Host cell-based screening assays for identification of molecules targeting Pseudomonas aeruginosa cyclic di-GMP signaling and biofilm formation.

The rapid emergence of bacterial resistance to antibiotics in current use is occurring worldwide and poses a significant threat to global healthcare systems. Recent research to identify new effective anti-bacterial agents has focused on regulatory pathways as targets for interference. Regulatory mechanisms employing intracellular Bis-(3',5') cyclic di-guanylate (c-di-GMP) as a secondary messenger represent a distinct category of subjects. This molecule, c-di-GMP, is present in nearly all bacterial species and plays a pivotal role in governing various biological processes, encompassing antibiotic resistance, biofilm formation, and virulence. Alteration of the cellular concentrations of the nucleotide through modulation of associated signaling pathways has the potential to reduce biofilm formation or increase susceptibility of the biofilm bacteria to antibiotics. Here, we have developed a screen for compounds that alter c-di-GMP levels in Pseudomonas aeruginosa in co-culture with bronchial epithelial cells. Through the assay of 200 natural compounds, we were able to identify several substances showing promising effects on P. aeruginosa in a host biofilm infection model. Importantly, we detected compounds that inhibit c-di-GMP levels and showed significant influence on biofilm formation and virulence in P. aeruginosa in vitro and in vivo. Consequently, we offer proof-of-concept information regarding swift and practical drug screening assays, suitable for medium- to high-throughput applications, which target the c-di-GMP signaling pathways in this significant Gram-negative pathogen.

Going virtual: a report from the sixth Young Microbiologists Symposium on 'Microbe Signalling, Organisation and Pathogenesis'.

The sixth Young Microbiologists Symposium on 'Microbe Signalling, Organisation and Pathogenesis' was scheduled to be held at the University of Southampton, UK, in late August 2020. However, due to the health and safety guidelines and travel restrictions as a response to the COVID-19 pandemic, the symposium was transitioned to a virtual format, a change embraced enthusiastically as the meeting attracted over 200 microbiologists from 40 countries. The event allowed junior scientists to present their work to a broad audience and was supported by the European Molecular Biology Organization, the Federation of European Microbiological Societies, the Society of Applied Microbiology, the Biochemical Society, the Microbiology Society and the National Biofilms Innovation Centre. Sessions covered recent advances in all areas of microbiology including: Secretion and transport across membranes, Gene regulation and signalling, Host-microbe interactions, and Microbial communities and biofilm formation. This report focuses on several of the highlights and exciting developments communicated during the talks and poster presentations.

Extracellular Amylase Is Required for Full Virulence and Regulated by the Global Posttranscriptional Regulator RsmA in Xanthomonas campestris Pathovar campestris.

As with many phytopathogenic bacteria, the virulence of Xanthomonas campestris pv. campestris, the causal agent of black rot disease in cruciferous plants, relies on secretion of a suite of extracellular enzymes that includes cellulase (endoglucanase), pectinase, protease, and amylase. Although the role in virulence of a number of these enzymes has been assessed, the contribution of amylase to X. campestris pv. campestris virulence has yet to be established. In this work, we investigated both the role of extracellular amylase in X. campestris pv. campestris virulence and the control of its expression. Deletion of XC3487 (here renamed amyAXcc), a putative amylase-encoding gene from the genome of X. campestris pv. campestris strain 8004, resulted in a complete loss of extracellular amylase activity and significant reduction in virulence. The extracellular amylase activity and virulence of the amyAXcc mutant could be restored to the wild-type level by expressing amyAXcc in trans. These results demonstrated that amyAXcc is responsible for the extracellular amylase activity of X. campestris pv. campestris and indicated that extracellular amylase plays an important role in X. campestris pv. campestris virulence. We also found that the expression of amyAXcc is strongly induced by starch and requires activation by the global posttranscriptional regulator RsmA. RsmA binds specifically to the 5'-untranslated region of amyAXcc transcripts, suggesting that RsmA regulates amyAXcc directly at the posttranscriptional level. Unexpectedly, in addition to posttranscriptional regulation, the use of a transcriptional reporter demonstrated that RsmA also regulates amyAXcc expression at the transcriptional level, possibly by an indirect mechanism.

