Luis E. N. Quadri

Quorum sensing by peptide pheromones and two component signal transduction systems in Gram-positive bacteria.

Cell‐density‐dependent gene expression appears to be widely spread in bacteria. This quorum‐sensing phenomenon has been well established in Gram‐negative bacteria, where N‐acyl homoserine lactones are the diffusible communication molecules that modulate cell‐density‐dependent phenotypes. Similarly, a variety of processes are known to be regulated in a cell‐density‐ or growth‐phase‐dependent manner in Gram‐positive bacteria. Examples of such quorum‐sensing modes in Gram‐positive bacteria are the development of genetic competence in Bacillus subtilis and Streptococcus pneumoniae, the virulence response in Staphylococcus aureus, and the production of antimicrobial peptides by several species of Gram‐positive bacteria including lactic acid bacteria. Cell‐density‐dependent regulatory modes in these systems appear to follow a common theme, in which the signal molecule is a post‐translationally processed peptide that is secreted by a dedicated ATP‐binding‐cassette exporter. This secreted peptide pheromone functions as the input signal for a specific sensor component of a two‐component signal‐transduction system. Moreover, genetic linkage of the common elements involved results in autoregulation of peptide‐pheromone production.

Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin.

BACKGROUND Many pathogenic bacteria secrete iron-chelating siderophores as virulence factors in the iron-limiting environments of their vertebrate hosts to compete for ferric iron. Mycobacterium tuberculosis mycobactins are mixed polyketide/nonribosomal peptides that contain a hydroxyaryloxazoline cap and two N-hydroxyamides that together create a high-affinity site for ferric ion. The mycobactin structure is analogous to that of the yersiniabactin and vibriobactin siderophores from the bacteria that cause plague and cholera, respectively. RESULTS A ten-gene cluster spanning 24 kilobases of the M. tuberculosis genome, designated mbtA-J, contains the core components necessary for mycobactin biogenesis. The gene products MbtB, MbtE and MbtF are proposed to be peptide synthetases, MbtC and MbtD polyketide synthases, MbtI an isochorismate synthase that provides a salicylate activated by MbtA, and MbtG a required hydroxylase. An aryl carrier protein (ArCP) domain is encoded in mbtB, and is probably the site of siderophore chain initiation. Overproduction and purification of the mbtB ArCP domain and MbtA in Escherichia coli allowed validation of the mycobactin initiation hypothesis, as sequential action of PptT (a phosphopantetheinyl transferase) and MbtA (a salicyl-AMP ligase) resulted in the mbtB ArCP domain being activated as salicyl-S-ArCP. CONCLUSIONS Mycobactins are produced in M. tuberculosis using a polyketide synthase/nonribosomal peptide synthetase strategy. The mycobactin gene cluster has organizational homologies to the yersiniabactin and enterobactin synthetase genes. Enzymatic targets for inhibitor design and therapeutic intervention are suggested by the similar ferric-ion ligation strategies used in the siderophores from Mycobacteria, Yersinia and E. coli pathogens.

Post-translational modification of polyketide and nonribosomal peptide synthases

The past year has witnessed a major advance in the study of polyketide and nonribosomal peptide biosynthesis with the identification of the phosphopantetheinyl transferase enzyme family, enzymes required to produce active, post-translationally modified polyketide and peptide synthases. Phosphopantetheinyl transferases required for fatty acid, peptide and siderophore biosynthesis have been characterized and a consensus sequence noted in order to facilitate future identification of additional proteins catalyzing phosphopantetheinyl transfer.

Peptide pheromone-dependent regulation of antimicrobial peptide production in Gram-positive bacteria: a case of multicellular behavior.

