Elio Rossi

The immunosuppressive drug azathioprine inhibits biosynthesis of the bacterial signal molecule cyclic-di-GMP by interfering with intracellular nucleotide pool availability

In Gram-negative bacteria, production of the signal molecule c-di-GMP by diguanylate cyclases (DGCs) is a key trigger for biofilm formation, which, in turn, is often required for the development of chronic bacterial infections. Thus, DGCs represent interesting targets for new chemotherapeutic drugs with anti-biofilm activity. We searched for inhibitors of the WspR protein, a Pseudomonas aeruginosa DGC involved in biofilm formation and production of virulence factors, using a set of microbiological assays developed in an Escherichia coli strain expressing the wspR gene. We found that azathioprine, an immunosuppressive drug used in the treatment of Crohn’s disease, was able to inhibit WspR-dependent c-di-GMP biosynthesis in bacterial cells. However, in vitro enzymatic assays ruled out direct inhibition of WspR DGC activity either by azathioprine or by its metabolic derivative 2-amino-6-mercapto-purine riboside. Azathioprine is an inhibitor of 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase, an enzyme involved in purine biosynthesis, which suggests that inhibition of c-di-GMP biosynthesis by azathioprine may be due to perturbation of intracellular nucleotide pools. Consistent with this hypothesis, WspR activity is abolished in an E. coli purH mutant strain, unable to produce AICAR transformylase. Despite its effect on WspR, azathioprine failed to prevent biofilm formation by P. aeruginosa; however, it affected production of extracellular structures in E. coli clinical isolates, suggesting efficient inhibition of c-di-GMP biosynthesis in this bacterium. Our results indicate that azathioprine can prevent biofilm formation in E. coli through inhibition of c-di-GMP biosynthesis and suggest that such inhibition might contribute to its anti-inflammatory activity in Crohn’s disease.

The Pyrimidine Nucleotide Biosynthetic Pathway Modulates Production of Biofilm Determinants in Escherichia coli

Bacteria are often found in multicellular communities known as biofilms, which constitute a resistance form against environmental stresses. Extracellular adhesion and cell aggregation factors, responsible for bacterial biofilm formation and maintenance, are tightly regulated in response to physiological and environmental cues. We show that, in Escherichia coli, inactivation of genes belonging to the de novo uridine monophosphate (UMP) biosynthetic pathway impairs production of curli fibers and cellulose, important components of the bacterial biofilm matrix, by inhibiting transcription of the csgDEFG operon, thus preventing production of the biofilm master regulator CsgD protein. Supplementing growth media with exogenous uracil, which can be converted to UMP through the pyrimidine nucleotide salvage pathway, restores csgDEFG transcription and curli production. In addition, however, exogenous uracil triggers cellulose production, particularly in strains defective in either carB or pyrB genes, which encode enzymes catalyzing the first steps of de novo UMP biosynthesis. Our results indicate the existence of tight and complex links between pyrimidine metabolism and curli/cellulose production: transcription of the csgDEFG operon responds to pyrimidine nucleotide availability, while cellulose production is triggered by exogenous uracil in the absence of active de novo UMP biosynthesis. We speculate that perturbations in the UMP biosynthetic pathways allow the bacterial cell to sense signals such as starvation, nucleic acids degradation, and availability of exogenous pyrimidines, and to adapt the production of the extracellular matrix to the changing environmental conditions.

An efficient rRNA removal method for RNA sequencing in GC-rich bacteria

BackgroundNext generation sequencing (NGS) technologies have revolutionized gene expression studies and functional genomics analysis. However, further improvement of RNA sequencing protocols is still desirable, in order to reduce NGS costs and to increase its accuracy. In bacteria, a major problem in RNA sequencing is the abundance of ribosomal RNA (rRNA), which accounts for 95-98% of total RNA and can therefore hinder sufficient coverage of mRNA, the main focus of transcriptomic studies. Thus, efficient removal of rRNA is necessary to achieve optimal coverage, good detection sensitivity and reliable results. An additional challenge is presented by microorganisms with GC-rich genomes, in which rRNA removal is less efficient.ResultsIn this work, we tested two commercial kits for rRNA removal, either alone or in combination, on Burkholderia thailandensis. This bacterium, chosen as representative of the important Burkholderia genus, which includes both pathogenic and environmental bacteria, has a rather large (6.72 Mb) and GC-rich (67.7%) genome. Each enriched mRNA sample was sequenced through paired-end Illumina GAIIx run in duplicate, yielding between 10 and 40 million reads. We show that combined treatment with both kits allows an mRNA enrichment of more than 238-fold, enabling the sequencing of almost all (more than 90%) B. thailandensis transcripts from less than 10 million reads, without introducing any bias in mRNA relative abundance, thus preserving differential expression profile.ConclusionsThe mRNA enrichment protocol presented in this work leads to an increase in detection sensitivity up to 770% compared to total RNA; such increased sensitivity allows for a corresponding reduction in the number of sequencing reads necessary for the complete analysis of whole transcriptome expression profiling. Thus we can conclude that the MICROBExpress/Ovation combined rRNA removal method could be suitable for RNA sequencing of whole transcriptomes of microorganisms with high GC content and complex genomes enabling at the same time an important scaling down of sequencing costs.

