attachment is not known. Here, we characterize CheY homologs' roles in swimming, swarming, and attachment to abiotic and biotic (wheat roots) surfaces and biofilm formation. We show that while strains lacking CheY1 and CheY6 are still able to navigate air gradients, strains lacking CheY4 and CheY7 are chemotaxis null. Expansion of swarming colonies in the presence of gradients requires chemotaxis. The induction of swarming depends on CheY4 and CheY7, but the cells' organization as dense clusters in productive swarms appear to depend on functional CheYs but not chemotaxis per se. Similarly, functional CheY homologs but not chemotaxis, contribute to attachment to both abiotic and root surfaces as well as to biofilm formation, although these effects are likely dependent on additional cell surface properties such as adhesiveness. Collectively, our data highlight distinct roles for multiple CheY homologs and for chemotaxis on swarming and attachment to surfaces.

Alpha-Tubulin Acetylation in Trypanosoma cruzi: A Dynamic Instability of Microtubules Is Required for Replication and Cell Cycle Progression.

Trypanosomatids have a cytoskeleton arrangement that is simpler than what is found in most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body. Subpellicular, mitotic and axonemal microtubules are extensively acetylated in Trypanosoma cruzi. Acetylation on lysine (K) 40 of α-tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that K40 acetylation occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle in eukaryotes. Several tubulin posttranslational modifications, including acetylation of K40, have been cataloged in trypanosomatids, but the functional importance of these modifications for microtubule dynamics and parasite biology remains largely undefined. The primary tubulin acetyltransferase was recently identified in several eukaryotes as Mec-17/ATAT, a Gcn5-related N-acetyltransferase. Here, we report that T. cruzi ATAT acetylates α-tubulin in vivo and is capable of auto-acetylation. TcATAT is located in the cytoskeleton and flagella of epimastigotes and colocalizes with acetylated α-tubulin in these structures. We have expressed TcATAT with an HA tag using the inducible vector pTcINDEX-GW in T. cruzi. Over-expression of TcATAT causes increased levels of the alpha tubulin acetylated species, induces morphological and ultrastructural defects, especially in the mitochondrion, and causes a halt in the cell cycle progression of epimastigotes, which is related to an impairment of the kinetoplast division. Finally, as a result of TcATAT over-expression we observed that parasites became more resistant to microtubule depolymerizing drugs. These results support the idea that α-tubulin acetylation levels are finely regulated for the normal progression of T. cruzi cell cycle.

Dual Control of Flagellar Synthesis and Exopolysaccharide Production by FlbD-FliX Class II Regulatory Proteins in Bradyrhizobium diazoefficiens.

Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean, has two independent flagellar systems: a single subpolar flagellum and several lateral flagella. Each flagellum is a very complex organelle composed of 30 to 40 different proteins located inside and outside the cell whereby flagellar gene expression must be tightly controlled. Such control is achieved by a hierarchy of regulators that ensure the timing of synthesis and the allocation of the different flagellar substructures. Previously, we analyzed the gene organization, expression, and function of the lateral flagellar system. Here, we studied the role of the response regulator FlbD and its trans-acting regulator FliX in the regulation of subpolar flagellar genes. We found that the LP-ring, distal rod, and hook of the subpolar flagellum were tightly controlled by FlbD and FliX. Furthermore, we obtained evidence for the existence of cross-regulation between these gene products and the expression of LafR, the master regulator of lateral flagella. In addition, we observed that extracellular polysaccharide production and biofilm formation also responded to these flagellar regulators. In this regard, FlbD might contribute to the switch between the planktonic and sessile states.IMPORTANCE Most environmental bacteria switch between two free-living states: planktonic, in which individual cells swim propelled by flagella, and sessile, in which bacteria form biofilms. Apart from being essential for locomotion, the flagellum has accessory functions during biofilm formation. The synthesis of flagella is a highly regulated process, and coordination with accessory functions requires the interconnection of various regulatory networks. Here, we show the role of class II regulators involved in the synthesis of the B. diazoefficiens subpolar flagellum and their possible participation in cross-regulation with the lateral flagellar system and exopolysaccharide production. These findings highlight the coordination of the synthetic processes of external structures, such as subpolar and lateral flagella, with exopolysaccharides, which are the main component of the biofilm matrix.

