Browsing by Subject "Bacteriophage"

S. aureus infections are prominent worldwide, and with the rapid increase in antimicrobial resistant variants such as methicillin-resistant MRSA, the need for new treatment alternatives is imminent (Monaco et al., 2017). Lytic bacteriophages are continually evolving new methods for the destruction of bacterial cells while avoiding their defence mechanisms. Screening hypothetical proteins of unknown function (HPUFs) from bacteriophages for toxic activity against bacteria may provide new and potentially life-saving approaches to combat bacterial infections (Liu et al., 2004, Singh et al., 2019). The Stab21 phage of Staphylococcus is a recently described lytic phage with over 85 % of its open reading frames annotated as HPUFs (Oduor et al., 2019). The successful identification of potentially toxic gene products could facilitate the discovery of novel bacterial targets for the development of new antimicrobials. It could also provide treatment options to multi-drug resistant S. aureus caused infections where no effective drugs are currently available. To reduce unnecessary screening of phage particle associated yet poorly annotated proteins, total proteins of phage particle were previously identified by LC-MS. Similar studies have previously been performed with Yersinia phage fR1-RT and Klebsiella phage fHe-Kpn01, where a handful of toxic proteins were discovered (Mohanraj et al., 2019, Spruit et al., 2020). To accelerate the screening process, a next-generation sequencing (NGS) high-throughput screening method was further developed by Kasurinen et al. (2021). In this study, 96 true HPUFs were selected and screened for their bactericidal activity in E. coli using the NGS-based approach. Fourteen potentially bacteriotoxic Stab-21 gene products were identified through toxicity screening in E. coli. Of these, three had a particularly low ratio of isolated plasmid after transformation while having a significant number of reads over each joint sequence, indicating their potentially high toxicity. The three most promising candidates were the gene products of g008, g081c and g175 of the Stab21 bacteriophage.

Bacteriophages are viruses that infect bacteria. With the ever-increasing threat of antibiotic resistance, they have emerged as a promising alternative treatment. Many phage genomes contain modified bases. They prevent digestion by restriction enzymes allowing the resistance of these viruses to bacterial defence mechanisms. YerA41, a phage that infects Yersinia ruckeri, contains a genome that could not be amplified using any of the DNA polymerases available in the market. Neither restrictions enzymes were able to digest it. These properties led to the assumption that YerA41 genome is not conventional and is likely to contain modified nucleotides. In order to replicate its genome, YerA41 should possess its own DNA polymerase that would be able to use, YerA41 genome as template. If so, it would be able to use other modified genomes as well. Hence, this DNA polymerase could become a very valuable biotechnological tool. In this study we isolated and optimised the purification of DNAP01, one of the putative DNA polymerases encoded by YerA41 genome. In addition, this work shows, with the help of different experiments, how DNAP01 is a novel DNA polymerase able to use YerA41 DNA as template. This is the first time an enzyme of this nature has been described and isolated.

The ever-increasing spread of antibiotic resistant bacteria creates a constant demand for new sources for antimicrobial drugs. Phages are a natural source for antibacterial proteins, but also produce a variety of unknown compounds, referred to as “hypothetical proteins of unknown function” (HPUF). HPUFs usually consist of structural proteins, but also small polypeptides that inhibit bacterial growth during infection. These peptides could be utilized in the discovery of new antimicrobial molecules. However, the current methods used for the screening of such proteins are time consuming and unreliable, making this a fairly unpopular option to utilize. In this study, a new NGS (Next Generation Sequencing) based assay for the screening of phage derived bacteriotoxic proteins was developed and tested by performing two separate experiments together with a previously used plating assay as a comparative method. A preliminary experiment was performed as a proof of principle, with five known toxic and five non-toxic genes. After this, the methods were compared by screening 23 previously identified HPUF genes of phage fHy-Eco03. In the plating assay genes were screened individually by observing growth of bacterial transformants upon gene expression. In the NGS assay genes we screened simultaneously by transforming them to E. coli cells as a pooled sample. Results were obtained with bioinformatics. Toxic genes were expected to be identified through a decrease in sequence read amount, as a consequence of bacterial growth inhibition. In the pre-experiment a difference between toxic and non- toxic proteins was not observed. The results between the NGS and plating assay in the screening of phage fHy-Eco03 genes, were similar and resulted in the identification of one toxic protein. The inconsistent results are probably an outcome of lac promoter repression by glucose supplementation, thus only highly toxic genes show an inhibitory effect. Despite this the NGS assay outperformed the plating assay in both accuracy and efficiency. The NGS assay has high potential to be used as a screening assay for phage derived toxic genes, however further optimization and validation is required, by firstly selecting compatible media and secondly by re- testing with different phages and host bacteria.

Suun mikrobiston bakteereista Streptococcus mutans on erityisen haitallinen aiheuttaen hammaskariesta. Tutkimukseni tavoitteena oli S. mutans spesifisten bakteriofagien eristäminen. Tutkimuksessa selvitettiin, löytyykö uusia faageja ja voidaanko niitä tulevaisuudessa käyttää faagiterapiassa kariesbakteerien hävittämiseen potilaista. Työ toteutettiin osana Helsingin yliopiston suurempaa bakteriofagitutkimusta, jonka lopputavoitteena on tuottaa uusia hoitomuotoja bakteeri-infektioiden hoitoon. Uudet hoitomuodot bakteeri-infektioissa ovat kasvavan tutkimuksen alla antibioottien tehon heikentyessä ja resistenttiyden lisääntyessä bakteerien keskuudessa. Tutkimus tehtiin yleisiä laboratoriotyöohjeita noudattaen bakteriofagien löytämiseksi. Tutkimuksessa käytettiin Hammaslääketieteenlaitokselta saatuja kantoja (Grönroos et al. 1998) sekä HUSLABista kerättyjä potilaskantoja. Tutkimukseni näytemateriaaleina käytettiin kaupallista bakteriofagiseosta, jätevesinäytteitä sekä sylkinäytteitä. Faageja etsittiin kaksikerrosmaljalta niin sanotulla Drop test- menetelmällä. Bakteriofagit voidaan eristää maljalla näkyvältä bakteerivapaalta alueelta tarkempaa tutkimusta varten. Suomen faagiterapiahanke on vasta alkuvaiheessa. Streptococcus mutansia infektoivia bakteriofageja ei löydetty tutkimukseni aikana yhdestäkään näytemateriaalista, joten soveltuvuutta faagiterapiaan ei voitu tutkia. Tästä syystä tutkimukseni toisessa vaiheessa yritettiin eristää myös uusia S. mutans kantoja vapaaehtoisten koehenkilöiden syljestä spesifisten kasvatusalustojen avulla. Ajan saatossa muuntuneet faagikannat voivat infektoida nykyisin esiintyviä bakteerikantoja vanhojen sijaan. Tutkimukseni aikana saatiin eristettyä 10 uutta S. mutans kantaa. Kaikkiaan S. mutans bakteriofageja on tieteellisten julkaisujen ja tietokantojen perusteella löydetty hyvin vähän tähän mennessä. Tutkimukseni tulos bakteriofagien osalta noudattaa siis aiempien tutkimusten linjaa. Jatkossa näytemääriä lisäämällä positiivisen faagilöydöksen todennäköisyys saattaisi kasvaa.