Genome-wide screen and functional analysis in Xanthomonas reveal a large number of mRNA-derived sRNAs, including the novel RsmA-sequester RsmU.

Although bacterial small noncoding RNAs (sRNAs) are known to play a critical role in various cellular processes, including pathogenesis, the identity and action of such sRNAs are still poorly understood in many organisms. Here we have performed a genome-wide screen and functional analysis of the sRNAs in Xanthomonas campestris pv. campestris (Xcc), an important phytopathogen. The 50-500-nt RNA fragments isolated from the wild-type strain grown in a virulence gene-inducing condition were sequenced and a total of 612 sRNA candidates (SRCs) were identified. The majority (82%) of the SRCs were derived from mRNA, rather than specific sRNA genes. A representative panel of 121 SRCs were analysed by northern blotting; 117 SRCs were detected, supporting the contention that the overwhelming majority of the 612 SRCs identified are indeed sRNAs. Phenotypic analysis of strains overexpressing different candidates showed that a particular sRNA, RsmU, acts as a negative regulator of virulence, the hypersensitive response, and cell motility in Xcc. In vitro electrophoretic mobility shift assay and in vivo coimmunoprecipitation analyses indicated that RsmU interacted with the global posttranscriptional regulator RsmA, although sequence analysis displayed that RsmU is not a member of the sRNAs families known to antagonize RsmA. Northern blotting analyses demonstrated that RsmU has two isoforms that are processed from the 3'-untranslated region of the mRNA of XC1332 predicted to encode ComEA, a periplasmic protein required for DNA uptake in bacteria. This work uncovers an unexpected major sRNA biogenesis strategy in bacteria and a hidden layer of sRNA-mediated virulence regulation in Xcc.

A SAM-I riboswitch with the ability to sense and respond to uncharged initiator tRNA.

All known riboswitches use their aptamer to senese one metabolite signal and their expression platform to regulate gene expression. Here, we characterize a SAM-I riboswitch (SAM-IXcc) from the Xanthomonas campestris that regulates methionine synthesis via the met operon. In vitro and in vivo experiments show that SAM-IXcc controls the met operon primarily at the translational level in response to cellular S-adenosylmethionine (SAM) levels. Biochemical and genetic data demonstrate that SAM-IXcc expression platform not only can repress gene expression in response to SAM binding to SAM-IXcc aptamer but also can sense and bind uncharged initiator Met tRNA, resulting in the sequestering of the anti-Shine-Dalgarno (SD) sequence and freeing the SD for translation initiation. These findings identify a SAM-I riboswitch with a dual functioning expression platform that regulates methionine synthesis through a previously unrecognized mechanism and discover a natural tRNA-sensing RNA element. This SAM-I riboswitch appears to be highly conserved in Xanthomonas species.

An improved bind-n-seq strategy to determine protein-DNA interactions validated using the bacterial transcriptional regulator YipR.

BACKGROUND: Interactions between transcription factors and DNA lie at the centre of many biological processes including DNA recombination, replication, repair and transcription. Most bacteria encode diverse proteins that act as transcription factors to regulate various traits. Several technologies for identifying protein-DNA interactions at the genomic level have been developed. Bind-n-seq is a high-throughput in vitro method first deployed to analyse DNA interactions associated with eukaryotic zinc-finger proteins. The method has three steps (i) binding protein to a randomised oligonucleotide DNA target library, (ii) deep sequencing of bound oligonucleotides, and (iii) a computational algorithm to define motifs among the sequences. The classical Bind-n-seq strategy suffers from several limitations including a lengthy wet laboratory protocol and a computational algorithm that is difficult to use. We introduce here an improved, rapid, and simplified Bind-n-seq protocol coupled with a user-friendly downstream data analysis and handling algorithm, which has been optimized for bacterial target proteins. We validate this new protocol by showing the successful characterisation of the DNA-binding specificities of YipR (YajQ interacting protein regulator), a well-known transcriptional regulator of virulence genes in the bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc). RESULTS: The improved Bind-n-seq approach identified several DNA binding motif sequences for YipR, in particular the CCCTCTC motif, which were located in the promoter regions of 1320 Xcc genes. Informatics analysis revealed that many of these genes regulate functions associated with virulence, motility, and biofilm formation and included genes previously found involved in virulence. Additionally, electromobility shift assays show that YipR binds to the promoter region of XC_2633 in a CCCTCTC motif-dependent manner. CONCLUSION: We present a new and rapid Bind-n-seq protocol that should be useful to investigate DNA-binding proteins in bacteria. The analysis of YipR DNA binding using this protocol identifies a novel DNA sequence motif in the promoter regions of target genes that define the YipR regulon.

