ABD-3, the confluence of powerful antibacterial modalities: ABDs delivering and expressing lss, the gene encoding lysostaphin.

In response to the antimicrobial resistance crisis, we have developed a powerful and versatile therapeutic platform, the Antibacterial Drone (ABD) system. The ABD consists of a highly mobile staphylococcal pathogenicity island re-purposed to deliver genes encoding antibacterial proteins. The chromosomally located island is induced by a co-resident helper phage, packaged in phage-like particles, and released in very high numbers upon phage-induced lysis. ABD particles specifically adsorb to bacteria causing an infection and deliver their DNA to these bacteria, where the bactericidal cargo genes are expressed, kill the bacteria, and cure the infection. Here, we report a major advance of the system, incorporation of the gene encoding a secreted, bactericidal, species-specific lytic enzyme, lysostsphin. This ABD not only kills the bacterium that has been attacked by the ABD, but also any surrounding bacteria that are sensitive to the lytic enzyme which is released by secretion and by lysis of the doomed cell. So while the killing field is thus expanded, there are no civilian casualties (bacteria that are insensitive to the ABD and its cargo protein(s) are not inadvertently killed). Without amplifying the number of ABD particles (which are not re-packaged), the expression and release of the cargo gene's product dramatically extend the effective reach of the ABD. A cargo gene that encodes a secreted bactericidal protein also enables the treatment of a mixed bacterial infection in which one of the infecting organisms is insensitive to the ABD delivery system but is sensitive to the ABD's secreted cargo protein.

Reconstitution of the S. aureus agr quorum sensing pathway reveals a direct role for the integral membrane protease MroQ in pheromone biosynthesis.

In Staphylococcus aureus, virulence is under the control of a quorum sensing (QS) circuit encoded in the accessory gene regulator (agr) genomic locus. Key to this pathogenic behavior is the production and signaling activity of a secreted pheromone, the autoinducing peptide (AIP), generated following the ribosomal synthesis and posttranslational modification of a precursor polypeptide, AgrD, through two discrete cleavage steps. The integral membrane protease AgrB is known to catalyze the first processing event, generating the AIP biosynthetic intermediate, AgrD (1-32) thiolactone. However, the identity of the second protease in this biosynthetic pathway, which removes an N-terminal leader sequence, has remained ambiguous. Here, we show that membrane protease regulator of agr QS (MroQ), an integral membrane protease recently implicated in the agr response, is directly involved in AIP production. Genetic complementation and biochemical experiments reveal that MroQ proteolytic activity is required for AIP biosynthesis in agr specificity group I and group II, but not group III. Notably, as part of this effort, the biosynthesis and AIP-sensing arms of the QS circuit were reconstituted together in vitro. Our experiments also reveal the molecular features guiding MroQ cleavage activity, a critical factor in defining agr specificity group identity. Collectively, our study adds to the molecular understanding of the agr response and Staphylococcus aureus virulence.

Dynamics of Antibacterial Drone Establishment in Staphylococcus aureus: Unexpected Effects of Antibiotic Resistance Genes.

The antibacterial drone (ABD) system is based on repurposing the phage-inducible staphylococcal pathogenicity islands (SaPIs) for use as antibacterial agents that are indifferent to antibiotic resistance. The ABDs were constructed by inserting tetM for tetracycline resistance (Tcr) selection, replacing the SaPI virulence genes with bactericidal or bacteriostatic genes such as CRISPR/cas9/agrA, whose expression kills by double-strand cleavage of agrA, or CRISPR/dcas9/agrP2P3, whose expression blocks the target organism's virulence. ABD DNA is packaged in phage-like particles that attack their staphylococcal targets in vivo as well as in vitro. We determine ABD titers by transfer frequency, enumerate surviving cells as a function of multiplicity, and analyze the fate of ABD DNA with green fluorescent protein. An initial study revealed surprisingly that many more cells were killed by the ABD than were measured by transduction. Our study of this phenomenon has revealed several important features of the ABD system: (i) a significant number of entering ABD DNA molecules do not go on to establish stable transductants (i.e., are abortive); (ii) ABD cargo genes are expressed immediately following entry, even by the abortive ABDs; (iii) immediate plating on Tc-containing agar seriously underestimates particle numbers, partly owing to Tc inhibition of protein synthesis; (iv) replacement of tetM with cadA (conferring resistance to CdCl2) provides more accurate particle enumeration; (v) ABDs expressing CRISPR/cas9/agrA kill ∼99.99% of infected cells and provide the most accurate measurement of particle numbers as well as proof of principle for the system; and (vi) surprisingly, TetM interferes with stable establishment of ABD DNA independently of Tcr. IMPORTANCE For a particulate therapeutic agent, such as the ABD, accurate enumeration of particles is critical to enable evaluation of preparative procedures and calculation of therapeutic dosages. It is equally important that a selective marker used for these two purposes be biologically inert. We have long used tetM for these purposes but show here that tetM not only underestimates particle titers, by over 20-fold in some experiments, but also seriously impedes stable establishment of the therapeutic particle DNA. Given that tetM is a very convenient and widely used selective marker, publication of these findings is of considerable importance to the microbiological community as well as an interesting illustration of the unpredictable biological effects of genes taken out of their native context.

