Overexpression of the flagellar motor protein MotB sensitizes Bacillus subtilis to aminoglycosides in a motility-independent manner.

The flagellar motor proteins, MotA and MotB, form a complex that rotates the flagella by utilizing the proton motive force (PMF) at the bacterial cell membrane. Although PMF affects the susceptibility to aminoglycosides, the effect of flagellar motor proteins on the susceptibility to aminoglycosides has not been investigated. Here, we found that MotB overexpression increased susceptibility to aminoglycosides, such as kanamycin and gentamicin, in Bacillus subtilis without affecting swimming motility. MotB overexpression did not affect susceptibility to ribosome-targeting antibiotics other than aminoglycosides, cell wall-targeting antibiotics, DNA synthesis-inhibiting antibiotics, or antibiotics inhibiting RNA synthesis. Meanwhile, MotB overexpression increased the susceptibility to aminoglycosides even in the motA-deletion mutant, which lacks swimming motility. Overexpression of the MotB mutant protein carrying an amino acid substitution at the proton-binding site (D24A) resulted in the loss of the enhanced aminoglycoside-sensitive phenotype. These results suggested that MotB overexpression sensitizes B. subtilis to aminoglycosides in a motility-independent manner. Notably, the aminoglycoside-sensitive phenotype induced by MotB requires the proton-binding site but not the MotA/MotB complex formation.

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.

Heterogeneous Flagellar Expression in Single Salmonella Cells Promotes Diversity in Antibiotic Tolerance.

Phenotypic heterogeneity among single cells in a genetically identical population leads to diverse environmental adaptation. The human and animal pathogen Salmonella enterica serovar Typhimurium exhibits heterogeneous expression of virulence genes, including flagellar and Salmonella pathogenicity island (SPI) genes. Little is known about how the differential expression of flagellar genes among single cells affects bacterial adaptation to stresses. Here, we have developed a triple-fluorescence reporter to simultaneously monitor the expression of flagellar and SPI-1 pathways. We show that the two pathways cross talk at the single-cell level. Intriguingly, cells expressing flagella (fliC-ON) exhibit decreased tolerance to antibiotics compared to fliC-OFF cells. Such variation depends on TolC-dependent efflux pumps. We further show that fliC-ON cells contain higher intracellular proton concentrations. This suggests that the assembly and rotation of flagella consume the proton motive force and decrease the efflux activity, resulting in antibiotic sensitivity. Such a trade-off between motility and efflux highlights a novel mechanism of antibiotic tolerance. IMPORTANCE Antibiotic resistance and tolerance pose a severe threat to human health. How bacterial pathogens acquire antibiotic tolerance is not clear. Here, we show that the human and animal pathogen Salmonella divides its population into subgroups that are different in their abilities to tolerate antibiotic treatments. In a Salmonella population that is genetically identical, some cells express flagella to move toward nutrients, while other cells do not express flagella. Interestingly, we show that Salmonella cells that do not express flagella are more tolerant to antibiotics. We have further determined the mechanism underlying such diverse responses to antibiotics. Flagellar motility uses cellular energy stored in the form of proton motive force and makes cells less efficient in pumping out toxic molecules such as antibiotics. The overall bacterial population therefore gains benefits from such diversity to quickly adapt to different environmental conditions.

Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells.

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.

Transcriptome analyses reveal the utilization of nitrogen sources and related metabolic mechanisms of Sporosarcina pasteurii.

In recent years, Sporosarcina pasteurii (S. pasteurii) has become one of the most popular bacteria in microbially induced calcium carbonate precipitation (MICP). Various applications have been developed based on the efficient urease that can induce the precipitation of calcium carbonate. However, the metabolic mechanism related to biomineralization of S. pasteurii has not been clearly elucidated. The process of bacterial culture and biomineralization consumes a large amount of urea or ammonium salts, which are usually used as agricultural fertilizers, not to mention probable environmental pollutions caused by the excessive use of these raw materials. Therefore, it is urgent to reveal the mechanism of nitrogen utilization and metabolism of S. pasteurii. In this paper, we compared the growth and gene expression of S. pasteurii under three different culture conditions through transcriptome analyses. GO and KEGG analyses revealed that both ammonium and urea were direct nitrogen sources of S. pasteurii, and the bacteria could not grow normally in the absence of ammonium or urea. To the best of our knowledge, this paper is the first one to reveal the nitrogen utilization mechanism of S. pasteurii through transcriptome methods. Furthermore, the presence of ammonium might promote the synthesis of intracellular ATP and enhance the motility of the bacteria. There should be an ATP synthesis mechanism associated with urea hydrolysis catalyzed by urease in S. pasteurii.

