Interaction d'Escherichia coli entérohémorragique (EHEC) avec Acanthamoeba castellanii et rôle du régulon Pho chez les EHEC

Les EHEC de sérotype O157:H7 sont des agents zoonotiques d’origine alimentaire ou hydrique. Ce sont des pathogènes émergeants qui causent chez l’humain des épidémies de gastro-entérite aiguë et parfois un syndrome hémolytique-urémique. Les EHEC réussissent leur transmission à l’humain à partir de leur portage commensal chez l’animal en passant par l’étape de survie dans l’environnement. L’endosymbiose microbienne est une des stratégies utilisées par les bactéries pathogènes pour survivre dans les environnements aquatiques. Les amibes sont des protozoaires vivants dans divers écosystèmes et connus pour abriter plusieurs agents pathogènes. Ainsi, les amibes contribueraient à transmettre les EHEC à l'humain. La première partie de mon projet de thèse est centrée sur l'interaction de l’amibe Acanthamoeba castellanii avec les EHEC. Les résultats montrent que la présence de cette amibe prolonge la persistance des EHEC, et ces dernières survivent à leur phagocytose par les amibes. Ces résultats démontrent le potentiel réel des amibes à héberger les EHEC et à contribuer à leur transmission. Cependant, l’absence de Shiga toxines améliore leur taux de survie intra-amibe. Par ailleurs, les Shiga toxines sont partiellement responsables de l’intoxication des amibes par les EHEC. Cette implication des Shiga toxines dans le taux de survie intracellulaire et dans la mortalité des amibes démontre l’intérêt d’utiliser les amibes comme modèle d'interaction hôte/pathogène pour étudier la pathogénicité des EHEC. Durant leur cycle de transmission, les EHEC rencontrent des carences en phosphate inorganique (Pi) dans l’environnement. En utilisant conjointement le système à deux composantes (TCS) PhoB-R et le système Pst (transport spécifique de Pi), les EHEC détectent et répondent à cette variation en Pi en activant le régulon Pho. La relation entre la virulence des EHEC, le PhoB-R-Pst et/ou le Pi environnemental demeure inconnue. La seconde partie de mon projet explore le rôle du régulon Pho (répondant à un stress nutritif de limitation en Pi) dans la virulence des EHEC. L’analyse transcriptomique montre que les EHEC répondent à la carence de Pi par une réaction complexe impliquant non seulement un remodelage du métabolisme général, qui est critique pour sa survie, mais aussi en coordonnant sa réponse de virulence. Dans ces conditions le régulateur PhoB contrôle directement l’expression des gènes du LEE et de l’opéron stx2AB. Ceci est confirmé par l’augmentation de la sécrétion de l’effecteur EspB et de la production et sécrétion de Stx2 en carence en Pi. Par ailleurs, l’activation du régulon Pho augmente la formation de biofilm et réduit la motilité chez les EHEC. Ceci corrèle avec l’induction des gènes régulant la production de curli et la répression de la voie de production d’indole et de biosynthèse du flagelle et du PGA (Polymère β-1,6-N-acétyle-D-glucosamine).

Global transcriptional responses of the toxic cyanobacterium, Microcystis aeruginosa, to nitrogen stress, phosphorus stress, and growth on organic matter

Whole transcriptome shotgun sequencing (RNA-seq) was used to assess the transcriptomic response of the toxic cyanobacterium Microcystis aeruginosa during growth with low levels of dissolved inorganic nitrogen (low N), low levels of dissolved inorganic phosphorus (low P), and in the presence of high levels of high molecular weight dissolved organic matter (HMWDOM). Under low N, one third of the genome was differentially expressed, with significant increases in transcripts observed among genes within the nir operon, urea transport genes (urtBCDE), and amino acid transporters while significant decreases in transcripts were observed in genes related to photosynthesis. There was also a significant decrease in the transcription of the microcystin synthetase gene set under low N and a significant decrease in microcystin content per Microcystis cell demonstrating that N supply influences cellular toxicity. Under low P, 27% of the genome was differentially expressed. The Pho regulon was induced leading to large increases in transcript levels of the alkaline phosphatase phoX, the Pst transport system (pstABC), and the sphX gene, and transcripts of multiple sulfate transporter were also significantly more abundant. While the transcriptional response to growth on HMWDOM was smaller (5–22% of genes differentially expressed), transcripts of multiple genes specifically associated with the transport and degradation of organic compounds were significantly more abundant within HMWDOM treatments and thus may be recruited by Microcystis to utilize these substrates. Collectively, these findings provide a comprehensive understanding of the nutritional physiology of this toxic, bloom-forming cyanobacterium and the role of N in controlling microcystin synthesis.

