Phage therapy is effective against infection by Mycobacterium ulcerans in a murine footpad model

Author Summary: Buruli Ulcer (BU), caused by Mycobacterium ulcerans, is a necrotizing disease of the skin, subcutaneous tissue and bone. Standard treatment of BU patients consists of a combination of the antibiotics rifampicin and streptomycin for 8 weeks. However, in advanced stages of the disease, surgical resection of the destroyed skin is still required. The use of bacterial viruses (bacteriophages) for the control of bacterial infections has been considered as an alternative or a supplement to antibiotic chemotherapy. By using a mouse model of M. ulcerans footpad infection, we show that mice treated with a single subcutaneous injection of the mycobacteriophage D29 present decreased footpad pathology associated with a reduction of the bacterial burden. In addition, D29 treatment induced increased levels of IFN-γ and TNF in M. ulcerans -infected footpads, correlating with a predominance of a mononuclear infiltrate. These findings suggest the potential use of phage therapy in BU, as a novel therapeutic approach against this disease, particularly in advanced stages where bacteria are found primarily in an extracellular location in the subcutaneous tissue, and thus immediately accessible by lytic phages.This work was supported by a grant from the Health Services of Fundacao Calouste Gulbenkian, and the Portuguese Science and Technology Foundation (FCT) fellowships SFRH/BPD/64032/2009, SFRH/BD/41598/2007, and SFRH/BPD/68547/2010 to GT, TGM, and AGF, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Bacteriophage-encoded depolymerases: their diversity and biotechnological applications

Bacteriophages (phages), natural enemies of bacteria, can encode enzymes able to degrade polymeric substances. These substances can be found in the bacterial cell surface, such as polysaccharides, or are produced by bacteria when they are living in biofilm communities, the most common bacterial lifestyle. Consequently, phages with depolymerase activity have a facilitated access to the host receptors, by degrading the capsular polysaccharides, and are believed to have a better performance against bacterial biofilms, since the degradation of extracellular polymeric substances by depolymerases might facilitate the access of phages to the cells within different biofilm layers. Since the diversity of phage depolymerases is not yet fully explored, this is the first review gathering information about all the depolymerases encoded by fully sequenced phages. Overall, in this study, 160 putative depolymerases, including sialidases, levanases, xylosidases, dextranases, hyaluronidases, peptidases as well as pectate/pectin lyases, were found in 143 phages (43 Myoviridae, 47 Siphoviridae, 37 Podoviridae, and 16 unclassified) infecting 24 genera of bacteria. We further provide information about the main applications of phage depolymerases, which can comprise areas as diverse as medical, chemical, or food-processing industry.DPP acknowledges the financial support from the Portuguese Foundation for Science and Technology (FCT) through the grant SFRH/BD/76440/2011. SS is an FCT investigator (IF/01413/2013). The authors also thank FCT for the Strategic Project of the UID/BIO/04469/2013 unit, FCT and European Union funds (FEDER/COMPETE) for the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER027462)

A role for accessory genes rI.-1 and rI.1 in the regulation of lysis inhibition by bacteriophage T4

Lysis inhibition (LIN) is a known feature of the T-even family of bacteriophages. Despite its historical role in the development of modern molecular genetics, many aspects of this phenomenon remain mostly unexplained. The key element of LIN is an interaction between two phage-encoded proteins, the T holin and the RI antiholin. This interaction is stabilized by RIII. In this report, we demonstrate the results of genetic experiments which suggest a synergistic action of two accessory proteins of bacteriophage T4, RI.-1, and RI.1 with RIII in the regulation of LIN

The Phenotype-Fitness Map in Experimental Evolution of Phages

Evolutionary biologists commonly interpret adaptations of organisms by reference to a phenotype-fitness map, a model of how different states of a phenotype affect fitness. Notwithstanding the popularity of this approach, it remains difficult to directly test these mappings, both because the map often describes only a small subset of phenotypes contributing to total fitness and because direct measures of fitness are difficult to obtain and compare to the map. Both limitations can be overcome for bacterial viruses (phages) grown in the experimental condition of unlimited hosts. A complete accounting of fitness requires 3 easily measured phenotypes, and total fitness is also directly measurable for arbitrary genotypes. Yet despite the presumed transparency of this system, directly estimated fitnesses often differ from fitnesses calculated from the phenotype-fitness map. This study attempts to resolve these discrepancies, both by developing a more exact analytical phenotype-fitness map and by exploring the empirical foundations of direct fitness estimates. We derive an equation (the phenotype-fitness map) for exponential phage growth that allows an arbitrary distribution of lysis times and burst sizes. We also show that direct estimates of fitness are, in many cases, plausibly in error because the population has not attained stable age distribution and thus violates the model underlying the phenotype-fitness map. In conjunction with data provided here, the new understanding appears to resolve a discrepancy between the reported fitness of phage T7 and the substantially lower value calculated from its phenotype-fitness map

War and peace:social interactions in infections

One of the most striking facts about parasites and microbial pathogens that has emerged in the fields of social evolution and disease ecology in the past few decades is that these simple organisms have complex social lives, indulging in a variety of cooperative, communicative and coordinated behaviours. These organisms have provided elegant experimental tests of the importance of relatedness, kin discrimination, cooperation and competition, in driving the evolution of social strategies. Here, we briefly review the social behaviours of parasites and microbial pathogens, including their contributions to virulence, and outline how inclusive fitness theory has helped to explain their evolution. We then take a mechanistically inspired ‘bottom-up’ approach, discussing how key aspects of the ways in which parasites and pathogens exploit hosts, namely public goods, mobile elements, phenotypic plasticity, spatial structure and multi-species interactions, contribute to the emergent properties of virulence and transmission. We argue that unravelling the complexities of within-host ecology is interesting in its own right, and also needs to be better incorporated into theoretical evolution studies if social behaviours are to be understood and used to control the spread and severity of infectious diseases