Mechanistic insights into host adaptation, virulence and epidemiology of the phytopathogen Xanthomonas.

Xanthomonas is a well-studied genus of bacterial plant pathogens whose members cause a variety of diseases in economically important crops worldwide. Genomic and functional studies of these phytopathogens have provided significant understanding of microbial-host interactions, bacterial virulence and host adaptation mechanisms including microbial ecology and epidemiology. In addition, several strains of Xanthomonas are important as producers of the extracellular polysaccharide, xanthan, used in the food and pharmaceutical industries. This polymer has also been implicated in several phases of the bacterial disease cycle. In this review, we summarise the current knowledge on the infection strategies and regulatory networks controlling virulence and adaptation mechanisms from Xanthomonas species and discuss the novel opportunities that this body of work has provided for disease control and plant health.

Modulation of antibiotic sensitivity and biofilm formation in Pseudomonas aeruginosa by interspecies signal analogues.

Pseudomonas aeruginosa, a significant opportunistic pathogen, can participate in inter-species communication through signaling by cis-2-unsaturated fatty acids of the diffusible signal factor (DSF) family. Sensing these signals leads to altered biofilm formation and increased tolerance to various antibiotics, and requires the histidine kinase PA1396. Here, we show that the membrane-associated sensory input domain of PA1396 has five transmembrane helices, two of which are required for DSF sensing. DSF binding is associated with enhanced auto-phosphorylation of PA1396 incorporated into liposomes. Further, we examined the ability of synthetic DSF analogues to modulate or inhibit PA1396 activity. Several of these analogues block the ability of DSF to trigger auto-phosphorylation and gene expression, whereas others act as inverse agonists reducing biofilm formation and antibiotic tolerance, both in vitro and in murine infection models. These analogues may thus represent lead compounds to develop novel adjuvants improving the efficacy of existing antibiotics.

Social behaviour and making attachments: a report from the fifth 'Young Microbiologists Symposium on Microbe Signalling, Organisation and Pathogenesis'.

The fifth Young Microbiologists Symposium was held in Queen's University Belfast, Northern Ireland, in late August 2018. The symposium, focused on 'Microbe signalling, organization and pathogenesis', attracted 121 microbiologists from 15 countries. The meeting allowed junior scientists to present their work to a broad audience, and was supported by the European Molecular Biology Organization, the Federation of European Microbiological Societies, the Society of Applied Microbiology, the Biochemical Society and the Microbiology Society. Sessions covered recent advances in areas of microbiology including gene regulation and signalling, secretion and transport across membranes, infection and immunity, and antibiotics and resistance mechanisms. In this Meeting Report, we highlight some of the most significant advances and exciting developments communicated during talks and poster presentations.

Diffusible signal factor signaling regulates multiple functions in the opportunistic pathogen Stenotrophomonas maltophilia.

OBJECTIVE: Stenotrophomonas maltophilia is a Gram-negative bacterium commonly isolated from nosocomial infections. Analysis of the genome of the clinical S. maltophilia isolate K279a indicates that it encodes a diffusible signal factor (DSF)-dependent cell-cell signaling mechanism that is highly similar to the system previously described in phytopathogens from the genera Xanthomonas and Xylella. Our objective was to study the function of DSF signaling in the clinical strain S. maltophilia K279a using genetic and functional genomic analyses. RESULTS: We compared the wild-type strain with a mutant deficient in the rpfF (regulation of pathogenicity factors) gene that is essential for the synthesis of DSF. The effects of disruption of DSF signaling were pleiotropic with an impact on virulence, biofilm formation and pathogenesis. The phenotypic effects of rpfF mutation in S. maltophilia could be reversed by addition of exogenous DSF. Taken together, we demonstrate that DSF signaling regulates factors contributing to virulence, biofilm formation and motility of this important opportunistic pathogen.

