Iron is a signal for Stenotrophomonas maltophilia biofilm formation, oxidative stress response, OMPs expression, and virulence.
ABSTRACT: Stenotrophomonas maltophilia is an emerging nosocomial pathogen. In many bacteria iron availability regulates, through the Fur system, not only iron homeostasis but also virulence. The aim of this work was to assess the role of iron on S. maltophilia biofilm formation, EPS production, oxidative stress response, OMPs regulation, quorum sensing (QS), and virulence. Studies were done on K279a and its isogenic fur mutant F60 cultured in the presence or absence of dipyridyl. This is the first report of spontaneous fur mutants obtained in S. maltophilia. F60 produced higher amounts of biofilms than K279a and CLSM analysis demonstrated improved adherence and biofilm organization. Under iron restricted conditions, K279a produced biofilms with more biomass and enhanced thickness. In addition, F60 produced higher amounts of EPS than K279a but with a similar composition, as revealed by ATR-FTIR spectroscopy. With respect to the oxidative stress response, MnSOD was the only SOD isoenzyme detected in K279a. F60 presented higher SOD activity than the wt strain in planktonic and biofilm cultures, and iron deprivation increased K279a SOD activity. Under iron starvation, SDS-PAGE profile from K279a presented two iron-repressed proteins. Mass spectrometry analysis revealed homology with FepA and another putative TonB-dependent siderophore receptor of K279a. In silico analysis allowed the detection of potential Fur boxes in the respective coding genes. K279a encodes the QS diffusible signal factor (DSF). Under iron restriction K279a produced higher amounts of DSF than under iron rich condition. Finally, F60 was more virulent than K279a in the Galleria mellonella killing assay. These results put in evidence that iron levels regulate, likely through the Fur system, S. maltophilia biofilm formation, oxidative stress response, OMPs expression, DSF production and virulence.
Project description:Iron has been shown to regulate biofilm formation, oxidative stress response and several pathogenic mechanisms in Stenotrophomonas maltophilia. Thus, the present study is aimed at identifying various iron acquisition systems and iron sources utilized during iron starvation in S. maltophilia. The annotations of the complete genome of strains K279a, R551-3, D457 and JV3 through Rapid Annotations using Subsystems Technology (RAST) revealed two putative subsystems to be involved in iron acquisition: the iron siderophore sensor and receptor system and the heme, hemin uptake and utilization systems/hemin transport system. Screening for these acquisition systems in S. maltophilia showed the presence of all tested functional genes in clinical isolates, but only a few in environmental isolates. NanoString nCounter Elements technology, applied to determine the expression pattern of the genes under iron-depleted condition, showed significant expression for FeSR (6.15-fold), HmuT (12.21-fold), Hup (5.46-fold), ETFb (2.28-fold), TonB (2.03-fold) and Fur (3.30-fold). The isolates, when further screened for the production and chemical nature of siderophores using CAS agar diffusion (CASAD) and Arnows's colorimetric assay, revealed S. maltophilia to produce catechol-type siderophore. Siderophore production was also tested through liquid CAS assay and was found to be greater in the clinical isolate (30.8%) compared to environmental isolates (4%). Both clinical and environmental isolates utilized hemoglobin, hemin, transferrin and lactoferrin as iron sources. All data put together indicates that S. maltophilia utilizes siderophore-mediated and heme-mediated systems for iron acquisition during iron starvation. These data need to be further confirmed through several knockout studies.
Project description:Stenotrophomonas maltophilia, a Gram-negative, multi-drug-resistant bacterium, is increasingly recognized as a key opportunistic pathogen. Thus, we embarked upon an investigation of S. maltophilia iron acquisition. To begin, we determined that the genome of strain K279a is predicted to encode a complete siderophore system, including a biosynthesis pathway, an outer-membrane receptor for ferrisiderophore, and other import and export machinery. Compatible with these data, K279a and other clinical isolates of S. maltophilia secreted a siderophore-like activity when grown at 25-37?°C in low-iron media, as demonstrated by a chrome azurol S assay, which detects iron chelation, and Arnow and Rioux assays, which detect catecholate structures. Importantly, these supernatants rescued the growth of iron-starved S. maltophilia, documenting the presence of a biologically active siderophore. A mutation in one of the predicted biosynthesis genes (entC) abolished production of the siderophore and impaired bacterial growth in low-iron conditions. Inactivation of the putative receptor gene (fepA) prevented the utilization of siderophore-containing supernatants for growth in low-iron conditions. Although the biosynthesis and import loci showed some similarity to those of enterobactin, a well-known catecholate made by enteric bacteria, the siderophore of K279a was unable to rescue the growth of an enterobactin-utilizing indicator strain, and conversely iron-starved S. maltophilia could not use purified enterobactin. Furthermore, the S. maltophilia siderophore displayed patterns of solubility in organic compounds and mobility upon thin-layer chromatography that were distinct from those of enterobactin and its derivative, salmochelin. Together, these data demonstrate that S. maltophilia secretes a novel catecholate siderophore.
