Biomedical subjects

Richard P Novick

Publications and source records attributed to Richard P Novick.

28 records · Page 2Linked to original sources

PubMed2003

Mobile genetic elements and bacterial toxinoses: the superantigen-encoding pathogenicity islands of Staphylococcus aureus.

It is a remarkable observation that virtually all bacterial toxins associated with specific clinical conditions (toxinoses) are encoded by mobile (and therefore variable) genetic elements. Remarkably, these rarely, if ever, carry determinants of antibiotic resistance. Examples are the toxins responsible for diphtheria, anthrax, tetanus, botulism, cholera, toxic shock, scarlet fever, exfoliative dermatitis, food poisoning, travelers' diarrhea, shigella dysentery, necrotizing pneumonia, and others. A recently discovered example of this phenomenon is the family of related staphylococcal pathogenicity islands encoding superantigens (SAgs). These are 15-20kb elements that occupy constant positions in the chromosomes of toxigenic strains, and are characterized by certain phage-related features, namely genes encoding integrases, helicases, and terminases, and the presence of flanking direct repeats. The prototype, SaPI1 of Staphylococcus aureus, encodes TSST-1 plus two newly described SAgs, SEK and SEL. Other members of the family encode enterotoxins B (SaPI3) and C (SaPI4), plus at least two other SAgs each. SaPI1 and SaPI2, also encoding TSST-1, are excised and induced to replicate by certain staphylococcal phages, and are then encapsidated at high efficiency into phage-like infectious particles with heads about 1/3 the size of the helper phage heads, commensurate with the sizes of the respective genomes. This results in transfer frequencies of the order of 10(8)/ml, and is presumably responsible for the spread of these elements as well as for their acquisition in the first place. In the absence of a helper phage, these two islands are highly stable; neither excision, loss, or transfer occurs at detectable frequency. Several general implications of this phenomenon will be discussed. One is that the determinants of these toxins have been imported from other species and therefore are not components of the basic genome of the extant producing organisms. This raises the question of the biological (adaptive?) roles of these toxins. Another is that the toxin-carrying units can spread among different (though probably related) species. An interesting question is that of the biological basis for the separation of toxin and resistance determinants.

Animals↗

PubMed2003

Autoinduction and signal transduction in the regulation of staphylococcal virulence.

The accessory genes of Staphylococcus aureus, including those involved in pathogenesis, are controlled by a complex regulatory network that includes at least four two-component systems, one of which, agr, is a quorum sensor, an alternative sigma factor and a large set of transcription factors, including at least two of the superantigen genes, tst and seb. These regulatory genes are hypothesized to act in a time- and population density-dependent manner to integrate signals received from the external environment with the internal metabolic machinery of the cell, in order to achieve the production of particular subsets of accessory/virulence factors at the time and in quantities that are appropriate to the needs of the organism at any given location. From the standpoint of pathogenesis, the regulatory agenda is presumably tuned to particular sites in the host organism. To address this hypothesis, it will be necessary to understand in considerable detail the regulatory interactions among the organism's numerous controlling systems. This review is an attempt to integrate a large body of data into the beginnings of a model that will hopefully help to guide research towards a full-scale test.

Amino Acid Sequence↗

PubMed2003

The staphylococcal saeRS system coordinates environmental signals with agr quorum sensing.

sae is a two-component signal transduction system in Staphylococcus aureus that regulates the expression of many virulence factors at the transcriptional level and appears to act synergistically with agr in some cases. In this study, the interactions between sae and agr have been characterized in some detail. It was found that the sae locus is larger and more complex than originally envisioned, in that it is expressed from several promoters, giving rise to four or five transcripts, at least three of which are initiated upstream of saeRS and contain two additional reading frames, here designated saeP and saeQ, which are likely to have important roles in sae function. The upstream transcripts are induced during exponential phase concomitantly with the onset of RNAIII synthesis and their induction requires the agr effector, RNAIII, but is blocked by several environmental signals that override the effects of RNAIII. saeR is also required for the induction of these transcripts, so that the sae locus contains an autoinduction circuit. It is suggested that sae is downstream of agr in the exoprotein activation pathway (and also epistatic with agr), that it coordinates the effects of environmental signals with the agr quorum-sensing system, and therefore that it is a key intermediary in the overall regulatory strategy by which S. aureus senses and responds to its environment.