Bacteriophage biodistribution and infectivity from honeybee to bee larvae using a T7 phage model

Bacteriophages (phages) or viruses that specifically infect bacteria have widely been studied as biocontrol agents against animal and plant bacterial diseases. They offer many advantages compared to antibiotics. The American Foulbrood (AFB) is a bacterial disease affecting honeybee larvae caused by Paenibacillus larvae. Phages can be very significant in fighting it mostly due to European restrictions to the use of antibiotics in beekeeping. New phages able to control P. larvae in hives have already been reported with satisfactory results. However, the efficacy and feasibility of administering phages indirectly to larvae through their adult workers only by providing phages in bees feeders has never been evaluated. This strategy is considered herein the most feasible as far as hive management is concerned. This in vivo study investigated the ability of a phage to reach larvae in an infective state after oral administration to honeybees. The screening (by direct PFU count) and quantification (by quantitative PCR) of the phage in bee organs and in larvae after ingestion allowed us to conclude that despite 104 phages reaching larvae only an average of 32 were available to control the spread of the disease. The fast inactivation of many phages in royal jelly could compromise this therapeutic approach. The protection of phages from hive-derived conditions should be thus considered in further developments for AFB treatment.This study was supported by the project APILYSE, PTDC/CVT-EPI/4008/2014 - POCI-01-0145-FEDER-016598, - funded by FEDER through COMPETE 2020 - Programa Operacional Competitividade e Internacionalização (POCI) and by national funds trough FCT - Fundação para a Ciência e a Tecnologia, I.P. The work was also supported by the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004), funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. HR was supported by FCT through the grant SFRH/BD/128859/2017. RC was founded by FCT and FEDER (POCI-010145-FEDER-007274).info:eu-repo/semantics/publishedVersio

Ongoing Phenotypic and Genomic Changes in Experimental Coevolution of RNA Bacteriophage Qβ and Escherichia coli

According to the Red Queen hypothesis or arms race dynamics, coevolution drives continuous adaptation and counter-adaptation. Experimental models under simplified environments consisting of bacteria and bacteriophages have been used to analyze the ongoing process of coevolution, but the analysis of both parasites and their hosts in ongoing adaptation and counter-adaptation remained to be performed at the levels of population dynamics and molecular evolution to understand how the phenotypes and genotypes of coevolving parasite–host pairs change through the arms race. Copropagation experiments with Escherichia coli and the lytic RNA bacteriophage Qβ in a spatially unstructured environment revealed coexistence for 54 days (equivalent to 163–165 replication generations of Qβ) and fitness analysis indicated that they were in an arms race. E. coli first adapted by developing partial resistance to infection and later increasing specific growth rate. The phage counter-adapted by improving release efficiency with a change in host specificity and decrease in virulence. Whole-genome analysis indicated that the phage accumulated 7.5 mutations, mainly in the A2 gene, 3.4-fold faster than in Qβ propagated alone. E. coli showed fixation of two mutations (in traQ and csdA) faster than in sole E. coli experimental evolution. These observations suggest that the virus and its host can coexist in an evolutionary arms race, despite a difference in genome mutability (i.e., mutations per genome per replication) of approximately one to three orders of magnitude

Nasty Viruses, Costly Plasmids, Population Dynamics, and the Conditions for Establishing and Maintaining CRISPR-Mediated Adaptive Immunity in Bacteria

Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR) abound in the genomes of almost all archaebacteria and nearly half the eubacteria sequenced. Through a genetic interference mechanism, bacteria with CRISPR regions carrying copies of the DNA of previously encountered phage and plasmids abort the replication of phage and plasmids with these sequences. Thus it would seem that protection against infecting phage and plasmids is the selection pressure responsible for establishing and maintaining CRISPR in bacterial populations. But is it? To address this question and provide a framework and hypotheses for the experimental study of the ecology and evolution of CRISPR, I use mathematical models of the population dynamics of CRISPR-encoding bacteria with lytic phage and conjugative plasmids. The results of the numerical (computer simulation) analysis of the properties of these models with parameters in the ranges estimated for Escherichia coli and its phage and conjugative plasmids indicate: (1) In the presence of lytic phage there are broad conditions where bacteria with CRISPR-mediated immunity will have an advantage in competition with non-CRISPR bacteria with otherwise higher Malthusian fitness. (2) These conditions for the existence of CRISPR are narrower when there is envelope resistance to the phage. (3) While there are situations where CRISPR-mediated immunity can provide bacteria an advantage in competition with higher Malthusian fitness bacteria bearing deleterious conjugative plasmids, the conditions for this to obtain are relatively narrow and the intensity of selection favoring CRISPR weak. The parameters of these models can be independently estimated, the assumption behind their construction validated, and the hypotheses generated from the analysis of their properties tested in experimental populations of bacteria with lytic phage and conjugative plasmids. I suggest protocols for estimating these parameters and outline the design of experiments to evaluate the validity of these models and test these hypotheses