Pseudolysogeny and sequential mutations build multiresistance to virulent bacteriophages in Pseudomonas aeruginosa

International audienceCoevolution between bacteriophages and their prey is the result of mutualistic interactions. Here we show that pseudolysogeny is a frequent outcome of infection by virulent phages of Pseudomonas aeruginosa, and that selection of resistant bacterial mutants is favored by continuous production of phages. We investigated the frequency and characteristics of P. aeruginosa strain PAO1 variants resisting infection by different combinations of virulent phages belonging to four genera. The frequency of resistant bacteria was 10-5 for single phage infection and 10-6 for infections with combinations of two or four phages. The genome of 27 variants was sequenced and the comparison with the genome of the parental PAO1 strain allowed the identification of point mutations or small indels. Four additional variants were characterized by a candidate gene approach. In total, 27 independent mutations were observed affecting 14 genes and a regulatory region. The mutations affected genes involved in biosynthesis of type IV pilus, alginate, LPS and O-antigen. Half of the variants possessed changes in homopolymer tracts responsible for frameshift mutations, and these phase variation mutants were shown to be unstable. Eleven double mutants were detected. The presence of free phage DNA was observed in association with exclusion of superinfection in half of the variants, and in three of them no chromosomal mutation could be found. Upon further growth of these pseudolysogens, some variants with new chromosomal mutations were recovered presumably due to continuous evolutionary pressure

La pseudolysogénie permet la sélection des mutations successives à la base de la résistance multiple de Pseudomonas aeruginosa aux bactériophages virulents

