Effect of a Depolymerase Encoded by Phage168 on a Carbapenem-Resistant Klebsiella pneumoniae and Its Biofilm.

Infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are becoming increasingly common within clinical settings, requiring the development of alternative therapies. In this study, we isolated, characterized, and sequenced the genome of a CRKP phage, Phage168. The total genomic DNA of Phage168 was 40,222 bp in length, encoding 49 predicted proteins. Among these proteins, Dep40, the gene product of ORF40, is a putative tail fiber protein that exhibits depolymerase activity based on the result of bioinformatics analyses. In vitro, we confirmed that the molecular weight of the Phage168 depolymerase protein was about 110 kDa, the concentration of the produced phage 168 depolymerase protein was quantified as being 1.2 mg/mL, and the depolymerase activity was still detectable after the dilution of 1.2 µg/mL. This recombinant depolymerase exhibited enzyme activity during the depolymerization of the formed CRKP biofilms. We also found that depolymerase, when combined with polymyxin B, was able to enhance the bactericidal effect of polymyxin B on CRKP strains by disrupting their biofilm. When recombinant depolymerase was used in combination with human serum, it enhanced the sensitivity of the CRKP strain UA168 to human serum, and the synergistic bactericidal effect reached the strongest level when the ratio of depolymerase to human serum was 3:1. Our results indicated that depolymerase encoded by Phage168 may be a promising strategy for combating infections caused by drug-resistant CRKP formed within the biofilm.

Permeability-Engineered Compartmentalization Enables In Vitro Reconstitution of Sustained Synthetic Biology Systems.

In nature, biological compartments such as cells rely on dynamically controlled permeability for matter exchange and complex cellular activities. Likewise, the ability to engineer compartment permeability is crucial for in vitro systems to gain sustainability, robustness, and complexity. However, rendering in vitro compartments such a capability is challenging. Here, a facile strategy is presented to build permeability-configurable compartments, and marked advantages of such compartmentalization are shown in reconstituting sustained synthetic biology systems in vitro. Through microfluidics, the strategy produces micrometer-sized layered microgels whose shell layer serves as a sieving structure for biomolecules and particles. In this configuration, the transport of DNAs, proteins, and bacteriophages across the compartments can be controlled an guided by a physical model. Through permeability engineering, a compartmentalized cell-free protein synthesis system sustains multicycle protein production; ≈100 000 compartments are repeatedly used in a five-cycle synthesis, featuring a yield of 2.2 mg mL-1 . Further, the engineered bacteria-enclosing compartments possess near-perfect phage resistance and enhanced environmental fitness. In a complex river silt environment, compartmentalized whole-cell biosensors show maintained activity throughout the 32 h pollutant monitoring. It is anticipated that permeability-engineered compartmentalization should pave the way for practical synthetic biology applications such as green bioproduction, environmental sensing, and bacteria-based therapeutics.

A Plasmid With Conserved Phage Genes Helps Klebsiella pneumoniae Defend Against the Invasion of Transferable DNA Elements at the Cost of Reduced Virulence.

Klebsiella pneumoniae exhibits extensive phenotypic and genetic diversity. Higher plasmid loads in the cell were supposed to play an key role in its genome diversity. Although some plasmids are widely distributed in Kp populations, they are poorly recognized. A plasmid named p2 in strain Kp1604 was predicted to be an intact prophage like Salmonella phage SSU5. However, our study showed that p2 was specifically packaged into membrane vesicles (MVs) rather than phage particles triggered by mitomycin C and subinhibitory concentrations of antibiotics. p2-minus mutant Kp1604Δp2 did not affect MV production. Compared with Kp1604, the capacity of plasmid uptake and the amount of phage burst of Kp1604Δp2 were improved. Moreover, virulence of Kp1604Δp2 also increased. Our results indicated that p2 could contribute to the host defense against the invasion of transferable DNA elements at the cost of reduced virulence. Further study on the mechanism will help us understand how it provides adaptive phenotypes to host evolution.