New ways to break an old bond: the bacterial carbon-phosphorus hydrolases and their role in biogeochemical phosphorus cycling

Phosphonates are organophosphorus molecules that contain the highly stable C-P bond, rather than the more common, and more labile, C-O-P phosphate ester bond. They have ancient origins but their biosynthesis is widespread among more primitive organisms and their importance in the contemporary biosphere is increasingly recognized; for example phosphonate-P is believed to play a particularly significant role in the productivity of the oceans. The microbial degradation of phosphonates was originally thought to occur only under conditions of phosphate limitation, mediated exclusively by the poorly characterized C-P lyase multienzyme system, under Pho regulon control. However, more recent studies have demonstrated the Pho-independent mineralization by environmental bacteria of three of the most widely distributed biogenic phosphonates: 2-aminoethylphosphonic acid (ciliatine), phosphonoacetic acid, and 2-amino-3-phosphonopropionic acid (phosphonoalanine). The three phosphonohydrolases responsible have unique specificities and are members of separate enzyme superfamilies; their expression is regulated by distinct members of the LysR family of bacterial transcriptional regulators, for each of which the phosphonate substrate of the respective degradative operon serves as coinducer. Previously no organophosphorus compound was known to induce the enzymes required for its own degradation. Whole-genome and metagenome sequence analysis indicates that the genes encoding these newly described C-P hydrolases are distributed widely among prokaryotes. As they are able to function under conditions in which C-P lyases are inactive, the three enzymes may play a hitherto-unrecognized role in phosphonate breakdown in the environment and hence make a significant contribution to global biogeochemical P-cycling.

Étude du rôle spécifique du système à deux composantes PhoBR et du système Pst (phosphate specific transport) dans la virulence d’une souche pathogène aviaire de Escherichia coli (APEC)

Les souches d’Escherichia coli pathogènes aviaires (APEC) sont responsables d’infections respiratoires et de septicémies chez la volaille. Le régulon Pho est contrôlé conjointement par le système à deux composantes PhoBR et par le système de transport spécifique du phosphate (Pst). Afin de déterminer l’implication de PhoBR et du système Pst dans la pathogenèse de la souche APEC O78 χ7122, différentes souche mutantes phoBR et pst ont été testées pour divers traits de virulence in vivo et in vitro. Les mutations menant à l’activation constitutive du régulon Pho rendaient les souches plus sensibles au peroxyde d’hydrogène et au sérum de lapin comparativement à la souche sauvage. De plus, l’expression des fimbriae de type 1 était affectée chez ces souches. L’ensemble des mutants Pho-constitutifs étaient aussi significativement moins virulents que la souche sauvage dans un modèle de coinfection de poulet, incluant les souches avec un système Pst fonctionnel. De plus, l’inactivation du régulateur PhoB chez un mutant Pst restaure la virulence. Par ailleurs, l’inactivation de PhoB n’affecte pas la virulence de la souche χ7122 dans notre modèle. De manière intéressante, le degré d’atténuation des souches mutantes corrèle directement avec le niveau d’activation du régulon Pho. Globalement, les résultats indiquent que l’activation du régulon Pho plutôt que le transport du phosphate via le système Pst joue un rôle majeur dans l’atténuation des APEC.