Bacteriophages are obligate parasites of bacteria that can be defined as virulent or temperate according to their lifestyle: virulent phages perform a lytic cycle by injecting their genome in the bacterial cell and immediately multiply. Temperate phages, instead, can either perform a lytic, or a lysogenic cycle by integrating their genome into the bacterial chromosome and persisting in a dormant state until the lytic cycle is resumed. The viral genome can also be maintained in the bacterial cell in an episomal form for an undetermined period of time in a stage known as pseudolysogeny. P. aeruginosa, a bacterium commonly found in the environment and in association with many hosts including plants and animals, is responsible for severe nosocomial infections. A proportion of clinical strains are multidrug-resistant, possessing a high ability to form biofilms which are very difficult to eradicate with conventional treatments. It is therefore essential to find new therapeutic approaches, such as phage therapy. Numerous clinical data obtained in Eastern Europe and Russia attest the effectiveness and safety of phage therapy. However, there remain uncertainties related to their therapeutic use and particularly the high frequency of natural resistance. Our project aimed to better understand the dynamic of phage/bacteria interactions by studying the resistance mechanisms acting in the reference strain P. aeruginosa PAO1, against virulent phages. Infections were performed by combining phages belonging to four different genera: Ab05, a ФKMV-like podovirus, Ab09, a LIT1-like podovirus, Ab27, a PB1-like myovirus and Ab17, a KPP10-like myovirus, all isolated in our laboratory. Single or multiple infections of P. aeruginosa PAO1 were performed, and a collection of phage-resistant variants was isolated and analysed. The frequency of phage-resistant variants selection was 10⁻⁵ for single phage infection, and 10⁻⁶ for infections with cocktails of two or four phages. The phenotype and mobility of the variants was often affected, as compared to the parental strain. The genome of 27 variants was entirely sequenced by Illumina technology in order to identify mutations responsible for the resistance. Other variants were analysed by a candidate gene approach. We identified point mutations or small indels: in total, 27 independent mutations affected 14 genes and 1 regulatory region. The affected genes encode proteins involved in biosynthesis of type IV pili (T4P) and lipopolysaccharide (LPS), frequently used as receptors by the phages. Other mutations were observed in genes necessary for alginate production. Of interest, we found that half of the variants with mutations in genes involved in LPS biosynthesis possessed unstable phase variation mutations, responsible for translation frameshift. In contrast, genes involved in pilus type IV biogenesis were mainly subjected to deletions. Surprisingly, the presence of free phage DNA was found in association with exclusion of superinfection in half of the variants and no chromosomal mutation could be found in three of them. Thus, we showed that pseudolysogeny is a frequent outcome of infection by virulent phages of P. aeruginosa. Moreover, double mutants were selected at high frequency and this could presumably due to evolutionary pressure exerted by re-activation of lytic cycle in some cells of the pseudolysogen population. In conclusion, if phage predation selects for variants with alterations in genes involved in biogenesis or regulation of virulence determinants such as LPS or alginate, the resulting phage-resistant variants could potentially exhibit altered levels of virulence in a beneficial or detrimental way. The use of cocktail does not lower significantly the frequency of phage-resistance and in addition we show that pseudolysogeny is a major actor in the selection of mutations.Les bactériophages sont des virus qui injectent leur génome dans une bactérie après fixation à des récepteurs sur la surface de celle-ci, puis effectuent un cycle de multiplication de leur ADN, la synthèse des protéines de structure, l’encapsidation du génome viral et la lyse de la bactérie. Les phages virulents réalisent uniquement des cycles lytiques alors que les phages tempérés peuvent également intégrer leur génome dans celui de la bactérie, donnant ainsi naissance à une bactérie dite lysogène. Les phages peuvent parfois être maintenus dans la bactérie sans effectuer un cycle lytique ni s’intégrer, dans un état encore peu compris, connu sous le nom de pseudolysogénie. Pseudomonas aeruginosa est une espèce bactérienne présente dans l’environnement et associée à de nombreux hôtes, végétaux et animaux. Elle est responsable de graves infections nosocomiales et on observe de plus en plus souvent des souches multirésistantes aux antibiotiques, ayant une grande capacité à former des biofilms, et en conséquence très difficiles à éradiquer. Il faut donc absolument trouver des approches thérapeutiques nouvelles telle que la phagothérapie. De nombreuses données cliniques obtenues dans les pays de l’est de l’Europe et en Russie attestent de l’efficacité et de l’innocuité de la phagothérapie, mais il reste des incertitudes en particulier concernant la nature et la fréquence des résistances naturelles. Notre projet vise à évaluer le potentiel thérapeutique des phages et à mieux comprendre la dynamique de leur interaction avec leur hôte. Nous avons étudié les mécanismes de résistance mis en place par la souche de P. aeruginosa, PAO1, à quatre bactériophages virulents appartenant à des genres différents: deux podovirus, Ab05 (ФKMV-like) et Ab09 (LIT1-like), et deux myovirus, Ab27 (PB1-like) et Ab17 (KPP10-like), tous isolés par notre laboratoire. Des infections simples ou multiples de PAO1 ont été réalisées, et une collection de variants résistants aux phages a été isolée et étudiée. La fréquence des bactéries résistantes était de 10⁻⁵ pour l'infection par un phage seul et 10⁻⁶ pour les infections par des combinaisons de deux ou quatre phages. Le phénotype et la mobilité des variants résistants étaient fréquemment affectés.Le génome de 27 variants a été entièrement séquencé par la technologie Illumina, et la comparaison avec le génome de la souche PAO1 a permis l'identification de mutations ponctuelles ou de petites indels. Quatre variants supplémentaires ont été caractérisés par une approche «gène candidat». Des mutations affectant 14 gènes différents et 1 région régulatrice ont été observées. Les gènes mutés codent pour des protéines impliquées dans la biosynthèse des pili de type IV (T4P) et des lipopolysacharides (LPS), très fréquemment utilisés comme récepteurs par les phages. Des mutations de la synthèse des alginates ont été également observées. La moitié des variants possède des mutations de variation de phase qui se sont révélées être instables. Par contre, les gènes impliqués dans la biosynthèse du T4P montrent des délétions stables. Nous avons aussi observé que la pseudolysogénie est une conséquence fréquente de l'infection par ces phages virulents et que la sélection de mutants (très souvent des mutants doubles) est favorisée par la production continue de phages par les pseudolysogènes. La présence d'ADN de phage libre a été observée en liaison avec l'exclusion de surinfection. Pour conclure, si les phages sélectionnent des bactéries résistantes possédant des altérations dans les gènes impliqués dans la biogenèse ou la régulation des déterminants de la virulence, celle-ci sera probablement modifiée, d'une manière bénéfique ou préjudiciable, ce qui reste à étudier. L'utilisation du cocktail par rapport à l’infection simple, ne réduit pas de manière significative la fréquence de la résistance aux phages et en outre, nous montrons que la pseudolysogénie est un acteur majeur de la sélection de mutations