Acetylation/deacetylation and microtubule associated proteins influence flagellar axonemal stability and sperm motility.

PTMs and microtubule-associated proteins (MAPs) are known to regulate microtubule dynamicity in somatic cells. Reported literature on modulation of α-tubulin acetyl transferase (αTAT1) and histone deacetylase 6 (HDAC6) in animal models and cell lines illustrate disparity in correlating tubulin acetylation status with stability of MT. Our earlier studies showed reduced acetyl tubulin in sperm of asthenozoospermic individuals. Our studies on rat sperm showed that on inhibition of HDAC6 activity, although tubulin acetylation increased, sperm motility was reduced. Studies were therefore undertaken to investigate the influence of tubulin acetylation/deacetylation on MT dynamicity in sperm flagella using rat and human sperm. Our data on rat sperm revealed that HDAC6 specific inhibitor Tubastatin A (T) inhibited sperm motility and neutralized the depolymerizing and motility debilitating effect of Nocodazole. The effect on polymerization was further confirmed in vitro using pure MT and recHDAC6. Also polymerized axoneme was less in sperm of asthenozoosperm compared to normozoosperm. Deacetylase activity was reduced in sperm lysates and axonemes exposed to T and N+T but not in axonemes of sperm treated similarly suggesting that HDAC6 is associated with sperm axonemes or MT. Deacetylase activity was less in asthenozoosperm. Intriguingly, the expression of MDP3 physiologically known to bind to HDAC6 and inhibit its deacetylase activity remained unchanged. However, expression of acetyl α-tubulin, HDAC6 and microtubule stabilizing protein SAXO1 was less in asthenozoosperm. These observations suggest that MAPs and threshold levels of MT acetylation/deacetylation are important for MT dynamicity in sperm and may play a role in regulating sperm motility.

Responses to transient receptor potential (TRP) channel agonists in Chlamydomonas reinhardtii.

Pungent substances, such as capsaicin and gingerol, activate the transient receptor potential (TRP)-V1 channel and affect the feeding behaviors of animals. To gain insight into how living organisms have acquired a sense for pungent substances, we explored the response to TRP agonists in a protist, Chlamydomonas reinhardtii When capsaicin or gingerol was applied to wild-type cells, they became immotile, with flagella detaching from the cell body. The degree of deflagellation was nearly halved in a mutant defective in the TRP channel ADF1. Deflagellation in the adf1 mutant was inhibited further by Ruthenium Red, indicating ADF1 and another TRP channel are involved in the deflagellation response. The response to capsaicin and gingerol was not inhibited by TRPV1-specific blockers such as 4-(3-Chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide (BCTC) and capsazepine. When capsaicin or gingerol was applied to wild-type cells in the presence of Ruthenium Red, a large proportion lost motility while flagella remained attached, suggesting that flagella stop contributing to motility, at least in part, through a TRP-channel-independent pathway. These results indicate that pungent compounds such as capsaicin and gingerol induce loss of flagellar motility and flagellar detachment in C.reinhardtii cells.

Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes.

Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.

Inhibitors of Bacterial Swarming Behavior.

Bacteria can migrate in groups of flagella-driven cells over semisolid surfaces. This coordinated form of motility is called swarming behavior. Swarming is associated with enhanced virulence and antibiotic resistance of various human pathogens and may be considered as favorable adaptation to the diverse challenges that microbes face in rapidly changing environments. Consequently, the differentiation of motile swarmer cells is tightly regulated and involves multi-layered signaling networks. Controlling swarming behavior is of major interest for the development of novel anti-infective strategies. In addition, compounds that block swarming represent important tools for more detailed insights into the molecular mechanisms of the coordination of bacterial population behavior. Over the past decades, there has been major progress in the discovery of small-molecule modulators and mechanisms that allow selective inhibition of swarming behavior. Herein, an overview of the achievements in the field and future directions and challenges will be presented.

