Opposing growth regulatory roles of protein kinase D isoforms in human keratinocytes.

PKD is a family of three serine/threonine kinases (PKD-1, -2, and -3) involved in the regulation of diverse biological processes including proliferation, migration, secretion, and cell survival. We have previously shown that despite expression of all three isoforms in mouse epidermis, PKD1 plays a unique and critical role in wound healing, phorbol ester-induced hyperplasia, and tumor development. In translating our findings to the human, we discovered that PKD1 is not expressed in human keratinocytes (KCs) and there is a divergence in the expression and function of other PKD isoforms. Contrary to mouse KCs, treatment of cultured human KCs with pharmacological inhibitors of PKDs resulted in growth arrest. We found that PKD2 and PKD3 are expressed differentially in proliferating and differentiating human KCs, with the former uniformly present in both compartments whereas the latter is predominantly expressed in the proliferating compartment. Knockdown of individual PKD isoforms in human KCs revealed contrasting growth regulatory roles for PKD2 and PKD3. Loss of PKD2 enhanced KC proliferative potential while loss of PKD3 resulted in a progressive proliferation defect, loss of clonogenicity and diminished tissue regenerative ability. This proliferation defect was correlated with up-regulation of CDK4/6 inhibitor p15(INK4B) and induction of a p53-independent G1 cell cycle arrest. Simultaneous silencing of PKD isoforms resulted in a more pronounced proliferation defect consistent with a predominant role for PKD3 in proliferating KCs. These data underline the importance and complexity of PKD signaling in human epidermis and suggest a central role for PKD3 signaling in maintaining human epidermal homeostasis.

Genetic characterization of Pseudomonas aeruginosa bacteriophage KPP10.

Bacteriophage (phage) KPP10 has been used in experimental phage therapies directed against P. aeruginosa infections. To examine the eligibility of phage KPP10 as a therapeutic phage, its genome was analyzed. The genomic DNA was shown to be 88,322 bp long, with 158 open reading frames (ORFs), and three tRNA genes were predicted. No ORF-encoded pathogenicity or lysogenization factor was predicted. A comparative genomic analysis revealed that phage KPP10, together with phage PAK_P3, can be grouped as a new type of lytic phage infecting P. aeruginosa. Phage KPP10 is considered to be suitable for therapeutic purposes because it is a lytic phage without ORF-encoded pathogenicity or a lysogenization factors.

Characteristics of a novel Pseudomonas aeruginosa bacteriophage, PAJU2, which is genetically related to bacteriophage D3.

Pseudomonas aeruginosa bacteriophage (phage) is one of the most taxonomically and genetically diverse phages. Although phage D3 is one of well-studied P. aeruginosa phages, no D3-related P. aeruginosa phage has been reported. We report a novel P. aeruginosa siphovirus, PAJU2, which is genetically related to but morphology distinct (highly elongated head) from phage D3. A PAJU2 capsid protein, Orf3, is thought to be synthesized as a protein fused to a prohead protease and is autocatalytically cleaved, which may form the head chain mail. Despite such morphological differences, PAJU2 is expected to be a useful genetic reference for phage D3.

A novel site-specific recombination system derived from bacteriophage phiMR11.

We report identification of a novel site-specific DNA recombination system that functions in both in vivo and in vitro, derived from lysogenic Staphylococcus aureus phage phiMR11. In silico analysis of the phiMR11 genome indicated orf1 as a putative integrase gene. Phage and bacterial attachment sites (attP and attB, respectively) and attachment junctions were determined and their nucleotide sequences decoded. Sequences of attP and attB were mostly different to each other except for a two bp common core that was the crossover point. We found several inverted repeats adjacent to the core sequence of attP as potential protein binding sites. The precise and efficient integration properties of phiMR11 integrase were shown on attP and attB in Escherichia coli and the minimum size of attP was found to be 34bp. In in vitro assays using crude or purified integrase, only buffer and substrate DNAs were required for the recombination reaction, indicating that other bacterially encoded factors are not essential for activity.

In silico and in vivo evaluation of bacteriophage phiEF24C, a candidate for treatment of Enterococcus faecalis infections.

