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Downregulation of autolysin-encoding genes by phage-derived lytic proteins inhibits biofilm formation in Staphylococcus aureus

ABSTRACT: Phage-derived lytic proteins are a promising alternative to conventional antimicrobials. One of their most interesting properties is that they do not readily select for resistant strains, which is likely due to the fact that their targets are essential for the viability of the bacterial cell. Moreover, genetic engineering allows the design of new "tailor-made" proteins that may exhibit improved antibacterial properties. One example of this is the chimeric protein CHAPSH3b, which consists of a catalytic domain from the virion-associated peptidoglycan (PG) hydrolase of phage vB_SauS-phiIPLA88 (HydH5) and the cell wall binding domain of lysostaphin. CHAPSH3b had previously shown the ability to kill S. aureus cells. Here, we demonstrate that this lytic protein also has potential for the control of biofilm-embedded S. aureus cells. Additionally, subinhibitory doses of CHAPSH3b can decrease biofilm formation by some S. aureus strains. Transcriptional analysis revealed that exposure of S. aureus cells to this enzyme leads to the downregulation of several genes coding for bacterial autolysins. One of these proteins, namely the major autolysin AtlA, is known to participate in staphylococcal biofilm development. Interestingly, an atl mutant strain did not display inhibition of biofilm development when grown at subinhibitory concentrations of CHAPSH3b, contrary to the observations made for the parental and complemented strains. Also, deletion of atl led to low-level resistance to CHAPSH3b and endolysin LysH5. Overall, our results reveal new aspects that should be considered when designing new phage-derived lytic proteins aimed for antimicrobial applications.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE94512 | GEO | 2017/03/16

SECONDARY ACCESSION(S): PRJNA370921

REPOSITORIES: GEO

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Project description:Staphylococcus aureus is a Gram-positive human pathogen causing a variety of human diseases in both hospital and community settings. This bacterium is so closely associated with prophages that it is rare to find S. aureus isolates without prophages. Two phages are known to be important for staphylococcal virulence: the beta-hemolysin (hlb) converting phage and the Panton-Valentine Leukocidin (PVL) converting phage. The hlb-converting phage is found in more than 90% of clinical isolates of S. aureus. This phage produces exotoxins and immune modulatory molecules, which inhibit human innate immune responses. The PVL-converting phage produces the two-component exotoxin PVL, which can kill human leucocytes. This phage is wide-spread among community-associated methicillin resistant S. aureus (CA-MRSA). It also shows strong association with soft tissue infections and necrotizing pneumonia. Several lines of evidence suggest that staphylococcal prophages increase bacterial virulence not only by providing virulence factors but also by altering bacterial gene expression: 1) Transposon insertion into prophage regulatory genes, but not into the genes of virulence factors, reduced S. aureus killing of Caenorhabditis elegans.; 2) Although the toxins and immune modulatory molecules encoded by the hlb- converting phages do not function in the murine system, deletion of ϕNM3, the hlb-converting phage in S. aureus Newman, reduced staphylococcal virulence in the murine abscess formation model. 3) In a preliminary microarray experiment, prophages in S. aureus Newman altered the expression of more than 300 genes. In this research proposal, using microarray and high-throughput quantitative RT-PCR (qRT-PCR) technologies, we will identify the effects of the two important staphylococcal phages on the gene expression of S. aureus in both in vitro and in vivo conditions. This project is intended to be completed within one year. All the data – microarray, qRT-PCR and all the primer sequences- will be made available to public 6 month after completion. Data from this project will help us to understand the role of prophages in the S. aureus pathogenesis and can lead to development of a strategy to interfere with the pathogenesis process. Following strains were grown in TSA broth: Staphylococcus aureus USA300 (reference) Staphylococcus aureus USA300 with deletion of ϕSa2usa (Query) Staphylococcus aureus USA300 with deletion of ϕSa3usa (Query) Staphylococcus aureus USA300 Prophage-free mutant (Query) Staphylococcus aureus USA300 Prophage-free mutant lysogenized with ϕSa2mw (Query) Staphylococcus aureus USA300 Prophage-free mutant lysogenized with ϕSa3usa (Query) strain: Staphylococcus aureus USA300 Prophage-free mutant lysogenized with both ϕSa2mw and ϕSa3usa (Query) RNA samples were harvested at early log, midlog and stationary phase.Samples were hybridized on aminosilane coated slides with 70-mer oligos.

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Downregulation of autolysin-encoding genes by phage-derived lytic proteins inhibits biofilm formation in Staphylococcus aureus

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