Samuel Kilcher - Academia.edu (original) (raw)

Papers by Samuel Kilcher

Nature Microbiology

At the end of a lytic bacteriophage replication cycle in Gram-positive bacteria, peptidoglycan-de... more At the end of a lytic bacteriophage replication cycle in Gram-positive bacteria, peptidoglycan-degrading endolysins that cause explosive cell lysis of the host can also attack non-infected bystander cells. Here we show that in osmotically stabilized environments, Listeria monocytogenes can evade phage predation by transient conversion to a cell wall-deficient L-form state. This L-form escape is triggered by endolysins disintegrating the cell wall from without, leading to turgor-driven extrusion of wall-deficient, yet viable L-form cells. Remarkably, in the absence of phage predation, we show that L-forms can quickly revert to the walled state. These findings suggest that L-form conversion represents a population-level persistence mechanism to evade complete eradication by phage attack. Importantly, we also demonstrate phage-mediated L-form switching of the urinary tract pathogen Enterococcus faecalis in human urine, which underscores that this escape route may be widespread and has ...

The rapid detection and species-level differentiation of bacterial pathogens facilitates antibiot... more The rapid detection and species-level differentiation of bacterial pathogens facilitates antibiotic stewardship and improves disease management. Here, we develop a rapid bacteriophage-based diagnostic assay to detect the most prevalent pathogens causing urinary tract infections:Escherichia coli, Klebsiellaspp., andEnterococcusspp. For each uropathogen, two virulent phages were genetically engineered to express a nanoluciferase reporter gene upon host infection. Using 206 patient urine samples, reporter phage-induced bioluminescence was quantified to identify bacteriuria and the assay was benchmarked against conventional urinalysis. Overall,E. coli, Klebsiellaspp., andEnterococcusspp. were each detected with high sensitivity (68%, 78%, 85%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 96%) at a resolution of ⩾103CFU/ml within 5 h. We further demonstrate how bioluminescence in urine can be used to predict phage antibacterial activity, demonstrating the future potential of rep...

Bacteriophages kill bacteria by osmotic lysis towards the end of the lytic cycle. In the case of ... more Bacteriophages kill bacteria by osmotic lysis towards the end of the lytic cycle. In the case of Gram-positive bacteria, peptidoglycan-degrading endolysins released at the end of infection cycle cause explosive cell lysis not only of the infected host, but can also attack non-infected bystander cells. Here, we show that in osmotically stabilized environments, Listeria monocytogenes can evade phage predation by transient conversion to a cell wall-deficient Lform state. This L-form escape is triggered by endolysins disintegrating the cell wall from without, leading to turgor-driven extrusion of wall-deficient, yet viable L-form cells. Remarkably, in absence of phage predation, we show that L-forms can quickly revert to the walled state. These findings suggest that L-form conversion represents a population-level persistence mechanism to evade complete eradication by phage attack. Importantly, we also demonstrate phage-mediated L-form switching of the urinary tract pathogen Enterococcus faecalis in human urine, which underscores that this escape route may be widespread and has important implications for phageand endolysin-based therapeutic interventions. .

Current Opinion in Virology, 2022

The alarming rise in antimicrobial resistance coupled with a lack of innovation in antibiotics ha... more The alarming rise in antimicrobial resistance coupled with a lack of innovation in antibiotics has renewed interest in the development of alternative therapies to combat bacterial infections. Despite phage therapy demonstrating success in various individual cases, a comprehensive and unequivocal demonstration of the therapeutic potential of phages remains to be shown. The co-evolution of phages and their bacterial hosts resulted in several inherent limitations for the use of natural phages as therapeutics such as restricted host range, moderate antibacterial efficacy, and frequent emergence of phage-resistance. However, these constraints can be overcome by leveraging recent advances in synthetic biology and genetic engineering to provide phages with additional therapeutic capabilities, improved safety profiles, and adaptable host ranges. Here, we examine different ways phages can be engineered to deliver heterologous therapeutic payloads to enhance their antibacterial efficacy and discuss their versatile applicability to combat bacterial pathogens.

Viruses, 2020

Fast and reliable detection of bacterial pathogens in clinical samples, contaminated food product... more Fast and reliable detection of bacterial pathogens in clinical samples, contaminated food products, and water supplies can drastically improve clinical outcomes and reduce the socio-economic impact of disease. As natural predators of bacteria, bacteriophages (phages) have evolved to bind their hosts with unparalleled specificity and to rapidly deliver and replicate their viral genome. Not surprisingly, phages and phage-encoded proteins have been used to develop a vast repertoire of diagnostic assays, many of which outperform conventional culture-based and molecular detection methods. While intact phages or phage-encoded affinity proteins can be used to capture bacteria, most phage-inspired detection systems harness viral genome delivery and amplification: to this end, suitable phages are genetically reprogrammed to deliver heterologous reporter genes, whose activity is typically detected through enzymatic substrate conversion to indicate the presence of a viable host cell. Infection...