Tandem mass tag-based quantitative proteomic analysis reveal the inhibition mechanism of thyme essential oil against flagellum of Listeria monocytogenes.

In this study, the antimicrobial mechanism of thyme essential oil (EO) against Listeria monocytogenes (LM) was investigated at the protein level using tandem mass tag-based quantitative proteomic analysis. The proteomic profiles of LM with 2 log CFU/ml reduction after thyme EO treatment (0.28 µl/ml, Treatment-1) were compared with those of 4 log CFU/ml reduction (0.31 µl/ml, Treatment-2) to identify key proteins involved in microbial inhibition. The results show that 100 and 745 differentially expressed proteins in LM subjected to Treatment-1 vs control and Treatment-2 vs control, respectively. The differentially expressed proteins were functionally categorized using gene ontology enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and STRING analyses. The differentially expressed proteins of LM in Treatment-1 vs control were involved in 45 biological processes, 18 cellular components, 48 molecular functions and 31 KEGG pathways. That of LM in Treatment-2 vs control were involved in 246 biological processes, 45 cellular components, 309 molecular functions and 86 KEGG pathways. It demonstrated that thyme EO treatment induced the cellular processes, environmental information processing, genetic information processing, human diseases, metabolism, organismal systems in LM according to the differently expression protein. Based on the known protein components of flagellar assembly and bacterial chemotaxis, the results suggest that treatment with thyme EO might inhibit flagellar synthesis, block the flagellar motility, and induce partial structural collapse in LM. The structure of flagella filament was damaged by thyme EO treatment. In addition, treatment with thyme EO might affect motility related to chemotaxis and adaptation in LM. This research contributes to the understanding of the molecular mechanisms underlying the inhibiting effects of thyme EO against foodborne pathogens and provide novel insights for further development of EO antimicrobial agents.

Multidrug Resistance Regulators MarA, SoxS, Rob, and RamA Repress Flagellar Gene Expression and Motility in Salmonella enterica Serovar Typhimurium.

Production of flagella is costly and subject to global multilayered regulation, which is reflected in the hierarchical control of flagellar production in many bacterial species. For Salmonella enterica serovar Typhimurium and its relatives, global regulation of flagellar production primarily occurs through the control of flhDC transcription and mRNA translation. In this study, the roles of the homologous multidrug resistance regulators MarA, SoxS, Rob, and RamA (constituting the mar-sox-rob regulon in S Typhimurium) in regulating flagellar gene expression were explored. Each of these regulators was found to inhibit flagellar gene expression, production of flagella, and motility. To different degrees, repression via these transcription factors occurred through direct interactions with the flhDC promoter, particularly for MarA and Rob. Additionally, SoxS repressed flagellar gene expression via a posttranscriptional pathway, reducing flhDC translation. The roles of these transcription factors in reducing motility in the presence of salicylic acid were also elucidated, adding a genetic regulatory element to the response of S Typhimurium to this well-characterized chemorepellent. Integration of flagellar gene expression into the mar-sox-rob regulon in S Typhimurium contrasts with findings for closely related species such as Escherichia coli, providing an example of plasticity in the mar-sox-rob regulon throughout the Enterobacteriaceae family.IMPORTANCE The mar-sox-rob regulon is a large and highly conserved stress response network in the Enterobacteriaceae family. Although it is well characterized in E. coli, the extent of this regulon in related species is unclear. Here, the control of costly flagellar gene expression is connected to the mar-sox-rob regulon of S Typhimurium, contrasting with the E. coli regulon model. These findings demonstrate the flexibility of the mar-sox-rob regulon to accommodate novel regulatory targets, and they provide evidence for its broader regulatory role within this family of diverse bacteria.

