Defects In Biofilm Formation Research Articles

Ferric uptake regulator (Fur) in Vibrio mimicus acts as an activator of citrate cycle and oxidative phosphorylation pathways, while enhancing virulence potential

Ferric uptake regulator (Fur) is a crucial bacterial regulator that controls the uptake of iron by bacteria, hence influencing their survival and pathogenicity. Vibrio mimicus is an epidemic pathogen that is harmful to aquatic animals and human health, with the characteristics of rapid onset and high mortality after infecting the fish. At present, the function and mechanism of Fur in V. mimicus are still unclear. In this study, the fur gene deletion strain and complementation strain of V. mimicus were constructed. Following fur deletion, proteomic analysis revealed that the abundance of 258 proteins was considerably up-regulated while the abundance of 307 proteins was dramatically down-regulated. The primary roles of these differentially expressed proteins were in a variety of pathways, including virulence, metabolism, enzymes, and so on. Among them, citrate cycle and oxidative phosphorylation were the main pathways affected by Fur. The activities of key enzymes in these two pathways were assessed. The results indicated a decrease in the activities of respiratory chain complexes II and IV, succinate dehydrogenase (SDH), and α-ketoglutarate dehydrogenase (α-KGDH) in the Δfur strain. Conversely, the activity of isocitrate dehydrogenase (NAD-IDH) was increased. Phenotypic characterization showed that the Δfur strain exhibited delayed growth, reduced motility, defective biofilm formation, decreased resistance to oxidative stress, increased adhesion, and decreased virulence. Besides, the LD50 of the Δfur strain was 31-fold higher than that of the WT strain. These results indicate that Fur serves as an activator for the citrate cycle and oxidative phosphorylation pathway in V. mimicus, and contributing to its virulence. Our findings enhance the understanding of the biological functions of Fur and lay the groundwork for further investigation into the regulatory mechanisms of Fur in V. mimicus.

Genetic determinants of increased sodium hypochlorite and ciprofloxacin susceptibility in Pseudomonas aeruginosa PA14 biofilms

Reactive chlorine species (RCS) like sodium hypochlorite (NaOCl) are potent oxidizing agents and widely used biocides in surface disinfection, water treatment, and biofilm elimination. Moreover, RCS are also produced by the human immune system to kill invading pathogens. However, bacteria have developed mechanisms to survive the damage caused by RCS. Using the comprehensive Pseudomonas aeruginosa PA14 transposon mutant library in a genetic screen, we identified a total of 28 P. aeruginosa PA14 mutants whose biofilms showed increased susceptibility to NaOCl in comparison to PA14 WT biofilms. Of these, ten PA14 mutants with a disrupted apaH, PA0793, acsA, PA1506, PA1547, PA3728, yajC, queA, PA3869, or PA14_32840 gene presented a 4-fold increase in NaOCl susceptibility compared to wild-type biofilms. While none of these mutants showed a defect in biofilm formation or attenuated susceptibility of biofilms toward the oxidant hydrogen peroxide (H2O2), all but PA14_32840 also exhibited a 2–4-fold increase in susceptibility toward the antibiotic ciprofloxacin. Further analyses revealed attenuated levels of intracellular ROS and catalase activity only for the apaH and PA1547 mutant, providing insights into the oxidative stress response in P. aeruginosa biofilms. The findings of this paper highlight the complexity of biofilm resistance and the intricate interplay between different mechanisms to survive oxidative stress. Understanding resistance strategies adopted by biofilms is crucial for developing more effective ways to fight resistant bacteria, ultimately contributing to better management of bacterial growth and resistance in clinical and environmental settings.

O-glycosylation of intrinsically disordered regions regulates homeostasis of membrane proteins in streptococci.

