Design of a targeted blood transcriptional panel for monitoring immunological changes accompanying pregnancy.

Background: Immunomodulatory processes exert steering functions throughout pregnancy. Detecting diversions from this physiologic immune clock may help identify pregnant women at risk for pregnancy-associated complications. We present results from a data-driven selection process to develop a targeted panel of mRNAs that may prove effective in detecting pregnancies diverting from the norm. Methods: Based on a de novo dataset from a resource-constrained setting and a dataset from a resource-rich area readily available in the public domain, whole blood gene expression profiles of uneventful pregnancies were captured at multiple time points during pregnancy. BloodGen3, a fixed blood transcriptional module repertoire, was employed to analyze and visualize gene expression patterns in the two datasets. Differentially expressed genes were identified by comparing their abundance to non-pregnant postpartum controls. The selection process for a targeted gene panel considered (i) transcript abundance in whole blood; (ii) degree of correlation with the BloodGen3 module; and (iii) pregnancy biology. Results: We identified 176 transcripts that were complemented with eight housekeeping genes. Changes in transcript abundance were seen in the early stages of pregnancy and similar patterns were observed in both datasets. Functional gene annotation suggested significant changes in the lymphoid, prostaglandin and inflammation-associated compartments, when compared to the postpartum controls. Conclusion: The gene panel presented here holds promise for the development of predictive, targeted, transcriptional profiling assays. Such assays might become useful for monitoring of pregnant women, specifically to detect potential adverse events early. Prospective validation of this targeted assay, in-depth investigation of functional annotations of differentially expressed genes, and assessment of common pregnancy-associated complications with the aim to identify these early in pregnancy to improve pregnancy outcomes are the next steps.

Vaginal Microbiota and Cytokine Levels Predict Preterm Delivery in Asian Women.

Preterm birth (PTB) is the most common cause of neonatal morbidity and mortality worldwide. Approximately half of PTBs is linked with microbial etiologies, including pathologic changes to the vaginal microbiota, which vary according to ethnicity. Globally more than 50% of PTBs occur in Asia, but studies of the vaginal microbiome and its association with pregnancy outcomes in Asian women are lacking. This study aimed to longitudinally analyzed the vaginal microbiome and cytokine environment of 18 Karen and Burman pregnant women who delivered preterm and 36 matched controls delivering at full term. Using 16S ribosomal RNA gene sequencing we identified a predictive vaginal microbiota signature for PTB that was detectable as early as the first trimester of pregnancy, characterized by higher levels of Prevotella buccalis, and lower levels of Lactobacillus crispatus and Finegoldia, accompanied by decreased levels of cytokines including IFNγ, IL-4, and TNFα. Differences in the vaginal microbial diversity and local vaginal immune environment were associated with greater risk of preterm birth. Our findings highlight new opportunities to predict PTB in Asian women in low-resource settings who are at highest risk of adverse outcomes from unexpected PTB, as well as in Burman/Karen ethnic minority groups in high-resource regions.

Organizing training workshops on gene literature retrieval, profiling, and visualization for early career researchers.

Early-career researchers must acquire the skills necessary to effectively search and extract information from biomedical literature. This ability is for instance crucial for evaluating the novelty of experimental results, and assessing potential publishing opportunities. Given the rapidly growing volume of publications in the field of biomedical research, new systematic approaches need to be devised and adopted for the retrieval and curation of literature relevant to a specific theme. In this context, we present a hands-on training curriculum aimed at retrieval, profiling, and visualization of literature associated with a given topic. The curriculum was implemented in a workshop in January 2021. Here we provide supporting material and step-by-step implementation guidelines with the ISG15 gene literature serving as an illustrative use case. Workshop participants can learn several skills, including: 1) building and troubleshoot PubMed queries in order to retrieve the literature associated with a gene of interest; 2) identifying key concepts relevant to given themes (such as cell types, diseases, and biological processes); 3) measuring the prevalence of these concepts in the gene literature; 4) extracting key information from relevant articles, and 5) developing a background section or summary on the basis of this information. Finally, trainees can learn to consolidate the structured information captured through this process for presentation via an interactive web application.

A Neutrophil-Driven Inflammatory Signature Characterizes the Blood Transcriptome Fingerprint of Psoriasis.