How Did the Archaellum Get Its Rotation?

How new functions evolve fascinates many evolutionary biologists. Particularly captivating is the evolution of rotation in molecular machines, as it evokes familiar machines that we have made ourselves. The archaellum, an archaeal analog of the bacterial flagellum, is one of the simplest rotary motors. It features a long helical propeller attached to a cell envelope-embedded rotary motor. Satisfyingly, the archaellum is one of many members of the large type IV filament superfamily, which includes pili, secretion systems, and adhesins, relationships that promise clues as to how the rotating archaellum evolved from a non-rotary ancestor. Nevertheless, determining exactly how the archaellum got its rotation remains frustratingly elusive. Here we review what is known about how the archaellum got its rotation, what clues exist, and what more is needed to address this question.

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

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.

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.

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.

Proteomics profiles of Cronobacter sakazakii and a fliF mutant: Adherence and invasion in mouse neuroblastoma cells.

Cronobacter sakazakii is an opportunistic foodborne pathogen associated with necrotizing enterocolitis, bacteremia, and meningitis in infants. A comparative proteomic study of C. sakazakii ATCC BAA-894 (CS WT) and a fliF::Tn5 mutant was performed, including the ability of both strains to adhere to and invade N1E-115 cells. To achieve this goal, a nonmotile C. sakazakii‬ ATCC BAA-894 fliF::Tn5 (CS fliF::Tn5) strain was generated using an EZ-Tn5 Tnp Transposome kit. Analysis of differential protein expression showed that 81.49% (361/443) of the proteins were expressed in both strains, 8.35% (37/443) were exclusively expressed in the CS WT strain, and 10.16% (45/443) were exclusively expressed in the CS fliF::Tn5 strain. The main exclusively expressed proteins in the CS WT strain were classified into the "cell motility" and "signal transduction mechanisms" subcategories. The proteins exclusively expressed in the CS fliF::Tn5 strain were classified into the following subcategories: "intracellular trafficking, secretion, and vesicular transport", "replication, recombination, and repair", "nucleotide transport and metabolism", "carbohydrate transport and metabolism", "coenzyme transport and metabolism", and "lipid transport and metabolism". Expression of the Cpa protein was detected in both strains, but Cpa was more abundant in the CS WT strain than in the CS fliF::Tn5 strain. A significant increase (p = 0.0001) in adherence to N1E-115 cells was observed in the nonmotile CS fliF::Tn5 strain (31.3 × 106 CFU/mL) compared to the CS WT strain (14.5 × 106 CFU/mL). Additionally, the CS WT strain showed a 0.17% invasion frequency in N1E-115 cells, which was significantly higher (p = 0.01) than that of the nonmotile CS fliF::Tn5 strain. In conclusion, the proteins involved in the motility were mainly identified by proteomic analysis in the CS WT strain compared to the CS fliF::Tn5 strain. Our data indicate that flagella are required to promote the invasion of N1E-115 cells and that the absence of flagella significantly increases the adherence to N1E-115 cells.

The structural organization of Giardia intestinalis cytoskeleton.

Giardia intestinalis, the causative agent of giardiasis, has complex cytoskeleton organization with structures involved in motility, adhesion, cell division, and cell differentiation. Microtubules are key components of the cytoskeleton and are the main elements of the ventral disc, median body, funis, in addition to four pairs of flagella. These cytoskeletal elements are basically stable microtubule arrangements. Although tubulins are the main proteins of these elements, molecular and biochemical analyses of Giardia trophozoites have revealed the presence of several new and not yet characterized proteins in these structures, which may contribute to their nanoarchitecture (mainly in the ventral disc). Despite these findings, morphological data are still required for understanding the organization and biogenesis of the cytoskeletal structures. In the study of this complex and specialized network of filaments in Giardia, two distinct and complementary approaches have been used in recent years: (a) transmission electron microscopy tomography of conventionally processed as well as cryo-fixed samples and (b) high-resolution scanning electron microscopy and helium ion microscopy in combination with new plasma membrane extraction protocols. In this review we include the most recent studies that have allowed better understanding of new Giardia components and their association with other filamentous structures of this parasite, thus providing new insights in the role of the cytoskeletal structures and their function in Giardia trophozoites.