Proteins harboring intrinsically disordered regions (IDRs) lacking stable secondary or tertiary structures are abundant across the three domains of life. These regions have not been systematically studied in prokaryotes. Our genome-wide analysis identifies extracytoplasmic serine/threonine-rich IDRs in several biologically important membrane proteins in streptococci. We demonstrate that these IDRs are O-glycosylated with glucose by glycosyltransferases GtrB and PgtC2 in Streptococcus pyogenes and Streptococcus pneumoniae, and with N-acetylgalactosamine by a Pgf-dependent mechanism in Streptococcus mutans. Absence of glycosylation leads to a defect in biofilm formation under ethanol-stressed conditions in S. mutans. We link this phenotype to the C-terminal IDR of a post-translocation secretion chaperone PrsA. O-glycosylation of the IDR protects this region from proteolytic degradation. The IDR length attenuates the efficiency of glycosylation and, consequently, the expression level of PrsA. Taken together, our data reveal that O-glycosylation of IDRs functions as a dynamic switch of protein homeostasis in streptococci.

The regulator FleQ both transcriptionally and post-transcriptionally regulates the level of RTX adhesins of Pseudomonas fluorescens.

Biofilm formation by the Gram-negative, Gammaproteobacteria Pseudomonas fluorescens relies on the repeats-in-toxin adhesins LapA and MapA in the cytoplasm, secretion of these adhesins through their respective type 1 secretion systems, and retention at the cell surface. Published work has shown that retention of the adhesins occurs via a post-translational mechanism involving the cyclic-di-GMP receptor LapD and the protease LapG. However, little is known about the underlying mechanisms that regulate the level of these adhesins. Here, we demonstrate that the master regulator FleQ modulates biofilm formation by both transcriptionally and post-transcriptionally regulating LapA and MapA. We find that a ΔfleQ mutant has a biofilm formation defect compared to the wild-type (WT) strain, which is attributed in part to a decrease in LapA and MapA abundance in the cell, despite the ΔfleQ mutant having increased levels of lapA and mapA transcripts compared to the WT strain. Through transposon mutagenesis and subsequent genetic analysis, we found that overstimulation of the Gac/Rsm pathway partially rescues biofilm formation in the ΔfleQ mutant background. Collectively, these findings provide evidence that FleQ regulates biofilm formation by both transcriptionally regulating the expression of the lapA and mapA genes and post-transcriptionally regulating the abundance of LapA and MapA, and that activation of the Gac/Rsm pathway can post-transcriptionally enhance biofilm formation by P. fluorescens. IMPORTANCE Biofilm formation is a highly coordinated process that bacteria undergo to colonize a variety of surfaces. For Pseudomonas fluorescens, biofilm formation requires the production and localization of repeats-in-toxin adhesins to the cell surface. To date, little is known about the underlying mechanisms that regulate biofilm formation by P. fluorescens. Here, we identify FleQ as a key regulator of biofilm formation that modulates both gene expression and abundance of LapA and MapA through both a transcriptional and post-transcriptional mechanism. We provide further evidence implicating activation of the Gac/Rsm system in FleQ-dependent regulation of biofilm formation. Together, our findings uncover evidence for a dual mechanism of transcriptional and post-transcriptional regulation of the LapA and MapA adhesins.

Two species with a peculiar evolution within the genus Pectobacterium suggest adaptation to a new environmental niche.

Historically, research on Soft Rot Pectobacteriacea (SRP) has focused on economically important crops and ornamentals and knowledge of these bacteria outside the plant context remains poorly investigated. Recently, two closely related species Pectobacterium aquaticum and Pectobacterium quasiaquaticum were isolated from water and have not been isolated from any plant yet. To identify the distinctive characteristics of these two species, we performed a comparative genomic analysis of 80 genomes representing 19 Pectobacterium species and performed an evolutionary reconstruction. Both water species underwent a reduction in genome size associated with a high pseudogene content. A high gene loss was predicted at the emergence of both species. Among the 199 gene families missing from both P. aquaticum and P. quasiaquaticum genomes but present in at least 80% of other Pectobacterium genomes, COG analysis identified many genes involved in nutrient transport systems. In addition, many type II secreted proteins were also missing in both species. Phenotypic analysis revealed that both species had reduced pectinolytic activity, a biofilm formation defect, were highly motile and had reduced virulence on several plants. These genomic and phenotypic data suggest that the ecological niche of P. aquaticum and P. quasiaquaticum may differ from that of other Pectobacterium species.