Transcriptome profiling approaches have been widely used to investigate the mechanisms underlying psoriasis pathogenesis. Most researchers have measured changes in transcript abundance in skin biopsies; relatively few have examined transcriptome changes in the blood. Although less relevant to the study of psoriasis pathogenesis, blood transcriptome profiles can be readily compared across various diseases. Here, we used a pre-established set of 382 transcriptional modules as a common framework to compare changes in blood transcript abundance in two independent public psoriasis datasets. We then compared the resulting "transcriptional fingerprints" to those obtained for a reference set of 16 pathological or physiological states. The perturbations in blood transcript abundance in psoriasis were relatively subtle compared to the changes we observed in other autoimmune and auto-inflammatory diseases. However, we did observe a consistent pattern of changes for a set of modules associated with neutrophil activation and inflammation; interestingly, this pattern resembled that observed in patients with Kawasaki disease. This similarity between the blood-transcriptome signatures in psoriasis and Kawasaki disease suggests that the immune mechanisms driving their pathogenesis might be partially shared.

Cohort profile: molecular signature in pregnancy (MSP): longitudinal high-frequency sampling to characterise cross-omic trajectories in pregnancy in a resource-constrained setting.

PURPOSE: A successful pregnancy relies on the interplay of various biological systems. Deviations from the norm within a system or intersystemic interactions may result in pregnancy-associated complications and adverse pregnancy outcomes. Systems biology approaches provide an avenue of unbiased, in-depth phenotyping in health and disease. The molecular signature in pregnancy (MSP) cohort was established to characterise longitudinal, cross-omic trajectories in pregnant women from a resource constrained setting. Downstream analysis will focus on characterising physiological perturbations in uneventful pregnancies, pregnancy-associated complications and adverse outcomes. PARTICIPANTS: First trimester pregnant women of Karen or Burman ethnicity were followed prospectively throughout pregnancy, at delivery and until 3 months post partum. Serial high-frequency sampling to assess whole blood transcriptomics and microbiome composition of the gut, vagina and oral cavity, in conjunction with assessment of gene expression and microbial colonisation of gestational tissue, was done for all cohort participants. FINDINGS TO DATE: 381 women with live born singletons averaged 16 (IQR 15-18) antenatal visits (13 094 biological samples were collected). At 5% (19/381) the preterm birth rate was low. Other adverse events such as maternal febrile illness 7.1% (27/381), gestational diabetes 13.1% (50/381), maternal anaemia 16.3% (62/381), maternal underweight 19.2% (73/381) and a neonate born small for gestational age 20.2% (77/381) were more often observed than preterm birth. FUTURE PLANS: Results from the MSP cohort will enable in-depth characterisation of cross-omic molecular trajectories in pregnancies from a population in a resource-constrained setting. Moreover, pregnancy-associated complications and unfavourable pregnancy outcomes will be investigated at the same granular level, with a particular focus on population relevant needs such as effect of tropical infections on pregnancy. More detailed knowledge on multiomic perturbations will ideally result in the development of diagnostic tools and ultimately lead to targeted interventions that may disproportionally benefit pregnant women from this resource-limited population. TRIAL REGISTRATION NUMBER: NCT02797327.

A prospective cohort for the investigation of alteration in temporal transcriptional and microbiome trajectories preceding preterm birth: a study protocol.

INTRODUCTION: Preterm birth (PTB) results from heterogeneous influences and is a major contributor to neonatal mortality and morbidity that continues to have adverse effects on infants beyond the neonatal period. This protocol describes the procedures to determine molecular signatures predictive of PTB through high-frequency sampling during pregnancy, at delivery and the postpartum period. METHODS AND ANALYSIS: Four hundred first trimester pregnant women from either Myanmar or Thailand of either Karen or Burman ethnicity, with a viable, singleton pregnancy will be enrolled in this non-interventional, prospective pregnancy birth cohort study and will be followed through to the postpartum period. Fortnightly finger prick capillary blood sampling will allow the monitoring of genome-wide transcript abundance in whole blood. Collection of stool samples and vaginal swabs each trimester, at delivery and postpartum will allow monitoring of intestinal and vaginal microbial composition. In a nested case-control analysis, perturbations of transcript abundance in capillary blood as well as longitudinal changes of the gut, vaginal and oral microbiome will be compared between mothers giving birth to preterm and matched cases giving birth to term neonates. Placenta tissue of preterm and term neonates will be used to determine bacterial colonisation as well as for the establishment of coding and non-coding RNA profiles. In addition, RNA profiles of circulating, non-coding RNA in cord blood serum will be compared with those of maternal peripheral blood serum at time of delivery. ETHICS AND DISSEMINATION: This research protocol that aims to detect perturbations in molecular trajectories preceding adverse pregnancy outcomes was approved by the ethics committee of the Faculty of Tropical Medicine, Mahidol University in Bangkok, Thailand (Ethics Reference: TMEC 15-062), the Oxford Tropical Research Ethics Committee (Ethics Reference: OxTREC: 33-15) and the local Tak Province Community Ethics Advisory Board. The results of this cooperative project will be disseminated in multiple publications staggered over time in international peer-reviewed scientific journals. TRIAL REGISTRATION NUMBER: NCT02797327; Pre-results.