Along with the increasing threat of nosocomial infections by vancomycin-resistant Enterococcus faecalis, bacteriophage (phage) therapy has been expected as an alternative therapy against infectious disease. Although genome information and proof of applicability are prerequisites for a modern therapeutic phage, E. faecalis phage has not been analyzed in terms of these aspects. Previously, we reported a novel virulent phage, phiEF24C, and its biology indicated its therapeutic potential against E. faecalis infection. In this study, the phiEF24C genome was analyzed and the in vivo therapeutic applicability of phiEF24C was also briefly assessed. Its complete genome (142,072 bp) was predicted to have 221 open reading frames (ORFs) and five tRNA genes. In our functional analysis of the ORFs by use of a public database, no proteins undesirable in phage therapy, such as pathogenic and integration-related proteins, were predicted. The noncompetitive directions of replication and transcription and the host-adapted translation of the phage were deduced bioinformatically. Its genomic features indicated that phiEF24C is a member of the SPO1-like phage genus and especially that it has a close relationship to the Listeria phage P100, which is authorized for prophylactic use. Thus, these bioinformatics analyses rationalized the therapeutic eligibility of phiEF24C. Moreover, the in vivo therapeutic potential of phiEF24C, which was effective at a low concentration and was not affected by host sensitivity to the phage, was proven by use of sepsis BALB/c mouse models. Furthermore, no change in mouse lethality was observed under either single or repeated phage exposures. Although further study is required, phiEF24C can be a promising therapeutic phage against E. faecalis infections.

Isolation and characterization of a novel Enterococcus faecalis bacteriophage phiEF24C as a therapeutic candidate.

Vancomycin-resistant Enterococcus faecalis (VRE) has become a significant threat in nosocomial settings. Bacteriophage (phage) therapy is frequently proposed as a potential alternative therapy for infections caused by this bacterium. To search for candidate therapeutic phages against Enterococcus faecalis infections, 30 Enterococcus faecalis phages were isolated from the environment. One of these, virulent phage phiEF24C, which has a broad host range, was selected for analysis. The plaque-forming ability of phiEF24C was virtually unaffected by differences in the clinical host strains. Furthermore, the phage had a shorter latent period and a larger burst size than ordinary tailed phages, indicating that phiEF24C has effective lytic activity against many Enterococcus faecalis strains, including VRE. Morphological and genomic analyses revealed that phiEF24C is a large myovirus (classified as family Myoviridae morphotype A1) with a linear double-stranded DNA genome of c. 143 kbp. Analyses of the N-terminal amino acid sequences of the virion proteins, together with the morphology and the genome size, speculated that phiEF24C is closely related to other myoviruses of Gram-positive bacteria that have been used experimentally or practically for therapy or prophylaxis. Considering these results, phiEF24C may be a potential candidate therapeutic phage against Enterococcus faecalis infections.

Tail-associated structural protein gp61 of Staphylococcus aureus phage phi MR11 has bifunctional lytic activity.

A tailed bacteriophage, phi MR11 (siphovirus), was selected as a candidate therapeutic phage against Staphylococcus aureus infections. Gene 61, one of the 67 ORFs identified, is located in the morphogenic module. The gene product (gp61) has lytic domains homologous to CHAP (corresponding to an amidase function) at its N-terminus and lysozyme subfamily 2 (LYZ2) at its C-terminus. Each domain of gp61 was purified as a recombinant protein. Both the amidase [amino acids (aa) 1-150] and the lysozyme (aa 401-624) domains but not the linker domain (aa 151-400) caused efficient lysis of S. aureus. Immunoelectron microscopy localized gp61 to the tail tip of the phi MR11 phage. These data strongly suggest that gp61 is a tail-associated lytic factor involved in local cell-wall degradation, allowing the subsequent injection of phi MR11 DNA into the host cytoplasm. Staphylococcus aureus lysogenized with phi MR11 was also lysed by both proteins. Staphylococcus aureus strains on which phi MR11 phage can only produce spots but not plaques were also lysed by each protein, indicating that gp61 may be involved in 'lysis from without'. This is the first report of the presence of a tail-associated virion protein that acts as a lysin, in an S. aureus phage.

Protein kinase D1 has a key role in wound healing and skin carcinogenesis.