PERSISTENCE OF CIRCULATING HBsAg/IgM COMPLEXES IN ACUTE VIRAL HEPATITIS, TYPE B: AN EARLY MARKER OF CHRONIC EVOLUTION

Serial serum samples from 110 patients with acute viral hepatitis type B were tested for HBsAg/IgM complexes by a newly developed solid-phase radioimmunoassay. In 102 patients the infection resolved and they recovered from the disease. In these patients, HBsAg/IgM complexes were either absent from the outset of disappeared from serum within four weeks of admission, long before HBsAg had cleared or serum alanine aminotransferase had returned to normal, 8 patients progressed to chronic HBsAg carrier state and chronic liver disease. In these patients, HBsAg/IgM complexes were detectable in the serum on admission, and never disappeared. These results indicate that persistence of circulating complexes containing HBsAg and IgM after the early phase of acute viral hepatitis type B is a predictor of disease chronicity. As early as the fifth week of illness those in whom chronic liver disease developed could be distinguished from those who recovered.

Gene and protein expression in response to different growth temperatures and oxygen availability in Burkholderia thailandensis.

Burkholderia thailandensis, although normally avirulent for mammals, can infect macrophages in vitro and has occasionally been reported to cause pneumonia in humans. It is therefore used as a model organism for the human pathogen B. pseudomallei, to which it is closely related phylogenetically. We characterized the B. thailandensis clinical isolate CDC2721121 (BtCDC272) at the genome level and studied its response to environmental cues associated with human host colonization, namely, temperature and oxygen limitation. Effects of the different growth conditions on BtCDC272 were studied through whole genome transcription studies and analysis of proteins associated with the bacterial cell surface. We found that growth at 37°C, compared to 28°C, negatively affected cell motility and flagella production through a mechanism involving regulation of the flagellin-encoding fliC gene at the mRNA stability level. Growth in oxygen-limiting conditions, in contrast, stimulated various processes linked to virulence, such as lipopolysaccharide production and expression of genes encoding protein secretion systems. Consistent with these observations, BtCDC272 grown in oxygen limitation was more resistant to phagocytosis and strongly induced the production of inflammatory cytokines from murine macrophages. Our results suggest that, while temperature sensing is important for regulation of B. thailandensis cell motility, oxygen limitation has a deeper impact on its physiology and constitutes a crucial environmental signal for the production of virulence factors.

Sulfate assimilation pathway intermediate phosphoadenosine 59-phosphosulfate acts as a signal molecule affecting production of curli fibres in Escherichia coli.

The enterobacterium Escherichia coli can utilize a variety of molecules as sulfur sources, including cysteine, sulfate, thiosulfate and organosulfonates. An intermediate of the sulfate assimilation pathway, adenosine 59-phosphosulfate (APS), also acts as a signal molecule regulating the utilization of different sulfur sources. In this work, we show that inactivation of the cysH gene, leading to accumulation of phosphoadenosine 59-phosphosulfate (PAPS), also an intermediate of the sulfate assimilation pathway, results in increased surface adhesion and cell aggregation by activating the expression of the curli-encoding csgBAC operon. In contrast, curli production was unaffected by the inactivation of any other gene belonging to the sulfate assimilation pathway. Overexpression of the cysH gene downregulated csgBAC transcription, further suggesting a link between intracellular PAPS levels and curli gene expression. In addition to curli components, the Flu, OmpX and Slp proteins were also found in increased amounts in the outer membrane compartment of the cysH mutant; deletion of the corresponding genes suggested that these proteins also contribute to surface adhesion and cell surface properties in this strain. Our results indicate that, similar to APS, PAPS also acts as a signal molecule, albeit with a distinct mechanism and role: whilst APS regulates organosulfonate utilization, PAPS would couple availability of sulfur sources to remodulation of the cell surface, as part of a more global effect on cell physiology.