The CtrA Regulon of Rhodobacter sphaeroides Favors Adaptation to a Particular Lifestyle.

Activation of the two-component system formed by CckA, ChpT, and CtrA (kinase, phosphotransferase, and response regulator, respectively) in Rhodobacter sphaeroides does not occur under the growth conditions commonly used in the laboratory. However, it is possible to isolate a gain-of-function mutant in CckA that turns the system on. Using massive parallel transcriptome sequencing (RNA-seq), we identified 321 genes that are differentially regulated by CtrA. From these genes, 239 were positively controlled and 82 were negatively regulated. Genes encoding the Fla2 polar flagella and gas vesicle proteins are strongly activated by CtrA. Genes involved in stress responses as well as several transcriptional factors are also positively controlled, whereas the photosynthetic and CO2 fixation genes are repressed. Potential CtrA-binding sites were bioinformatically identified, leading to the proposal that at least 81 genes comprise the direct regulon. Based on our results, we ponder that the transcriptional response orchestrated by CtrA enables a lifestyle in which R. sphaeroides will effectively populate the surface layer of a water body enabled by gas vesicles and will remain responsive to chemotactic stimuli using the chemosensoring system that controls the Fla2 flagellum. Simultaneously, fine-tuning of photosynthesis and stress responses will reduce the damage caused by heat and high light intensity in this water stratum. In summary, in this bacterium CtrA has evolved to control physiological responses that allow its adaptation to a particular lifestyle instead of controlling the cell cycle as occurs in other species.IMPORTANCE Cell motility in Alphaproteobacteria is frequently controlled by the CckA, ChpT, and CtrA two-component system. Under the growth conditions commonly used in the laboratory, ctrA is transcriptionally inactive in Rhodobacter sphaeroides, and motility depends on the Fla1 flagellar system that was acquired by a horizontal transfer event. Likely, the incorporation of this flagellar system released CtrA from the strong selective pressure of being the main motility regulator, allowing this two-component system to specialize and respond to some specific conditions. Identifying the genes that are directly regulated by CtrA could help us understand the conditions in which the products of this regulon are required. Massive parallel transcriptome sequencing (RNA-seq) revealed that CtrA orchestrates an adaptive response that contributes to the colonization of a particular environmental niche.

Characterization of FliL Proteins in Bradyrhizobium diazoefficiens: Lateral FliL Supports Swimming Motility, and Subpolar FliL Modulates the Lateral Flagellar System.

Bradyrhizobium diazoefficiens is a soil alphaproteobacterium that possesses two evolutionarily distinct flagellar systems, a constitutive subpolar flagellum and inducible lateral flagella that, depending on the carbon source, may be expressed simultaneously in liquid medium and used interactively for swimming. In each system, more than 30 genes encode the flagellar proteins, most of which are well characterized. Among the exceptions is FliL, which has been scarcely studied in alphaproteobacteria and whose function in other bacterial classes is somewhat controversial. Because each B. diazoefficiens flagellar system contains its own fliL paralog, we obtained the respective deletions ΔfliLS (subpolar) and ΔfliLL (lateral) to study their functions in swimming. We determined that FliLL was essential for lateral flagellum-driven motility. FliLS was dispensable for swimming in either liquid or semisolid medium; however, it was found to play a crucial role in upregulation of the lateral flagellum regulon under conditions of increased viscosity/flagellar load. Therefore, although FliLS seems to be not essential for swimming, it may participate in a mechanosensor complex that controls lateral flagellum induction.IMPORTANCE Bacterial motility propelled by flagella is an important trait in most environments, where microorganisms must explore the habitat toward beneficial resources and evade toxins. Most bacterial species have a unique flagellar system, but a few species possess two different flagellar systems in the same cell. An example is Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean, which uses both systems for swimming. Among the less-characterized flagellar proteins is FliL, a protein typically associated with a flagellum-driven surface-based collective motion called swarming. By using deletion mutants in each flagellar system's fliL, we observed that one of them (lateral) was required for swimming, while the other (subpolar) took part in the control of lateral flagellum synthesis. Hence, this protein seems to participate in the coordination of activity and production of both flagellar systems.