PQS Produced by the Pseudomonas aeruginosa Stress Response Repels Swarms Away from Bacteriophage and Antibiotics.

We investigate the effect of bacteriophage infection and antibiotic treatment on the coordination of swarming, a collective form of flagellum- and pilus-mediated motility in bacteria. We show that phage infection of the opportunistic bacterial pathogen Pseudomonas aeruginosa abolishes swarming motility in the infected subpopulation and induces the release of the Pseudomonas quinolone signaling molecule PQS, which repulses uninfected subpopulations from approaching the infected area. These mechanisms have the overall effect of limiting the infection to a subpopulation, which promotes the survival of the overall population. Antibiotic treatment of P. aeruginosa elicits the same response, abolishing swarming motility and repulsing approaching swarms away from the antibiotic-treated area through a PQS-dependent mechanism. Swarms are entirely repelled from the zone of antibiotic-treated P. aeruginosa, consistent with a form of antibiotic evasion, and are not repelled by antibiotics alone. PQS has multiple functions, including serving as a quorum-sensing molecule, activating an oxidative stress response, and regulating the release of virulence and host-modifying factors. We show that PQS serves additionally as a stress warning signal that causes the greater population to physically avoid cell stress. The stress response at the collective level observed here in P. aeruginosa is consistent with a mechanism that promotes the survival of bacterial populations.IMPORTANCE We uncover a phage- and antibiotic-induced stress response in the clinically important opportunistic pathogen Pseudomonas aeruginosa Phage-infected P. aeruginosa subpopulations are isolated from uninfected subpopulations by the production of a stress-induced signal. Activation of the stress response by antibiotics causes P. aeruginosa to physically be repelled from the area containing antibiotics altogether, consistent with a mechanism of antibiotic evasion. The stress response observed here could increase P. aeruginosa resilience against antibiotic treatment and phage therapy in health care settings, as well as provide a simple evolutionary strategy to avoid areas containing stress.

Ethanol Decreases Pseudomonas aeruginosa Flagellar Motility through the Regulation of Flagellar Stators.

Pseudomonas aeruginosa frequently encounters microbes that produce ethanol. Low concentrations of ethanol reduced P. aeruginosa swim zone area by up to 45% in soft agar. The reduction of swimming by ethanol required the flagellar motor proteins MotAB and two PilZ domain proteins (FlgZ and PilZ). PilY1 and the type 4 pilus alignment complex (comprising PilMNOP) were previously implicated in MotAB regulation in surface-associated cells and were required for ethanol-dependent motility repression. As FlgZ requires the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) to represses motility, we screened mutants lacking genes involved in c-di-GMP metabolism and found that mutants lacking diguanylate cyclases SadC and GcbA were less responsive to ethanol. The double mutant was resistant to its effects. As published previously, ethanol also represses swarming motility, and the same genes required for ethanol effects on swimming motility were required for its regulation of swarming. Microscopic analysis of single cells in soft agar revealed that ethanol effects on swim zone area correlated with ethanol effects on the portion of cells that paused or stopped during the time interval analyzed. Ethanol increased c-di-GMP in planktonic wild-type cells but not in ΔmotAB or ΔsadC ΔgcbA mutants, suggesting c-di-GMP plays a role in the response to ethanol in planktonic cells. We propose that ethanol produced by other microbes induces a regulated decrease in P. aeruginosa motility, thereby promoting P. aeruginosa colocalization with ethanol-producing microbes. Furthermore, some of the same factors involved in the response to surface contact are involved in the response to ethanol.IMPORTANCE Ethanol is an important biologically active molecule produced by many bacteria and fungi. It has also been identified as a potential marker for disease state in cystic fibrosis. In line with previous data showing that ethanol promotes biofilm formation by Pseudomonas aeruginosa, here we report that ethanol reduces swimming motility using some of the same proteins involved in surface sensing. We propose that these data may provide insight into how microbes, via their metabolic byproducts, can influence P. aeruginosa colocalization in the context of infection and in other polymicrobial settings.

Methanol extract of Lonicera caerulea var. emphyllocalyx fruit has anti-motility and anti-biofilm activity against enteropathogenic Escherichia coli.

