Paolo Visca

Iron transport and regulation, cell signalling and genomics: lessons from Escherichia coli and Pseudomonas

A variety of bacterial species secrete and take up chelating compounds that enable acquisition of iron (siderophores). It has become clear that a common feature in regulation of different iron acquisition systems is the involvement of alternative sigma factor proteins of the extracytoplasmic function (ECF) family. Two of these proteins, PvdS from Pseudomonas aeruginosa and FecI from Escherichia coli K‐12, have been studied extensively. PvdS directs transcription of genes required for the biosynthesis of a siderophore, pyoverdine, and FecI causes ex‐pression of genes for uptake of ferric citrate. FecI forms part of a signalling system that responds to the presence of ferric citrate. Here, we review recent advances in understanding of PvdS and of the Fec signalling system. PvdS and FecI are part of a distinct subfamily of ECF sigma factors involved in iron acquisition and hence named the iron‐starvation sigmas. Analysis of microbial genome sequences shows that Fec‐like signalling systems are present in a wide range of species and many such systems may be present in a single species. The availability of tools for large‐scale genome analysis is likely to lead to rapid advances in our understanding of this expanding family of proteins.

The response to stationary-phase stress conditions in Escherichia coli : role and regulation of the glutamic acid decarboxylase system

Inducible bacterial amino acid decarboxylases are expressed at the end of active cell division to counteract acidification of the extracellular environment during fermentative growth. It has been proposed that acid resistance in some enteric bacteria strictly relies on a glutamic acid‐dependent system. The Escherichia coli chromosome contains distinct genes encoding two biochemically identical isoforms of glutamic acid decarboxylase, GadA and GadB. The gadC gene, located downstream of gadB, has been proposed to encode a putative antiporter implicated in the export of γ‐aminobutyrate, the glutamic acid decarboxylation product. In the present work, we provide in vivo evidence that gadC is co‐transcribed with gadB and that the functional glutamic acid‐dependent system requires the activities of both GadA/B and GadC. We also found that expression of gad genes is positively regulated by acidic shock, salt stress and stationary growth phase. Mutations in hns, the gene for the histone‐like protein H‐NS, cause derepressed expression of the gad genes, whereas the rpoS mutation abrogates gad transcription even in the hns background. According to our results, the master regulators H‐NS and RpoS are hierarchically involved in the transcriptional control of gad expression: H‐NS prevents gad expression during the exponential growth whereas the alternative sigma factor RpoS relieves H‐NS repression during the stationary phase, directly or indirectly accounting for transcription of gad genes.

Whole-Genome Pyrosequencing of an Epidemic Multidrug-Resistant Acinetobacter baumannii Strain Belonging to the European Clone II Group

ABSTRACT The whole-genome sequence of an epidemic, multidrug-resistant Acinetobacter baumannii strain (strain ACICU) belonging to the European clone II group and carrying the plasmid-mediated blaOXA-58 carbapenem resistance gene was determined. The A. baumannii ACICU genome was compared with the genomes of A. baumannii ATCC 17978 and Acinetobacter baylyi ADP1, with the aim of identifying novel genes related to virulence and drug resistance. A. baumannii ACICU has a single chromosome of 3,904,116 bp (which is predicted to contain 3,758 genes) and two plasmids, pACICU1 and pACICU2, of 28,279 and 64,366 bp, respectively. Genome comparison showed 86.4% synteny with A. baumannii ATCC 17978 and 14.8% synteny with A. baylyi ADP1. A conspicuous number of transporters belonging to different superfamilies was predicted for A. baumannii ACICU. The relative number of transporters was much higher in ACICU than in ATCC 17978 and ADP1 (76.2, 57.2, and 62.5 transporters per Mb of genome, respectively). An antibiotic resistance island, AbaR2, was identified in ACICU and had plausibly evolved by reductive evolution from the AbaR1 island previously described in multiresistant strain A. baumannii AYE. Moreover, 36 putative alien islands (pAs) were detected in the ACICU genome; 24 of these had previously been described in the ATCC 17978 genome, 4 are proposed here for the first time and are present in both ATCC 17978 and ACICU, and 8 are unique to the ACICU genome. Fifteen of the pAs in the ACICU genome encode genes related to drug resistance, including membrane transporters and ex novo acquired resistance genes. These findings provide novel insight into the genetic basis of A. baumannii resistance.

