Inês N. Silva

Distribution of Cepacian Biosynthesis Genes among Environmental and Clinical Burkholderia Strains and Role of Cepacian Exopolysaccharide in Resistance to Stress Conditions

ABSTRACT The genus Burkholderia includes strains pathogenic to animals and plants, bioremediators, or plant growth promoters. Genome sequence analyses of representative Burkholderia cepacia complex (Bcc) and non-Bcc strains for the presence of the bce-I gene cluster, directing the biosynthesis of the exopolysaccharide (EPS) cepacian, further extended this previously described cluster by another 9 genes. The genes in the bce-II cluster were named bceM to bceU and encode products putatively involved in nucleotide sugar precursor biosynthesis and repeat unit assembly, modification, and translocation across the cytoplasmic membrane. Disruption of the B. cepacia IST408 bceQ and bceR genes, encoding a putative repeat unit flippase and a glycosyltransferase, respectively, resulted in the abolishment of cepacian biosynthesis. A mutation in the bceS gene, encoding a putative acyltransferase, did not affect EPS production yield significantly but decreased its acetylation content by approximately 20%. Quantitative real-time reverse transcription-PCR experiments confirmed the induction of genes in the bce-I and bce-II clusters in a Burkholderia multivorans EPS producer clinical isolate in comparison to the level for its isogenic EPS-defective strain. Fourier Transform infrared spectroscopy analysis confirmed that the exopolysaccharide produced by 10 Burkholderia isolates tested was cepacian. The ability of Burkholderia strains to withstand desiccation and metal ion stress was higher when bacteria were incubated in the presence of 2.5 g/liter of cepacian, suggesting that this EPS plays a role in the survival of these bacteria by contributing to their ability to thrive in different environments.

Pathogenicity, virulence factors, and strategies to fight against Burkholderia cepacia complex pathogens and related species

The Burkholderia cepacia complex (Bcc) is a group of 17 closely related species of the β-proteobacteria subdivision that emerged in the 1980s as important human pathogens, especially to patients suffering from cystic fibrosis. Since then, a remarkable progress has been achieved on the taxonomy and molecular identification of these bacteria. Although some progress have been achieved on the knowledge of the pathogenesis traits and virulence factors used by these bacteria, further work envisaging the identification of potential targets for the scientifically based design of new therapeutic strategies is urgently needed, due to the very difficult eradication of these bacteria with available therapies. An overview of these aspects of Bcc pathogenesis and opportunities for the design of future therapies is presented and discussed in this work.

Insights into the Role of Extracellular Polysaccharides in Burkholderia Adaptation to Different Environments

The genus Burkholderia comprises more than 60 species able to adapt to a wide range of environments such as soil and water, and also colonize and infect plants and animals. They have large genomes with multiple replicons and high gene number, allowing these bacteria to thrive in very different niches. Among the properties of bacteria from the genus Burkholderia is the ability to produce several types of exopolysaccharides (EPSs). The most common one, cepacian, is produced by the majority of the strains examined irrespective of whether or not they belong to the Burkholderia cepacia complex (Bcc). Cepacian biosynthesis proceeds by a Wzy-dependent mechanism, and some of the B. cepacia exopolysaccharide (Bce) proteins have been functionally characterized. In vitro studies showed that cepacian protects bacterial cells challenged with external stresses. Regarding virulence, bacterial cells with the ability to produce EPS are more virulent in several animal models of infection than their isogenic non-producing mutants. Although the production of EPS within the lungs of cystic fibrosis (CF) patients has not been demonstrated, the in vitro assessment of the mucoid phenotype in serial Bcc isolates from CF patients colonized for several years showed that mucoid to non-mucoid transitions are relatively frequent. This morphotype variation can be induced under laboratory conditions by exposing cells to stress such as high antibiotic concentration. Clonal isolates where mucoid to non-mucoid transition had occurred showed that during lung infection, genomic rearrangements, and mutations had taken place. Other phenotypic changes include variations in motility, chemotaxis, biofilm formation, bacterial survival rate under nutrient starvation and virulence. In this review, we summarize major findings related to EPS biosynthesis by Burkholderia and the implications in broader regulatory mechanisms important for cell adaptation to the different niches colonized by these bacteria.

