C-di-GMP regulates <i>Pseudomonas aeruginosa</i> stress response to tellurite during both planktonic and biofilm modes of growth

Stress response plays an important role on microbial adaptation under hostile environmental conditions. It is generally unclear how the signaling transduction pathway mediates a stress response in planktonic and biofilm modes of microbial communities simultaneously. Here, we showed that metalloid tellurite (TeO(3)(2–)) exposure induced the intracellular content of the secondary messenger cyclic di-GMP (c-di-GMP) of Pseudomonas aeruginosa. Two diguanylate cyclases (DGCs), SadC and SiaD, were responsible for the increased intracellular content of c-di-GMP. Enhanced c-di-GMP levels by TeO(3)(2–) further increased P. aeruginosa biofilm formation and resistance to TeO(3)(2–). P. aeruginosa ΔsadCΔsiaD and PAO1/p(lac)-yhjH mutants with low intracellular c-di-GMP content were more sensitive to TeO(3)(2–) exposure and had low relative fitness compared to the wild-type PAO1 planktonic and biofilm cultures exposed to TeO(3)(2–). Our study provided evidence that c-di-GMP level can play an important role in mediating stress response in microbial communities during both planktonic and biofilm modes of growth

Quorum Sensing and Virulence of Pseudomonas aeruginosa during Lung Infection of Cystic Fibrosis Patients

Pseudomonas aeruginosa is the predominant microorganism in chronic lung infection of cystic fibrosis patients. The chronic lung infection is preceded by intermittent colonization. When the chronic infection becomes established, it is well accepted that the isolated strains differ phenotypically from the intermittent strains. Dominating changes are the switch to mucoidity (alginate overproduction) and loss of epigenetic regulation of virulence such as the Quorum Sensing (QS). To elucidate the dynamics of P. aeruginosa QS systems during long term infection of the CF lung, we have investigated 238 isolates obtained from 152 CF patients at different stages of infection ranging from intermittent to late chronic. Isolates were characterized with regard to QS signal molecules, alginate, rhamnolipid and elastase production and mutant frequency. The genetic basis for change in QS regulation were investigated and identified by sequence analysis of lasR, rhlR, lasI and rhlI. The first QS system to be lost was the one encoded by las system 12 years (median value) after the onset of the lung infection with subsequent loss of the rhl encoded system after 17 years (median value) shown as deficiencies in production of the 3-oxo-C12-HSL and C4-HSL QS signal molecules respectively. The concomitant development of QS malfunction significantly correlated with the reduced production of rhamnolipids and elastase and with the occurrence of mutations in the regulatory genes lasR and rhlR. Accumulation of mutations in both lasR and rhlR correlated with development of hypermutability. Interestingly, a higher number of mucoid isolates were found to produce C4-HSL signal molecules and rhamnolipids compared to the non-mucoid isolates. As seen from the present data, we can conclude that P. aeruginosa and particularly the mucoid strains do not lose the QS regulation or the ability to produce rhamnolipids until the late stage of the chronic infection

A broad range quorum sensing inhibitor working through sRNA inhibition

Abstract For the last decade, chemical control of bacterial virulence has received considerable attention. Ajoene, a sulfur-rich molecule from garlic has been shown to reduce expression of key quorum sensing regulated virulence factors in the opportunistic pathogen Pseudomonas aeruginosa. Here we show that the repressing effect of ajoene on quorum sensing occurs by inhibition of small regulatory RNAs (sRNA) in P. aeruginosa as well as in Staphylococcus aureus, another important human pathogen that employs quorum sensing to control virulence gene expression. Using various reporter constructs, we found that ajoene lowered expression of the sRNAs RsmY and RsmZ in P. aeruginosa and the small dual-function regulatory RNA, RNAIII in S. aureus, that controls expression of key virulence factors. We confirmed the modulation of RNAIII by RNA sequencing and found that the expression of many QS regulated genes encoding virulence factors such as hemolysins and proteases were lowered in the presence of ajoene in S. aureus. Importantly, our findings show that sRNAs across bacterial species potentially may qualify as targets of anti-virulence therapy and that ajoene could be a lead structure in search of broad-spectrum compounds transcending the Gram negative-positive borderline

The Pseudomonas putida CsrA/RsmA homologues negatively affect c-di-GMP pools and biofilm formation through the GGDEF/EAL response regulator CfcR