Foodborne diseases have become a worldwide problem that threatens public health and welfare. Enteropathogenic Escherichia coli (EPEC) is one of major pathogens of moderate to severe diarrhea. The increased prevalence of EPEC strains that produce extended spectrum ß-lactamase (ESBL) has deepened the problem. The fruit of Lonicera caerulea var. emphyllocalyx (LCE) has been used as a traditional food preservative and medicine in northern temperate zones such as Hokkaido Island, Japan. In this study, we investigated the antibacterial effect of LCE fruit extract (LCEE) against EPEC. The antibacterial activities of LCEE were examined by bacterial growth, time-kill curve, soft-agar motility, electron microscopy, and 96 well-microplate biofilm assays. We also investigated the bacterial mRNA expression of biofilm-associated genes (fliC, csgA, and fimA) by quantitative real-time PCR assays. LCEE was found to suppress the growth, time-kill curve, and spread of EPEC. It also reduced the biofilm formation in a dose-dependent manner. Morphological analysis using transmission and scanning electron microscopy revealed that LCEE diminished the function of flagella resulting in reduced motility and biofilm formation. The mRNA expression of all three biofilm associated genes was downregulated under LCEE treatment. Extracts of the fruit of LCE inhibit the motility and biofilm formation of EPEC as a result of the inhibition of flagella development and function. We propose LCEE as a therapeutic candidate for the effective therapy of EPEC-associated infectious diseases.

Sulfated vizantin suppresses mucin layer penetration dependent on the flagella motility of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections, such as pneumonia and bacteremia. Several studies demonstrated that flagellar motility is an important virulence factor for P. aeruginosa infection. In this study, we determined whether sulfated vizantin affects P. aeruginosa flagellar motility in the absence of direct antimicrobial activity. We found that 100 µM sulfated vizantin suppressed P. aeruginosa PAO1 from penetrating through an artificial mucin layer by affecting flagellar motility, although it did not influence growth nor bacterial protease activity. To further clarify the mechanism in which sulfated vizantin suppresses the flagellar motility of P. aeruginosa PAO1, we examined the effects of sulfated vizantin on the composition of the flagellar filament and mRNA expression of several flagella-related genes, finding that sulfated vizantin did not influence the composition of the flagellar complex (fliC, motA, and motB) in P. aeruginosa PAO1, but significantly decreased mRNA expression of the chemotaxis-related genes cheR1, cheW, and cheZ. These results indicated that sulfated vizantin is an effective inhibitor of flagellar motility in P. aeruginosa.

Gametocytes from K13 Propeller Mutant Plasmodium falciparum Clinical Isolates Demonstrate Reduced Susceptibility to Dihydroartemisinin in the Male Gamete Exflagellation Inhibition Assay.

Mutations in the kelch propeller domain (K13 propeller) of Plasmodium falciparum parasites from Southeast Asia are associated with reduced susceptibility to artemisinin. We exposed in vitro-cultured stage V gametocytes from Cambodian K13 propeller mutant parasites to dihydroartemisinin and evaluated the inhibition of male gamete formation in an in vitro exflagellation inhibition assay (EIA). Gametocytes with the R539T and C580Y K13 propeller alleles were less susceptible to dihydroartemisinin and had significantly higher 50% inhibitory concentrations (IC50s) than did gametocytes with wild-type alleles.

Feedback regulation of Caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor HfiA.

To permanently attach to surfaces, Caulobacter crescentusproduces a strong adhesive, the holdfast. The timing of holdfast synthesis is developmentally regulated by cell cycle cues. When C. crescentusis grown in a complex medium, holdfast synthesis can also be stimulated by surface sensing, in which swarmer cells rapidly synthesize holdfast in direct response to surface contact. In contrast to growth in complex medium, here we show that when cells are grown in a defined medium, surface contact does not trigger holdfast synthesis. Moreover, we show that in a defined medium, flagellum synthesis and regulation of holdfast production are linked. In these conditions, mutants lacking a flagellum attach to surfaces over time more efficiently than either wild-type strains or strains harboring a paralyzed flagellum. Enhanced adhesion in mutants lacking flagellar components is due to premature holdfast synthesis during the cell cycle and is regulated by the holdfast synthesis inhibitor HfiA. hfiA transcription is reduced in flagellar mutants and this reduction is modulated by the diguanylate cyclase developmental regulator PleD. We also show that, in contrast to previous predictions, flagella are not necessarily required for C. crescentus surface sensing in the absence of flow, and that arrest of flagellar rotation does not stimulate holdfast synthesis. Rather, our data support a model in which flagellum assembly feeds back to control holdfast synthesis via HfiA expression in a c-di-GMP-dependent manner under defined nutrient conditions.

