Discovery of Three Toxic Proteins of Klebsiella Phage fHe-Kpn01

Abstract

The lytic phage, fHe-Kpn01 was isolated from sewage water using an extended-spectrum beta-lactamase-producing strain of Klebsiella pneumoniae as a host. The genome is 43,329 bp in size and contains direct terminal repeats of 222 bp. The genome contains 56 predicted genes, of which proteomics analysis detected 29 different proteins in purified phage particles. Comparison of fHe-Kpn01 to other phages, both morphologically and genetically, indicated that the phage belongs to the family Podoviridae and genus Drulisvirus. Because fHe-Kpn01 is strictly lytic and does not carry any known resistance or virulence genes, it is suitable for phage therapy. It has, however, a narrow host range since it infected only three of the 72 tested K. pneumoniae strains, two of which were of capsule type KL62. After annotation of the predicted genes based on the similarity to genes of known function and proteomics results on the virion-associated proteins, 22 gene products remained annotated as hypothetical proteins of unknown function (HPUF). These fHe-Kpn01 HPUFs were screened for their toxicity in Escherichia coli. Three of the HPUFs, encoded by the genes g10, g22, and g38, were confirmed to be toxic.

Characterization and Full Genome Sequence of Novel KPP-5 Lytic Phage against Klebsiella pneumoniae Responsible for Recalcitrant Infection

“…YP_009286649) contained double TMD regions via the prediction of the TMHMM server ( Figure 10 ). In phage genomes, 70% of the predicted genes are annotated to encode hypothetical proteins, most of which range from 30 to 200 amino acids with unknown functions [ 18 , 94 , 95 , 96 ]. The high potential for the use of these proteins as toxic proteins has already been demonstrated, where they can be toxic through unknown mechanisms and have the potential to develop into novel antimicrobial peptides [ 18 ].…”

“…In addition, phages have been shown to be active not only against plank-tonic bacteria but also against bacteria organized in biofilms by lysing bacterial cells or the biofilm matrix [ 16 , 17 ]. Phage proteins, such as endolysins, hydrolases, depolymerases, and holins, are already extensively investigated in the development of promising new antibiotics, where phages use these enzymes to infect or lyse bacteria [ 18 , 19 ]. Even more phage proteins are directly linked to bacterial proteins in order to sustain a successful infection cycle, and, surprisingly, the functional mechanisms of these associations are largely unknown [ 18 , 20 ].…”

Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay

“…Phage lysates were produced from semiconfluent soft-agar plates as described elsewhere [ 17 ]. The phage lysates were then concentrated, purified using ultracentrifugation with a glycerol step gradient [ 18 ], and stored at 4 °C as described earlier [ 10 ].…”

“…Based on previous studies, many HPUFs include small molecules with toxic properties (toxHPUFs) that phages utilize during the infection cycle to hamper the cellular functions and defense mechanisms of the bacterial host [ 6 ]. Traditionally, toxic gene products have been detected by the inefficient transformation of bacterial cells with a plasmid carrying the toxin-encoding gene [ 6 , 7 , 8 , 9 , 10 , 11 ]. In the plating efficiency assay, the number of transformants obtained with the HPUF-encoding genes is compared to the number of transformants obtained with control genes encoding known toxic and non-toxic proteins.…”

“…Using NGS read coverages, the relative abundance of correctly ligated inserts can be determined for a pooled ligation mixture and for the plasmids isolated from the pooled transformants. We describe and validate here the NGS screening assay by carrying out screening with five known toxic and four non-toxic genes of phages fHe-Kpn01 [ 10 ], ϕR1-RT [ 8 ], and T4 [ 12 ] and 32 HPUF-encoding genes of Escherichia phage fHy-Eco03 in a comparative study with the plating assay [ 10 ].…”