Étude transcriptionnelle d'une souche pathogène aviaire de Escherichia coli (APEC) et son mutant Pst (phosphate specific transport)

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

The pst operon of enteropathogenic Escherichia coli enhances bacterial adherence to epithelial cells

Enteropathogenic Escherichia coli (EPEC) adheres in vivo and in vitro to epithelial cells. Two main adhesins, the bundle-forming pilus and intimin, encoded by the Up operon and eae, respectively, are responsible for the localized and the intimate adherence phenotypes. Deletion of the pst operon of EPEC abolishes the transport of inorganic phosphate through the phosphate-specific transport system and causes the constitutive expression of the PHO regulon genes. In the absence of pst there is a decrease in the expression of the main EPEC adhesins and a reduction in bacterial adherence to epithelial cells in vitro. This effect is not related to PHO constitutivity, because a Delta pst phoB double mutant that is defective in the transcription of the PHO genes also displayed low levels of adherence and expression of adhesins. Likewise, a PHO-constitutive phoR mutation did not affect bacterial adherence. The expression of the per operon, which encodes the Up and ler regulators PerA and PerC, is also negatively affected by the pst deletion. Overall, the data presented here demonstrate that the pst operon of EPEC plays a positive role in the bacterial adherence mechanism by increasing the expression of perA and perC and consequently the transcription of bfp and eae.

Alkaline phosphatase as a reporter of sigma(S) levels and rpoS polymorphisms in different E-coli strains

sigma(S) is responsible for the transcriptional regulation of genes related to protection against stresses and bacterial survival and it accumulates in the cell under conditions of stress, such as nutrient limitation. An increase in the levels of sigma(S) causes a reduction in the expression of genes that are transcribed by RNA polymerase associated with the principal sigma factor, sigma(70). phoA, that encodes alkaline phosphatase (AP) is expressed under phosphate shortage conditions, and is also repressed by sigma(S). Here we show that in a Pi-limited chemostat, accumulation of rpoS mutations is proportional to the intrinsic level of sigma(S) in the cells. Acquisition of mutations in rpoS relieves repression of the PHO genes. We also devised a non-destructive method based on the rpoS effect on AP that differentiates between rpo(S+) and rpoS mutants, as well as between high and low-sigma(S) producers. Using this method, we provide evidence that sigma(S) contributes to the repression of AP under conditions of Pi excess and that AP variation among different strains is at least partly due to intrinsic variation in sigma(S) levels. Consequently, a simple and non-destructive AP assay can be employed to differentiate between strains expressing different levels of sigma(S) on agar plates.

Transcriptional Processing of the pst Operon of Escherichia coli

The pst operon of Escherichia coli is composed of five genes that encode a high-affinity phosphate transport system. pst belongs to the PHO regulon, which is a group of genes and operons that are induced in response to phosphate limitation. The pst operon also has a regulatory role in the repression of PHO genes` transcription under phosphate excess conditions. Transcription of pst is initiated at the promoter located upstream to the first gene, pstS. Immediately after its synthesis, the primary transcript of pst is cleaved into shorter mRNA molecules in a ribonuclease E-dependent manner. Other ribonucleases, such as RNase III and MazF, do not play a role in pst mRNA processing. RNase E is thus at least partially responsible for processing the pst primary transcript.

Stability of the pstS transcript of Escherichia coli

The pst operon of Escherichia coli is composed of five genes that encode a high-affinity phosphate transport system. As a member of the PHO regulon, pst transcription is activated under phosphate shortage conditions. Under phosphate-replete conditions, the pst operon also functions as a negative regulator of the PHO genes. Transcription of pst is initiated at the promoter located upstream to the first gene, pstS. Immediately after its synthesis, the primary transcript of pst is cleaved into shorter mRNA molecules. The transcription unit corresponding to pstS is significantly more abundant than the transcripts of the other pst genes due to stabilisation of pstS mRNA by a repetitive extragenic palindrome (REP) structure downstream to the pstS locus. The presence of the REP sequence also results in an increased level of PstS proteins. However, the surplus level of PstS proteins produced in the presence of REP does not contribute to the repressive role of Pst in PHO expression.