Project description:Stenotrophomonas maltophilia is a non-fermenting Gram-negative bacterium that is ubiquitous in the environment. In humans, this opportunistic multi-drug-resistant pathogen is responsible for a plethora of healthcare-associated infections. Here, we utilized a whole genome sequencing (WGS)-based phylogenomic core single nucleotide polymorphism (SNP) approach to characterize S. maltophilia subgroups, their potential association with human infection, and to detect any possible transmission events. In total, 89 isolates (67 clinical and 22 environmental) from Germany were sequenced. Fully finished genomes of five strains were included in the dataset for the core SNP phylogenomic analysis. WGS data were compared with conventional genotyping results as well as with underlying disease, biofilm formation, protease activity, lipopolysaccharide (LPS) SDS-PAGE profiles, and serological specificity of an antibody raised against the surface-exposed O-antigen of strain S. maltophilia K279a. The WGS-based phylogenies grouped the strains into 12 clades, out of which 6 contained exclusively human and 3 exclusively environmental isolates. Biofilm formation and proteolytic activity did correlate neither with the phylogenetic tree, nor with the origin of isolates. In contrast, the genomic classification correlated well with the reactivity of the strains against the K279a O-specific antibody, as well as in part with the LPS profiles. Three clusters of clinical strains had a maximum distance of 25 distinct SNP positions, pointing to possible transmission events or acquisition from the same source. In conclusion, these findings indicate the presence of specific subgroups of S. maltophilia strains adapted to the human host.
Project description:Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can infect the lungs of people with cystic fibrosis (CF). The highly viscous mucus in the CF lung, expectorated as sputum, serves as the primary nutrient source for microbes colonizing this site and induces virulence-associated phenotypes and gene expression in several CF pathogens. Here, we characterized the transcriptional responses of three S. maltophilia strains during exposure to synthetic CF sputum medium (SCFM2) to gain insight into how this organism interacts with the host in the CF lung. These efforts led to the identification of 881 transcripts differentially expressed by all three strains, many of which reflect the metabolic pathways used by S. maltophilia in sputum, as well as altered stress responses. The latter correlated with increased resistance to peroxide exposure after pregrowth in SCFM2 for two of the strains. We also compared the SCFM2 transcriptomes of two S. maltophilia CF isolates to that of the acute infection strain, S. maltophilia K279a, allowing us to identify CF isolate-specific signatures in differential gene expression. The expression of genes from the accessory genomes was also differentially altered in response to SCFM2. Finally, a number of biofilm-associated genes were differentially induced in SCFM2, particularly in K279a, which corresponded to increased aggregation and biofilm formation in this strain relative to both CF strains. Collectively, this work details the response of S. maltophilia to an environment that mimics important aspects of the CF lung, identifying potential survival strategies and metabolic pathways used by S. maltophilia during infections.IMPORTANCE Stenotrophomonas maltophilia is an important infecting bacterium in the airways of people with cystic fibrosis (CF). However, compared to the other CF pathogens, S. maltophilia has been relatively understudied. The significance of our research is to provide insight into the global transcriptomic changes of S. maltophilia in response to a medium that was designed to mimic important aspects of the CF lung. This study elucidates the overall metabolic changes that occur when S. maltophilia encounters the CF lung and generates a road map of candidate genes to test using in vitro and in vivo models of CF.