Antibacterial particles and predatory bacteria as alternatives to antibacterial chemicals in the era of antibiotic resistance.

This review is focused on the subset of antibacterial agents whose action involves one-on-one targeting of infecting bacteria. These agents target individual bacteria and their efficacy is based on particle numbers in contrast to chemical agents such as antibiotics, whose efficacy is based on minimal inhibitory concentrations. Four extant members of this class are predatory bacteria, functional (plaque-forming) phages, and engineered particulate systems, phagemids (plasmids that contain a phage packaging signal) and antibacterial drones (ABDs) that package chromosomal island DNA carrying antibacterial genes. We differentiate the natural predators, phages and predatory bacteria, from the engineered delivery vehicles, phagemids and ABDs, because the latter are much more versatile and can largely bypass the historical warfare that informs the predator-prey interactions.

A regulatory cascade controls Staphylococcus aureus pathogenicity island activation.

Staphylococcal pathogenicity islands (SaPIs) are a family of closely related mobile chromosomal islands that encode and disseminate the superantigen toxins, toxic shock syndrome toxin 1 and superantigen enterotoxin B (SEB). They are regulated by master repressors, which are counteracted by helper phage-encoded proteins, thereby inducing their excision, replication, packaging and intercell transfer. SaPIs are major components of the staphylococcal mobilome, occupying five chromosomal att sites, with many strains harbouring two or more. As regulatory interactions between co-resident SaPIs could have profound effects on the spread of superantigen pathobiology, we initiated the current study to search for such interactions. Using classical genetics, we found that, with one exception, their regulatory systems do not cross-react. The exception was SaPI3, which was originally considered defective because it could not be mobilized by any known helper phage. We show here that SaPI3 has an atypical regulatory module and is induced not by a phage but by many other SaPIs, including SaPI2, SaPIbov1 and SaPIn1, each encoding a conserved protein, Sis, which counteracts the SaPI3 repressor, generating an intracellular regulatory cascade: the co-resident SaPI, when conventionally induced by a helper phage, expresses its sis gene which, in turn, induces SaPI3, enabling it to spread. Using bioinformatics analysis, we have identified more than 30 closely related coancestral SEB-encoding SaPI3 relatives occupying the same att site and controlled by a conserved regulatory module, immA-immR-str'. This module is functionally analogous but unrelated to the typical SaPI regulatory module, stl-str. As SaPIs are phage satellites, SaPI3 and its relatives are SaPI satellites.

Discovery of quorum quenchers targeting the membrane-embedded sensor domain of the Staphylococcus aureus receptor histidine kinase, AgrC.

We combined mRNA display technology with lipid-nanodisc based selections and identified high-affinity ligands targeting the integral membrane sensor domain of the histidine kinase AgrC as potent inhibitors of Staphylococcus aureus quorum sensing-modulated virulence. Our study highlights the potential of this integrated approach for identifying functional modulators of integral membrane proteins.

Pathogenicity Islands and Their Role in Staphylococcal Biology.