Current Opinion in Biotechnology, 2021

The antimicrobial and therapeutic efficacy of bacteriophages is currently limited, mostly due to ... more The antimicrobial and therapeutic efficacy of bacteriophages is currently limited, mostly due to rapid emergence of phage-resistance and the inability of most phage isolates to bind and infect a broad range of clinical strains. Here, we discuss how phage therapy can be improved through recent advances in genetic engineering. First, we outline how receptor-binding proteins and their relevant structural domains are engineered to redirect phage specificity and to avoid resistance. Next, we summarize how phages are reprogrammed as prokaryotic gene therapy vectors that deliver antimicrobial 'payload' proteins, such as sequence-specific nucleases, to target defined cells within complex microbiomes. Finally, we delineate big data- and novel artificial intelligence-driven approaches that may guide the design of improved synthetic phage in the future.

Cell Host & Microbe, 2020

Highlights d Listeria anti-CRISPR protein AcrIIA1 serves as an anti-CRISPR and a vital autorepres... more Highlights d Listeria anti-CRISPR protein AcrIIA1 serves as an anti-CRISPR and a vital autorepressor d The strong early acr promoter must be repressed for maximal phage fitness d AcrIIA1 allows prophages to tune Acr expression to Cas9 levels d AcrIIA1 homologs have been co-opted by host bacteria as ''anti-anti-CRISPRs''

Applied and Environmental Microbiology, 2020

Culture-dependent methods are the gold standard for sensitive and specific detection of pathogeni... more Culture-dependent methods are the gold standard for sensitive and specific detection of pathogenic bacteria within the food production chain. In contrast to molecular approaches, these methods detect viable cells, which is a key advantage for foods generated from heat-inactivated source material. However, culture-based diagnostics are typically much slower than molecular or proteomic strategies. Reporter phage assays combine the best of both worlds and allow for near online assessment of microbial safety because phage replication is extremely fast, highly target specific, and restricted to metabolically active host cells. In addition, reporter phage assays are inexpensive and do not require highly trained personnel, facilitating their on-site implementation. The reporter phages presented in this study not only allow for rapid detection but also enable an early estimation of the potential virulence ofListeriaisolates from food production and processing sites.

Cell Reports, 2019

Highlights d Adaptation of Listeria phage serovar specificity through targeted RBP variations d H... more Highlights d Adaptation of Listeria phage serovar specificity through targeted RBP variations d High-resolution crystal structure of a Listeria phage receptor binding protein d Structure-guided design of RBP chimeras yields phages with predictable host ranges d Synthetic RBPs extend phage binding specificity (from SV 4b to 4a, 4b, 4d, 5, 6b)

PLOS Pathogens, 2019

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and r... more The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen.

SUMMARYBacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy foreign DNA. Bacteriop... more SUMMARYBacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy foreign DNA. Bacteriophages, in turn, have evolved diverse “anti-CRISPR” proteins (Acrs) to counteract acquired immunity. InListeria monocytogenes, prophages encode 2-3 distinct anti-Cas9 proteins, withacrIIA1always present; however, its mechanism is unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain and, inListeria, triggers Cas9 degradation. AcrIIA1 displays broad-spectrum inhibition of Type II-A and II-C Cas9s, including an additional highly-divergedListeriaCas9. During lytic infection, AcrIIA1 is insufficient for rapid Cas9 inactivation, thus phages require an additional “partner” Acr that rapidly blocks Cas9-DNA-binding. The AcrIIA1 N-terminal domain (AcrIIA1NTD) is dispensable for anti-CRISPR activity; instead it is required for optimal phage replication through direct transcriptional repression of the anti-CRISPR locus. AcrIIA1NTDis widespread amongstFir...

