Marie-Christine Groleau

The various lifestyles of the Burkholderia cepacia complex species: a tribute to adaptation

The Burkholderia cepacia complex (Bcc) is composed of 17 closely related species. These bacteria are widely but heterogeneously distributed in the natural environment, such as soil, water and rhizosphere. Bcc strains are able to colonize various ecological niches by adopting versatile lifestyles, including saprophytism and (positive or deleterious) association with eukaryotic cells. Bcc strains have proven to be very efficient in biocontrol, plant growth promotion and bioremediation. However, they also are important opportunistic pathogens that can cause severe respiratory infections among individuals suffering from cystic fibrosis or chronic granulomatous disease. Therefore, considering that the distinction between plant beneficial and clinical strains is not obvious, biotechnological applications of Bcc strains are currently not allowed. This minireview provides an overview of the wide range of lifestyles that Bcc bacteria can adopt, leading to glimpses into their tremendous adaptation potential and highlighting remaining questions concerning potential implicated mechanisms.

The End of an Old Hypothesis: The Pseudomonas Signaling Molecules 4-Hydroxy-2-Alkylquinolines Derive from Fatty Acids, Not 3-Ketofatty Acids

Groups of pathogenic bacteria use diffusible signals to regulate their virulence in a concerted manner. Pseudomonas aeruginosa uses 4-hydroxy-2-alkylquinolines (HAQs), including 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), as unique signals. We demonstrate that octanoic acid is directly incorporated into HHQ. This finding rules out the long-standing hypothesis that 3-ketofatty acids are the precursors of HAQs. We found that HAQ biosynthesis, which requires the PqsABCD enzymes, proceeds by a two-step pathway: (1) PqsD mediates the synthesis of 2-aminobenzoylacetate (2-ABA) from anthraniloyl-coenzyme A (CoA) and malonyl-CoA, then (2) the decarboxylating coupling of 2-ABA to an octanoate group linked to PqsC produces HHQ, the direct precursor of PQS. PqsB is tightly associated with PqsC and required for the second step. This finding uncovers promising targets for the development of specific antivirulence drugs to combat this opportunistic pathogen.

Vfm a new quorum sensing system controls the virulence of Dickeya dadantii

Dickeya dadantii is a plant pathogen that secretes cell wall-degrading enzymes (CWDE) that are responsible for soft-rot symptoms. Virulence genes are expressed in a concerted manner and culminate when bacterial multiplication slows. We identify a 25 kb vfm cluster required for D. dadantii CWDE production and pathogenesis. The vfm cluster encodes proteins displaying similarities both with enzymes involved in amino acid activation and with enzymes involved in fatty acid biosynthesis. These similarities suggest that the vfm genes direct the production of a metabolite. Cell-free supernatant from the D. dadantii wild-type strain restores CWDE production in vfm mutants. Collectively, our results indicate that vfm genes direct the synthesis of an extracellular signal and constitute a new quorum sensing system. Perception of the signal is achieved by the two-component system VfmH-VfmI, which activates the expression of the vfmE gene encoding an AraC regulator. VfmE then activates both the transcription of the CWDE genes and the expression of the vfm operons. The vfm gene cluster does not seem to be widespread among bacterial species but is conserved in other Dickeya species and could have been laterally transferred to Rahnella. This work highlights that entirely new families of bacterial languages remain to be discovered.

Phase variation has a role in Burkholderia ambifaria niche adaptation

Members of the Burkholderia cepacia complex (Bcc), such as B. ambifaria, are effective biocontrol strains, for instance, as plant growth-promoting bacteria; however, Bcc isolates can also cause severe respiratory infections in people suffering from cystic fibrosis (CF). No distinction is known between isolates from environmental and human origins, suggesting that the natural environment is a potential source of infectious Bcc species. While investigating the presence and role of phase variation in B. ambifaria HSJ1, an isolate recovered from a CF patient, we identified stable variants that arose spontaneously irrespective of the culture conditions. Phenotypic and proteomic approaches revealed that the transition from wild-type to variant types affects the expression of several putative virulence factors. By using four different infection models (Drosophila melanogaster, Galleria mellonella, macrophages and Dictyostelium discoideum), we showed that the wild-type was more virulent than the variant. It may be noted that the variant showed reduced replication in a human monocyte cell line when compared with the wild-type. On the other hand, the variant of isolate HSJ1 was more competitive in colonizing plant roots than the wild-type. Furthermore, we observed that only clinical B. ambifaria isolates generated phase variants, and that these variants showed the same phenotypes as observed with the HSJ1 variant. Finally, we determined that environmental B. ambifaria isolates showed traits that were characteristic of variants derived from clinical isolates. Our study therefore suggest that B. ambifaria uses phase variation to adapt to drastically different environments: the lung of patients with CF or the rhizosphere.