Hemoglobin and heme scavenging

Release of hemoglobin into plasma is a physiological phenomenon associated with intravascular hemolysis. In plasma, stable haptoglobin‐hemoglobin complexes are formed and these are subsequently delivered to the reticulo‐endothelial system by CD163 receptor‐mediated endocytosis. Heme arising from the degradation of hemoglobin, myoglobin, and of enzymes with heme prosthetic groups could be delivered in plasma. Albumin, haptoglobin, hemopexin, and high and low density lipoproteins cooperate to trap the plasma heme, thereby ensuring its complete clearance. Then hemopexin releases the heme into hepatic parenchymal cells only after internalization of the hemopexin‐heme complex by CD91 receptor‐mediated endocytosis. Moreover, α1‐microglobulin contributes to heme degradation by a still unknown mechanism, with the concomitant formation of heterogeneous yellow‐brown kynurenine‐derived chromophores which are very tightly bound to amino acid residues close to the rim of the lipocalin pocket. During hemoglobin synthesis, the erythroid α‐chain hemoglobin‐stabilizing protein specifically binds free α‐hemoglobin subunits limiting the free protein toxicity. Although highly toxic because capable of catalyzing free radical formation, heme is also a major and readily available source of iron for pathogenic organisms. Gram‐negative bacteria pick up the heme‐bound iron through the secretion of a hemophore that takes up either free heme or heme bound to heme‐proteins and transports it to a specific receptor, which, in turn, releases the heme and hence iron into the bacterium. Here, hemoglobin and heme trapping mechanisms are summarized. IUBMB Life, 57: 749‐759, 2005

Structural genes for salicylate biosynthesis from chorismate in Pseudomonas aeruginosa

Salicylate is a precursor of pyochelin in Pseudomonas aeruginosa and both compounds display siderophore activity. To elucidate the salicylate biosynthetic pathway, we have cloned and sequenced a chromosomal region of P. aeruginosa PAO1 containing two adjacent genes, designated pchB and pchA, which are necessary for salicylate formation. The pchA gene encodes a protein of 52 kDa with extensive similarity to the chorismate-utilizing enzymes isochorismate synthase, anthranilate synthase (component I) and p-aminobenzoate synthase (component I), whereas the 11 kDa protein encoded by pchB does not show significant similarity with other proteins. The pchB stop codon overlaps the presumed pchA start codon. Expression of the pchA gene in P. aeruginosa appears to depend on the transcription and translation of the upstream pchB gene. The pchBA genes are the first salicylate biosynthetic genes to be reported. Salicylate formation was demonstrated in an Escherichia coli entC mutant lacking isochorismate synthase when this strain expressed both the pchBA genes, but not when it expressed pchB alone. By contrast, an entB mutant of E. coli blocked in the conversion of isochorismate to 2,3-dihydro-2,3-dihydroxybenzoate formed salicylate when transformed with a pchB expression construct. Salicylate formation could also be demonstrated in vitro when chorismate was incubated with a crude extract of P. aeruginosa containing overproduced PchA and PchB proteins; salicylate and pyruvate were formed in equimolar amounts. Furthermore, salicylate-forming activity could be detected in extracts from a P. aeruginosa pyoverdin-negative mutant when grown under iron limitation, but not with iron excess. Our results are consistent with a pathway leading from chorismate to isochorismate and then to salicylate plus pyruvate, catalyzed consecutively by the iron-repressible PchA and PchB proteins in P. aeruginosa.

Acinetobacter baumannii: evolution of a global pathogen.

Acinetobacter baumannii is an opportunistic nosocomial pathogen and one of the six most important multidrug-resistant microorganisms in hospitals worldwide. This human pathogen is responsible for a vast array of infections, of which ventilator-associated pneumonia and bloodstream infections are the most common, and mortality rates can reach 35%. Community-acquired infections have also been reported, but few strains have been recovered from environmental sources and infection reservoirs external to the hospital have not been identified. The majority of A. baumannii infections are caused by two main population clones with worldwide distribution. Infection outbreaks are often associated with multidrug resistance, including the recent emergence of strains resistant to all available antibiotics. Nevertheless, A. baumannii virulence traits and pathogenic potential have mostly remained elusive. The recent expansion of A. baumannii sequenced genomes has permitted the development of large-array phylogenomic and phenotypic analyses, which can offer valuable insights into the evolution and adaptation of A. baumannii as a human pathogen. This review summarises these recent advances, with particular focus on A. baumannii evolutionary and genomic aspects, and proposes new avenues of research.

