Richard P. Novick

Autoinduction and signal transduction in the regulation of staphylococcal virulence

The accessory genes of Staphylococcus aureus, in‐cluding 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 organisms 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.

Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule.

The production of most toxins and other exoproteins in Staphylococcus aureus is controlled globally by a complex polycistronic regulatory locus, agr. Secretory proteins are up‐regulated by agr whereas surface proteins are down‐regulated. agr contains two divergent promoters, one of which directs the synthesis of a 514 nucleotide (nt) transcript, RNAIII. In this report, we show that the cloned RNAIII determinant restores both positive and negative regulatory functions of agr to an agr‐null strain and that the RNA itself, rather than any protein, is the effector molecule. RNAIII acts primarily on the initiation of transcription and, secondarily in some cases, at the level of translation. In these cases, translation and transcription are regulated independently. RNAIII probably regulates translation directly by interacting with target gene transcripts and transcription indirectly by means of intermediary protein factors.

Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus

Abstract A high-frequency transducing element for erythromycin resistance in Staphylococcus aureus has been found. This element, P11de, is apparently the result of a recombinational event between a temperate phage, P11, and a penicillinase plasmid, γ; it lacks substantial sections both of the plasmid and of the bacteriophage. The phage moiety is cryptic, conferring neither lysogeny nor superinfection immunity upon host cells carrying it; helper phage is required for the production of transducing particles but not for the transduction process. Demonstrable phage-related properties include reactivation of ultraviolet-inactivated homologous phage and rescue of temperature-sensitive phage mutants. The plasmic moiety includes an erythromycin resistance marker, and the mc region, which is responsible for plasmid compatibility, is involved in a specific host-plasmid interaction and is required for plasmid replication; all the other known plasmid markers are missing. The autonomous replication of P11de as well as its intracellular behavior vis-a-vis prophages and other plasmids appear to be governed by its mc determinant. In these respects P11de is similar to the parental γ plasmid. This plasmid control, however, is superseded by active P11, which induces P11de to multiply extensively. P11de can recombine with wild-type plasmids; the resulting recombinants are not transduced at high frequency, nor do they reactivate UV inactivated phage particles. Based on these findings, a possible genetic structure for P11de is presented and compared with maps of naturally occurring plasmids.

Quorum Sensing in Staphylococci

The staphylococcal agr locus encodes a quorum sensing (QS) system that controls the expression of virulence and other accessory genes by a classical two-component signaling module. Like QS modalities in other Gram-positive bacteria, agr encodes an autoactivating peptide (AIP) that is the inducing ligand for AgrC, the agr signal receptor. Unlike other such systems, agr variants have arisen that show strong cross-inhibition in heterologous combinations, with important evolutionary implications. Also unlike other systems, the effector of global gene regulation in the agr system is a major regulatory RNA, RNAIII. In this review, we describe the functions of the agr systems elements, show how they interact to bring about the regulatory response, and discuss the role of QS in staphylococcal pathobiology. We conclude with the suggestion that agr autoactivation, unlike classical enzyme induction, can occur under suboptimal conditions and can distinguish self from non-self by inducing an exclusive and coordinated population wide response.

The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus

Tst, the gene for toxic shock syndrome toxin‐1 (TSST‐1), is part of a 15.2 kb genetic element in Staphylococcus aureus that is absent in TSST‐1‐negative strains. The prototype, in RN4282, is flanked by a 17 nucleotide direct repeat and contains genes for a second possible superantigen toxin, a Dichelobacter nodosus VapE homologue and a putative integrase. It is readily transferred to a recA− recipient, and it always inserts into a unique chromosomal copy of the 17 nucleotide sequence in the same orientation. It is excised and circularized by staphylococcal phages φ13 and 80α and replicates during the growth of the latter, which transduces it at very high frequency. Because of its site and orientation specificity and because it lacks other identifiable phage‐like genes, we consider it to be a pathogenicity island (PI) rather than a transposon or a defective phage. The tst element in RN4282, near tyrB, is designated SaPI1. That in RN3984 in the trp region is only partially homologous to SaPI1 and is excised by phage 80 but not by 80α. It is designated SaPI2. These PIs are the first in any Gram‐positive species and the first for which mobility has been demonstrated. Their mobility may be responsible for the spread of TSST‐1 production among S. aureus strains.

Accessory Gene Regulator (agr) Locus in Geographically Diverse Staphylococcus aureus Isolates with Reduced Susceptibility to Vancomycin

ABSTRACT 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.