Recovery and Characterization of Bacteria Resisting Infection by Lytic Bacteriophage

Bacteria and bacteriophages coexist and coevolve, bacteriophages being obligatory predators exerting an evolutionary pressure on their prey. Mechanisms in action vary depending on the bacterial genomic content and on the regulation of the bacteriophage cycle. To assess the multiplicity of bacterial genes involved in resistance as well as the changes in the bacteriophage interactions with the bacteria, it is necessary to isolate and investigate large numbers of independent resistant variants. Here we describe protocols that have been applied to the study of Pseudomonas aeruginosa and four of its virulent bacteriophages belonging to the Podoviridae and Myoviridae bacteriophage families. Mutations are identified using whole genome sequencing of resistant variants. Phenotypic analyses are performed to describe the changes conferred by the mutations

Fine structure analysis of lipopolysaccharides in bacteriophage-resistant Pseudomonas aeruginosa PAO1 mutants

Pseudomonas aeruginosa lipopolysaccharides (LPS) serve as primary receptors for many bacteriophages and, consequently, their biosynthesis is frequently affected in phage-resistant mutants. We previously isolated phage-resistant PAO1 mutants using three different phages, and showed that they were affected in the synthesis of LPS. Here we have investigated in detail the effect of mutations in seven genes involved in different steps of the production of core and oligosaccharide chains. The band profile of purified LPS was analysed by PAGE, and we further characterized the O-chains and core structures by MALDI mass spectrometry (MS). Mild LPS extraction conditions and native LPS MS analyses helped unveil lipid A molecular species with three phosphate residues in the close vicinity of the already highly charged inner-core region. No other MS direct analysis has allowed this peculiarity to be demonstrated for native lipid A high-molecular-weight molecular species, in normal growth conditions and without involving separation techniques. The present results shed light on the possible interactions between the phages and the LPS structures in the early phase of infection

The Basis for Natural Multiresistance to Phage in Pseudomonas aeruginosa

Pseudomonas aeruginosa is responsible for long-term infections and is particularly resistant to treatments when hiding inside the extracellular matrix or biofilms. Phage therapy might represent an alternative to antibiotic treatment, but up to 10% of clinical strains appear to resist multiple phages. We investigated the characteristics of P. aeruginosa clinical strains naturally resistant to phages and compared them to highly susceptible strains. The phage-resistant strains were defective in lipopolysaccharide (LPS) biosynthesis, were nonmotile and displayed an important degree of autolysis, releasing phages and pyocins. Complete genome sequencing of three resistant strains showed the existence of a large accessory genome made of multiple insertion elements, genomic islands, pyocins and prophages, including two phages performing lateral transduction. Mutations were found in genes responsible for the synthesis of LPS and/or type IV pilus, the major receptors for most phages. CRISPR-Cas systems appeared to be absent or inactive in phage-resistant strains, confirming that they do not play a role in the resistance to lytic phages but control the insertion of exogenous sequences. We show that, despite their apparent weakness, the multiphage-resistant strains described in this study displayed selective advantages through the possession of various functions, including weapons to eliminate other strains of the same or closely related species

Investigation of Pseudomonas aeruginosa strain PcyII-10 variants resisting infection by N4-like phage Ab09 in search for genes involved in phage adsorption

International audienceBacteria and their bacteriophages coexist and coevolve for the benefit of both in a mutualistic association. Multiple mechanisms are used by bacteria to resist phages in a trade-off between survival and maintenance of fitness. In vitro studies allow inquiring into the fate of virus and host in different conditions aimed at mimicking natural environment. We analyse here the mutations emerging in a clinical Pseudomonas aeruginosa strain in response to infection by Ab09, a N4-like lytic podovirus and describe a variety of chromosomal deletions and mutations conferring resistance. Some deletions result from illegitimate recombination taking place during long-term maintenance of the phage genome. Phage variants with mutations in a tail fiber gene are selected during pseudolysogeny with the capacity to infect resistant cells and produce large plaques. These results highlight the complex host/phage association and suggest that phage Ab09 promotes bacterial chromosome rearrangements. Finally this study points to the possible role of two bacterial genes in Ab09 phage adhesion to the cell, rpsB encoding protein S2 of the 30S ribosomal subunit and ORF1587 encoding a Wzy-like membrane protein involved in LPS biosynthesis