Project description:The activities of fumarase- and manganese-cofactored superoxide dismutase (SOD), encoded by the fumC and sodA genes in Pseudomonas aeruginosa, are elevated in mucoid, alginate-producing bacteria and in response to iron deprivation (D. J. Hassett, M. L. Howell, P. A. Sokol, M. L. Vasil, and G. E. Dean, J. Bacteriol. 179:1442-1451, 1997). In this study, a 393-bp open reading frame, fagA (Fur-associated gene), was identified immediately upstream of fumC, in an operon with orfX and sodA. Two iron boxes or Fur (ferric uptake regulatory protein) binding sites were discovered just upstream of fagA. Purified P. aeruginosa Fur caused a gel mobility shift of a PCR product containing these iron box regions. DNA footprinting analysis revealed a 37-bp region that included the Fur binding sites and was protected by Fur. Primer extension analysis and RNase protection assays revealed that the operon is composed of at least three major iron-regulated transcripts. Four mucoid fur mutants produced 1.7- to 2.6-fold-greater fumarase activity and 1.7- to 2.3-greater amounts of alginate than wild-type organisms. A strain devoid of the alternative sigma factor AlgT(U) produced elevated levels of one major transcript and fumarase C and manganase-cofactored SOD activity, suggesting that AlgT(U) may either play a role in regulating this transcript or function in some facet of iron metabolism. These data suggest that the P. aeruginosa fagA, fumC, orfX, and sodA genes reside together on a small operon that is regulated by Fur and is transcribed in response to iron limitation in mucoid, alginate-producing bacteria.
Project description:The Gram-negative bacterium Stenotrophomonas maltophilia is increasingly identified as a multidrug-resistant pathogen, being associated with pneumonia, among other infections. Despite this increasing clinical problem, the genetic and molecular basis of S. maltophilia virulence is quite minimally defined. We now report that strain K279a, the first clinical isolate of S. maltophilia to be sequenced, encodes a functional type II protein secretion (T2S) system. Indeed, mutants of K279a that contain a mutation in the xps locus exhibit a loss of at least seven secreted proteins and three proteolytic activities. Unlike culture supernatants from the parental K279a, supernatants from multiple xps mutants also failed to induce the rounding, detachment, and death of A549 cells, a human lung epithelial cell line. Supernatants of the xps mutants were also unable to trigger a massive rearrangement in the host cell's actin cytoskeleton that was associated with K279a secretion. In all assays, a complemented xpsF mutant behaved as the wild type did, demonstrating that Xps T2S is required for optimal protein secretion and the detrimental effects on host cells. The activities that were defined as being Xps dependent in K279a were evident among other respiratory isolates of S. maltophilia. Utilizing a similar type of genetic analysis, we found that a second T2S system (Gsp) encoded by the K279a genome is cryptic under all of the conditions tested. Overall, this study represents the first examination of T2S in S. maltophilia, and the data obtained indicate that Xps T2S likely plays an important role in S. maltophilia pathogenesis.
Project description:There is an increasing appreciation of the polymicrobial nature of many bacterial infections such as those associated with cystic fibrosis (CF) and of the potentially important role for interspecies interactions in influencing both bacterial virulence and response to therapy. Patients with CF are often co-infected with Pseudomonas aeruginosa and other pathogens including Burkholderia cenocepacia and Stenotrophomonas maltophilia. These latter bacteria produce signal molecules of the diffusible signal factor (DSF) family, which are cis-2-unsaturated fatty acids. We have previously shown by in vitro studies that DSF from S. maltophilia leads to altered biofilm formation and increased resistance to antibiotics by P. aeruginosa; these responses of P. aeruginosa require the sensor kinase PA1396. Here we show that DSF signals are present in sputum taken from patients with CF. Presence of these DSF signals was correlated with patient colonization by S. maltophilia and/or B. cenocepacia. Analysis of 50 clinical isolates of P. aeruginosa showed that each responded to the presence of synthetic DSF by increased antibiotic resistance and these strains demonstrated little sequence variation in the PA1396 gene. In animal experiments using CF transmembrane conductance regulator knockout mice, the presence of DSF promoted P. aeruginosa persistence. Furthermore, antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells was enhanced in the presence of DSF. Taken together, these data provide substantial evidence that interspecies DSF-mediated bacterial interactions occur in the CF lung and may influence the efficacy of antibiotic treatment, particularly for chronic infections involving persistence of bacteria.