LasR Variant Cystic Fibrosis Isolates Reveal an Adaptable Quorum-Sensing Hierarchy in Pseudomonas aeruginosa

ABSTRACT Chronic Pseudomonas aeruginosa infections cause significant morbidity in patients with cystic fibrosis (CF). Over years to decades, P. aeruginosa adapts genetically as it establishes chronic lung infections. Nonsynonymous mutations in lasR, the quorum-sensing (QS) master regulator, are common in CF. In laboratory strains of P. aeruginosa, LasR activates transcription of dozens of genes, including that for another QS regulator, RhlR. Despite the frequency with which lasR coding variants have been reported to occur in P. aeruginosa CF isolates, little is known about their consequences for QS. We sequenced lasR from 2,583 P. aeruginosa CF isolates. The lasR sequences of 580 isolates (22%) coded for polypeptides that differed from the conserved LasR polypeptides of well-studied laboratory strains. This collection included 173 unique lasR coding variants, 116 of which were either missense or nonsense mutations. We studied 31 of these variants. About one-sixth of the variant LasR proteins were functional, including 3 with nonsense mutations, and in some LasR-null isolates, genes that are LasR dependent in laboratory strains were nonetheless expressed. Furthermore, about half of the LasR-null isolates retained RhlR activity. Therefore, in some CF isolates the QS hierarchy is altered such that RhlR quorum sensing is independent of LasR regulation. Our analysis challenges the view that QS-silent P. aeruginosa is selected during the course of a chronic CF lung infection. Rather, some lasR sequence variants retain functionality, and many employ an alternate QS strategy involving RhlR. IMPORTANCE Chronic Pseudomonas aeruginosa infections, such as those in patients with the genetic disease cystic fibrosis, are notable in that mutants with defects in the quorum-sensing transcription factor LasR frequently arise. In laboratory strains of P. aeruginosa, quorum sensing activates transcription of dozens of genes, many of which encode virulence factors, such as secreted proteases and hydrogen cyanide synthases. In well-studied laboratory strains, LasR-null mutants have a quorum-sensing-deficient phenotype. Therefore, the presence of LasR variants in chronic infections has been interpreted to indicate that quorum-sensing-regulated products are not important for those infections. We report that some P. aeruginosa LasR variant clinical isolates are not LasR-null mutants, and others have uncoupled a second quorum-sensing system, the RhlR system, from LasR regulation. In these uncoupled isolates, RhlR independently activates at least some quorum-sensing-dependent genes. Our findings suggest that quorum sensing plays a role in chronic P. aeruginosa infections, despite the emergence of LasR coding variants. Chronic Pseudomonas aeruginosa infections, such as those in patients with the genetic disease cystic fibrosis, are notable in that mutants with defects in the quorum-sensing transcription factor LasR frequently arise. In laboratory strains of P. aeruginosa, quorum sensing activates transcription of dozens of genes, many of which encode virulence factors, such as secreted proteases and hydrogen cyanide synthases. In well-studied laboratory strains, LasR-null mutants have a quorum-sensing-deficient phenotype. Therefore, the presence of LasR variants in chronic infections has been interpreted to indicate that quorum-sensing-regulated products are not important for those infections. We report that some P. aeruginosa LasR variant clinical isolates are not LasR-null mutants, and others have uncoupled a second quorum-sensing system, the RhlR system, from LasR regulation. In these uncoupled isolates, RhlR independently activates at least some quorum-sensing-dependent genes. Our findings suggest that quorum sensing plays a role in chronic P. aeruginosa infections, despite the emergence of LasR coding variants.