Dynamic Sessile-Droplet Habitats for Controllable Cultivation of Bacterial Biofilm.

Bacterial biofilms play essential roles in biogeochemical cycling, degradation of environmental pollutants, infection diseases, and maintenance of host health. The lack of quantitative methods for growing and characterizing biofilms remains a major challenge in understanding biofilm development. In this study, a dynamic sessile-droplet habitat is introduced, a simple method which cultivates biofilms on micropatterns with diameters of tens to hundreds of micrometers in a microfluidic channel. Nanoliter plugs are utilized, spaced by immiscible carrier oil to initiate and support the growth of an array of biofilms, anchored on and spatially confined to the micropatterns arranged on the bottom surface of the microchannel, while planktonic or dispersal cells are flushed away by shear force of aqueous plugs. The performance of the aforementioned method of cultivating biofilms is demonstrated by Pseudomonas aeruginosa PAO1 and its derived mutants, and quantitative antimicrobial susceptibility testing of PAO1 biofilms. This method could significantly eliminate corner effects, avoid microchannel clogging, and constrain the growth of biofilms for long-term observations. The controllable sessile droplet-based biofilm cultivation presented in this study should shed light on more quantitative and long-term studies of biofilms, and open new avenues for investigation of biofilm attachment, growth, expansion, and eradication.

Chemical Screening for Flagella-Associated Phenotypes in Chlamydomonas reinhardtii.

Flagella of the unicellular green alga Chlamydomonas reinhardtii are nearly identical to cilia of vertebrate cells and provide an excellent model to study ciliogenesis. Cilia and flagella are important organelles used for motility and sensing the extracellular environment. Abnormalities in cilia structure or ciliary dysfunction can have devastating consequences ranging from diabetes and obesity to polycystic kidney disease and mental retardation. Small-molecule inhibitor libraries can be used to screen for flagellum-associated phenotypes in assembly, length, motility, deflagellation, and cellular toxicity. These phenotypes can be assessed from direct microscopic visualization and custom-designed assays. These methods identify fundamental regulators of ciliary biology as well as potential therapeutic interventions for ciliopathies.

In Vitro Antileishmanial Activity of a Black Morel, Morchella importuna (Ascomycetes).

We studied the anti-Leishmania activity of a fractionated extract from the mushroom Morchella importuna in an in vitro system. Leishmaniasis is an important infectious disease caused by a range of Leishmania species, which are multihost protozoa parasites transmitted to humans by the sand fly and infecting macrophages. Leishmaniasis is an increasing worldwide health problem, including in the Mediterranean basin. Current chemotherapy treatments are limited by their toxic effects, the need for long-term treatment, and the increasing development of resistance by the parasite cells. Thus, alternative therapies are being considered, including herbal and mushroom products. We studied the effect of extracts from M. importuna on L. tropica promastigote cell proliferation and survival, and on their toxicity against human macrophages. The aqueous mushroom extract was compared with 3 successive extracted fractions: an 80% ethanol fraction, a water-soluble polysaccharide fraction, and a polyphenolic fraction. All 4 extracts showed anti-Leishmania activity; the aqueous extract was most active. The inhibition activity was dose dependent in killing Leishmania. No cell recovery was recorded after exposure to the mushroom extract. Microscopic observation showed morphological changes and the loss of flagella on the parasites. No cytotoxic activity was recorded against human macrophages at the same extract concentrations. The findings suggest the potential use of extracts of an edible Morchella mushroom against the Leishmania parasite in humans.