QCR7 affects the virulence of Candida albicans and the uptake of multiple carbon sources present in different host niches.

Candida albicans is a commensal yeast that may cause life-threatening infections. Studies have shown that the cytochrome b-c1 complex subunit 7 gene (QCR7) of C. albicans encodes a protein that forms a component of the mitochondrial electron transport chain complex III, making it an important target for studying the virulence of this yeast. However, to the best of our knowledge, the functions of QCR7 have not yet been characterized. A QCR7 knockout strain was constructed using SN152, and BALb/c mice were used as model animals to determine the role of QCR7 in the virulence of C. albicans. Subsequently, the effects of QCR7 on mitochondrial functions and use of carbon sources were investigated. Next, its mutant biofilm formation and hyphal growth maintenance were compared with those of the wild type. Furthermore, the transcriptome of the qcr7Δ/Δ mutant was compared with that of the WT strain to explore pathogenic mechanisms. Defective QCR7 reduced recruitment of inflammatory cells and attenuated the virulence of C. albicans infection in vivo. Furthermore, the mutant influenced the use of multiple alternative carbon sources that exist in several host niches (GlcNAc, lactic acid, and amino acid, etc.). Moreover, it led to mitochondrial dysfunction. Furthermore, the QCR7 knockout strain showed defects in biofilm formation or the maintenance of filamentous growth. The overexpression of cell-surface-associated genes (HWP1, YWP1, XOG1, and SAP6) can restore defective virulence phenotypes and the carbon-source utilization of qcr7Δ/Δ. This study provides new insights into the mitochondria-based metabolism of C. albicans, accounting for its virulence and the use of variable carbon sources that promote C. albicans to colonize host niches.

Analysis of the role of the QseBC two-component sensory system in epinephrine-induced motility and intracellular replication of Burkholderiapseudomallei.

Burkholderia pseudomallei is a facultative intracellular bacterial pathogen that causes melioidosis, a severe invasive disease of humans. We previously reported that the stress-related catecholamine hormone epinephrine enhances motility of B. pseudomallei, transcription of flagellar genes and the production of flagellin. It has been reported that the QseBC two-component sensory system regulates motility and virulence-associated genes in other Gram-negative bacteria in response to stress-related catecholamines, albeit disparities between studies exist. We constructed and whole-genome sequenced a mutant of B. pseudomallei with a deletion spanning the predicted qseBC homologues (bpsl0806 and bpsl0807). The ΔqseBC mutant exhibited significantly reduced swimming and swarming motility and reduced transcription of fliC. It also exhibited a defect in biofilm formation and net intracellular survival in J774A.1 murine macrophage-like cells. While epinephrine enhanced bacterial motility and fliC transcription, no further reduction in these phenotypes was observed with the ΔqseBC mutant in the presence of epinephrine. Plasmid-mediated expression of qseBC suppressed bacterial growth, complicating attempts to trans-complement mutant phenotypes. Our data support a role for QseBC in motility, biofilm formation and net intracellular survival of B. pseudomallei, but indicate that it is not essential for epinephrine-induced motility per se.

Effect of Exogenous Spermine on Biofilm Formation in Mycobacteria by Stimulating the Synthesis of Glycopeptidolipids

Biofilm formation is of great interest by its ability to increase bacterial tolerance to antibiotics that represent a serious problem for modern medicine. Among mycobacteria, which are also capable of forming biofilms, there are pathogens of socially dangerous infections, including tuberculosis. Basing on these data, the strains of Mycolicibacterium smegmatis mc2 155 were chosen as the objects of this study, including the parent strain without deletions and its mutants with one (ΔrelMsm) and double (ΔrelMsmΔrelZ) chromosomal deletions of the genes responsible for the synthesis of alarmone synthetase enzymes. Biofilms of mutant strains exhibited defects in biofilm formation. We have shown that the integrity, hydrophobicity, and the level of biomass of surface mycobacterial biofilms are dependent on the amount of glycopeptidolipids (GPL) in cells. The level of GPL depends on the activity of alarmone synthetases. The biogenic polyamine spermine is able to enhance the production of GPLs, restoring the integrity of biofilms of mutant strains. It is possible that this effect of spermine is caused by the influence on the activity of mycobacterial alarmone synthetases, which makes promising the further studying the molecular mechanisms of its action.