Interplay of the modified nucleotide phosphoadenosine 5′-phosphosulfate (PAPS) with global regulatory proteins in Escherichia coli: modulation of cyclic AMP (cAMP)-dependent gene expression and interaction with the HupA regulatory protein

In the bacterium Escherichia coli, some intermediates of the sulfate assimilation and cysteine biosynthesis pathway can act as signal molecules and modulate gene expression. In addition to sensing and utilization of sulphur sources, these signaling mechanisms also impact more global cell processes, such as resistance to antimicrobial agents and biofilm formation. In a recent work, we have shown that inactivation of the cysH gene, encoding phosphoadenosine-phosphosulfate (PAPS) reductase, and the consequent increase in intracellular PAPS concentration, strongly affect production of several cell surface-associated structures, enhancing surface adhesion and cell aggregation. In order to identify the molecular mechanism relaying intracellular PAPS concentration to regulation of cell surface-associated structures, we looked for mutations able to suppress the effects of cysH inactivation. We found that mutations in the adenylate cyclase-encoding cyaA gene abolished the effects of PAPS accumulation; consistent with this result, cyclic AMP (cAMP)-dependent gene expression appears to be increased in the cysH mutant. Experiments aimed at the direct identification of proteins interacting with either CysC or CysH, i.e. the PAPS-related proteins APS kinase and PAPS reductase, allowed us to identify several regulators, namely, CspC, CspE, HNS and HupA. Protein-protein interaction between HupA and CysH was confirmed by a bacterial two hybrid system, and inactivation of the hupA gene enhanced the effects of the cysH mutation in terms of production of cell surface-associated factors. Our results indicate that PAPS can modulate different regulatory systems, providing evidence that this molecule acts as a global signal molecule in E. coli.

“It’s a gut feeling” – Escherichia coli biofilm formation in the gastrointestinal tract environment

Abstract Escherichia coli can commonly be found, either as a commensal, probiotic or a pathogen, in the human gastrointestinal (GI) tract. Biofilm formation and its regulation is surprisingly variable, although distinct regulatory pattern of red, dry and rough (rdar) biofilm formation arise in certain pathovars and even clones. In the GI tract, environmental conditions, signals from the host and from commensal bacteria contribute to shape E. coli biofilm formation within the multi-faceted multicellular communities in a complex and integrated fashion. Although some major regulatory networks, adhesion factors and extracellular matrix components constituting E. coli biofilms have been recognized, these processes have mainly been characterized in vitro and in the context of interaction of E. coli strains with intestinal epithelial cells. However, direct observation of E. coli cells in situ, and the vast number of genes encoding surface appendages on the core or accessory genome of E. coli suggests the complexity of the biofilm process to be far from being fully understood. In this review, we summarize biofilm formation mechanisms of commensal, probiotic and pathogenic E. coli in the context of the gastrointestinal tract. Graphical Abstract

Glucose availability enhances lipopolysaccharide production and immunogenicity in the opportunistic pathogen Acinetobacter baumannii

AIM Acinetobacter baumannii can cause sepsis with high mortality rates. We investigated whether glucose sensing might play a role in A. baumannii pathogenesis. MATERIALS & METHODS We carried out transcriptome analysis and extracellular polysaccharide determination in an A. baumannii clinical isolate grown on complex medium with or without glucose supplementation, and assessed its ability to induce production of inflammatory cytokines in human macrophages. RESULTS Growth in glucose-supplemented medium strongly enhanced A. baumannii sugar anabolism, resulting in increasing lipopolysaccharide biosynthesis. In addition, glucose induced active shedding of lipopolysaccharide, in turn triggering a strong induction of inflammatory cytokines in human macrophages. Finally, hemolytic activity was strongly enhanced by growth in glucose-supplemented medium. CONCLUSION We propose that sensing of exogenous glucose might trigger A. baumannii pathogenesis during sepsis.

Cellulose production is coupled to sensing of the pyrimidine biosynthetic pathway via c‐di‐GMP production by the DgcQ protein of Escherichia coli

Production of cellulose, a stress response-mediated process in enterobacteria, is modulated in Escherichia coli by the activity of the two pyrimidine nucleotide biosynthetic pathways, namely, the de novo biosynthetic pathway and the salvage pathway, which relies on the environmental availability of pyrimidine nitrogenous bases. We had previously reported that prevalence of the salvage over the de novo pathway triggers cellulose production via synthesis of the second messenger c-di-GMP by the DgcQ (YedQ) diguanylate cyclase. In this work, we show that DgcQ enzymatic activity is enhanced by UTP, whilst being inhibited by N-carbamoyl-aspartate, an intermediate of the de novo pathway. Thus, direct allosteric control by these ligands allows full DgcQ activity exclusively in cells actively synthesizing pyrimidine nucleotides via the salvage pathway. Inhibition of DgcQ activity by N-carbamoyl-aspartate appears to be favoured by protein-protein interaction between DgcQ and PyrB, a subunit of aspartate transcarbamylase, which synthesizes N-carbamoyl-aspartate. Our results suggest that availability of pyrimidine bases might be sensed, somehow paradoxically, as an environmental stress by E. coli. We hypothesize that this link might have evolved since stress events, leading to extensive DNA/RNA degradation or lysis of neighbouring cells, can result in increased pyrimidine concentrations and activation of the salvage pathway.