Pathogenicity islands are members of a vast collection of genomic islands that encode important virulence, antibiotic resistance and other accessory functions and have a critical role in bacterial gene transfer. Staphylococcus aureus is host to a large family of such islands, known as SaPIs, which encode super antigen and other virulence determinants, are mobilized by helper phages and transferred at extremely high frequencies. They benefit their host cells by interfering with phage predation and enhancing horizontal gene transfer. This chapter describes their life cycle, the bases of their phage interference mechanisms, their transfer system and their conversion to antibacterial agents for treatment ofstaphylococcal infections.

Identification of a Molecular Latch that Regulates Staphylococcal Virulence.

Virulence induction in the Staphylococcus aureus is under the control of a quorum sensing (QS) circuit encoded by the accessory gene regulator (agr) locus. Allelic variation within agr produces four QS specificity groups, each producing a unique secreted autoinducer peptide (AIP) and receptor histidine kinase (RHK), AgrC. Cognate AIP-AgrC interactions activate virulence through a two-component signaling cascade, whereas non-cognate pairs are generally inhibitory. Here we pinpoint a key hydrogen-bonding interaction within AgrC that acts as a switch to convert helical motions propagating from the receptor sensor domain into changes in inter-domain association within the kinase module. AgrC mutants lacking this interaction are constitutively active in vitro and in vivo, the latter leading to a pronounced attenuation of S. aureus biofilm formation. Thus, our work sheds light on the regulation of this biomedically important RHK.

Conversion of staphylococcal pathogenicity islands to CRISPR-carrying antibacterial agents that cure infections in mice.

Staphylococcus aureus and other staphylococci continue to cause life-threatening infections in both hospital and community settings. They have become increasingly resistant to antibiotics, especially ß-lactams and aminoglycosides, and their infections are now, in many cases, untreatable. Here we present a non-antibiotic, non-phage method of treating staphylococcal infections by engineering of the highly mobile staphylococcal pathogenicity islands (SaPIs). We replaced the SaPIs' toxin genes with antibacterial cargos to generate antibacterial drones (ABDs) that target the infecting bacteria in the animal host, express their cargo, kill or disarm the bacteria and thus abrogate the infection. Here we have constructed ABDs with either a CRISPR-Cas9 bactericidal or a CRISPR-dCas9 virulence-blocking module. We show that both ABDs block the development of a murine subcutaneous S. aureus abscess and that the bactericidal module rescues mice given a lethal dose of S. aureus intraperitoneally.

Staphylococcal pathogenicity islands-movers and shakers in the genomic firmament.

The staphylococcal pathogenicity islands (SaPIs) are highly mobile 15kb genomic islands that carry superantigen genes and other virulence factors and are mobilized by helper phages. Helper phages counteract the SaPI repressor to induce the SaPI replication cycle, resulting in encapsidation in phage like particles, enabling high frequency transfer. The SaPIs split from a protophage lineage in the distant past, have evolved a variety of novel and salient features, and have become an invaluable component of the staphylococcal genome. This review focuses on recent studies describing three different mechanisms of SaPI interference with helper phage reproduction and other studies demonstrating that helper phage mutations to resistance against this interference impact phage evolution. Also described are recent results showing that SaPIs contribute in a major way to lateral transfer of host genes as well as enabling their own transfer. SaPI-like elements, readily identifiable in the bacterial genome, are widespread throughout the Gram-positive cocci, though functionality has thus far been demonstrated for only a single one of these.

Functional Plasticity of the AgrC Receptor Histidine Kinase Required for Staphylococcal Virulence.

Staphylococcus aureus employs the receptor histidine kinase (RHK), AgrC, to detect quorum-sensing (QS) pheromones, the autoinducer peptides (AIPs), which regulate the virulence of the bacterium. Variation in the QS circuit divides S. aureus into four subgroups, each producing a specific AIP-AgrC pair. While the timing of QS induction is known to differ among these subgroups, the molecular basis of this phenomenon is unknown. Here, we report the successful reconstitution of several AgrC variants and show that the agonist-induced activity of the receptors varies in a manner that accounts for these temporal differences in QS induction. Our studies also reveal a key regulatory hotspot on AgrC that controls the basal activity of RHK as well as the responsiveness of the system to ligand inputs. Collectively, these studies offer insights into the capacity of the RHK for adaptive evolution.

Phage-inducible islands in the Gram-positive cocci.