Quorum sensing enables unicellular organisms to behave in a multicellular way by allowing population-wide synchronized adaptive responses that involve modulation of a wide range of physiological responses in a cell density-, cell proximity- or growth phase-dependent manner. Examples of processes modulated by quorum sensing are the development of genetic competence, conjugative plasmid transfer, sporulation and cell differentiation, biofilm formation, virulence response, production of antibiotics, antimicrobial peptides and toxins, and bioluminescence (for reviews see [38]). The cell-to-cell communication strategies involved in these processes are based on the utilization of small signal molecules produced and released into the environment by the microorganisms. These communication molecules are referred to as pheromones and act as chemical messengers that transmit information across space. The extracellular pheromones accumulate in the environment and trigger a response in the target cells when its concentration reaches a certain threshold value. Elucidation of the chemical nature of the pheromones modulating the processes mentioned above reveals that most of them are unmodified peptides, post-translationally modified peptides, N-acyl homoserine lactones, or butyrolactones. Lactone-based pheromones are the preferred communication signals in Gram-negative bacteria (for review see [47,48]), whereas peptide-based pheromones are the predominant extracellular signals among Gram-positive bacteria (for review see [37,61]). However, lactone-based pheromones are utilized as signals that modulate differentiation and secondary metabolism production in Streptomyces (for review see [20]). This review focuses on the major advances and current views of the peptide-pheromone dependent regulatory circuits involved in production of antimicrobial peptides in Gram-positive bacteria.

Transcriptome Analysis of the Response of Pseudomonas aeruginosa to Hydrogen Peroxide

Pseudomonas aeruginosa must often overcome a high concentration of oxidants to successfully infect the human host. We report here the results of a transcriptome profiling comparing cells treated with H(2)O(2) and untreated controls. The data indicate that the early response of P. aeruginosa to H(2)O(2) consists of an upregulation of protective mechanisms and a downregulation of primary metabolism.

Transcriptome analysis of the Pseudomonas aeruginosa response to iron

To successfully infect humans, Pseudomonas aeruginosa (Pa) must overcome the low iron availability in host tissues. A transcriptome comparison was carried out between iron-starved cells of Pa treated with iron and untreated controls. The present study is the first global analysis of the early transcriptional response of exponentially growing Pa to iron. Approximately 1.3% of the Pa genes displayed ≥5.0-fold changes in mRNA levels in iron-treated cells. Treatment affected the mRNA levels of many genes required for iron acquisition as well as several genes with relevance to virulence previously known to be regulated by iron. More importantly, the analysis permitted identification of 107 Pa genes whose mRNA levels were not previously known to be affected by iron. These genes are good candidates for mutagenesis studies aimed at identifying novel functions relevant to iron metabolism in Pa. Some of these genes encode predicted siderophore receptors, iron transport systems, TonB-dependent receptors, regulatory proteins, and proteins relevant to virulence. Notably, 49 genes encode hypothetical or conserved hypothetical proteins of unknown function, suggesting that they are involved directly or indirectly in iron metabolism or metabolic adaptation to different iron-availability conditions.

Pseudomonas aeruginosa SoxR Does Not Conform to the Archetypal Paradigm for SoxR-Dependent Regulation of the Bacterial Oxidative Stress Adaptive Response

ABSTRACT SoxR is a transcriptional regulator that controls an oxidative stress response in Escherichia coli. The regulator is primarily activated by superoxide anion-dependent oxidation. Activated SoxR turns on transcription of a single gene, soxS, which encodes a transcriptional regulator that activates a regulon that includes dozens of oxidative stress response genes. SoxR homologues have been identified in many bacterial species, including the opportunistic pathogen Pseudomonas aeruginosa. However, the expected SoxR partner, SoxS, has not been found in P. aeruginosa. Thus, the primary gene target(s) of P. aeruginosa SoxR is unknown and the involvement of this regulator in the oxidative stress response of the bacterium remains unclear. We utilized transcriptome profiling to identify the P. aeruginosa SoxR regulon and constructed and characterized an unmarked P. aeruginosa ΔsoxR mutant. We provide evidence indicating that P. aeruginosa SoxR activates a six-gene regulon in response to O2·−-induced stress. The regulon includes three transcriptional units: (i) the recently identified mexGHI-ompD four-gene operon, which encodes a multidrug efflux pump system involved in quorum-sensing signal homeostasis; (ii) gene PA3718, encoding a probable efflux pump; and (iii) gene PA2274, encoding a probable monooxygenase. We also demonstrate that P. aeruginosa SoxR is not a key regulatory player in the oxidative stress response. Finally, we show that P. aeruginosa SoxR is required for virulence in a mouse model of intrapulmonary infection. These results demonstrate that the E. coli-based SoxRS paradigm does not hold in P. aeruginosa and foster new hypotheses for the possible physiological role of P. aeruginosa SoxR.