Single-Nucleotide Variations of the Human Nuclear Hormone Receptor Genes in 60,000 Individuals.

Nuclear hormone receptors (NRs) mediate biologic actions of lipophilic molecules to gene transcription and are phylogenetically and functionally categorized into seven subfamilies and three groups, respectively. Single-nucleotide variations (SNVs) or polymorphisms are genetic changes influencing individual response to environmental factors and susceptibility to various disorders, and are part of the genetic diversification and basis for evolution. We sorted out SNVs of the human NR genes from 60,706 individuals, calculated three parameters (percentage of all variants, percentage of loss-of-function variants, and ratio of nonsynonymous/synonymous variants in their full protein-coding or major domain-coding sequences), and compared them with several valuables. Comparison of these parameters between NRs and control groups identified that NRs form a highly conserved gene family. The three parameters for the full coding sequence are positively correlated with each other, whereas four NR genes are distinct from the others with much higher tolerance to protein sequence-changing variants. DNA-binding domain and N-terminal domain are respectively those bearing the least and the most variation. NR subfamilies based on their phylogenetic proximity or functionality as well as diversity of tissue distribution and numbers of partner molecules are all not correlated with the variation parameters, whereas their gene age demonstrates an association. Our results suggest that the natural selection driving the NR family evolution still operates in humans. Gene age and probably the potential to adapt to various new ligands, but not current functional diversity, are major determinants for SNVs of the human NR genes.

C2H2-Type Zinc Finger Proteins: Evolutionarily Old and New Partners of the Nuclear Hormone Receptors.

Nuclear hormone receptors (NRs) are evolutionarily conserved ligand-dependent transcription factors. They are essential for human life, mediating the actions of lipophilic molecules, such as steroid hormones and metabolites of fatty acid, cholesterol, and external toxic compounds. The C2H2-type zinc finger proteins (ZNFs) form the largest family of the transcription factors in humans and are characterized by multiple, tandemly arranged zinc fingers. Many of the C2H2-type ZNFs are conserved throughout evolution, suggesting their involvement in preserved biological activities, such as general transcriptional regulation and development/differentiation of organs/tissues observed in the early embryonic phase. However, some C2H2-type ZNFs, such as those with the Krüppel-associated box (KRAB) domain, appeared relatively late in evolution and have significantly increased family members in mammals including humans, possibly modulating their complicated transcriptional network and/or supporting the morphological development/functions specific to them. Such evolutional characteristics of the C2H2-type ZNFs indicate that these molecules influence the NR functions conserved through evolution, whereas some also adjust them to meet with specific needs of higher organisms. We review the interaction between NRs and C2H2-type ZNFs by focusing on some of the latter molecules.

A curated transcriptome dataset collection to investigate the development and differentiation of the human placenta and its associated pathologies.

Compendia of large-scale datasets made available in public repositories provide a precious opportunity to discover new biomedical phenomena and to fill gaps in our current knowledge. In order to foster novel insights it is necessary to ensure that these data are made readily accessible to research investigators in an interpretable format. Here we make a curated, public, collection of transcriptome datasets relevant to human placenta biology available for further analysis and interpretation via an interactive data browsing interface. We identified and retrieved a total of 24 datasets encompassing 759 transcriptome profiles associated with the development of the human placenta and associated pathologies from the NCBI Gene Expression Omnibus (GEO) and present them in a custom web-based application designed for interactive query and visualization of integrated large-scale datasets ( http://placentalendocrinology.gxbsidra.org/dm3/landing.gsp). We also performed quality control checks using relevant biological markers. Multiple sample groupings and rank lists were subsequently created to facilitate data query and interpretation. Via this interface, users can create web-links to customized graphical views which may be inserted into manuscripts for further dissemination, or e-mailed to collaborators for discussion. The tool also enables users to browse a single gene across different projects, providing a mechanism for  developing new perspectives on the role of a molecule of interest across multiple biological states. The dataset collection we created here is available at: http://placentalendocrinology.gxbsidra.org/dm3.

