Michael J. Federle

Interspecies communication in bacteria

Until recently, bacteria were considered to live rather asocial, reclusive lives. New research shows that, in fact, bacteria have elaborate chemical signaling systems that enable them to communicate within and between species. One signal, termed AI-2, appears to be universal and facilitates interspecies communication. Many processes, including virulence factor production, biofilm formation, and motility, are controlled by AI-2. Strategies that interfere with communication in bacteria are being explored in the biotechnology industry with the aim of developing novel antimicrobials. AI-2 is a particularly attractive candidate for such studies because of its widespread use in the microbial kingdom.

Virulence control in group A Streptococcus by a two-component gene regulatory system: Global expression profiling and in vivo infection modeling

Two-component gene regulatory systems composed of a membrane-bound sensor and cytoplasmic response regulator are important mechanisms used by bacteria to sense and respond to environmental stimuli. Group A Streptococcus, the causative agent of mild infections and life-threatening invasive diseases, produces many virulence factors that promote survival in humans. A two-component regulatory system, designated covRS (cov, control of virulence; csrRS), negatively controls expression of five proven or putative virulence factors (capsule, cysteine protease, streptokinase, streptolysin S, and streptodornase). Inactivation of covRS results in enhanced virulence in mouse models of invasive disease. Using DNA microarrays and quantitative RT-PCR, we found that CovR influences transcription of 15% (n = 271) of all chromosomal genes, including many that encode surface and secreted proteins mediating host–pathogen interactions. CovR also plays a central role in gene regulatory networks by influencing expression of genes encoding transcriptional regulators, including other two-component systems. Differential transcription of genes influenced by covR also was identified in mouse soft-tissue infection. This analysis provides a genome-scale overview of a virulence gene network in an important human pathogen and adds insight into the molecular mechanisms used by group A Streptococcus to interact with the host, promote survival, and cause disease.

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

SUMMARY Cell-cell communication, or quorum sensing, is a widespread phenomenon in bacteria that is used to coordinate gene expression among local populations. Its use by bacterial pathogens to regulate genes that promote invasion, defense, and spread has been particularly well documented. With the ongoing emergence of antibiotic-resistant pathogens, there is a current need for development of alternative therapeutic strategies. An antivirulence approach by which quorum sensing is impeded has caught on as a viable means to manipulate bacterial processes, especially pathogenic traits that are harmful to human and animal health and agricultural productivity. The identification and development of chemical compounds and enzymes that facilitate quorum-sensing inhibition (QSI) by targeting signaling molecules, signal biogenesis, or signal detection are reviewed here. Overall, the evidence suggests that QSI therapy may be efficacious against some, but not necessarily all, bacterial pathogens, and several failures and ongoing concerns that may steer future studies in productive directions are discussed. Nevertheless, various QSI successes have rightfully perpetuated excitement surrounding new potential therapies, and this review highlights promising QSI leads in disrupting pathogenesis in both plants and animals.

Ligand-induced asymmetry in histidine sensor kinase complex regulates quorum sensing.

Bacteria sense their environment using receptors of the histidine sensor kinase family, but how kinase activity is regulated by ligand binding is not well understood. Autoinducer-2 (AI-2), a secreted signaling molecule originally identified in studies of the marine bacterium Vibrio harveyi, regulates quorum-sensing responses and allows communication between different bacterial species. AI-2 signal transduction in V. harveyi requires the integral membrane receptor LuxPQ, comprised of periplasmic binding protein (LuxP) and histidine sensor kinase (LuxQ) subunits. Combined X-ray crystallographic and functional studies show that AI-2 binding causes a major conformational change within LuxP, which in turn stabilizes a quaternary arrangement in which two LuxPQ monomers are asymmetrically associated. We propose that formation of this asymmetric quaternary structure is responsible for repressing the kinase activity of both LuxQ subunits and triggering the transition of V. harveyi into quorum-sensing mode.

A novel double‐tryptophan peptide pheromone controls competence in Streptococcus spp. via an Rgg regulator

All streptococcal genomes encode the alternative sigma factor SigX and 21 SigX‐dependent proteins required for genetic transformation, yet no pyogenic streptococci are known to develop competence. Resolving this paradox may depend on understanding the regulation of sigX. We report the identification of a regulatory circuit linked to the sigX genes of mutans, pyogenic, and bovis streptococci that uses a novel small, double‐tryptophan‐containing sigX‐inducing peptide (XIP) pheromone. In all three groups, the XIP gene (comS), and sigX have identical, non‐canonical promoters consisting of 9 bp inverted repeats separated from a −10 hexamer by 19 bp. comS is adjacent to a gene encoding a putative transcription factor of the Rgg family and is regulated by its product, which we designate ComR. Deletion of comR or comS in Streptococcus mutans abolished transformability, as did deletion of the oligopeptide permease subunit oppD, suggesting that XIP is imported. Providing S. mutans with synthetic fragments of ComS revealed that seven C‐terminal residues, including the WW motif, cause robust induction of both sigX and the competent state. We propose that this circuit is the proximal regulator of sigX in S. mutans, and we infer that it controls competence in a parallel way in all pyogenic and bovis streptococci.

