Periodic Reporting for period 3 - CoPathoPhage (Pathogen-phage cooperation during mammalian infection)
Periodo di rendicontazione: 2022-10-01 al 2024-03-31
Most bacterial pathogens are lysogens, namely carry DNA of phages within their genome, referred to as prophages. While these prophages have the potential to turn under stress into infective viruses which kill their host bacterium in a matter of minutes, it is unclear how pathogens manage to survive this internal threat under the stresses imposed by their invasion into mammalian cells. In the proposed project, we aim to study the hypothesis that phages cooperate with the bacterial hosts in the mammalian environment, and thus support their virulence. Several years ago, we uncovered a novel pathogen-phage interaction, in which an infective phage promotes the virulence of its host, the bacterial pathogen Listeria monocytogenes (Lm), via adaptive behaviour. More recently, we discovered that the prophage, though fully infective, is non-autonomous, and is completely dependent on another phage element (cryptic) that inhabit the Lm chromosome. These findings lead us to propose that the intimate cross-regulatory interactions between all phage elements within the bacterial chromosome (infective and cryptic), are crucial in promoting bacteria-phage patho-adaptive behaviours in the mammalian niche and thereby bacterial virulence. In this project, we investigate specific cross-regulatory and cooperative interactions between the phage elements, study the domestication of phage derived regulatory factors, and examine the hypothesis that they collectively form an auxiliary regulatory system that tempers infective phages. Finally, we examine the idea that the mammalian niche drives the evolution of temperate phages into patho-adaptive phages, and phages that lack this adaptation kill their hosts during mammalian infection. This work is expected to provide novel insights into bacteria-phage coexistence in mammalian environments and to facilitate the development of innovative phage therapy strategies.
In this project, we identified a novel and intriguing cross regulatory interaction between the two phage elements that inhabit the Lm chromosome. We identified and characterized a new phage regulator, named AriS, that controls the two phage elements under SOS conditions, bearing the capacity to concomitantly inhibit their lytic induction. In the course of these studies, we found that RecA is directly involved in the induction of the phage elements, and that AriS inhibits RecA, thereby preventing the induction of the phage elements and SOS stress. Further we found that AriS is a two-domain protein that possesses two distinct activities, one regulating the genes of its encoding phage and the other downregulating the bacterial SOS response. While the first activity associated with its N-terminal AntA/AntB domain, the second associated with its C-terminal ANT/KilAC domain. Interestingly, the ANT/KilAC domain was found to be conserved in many AriS-like proteins of listerial and non-listerial prophages, suggesting that temperate phages acquired such dual-function regulators to align their response with the other phage elements that reside the genome.
Exploring other phage remnant elements of Lm, we investigated a conserved genetic locus that contains phage related genes. We found these genes to be transcriptionally upregulated during Lm’s growth in intracellular conditions. We found that this element encodes a metzincin and TIMP-like proteins, that interact in a manner rarely recorded—the TIMP-like lipoprotein is adapted to be cleaved and released to the culture medium by the metzincin. The release of the TIMP-like leads to changes in the bacterial cell morphology and to an increase in bacterial sensitivity to phage lysins and drugs that target the cell wall. This locus may therefore have therapeutic use: to make otherwise resistant bacteria more susceptible to lytic factors and compounds, such as penicillin and phage-encoded lysins, which play an important role in phage therapy.
Exploring other phage remnant elements of Lm, we investigated a conserved genetic locus that contains phage related genes. We found these genes to be transcriptionally upregulated during Lm’s growth in intracellular conditions. We found that this element encodes a metzincin and TIMP-like proteins, that interact in a manner rarely recorded—the TIMP-like lipoprotein is adapted to be cleaved and released to the culture medium by the metzincin. The release of the TIMP-like leads to changes in the bacterial cell morphology and to an increase in bacterial sensitivity to phage lysins and drugs that target the cell wall. This locus may therefore have therapeutic use: to make otherwise resistant bacteria more susceptible to lytic factors and compounds, such as penicillin and phage-encoded lysins, which play an important role in phage therapy.
Our findings brought us to suggest that temperate phages acquired mechanisms to manipulate host processes, including the SOS response (and RecA specifically), for their own benefit. Our data support the premise that regulation of the SOS system by temperate phages and other mobile genetic elements, is a common strategy that support the interaction with the host. In this respect, AriS represents yet another player in the intricate interaction between bacteria and temperate phages.
By the end of the project we expect to uncover additional determinants and mechanisms that are involved in the regulation of the phage elements, and make a true progress in understanding the biological impact of inter-phage cross-talk in supporting bacterial virulence.
By the end of the project we expect to uncover additional determinants and mechanisms that are involved in the regulation of the phage elements, and make a true progress in understanding the biological impact of inter-phage cross-talk in supporting bacterial virulence.