Transcript profiling reveals the role of PDB1, a subunit of the pyruvate dehydrogenase complex, in Candida albicans biofilm formation

Candida albicans, the most prevalent fungal pathogen in the human microbiota can form biofilms on implanted medical devices. These biofilms are tolerant to conventional antifungal drugs and the host immune system as compared to the free-floating planktonic cells. Several in vitro models of biofilm formation have been used to determine the C. albicans biofilm-forming process, regulatory networks, and their properties. Here, we performed a genome-wide transcript profiling with C. albicans cells grown in YPD medium both in planktonic and biofilm condition. Transcript profiling of YPD-grown biofilms was further compared with published Spider medium-grown biofilm transcriptome data. This comparative analysis highlighted the differentially expressed genes and the pathways altered during biofilm formation. In addition, we demonstrated that overexpression of the PDB1 gene encoding a subunit of the pyruvate dehydrogenase resulted in defective biofilm formation. Altogether, this comparative analysis of transcript profiles from two different studies provides a robust reading on biofilm-altered genes and pathways during C. albicans biofilm development.

The galU gene is required for in vivo survival of pseudomonas plecoglossicida in large yellow croaker (Larimichthys crocea).

Pseudomonas plecoglossicida is an important pathogenic bacterium in aquaculture that causes visceral granulomas in large yellow croaker (Larimichthys crocea). Uridine diphosphate glucose phosphorylase encoded by galU plays a key role in biosynthesis of the bacterial envelope, particularly lipopolysaccharide and the capsule. In this study, we inactivated the galU gene in the P. plecoglossicida isolate XSDHY-P. The galU mutant strain showed impaired growth in the early exponential stage and lacked the O polysaccharide side chain in lipopolysaccharide, but almost no defect in biofilm formation was detected. The galU mutant strain also exhibited significantly more sensitivity to the bactericidal action of normal fish serum mediated by the complement system compared to the wild-type strain. In a cell model originating from the head kidney of large yellow croaker, the galU mutant strain showed lower capacities of adhesion, invasion, and intracellular survival compared to the wild-type strain. In addition, the deficiency of the galU mutant drastically decreased bacterial loads in tissues and attenuated P. plecoglossicida virulence in fish. These results suggest that the galU gene of P. plecoglossicida is required for in vivo survival in large yellow croaker.

The novel type II toxin-antitoxin PacTA modulates Pseudomonas aeruginosa iron homeostasis by obstructing the DNA-binding activity of Fur.

Type II toxin–antitoxin (TA) systems are widely distributed in bacterial and archaeal genomes and are involved in diverse critical cellular functions such as defense against phages, biofilm formation, persistence, and virulence. GCN5-related N-acetyltransferase (GNAT) toxin, with an acetyltransferase activity-dependent mechanism of translation inhibition, represents a relatively new and expanding family of type II TA toxins. We here describe a group of GNAT-Xre TA modules widely distributed among Pseudomonas species. We investigated PacTA (one of its members encoded by PA3270/PA3269) from Pseudomonas aeruginosa and demonstrated that the PacT toxin positively regulates iron acquisition in P. aeruginosa. Notably, other than arresting translation through acetylating aminoacyl-tRNAs, PacT can directly bind to Fur, a key ferric uptake regulator, to attenuate its DNA-binding affinity and thus permit the expression of downstream iron-acquisition-related genes. We further showed that the expression of the pacTA locus is upregulated in response to iron starvation and the absence of PacT causes biofilm formation defect, thereby attenuating pathogenesis. Overall, these findings reveal a novel regulatory mechanism of GNAT toxin that controls iron-uptake-related genes and contributes to bacterial virulence.