Project description:Stenotrophomonas maltophilia is an emerging global opportunistic pathogen that has been appearing with increasing prevalence in cystic fibrosis (CF). A secreted protease from S. maltophilia has been reported as its chief potential virulence factor. Here, using the reference clinical strain S. maltophilia K279a, the major secreted proteases were identified. Protein biochemistry and mass spectrometry were carried out on K279a culture supernatant. The effect of K279a culture supernatant on cleavage and anti-neutrophil elastase activity of the three majors pulmonary antiproteases was quantified. A deletion mutant of S. maltophilia lacking expression of a protease was constructed. The serine proteases StmPR1, StmPR2 and StmPR3, in addition to chitinase A and an outer membrane esterase were identified in culture supernatants. Protease activity was incompletely abrogated in a K279a-?StmPR1: Erm mutant. Wild type K279a culture supernatant degraded alpha-1 antitrypsin (AAT), secretory leucoprotease inhibitor (SLPI) and elafin, important components of the lung's innate immune defences. Meanwhile SLPI and elafin, but not AAT, retained their ability to inhibit neutrophil elastase. StmPR3 together with StmPR1 and StmPR2, is likely to contribute to protease-mediated innate immune dysfunction in CF.
Project description:The interaction of the Gram-negative bacterium Stenotrophomonas maltophilia with eukaryotes can improve overall plant growth and health, but can also cause opportunistic infections in humans. While the quorum sensing molecule DSF (diffusible signal factor) is responsible for the regulation of phenotypes in pathogenic Stenotrophomonas, up until now, no beneficial effects were reported to be controlled by it. Our objective was to study the function of DSF in the plant growth promoting model strain S. maltophilia R551-3 using functional and transcriptomic analyses. For this purpose, 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. Oilseed rape seeds treated with the wild-type strain showed a statistically significant increase in germination rate compared with those treated with the rpfF mutant. Similarly, the wild-type strain exhibited better plant growth promotion and a greater efficiency in colonizing oilseed rape compared to the mutant strain. Moreover, only the wild-type was capable of forming structured cell aggregates both in vitro and in the rhizosphere, a characteristic mediated by DSF. Gene transcription analyses showed that numerous genes known to play a role in plant colonization (e.g. chemotaxis, cell motility, biofilm formation, multidrug efflux pumps) are controlled by the rpf/DSF system in S. maltophilia. In addition, we detected new potential functions of spermidine, primarily for both growth promotion and stress protection. Overall, our results showed a correspondence between the regulation of DSF and the positive interaction effect with the plant host.
Project description:Stenotrophomonas maltophilia K279a diverges into subpopulations with distinct but reversible phenotypes of small and big colonies when challenged with ampicillin. This observation is consistent with the formation of long cell chains during exponential growth phase and the occurrence of mainly coccoid– or rod-shaped cells in liquid media. Further, scanning electron micrographs of SMK279a revealed that cells formed gigantic outer membrane vesicles in response to β-lactam treatment. RNA-seq analysis of small vs. big colonies unveiled that cells regulate at least seven genes differentially among colony morphotypes. Among those were the blaL1 and blaL2 genes the most strongly regulated ones with an eleven- and six-fold increased transcription, respectively. Further studies with promoter fusions of blaL1 and blaL2 genes implied that expression of both genes is also subject to high levels of phenotypic heterogeneous expression on a single cell level. Additional RNA-seq analysis of this homogenously versus heterogeneously blaL2 expressing cells identified comE homologue as differentially expressed, in which by the expression of extra copies of comE in S. maltophilia K279a reduced the level of those cells that were in a blaL2-ON model to 1% or lower. Together with genome-wide sequence analysis of cells from the different colony morphotypes, the data presented here suggests that phenotypic heterogeneity in S. maltophilia K279a is a result of non-genetic variations within isogenic populations and also polymorphisms in this strain do not influence β-lactamase resistance phenotype. Overall design: In total 10 samples were analyzed (5 different conditions with two independent samples), condition K279a small colonies: S. maltophilia K279a small colonies from LB agar plates grown on ampicillin for 48 h, condition K279a big colonies: S. maltophilia K279a big colonies from LB agar plates grown on ampicillin for 48 h, condition K279a without ampicillin: S. maltophilia K279a colonies from LB agar plates grown without ampicillin for 48 h as a control, condition K279a liquid culture 27 h: S. maltophilia K279a grown in liquid medium for 27 h, condition K279a liquid culture 32 h: S. maltophilia K279a grown in liquid medium for 32 h