Protein kinase D (PKD) is a family of stress-responsive serine/threonine kinases implicated in the regulation of diverse cellular functions including cell growth, differentiation, apoptosis, and cell motility. Although all three isoforms are expressed in keratinocytes, their role in skin biology and pathology is poorly understood. We recently identified a critical role for PKD1 during reversal of keratinocyte differentiation in culture, suggesting a potential proproliferative role in epidermal adaptive responses. Here, we generated mice with targeted deletion of PKD1 in the epidermis to evaluate the significance of PKD1 in normal and hyperplastic conditions. These mice displayed a normal skin phenotype, indicating that PKD1 is dispensable for skin development and homeostasis. Upon wounding, however, PKD1-deficient mice exhibited delayed wound re-epithelialization correlated with a reduced proliferation and migration of keratinocytes at the wound edge. In addition, the hyperplastic and inflammatory responses to topical phorbol ester were significantly suppressed, suggesting involvement of PKD1 in tumor promotion. Consistently, when subjected to the two-stage chemical skin carcinogenesis protocol, PKD1-deficient mice were resistant to papilloma formation when compared with control littermates. These results revealed a critical proproliferative role for PKD1 in epidermal adaptive responses, suggesting a potential therapeutic target in skin wound and cancer treatment.

Pseudomonas aeruginosa keratitis in mice: effects of topical bacteriophage KPP12 administration.

The therapeutic effects of bacteriophage (phage) KPP12 in Pseudomonas aeruginosa keratitis were investigated in mice. Morphological analysis showed that phage KPP12 is a member of the family Myoviridae, morphotype A1, and DNA sequence analysis revealed that phage KPP12 is similar to PB1-like viruses. Analysis of the phage KPP12 genome did not identify any genes related to drug resistance, pathogenicity or lysogenicity, and so phage KPP12 may be a good candidate for therapeutic. KPP12 showed a broad host range for P. aeruginosa strains isolated from clinical ophthalmic infections. Inoculation of the scarified cornea with P. aeruginosa caused severe keratitis and eventual corneal perforation. Subsequent single-dose administration of KPP12 eye-drops significantly improved disease outcome, and preserved the structural integrity and transparency of the infected cornea. KPP12 treatment resulted in the suppression of neutrophil infiltration and greatly enhanced bacterial clearance in the infected cornea. These results indicate that bacteriophage eye-drops may be a novel adjunctive or alternative therapeutic agent for the treatment of infectious keratitis secondary to antibiotic-resistant bacteria.

Efficient elimination of multidrug-resistant Staphylococcus aureus by cloned lysin derived from bacteriophage phi MR11.

We report the successful purification of a cloned lysin encoded by the novel Staphylococcus aureus bacteriophage phi MR11. The lysin, designated MV-L, rapidly and completely lysed cells of a number of S. aureus strains tested, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus and a subset of vancomycin-intermediate S. aureus (VISA) in growing conditions. MV-L-mediated killing is specific to S. aureus and not to other species, except for S. simulans. MV-L exerted its staphylocidal effect synergistically with glycopeptide antibiotics against VISA. MV-L efficiently eliminated MRSA that had been artificially inoculated into the nares of mice. The intraperitoneal administration of MV-L also protected mice against MRSA septic death, without any harmful effects. Although MV-L evoked detectable levels of a humoral response in mice, the antibodies did not abolish the bacteriolytic activity. These results indicate that MV-L might be useful as a powerful therapeutic agent against multidrug-resistant S. aureus infections.

Bacteriophage therapy: a revitalized therapy against bacterial infectious diseases.

Bacteriophage (phage) therapy involves using phages or their products as bioagents for the treatment or prophylaxis of bacterial infectious diseases. Much evidence in support of the effectiveness of phage therapy against bacterial infectious diseases has accumulated since 1980 from animal model studies conducted in Western countries. Reports indicate that appropriate administration of living phages can be used to treat lethal infectious diseases caused by gram-negative bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Vibrio vulnificus, and Salmonella spp., and gram-positive bacteria, such as Enterococcus faecium and Staphylococcus aureus. The phage display system and genetically modified nonreplicating phages are also effective for treatment of Helicobacter pylori and P. aeruginosa, respectively. In addition to phage particles per se, purified phage-encoded peptidoglycan hydrolase (lysin) is also reported to be effective for the treatment of bacterial infectious diseases caused by gram-positive bacteria such as Streptococcus pyogenes, S. pneumoniae, Bacillus anthracis, and group B streptococci. All phage lysins that have been studied to date exhibit immediate and strong bacteriolytic activity when applied exogenously. Furthermore, phage-coded inhibitors of peptidoglycan synthesis (protein antibiotics), search methods for novel antibacterial agents using phage genome informatics, and vaccines utilizing phages or their products are being developed. Phage therapy will compensate for unavoidable complications of chemotherapy such as the appearance of multidrug resistance or substituted microbism.