Assembly of aryl‐capped siderophores by modular peptide synthetases and polyketide synthases

Bacterial siderophores assist pathogens in iron acquisition inside their hosts. They are often essential for achieving a successful infection, and their biosynthesis represents an attractive antibiotic target. Recently, several siderophore biosynthetic loci have been identified, and in vitro studies have advanced our knowledge of the biosynthesis of aryl‐capped peptide and peptide–polyketide siderophores from Mycobacterium spp., Pseudomonas spp., Yersinia spp. and other bacteria. These studies also provided insights into the assembly of related siderophores and many secondary metabolites of medical relevance. Assembly of aryl‐capped peptide and peptide–polyketide siderophores involves non‐ribosomal peptide synthetase, polyketide synthase and non‐ribosomal‐peptide polyketide hybrid subunits. Analysis of these subunits suggests that their domains and modules are functionally and structurally independent. It appears that nature has selected a set of functional domains and modules that can be rearranged in different order and combinations to biosynthesize different products. Although much remains to be learned about modular synthetases and synthases, it is already possible to conceive strategies to engineer these enzymes to generate novel products.

Effect of Amino Acid Substitutions on the Activity of Carnobacteriocin B2 OVERPRODUCTION OF THE ANTIMICROBIAL PEPTIDE, ITS ENGINEERED VARIANTS, AND ITS PRECURSOR IN ESCHERICHIA COLI

Carnobacteriocin B2, a 48-amino acid antimicrobial peptide containing a YGNGV motif that is produced by the lactic acid bacterium Carnobacterium piscicola LV17B, was overexpressed as fusion with maltose-binding protein in Escherichia coli. This fusion protein was cleaved with Factor Xa to allow isolation of the mature bacteriocin that was identical in all respects to that obtained from C. piscicola Similar methodology permitted production of the precursor precarnobacteriocin B2 (CbnB2P), which has an 18-amino acid leader, as well as six mutants of the mature peptide: CbnF3 (Tyr3 → Phe), CbnS33 (Phe33 → Ser), CbnI34 (Val34 → Ile), CbnI37 (Val37 → Ile), CbnG46 (Arg46 → Gly), and Cbn28 (truncated frameshift mutation: (carnobacteriocin B2 1-28) + ELTHL). Examination of these compounds for antimicrobial activity showed that although CbnI34, CbnI37, and CbnG46 were fully active, CbnB2P, CbnF3, CbnS33, Cbn28, and all of the fusion proteins had greatly reduced or no antimicrobial activity. Expression of the immunity protein that protects against the action of the parent carnobacteriocin B2 in a previously sensitive organism also protects against the active mutants. Because carnobacteriocin B2 also acts as an inducer of bacteriocin production in C. piscicola, the ability of the precursor CbnB2P and the mutants to exert this effect was examined. All were able to induce Bac− cultures and reestablish the Bac+ phenotype except for the truncated Cbn28. The results demonstrate that very minor changes in the peptide sequence may drastically alter antimicrobial activity but that the induction of bacteriocin production is much more tolerant of structural modification, especially at the N terminus.

Strategic Paradigm Shifts in the Antimicrobial Drug Discovery Process of the 21st Century

The numbers of global infections produced by bacterial strains that are resistant to single and multiple antimicrobial drugs are on the rise. Concomitant with this alarming upward trend, there is a clear downward trend in the intent and determination of pharmaceutical companies to develop novel antimicrobials. One of the pressing goals to confront the twenty first centurys public health challenges brought about by the escalating antibacterial drug resistance problem is the development of an armamentarium of new chemotherapeutic agents. Two interconnected strategic paradigm shifts in the drug discovery process that are anticipated to facilitate the achievement of this goal are discussed herein. One is an antimicrobial to anti-infective (ATA) paradigm shift. The other is a shift from a target candidate prioritization (TCP) paradigm that is dominated by an essential target preference criterion to an alternative paradigm that relies on a less restrictive criterion, one that does not exclude conditionally essential targets. Examples of conditionally essential targets for the development of anti infectives include the enzymes involved in the biosynthesis of small molecule virulence effectors such as non ribosomal peptide polyketide derived iron scavenging siderophores. Siderophores are utilized for iron uptake by many pathogenic bacteria, including Mycobacterium and Yersinia species. The recent progress towards developing inhibitors of siderophore biosynthesis is discussed herein.