Bacterial Proteins↗

PubMed2002

Key determinants of receptor activation in the agr autoinducing peptides of Staphylococcus aureus.

Staphylococcal pathogenesis is regulated by a two-component quorum-sensing system, agr, activated upon binding of a self-coded autoinducing peptide (AIP) to the receptor-histidine kinase, AgrC. The AIPs consist of a thiolactone macrocyle and an exocyclic "tail", both of which are important for function. In this report, characterization of the unique AIPs from the four known agr specificity groups of Staphylococcus aureus has been completed, along with analysis of cross-group inhibition of AgrC activation by each of the four AIPs. The following conclusions have been drawn: (i) The native thiolactone macrocyle and tail are necessary and sufficient for full activation by the AIPs, whereas the AIP-I macrocycle alone is a partial agonist. (ii) The native N-terminus is less critical, as that of AIP-I can be modified without affecting bioactivity, although that of AIP-III cannot. (iii) The ring and tail may function differently in different AIPs. Thus the group I and IV AIPs differ at a single (endocyclic) residue, which is the determinant of AIP specificity for these two groups and is essential for function. A similarly critical residue in AIP-II, however, is exocyclic. (iv) Cross-inhibition is more tolerant of sequence and structural diversity than is activation, suggesting that the AIPs interact differently with cognate than with heterologous receptors. (v) Chimeric peptides, in which the tails and macrocycles are switched, do not activate and instead inhibit receptor activation. These data suggest a model in which activation and inhibition involves different binding orientations within the ligand binding pocket of each receptor.

Amino Acid Sequence↗

PubMed2002

Transmembrane topology of AgrB, the protein involved in the post-translational modification of AgrD in Staphylococcus aureus.

The accessory gene regulator (agr) of Staphylococcus aureus is the central regulatory system that controls the gene expression for a large set of virulence factors. This global regulatory locus consists of two transcripts: RNAII and RNAIII. RNAII encodes four genes (agrA, B, C, and D) whose gene products assemble a quorum sensing system. RNAIII is the effector of the Agr response. Both the agrB and agrD genes are essential for the production of the autoinducing peptide, which functions as a signal for the quorum sensing system. In this study, we demonstrated the transmembrane nature of AgrB protein in S. aureus. A transmembrane topology model of AgrB was proposed based on AgrB-PhoA fusion analyses in Escherichia coli. Two hydrophilic regions with several highly conserved positively charged amino acid residues among various AgrBs were found to be located in the cytoplasmic membrane as suggested by PhoA-AgrB fusion studies. However, this finding is inconsistent with the putative transmembrane profile of AgrB by computer analysis. Furthermore, we detected an intermediate peptide of processed AgrD from S. aureus cells expressing AgrB and a 6 histidine-tagged AgrD. These results provide direct evidence that AgrB is involved in the proteolytic processing of AgrD. We speculate that AgrB is a novel protein with proteolytic enzyme activity and a transporter facilitating the export of the processed AgrD peptide.

Amino Acid Sequence↗

PubMed2002

Global repression of exotoxin synthesis by staphylococcal superantigens.

Virulent Staphylococcus aureus strains typically produce and secrete large quantities of many extracellular proteins involved in pathogenesis. Such strains cause the classical staphylococcal lesion--local tissue destruction and aggressive inflammation accompanied by the massive influx of polymorphonuclear leukocytes, leading to the formation of pus. Most strains causing toxic shock syndrome, however, produce and secrete very small quantities of most exoproteins although they elaborate high levels of toxic shock syndrome toxin-1 (TSST-1). These strains cause local infections that are remarkably apurulent although potentially fatal owing to the superantigen. We have analyzed this disparity and have found that TSST-1 itself is a negative global regulator of exoprotein gene transcription. TSST-1 not only represses most exoprotein genes but determines its own high expression level by autorepression. We report also that a second superantigen, enterotoxin B, has similar regulatory properties.

Abscess↗

PubMed2002

Accessory gene regulator (agr) locus in geographically diverse Staphylococcus aureus isolates with reduced susceptibility to vancomycin.