Long-Term Evolution of Burkholderia multivorans during a Chronic Cystic Fibrosis Infection Reveals Shifting Forces of Selection

Bacteria may become genetically and phenotypically diverse during long-term colonization of cystic fibrosis (CF) patient lungs, yet our understanding of within-host evolutionary processes during these infections is lacking. Here we combined current genome sequencing technologies and detailed phenotypic profiling of the opportunistic pathogen Burkholderia multivorans using sequential isolates sampled from a CF patient over 20 years. The evolutionary history of these isolates highlighted bacterial genes and pathways that were likely subject to strong selection within the host and were associated with altered phenotypes, such as biofilm production, motility, and antimicrobial resistance. Importantly, multiple lineages coexisted for years or even decades within the infection, and the period of diversification within the dominant lineage was associated with deterioration of the patient’s lung function. Identifying traits under strong selection during chronic infection not only sheds new light onto Burkholderia evolution but also sets the stage for tailored therapeutics targeting the prevailing lineages associated with disease progression. ABSTRACT Burkholderia multivorans is an opportunistic pathogen capable of causing severe disease in patients with cystic fibrosis (CF). Patients may be chronically infected for years, during which the bacterial population evolves in response to unknown forces. Here we analyze the genomic and functional evolution of a B. multivorans infection that was sequentially sampled from a CF patient over 20 years. The population diversified into at least four primary, coexisting clades with distinct evolutionary dynamics. The average substitution rate was only 2.4 mutations/year, but notably, some lineages evolved more slowly, whereas one diversified more rapidly by mostly nonsynonymous mutations. Ten loci, mostly involved in gene expression regulation and lipid metabolism, acquired three or more independent mutations and define likely targets of selection. Further, a broad range of phenotypes changed in association with the evolved mutations; they included antimicrobial resistance, biofilm regulation, and the presentation of lipopolysaccharide O-antigen repeats, which was directly caused by evolved mutations. Additionally, early isolates acquired mutations in genes involved in cyclic di-GMP (c-di-GMP) metabolism that associated with increased c-di-GMP intracellular levels. Accordingly, these isolates showed lower motility and increased biofilm formation and adhesion to CFBE41o− epithelial cells than the initial isolate, and each of these phenotypes is an important trait for bacterial persistence. The timing of the emergence of this clade of more adherent genotypes correlated with the period of greatest decline in the patient’s lung function. All together, our observations suggest that selection on B. multivorans populations during long-term colonization of CF patient lungs either directly or indirectly targets adherence, metabolism, and changes in the cell envelope related to adaptation to the biofilm lifestyle. IMPORTANCE Bacteria may become genetically and phenotypically diverse during long-term colonization of cystic fibrosis (CF) patient lungs, yet our understanding of within-host evolutionary processes during these infections is lacking. Here we combined current genome sequencing technologies and detailed phenotypic profiling of the opportunistic pathogen Burkholderia multivorans using sequential isolates sampled from a CF patient over 20 years. The evolutionary history of these isolates highlighted bacterial genes and pathways that were likely subject to strong selection within the host and were associated with altered phenotypes, such as biofilm production, motility, and antimicrobial resistance. Importantly, multiple lineages coexisted for years or even decades within the infection, and the period of diversification within the dominant lineage was associated with deterioration of the patient’s lung function. Identifying traits under strong selection during chronic infection not only sheds new light onto Burkholderia evolution but also sets the stage for tailored therapeutics targeting the prevailing lineages associated with disease progression.

Comparative Transcriptomic Analysis of the Burkholderia cepacia Tyrosine Kinase bceF Mutant Reveals a Role in Tolerance to Stress, Biofilm Formation, and Virulence

ABSTRACT The bacterial tyrosine-kinase (BY-kinase) family comprises the major group of bacterial enzymes endowed with tyrosine kinase activity. We previously showed that the BceF protein from Burkholderia cepacia IST408 belongs to this BY-kinase family and is involved in the biosynthesis of the exopolysaccharide cepacian. However, little is known about the extent of regulation of this protein kinase activity. In order to examine this regulation, we performed a comparative transcriptome profile between the bceF mutant and wild-type B. cepacia IST408. The analyses led to identification of 630 genes whose expression was significantly changed. Genes with decreased expression in the bceF mutant were related to stress response, motility, cell adhesion, and carbon and energy metabolism. Genes with increased expression were related to intracellular signaling and lipid metabolism. Mutation of bceF led to reduced survival under heat shock and UV light exposure, reduced swimming motility, and alteration in biofilm architecture when grown in vitro. Consistent with some of these phenotypes, the bceF mutant demonstrated elevated levels of cyclic-di-GMP. Furthermore, BceF contributed to the virulence of B. cepacia for larvae of the Greater wax moth, Galleria mellonella. Taken together, BceF appears to play a considerable role in many cellular processes, including biofilm formation and virulence. As homologues of BceF occur in a number of pathogenic and plant-associated Burkholderia strains, the modulation of bacterial behavior through tyrosine kinase activity is most likely a widely occurring phenomenon.

