Phage infection and sub-lethal antibiotic exposure mediateEnterococcus faecalistype VII secretion system dependent inhibition of bystander bacteria

Abstract

Enterococci constitute a minor component of the healthy human microbiota (1). Enterococci, 54 including Enterococcus faecalis, are also nosocomial pathogens that cause a variety of diseases, 55 including sepsis, endocarditis, surgical-site, urinary tract and mixed bacterial infections (2, 3). 56Over recent decades, enterococci have acquired extensive antibiotic resistance traits, including 57 resistance to "last-resort" antibiotics such as vancomycin, daptomycin, and linezolid (4-8). 58Following antibiotic therapy, multi-drug resistant (MDR) enterococci can outgrow to become a 59 dominant member of the intestinal microbiota, resulting in intestinal barrier invasion and blood 60 stream infection (7, 9). The ongoing evolution of MDR enterococci in healthcare settings (4-6, 10, 61 11) and their ability to transmit antibiotic resistance among diverse bacteria (9, 12-15), emphasize 62 the immediate need for novel therapeutic approaches to control enterococcal infections. 63Viruses that infect and kill bacteria (bacteriophages or phages) are receiving attention for their 64 use as antibacterial agents (16). Recent studies have demonstrated the efficacy of anti-65 enterococcal phages in murine models of bacteremia (17)(18)(19) and the administration of phages to 66 reduce E. faecalis burden in the intestine gives rise to phage resistant isolates that are 67 resensitized to antibiotics (20). Considering phages are highly specific for their target bacterium, 68 coupled with the self-limiting nature of their host-dependent replication, suggests that unlike 69 antibiotics which have broad off-target antimicrobial activity, phages are likely to have nominal 70 impact on bacteria outside of their intended target strain (21-23). However, our understanding of 71 how phages interact with bacteria and the bacterial response to phage infection is limited. 72While studying the transcriptional response of phage infected E. faecalis cells, we discovered 73 that phage infection induced the expression of genes involved in the biosynthesis of a type VIIb 74 secretion system (T7SS) (24). Firmicutes, including the enterococci, harbor diverse T7SS genes 75 encoding transmembrane and cytoplasmic proteins involved in the secretion of protein substrates 76 (25), and T7SSs promote antagonism of non-kin bacterial cells through contact-dependent 77 secretion of antibacterial effectors and/or toxins (26, 27). The antibacterial activity of T7SSs from 78 96 Results 97 Phage mediated induction of E. faecalis T7SS leads to interspecies antagonism. 98A hallmark feature of phage therapy is that because phages often have a narrow host range, 99 they will not influence the growth of non-susceptible bacteria occupying the same niche (22). We 100 discovered that infection of E. faecalis OG1RF by phage VPE25, induces the expression of T7SS 101 genes (24). The E. faecalis OG1RF T7SS locus is absent in the commonly studied vancomycin-102 resistant strain V583, despite conservation of flanking genes (Fig. 1A) (32, 33). Homologs of the 103 E. faecalis T7SS ...

Activation of a bacterial killing machine

“…While cell wall-targeting antibiotics had little effect on T7SS expression, daptomycin which targets the cell membrane resulted in T7SS gene up-regulation. Curiously, a similar up-regulation was also seen when cells were treated with DNA-targeting antibiotics ciprofloxacin or mitomycin C. In agreement with the observed up-regulation of the T7SS genes, treatment with daptomycin was also shown to induce T7SS-dependent inhibition of bystander bacteria ( [19]; Fig 1).…”

“…Among the observations made by Chatterjee and colleauges, it was surprising to note that cell wall targeting antibiotics did not affect regulation of the T7 genes [ 19 ]. This is perhaps unexpected since it is well documented that IreK responds to cell wall stresses [ 24 ].…”

“…The study by Chaterjee and colleagues has investigated environmental cues that activate the T7SS in the firmicute Enterococcus faecalis [19], a bacterium found in the gut microbiota of healthy individuals, which is an opportunistic pathogen capable of causing severe infection [20]. Previous work investigating phage therapy as an alternative treatment for drug-resistant E. faecalis noted that phage-infected cells induce expression of the T7SS locus [21].…”

Structure of the Extracellular Region of the Bacterial Type VIIb Secretion System Subunit EsaA

“…The interkingdom-targeting capability of T7SSb has also been demonstrated in the opportunistic pathogen Streptococcus intermedius, with the antibacterial activity being attributed to the NAD + hydrolase effector TelB and the cell wall precursor degrading effector TelC (Hasegawa et al, 2017;Klein et al, 2018;Whitney et al, 2017). The T7SSb pathways of Bacillus subtilis and Enterococcus faecalis were also recently shown to antagonize competitor bacteria (Tassinari et al, 2020;Chatterjee et al, 2020).…”

Structure of the extracellular region of the bacterial type VIIb secretion system subunit EsaA

“…The interkingdom-targeting capability of the T7SSb has also been demonstrated in the opportunistic pathogen Streptococcus intermedius with the antibacterial activity being attributed to the NAD + hydrolase effector TelB and the cell wall precursor degrading effector TelC (Hasegawa et al, 2017;Klein et al, 2018;Whitney et al, 2017). The T7SSb pathways of Bacillus subtilis and Enterococcus faecalis were also recently shown to antagonize competitor bacteria (Tassinari et al, 2020;Chatterjee et al, 2020).…”