Implications of bacterial viruses on pathogenic bacteria : from natural microbial communities to therapeutic applications

Bacterial viruses (i.e. phages) are ubiquitous intracellular parasites of bacteria, that along with protist grazers account for majority of bacterial mortality in nature. Phages impose strong selection for bacterial phage-resistance, which is often coupled with fitness costs on bacterial traits such as growth ability, virulence or motility. Traditionally phage-host interactions have been studied with two species systems in the laboratory, neglecting the complex web of interactions present in natural communities. The ability of phages to selectively kill bacteria has ignited an interest on phages as alternative antibacterials. However, in order to develop phage therapy, understanding of phage-host interactions in the eco-evolutionary context is essential. In this thesis I studied the implications of lytic phages on opportunistic pathogenic bacteria, as opportunists often have the ability reproduce and reside in outside-host environments, where they are predisposed to a variety of selection pressures. The role of phages in top-down control of bacterial biomass and the evolution of bacterial phage-resistance were studied in the presence of protist predators with differing feeding modes, in low-resource systems mimicking natural pond environment. Hypothesis of coincidental evolution suggests that virulence is a by-product of selection for traits that maximize bacterial fitness in environmental reservoirs. Yet, disease outbreaks by opportunists are relatively rare, suggesting that something constrains the selection for virulence. To assess the role of lytic phages on the evolution of virulence, bacteria were cultured in low-resource environment, accompanied with changes in temperature regime or changes in composition of the community of interacting bacterial enemy species, and the virulence of bacteria was measured in vivo. To study whether the potential phage-resistant bacteria surviving phage therapy would be coupled with lowered virulence, due to costs associated with phage-resistance, a clinical bacterial isolate was exposed to phage cocktails and the virulence of the phage-resistant bacteria was measured in vivo. given the strong selection for phage- resistance, the prospects of phage therapy depend a great deal on whether new phages infecting pathogenic bacteria can be readily isolated from environment. To address this, an attempt was made to isolate phages against clinical bacterial isolates harboring resistance genes to multiple antibiotics. A single lytic phage was shown to be a non- efficient top-down regulator of bacterial biomass. Rapidly emerging phage-resistant bacteria took over the bacterial populations after initial lysis by phages and protist grazers accounted for most of the long-term negative trophic effects on bacterial biomass. The presence of protist predators selected for bacteria that were less susceptible to infection by lytic phages, which suggests an overlap in the bacterial defense against a parasite and predatory protists. In general, the presence of lytic phages selected for lowered virulence in bacteria. High temperature selected for more virulent and more motile bacteria, but this was constrained by the presence of a lytic phage. In the multispecies communities the presence of all bacterial enemies led to decreased virulence in vivo. Altogether, these results contrast the hypothesis of coincidental evolution, and suggest that the presence of phages in natural reservoirs constrains the evolution of virulence, most likely through fitness costs associated with phage-resistance. Exposure to phage cocktails was also shown to be associated with decreased bacterial virulence in the phage-resistant bacteria. However, exposure to some individual phages resulted in more virulent bacteria, suggesting that the outcome of therapy could depend on the identity of the phage cocktail. Finally, a phage cocktail lysing a wide range of clinical strains was isolated from sewage. This, along with geographical patterns of phage infections suggest that new phages are available in environmental reservoirs for therapy, and the emergence of phage-resistance should not hinder the prospects of phage therapy in the global perspective.Bakteerivirukset (faagit) ovat bakteerien solunsisäisiä loisia, jotka yhdessä alkueliösaalistajien kanssa aiheuttavat suurimman osan bakteerien kuolleisuudesta luonnossa. Faagit luovat voimakkaan valintapaineen faagivastustuskyvylle ja vastustuskyvystä seuraa usein kustannus esimerkiksi bakteerin kasvu-, taudinaiheuttamis- tai liikkumiskyvylle. Perinteisesti faagin ja isännän välisiä vuorovaikutuksia on tutkittu kahden lajin kokeissa laboratoriossa, mutta lähestymistapa jättää huomiotta luonnollisissa yhteisöissä vallitsevien vuorovaikutusten kirjon. Koska faagit tappavat kohdennetusti tiettyjä bakteereita, on ehdotettu, että faageja voitaisiin käyttää antibiootteina. Faagiterapian kehittäminen vaatii kuitenkin faagi-isäntä vuorovaikutusten tuntemusta niin ekologisesta kuin evolutiivisestakin näkökulmasta. Tässä väitöskirjassa olen tutkinut lyyttisten bakteerivirusten vaikutuksia opportunistisiin taudinaiheuttajabakteereihin. Osa opportunistisista bakteereista kykenee elämään ja lisääntymään isäntänsä ulkopuolisissa ympäristöissä, missä ne altistuvat lukuisille erilaisille valintapaineille. Tutkin faagien merkitystä bakteeribiomassan vähentäjinä, sekä bakteerien faagivastustuskyvyn evoluutiota matalaravinteisissa lampea muistuttavassa ympäristössä, joissa oli faagien lisäksi läsnä ravinnonhankintatavoiltaan toisistaan poikkeavia alkueliöitä. Sattumanvaraisen evoluution hypoteesin (coincidental evolution hypothesis) mukaan taudinaiheuttamiskyky on seurausta sellaisiin elinkiertopiirteisiin kohdistuvasta valinnasta, jotka parantavat kelpoisuutta isännän ulkopuolisissa ympäristöissä. Opportunistien aiheuttamat tautiepidemiat ovat kuitenkin verraten harvinaisia, mikä viittaa siihen että jokin tekijä ympäristössä toimii vastavoimana taudinaiheuttamiskykyyn kohdistuvalle valinnalle. Faagien vaikutuksia opportunistibakteerien taudinaiheuttamiskyvyn evoluutioon tutkittiin niinikään matalaravinteisessa ympäristössä, joissa vaihtelevina ympäristötekijöinä olivat joko lämpötila tai bakteereja ravinnokseen käyttävien vihollisten eri yhdistelmät. Kokeiden päätteeksi taudinaiheuttamiskyky mitattiin hyönteisissä. Kliinistä taudinaiheuttajabakteerikantaa altistettiin faagi-koktaileille (phage cocktails) kokeessa, jonka tarkoituksena oli selvittää onko faageille vastustuskykyisten bakteerin taudinaiheuttamiskyky alentunut faagipuolustuksen aiheuttamien kustannusten seurauksena, ja mahdolliset muutokset taudinaiheuttamiskyvyssä mitattiin hyönteisissä. Faagivastustuskykyyn kohdistuvan voimakkaan valintapaineen huomioonottaen faagiterapian onnistumisen kannalta on merkittävää, voidaanko uusia faageja tarvittaessa eristää ympäristöstä. Tavoitteeksi asetettiin eristää uusia faageja, jotka estävät kliinisten antibiooteille vastustuskykyisten bakteereiden kasvua. Yksittäinen faagityyppi osoittautui tehottomaksi säätelemään bakteeribiomassan määrää pitkällä aikavälillä ja faageille vastustuskykyiset bakteerit palauttivat populaatiot nopeasti lähes kontrollitasolle. Alkueläimet näin ollen vastasivat pitkällä aikavälillä käytännössä kaikesta bakteeribiomassan vähentymisestä. Alkueliöt vaikuttivat myös bakteerien faagivastustuskyvyn evoluutioon: alkueliöille altistuneet bakteerit olivat vähemmän alttiita faagi-infektioille. Korkea lämpötila johti taudinaiheuttamiskykyvyn nousuun bakteereissa, mutta ilmiö kumoutui faagien vaikutuksesta. Monilajisissa yhteisöissä kaikkien bakteeripetojen läsnäolo taas alensi bakteerien taudinaiheuttamiskykyä. Nämä tulokset puhuvat sattumanvaraisen evoluution hypoteesia vastaan ja faagit näyttäisivät sen sijaan luovan valintapaineen vähemmän taudinaiheuttamiskykyisille bakteereille. Myös faagikoktailit aiheuttivat taudinaiheuttamiskyvyn laskua bakteereissa. Jotkut yksittäiset virukset näyttivät kuitenkin nostavan bakteerien taudinaiheuttamiskykyä vastustuskykyisissä bakteereissa, minkä vuoksi tuleekin noudattaa erityistä varovaisuutta valittaessa faageja koktaileihin ja faagiterapiaan. Jätevedestä onnistuttiin eristämään kokoelma faageja, jotka estivät kasvua laajassa joukossa kliinisiä antibiooteille vastustuskykyisiä bakteerikantoja. Tämä tulos yhdessä faagi-infektioiden maantiedettä koskevien havaintojen kanssa antaa olettaa, että uusia faageja on eristettävissä ympäristövarannoista ja että laaja-alaisen faagivastustuskyvyn syntyminen ei ole este faagiterapian kehittämiselle tulevaisuudessa

