Deciphering Gram-negative phage-inducible chromosomal island strategies for spreading in nature

The concept that bacterial genomes within a single species can vary widely in gene content is not new. However, it was only with the arrival of the genomic era that the phenomenon has been properly understood. Not only was the genome size different; a significant number of the genes present in different strains from a specific species were not even related i.e. had no homologous genes in the others. Most of these genes were carried on mobile genetics elements (MGEs), including plasmids, bacteriophages, pathogenicity islands, transposons or integrons, which can be potentially transferred among bacteria. Thus, it is now assumed that horizontal gene transfer (HGT) has had an extremely important role in bacterial evolution. Indeed it has been estimated that some 20% of the extant genetic content of any given bacterial species has been acquired from other organisms. Perhaps half of this consists of MGEs, which have moved freely within and between species, and have occasionally crossed intergeneric boundaries.
In facultative pathogens, MGEs are largely responsible for antibiotic resistance, environmental adaptations and the wide variety of adaptations to life in host tissues that we perceive as pathogenesis. In most pathogenic bacteria, all known classes of bacterial MGEs may contribute to pathogenesis, and it is particularly striking that essentially all of the bacterial toxins that cause specific toxin-mediated diseases - toxinoses - such as PVL pneumonia, diphtheria, dysentery, toxic shock syndrome, food poisoning, necrotizing pneumonia, scalded skin syndrome, botulism, hemolytic-uremic syndrome or necrotizing fasciitis, are encoded by MGEs.
This application represents the culmination of a long and highly productive research program starting in 2003 and extending to the present. During this time we have characterised a novel family of mobile staphylococcal pathogenicity islands, the SaPIs, which are the only source of several important superantigens, including toxic shock syndrome toxin-1 and enterotoxins B and C, as well as the source for other virulence factors related to host adaptation. Not surprisingly, these elements are not just confined to the Staphylococci, but are widespread within Gram-positive bacteria. Recently, we have also demonstrated that similar elements occur widely in Gram-negative bacteria, conforming a unique class of MGEs, the phage-inducible chromosomal islands (PICIs). We suggest that the PICIs have spread widely throughout the bacterial world, and have diverged much more slowly than their host organisms. If true, these findings represent the discovery of a new class of MGE, which have a broad impact on lateral gene transfer and virulence in the bacterial world.
Although our previous studies have deciphered how the PICI elements present in the Gram-positive bacteria are induced and horizontally transferred, two main questions remain to be determined in the biology of the Gram-negative PICIs: i) how these elements sense the presence of their helper phages, and ii) how they hijack the phage machinery for their own specific packaging, blocking phage reproduction. To decipher these two processes is of vital importance to understand how these elements spread in nature. In this project we will answer these questions. By achieving these objectives we will establish new paradigms involving pathogenicity islands in bacterial evolution and virulence, and will provide strategies to block pathogenicity island dissemination and the emergence of novel bacterial virulent clones.

Bacteria are successful as commensal organisms or pathogens partly because they rapidly adapt to selective pressures imparted by the human host. Mobile genetic elements (MGEs) play a central role in this adaptation process and are a means to transfer genetic information (DNA) among and within bacterial species. Importantly, MGEs encode putative virulence factors and molecules that confer resistance to antibiotics. Inasmuch as bacterial infections are a significant problem worldwide and continue to emerge in epidemic waves, there has been significant effort to understand the agents that effect DNA movement.
In recent years, we have extensively characterised a family of pathogenicity islands in Staphylococcus aureus, SaPIs, which contribute substantively to horizontal gene transfer, host adaptation and virulence. We have recently demonstrated that similar elements occur widely in bacteria, that these are not defective prophages, despite their annotations in GenBank, but are rather a unique class of MGEs, the phage-inducible chromosomal islands (PICIs).
Although our studies have confirmed the universality of the PICIs, there are two important questions related to the biology of the PICIs present in the Gram-negative bacteria that remain to be answered: i) how these elements sense the presence of their helper phages; and ii) how they hijack the phage machinery for their own specific packaging, blocking phage reproduction. Note that these are the two key processes that define the ability of these elements to spread in nature, generating novel virulent and resistant clones. Answering these questions will be the main goal of the current proposal. Our studies will greatly enhance understanding of the evolution of pathogenic bacteria and apparition of novel virulence clones through the acquisition of MGEs, especially across genera.

Bacterial genomes are in constant evolution, with the continual emergence of new virulent clones. However, the mechanistic basis for this process is not well understood. In spite of their evident role in bacterial pathogenesis, environmental and genetic factors that regulate transfer of MGEs in bacterial populations are largely unknown. In fact, and paradoxically, the hospital environment, where the pressure to eliminate pathogenic bacteria is highest, has evolved as one of the major forces promoting antibiotic resistance and increasing the arsenal of virulence genes in the most clinically relevant pathogenic bacteria.
We propose that the novel PICI family of MGEs is a key factor in the pathogenesis of most clinically-relevant bacterial pathogens and in the emergence of novel virulent clones. In support of this, the prototypical member of the family, the SaPIs, have a high impact on the virulence of S. aureus. In addition, these elements have been also involved in the adaptation of S. aureus to new hosts and in the development of new virulent clones, clearly confirming their role in pathogenesis. Understanding the biology of the members of this family, how they evolve and how they are transferred, will provide new strategies to prevent the emergence of new virulent clones as well as new strategies to combat bacterial infections.
Because of the multi-faceted, cross-disciplinary and wide-ranging nature of the proposed project, a relatively large number of academic disciplines will benefit, both nationally and internationally, from the work; these include bacteriology, virology, immunology, cell biology, vaccinology or pharmacology.
In addition, the development of novel approaches for studying bacterial evolution and virulence, together with the identification of potential vaccine antigens and drug targets, will make a significant contribution to combating an important human infectious diseases and will have a major impact on improving human health, a MRC priority area. These data may be of value to companies with an interest in treating bacterial infections. In this way, we feel the project could be of great value to industry.