Microbiome characteristics of induced sputum compared to bronchial fluid and upper airway samples.

OBJECTIVE: The study of the community of microorganisms (the microbiota) in the lower airways in children is restricted to opportunistic sampling in children undergoing elective general anaesthetic. Here we tested the hypothesis that induced sputum is a valid alternative to directly sampling the lower airways to study lower airway microbiota. METHODS: Children scheduled for elective operations were recruited. Pre-operatively a sample of induced sputum was obtained. After anaesthesia was induced, a bronchial brushing and swabs of the upper respiratory tract were obtained. Bacterial community analysis was performed by amplification of the V3-V4 16S rRNA gene region. RESULTS: Twenty children were recruited, mean age 10.7 years. Induced sputum samples were obtained from 12 children, bronchial brushing from 14 and nasal, mouth, and throat samples in 15, 16, and 17 children. The profile of bacterial communities was similar in the mouth, throat, and sputum samples with the nose and bronchial samples being different. Actinobacteria species dominated the nose and mouth, Fusobacteria were the dominant species in the throat and sputum while Proteobacteria species dominated in bronchial samples. Forty-one percent of detected bacteria in bronchial samples were unclassified. Bacterial communities from the mouth, throat, and induced sputum were tightly clustered and were distinct from nose and those found in bronchial communities. CONCLUSIONS: Induced sputum may not be a valid surrogate for microbiome assessment of the lower airways in all individuals. Many bacteria in bronchial samples were not recognized by standard testing, suggesting that our understanding of the lower airway microbiota in children remains rudimentary.

Stenotrophomonas maltophilia.

This infographic describes the key regulated traits of Stenotrophomonas maltophilia, important for beneficial plant interactions, and also its increasing incidence as a nosocomial and community-acquired infection. Stenotrophomonas maltophilia is a cosmopolitan and ubiquitous bacterium found in a range of environmental habitats, including extreme ones, although in nature it is mainly associated with plants. S. maltophilia fulfils important ecosystem functions in the sulfur and nitrogen cycles, in degradation of complex compounds and pollutants, and in promoti on of plant growth and health. Stenotrophomonas can also colonize extreme man-made niches in hospitals, space shuttles, and clean rooms. S. maltophilia has emerged as a global opportunistic human pathogen, which does not usually infect healthy hosts but is associated with high morbidity and mortality in severely immunocompromised and debilitated individuals. S. maltophilia can also be recovered from polymicrobial infections, most notably from the respiratory tract of cystic fibrosis patients. Close relatives of S. maltophilia, for example, S. rhizophila, provide a harmless alternative for biotechnological applications without human health risks.

The Ax21 protein influences virulence and biofilm formation in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an antibiotic-resistant Gram-negative pathogen, which is associated with hospital-acquired infection. The genome encodes a protein highly related to the Ax21 protein of Xanthomonas oryzae that is implicated in interactions of this plant pathogen with rice. Here, we report on the pleiotropic nature of ax21 mutation in S. maltophilia and the effects of addition of the Ax21 protein on the restoration of the wild-type phenotype. We show that loss by mutation of Ax21 leads to reduced motility, reduced biofilm formation, reduced tolerance to the antibiotic tobramycin and reduced virulence to larvae of Galleria mellonella, as well as alteration in the expression of specific genes associated with virulence or antibiotic resistance. Addition of the Ax21protein restored motility and the level of gene expression towards wild type. These findings are consistent with the notion that the Ax21 protein is involved in intraspecies communication, although other interpretations cannot be discounted.

Probing the Role of Cyclic di-GMP Signaling Systems in Disease Using Chinese Radish.

The determination of the genome sequences of pathogenic bacteria has facilitated functional analyses that aim to understand the molecular basis of virulence. In particular, genome sequence information of the pathogen Xanthomonas campestris pathovar campestris has allowed researchers to identify and functionally analyze the role of intracellular signaling involving cyclic di-GMP in black rot disease of crucifers. Here, we describe leaf clipping and spraying methods for testing the virulence of wild type and derived mutants of X. campestris in Chinese radish. These methods address different facets of the disease cycle, which requires the ability to survive epiphytically before entry into the plant and growth and systemic spread within the xylem.