Nucleic Acids Research, 2018

CRISPR-Cas systems provide bacteria with adaptive immunity against invading DNA elements includin... more CRISPR-Cas systems provide bacteria with adaptive immunity against invading DNA elements including bacteriophages and plasmids. While CRISPR technology has revolutionized eukaryotic genome engineering, its application to prokaryotes and their viruses remains less well established. Here we report the first functional CRISPR-Cas system from the genus Listeria and demonstrate its native role in phage defense. LivCRISPR-1 is a type II-A system from the genome of L. ivanovii subspecies londoniensis that uses a small, 1078 amino acid Cas9 variant and a unique NNACAC protospacer adjacent motif. We transferred LivCRISPR-1 cas9 and trans-activating crRNA into Listeria monocytogenes. Along with crRNA encoding plasmids, this programmable interference system enables efficient cleavage of bacterial DNA and incoming phage genomes. We used LivCRISPR-1 to develop an effective engineering platform for large, non-integrating Listeria phages based on allelic replacement and CRISPR-Cas-mediated counterselection. The broad host-range Listeria phage A511 was engineered to encode and express lysostaphin, a cell wall hydrolase that specifically targets Staphylococcus peptidoglycan. In bacterial co-culture, the armed phages not only killed Listeria hosts but also lysed Staphylococcus cells by enzymatic collateral damage. Simultaneous killing of unrelated bacteria by a single phage demonstrates the potential of CRISPR-Casassisted phage engineering, beyond single pathogen control.

Proceedings of the National Academy of Sciences, 2018

Significance The unique host specificity and antimicrobial activity of bacterial viruses have ins... more Significance The unique host specificity and antimicrobial activity of bacterial viruses have inspired many diagnostic and antibacterial applications in industry, agriculture, and medicine. Because of the rise in antibiotic-resistant infections, phage therapy is a reemerging field of interest. Many restrictions that are associated with the use of native, isolated phages can be overcome by genetic engineering. Thus, efficient genome-editing tools are needed to unleash the full potential of phage therapy and biotechnology. In vitro assembly of synthetic virus genomes and the rapid isolation of corresponding phages is an important step in this direction. By using L-form bacteria as rebooting compartments of synthetic genomes, we report a simple, highly efficient, and broadly applicable technology that enables engineering of diverse phage families.

Trends in Microbiology, 2018

Viruses of bacteria (bacteriophages or phages) are highly evolved nanomachines that recognize bac... more Viruses of bacteria (bacteriophages or phages) are highly evolved nanomachines that recognize bacterial cell walls, deliver genetic information, and kill or transform their targets with unparalleled specificity. For a long time, the use of genetically modified phages was limited to phage display approaches and fundamental research. This is mostly because phage engineering has been a complex and time-consuming task, applicable for only a few well characterized model phages. Recent advances in sequencing technology and molecular biology gave rise to rapid and precise tools that enable modification of lesswell-characterized phages. These methods will pave the way for the development of modular designer-phages as versatile biologics that efficiently control multidrug-resistant bacteria and provide novel tools for pathogen detection, drug development, and beyond. Bacteriophages as Antimicrobials Phages are the most abundant biological entities in the environment [1-3], are relatively easy to isolate and propagate, and are highly evolved to kill specific bacterial strains, species, or sometimes even genera. Their antimicrobial and therapeutic potential was recognized immediately after their discovery, until the introduction of antibiotics displaced the application of phages shortly after World War II. In eastern Europe and the Soviet Union, phage therapy (see Glossary) was further developed, and it currently constitutes a standard medical practice in the Republic of Georgia [4]. More than 100 years after their discovery [5,6], the number of commercially available phage products is still limited, at least in Western countries. This is partly due to the availability of antibiotics as a cheap and effective alternative, to the lack of controlled studies on therapeutic phage efficacy, and to uncertainties with respect to intellectual property (IP) rights and the approval of phage-based treatments [7-9]. Nevertheless, the increasing incidence of infections with antibiotic-resistant bacteria and the decreasing rate of discovery of conventional antibiotics have revived a strong interest in antimicrobial phage applications [9-11]. In addition, undesirable side-effects associated with the antibiotic-mediated removal of commensal bacteria (e.g., gut dysbiosis) [12] demonstrate that pathogenspecific therapeutic options will be an important avenue for future drug development. Here, we outline the potential of novel phage engineering approaches to improve, customize, and market phage-based antimicrobials. Enhancing the Properties of Natural Bacteriophages In this section we discuss some of the inherent limitations associated with the clinical use of native, nonmodified phages and how they could be overcome by genetic engineering. Avoiding Phage Resistance Bacteria have evolved a large number of sophisticated phage-resistance mechanisms to prevent virus binding, infection, and replication [13-16] which can negatively affect phage Highlights The increasing prevalence of antibiotic-resistant pathogenic bacteria has sparked renewed interest in bacteriophage therapy. Synthetic biology methods allow design, straightforward construction, and testing of engineered bacteriophages that target both Gram-positive and Gram-negative bacteria. Phages with small genomes are easier to engineer using synthetic methods, while recombination-based approaches are currently the method of choice for larger phages.