The SaPIs are a cohesive subfamily of extremely common phage-inducible chromosomal islands (PICIs) that reside quiescently at specific att sites in the staphylococcal chromosome and are induced by helper phages to excise and replicate. They are usually packaged in small capsids composed of phage virion proteins, giving rise to very high transfer frequencies, which they enhance by interfering with helper phage reproduction. As the SaPIs represent a highly successful biological strategy, with many natural Staphylococcus aureus strains containing two or more, we assumed that similar elements would be widespread in the Gram-positive cocci. On the basis of resemblance to the paradigmatic SaPI genome, we have readily identified large cohesive families of similar elements in the lactococci and pneumococci/streptococci plus a few such elements in Enterococcus faecalis. Based on extensive ortholog analyses, we found that the PICI elements in the four different genera all represent distinct but parallel lineages, suggesting that they represent convergent evolution towards a highly successful lifestyle. We have characterized in depth the enterococcal element, EfCIV583, and have shown that it very closely resembles the SaPIs in functionality as well as in genome organization, setting the stage for expansion of the study of elements of this type. In summary, our findings greatly broaden the PICI family to include elements from at least three genera of cocci.

The Floating (Pathogenicity) Island: A Genomic Dessert.

Among the prokaryotic genomic islands (GIs) involved in horizontal gene transfer (HGT) are the classical pathogenicity islands, including the integrative and conjugative elements (ICEs), the gene-transfer agents (GTAs), and the staphylococcal pathogenicity islands (SaPIs), the primary focus of this review. While the ICEs and GTAs mediate HGT autonomously, the SaPIs are dependent on specific phages. The ICEs transfer primarily their own DNA, the GTAs exclusively transfer unlinked host DNA, and the SaPIs combine the capabilities of both. Thus the SaPIs derive their importance from the genes they carry (their genetic cargo) and the genes they move. They act not only as versatile high-frequency mobilizers but also as mediators of phage interference and consequently are major benefactors of their host bacteria.

An insight into staphylococcal pathogenicity island-mediated interference with phage late gene transcription.

Staphylococcal pathogenicity islands (SaPIs) are ∼15 kb chromosomally located mobile elements that parasitize "helper" phages which provide a de-repressor protein plus virion and lysis proteins which enable the release of infectious SaPI particles in very high titers. All SaPIs interfere with the reproduction of their helper phages, using 3 different mechanisms. The logic of SaPI reproduction requires that these interference mechanisms do not totally block phage production, as this would be lethal for them as well as for the phage. The discovery of 2 SaPI2 proteins that totally block phage 80 by interfering with late phage transcription was inconsistent with this principle and led to the discovery of a third protein that binds to one of the interference proteins and modulates its activity, thus preventing complete inhibition of the phage. These systems permit the SaPIs to engage in horizontal transfer of unlinked chromosomal genes as well as their own.

Virus Satellites Drive Viral Evolution and Ecology.

Virus satellites are widespread subcellular entities, present both in eukaryotic and in prokaryotic cells. Their modus vivendi involves parasitism of the life cycle of their inducing helper viruses, which assures their transmission to a new host. However, the evolutionary and ecological implications of satellites on helper viruses remain unclear. Here, using staphylococcal pathogenicity islands (SaPIs) as a model of virus satellites, we experimentally show that helper viruses rapidly evolve resistance to their virus satellites, preventing SaPI proliferation, and SaPIs in turn can readily evolve to overcome phage resistance. Genomic analyses of both these experimentally evolved strains as well as naturally occurring bacteriophages suggest that the SaPIs drive the coexistence of multiple alleles of the phage-coded SaPI inducing genes, as well as sometimes selecting for the absence of the SaPI depressing genes. We report similar (accidental) evolution of resistance to SaPIs in laboratory phages used for Staphylococcus aureus typing and also obtain the same qualitative results in both experimental evolution and phylogenetic studies of Enterococcus faecalis phages and their satellites viruses. In summary, our results suggest that helper and satellite viruses undergo rapid coevolution, which is likely to play a key role in the evolution and ecology of the viruses as well as their prokaryotic hosts.

Key driving forces in the biosynthesis of autoinducing peptides required for staphylococcal virulence.