Anti-leishmanial, anti-inflammatory and antimicrobial activities of phenolic derivatives from Tibouchina paratropica.

A new phenolic derivative, 2,8-dihydroxy-7H-furo[2,3-f]chromen-7-one (1), together with isoquercitrin (2), was isolated from the aerial parts of Tibouchina paratropica. Compound structures were elucidated by spectroscopic methods. Both compounds show antimicrobial activity towards a panel of bacterial and fungal pathogens, and compound 1 displayed potent anti-parasitic activity against Leishmania donovani (IC50 = 0.809 µg/mL). In addition, an 85% reduction in the secretion of the pro-inflammatory cytokine IL-6 was recorded when macrophages challenged with lipopolysaccharide were exposed to compound 1, but no effect on the anti-inflammatory IL-10 was observed. Compound 2 showed neither anti-parasitic nor anti-inflammatory properties. In addition, no cytotoxic activities were observed against the human-derived macrophage THP-1 cells.

Leishmania donovani infection causes distinct epigenetic DNA methylation changes in host macrophages.

Infection of macrophages by the intracellular protozoan Leishmania leads to down-regulation of a number of macrophage innate host defense mechanisms, thereby allowing parasite survival and replication. The underlying molecular mechanisms involved remain largely unknown. In this study, we assessed epigenetic changes in macrophage DNA methylation in response to infection with L. donovani as a possible mechanism for Leishmania driven deactivation of host defense. We quantified and detected genome-wide changes of cytosine methylation status in the macrophage genome resulting from L. donovani infection. A high confidence set of 443 CpG sites was identified with changes in methylation that correlated with live L. donovani infection. These epigenetic changes affected genes that play a critical role in host defense such as the JAK/STAT signaling pathway and the MAPK signaling pathway. These results provide strong support for a new paradigm in host-pathogen responses, where upon infection the pathogen induces epigenetic changes in the host cell genome resulting in downregulation of innate immunity thereby enabling pathogen survival and replication. We therefore propose a model whereby Leishmania induced epigenetic changes result in permanent down regulation of host defense mechanisms to protect intracellular replication and survival of parasitic cells.

Hair follicle mesenchyme-associated PD-L1 regulates T-cell activation induced apoptosis: a potential mechanism of immune privilege.

The immune privilege (IP) of hair follicles (HFs) has been well established in previous studies. However, whether cultured HF cells still exhibit IP properties, the individual factors involved in this process, and the detailed mechanisms that drive and maintain IP, are largely unidentified. We found preferential expression of IP-associated genes in cultured HF dermal papilla and dermal sheath cup cells (DSCCs) compared with non-follicular fibroblasts (FBs) at passage 4, suggesting a potential for functional IP. Notably, programmed cell death 1 ligand 1 (PD-L1) was significantly upregulated in DSCCs and dermal papilla cells relative to FBs. IFNγ secretion from peripheral blood mononuclear cells (PBMCs) co-cultured with histoincompatible DSCCs was significantly lower than with FB and higher percentages of early apoptotic, Annexin V+ cells were observed in PBMC co-cultured with DSCCs. Knockdown of PD-L1 translation by silencing interfering RNA in DSCCs enabled increased IFNγ secretion by PBMCs, whereas transfection of pCMV6-XL4/hPD-L1 in FB significantly reduced IFNγ secretion and increased apoptosis in co-cultured PBMCs. We also found that apoptosis in allogeneic T cells induced by DSCCs was largely dependent on the mitochondrial pathway. Our study suggests IP properties are exhibited in cultured DSCCs in part through expression of negative co-signaling molecule PD-L1.

Differential quantitative proteomic profiling of Leishmania infantum and Leishmania mexicana density gradient separated membranous fractions.