Interplay between 4-Hydroxy-3-Methyl-2-Alkylquinoline and N-Acyl-Homoserine Lactone Signaling in a Burkholderia cepacia Complex Clinical Strain

Species from the Burkholderia cepacia complex (Bcc) share a canonical LuxI/LuxR quorum sensing (QS) regulation system named CepI/CepR, which mainly relies on the acyl-homoserine lactone (AHL), octanoyl-homoserine lactone (C8-HSL) as signaling molecule. Burkholderia ambifaria is one of the least virulent Bcc species, more often isolated from rhizospheres where it exerts a plant growth-promoting activity. However, clinical strains of B. ambifaria display distinct features, such as phase variation and higher virulence properties. Notably, we previously reported that under laboratory conditions, only clinical strains of the B. ambifaria species produced 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs) via expression of the hmqABCDEFG operon. HMAQs are the methylated counterparts of the 4-hydroxy-2-alkylquinolines (HAQs) produced by the opportunistic human pathogen Pseudomonas aeruginosa, in which they globally contribute to the bacterial virulence and survival. We have found that unlike P. aeruginosa’s HAQs, HMAQs do not induce their own production. However, they indirectly regulate the expression of the hmqABCDEFG operon. In B. ambifaria, a strong link between CepI/CepR-based QS and HMAQs is proposed, as we have previously reported an increased production of C8-HSL in HMAQ-negative mutants. Here, we report the identification of all AHLs produced by the clinical B. ambifaria strain HSJ1, namely C6-HSL, C8-HSL, C10-HSL, 3OHC8-HSL, 3OHC10-HSL, and 3OHC12-HSL. Production of significant levels of hydroxylated AHLs prompted the identification of a second complete LuxI/LuxR-type QS system relying on 3OHC10-HSL and 3OHC12-HSL, that we have named CepI2/CepR2. The connection between these two QS systems and the hmqABCDEFG operon, responsible for HMAQs biosynthesis, was investigated. The CepI/CepR system strongly induced the operon, while the second system appears moderately involved. On the other hand, a HMAQ-negative mutant overproduces AHLs from both QS systems. Even if HMAQs are not classical QS signals, their effect on AHL-based QS system still gives them a part to play in the QS circuitry in B. ambifaria and thus, on regulation of various phenotypes.

Genomic characterisation of environmental Pseudomonas aeruginosa isolated from dental unit waterlines revealed the insertion sequence ISPa11 as a chaotropic element

ABSTRACT The bacterium Pseudomonas aeruginosa is well known to have a remarkable adaptive capacity allowing it to colonise many environments. A recent study on environmental isolates from dental unit waterlines (DUWLs) hinted at a genetic clustering into two groups. Isolates from one of these groups, named cluster III, were shown to have unusual phenotypes for environmental isolates, such as an increased biofilm production. To have a better ecological view, more specifically on isolates from cluster III, the complete genomes of 39 isolates including 16 from DUWLs were sequenced. In addition to an investigation of antibiotic resistance and secretion system gene content, a molecular phylogeny allowed confirmation of the split of the 16 environmental isolates in two groups and also sheds light on a correlation between the phylogenetic positions and the serotypes of the isolates. Isolates from cluster III harboured insertion sequences ISPa11 inserted into the O‐specific antigen biosynthesis clusters and the gene lasR, encoding for a master regulator of the quorum sensing. Investigation of key regulators revealed another truncated gene, gacS. Alteration in lasR and gacS genes was consistent with phenotypic assays confirming their inactivation. These results bring new perspectives regarding the ecological adaptive potential of P. aeruginosa. &NA; Graphical Abstract Figure. This study sheds light on a phylogenomics dichotomy between dental unit waterlines and clinical isolates of Pseudomonas aeruginosa.

Liquid Chromatography/Mass Spectrometry (LC/MS) for the Detection and Quantification of N-Acyl-L-Homoserine Lactones (AHLs) and 4-Hydroxy-2-Alkylquinolines (HAQs)

High-performance liquid chromatography (HPLC) coupled in-line with mass spectrometry (MS) permits rapid and specific identification and quantification of N-acyl-L-homoserine lactones (AHLs) and 4-hydroxy-2-alkylquinolines (HAQs). We are presenting here methods for the analysis of these molecules directly from biological samples using LC/MS.