Interaction and signalling networks: a report from the fourth 'Young Microbiologists Symposium on Microbe Signalling, Organisation and Pathogenesis'.

At the end of June, over 120 microbiologists from 18 countries gathered in Dundee, Scotland for the fourth edition of the Young Microbiologists Symposium on 'Microbe Signalling, Organisation and Pathogenesis'. The aim of the symposium was to give early career microbiologists the opportunity to present their work in a convivial environment and to interact with senior world-renowned scientists in exciting fields of microbiology research. The meeting was supported by the Microbiology Society, the Society of Applied Microbiology and the American Society for Microbiology with further sponsorship from the European Molecular Biology Organisation and the Royal Society of Edinburgh. In this report, we highlight some themes that emerged from the many interesting talks and poster presentations, as well as some of the other activities that were on offer at this energetic meeting.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Combating chronic bacterial infections by manipulating cyclic nucleotide-regulated biofilm formation.

Many pathogenic bacteria can form biofilms in clinical settings with major consequences for the progression of infections. Bacterial biofilm communities are typically much more resistant to both antibiotic treatment and clearance by the immune system in comparison to free-living cells. Therefore, drugs that specifically target the formation or maintenance of biofilms would be very valuable additions to current clinical options. Cyclic nucleotide second messengers, in particular cyclic-diguanosine-GMP (c-di-GMP), are now known to play a major role in biofilm formation, and maintenance, in many bacterial species. In this special report, we will review recent progress toward the development of drugs that interfere with c-di-GMP signaling as a route to control biofilm infections. We will focus on the chronic infections associated with the notorious opportunistic pathogen Pseudomonas aeruginosa, although the principles outlined here are also relevant to most bacterial pathogens.

Functional and genomic insights into the pathogenesis of Burkholderia species to rice.

A number of species of bacteria from the genus Burkholderia have been shown to be causal agents of diseases of rice. These diseases, caused by Burkholderia glumae, B. gladioli and B. plantarii, are becoming increasingly common across the globe. This is particularly so for B. glumae, whose ability to grow at elevated temperatures suggests that it may become a prevalent problem in an era of global warming. Despite the increasing threat to rice, relatively little is known about the virulence mechanisms employed by these pathogens. Work over the last 5 years has provided an increasing insight into these factors and their control by environmental and other cues. In addition, the determination of a number of genome sequences has allowed bioinformatic predictions of further possible mechanisms, which can now be investigated experimentally. Here, we review recent advances in the understanding of virulence of Burkholderia to rice, to include discussion of the roles of toxins, type II secreted enzymes, type III secreted effectors and motility as well as their regulation by quorum sensing, two-component systems and cyclic di-GMP signalling. Finally, we consider a number of approaches for the control of bacterial virulence through the modulation of quorum sensing and toxin degradation.

The DSF Family of Cell-Cell Signals: An Expanding Class of Bacterial Virulence Regulators.

Many pathogenic bacteria use cell-cell signaling systems involving the synthesis and perception of diffusible signal molecules to control virulence as a response to cell density or confinement to niches. Bacteria produce signals of diverse structural classes. Signal molecules of the diffusible signal factor (DSF) family are cis-2-unsaturated fatty acids. The paradigm is cis-11-methyl-2-dodecenoic acid from Xanthomonas campestris pv. campestris (Xcc), which controls virulence in this plant pathogen. Although DSF synthesis was thought to be restricted to the xanthomonads, it is now known that structurally related molecules are produced by the unrelated bacteria Burkholderia cenocepacia and Pseudomonas aeruginosa. Furthermore, signaling involving these DSF family members contributes to bacterial virulence, formation of biofilms and antibiotic tolerance in these important human pathogens. Here we review the recent advances in understanding DSF signaling and its regulatory role in different bacteria. These advances include the description of the pathway/mechanism of DSF biosynthesis, identification of novel DSF synthases and new members of the DSF family, the demonstration of a diversity of DSF sensors to include proteins with a Per-Arnt-Sim (PAS) domain and the description of some of the signal transduction mechanisms that impinge on virulence factor expression. In addition, we address the role of DSF family signals in interspecies signaling that modulates the behavior of other microorganisms. Finally, we consider a number of recently reported approaches for the control of bacterial virulence through the modulation of DSF signaling.