Journal of Bacteriology, 2010

ABSTRACTBrochothrixbelongs to the low-GC branch of Gram-positive bacteria (Firmicutes), closely r... more ABSTRACTBrochothrixbelongs to the low-GC branch of Gram-positive bacteria (Firmicutes), closely related toListeria,Staphylococcus,Clostridium, andBacillus. Brochothrix thermosphactais a nonproteolytic food spoilage organism, adapted to growth in vacuum-packaged meats. We report the first genome sequences and characterization ofBrochothrixbacteriophages. Phage A9 is a myovirus with an 89-nm capsid diameter and a 171-nm contractile tail; it belongs to theSpounavirinaesubfamily and shares significant homologies withListeriaphage A511,Staphylococcusphage Twort, and others. The A9 unit genome is 127 kb long with 11-kb terminal redundancy; it encodes 198 proteins and 6 tRNAs. Phages BL3 and NF5 are temperate siphoviruses with a head diameter of 56 to 59 nm. The BL3 tail is 270 nm long, whereas NF5 features a short tail of only 94 nm. The NF5 genome (36.95 kb) encodes 57 gene products, BL3 (41.52 kb) encodes 65 products, and both are arranged in life cycle-specific modules. Surprisingly, B...

Methods in Molecular Biology, 2019

RNA interference (RNAi) allows for transient, targeted depletion of cellular or viral proteins. P... more RNA interference (RNAi) allows for transient, targeted depletion of cellular or viral proteins. Previously, small interfering RNA (siRNA) screens targeting cellular factors successfully identified several host genes that are required for VACV infection, and other viruses such as HIV. In this chapter, we outline how RNAi can be adapted to unravel the functions of poxvirus genes, using a 96-well format. Additionally, we describe two different high-throughput methods (flow cytometry and automated microscopy) to assess infection levels of an engineered VACV that encodes a fluorescent reporter protein under an early and/or late viral gene promoter.

Nature Microbiology, 2018

Introductory paragraph Cell motility is essential for viral dissemination 1. Vaccinia virus (VACV... more Introductory paragraph Cell motility is essential for viral dissemination 1. Vaccinia virus (VACV), a close relative of smallpox virus, is thought to exploit cell motility as a means to enhance the spread of infection 1. A single viral protein, F11L, contributes to this by blocking RhoA signalling to facilitate cell retraction 2. However, F11L alone is not sufficient for VACV induced cell motility, indicating that additional viral factors must be involved. Here we show that the VACV epidermal growth factor homolog, VGF, promotes infected cell motility and the spread of viral infection. We found that VGF secreted from early infected cells is cleaved by ADAM10 whereupon it acts largely in a paracrine fashion to direct cell motility at the leading edge of infection. Real-time tracking of cells infected in the presence of EGFR/MAPK/FAK/ADAM10 inhibitors, or with VGF and F11 deleted viruses, revealed defects in radial velocity and directional migration efficiency leading to impaired cell-to-cell spread of infection. Furthermore, intravital imaging showed that virus spread and lesion formation are attenuated in the absence of VGF. Our results demonstrate how poxviruses hijack epidermal growth factor receptor induced cell motility to promote rapid and efficient spread of infection in vitro and in vivo.

Nature microbiology, Jan 9, 2018

To orchestrate context-dependent signalling programmes, poxviruses encode two dual-specificity en... more To orchestrate context-dependent signalling programmes, poxviruses encode two dual-specificity enzymes, the F10 kinase and the H1 phosphatase. These signalling mediators are essential for poxvirus production, yet their substrate profiles and systems-level functions remain enigmatic. Using a phosphoproteomic screen of cells infected with wild-type, F10 and H1 mutant vaccinia viruses, we systematically defined the viral signalling network controlled by these enzymes. Quantitative cross-comparison revealed 33 F10 and/or H1 phosphosites within 17 viral proteins. Using this proteotype dataset to inform genotype-phenotype relationships, we found that H1-deficient virions harbour a hidden hypercleavage phenotype driven by reversible phosphorylation of the virus protease I7 (S134). Quantitative phosphoproteomic profiling further revealed that the phosphorylation-dependent activity of the viral early transcription factor, A7 (Y367), underlies the transcription-deficient phenotype of H1 mutan...