The majority of infections with glycopeptide intermediate-level resistant Staphylococcus aureus (GISA) originate in biomedical devices, suggesting a possible increased ability of these strains to produce biofilm. Loss of function of the accessory gene regulator (agr) of S. aureus has been suggested to confer an enhanced ability to bind to polystyrene. We studied agr in GISA, hetero-GISA, and related glycopeptide-susceptible S. aureus isolates. All GISA strains from diverse geographic origins belong to agr group II. All GISA strains were defective in agr function, as demonstrated by their inability to produce delta-hemolysin. Hetero-GISA isolate A5940 demonstrated a nonsense mutation in agrA that was not present in a pulsed-field gel electrophoresis-indistinguishable vancomycin-susceptible isolate from the same patient. Various other agr point mutations were noted in several clinical GISA and hetero-GISA isolates. A laboratory-generated agr-null strain demonstrated a small but reproducible increase in vancomycin heteroresistance after growth in vitro in subinhibitory concentrations of vancomycin. This was not seen in the isogenic agr group II parent strain in which agr was intact. The in vitro bactericidal activity of vancomycin was attenuated in the agr-null strain compared to the parent strain. These findings imply that compromised agr function is advantageous to clinical isolates of S. aureus toward the development of vancomycin heteroresistance, perhaps through the development of vancomycin tolerance.

Amino Acid Sequence↗

PubMed2002

Responses of endoscopy patients in Ladakh, India, to Helicobacter pylori whole-cell and Cag A antigens.

Although Helicobacter pylori is a cosmopolitan colonizer of the human stomach, the responses among persons in remote populations from whom H. pylori was cultured have not been studied. We report on studies of 189 persons in the Ladakh region of India in whom serum immunoglobulin G responses to H. pylori whole-cell and Cag A antigens were measured. H. pylori was isolated from 68 of these patients. An H. pylori whole-cell antigen derived from Ladakhi strains outperformed a similar antigen from U.S. strains, as determined by antigen-specific enzyme-linked immunosorbent assays. In total, 95% of the population was seropositive, including individuals responding only to the Cag A antigen. Correlation with culture results showed that these were true positives and, therefore, that the H. pylori whole-cell serology was falsely negative in some cases. In addition to establishing a collection of H. pylori isolates from a remote area in the world, we show that use of H. pylori whole-cell and Cag A serology together increases the sensitivity for the detection of colonization.

Adolescent↗

PubMed2002

High genetic variability of the agr locus in Staphylococcus species.

The agr quorum-sensing and signal transduction system was initially described in Staphylococcus aureus, where four distinct allelic variants have been sequenced. Western blotting suggests the presence of homologous loci in many other staphylococci, and this has been confirmed for S. epidermidis and S. lugdunensis. In this study we isolated agr-like loci from a range of staphylococci by using PCR amplification from primers common to the six published agr sequences and bracketing the most variable region, associated with quorum-sensing specificity. Positive amplifications were obtained from 14 of 34 staphylococcal species or subspecies tested. Sequences of the amplicons identified 24 distinct variants which exhibited extensive sequence divergence with only 10% of the nucleotides absolutely conserved on multiple alignment. This variability involved all three open reading frames involved in quorum sensing and signal transduction. However, these variants retained several protein signatures, including the conserved cysteine residue of the autoinducing peptide, with the exception of S. intermedius of pigeon origin, which contained a serine in place of cysteine at this position. We discuss hypotheses on the mode of action and the molecular evolution of the agr locus based on comparisons between the newly determined sequences.

Bacterial Proteins↗

PubMed2001

Reversible and specific extracellular antagonism of receptor-histidine kinase signaling.

Staphylococcal pathogenesis is regulated by a two-component quorum-sensing system, agr, activated by a self-coded autoinducing peptide (AIP). The agr system is widely divergent and is unique in that variant AIPs cross-inhibit agr activation in heterologous combinations. Cross-inhibition, but not self-activation, is widely tolerant of structural diversity in the AIPs so that these two processes must involve different mechanisms of interaction with the respective receptors. Herein, we have utilized this naturally occurring antagonism to demonstrate that both activation and inhibition are reversible and that activators and inhibitors interact at a common site on the receptor. These results suggest that molecules designed to compete with natural agonists for binding at receptor-histidine kinase sensor domains could represent a general approach to the inhibition of receptor-histidine kinase signaling.

Amino Acid Sequence↗