Stress Conditions Triggering Mucoid Morphotype Variation in Burkholderia Species and Effect on Virulence in Galleria mellonella and Biofilm Formation In Vitro

Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens causing chronic respiratory infections particularly among cystic fibrosis patients. During these chronic infections, mucoid-to-nonmucoid morphotype variation occurs, with the two morphotypes exhibiting different phenotypic properties. Here we show that in vitro, the mucoid clinical isolate Burkholderia multivorans D2095 gives rise to stable nonmucoid variants in response to prolonged stationary phase, presence of antibiotics, and osmotic and oxidative stresses. Furthermore, in vitro colony morphotype variation within other members of the Burkholderia genus occurred in Bcc and non-Bcc strains, irrespectively of their clinical or environmental origin. Survival to starvation and iron limitation was comparable for the mucoid parental isolate and the respective nonmucoid variant, while susceptibility to antibiotics and to oxidative stress was increased in the nonmucoid variants. Acute infection of Galleria mellonella larvae showed that, in general, the nonmucoid variants were less virulent than the respective parental mucoid isolate, suggesting a role for the exopolysaccharide in virulence. In addition, most of the tested nonmucoid variants produced more biofilm biomass than their respective mucoid parental isolate. As biofilms are often associated with increased persistence of pathogens in the CF lungs and are an indicative of different cell-to-cell interactions, it is possible that the nonmucoid variants are better adapted to persist in this host environment.

The Tyrosine Kinase BceF and the Phosphotyrosine Phosphatase BceD of Burkholderia contaminans Are Required for Efficient Invasion and Epithelial Disruption of a Cystic Fibrosis Lung Epithelial Cell Line

ABSTRACT Bacterial tyrosine kinases and their cognate protein tyrosine phosphatases are best known for regulating the biosynthesis of polysaccharides. Moreover, their roles in the stress response, DNA metabolism, cell division, and virulence have also been documented. The aim of this study was to investigate the pathogenicity and potential mechanisms of virulence dependent on the tyrosine kinase BceF and phosphotyrosine phosphatase BceD of the cystic fibrosis opportunistic pathogen Burkholderia contaminans IST408. The insertion mutants bceD::Tp and bceF::Tp showed similar attenuation of adhesion and invasion of the cystic fibrosis lung epithelial cell line CFBE41o- compared to the parental strain B. contaminans IST408. In the absence of bceD or bceF genes, B. contaminans also showed a reduction in the ability to translocate across polarized epithelial cell monolayers, demonstrated by a higher transepithelial electrical resistance, reduced flux of fluorescein isothiocyanate-labeled bovine serum albumin, and higher levels of tight junction proteins ZO-1, occludin, and claudin-1 present in monolayers exposed to these bacterial mutants. Furthermore, bceD::Tp and bceF::Tp mutants induced lower levels of interleukin-6 (IL-6) and IL-8 release than the parental strain. In conclusion, although the mechanisms of pathogenicity dependent on BceD and BceF are not understood, these proteins contribute to the virulence of Burkholderia by enhancement of cell attachment and invasion, disruption of epithelial integrity, and modulation of the proinflammatory response.

Draft Genome Sequences of Two Burkholderia multivorans Sequential Isolates from a Chronic Lung Infection of a Cystic Fibrosis Patient

ABSTRACT Burkholderia multivorans belongs to the Burkholderia cepacia complex, which comprises opportunistic pathogens infecting cystic fibrosis (CF) patients. Here, we report the genome sequences and annotations of two sequential B. multivorans clinical isolates (D2095 and D2214) displaying different traits. The differences in the genomic contents of these isolates may provide clues regarding the evolution of B. multivorans within the airways of a CF patient.