Alternative promoters in the pst operon of Escherichia coli

The pst operon of Escherichia coli is composed of five genes pstS, pstC, pstA, pstB and phoU, that encode a high-affinity phosphate transport system and a negative regulator of the PHO regulon. Transcription of pst is induced under phosphate shortage and is initiated at the promoter located upstream of the first gene of the operon, pstS. Here, we show by four different technical approaches the existence of additional internal promoters upstream of pstC, pstB and phoU. These promoters are not induced by Pi-limitation and do not possess PHO-box sequences. Plasmids carrying the pst internal genes partially complement chromosomal mutations in their corresponding genes, indicating that they are translated into functional proteins.

Elucidating the Regulon of the Multidrug Resistance Regulator RarA in Klebsiella pneumoniae

RarA is an AraC-type regulator in Klebsiella pneumoniae, which, when overexpressed, confers a low-level multidrug-resistant (MDR) phenotype linked to the upregulation of both the acrAB and oqxAB efflux genes. Increased rarA expression has also been shown to be integral in the development of tigecycline resistance in the absence of ramA in K. pneumoniae. Given its phenotypic role in MDR, microarray analyses were performed to determine the RarA regulon. Transcriptome analysis was undertaken using strains Ecl8?rarA/pACrarA-2 (rarA-expressing construct) and Ecl8?rarA/pACYC184 (vector-only control) using bespoke microarray slides consisting of probes derived from the genomic sequences of K. pneumoniae MGH 78578 (NC_009648.1) and Kp342 (NC_011283.1). Our results show that rarA overexpression resulted in the differential expression of 66 genes (42 upregulated and 24 downregulated). Under the COG (clusters of orthologous groups) functional classification, the majority of affected genes belonged to the category of cell envelope biogenesis and posttranslational modification, along with genes encoding the previously uncharacterized transport proteins (e.g., KPN_03141, sdaCB, and leuE) and the porin OmpF. However, genes associated with energy production and conversion and amino acid transport/metabolism (e.g., nuoA, narJ, and proWX) were found to be downregulated. Biolog phenotype analyses demonstrated that rarA overexpression confers enhanced growth of the overexpresser in the presence of several antibiotic classes (i.e., beta-lactams and fluoroquinolones), the antifungal/antiprotozoal compound clioquinol, disinfectants (8-hydroxyquinoline), protein synthesis inhibitors (i.e., minocycline and puromycin), membrane biogenesis agents (polymyxin B and amitriptyline), DNA synthesis (furaltadone), and the cytokinesis inhibitor (sanguinarine). Both our transcriptome and phenotypic microarray data support and extend the role of RarA in the MDR phenotype of K. pneumoniae.

Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation

Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.

Identification of components of the Sigma B Regulon in Listeria monocytogenes that contribute to acid and salt tolerance

Sigma B (σB) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the ΔsigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The ΔsigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that σB contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the ΔsigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of σB in L. monocytogenes, we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the ΔsigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of σB. It also demonstrated clear roles for σB in both osmotic and low-pH stress tolerance and identified specific components of the σB regulon that contribute to the responses observed.

Proteomic analyses of a Listeria monocytogenes mutant lacking σB identify new components of the σB regulon and highlight a role for σB in the utilization of glycerol

In Listeria monocytogenes the alternative sigma factor σB plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the σB regulon in L. monocytogenes 10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic ΔsigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was σB dependent; 17 of these proteins were found to require the presence of σB for full expression, while 21 were expressed at a higher level in the ΔsigB mutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for σB in the general stress response and highlighted a probable role for σB in metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of σB in glycerol metabolism. Five newly identified members of the σB regulon were shown to be under direct regulation of σB using reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four σB-dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified σB-dependent genes, as well as proteomic analyses, σB was shown to play a major role in the stationary phase of growth in complex media.