Recognition of extracellular DNA by type IV pili promotes biofilm formation by Clostridioides difficile

Clostridioides difficile is a Gram-positive bacillus, which is a frequent cause of gastrointestinal infections triggered by the depletion of the gut microbiome. Because of the frequent recurrence of these infections after antibiotic treatment, mechanisms of C. difficile persistence and recurrence, including biofilm formation, are of increasing interest. Previously, our group and others found that type IV pili, filamentous helical appendages polymerized from protein subunits, promoted microcolony and biofilm formation in C. difficile. In Gram-negative bacteria, the ability of type IV pili to mediate bacterial self-association has been explained through interactions between the pili of adjacent cells, but type IV pili from several Gram-negative species are also required for natural competence through DNA uptake. Here, we report the ability of two C. difficile pilin subunits, PilJ and PilW, to bind to DNA in vitro, as well as the defects in biofilm formation in the pilJ and pilW gene-interruption mutants. Additionally, we have resolved the X-ray crystal structure of PilW, which we use to model possible structural mechanisms for the formation of C. difficile biofilm through interactions between type IV pili and the DNA of the extracellular matrix. Taken together, our results provide further insight into the relationship between type IV pilus function and biofilm formation in C. difficile and, more broadly, suggest that DNA recognition by type IV pili and related structures may have functional importance beyond DNA uptake for natural competence.

M. tuberculosis AlkX Encoded by rv3249c Regulates a Conserved Alkane Hydroxylase System That Is Important for Replication in Macrophages and Biofilm Formation.

ABSTRACTMycobacterium tuberculosis is a highly specialized human pathogen. The success of M. tuberculosis is due to its ability to replicate within host macrophages, resist host immune responses, and ultimately enter a persistent state during a latent tuberculosis infection. Understanding how M. tuberculosis adapts to and replicates in the intracellular environment of the host is crucial for the development of novel, targeted therapeutics. We report the characterization of an M. tuberculosis mutant lacking Rv3249c, a TetR transcriptional regulator. We show that Rv3249c directly represses the adjacent alkB-rubA-rubB operon encoding an alkane hydroxylase/rubredoxin system. For consistency with related systems, we have named the rv3249c gene alkX. The alkX mutant survived better than wild-type M. tuberculosis inside macrophages. This could be phenocopied by overexpression of the alkB-rubA-rubB locus. We hypothesized that the improved intracellular survival phenotype is a result of increased fitness of the mutant; however, we found that the alkX mutant had a defect when grown on some host-associated carbon sources in vitro. We also found that the alkX mutant had a defect in biofilm formation, also linked to the overexpression of the alkB-rubAB genes. Combined, these results define the primary role of AlkX as a transcriptional repressor of the alkB-rubAB operon and suggest the operon contributes to intracellular survival of the pathogen.IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is the leading cause of death worldwide due to a single infectious agent. It is important to understand how M. tuberculosis adapts to and replicates in the intracellular environment of the host. In this study, we characterized the TetR transcriptional regulator Rv3249c and show that it regulates a highly conserved alkane hydroxylase/rubredoxin system. Our data demonstrate that the AlkBRubAB system contributes to the success of the bacterium in host macrophages.

SigB regulates stress resistance, glucose starvation, MnSOD production, biofilm formation, and root colonization in Bacillus cereus 905.

Bacillus cereus 905, originally isolated from wheat rhizosphere, exhibits strong colonization ability on wheat roots. Our previous studies showed that root colonization is contributed by the ability of the bacterium to efficiently utilize carbon sources and form biofilms and that the sodA2 gene-encoded manganese-containing superoxide dismutase (MnSOD2) plays an indispensable role in the survival of B. cereus 905 in the wheat rhizosphere. In this investigation, we further demonstrated that the ability of B. cereus 905 to resist adverse environmental conditions is partially attributed to activation of the alternative sigma factor σB, encoded by the sigB gene. The sigB mutant experienced a dramatic reduction in survival when cells were exposed to ethanol, acid, heat, and oxidative stress or under glucose starvation. Analysis of the sodA2 gene transcription revealed a partial, σB-dependent induction of the gene during glucose starvation or when treated with paraquat. In addition, the sigB mutant displayed a defect in biofilm formation under stress conditions. Finally, results from the root colonization assay indicated that sigB and sodA2 collectively contribute to B. cereus 905 colonization on wheat roots. Our study suggests a diverse role of SigB in rhizosphere survival and root colonization of B. cereus 905 under stress conditions. KEY POINTS : • SigB confers resistance to environmental stresses in B. cereus 905. • SigB plays a positive role in glucose utilization and biofilm formation in B. cereus. • SigB and SodA2 collectively contribute to colonization on wheat roots by B. cereus.