Differences in Virulence Between Legionella pneumophila Isolates From Human and Non-human Sources Determined in Galleria mellonella Infection Model

Legionella pneumophila is a ubiquitous bacterium in freshwater environments and in many man-made water systems capable of inducing pneumonia in humans. Despite its ubiquitous character most studies on L. pneumophila virulence focused on clinical strains and isolates from man-made environments, so little is known about the nature and extent of virulence variation in strains isolated from natural environments. It has been established that clinical isolates are less diverse than man-made and natural environmental strains, suggesting that only a subset of environmental isolates is specially adapted to infect humans. In this work we intended to determine if unrelated L. pneumophila strains, isolated from different environments and with distinct virulence-related genetic backgrounds, displayed differences in virulence, using the Wax Moth Galleria mellonella infection model. We found that all tested strains were pathogenic in G. mellonella, regardless of their origin. Indeed, a panoply of virulence-related phenotypes was observed sustaining the existence of significant differences on the ability of L. pneumophila strains to induce disease. Taken together our results suggest that the occurrence of human infection is not related with the increased capability of some strains to induce disease since we also found a concentration threshold above which L. pneumophila strains are equally able to cause disease. In addition, no link could be established between the sequence-type (ST) and L. pneumophila pathogenicity. We envision that in man-made water distribution systems environmental filtering selection and biotic competition acts structuring L. pneumophila populations by selecting more resilient and adapted strains that can rise to high concentration if no control measures are implemented. Therefore, public health strategies based on the sequence based typing (STB) scheme analysis should take into account that the major disease-associated clones of L. pneumophila were not related with higher virulence in G. mellonella infection model, and that potential variability of virulence-related phenotypes was found within the same ST.

Mechanisms Controlling the Expression of the Exopolysaccharide of Burkholderia and Role in Niche Adaptation

Bacteria from the genus Burkholderia are widespread in nature, with strains being isolated from rhizosphere, aquatic environments, man-made environments and in association with hosts causing disease or beneficial interactions. One common feature across the genus is the ability to produce an exopolysaccharide (EPS) termed cepacian. This ability is confirmed by the presence of the bce biosynthetic genes in all Burkholderia sequenced genomes with the exception for Burkholderia mallei (Ferreira et al., 2010). Cepacian is composed of a branched acetylated heptasaccharide repeat-unit with D-glucose, D-rhamnose, D-mannose, D-galactose and D-glucuronic acid in the ratio of 1:1:1:3:1 (Cescutti et al., 2000). Cepacian biosynthesis starts with the synthesis of the nucleotide sugar precursors, catalyzed by isomerases, mutases, epimerases, among other enzymes (Ferreira et al., 2010). This step is followed by the assembly of the repeat-units by the sequential addition of sugars to an isoprenoid lipid by dedicated glycosyltransferases. The last step of cepacian biosynthesis comprises polymerization and export of the polysaccharide to the extracellular environment. A multienzyme complex including a repeat-unit translocase, a polysaccharide polymerase, an outer membrane protein, among others are involved in this process (Ferreira et al., 2010; Moreira et al., 2003). Insights into the biosynthetic steps will be covered in this chapter. Within the genus Burkholderia, a particular group of strains belonging to the so called Burkholderia cepacia complex (Bcc) have been isolated from the lungs of patients suffering of cystic fibrosis (CF). The analysis of sequential isolates from the same patient revealed that during the bacterial process of lung infection and colonization, a transition between mucoid to nonmucoid phenotype occurs (Zlosnik et al., 2008). This phenotypic conversion is predominantly from mucoid to nonmucoid and the relevance of it in disease progression is still under debate. It is therefore of importance not only to understand the factors triggering the mucoid phenotype conversion, but also the molecular mechanisms involved in cepacian biosynthesis regulation. Concerning EPS biosynthesis regulation, the present knowledge includes regulation at transcriptional level, namely through quorum sensing mechanisms. Indeed, it is known that exopolysaccharide production in plant-associated Burkholderia is regulated by N-acyl homoserine lactones (Suarez-Moreno et al., 2010). Another Burkholderia gene recently characterized and implicated in cepacian production is hfq encoding a RNA chaperone involved in the riboregulation of target mRNAs by small regulatory non-coding