Biology subjects

Duerkop, B. A.

Publications and source records attributed to Duerkop, B. A..

21 records · Page 2Linked to original sources

bioRxiv2019

Parallel genomics uncover novel enterococcal-bacteriophage interactions

Bacteriophages (phages) have been proposed as alternative therapeutics for the treatment of multidrug resistant bacterial infections. However, there are major gaps in our understanding of the molecular events in bacterial cells that control how bacteria respond to phage predation. Using the model organism Enterococcus faecalis, we employed two distinct genomic approaches, transposon (Tn) library screening and RNA sequencing, to investigate the interaction of E. faecalis with a virulent phage. We discovered that a transcription factor encoding a LytR family response regulator controls the expression of enterococcal polysaccharide antigen (epa) genes that are involved in phage infection and bacterial fitness. In addition, we discovered that DNA mismatch repair mutants rapidly evolve phage adsorption deficiencies, underpinning a molecular basis for epa mutation during phage infection. Transcriptomic profiling of phage infected E. faecalis revealed broad transcriptional changes influencing viral replication and progeny burst size. We also demonstrate that phage infection alters the expression of bacterial genes associated with intra and inter-bacterial interactions, including genes involved in quorum sensing and polymicrobial competition. Together our results suggest that phage predation has the potential to influence complex microbial behavior and may dictate how bacteria respond to external environmental stimuli. These responses could have collateral effects (positive or negative) on microbial communities such as the host microbiota during phage therapy. ImportanceWe lack fundamental understanding of how phage infection influences bacterial gene expression and consequently how bacterial responses to phage infection affect the assembly of polymicrobial communities. Using parallel genomic approaches, we have discovered novel transcriptional regulators and metabolic genes that influence phage infection. The integration of whole genome transcriptomic profiling during phage infection has revealed the differential regulation of genes important for group behaviors and polymicrobial interactions. Our work suggests that therapeutic phages could more broadly influence bacterial community composition outside of their intended host targets.

microbiology↗

bioRxiv2019

Conjugative delivery of CRISPR-Cas9 for the selective depletion of antibiotic-resistant enterococci

The innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiont Enterococcus faecalis is associated with HAIs and some strains are MDR. Therefore, novel strategies to control E. faecalis populations are needed. We previously characterized an E. faecalis Type II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers to E. faecalis for the selective removal of antibiotic resistance genes. Using in vitro competition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistant E. faecalis by several orders of magnitude. Finally, we show that E. faecalis donor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinants in vivo. Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.\n\nImportanceCRISPR-Cas nucleic acid targeting systems hold promise for the amelioration of multidrug-resistant enterococci, yet the utility of such tools in the context of the intestinal environment where enterococci reside is understudied. We describe the development of a CRISPR-Cas antimicrobial, deployed on a conjugative plasmid, for the targeted removal of antibiotic resistance genes from intestinal Enterococcus faecalis. We demonstrate that CRISPR-Cas targeting reduces antibiotic resistance of E. faecalis by several orders of magnitude in the intestine. Although barriers exist that influence the penetrance of the conjugative CRISPR-Cas antimicrobial among target recipient E. faecalis cells, the removal of antibiotic resistance genes in E. faecalis upon uptake of the CRISPR-Cas antimicrobial is absolute. In addition, cells that obtain the CRISPR-Cas antimicrobial are immunized against the acquisition of new antibiotic resistance genes. This study suggests a potential path toward plasmid based CRISPR-Cas therapies in the intestine.

microbiology↗

bioRxiv2019

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 multi-drug 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 non-conserved 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 the cell wall targeting antibiotics, vancomycin and daptomycin. Finally, we show that an E. faecalis epa 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.

microbiology↗