Characterization of key enzymes involved in triacylglycerol biosynthesis in mycobacteria

Phosphatidic acid phosphatase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. PAP activity has a key role in the regulation of PA flux towards TAG or glycerophospholipid synthesis. In this work we have characterized two Mycobacterium smegmatis genes encoding for functional PAP proteins. Disruption of both genes provoked a sharp reduction in de novo TAG biosynthesis in early growth phase cultures under stress conditions. In vivo labeling experiments demonstrated that TAG biosynthesis was restored in the ∆PAP mutant when bacteria reached exponential growth phase, with a concomitant reduction of phospholipid synthesis. In addition, comparative lipidomic analysis showed that the ∆PAP strain had increased levels of odd chain fatty acids esterified into TAGs, suggesting that the absence of PAP activity triggered other rearrangements of lipid metabolism, like phospholipid recycling, in order to maintain the wild type levels of TAG. Finally, the lipid changes observed in the ∆PAP mutant led to defective biofilm formation. Understanding the interaction between TAG synthesis and the lipid composition of mycobacterial cell envelope is a key step to better understand how lipid homeostasis is regulated during Mycobacterium tuberculosis infection.

MmbR, a master transcription regulator that controls fatty acid β-oxidation genes in Mycolicibacterium smegmatis.

An unusually high lipid content and a complex lipid profile are the most distinctive features of the mycobacterial cell envelope. However, our understanding of the regulatory mechanism underlying mycobacterial lipid metabolism is limited, and the major regulators responsible for lipid homeostasis remain to be characterized. Here, we identified MmbR as a novel master regulator that is essential for maintaining lipid homeostasis in Mycolicibacterium smegmatis. We found that MmbR controls fatty acid β-oxidation and modulates biofilm formation in Mycolicibacterium smegmatis. Although MmbR possesses the properties of nucleoid-associated proteins, it acts as a TetR-like transcription factor, directly regulating and intensively repressing the expression of a group of core genes involved in fatty acid β-oxidation. Furthermore, both long-chain acyl-Coenzyme A and fatty acids appear to regulate the signal molecules modulated by MmbR. The deletion of mmbR led to a significant reduction in intracellular fatty acid content and a decrease in the relative lipid composition of the biofilm. The lack of mmbR led to morphological changes in the mycobacterial colony, defects in biofilm formation and enhanced sensitivity to anti-tuberculosis drugs. Our study is the first to establish a link between the transcriptional regulation of fatty acid β-oxidation genes and lipid homeostasis in mycobacteria.

The Two-Component Locus MSMEG_0244/0246 Together With MSMEG_0243 Affects Biofilm Assembly in M. smegmatis Correlating With Changes in Phosphatidylinositol Mannosides Acylation.

Ferric and ferrous iron is an essential transition metal for growth of many bacterial species including mycobacteria. The genomic region msmeg_0234 to msmeg_0252 from Mycobacterium smegmatis is putatively involved in iron/heme metabolism. We investigate the genes encoding the presumed two component system MSMEG_0244/MSMEG_0246, the neighboring gene msmeg_0243 and their involvement in this process. We show that purified MSMEG_0243 indeed is a heme binding protein. Deletion of msmeg_0243/msmeg_0244/msmeg_0246 in Mycobacterium smegmatis leads to a defect in biofilm formation and colony growth on solid agar, however, this phenotype is independent of the supplied iron source. Further, analysis of the corresponding mutant and its lipids reveals that changes in morphology and biofilm formation correlate with altered acylation patterns of phosphatidylinositol mannosides (PIMs). We provide the first evidence that msmeg_0244/msmeg_0246 work in concert in cellular lipid homeostasis, especially in the maintenance of PIMs, with the heme-binding protein MSMEG_0243 as potential partner.