Evasion of human innate and acquired immunity by a bacterial homolog of CD11b that inhibits opsonophagocytosis

Microbial pathogens must evade the human immune system to survive, disseminate and cause disease. By proteome analysis of the bacterium Group A Streptococcus (GAS), we identified a secreted protein with homology to the α-subunit of Mac-1, a leukocyte β2 integrin required for innate immunity to invading microbes. The GAS Mac-1–like protein (Mac) was secreted by most pathogenic strains, produced in log-phase and controlled by the covR-covS two-component gene regulatory system, which also regulates transcription of other GAS virulence factors. Patients with GAS infection had titers of antibody specific to Mac that correlated with the course of disease, demonstrating that Mac was produced in vivo. Mac bound to CD16 (FcγRIIIB) on the surface of human polymorphonuclear leukocytes and inhibited opsonophagocytosis and production of reactive oxygen species, which resulted in significantly decreased pathogen killing. Thus, by mimicking a host-cell receptor required for an innate immune response, the GAS Mac protein inhibits professional phagocyte function by a novel strategy that enhances pathogen survival, establishment of infection and dissemination.

Peptide pheromone signaling in Streptococcus and Enterococcus

Intercellular chemical signaling in bacteria, commonly referred to as quorum sensing (QS), relies on the production and detection of compounds known as pheromones to elicit coordinated responses among members of a community. Pheromones produced by Gram-positive bacteria are comprised of small peptides. Based on both peptide structure and sensory system architectures, Gram-positive bacterial signaling pathways may be classified into one of four groups with a defining hallmark: cyclical peptides of the Agr type, peptides that contain Gly-Gly processing motifs, sensory systems of the RNPP family, or the recently characterized Rgg-like regulatory family. The recent discovery that Rgg family members respond to peptide pheromones increases substantially the number of species in which QS is likely a key regulatory component. These pathways control a variety of fundamental behaviors including conjugation, natural competence for transformation, biofilm development, and virulence factor regulation. Overlapping QS pathways found in multiple species and pathways that utilize conserved peptide pheromones provide opportunities for interspecies communication. Here we review pheromone signaling identified in the genera Enterococcus and Streptococcus, providing examples of all four types of pathways.

Identification of binding sites for the group A streptococcal global regulator CovR

The CovRS two‐component system (also called CsrRS) of the group A streptococcus (GAS) acts as a global regulator, influencing the transcription of at least six virulence factors. The synthesis of the hyaluronic acid capsule, a virulence factor encoded by the hasABC operon, is negatively regulated by CovRS. We confirmed that phosphorylation of CovR increases its binding to a DNA fragment containing the hasA promoter. Using DNase I footprinting, we identified five binding sites surrounding the hasA promoter from bases –79 to +73 (where +1 is the start of transcription). One pair of thymines within each binding site appears to be necessary for CovR binding in vitro, as shown by uracil interference analysis. When each of these thymine pairs was altered by site‐directed mutagenesis, CovR binding was reduced in vitro, confirming the role of each thymine pair in binding. Using a transcriptional reporter system with a single chromosomal copy of PhasA–gusA, we demonstrated the importance of each of four of these binding sites for CovR repression of the hasA promoter. Based on this information, we propose a consensus sequence for CovR binding to DNA.

Autoinducer-2-Based Chemical Communication in Bacteria: Complexities of Interspecies Signaling

Cell-cell communication in bacteria, called quorum sensing, relies on production, release, and detection of signaling molecules, termed autoinducers. Communication enables populations of cells to synchronize gene expression and therefore behave as a group in a manner akin to cells in multicellular organisms. Most quorum-sensing systems allow communication within an individual species of bacteria. However, one autoinducer, called AI-2, is produced and recognized by many different bacterial species, indicating that some bacteria communicate across species boundaries. Current studies are aimed at discovering the role that AI-2 plays in gene regulation. Differential gene expression in response to AI-2 may cause bacterial behavioral changes, such as biofilm formation or transition to a pathogenic state. Interestingly, multiple mechanisms to detect AI-2 exist. These differences likely reflect variations in the role that AI-2 plays for different bacteria. Additionally, structural analyses of the AI-2 receptor in V. harveyi have provided insight into bacterial trans-membrane signal transduction. A further understanding of bacterial quorum-sensing processes may facilitate development of new technologies aimed at interfering with bacterial communication and virulence.

Development of Competence for Genetic Transformation of Streptococcus mutans in a Chemically Defined Medium

Streptococcus mutans develops competence for genetic transformation in response to regulatory circuits that sense at least two peptide pheromones. One peptide, known as CSP, is sensed by a two-component signal transduction system through a membrane receptor, ComD. The other, derived from the primary translation product ComS, is thought to be sensed by an intracellular receptor, ComR, after uptake by oligopeptide permease. To allow study of this process in a medium that does not itself contain peptides, development of competence was examined in the chemically defined medium (CDM) described by van de Rijn and Kessler (Infect. Immun. 27:444, 1980). We confirmed a previous report that in this medium comS mutants of strain UA159 respond to a synthetic peptide comprising the seven C-terminal residues of ComS (ComS(11-17)) by increasing expression of the alternative sigma factor SigX, which in turn allows expression of competence effector genes. This response provided the basis for a bioassay for the ComS pheromone in the 100 to 1,000 nM range. It was further observed that comS(+) (but not comS mutant) cultures developed a high level of competence in the late log and transition phases of growth in this CDM without the introduction of any synthetic stimulatory peptide. This endogenous competence development was accompanied by extracellular release of one or more signals that complemented a comS mutation at levels equivalent to 1 μM synthetic ComS(11-17).