Analysis by Transduction of Mutations affecting Penicillinase Formation in Staphylococcus aureus

SUMMARY: Seventy-five mutants with alterations in penicillinase formation were isolated from a strain of Staphylococcus aureus inducible for penicillinase. The mutants fell into three main categories on the basis of penicillinase activity and inducibility: (i) microinducible mutants which formed decreased amounts of penicillinase but retained the property of inducibility; (ii) penicillinase-negative variants which produced no detectable penicillinase and which showed no effect of inoculum size on penicillin resistance; (iii) strains with a wide range of penicillinase activities that produced the enzyme constitutively. Treatment of the wild-type strain with ethylmethane sulphonate increased the frequency of occurrence of microinducible and constitutive mutants but did not alter the incidence of the penicillinase-negative variants which were present in all cultures at a frequency of about 10-3. Representative mutants of each class were examined for ability to revert to wild type and to give wild-type recombinants in transductional crosses. The constitutive strains and the microinducible strains behaved like point mutants in that they reverted and in that they gave wild-type recombinants. The penicillinase-negative mutants, however, behaved differently in that they were not observed to revert nor did they give wild-type recombinants in crosses, either with one another or with microinducible or constitutive mutants. A naturally occurring penicillinase-negative strain of S. aureus behaved similarly to the penicillinase-negative mutants in these respects. The possibility that the penicillinase region in S. aureus is associated with a plasmid and thus inherited extrachromosomally is considered and discussed. The properties of the penicillinase-negative variants could be explained as resulting from the loss of such a plasmid. Consistent with the plasmid hypothesis is the finding that ultraviolet irradiation of transducing phage produced an exponential decline of transducing titre for penicillinase; against it is the failure of acridine orange to increase the frequency of the penicillinase-negative variants.

Novel Cassette-Based Shuttle Vector System for Gram-Positive Bacteria

ABSTRACT Our understanding of staphylococcal pathogenesis depends on reliable genetic tools for gene expression analysis and tracing of bacteria. Here, we have developed and evaluated a series of novel versatile Escherichia coli-staphylococcal shuttle vectors based on PCR-generated interchangeable cassettes. Advantages of our module system include the use of (i) staphylococcal low-copy-number, high-copy-number, thermosensitive and theta replicons and selectable markers (choice of erythromycin, tetracycline, chloramphenicol, kanamycin, or spectinomycin); (ii) an E. coli replicon and selectable marker (ampicillin); and (iii) a staphylococcal phage fragment that allows high-frequency transduction and an SaPI fragment that allows site-specific integration into the Staphylococcus aureus chromosome. The staphylococcal cadmium-inducible Pcad-cadC and constitutive PblaZ promoters were designed and analyzed in transcriptional fusions to the staphylococcal β-lactamase blaZ, the Vibrio fischeri luxAB, and the Aequorea victoria green fluorescent protein reporter genes. The modular design of the vector system provides great flexibility and variety. Questions about gene dosage, complementation, and cis-trans effects can now be conveniently addressed, so that this system constitutes an effective tool for studying gene regulation of staphylococci in various ecosystems.

Exfoliatin-Producing Strains Define a Fourth agr Specificity Group in Staphylococcus aureus

The staphylococcal virulon is activated by the density-sensing agr system, which is autoinduced by a short peptide (autoinducing peptide [AIP]) processed from a propeptide encoded by agrD. A central segment of the agr locus, consisting of the C-terminal two-thirds of AgrB (the putative processing enzyme), AgrD, and the N-terminal half of AgrC (the receptor), shows striking interstrain variation. This finding has led to the division of Staphylococcus aureus isolates into three different agr specificity groups and to the division of non-aureus staphylococci into a number of others. The AIPs cross-inhibit the agr responses between groups. We have previously shown that most menstrual toxic shock strains belong to agr specificity group III but that no strong clinical identity has been associated with strains of the other two groups. In the present report, we demonstrate a fourth agr specificity group among S. aureus strains and show that most exfoliatin-producing strains belong to this group. A striking common feature of group IV strains is activation of the agr response early in exponential phase, at least 2 h earlier than in strains of the other groups. This finding raises the question of the biological significance of the agr autoinduction threshold.

The phage-related chromosomal islands of Gram-positive bacteria

The phage-related chromosomal islands (PRCIs) were first identified in Staphylococcus aureus as highly mobile, superantigen-encoding genetic elements known as the S. aureus pathogenicity islands (SaPIs). These elements are characterized by a specific set of phage-related functions that enable them to use the phage reproduction cycle for their own transduction and inhibit phage reproduction in the process. SaPIs produce many phage-like infectious particles; their streptococcal counterparts have a role in gene regulation but may not be infectious. These elements therefore represent phage satellites or parasites, not defective phages. In this Review, we discuss the shared genetic content of PRCIs, their life cycle and their ability to be transferred across large phylogenetic distances.