Candida albicans Mrv8, is involved in epithelial damage and biofilm formation

ABSTRACT Candida albicans is the most common human fungal pathogen that can cause superficial and deep-seated infections in susceptible individuals. Despite its medical importance, the vast majority of C. albicans genes remain of unknown function. Here, we report a role for the lineage-specific gene, MRV8, in host pathogen interactions, mycelial microcolony maturation and biofilm formation. In silico analysis indicated that MRV8 encodes a four-pass transmembrane protein unique to the closely related pathogens C. albicans and Candida dubliniensis. Deletion of MRV8 did not affect C. albicans adherence to, or initial invasion into human oral epithelia, but inhibited mycelial development and strongly reduced epithelial damage. mrv8Δ/Δ cells exhibited a media-dependent defect in biofilm formation and mutant biofilm metabolic activity was enhanced by cyclosporin A. mrv8Δ/Δ biofilms were more tolerant to treatment with caspofungin, but not to fluconazole or amphotericin B. Co-stimulation with calcium chloride and calcofluor white rescued biofilm growth in the presence of caspofungin, and this rescue-effect was Mrv8-dependent. Together, our data demonstrate an important role for a lineage-specific gene (MRV8) in C. albicans biofilm formation, drug tolerance and host–pathogen interactions.

Investigating the Role of the Med Protein in Biofilm Formation

Bacillus subtilis is a soil‐dwelling bacterium that serves as a good model organism for the study of different adaptive responses to various environmental conditions. These adaptive responses, including spore formation, cannibalism, and biofilm formation, are controlled by the master regulator Spo0A whose phosphorylation is under the control of five kinases. Initial interest in the protein Med came from its role in the transcription of operons related to cannibalism, and studies show the effect of Med is dependent on one of the five kinases, KinD. These same studies suggest Med may play a role in biofilm formation in a KinD‐dependent manner. The goal of our study of the Med protein is to determine whether it plays a direct role in biofilm formation by directly detecting environmental signals or indirectly through interactions with KinD. To determine whether Med is involved in detecting environmental signals, Δmed and ΔkinD bacteria were compared to wildtype B. subtilis using luciferase reporter assays that quantify the effect of different small molecules on the transcription of biofilm genes. Luciferase assays were compared to biofilm assays under the same conditions to visualize the effect on the biofilm phenotypes. We find that Δmed mutants had a defect in biofilm formation and reduced transcription of biofilm genes compared with wildtype bacteria. However, Δmed mutants were the same as wildtype bacteria in terms of their pattern of stimulation by small molecules. These results indicate that Med is not directly sensing environmental signals and instead is likely to be involved in the biofilm formation pathway indirectly through KinD, which has been shown to bind small molecules through its extracellular CACHE domain. We therefore shifted our focus to investigating interactions between Med and KinD and how Med may affect KinD’s ability to sense small molecules. Med and KinD were purified by tagging them with GST and His tags respectively. Additionally, in the case of KinD we purified constructs of the extracellular and intracellular domains to more precisely determine where the Med protein may be interacting with KinD. Presence of the proteins was determined by SDS‐PAGE and interactions between them was determined by Western blot analysis.Support or Funding InformationResearch was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM135861.

How to Use a Mutant Library to Identify Genes Required for Biofilm Formation in the Pathogenic Fungus Candida albicans.

With over 1 billion infections and the causative agents showing critical diseases such as pancreatic cancer, the study of pathogenic fungi has never been more critical. In 2017, the United States spent $7.2 billion on fungal diseases. $4.5 billion was allocated to 75,055 hospitalizations, while $2.6 billion went to 8,993,230 outpatient visits. For Candida infections specifically, the cost was $1.4 billion. Currently, there are few classes of antifungals available, and resistance is growing. The identification of genes required for biofilm formation is essential for new antifungal development. This review details how to identify, verify, and characterize defective biofilm formation mutants in C. albicans. This includes how to run an in vitro biofilm formation assay, how to create clean deletions using the modified CRISPR-Cas9 system, how to assay to identify the potential causes of the defect, and how to create complementation strains to confirm the mutant defect.