Protist predation can select for bacteria with lowered susceptibility to infection by lytic phages

Background: Consumer-resource interactions constitute one of the most common types of interspecific antagonistic interaction. In natural communities, complex species interactions are likely to affect the outcomes of reciprocal co-evolution between consumers and their resource species. Individuals face multiple enemies simultaneously, and consequently they need to adapt to several different types of enemy pressures. In this study, we assessed how protist predation affects the susceptibility of bacterial populations to infection by viral parasites, and whether there is an associated cost of defence on the competitive ability of the bacteria. As a study system we used Serratia marcescens and its lytic bacteriophage, along with two bacteriovorous protists with distinct feeding modes: Tetrahymena thermophila (particle feeder) and Acanthamoeba castellanii (surface feeder). The results were further confirmed with another study system with Pseudomonas and Tetrahymena thermophila. Results: We found that selection by protist predators lowered the susceptibility to infections by lytic phages in Serratia and Pseudomonas. In Serratia, concurrent selection by phages and protists led to lowered susceptibility to phage infections and this effect was independent from whether the bacteria shared a co-evolutionary history with the phage population or not. Bacteria that had evolved with phages were overall more susceptible to phage infection (compared to bacteria with history with multiple enemies) but they were less vulnerable to the phages they had co-evolved with than ancestral phages. Selection by bacterial enemies was costly in general and was seen as a lowered fitness in absence of phages, measured as a biomass yield. Conclusions: Our results show the significance of multiple species interactions on pairwise consumer-resource interaction, and suggest potential overlap in defending against predatory and parasitic enemies in microbial consumer-resource communities. Ultimately, our results could have larger scale effects on eco-evolutionary community dynamics.Peer reviewe

Sequence Variability of P2-Like Prophage Genomes Carrying the Cytolethal Distending Toxin V Operon in Escherichia coli O157

Cytolethal distending toxins (CDT) are potent cytotoxins of several Gram-negative pathogenic bacteria, including Escherichia coli, in which five types (CDT-I to CDT-V) have been identified so far. CDT-V is frequently associated with Shiga-toxigenic E. coli (STEC), enterohemorrhagic E. coli (EHEC) O157 strains, and strains not fitting any established pathotypes. In this study, we were the first to sequence and annotate a 31.2-kb-long, noninducible P2-like prophage carrying the cdt-V operon from an stx- and eae-negative E. coli O157:H43 strain of bovine origin. The cdt-V operon is integrated in the place of the tin and old phage immunity genes (termed the TO region) of the prophage, and the prophage itself is integrated into the bacterial chromosome between the housekeeping genes cpxP and fieF. The presence of P2-like genes (n = 20) was investigated in a further five CDT-V-positive bovine E. coli O157 strains of various serotypes, three EHEC O157:NM strains, four strains expressing other variants of CDT, and eight CDT-negative strains. All but one CDT-V-positive atypical O157 strain uniformly carried all the investigated genomic regions of P2-like phages, while the EHEC O157 strains missed three regions and the CDT-V-negative strains carried only a few P2-like sequences. Our results suggest that P2-like phages play a role in the dissemination of cdt-V between E. coli O157 strains and that after integration into the bacterial chromosome, they adapted to the respective hosts and became temperate

Top-down effects of a lytic bacteriophage and protozoa on bacteria in aqueous and biofilm phases

Peer reviewe

Systematic Comparison of Epidemic and Non-Epidemic Carbapenem Resistant Klebsiella pneumoniae Strains

Over the past few decades, extensively drug resistant (XDR) resistant Klebsiella pneumoniae has become a notable burden to healthcare all over the world. Especially carbapenemase-producing strains are problematic due to their capability to withstand even last resort antibiotics. Some sequence types (STs) of K. pneumoniae are significantly more prevalent in hospital settings in comparison to other equally resistant strains. This provokes the question whether or not there are phenotypic characteristics that may render certain K. pneumoniae more suitable for epidemic dispersal between patients, hospitals, and different environments. In this study, we selected seven epidemic and non-epidemic carbapenem resistant K. pneumoniae isolates for extensive systematic characterization for phenotypic and genotypic qualities in order to identify potential factors that precede or emerge from epidemic successfulness. Studied characteristics include growth rates and densities in different conditions (media, temperature, pH, resource levels), tolerance to alcohol and drought, inhibition between strains, ability to compensate pH, as well as various genomic features. Overall, there are clear differences between isolates, yet, only drought tolerance was found to notably associate with non-epidemic K. pneumoniae strains. We further report a preliminary study on the potential to control K. pneumoniae ST11 with an antimicrobial component produced by a non-epidemic K. pneumoniae. This component initially restricts bacterial growth, but stable resistance develops rapidly in vitro