Staphylococci produce autoinducing peptides (AIPs) as quorum-sensing signals that regulate virulence. These AIPs feature a thiolactone macrocycle that connects the peptide C terminus to the side chain of an internal cysteine. AIPs are processed from ribosomally synthesized precursors [accessory gene regulator D (AgrD)] through two proteolytic events. Formation of the thiolactone is coupled to the first of these and involves the activity of the integral membrane protease AgrB. This step is expected to be thermodynamically unfavorable, and therefore, it is unclear how AIP-producing bacteria produce sufficient amounts of the thiolactone-containing intermediate to drive quorum sensing. Herein, we present the in vitro reconstitution of the AgrB-dependent proteolysis of an AgrD precursor from Staphylococcus aureus. Our data show that efficient thiolactone production is driven by two unanticipated features of the system: (i) membrane association of the thiolactone-containing intermediate, which stabilizes the macrocycle, and (ii) rapid degradation of the C-terminal proteolysis fragment AgrD(C), which affects the reaction equilibrium position. Cell-based studies confirm the intimate link between AIP production and intracellular AgrD(C) levels. Thus, our studies explain the chemical principles that drive AIP production, including uncovering a hitherto unknown link between quorum sensing and peptide turnover.

Increasing AIP Macrocycle Size Reveals Key Features of agr Activation in Staphylococcus aureus.

The agr locus in the commensal human pathogen, Staphylococcus aureus, is a two-promoter regulon with allelic variability that produces a quorum-sensing circuit involved in regulating virulence within the bacterium. Secretion of unique autoinducing peptides (AIPs) and detection of their concentrations by AgrC, a transmembrane receptor histidine kinase, coordinates local bacterial population density with global changes in gene expression. The finding that staphylococcal virulence can be inhibited through antagonism of this quorum-sensing pathway has fueled tremendous interest in understanding the structure-activity relationships underlying the AIP-AgrC interaction. The defining structural feature of the AIP is a 16-membered, thiolactone-containing macrocycle. Surprisingly, the importance of ring size on agr activation or inhibition has not been explored. In this study, we address this deficiency through the synthesis and functional analysis of AIP analogues featuring enlarged and reduced macrocycles. Notably, this study is the first to interrogate AIP function by using both established cell-based reporter gene assays and newly developed in vitro AgrC-I binding and autophosphorylation activity assays. Based on our data, we present a model for robust agr activation involving a cooperative, three-points-of-contact interaction between the AIP macrocycle and AgrC.

An rpsL-based allelic exchange vector for Staphylococcus aureus.

Staphylococcus aureus is one of the most successful bacterial pathogens, harboring a vast repertoire of virulence factors in its arsenal. As such, the genetic manipulation of S. aureus chromosomal DNA is an important tool for the study of genes involved in virulence and survival in the host. Previously reported allelic exchange vectors for S. aureus are shuttle vectors that can be propagated in Escherichia coli, so that standard genetic manipulations can be carried out. Most of the vectors currently in use carry the temperature-sensitive replicon (pE194ts) that was originally developed for use in Bacillus subtilis. Here we show that in S. aureus, the thermosensitivity of a pE194ts vector is incomplete at standard non-permissive temperatures (42 °C), and replication of the plasmid is impaired but not abolished. We report rpsL-based counterselection vectors, with an improved temperature-sensitive replicon (pT181 repC3) that is completely blocked for replication in S. aureus at non-permissive and standard growth temperature (37 °C). We also describe a set of temperature-sensitive vectors that can be cured at standard growth temperature. These vectors provide highly effective tools for rapidly generating allelic replacement mutations and curing expression plasmids, and expand the genetic tool set available for the study of S. aureus.

Bacteriophage-mediated spread of bacterial virulence genes.

Bacteriophages are types of viruses that infect bacteria. They are the most abundant and diverse entities in the biosphere, and influence the evolution of most bacterial species by promoting gene transfer, sometimes in unexpected ways. Although pac-type phages can randomly package and transfer bacterial DNA by a process called generalized transduction, some mobile genetic elements have developed elegant and sophisticated strategies to hijack the phage DNA-packaging machinery for their own transfer. Moreover, phage-like particles (gene transfer agents) have also evolved, that can package random pieces of the producing cell's genome. The purpose of this review is to give an overview of some of the various ways by which phages and phage-like particles can transfer bacterial genes, driving bacterial evolution and promoting the emergence of novel pathogens.