Leishmaniasis, caused by infection with Leishmania, is a major public health concern affecting more than 20million people globally. Leishmania has a digenetic lifecycle consisting of an extracellular flagellated promastigote, adapted to live in the mid-gut of the sand fly host and an aflagellated intracellular amastigote that resides within the macrophage of the mammalian host. Leishmania mexicana and Leishmania infantum are causative agents of cutaneous and visceral leishmaniasis, respectively. Membrane proteins play a pivotal role in host-pathogen interactions and in regulatory pathways. As the genome of Leishmania is essentially constitutively expressed, regulation of protein expression during differentiation occurs post-transcriptionally and/or post-translationally. Quantitative mass spectrometry using iTRAQ labeling identified differences in the proteomes of density gradient separated membranous fractions of promastigote and amastigote life-stages. We identified 189 L. infantum and 107 L. mexicana non-redundant proteins of which 20-40% showed differential expression levels between promastigote and amastigote lifecycle stages. Differentially expressed proteins mapped to several pathways including cell motility, metabolism, and infectivity as well as virulence factors such as eEF-1α, amastin and leishmanolysin (GP63). Western blot analysis validated iTRAQ quantitation for leishmanolysin. Focusing on differentially expressed proteins essential for pathogenesis, may ultimately lead to the identification of novel potential therapeutic targets. BIOLOGICAL SIGNIFICANCE: Leishmania, protozoan parasites of the Trypanosomatidae family, are the causative agents of leishmaniasis that represents a major public health concern affecting more than 20million people globally Membrane associated proteins play a pivotal role in host-pathogen interactions and in regulatory pathways. Quantitative proteomic analysis of the membranous fractions from L. mexicana and L. infantum (causative agents of cutaneous and visceral leishmaniasis, respectively) identified a number of proteins that may have important stage-specific functions in either the sand fly or mammalian host. The function of these proteins includes roles in virulence, as well as differences in metabolic process between life stages. Many of the proteins identified may act as virulence factors playing significant roles in parasite invasion, host-parasite interaction or parasite survival and thus may have therapeutic potential as drug target candidates.

Modes of action of Leishmanicidal antimicrobial peptides.

Leishmaniasis is one of the major neglected tropical diseases of the world. It is present in 88 countries with an estimated number of 500,000 cases of visceral leishmaniasis and 1.5 million cases of cutaneous disease. No effective vaccinations are available against leishmaniasis and the efficacy of existing treatments is compromised due to the emergence of drug resistance. Thus, there is an urgent need to develop new compounds with antileishmanial activity. Antimicrobial peptides have potential as novel antileishmanial therapy, either for use alone or in combination with current drug regimens. The modes of action of these peptides against Leishmania includes: membrane disruption leading to necrotic cell death; induction of apoptosis; binding to intracellular target(s); and indirect effects via immunomodulation of host immune cells. This article reviews the mechanisms of action of antimicrobial peptides with leishmanicidal activity.

Effect of BMAP-28 antimicrobial peptides on Leishmania major promastigote and amastigote growth: role of leishmanolysin in parasite survival.

BACKGROUND: Protozoan parasites, such as Leishmania, still pose an enormous public health problem in many countries throughout the world. Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies. Several innovative approaches are under investigation, including the utilization of host defence peptides (HDPs) as emerging anti-parasitic therapies. HDPs are characterised by their small size, amphipathic nature and cationicity, which induce permeabilization of cell membranes, whilst modulating the immune response of the host. Recently, members of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) has broad antimicrobial activities and confers protection in animal models of bacterial infection or sepsis. We tested the effectiveness of the use of BMAP-28 and two of its isomers the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), as anti-leishmanial agents against the promastigote and amastigote intracellular Leishmania major lifecycle stages. METHODOLOGY/PRINCIPAL FINDINGS: An MTS viability assay was utilized to show the potent antiparasitic activity of BMAP-28 and its protease resistant isomers against L. major promastigotes in vitro. Cell membrane permeability assays, caspase 3/7, Tunel assays and morphologic studies suggested that this was a late stage apoptotic cell death with early osmotic cell lysis caused by the antimicrobial peptides. Furthermore, BMAP-28 and its isomers demonstrated anti-leishmanial activities against intracellular amastigotes within a macrophage infection model. CONCLUSIONS/SIGNIFICANCE: Interestingly, D-BMAP-28 appears to be the most potent antiparasitic of the three isomers against wild type L. major promastigotes and amastigotes. These exciting results suggest that BMAP-28 and its protease resistant isomers have significant therapeutic potential as novel anti-leishmanials.

Nuclear envelope disruption involving host caspases plays a role in the parvovirus replication cycle.