A carrier state is established in Pseudomonas aeruginosa by phage LeviOr01, a newly isolated ssRNA levivirus

International audiencessRNA bacteriophages are very abundant but poorly studied, particularly in relation to their effect on bacterial evolution. We isolated a new Pseudomonas aeruginosa levivirus, vB_PaeL_PcyII-10_LeviOr01, from hospital waste water. Its genome comprises 3669 nucleotides and encodes four putative proteins. Following bacterial infection, a carrier state is established in a fraction of the cells, conferring superinfection immunity. Such cells also resist other phages that use type IV pili as a receptor. The carrier population is composed of a mixture of cells producing phage, and susceptible cells that are non-carriers. Carrier cells accumulate phage until they burst, releasing large quantities of virions. The continuous presence of phage favours the emergence of host variants bearing mutations in genes involved in type IV pilus biogenesis, but also in genes affecting lipopolysaccharide (LPS) synthesis. The establishment of a carrier state in which phage particles are continuously released was previously reported for some dsRNA phages, but has not previously been described for a levivirus. The present results highlight the importance of the carrier state, an association that benefits both phages and bacteria and plays a role in bacterial evolution

Large Preferred Region for Packaging of Bacterial DNA by phiC725A, a Novel Pseudomonas aeruginosa F116-Like Bacteriophage.

Bacteriophage vB_PaeP_PAO1_phiC725A (short name phiC725A) was isolated following mitomycin C induction of C7-25, a clinical Pseudomonas aeruginosa strain carrying phiC725A as a prophage. The phiC725A genome sequence shows similarity to F116, a P. aeruginosa podovirus capable of generalized transduction. Likewise, phiC725A is a podovirus with long tail fibers. PhiC725A was able to lysogenize two additional P. aeruginosa strains in which it was maintained both as a prophage and in an episomal state. Investigation by deep sequencing showed that bacterial DNA carried inside phage particles originated predominantly from a 700-800kb region, immediately flanking the attL prophage insertion site, whether the phages were induced from a lysogen or recovered after infection. This indicates that during productive replication, recombination of phage genomes with the bacterial chromosome at the att site occurs occasionally, allowing packaging of adjacent bacterial DNA

Complete Genome Sequences of Pseudomonas aeruginosa Phages vB_PaeP_PcyII-10_P3P1 and vB_PaeM_PcyII-10_PII10A.

International audiencevB_PaeP_PcyII-10_P3P1 and vB_PaeM_PcyII-10_PII10A are Pseudomonas aeruginosa bacteriophages belonging, respectively, to the Lit1virus genus of the Podoviridae family and the Pbunavirus genus of the Myoviridae family. Their genomes are 72,778 bp and 65,712 bp long, containing 94 and 93 predicted open reading frames, respectively

A novel Pseudomonas aeruginosa bacteriophage, Ab31, a chimera formed from temperate phage PAJU2 and P. putida lytic phage AF: characteristics and mechanism of bacterial resistance.

A novel temperate bacteriophage of Pseudomonas aeruginosa, phage vB_PaeP_Tr60_Ab31 (alias Ab31) is described. Its genome is composed of structural genes related to those of lytic P. putida phage AF, and regulatory genes similar to those of temperate phage PAJU2. The virion structure resembles that of phage AF and other lytic Podoviridae (S. enterica Epsilon 15 and E. coli phiv10) with similar tail spikes. Ab31 was able to infect P. aeruginosa strain PA14 and two genetically related strains called Tr60 and Tr162, out of 35 diverse strains from cystic fibrosis patients. Analysis of resistant host variants revealed different phenotypes, including induction of pigment and alginate overproduction. Whole genome sequencing of resistant variants highlighted the existence of a large deletion of 234 kbp in two strains, encompassing a cluster of genes required for the production of CupA fimbriae. Stable lysogens formed by Ab31 in strain Tr60, permitted the identification of the insertion site. During colonization of the lung in cystic fibrosis patients, P. aeruginosa adapts by modifying its genome. We suggest that bacteriophages such as Ab31 may play an important role in this adaptation by selecting for bacterial characteristics that favor persistence of bacteria in the lung