ProxiMAX randomisation:a new technology for non-degenerate saturation mutagenesis of contiguous codons

Back in 2003, we published ‘MAX’ randomisation, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. ‘MAX’ randomisation saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an alpha-helix, as in zinc finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple, contiguous codons in a non-degenerate manner. We have now developed ‘ProxiMAX’ randomisation, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomisation uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialised chemistry, reagents nor equipment and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in pre-defined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomisation is particularly relevant to antibody engineering

Beyond the natural proteome:nondegenerate saturation mutagenesis - methodologies and advantages

Beyond the natural proteome, high-throughput mutagenesis offers the protein engineer an opportunity to “tweak” the wild-type activity of a protein to create a recombinant protein with required attributes. Of the various approaches available, saturation mutagenesis is one of the core techniques employed by protein engineers and in recent times, nondegenerate saturation mutagenesis is emerging as the approach of choice. This review compares the current methodologies available for conducting nondegenerate saturation mutagenesis with traditional, degenerate saturation and briefly outlines the options available for screening the resulting libraries, to discover a novel protein with the required activity and/or specificity

A diversity of uncharacterized reverse transcriptases in bacteria

Retroelements are usually considered to be eukaryotic elements because of the large number and variety in eukaryotic genomes. By comparison, reverse transcriptases (RTs) are rare in bacteria, with only three characterized classes: retrons, group II introns and diversity-generating retroelements (DGRs). Here, we present the results of a bioinformatic survey that aims to define the landscape of RTs across eubacterial, archaeal and phage genomes. We identify and categorize 1021 RTs, of which the majority are group II introns (73%). Surprisingly, a plethora of novel RTs are found that do not belong to characterized classes. The RTs have 11 domain architectures and are classified into 20 groupings based on sequence similarity, phylogenetic analyses and open reading frame domain structures. Interestingly, group II introns are the only bacterial RTs to exhibit clear evidence for independent mobility, while five other groups have putative functions in defense against phage infection or promotion of phage infection. These examples suggest that additional beneficial functions will be discovered among uncharacterized RTs. The study lays the groundwork for experimental characterization of these highly diverse sequences and has implications for the evolution of retroelements

Modular Mass Spectrometric Tool for Analysis of Composition and Phosphorylation of Protein Complexes

The combination of high accuracy, sensitivity and speed of single and multiple-stage mass spectrometric analyses enables the collection of comprehensive sets of data containing detailed information about complex biological samples. To achieve these properties, we combined two high-performance matrix-assisted laser desorption ionization mass analyzers in one modular mass spectrometric tool, and applied this tool for dissecting the composition and post-translational modifications of protein complexes. As an example of this approach, we here present studies of the Saccharomyces cerevisiae anaphase-promoting complexes (APC) and elucidation of phosphorylation sites on its components. In general, the modular concept we describe could be useful for assembling mass spectrometers operating with both matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) ion sources into powerful mass spectrometric tools for the comprehensive analysis of complex biological samples

A C-terminal cysteine residue is required for peptide-based inhibition of the NGF/TrkA interaction at nM concentrations:implications for peptide-based analgesics

Inhibition of the NGF/TrkA interaction presents an interesting alternative to the use of non-steroidal anti-inflammatories and/or opioids for the control of inflammatory, chronic and neuropathic pain. Most prominent of the current approaches to this therapy is the antibody Tanezumab, which is a late-stage development humanized monoclonal antibody that targets NGF. We sought to determine whether peptides might similarly inhibit the NGF/TrkA interaction and so serve as future therapeutic leads. Starting from two peptides that inhibit the NGF/TrkA interaction, we sought to eliminate a cysteine residue close to the C-terminal of both sequences, by an approach of mutagenic analysis and saturation mutagenesis of mutable residues. Elimination of cysteine from a therapeutic lead is desirable to circumvent manufacturing difficulties resulting from oxidation. Our analyses determined that the cysteine residue is not required for NGF binding, but is essential for inhibition of the NGF/TrkA interaction at pharmacologically relevant peptide concentrations. We conclude that a cysteine residue is required within potential peptide-based therapeutic leads and hypothesise that these peptides likely act as dimers, mirroring the dimeric structure of the TrkA receptor