An enterococcal phage protein inhibits type IV restriction enzymes involved in antiphage defense.

The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of antibiotics needed to combat these infections remains stagnant. MDR enterococci are a major contributor to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which uses lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of resistance and the mechanisms underlying this resistance are poorly defined. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) that we show restricts the replication of phage phi47 in vancomycin-resistant E. faecalis. We further find that phi47 evolves to overcome restriction by acquiring a missense mutation in a TIV-RE inhibitor protein. We show that this inhibitor, termed type IV restriction inhibiting factor A (tifA), binds and inactivates diverse TIV-REs. Overall, our findings advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages evolve to overcome antiphage defense systems.

A 44-Nucleotide Region in the Chikungunya Virus 3' UTR Dictates Viral Fitness in Disparate Host Cells.

We previously reported that deletion of a 44-nucleotide element in the 3' untranslated region (UTR) of the Chikungunya virus (CHIKV) genome enhances the virulence of CHIKV infection in mice. Here, we find that while this 44-nucleotide deletion enhances CHIKV fitness in murine embryonic fibroblasts in a manner independent of the type I interferon response, the same mutation decreases viral fitness in C6/36 mosquito cells. Further, the fitness advantage conferred by the UTR deletion in mammalian cells is maintained in vivo in a mouse model of CHIKV dissemination. Finally, SHAPE-MaP analysis of the CHIKV 3' UTR revealed this 44-nucleotide element forms a distinctive two-stem-loop structure that is ablated in the mutant 3' UTR without altering additional 3' UTR RNA secondary structures.

Staphylococcus aureus oleate hydratase produces ligands that activate host PPARα.

Commensal gut bacteria use oleate hydratase to release a spectrum of hydroxylated fatty acids using host-derived unsaturated fatty acids. These compounds are thought to attenuate the immune response, but the underlying signaling mechanism(s) remain to be established. The pathogen Staphylococcus aureus also expresses an oleate hydratase and 10-hydroxyoctadecanoic acid (h18:0) is the most abundant oleate hydratase metabolite found at Staphylococcal skin infection sites. Here, we show h18:0 stimulates the transcription of a set of lipid metabolism genes associated with the activation of peroxisome proliferator activated receptor (PPAR) in the RAW 264.7 macrophage cell line and mouse primary bone marrow-derived macrophages. Cell-based transcriptional reporter assays show h18:0 selectively activates PPARα. Radiolabeling experiments with bone marrow-derived macrophages show [1-14C]h18:0 is not incorporated into cellular lipids, but is degraded by ß-oxidation, and mass spectrometry detected shortened fragments of h18:0 released into the media. The catabolism of h18:0 was >10-fold lower in bone marrow-derived macrophages isolated from Ppara -/- knockout mice, and we recover 74-fold fewer S. aureus cells from the skin infection site of Ppara -/- knockout mice compared to wildtype mice. These data identify PPARα as a target for oleate hydratase-derived hydroxy fatty acids and support the existence of an oleate hydratase-PPARα signaling axis that functions to suppress the innate immune response to S. aureus.

Convergent impact of vaccination and antibiotic pressures on pneumococcal populations.

Streptococcus pneumoniae is a remarkably adaptable and successful human pathogen, playing dual roles of both asymptomatic carriage in the nasopharynx and invasive disease including pneumonia, bacteremia, and meningitis. Efficacious vaccines and effective antibiotic therapies are critical to mitigating morbidity and mortality. However, clinical interventions can be rapidly circumvented by the pneumococcus by its inherent proclivity for genetic exchange. This leads to an underappreciated interplay between vaccine and antibiotic pressures on pneumococcal populations. Circulating populations have undergone dramatic shifts due to the introduction of capsule-based vaccines of increasing valency imparting strong selective pressures. These alterations in population structure have concurrent consequences on the frequency of antibiotic resistance profiles in the population. This review will discuss the interactions of these two selective forces. Understanding and forecasting the drivers of antibiotic resistance and capsule switching are of critical importance for public health, particularly for such a genetically promiscuous pathogen as S. pneumoniae.

Evolution of a small phage protein overcomes antiphage defense in Enterococcus faecalis.

