Endolysin EN572-5 as an alternative to treat urinary tract infection caused by Streptococcus agalactiae.

Streptococcus agalactiae (Group B Streptococcus, GBS) is an opportunistic pathogen causing urinary tract infection (UTI). Endolysin EN572-5 was identified in prophage KMB-572-E of the human isolate Streptococcus agalactiae KMB-572. The entire EN572-5 gene was cloned into an expression vector and the corresponding recombinant protein EN572-5 was expressed in Escherichia coli in a soluble form, isolated by affinity chromatography, and characterized. The isolated protein was highly active after 30 min incubation in a temperature range of - 20 °C to 37 °C and in a pH range of 5.5-8.0. The endolysin EN572-5 lytic activity was tested on different Streptococcus spp. and Lactobacillus spp. The enzyme lysed clinical GBS (n = 31/31) and different streptococci (n = 6/8), and also exhibited moderate lytic activity against UPEC (n = 4/4), but no lysis of beneficial vaginal lactobacilli (n = 4) was observed. The ability of EN572-5 to eliminate GBS during UTI was investigated using an in vitro model of UPSA. After the administration of 3 µM EN572-5, a nearly 3-log decrease of urine bacterial burden was detected within 3 h. To date, no studies have been published on the use of endolysins against S. agalactiae during UTI. KEY POINTS: • A lytic protein, EN572-5, from a prophage of a human GBS isolate has been identified. • This protein is easily produced, simple to prepare, and stable after lyophilization. • The bacteriolytic activity of EN572-5 was demonstrated for the first time in human urine.

Characterization of a newly discovered putative DNA replication initiator from Paenibacillus polymyxa phage phiBP.

The bacteriophage phiBP contains a newly discovered putative replisome organizer, a helicase loader, and a beta clamp, which together may serve to replicate its DNA. Bioinformatics analysis of the phiBP replisome organizer sequence showed that it belongs to a recently identified family of putative initiator proteins. We prepared and isolated a wild type-like recombinant protein, gpRO-HC, and a mutant protein gpRO-HCK8A, containing a lysine to alanine substitution at position 8. gpRO-HC had low ATPase activity regardless of the presence of DNA, while the ATPase activity of the mutant was significantly higher. gpRO-HC bound to both single- and double-stranded DNA substrates. Different methods showed that gpRO-HC forms higher oligomers containing about 12 subunits. This work provides the first information about another group of phage initiator proteins, which trigger DNA replication in phages infecting low GC Gram-positive bacteria.

A novel phage-encoded endolysin EN534-C active against clinical strain Streptococcus agalactiae GBS.

Streptococcus agalactiae (Group B Streptococcus, GBS) is primarily known as a major neonatal pathogen. In adults, these bacteria often colonize the gastrointestinal and urogenital tracts. Treatment of infections using antibiotics is often complicated by recurrences caused by multi-resistant streptococci. Endolysin EN534 from prophage A2 of human isolate Streptococcus agalactiae KMB-534 has a modular structure consisting of two terminal catalytic domains, amidase_3 and CHAP, and one central binding domain, LysM. The EN534 gene was cloned into an expression vector, and the corresponding recombinant protein EN534-C was expressed in Escherichia coli in a soluble form and isolated by affinity chromatography. The lytic activity of this endolysin was tested on cell wall substrates from different GBS serotypes, B. subtilis, L. jensenii, and E. coli. The enzyme lysed streptococci, but not beneficial vaginal lactobacilli. The isolated protein is stable in a temperature range of 20-37 °C. Calcium ions enhanced the activity of the enzyme in the pH range from 5.0 to 8.0. The exolytic activity of EN534-C was observed by time-lapse fluorescence microscopy on a S. agalactiae CCM 6187 substrate. Recombinant endolysin EN534-C may have the potential to become an antimicrobial agent for the treatment of S. agalactiae infections.

Identification of Brevibacterium flavum genes related to receptors involved in bacteriophage BFK20 adsorption.

Phage infection of bacterial cells is a process requiring the interaction between phage receptor binding proteins and receptors on the bacterial cell surface. We prepared a Brevibacterium flavum CCM 251 EZ-Tn5 transposon insertional library and isolated phage-resistant mutants. Analysis of the DNA fragments produced by single-primer PCR was used to determine the EZ-Tn5 transposon insertion sites in the genomes of phage-resistant B. flavum mutants. Seven disrupted genes were identified in forty B. flavum mutants. The phage resistance of these mutants was demonstrated by cultivation analysis in the presence of BFK20, and the adsorption rate of BFK20 to these mutants was tested. B. flavum mutants displayed significantly reduced adsorption rates; the lowest rate was observed for mutants containing interrupted major facilitator superfamily (MFS) protein and glycosyltransferase genes. Uninterrupted forms of these genes were cloned into corynebacterial vector pJUP06 and used for in trans complementation of the corresponding B. flavum mutants. The growth of these complemented mutants when infected with BFK20 closely resembled that of wild-type B. flavum. These complemented mutants also exhibited similar BFK20 adsorption as the wild-type control. We infer that the disrupted MFS protein and glycosyltransferase genes are responsible for the phage-resistant phenotype of these B. flavum transposition mutants.

The BFK20 phage replication origin confers a phage-encoded resistance phenotype to the industrial strain Brevibacterium flavum.

The phage BFK20 replication origin was identified using bioinformatics tools and a fragment with the origin nucleotide sequence was cloned into the tetracycline resistance gene of Escherichia coli vector pBR328, to make the plasmid pBOS. After transformation into the host strain Brevibacterium flavum CCM 251, pBOS was able to replicate, showing that the cloned region may function as a replication origin. The presence of the BFK20 origin sequence in a pBOS plasmid isolated from B. flavum CCM 251 was confirmed by Southern hybridisation. Monitoring pBOS stability in corynebacterial hosts showed that pBOS was stable in Corynebacterium glutamicum RM3 for 20 generations and in B. flavum CCM 251 for 10 generations. The effect of the cloned BFK20 replication origin on host resistance to BFK20 infection was tested. Growth of a B. flavum CCM 251 strain harbouring pBOS stopped after phage infection, but without complete lysis. Five hours after infection, the viability of the modified strain was about five times higher than the viability of wild-type B. flavum CCM 251. Thus, the ability of the BFK20 replication origin to confer the origin-derived phage-encoded resistance phenotype to B. flavum CCM 251 was confirmed.