Expression of cfcR, encoding the only GGDEF/EAL response regulator in Pseudomonas putida, is transcriptionally regulated by RpoS, ANR and FleQ, and the functionality of CfcR as a diguanylate cyclase requires the multisensor CHASE3/GAF hybrid histidine kinase named CfcA. Here an additional level of cfcR control, operating post-transcriptionally via the RNA-binding proteins RsmA, RsmE and RsmI, is unraveled. Specific binding of the three proteins to an Rsm binding motif (5’CANGGANG3’) encompassing the translational start codon of cfcR was confirmed. Although RsmA exhibited the highest binding affinity to the cfcR transcript, single deletions of rsmA, rsmE or rsmI, caused minor derepression in CfcR translation compared to a ∆rsmIEA triple mutant. RsmA also showed a negative impact on c-di-GMP levels in a double mutant ∆rsmIE through the control of cfcR, which is responsible for most of the free c-di-GMP during stationary phase in static conditions. In addition, a CfcR-dependent c-di-GMP boost was observed during this stage in ∆rsmIEA confirming the negative effect of Rsm proteins on CfcR translation and explaining the increased biofilm formation in this mutant compared to the wild type. Overall these results suggest that CfcR is a key player in biofilm formation regulation by the Rsm proteins in P. putida

Role of extracellular matrix components in biofilm formation and adaptation of Pseudomonas ogarae F113 to the rhizosphere environment

Regulating the transition of bacteria from motile to sessile lifestyles is crucial for their ability to compete effectively in the rhizosphere environment. Pseudomonas are known to rely on extracellular matrix (ECM) components for microcolony and biofilm formation, allowing them to adapt to a sessile lifestyle. Pseudomonas ogarae F113 possesses eight gene clusters responsible for the production of ECM components. These gene clusters are tightly regulated by AmrZ, a major transcriptional regulator that influences the cellular levels of c-di-GMP. The AmrZ-mediated transcriptional regulation of ECM components is primarily mediated by the signaling molecule c-di-GMP and the flagella master regulator FleQ. To investigate the functional role of these ECM components in P. ogarae F113, we performed phenotypic analyses using mutants in genes encoding these ECM components. These analyses included assessments of colony morphology, dye-staining, static attachment to abiotic surfaces, dynamic biofilm formation on abiotic surfaces, swimming motility, and competitive colonization assays of the rhizosphere. Our results revealed that alginate and PNAG polysaccharides, along with PsmE and the fimbrial low molecular weight protein/tight adherence (Flp/Tad) pilus, are the major ECM components contributing to biofilm formation. Additionally, we found that the majority of these components and MapA are needed for a competitive colonization of the rhizosphere in P. ogarae F113

AmrZ is a major determinant of c-di-GMP levels in Pseudomonas fluorescens F113

The transcriptional regulator AmrZ is a global regulatory protein conserved within the pseudomonads. AmrZ can act both as a positive and a negative regulator of gene expression, controlling many genes implicated in environmental adaption. Regulated traits include motility, iron homeostasis, exopolysaccharides production and the ability to form biofilms. In Pseudomonas fluorescens F113, an amrZ mutant presents a pleiotropic phenotype, showing increased swimming motility, decreased biofilm formation and very limited ability for competitive colonization of rhizosphere, its natural habitat. It also shows different colony morphology and binding of the dye Congo Red. The amrZ mutant presents severely reduced levels of the messenger molecule cyclic-di-GMP (c-di-GMP), which is consistent with the motility and biofilm formation phenotypes. Most of the genes encoding proteins with diguanylate cyclase (DGCs) or phosphodiesterase (PDEs) domains, implicated in c-di-GMP turnover in this bacterium, appear to be regulated by AmrZ. Phenotypic analysis of eight mutants in genes shown to be directly regulated by AmrZ and encoding c-di-GMP related enzymes, showed that seven of them were altered in motility and/or biofilm formation. The results presented here show that in P. fluorescens, AmrZ determines c-di-GMP levels through the regulation of a complex network of genes encoding DGCs and PDEs

Chemoperception of Specific Amino Acids Controls Phytopathogenicity in Pseudomonas syringae pv. tomato

IMPORTANCE There is substantive evidence that chemotaxis is a key requisite for efficient pathogenesis in plant pathogens. However, information regarding particular bacterial chemoreceptors and the specific plant signal that they sense is scarce. Our work shows that the phytopathogenic bacterium Pseudomonas syringae pv. tomato mediates not only chemotaxis but also the control of pathogenicity through the perception of the plant abundant amino acids Asp and Glu. We describe the specificity of the perception of L- and D-Asp and L-Glu by the PsPto-PscA chemoreceptor and the involvement of this perception in the regulation of pathogenicity-related traits. Moreover, a saturating concentration of D-Asp reduces bacterial virulence, and we therefore propose that ligand-mediated interference of key chemoreceptors may be an alternative strategy to control virulence.Supplemental material for this article may be found at https://doi.org/10.1128/mBio .01868-19.We acknowledge M. Trini Gallegos for kindly provide plasmid pCdrA::gfpS and S. Nebreda for technical assistance.Chemotaxis has been associated with the pathogenicity of bacteria in plants and was found to facilitate bacterial entry through stomata and wounds. However, knowledge regarding the plant signals involved in this process is scarce. We have addressed this issue using Pseudomonas syringae pv. tomato, which is a foliar pathogen that causes bacterial speck in tomato. We show that the chemoreceptor P. syringae pv. tomato PscA (PsPto-PscA) recognizes specifically and with high affinity L-Asp, L-Glu, and D-Asp. The mutation of the chemoreceptor gene largely reduced chemotaxis to these ligands but also altered cyclic di-GMP (c-di-GMP) levels, biofilm formation, and motility, pointing to cross talk between different chemosensory pathways. Furthermore, the PsPto-PscA mutant strain showed reduced virulence in tomato. Asp and Glu are the most abundant amino acids in plants and in particular in tomato apoplasts, and we hypothesize that this receptor may have evolved to specifically recognize these compounds to facilitate bacterial entry into the plant. Infection assays with the wild-type strain showed that the presence of saturating concentrations of D-Asp also reduced bacterial virulence.This work was supported by grants AGL2015-63851-R and RTI2018-095222-B100 (to E.L.-S.) and BIO2016-76779-P (to T.K.) from the Ministerio de Economía y Competitividad, Spain. J.P.C.-V. was supported by the FPI program (BES-2016-076452, MINECOSpain)