The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of new antibiotics needed to combat these infections remains stagnant. MDR enterococci, which are a common cause of hospital-acquired infections, are emerging as one of the major contributors to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which entails the use of lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of phage resistance and the mechanisms underlying this resistance are unknown. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from vancomycin-resistant Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) and we show that this enzyme is sufficient to restrict the replication of the lytic phage in E. faecalis. We further find that phages can evolve to overcome restriction by acquiring a missense mutation in a novel TIV-RE inhibitor protein encoded by many enterococcal phages. We show that this inhibitor, which we have named anti-restriction-factor A (arfA), directly binds to and inactivates diverse TIV-REs. Overall, our findings significantly advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages can evolve to overcome antiphage defense systems.

Genetically distant bacteriophages select for unique genomic changes in Enterococcus faecalis.

The human microbiota harbors diverse bacterial and bacteriophage (phage) communities. Bacteria evolve to overcome phage infection, thereby driving phage evolution to counter bacterial resistance. Understanding how phages select for genetic alterations in medically relevant bacteria is important as phages become established biologics for the treatment of multidrug-resistant (MDR) bacterial infections. Before phages can be widely used as standalone or combination antibacterial therapies, we must obtain a deep understanding of the molecular mechanisms of phage infection and how host bacteria alter their genomes to become resistant. We performed coevolution experiments using a single Enterococcus faecalis strain and two distantly related phages to determine how phage pressure impacts the evolution of the E. faecalis genome. Whole-genome sequencing of E. faecalis following continuous exposure to these two phages revealed mutations previously demonstrated to be essential for phage infection. We also identified mutations in genes previously unreported to be associated with phage infection in E. faecalis. Intriguingly, there was only one shared mutation in the E. faecalis genome that was selected by both phages tested, demonstrating that infection by two genetically distinct phages selects for diverse variants. This knowledge serves as the basis for the continued study of E. faecalis genome evolution during phage infection and can be used to inform the design of future therapeutics, such as phage cocktails, intended to target MDR E. faecalis.

Let Me Upgrade You: Impact of Mobile Genetic Elements on Enterococcal Adaptation and Evolution.

Enterococci are Gram-positive bacteria that have evolved to thrive as both commensals and pathogens, largely due to their accumulation of mobile genetic elements via horizontal gene transfer (HGT). Common agents of HGT include plasmids, transposable elements, and temperate bacteriophages. These vehicles of HGT have facilitated the evolution of the enterococci, specifically Enterococcus faecalis and Enterococcus faecium, into multidrug-resistant hospital-acquired pathogens. On the other hand, commensal strains of Enterococcus harbor CRISPR-Cas systems that prevent the acquisition of foreign DNA, restricting the accumulation of mobile genetic elements. In this review, we discuss enterococcal mobile genetic elements by highlighting their contributions to bacterial fitness, examine the impact of CRISPR-Cas on their acquisition, and identify key areas of research that can improve our understanding of enterococcal evolution and ecology.

Bacteriophage Resistance Alters Antibiotic-Mediated Intestinal Expansion of Enterococci.

Enterococcus faecalis is a human intestinal pathobiont with intrinsic and acquired resistance to many antibiotics, including vancomycin. Nature provides a diverse and virtually untapped repertoire of bacterial viruses, or bacteriophages (phages), that could be harnessed to combat multidrug-resistant enterococcal infections. Bacterial phage resistance represents a potential barrier to the implementation of phage therapy, emphasizing the importance of investigating the molecular mechanisms underlying the emergence of phage resistance. Using a cohort of 19 environmental lytic phages with tropism against E. faecalis, we found that these phages require the enterococcal polysaccharide antigen (Epa) for productive infection. Epa is a surface-exposed heteroglycan synthesized by enzymes encoded by both conserved and strain-specific genes. We discovered that exposure to phage selective pressure favors mutation in nonconserved epa genes both in culture and in a mouse model of intestinal colonization. Despite gaining phage resistance, epa mutant strains exhibited a loss of resistance to cell wall-targeting antibiotics. Finally, we show that an E. faecalisepa mutant strain is deficient in intestinal colonization, cannot expand its population upon antibiotic-driven intestinal dysbiosis, and fails to be efficiently transmitted to juvenile mice following birth. This study demonstrates that phage therapy could be used in combination with antibiotics to target enterococci within a dysbiotic microbiota. Enterococci that evade phage therapy by developing resistance may be less fit at colonizing the intestine and sensitized to vancomycin, preventing their overgrowth during antibiotic treatment.