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Engineered bacteriophages provide powerful tools for biotechnology, diagnostics, pathogen control, and therapy. However, current techniques for phage editing are experimentally challenging and limited to few phages and host organisms. Viruses that target Gram-positive bacteria are particularly difficult to modify. Here, we present a platform technology that enables rapid, accurate, and selection-free construction of synthetic, tailor-made phages that infect Gram-positive bacteria. To this end, custom-designed, synthetic phage genomes were assembled in vitro from smaller DNA fragments. We show that replicating, cell wall-deficient Listeria monocytogenes L-form bacteria can reboot synthetic phage genomes upon transfection, i.e., produce virus particles from naked, synthetic DNA. Surprisingly, Listeria L-form cells not only support rebooting of native and synthetic Listeria phage genomes but also enable cross-genus reactivation of Bacillus and Staphylococcus phages from their DNA, thereby broadening the approach to phages that infect other important Gram-positive pathogens. We then used this platform to generate virulent phages by targeted modification of temperate phage genomes and demonstrated their superior killing efficacy. These synthetic, virulent phages were further armed by incorporation of enzybiotics into their genomes as a genetic payload, which allowed targeting of phage-resistant bystander cells. In conclusion, this straightforward and robust synthetic biology approach redefines the possibilities for the development of improved and completely new phage applications, including phage therapy.

The human intestinal microbiota is a crucial factor in the pathogenesis of various diseases, such as metabolic syndrome or inflammatory bowel disease (IBD). Yet, knowledge about the role of environmental factors such as smoking (which is known to influence theses aforementioned disease states) on the complex microbial composition is sparse. We aimed to investigate the role of smoking cessation on intestinal microbial composition in 10 healthy smoking subjects undergoing controlled smoking cessation. During the observational period of 9 weeks repetitive stool samples were collected. Based on abundance of 16S rRNA genes bacterial composition was analysed and compared to 10 control subjects (5 continuing smokers and 5 non-smokers) by means of Terminal Restriction Fragment Length Polymorphism analysis and high-throughput sequencing. Profound shifts in the microbial composition after smoking cessation were observed with an increase of Firmicutes and Actinobacteria and a lower proportion of Bacteroidetes and Proteobacteria on the phylum level. In addition, after smoking cessation there was an increase in microbial diversity. These results indicate that smoking is an environmental factor modulating the composition of human gut microbiota. The observed changes after smoking cessation revealed to be similar to the previously reported differences in obese compared to lean humans and mice respectively, suggesting a potential pathogenetic link between weight gain and smoking cessation. In addition they give rise to a potential association of smoking status and the course of IBD.

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.

Endolysins are enzymes used by bacteriophages at the end of their replication cycle to degrade the peptidoglycan of the bacterial host from within, resulting in cell lysis and release of progeny virions. Due to the absence of an outer membrane in the Gram-positive bacterial cell wall, endolysins can access the peptidoglycan and destroy these organisms when applied externally, making them interesting antimicrobial candidates, particularly in light of increasing bacterial drug resistance. This article reviews the modular structure of these enzymes, in which cell wall binding and catalytic functions are separated, as well as their mechanism of action, lytic activity and potential as antimicrobials. It particularly focuses on molecular engineering as a means of optimizing endolysins for specific applications, highlights new developments that may render these proteins active against Gram-negative and intracellular pathogens and summarizes the most recent applications of endolysins in the fields of medicine, food safety, agriculture and biotechnology.

The inherent ability of bacteriophages (phages) to infect specific bacterial hosts makes them ideal candidates to develop into antimicrobial agents for pathogen-specific remediation in food processing, biotechnology, and medicine (e.g., phage therapy). Conversely, phage contaminations of fermentation processes are a major concern to dairy and bioprocessing industries. The first stage of any successful phage infection is adsorption to a bacterial host cell, mediated by receptor-binding proteins (RBPs). As the first point of contact, the binding specificity of phage RBPs is the primary determinant of bacterial host range, and thus defines the remediative potential of a phage for a given bacterium. Co-evolution of RBPs and their bacterial receptors has forced endless adaptation cycles of phage-host interactions, which in turn has created a diverse array of phage adsorption mechanisms utilizing an assortment of RBPs. Over the last decade, these intricate mechanisms have been studied intensely using electron microscopy and X-ray crystallography, providing atomic-level details of this fundamental stage in the phage infection cycle. This review summarizes current knowledge surrounding the molecular basis of host interaction for various socioeconomically important Gram-positive targeting phage RBPs to their protein- and saccharide-based receptors. Special attention is paid to the abundant and best-characterized Siphoviridae family of tailed phages. Unravelling these complex phage-host dynamics is essential to harness the full potential of phage-based technologies, or for generating novel strategies to combat industrial phage contaminations.