Effects of antibiotic treatment and phagocyte infiltration on development of Pseudomonas aeruginosa biofilm—Insights from the application of a novel PF hydrogel model in vitro and in vivo

Background and purposeBacterial biofilm infections are major health issues as the infections are highly tolerant to antibiotics and host immune defenses. Appropriate biofilm models are important to develop and improve to make progress in future biofilm research. Here, we investigated the ability of PF hydrogel material to facilitate the development and study of Pseudomonas aeruginosa biofilms in vitro and in vivo.MethodsWild-type P. aeruginosa PAO1 bacteria were embedded in PF hydrogel situated in vitro or in vivo, and the following aspects were investigated: 1) biofilm development; 2) host immune response and its effect on the bacteria; and 3) efficacy of antibiotic treatment.ResultsMicroscopy demonstrated that P. aeruginosa developed typical biofilms inside the PF hydrogels in vitro and in mouse peritoneal cavities where the PF hydrogels were infiltrated excessively by polymorphonuclear leukocytes (PMNs). The bacteria remained at a level of ~106 colony-forming unit (CFU)/hydrogel for 7 days, indicating that the PMNs could not eradicate the biofilm bacteria. β-Lactam or aminoglycoside mono treatment at 64× minimal inhibitory concentration (MIC) killed all bacteria in day 0 in vitro biofilms, but not in day 1 and older biofilms, even at a concentration of 256× MIC. Combination treatment with the antibiotics at 256× MIC completely killed the bacteria in day 1 in vitro biofilms, and combination treatment in most of the cases showed significantly better bactericidal effects than monotherapies. However, in the case of the established in vivo biofilms, the mono and combination antibiotic treatments did not efficiently kill the bacteria.ConclusionOur results indicate that the bacteria formed typical biofilms in PF hydrogel in vitro and in vivo and that the biofilm bacteria were tolerant against antibiotics and host immunity. The PF hydrogel biofilm model is simple and easy to fabricate and highly reproducible with various application possibilities. We conclude that the PF hydrogel biofilm model is a new platform that will facilitate progress in future biofilm investigations, as well as studies of the efficacy of new potential medicine against biofilm infections

The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms

Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive “non-producing” cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development.National Institutes of Health (U.S.). National Institute of Environmental Health Sciences (Training Grant in Toxicology 5 T32 ES7020-37

The Airway Microbiota in Cystic Fibrosis: A Complex Fungal and Bacterial Community—Implications for Therapeutic Management

International audienceBackground Given the polymicrobial nature of pulmonary infections in patients with cystic fibrosis (CF), it is essential to enhance our knowledge on the composition of the microbial community to improve patient management. In this study, we developed a pyrosequencing approach to extensively explore the diversity and dynamics of fungal and prokaryotic populations in CF lower airways. Methodology and Principal Findings Fungi and bacteria diversity in eight sputum samples collected from four adult CF patients was investigated using conventional microbiological culturing and high-throughput pyrosequencing approach targeting the ITS2 locus and the 16S rDNA gene. The unveiled microbial community structure was compared to the clinical profile of the CF patients. Pyrosequencing confirmed recently reported bacterial diversity and observed complex fungal communities, in which more than 60% of the species or genera were not detected by cultures. Strikingly, the diversity and species richness of fungal and bacterial communities was significantly lower in patients with decreased lung function and poor clinical status. Values of Chao1 richness estimator were statistically correlated with values of the Shwachman-Kulczycki score, body mass index, forced vital capacity, and forced expiratory volume in 1 s (p = 0.046, 0.047, 0.004, and 0.001, respectively for fungal Chao1 indices, and p = 0.010, 0.047, 0.002, and 0.0003, respectively for bacterial Chao1 values). Phylogenetic analysis showed high molecular diversities at the sub-species level for the main fungal and bacterial taxa identified in the present study. Anaerobes were isolated with Pseudomonas aeruginosa, which was more likely to be observed in association with Candida albicans than with Aspergillus fumigatus