Parvoviruses are small, nonenveloped, single-stranded DNA viruses which replicate in the nucleus of the host cell. We have previously found that early during infection the parvovirus minute virus of mice (MVM) causes small, transient disruptions of the nuclear envelope (NE). We have now investigated the mechanism used by MVM to disrupt the NE. Here we show that the viral phospholipase A2, the only known enzymatic domain on the parvovirus capsid, is not involved in causing NE disruption. Instead, the virus utilizes host cell caspases, which are proteases involved in causing NE breakdown during apoptosis, to facilitate these nuclear membrane disruptions. Studies with pharmacological inhibitors indicate that caspase-3 in particular is involved. A caspase-3 inhibitor prevents nuclear lamin cleavage and NE disruption in MVM-infected mouse fibroblast cells and reduces nuclear entry of MVM capsids and viral gene expression. Caspase-3 is, however, not activated above basal levels in MVM-infected cells, and other aspects of apoptosis are not triggered during early MVM infection. Instead, basally active caspase-3 is relocalized to the nuclei of infected cells. We propose that NE disruption involving caspases plays a role in (i) parvovirus entry into the nucleus and (ii) alteration of the compartmentalization of host proteins in a way that is favorable for the virus.

Novel genetic determinants of low-level aminoglycoside resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa strains isolated from patients with persistent lung infections and cystic fibrosis have been found to gradually develop aminoglycoside resistance over time. The aim of this study was to identify potential contributors to low-level aminoglycoside resistance, which may cause such graduated increases in resistance. The Harvard P. aeruginosa PA14 nonredundant library, consisting of approximately 5,800 mutants, was screened for twofold or greater increases in tobramycin resistance. Mutants carrying mutations in a total of 135 unique genes were identified and confirmed to have reduced susceptibility to tobramycin. Many of these genes were involved predominantly in energy metabolism; however, most of these mutants did not exhibit growth defects under the conditions tested, although some exhibited the small-colony phenotype and/or defects in growth under anaerobic conditions. Lipopolysaccharide mutants were also identified, and it was found that tobramycin had a reduced ability to permeabilize the outer membranes of these mutants. The results of this study emphasize the complexity of the interactions that tobramycin may have within the bacterial cell and introduce a large number of novel genes which may play a role in tobramycin resistance.

Mutator genes giving rise to decreased antibiotic susceptibility in Pseudomonas aeruginosa.

Screening of the PA14 genomic transposon mutant library for resistance to ceftazidime, tobramycin, and ciprofloxacin led to the discovery of several mutants that appeared in more than one screen. Testing of the frequency of mutation to ciprofloxacin resistance revealed previously known mutator genes, including mutS and mutL, as well as mutators that have not yet been described for P. aeruginosa, including PA3958 and RadA (PA4609).

The Lon protease of Pseudomonas aeruginosa is induced by aminoglycosides and is involved in biofilm formation and motility.

Pseudomonas aeruginosa is an important nosocomial opportunistic human pathogen and a major cause of chronic lung infections in individuals with cystic fibrosis. Serious infections by this organism are often treated with a combination of aminoglycosides and semi-synthetic penicillins. Subinhibitory concentrations of antibiotics are now being recognized for their role in microbial persistence and the development of antimicrobial resistance, two very important clinical phenomena. An extensive screen of a P. aeruginosa PAO1 luciferase gene fusion library was performed to identify genes that were differentially regulated during exposure to subinhibitory gentamicin. It was demonstrated that subinhibitory concentrations of gentamicin and tobramycin induced a set of genes that are likely to affect the interaction of P. aeruginosa with host cells, including the gene encoding Lon protease, which is known to play a major role in protein quality control. Studies with a lon mutant compared to its parent and a complemented strain indicated that this protein was essential for biofilm formation and motility in P. aeruginosa.

Identification of genes involved in swarming motility using a Pseudomonas aeruginosa PAO1 mini-Tn5-lux mutant library.

During a screening of a mini-Tn5-luxCDABE transposon mutant library of Pseudomonas aeruginosa PAO1 for alterations in swarming motility, 36 mutants were identified with Tn5 insertions in genes for the synthesis or function of flagellin and type IV pilus, in genes for the Xcp-related type II secretion system, and in regulatory, metabolic, chemosensory, and hypothetical genes with unknown functions. These mutants were differentially affected in swimming and twitching motility but in most cases had only a minor additional motility defect. Our data provide evidence that swarming is a more complex type of motility, since it is influenced by a large number of different genes in P. aeruginosa. Conversely, many of the swarming-negative mutants also showed an impairment in biofilm formation, indicating a strong relationship between these types of growth states.