The host range of bacteriophages defines their impact on bacterial communities and genome diversity. Here, we characterize 94 novel staphylococcal phages from wastewater and establish their host range on a diversified panel of 117 staphylococci from 29 species. Using this high-resolution phage-bacteria interaction matrix, we unveil a multi-species host range as a dominant trait of the isolated staphylococcal phages. Phage genome sequencing shows this pattern to prevail irrespective of taxonomy. Network analysis between phage-infected bacteria reveals that hosts from multiple species, ecosystems, and drug-resistance phenotypes share numerous phages. Lastly, we show that phages throughout this network can package foreign genetic material enclosing an antibiotic resistance marker at various frequencies. Our findings indicate a weak host specialism of the tested phages, and therefore their potential to promote horizontal gene transfer in this environment. The host range of bacteriophages defines their impact on bacterial ecology and diversity. Here, Göller et al. isolate 94 staphylococcal phages from wastewater and determine their host range on 117 staphylococci from 29 species, revealing a predominant multi-species host range and thus great potential for horizontal gene transfer.

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 with such engineered reporter phages typically leads to a rapid burst of reporter protein production that enables highly sensitive detection. In this review, we highlight recent advances in infection-based detection methods, present guidelines for reporter phage construction, outline technical aspects of reporter phage engineering, and discuss some of the advantages and pitfalls of phage-based pathogen detection. Recent improvements in reporter phage construction and engineering further substantiate the potential of these highly evolved nanomachines as rapid and inexpensive detection systems to replace or complement traditional diagnostic approaches.

Bacteriophages operate via pathogen-specific mechanisms of action distinct from conventional, broad-spectrum antibiotics and are emerging as promising alternative antimicrobials. However, phage-mediated killing is often limited by bacterial resistance development. Here, we engineer phages for target-specific effector gene delivery and host-dependent production of colicin-like bacteriocins and cell wall hydrolases. Using urinary tract infection (UTI) as a model, we show how heterologous effector phage therapeutics (HEPTs) suppress resistance and improve uropathogen killing by dual phage- and effector-mediated targeting. Moreover, we designed HEPTs to control polymicrobial uropathogen communities through production of effectors with cross-genus activity. Using phage-based companion diagnostics, we identified potential HEPT responder patients and treated their urine ex vivo. Compared to wildtype phage, a colicin E7-producing HEPT demonstrated superior control of patient E. coli bacteriuria. Arming phages with heterologous effectors paves the way for successful UTI treatment and represents a versatile tool to enhance and adapt phage-based precision antimicrobials. Du et al . genetically engineer bacteriophages into heterologous effector phage therapeutics, enabling dual phage- and effector-mediated targeting for a two-pronged attack against bacterial pathogens.

The incidence of foodborne infectious diseases is stable or has even increased in many countries. Consequently, our awareness regarding hygiene measures in food production has also increased dramatically over the last decades. However, even today's modern production techniques and intensive food-monitoring programs have not been able to effectively control the problem. At the same time, increased production volumes are distributed to more consumers, and if contaminated, potentially cause mass epidemics. Accordingly, research directed to improve food safety has also been taken forward, also exploring novel methods and technologies. Such an approach is represented by the use of bacteriophage for specific killing of unwanted bacteria. The extreme specificity of phages renders them ideal candidates for applications designed to increase food safety during the production process. Phages are the natural enemies of bacteria, and can be used for biocontrol of bacteria without interfering with the natural microflora or the cultures in fermented products. Moreover, phages or phage-derived proteins can also be used to detect the presence of unwanted pathogens in food or the production environments, which allows quick and specific identification of viable cells. This review intends to briefly summarize and explain the principles and current standing of these approaches.

The foodborne pathogen Listeria monocytogenes (Lm) is a highly heterogeneous species and currently comprises of 4 evolutionarily distinct lineages. Here, we characterize isolates from severe ovine listeriosis outbreaks that represent a hybrid sub-lineage of the major lineage II (HSL-II) and serotype 4h. HSL-II isolates are highly virulent and exhibit higher organ colonization capacities than well-characterized hypervirulent strains of Lm in an orogastric mouse infection model. The isolates harbour both the Lm Pathogenicity Island (LIPI)-1 and a truncated LIPI-2 locus, encoding sphingomyelinase (SmcL), a virulence factor required for invasion and bacterial translocation from the gut, and other non-contiguous chromosomal segments from another pathogenic species, L . ivanovii . HSL-II isolates exhibit a unique wall teichoic acid (WTA) structure essential for resistance to antimicrobial peptides, bacterial invasion and virulence. The discovery of isolates harbouring pan-species virulence genes of the genus Listeria warrants global efforts to identify further hypervirulent lineages of Lm. Listeria monocytogenes isolates are highly heterogeneous and exhibit different levels of virulence. Here, the authors identify hypervirulent isolates that represent a hybrid sub-lineage of the major lineage II harbouring virulence factors from Listeria ivanovii and wall teichoic acids found in major lineage I.