Anthrax toxin: a tripartite lethal combination1

Anthrax is a severe bacterial infection that occurs when Bacillus anthracis spores gain access into the body and germinate in macrophages, causing septicemia and toxemia. Anthrax toxin is a binary A–B toxin composed of protective antigen (PA), lethal factor (LF), and edema factor (EF). PA mediates the entry of either LF or EF into the cytosol of host cells. LF is a zinc metalloprotease that inactivates mitogen‐activated protein kinase kinase inducing cell death, and EF is an adenylyl cyclase impairing host defences. Inhibitors targeting different steps of toxin activity have recently been developed. Anthrax toxin has also been exploited as a therapeutic agent against cancer.

Deciphering the Multifactorial Nature of Acinetobacter baumannii Pathogenicity

Background Acinetobacter baumannii is an emerging bacterial pathogen that causes a broad array of infections, particularly in hospitalized patients. Many studies have focused on the epidemiology and antibiotic resistance of A. baumannii, but little is currently known with respect to its virulence potential. Methodology/Principal Findings The aim of this work was to analyze a number of virulence-related traits of four A. baumannii strains of different origin and clinical impact for which complete genome sequences were available, in order to tentatively identify novel determinants of A. baumannii pathogenicity. Clinical strains showed comparable virulence in the Galleria mellonella model of infection, irrespective of their status as outbreak or sporadic strains, whereas a non-human isolate was avirulent. A combined approach of genomic and phenotypic analyses led to the identification of several virulence factors, including exoproducts with hemolytic, phospholipase, protease and iron-chelating activities, as well as a number of multifactorial phenotypes, such as biofilm formation, surface motility and stress resistance, which were differentially expressed and could play a role in A. baumannii pathogenicity. Conclusion/Significance This work provides evidence of the multifactorial nature of A. baumannii virulence. While A. baumannii clinical isolates could represent a selected population of strains adapted to infect the human host, subpopulations of highly genotypically and phenotypically diverse A. baumannii strains may exist outside the hospital environment, whose relevance and distribution deserve further investigation.

Acinetobacter infection--an emerging threat to human health.

The genus Acinetobacter comprises a complex and heterogeneous group of bacteria, many of which are capable of causing a range of opportunistic, often catheter‐related, infections in humans. However, Acinetobacter baumannii, as well as its close relatives belonging to genomic species 3 (“Acinetobacter pittii”) and 13TU (“Acinetobacter nosocomialis”), are important nosocomial pathogens, often associated with epidemic outbreaks of infection, that are only rarely found outside of a clinical setting. These organisms are frequently pandrug‐resistant and are capable of causing substantial morbidity and mortality in patients with severe underlying disease, both in the hospital and in the community. Several epidemic clonal lineages of A. baumannii have disseminated worldwide and seem to have a selective advantage over non‐epidemic strains. The reasons for the success of these epidemic lineages remain to be elucidated, but could be related to the potential of these organisms to achieve very dynamic reorganization and rapid evolution of their genome, including the acquisition and expression of additional antibiotic resistance determinants, under fluctuating environmental and selective conditions.

Common themes and variations in the rhodanese superfamily

The rhodanese homology domain is a ubiquitous fold found in several phylogenetically related proteins encoded by eubacterial, archeal, and eukaryotic genomes. Although rhodanese‐like proteins share evolutionary relationships, analysis of their sequences highlights that they are so heterogeneous to form the rhodanese superfamily. The variability occurs at different levels including sequence, active site loop length, presence of a critical catalytic Cys residue, and domain arrangement. Even within the same genome, multiple genes encode rhodanese‐like proteins presenting with variably arranged rhodanese domain(s): as single or tandem domain(s), or combined with other protein domain(s). Given the highly variable organization of the rhodanese domain(s) and the context where it is found, here we review the structural organization and function of the rhodanese‐like proteins. The overview of the most recent findings about rhodanese allow us to depict a superfamily of versatile proteins relying on persulfide chemistry to accomplish cellular functions spanning from resistance to environmental threats, such as cyanide, and key cellular reactions related to sulfur metabolism and progression of cell cycle. IUBMB Life, 59: 51‐59, 2007