Search

Aims: Polymyxin B (PMB) is known to require reactive oxygen species (ROS) for its bactericidal activity, but the mechanism of PMB resistance in various Pseudomonas aeruginosa strains has been poorl...

PP7 is a leviphage, with a single-stranded RNA genome, that infects Pseudomonas aeruginosa PAO1. A reverse genetic system for PP7 was previously created by using reverse-transcribed cDNA (PP7(O)) from a virion-derived RNA genome. Here, we have found that the PP7(O) cDNA contained 20 nucleotide differences from the PP7 genome sequence deposited in the database. We created another reverse genetic system exploiting chemically synthesized cDNA (PP7(S)) based on the database sequence. Unlike PP7(O), which yielded infectious PP7 virions, PP7(S)-derived particles were incapable of plaque formation on PAO1 cells, which was restored in the PAO1 cells expressing the maturation protein (MP) from PP7(O). Using this reverse genetic system, we revealed two amino acid residues involved in the known roles of MP (i.e., adsorption and genome replication), fortuitously providing a lesson that the viral RNA genome sequencing needs functional verification, possibly by a reverse genetic system. IMPORTANCE The biological significance of RNA phages has been largely ignored, ironically, because few studies have focused on RNA phages. As an initial attempt to properly represent RNA phages in the phageome, we previously created, by using reverse-transcribed cDNA, a reverse genetic system for the small RNA phage PP7, which infects the opportunistic human pathogen Pseudomonas aeruginosa. We report another system by using chemically synthesized cDNA based on the database genome that has 20 nucleotide differences from the previous cDNA. Investigation of those cDNA-derived phage virions revealed that two amino acids of the maturation protein are crucial for the normal phage lifecycle at different steps. Our study provides insight into the molecular basis for the RNA phage lifecycle and a lesson that the RNA genome sequencing needs to be carefully validated by cDNA-based phage assembly systems.

Reverse genetic systems for RNA viruses are the platforms to introduce mutations into the RNA genomes and thus have helped understand their life cycle and harness them for human purposes to develop vaccines and delivery systems. These systems are based on the complementary DNA (cDNA) of the RNA viruses, whose transcripts derived from bacterial RNA polymerases act not only as the primary mRNA for phage protein synthesis, but also as the template for phage RNA replicases (aka. RNA-dependent RNA polymerases). Here, we present a protocol optimized for the small RNA phages of Leviviridae (i.e., leviphages) infecting Pseudomonas aeruginosa. This protocol includes three fundamental steps: (i) Creation of a promoter-fused cDNA, (ii) generation of a clone into mini-Tn7-based vector, and (iii) introduction of the clone into non-susceptible hosts. As the representative example, we describe the reverse genetic system for PP7, which infects a set of P. aeruginosa strains such as PAO1. The cDNA was fused to the T7 promoter, which was cloned in mini-Tn7-Gm. This construct was introduced into P. aeruginosa PAK and E. coli HB101. Functional assembly of PP7 phages from the culture supernatants were assessed by plaque formation on PAO1 and the phage particles were observed under transmission microscope. We found that the host cells should be cultured at 30 °C for the maximal phage production (~1012 pfu/mL). The reverse genetic systems will provide a new insight into the life cycle of the RNA phages and help develop engineered variants with new traits for phage applications regarding selective diagnosis and efficient therapy.

Despite the successful use of antibacterials, the emergence of multidrug-resistant bacteria has become a serious threat to global healthcare. In this era of antibacterial crisis, bacteriophages (phages) are being explored as an antibacterial treatment option since they possess a number of advantages over conventional antibacterials, especially in terms of specificity and biosafety; phages specifically lyse target bacteria while not affecting normal and/or beneficial bacteria and display little or no toxicity in that they are mainly composed of proteins and nucleic acids, which consequently significantly reduces the time and cost involved in antibacterial development. However, these benefits also create potential issues regarding antibacterial spectra and host immunity; the antibacterial spectra being very narrow when compared to those of chemicals, with the phage materials making it possible to trigger host immune responses, which ultimately disarm antibacterial efficacy upon successive treatments. In addition, phages play a major role in horizontal gene transfer between bacterial populations, which poses serious concerns for the potential of disastrous consequences regarding antibiotic resistance. Fortunately, however, recent advancements in synthetic biology tools and the speedy development of phage genome resources have allowed for research on methods to circumvent the potentially disadvantageous aspects of phages. These novel developments empower research which goes far beyond traditional phage therapy approaches, opening up a new chapter for phage applications with new antibacterial platforms. Herein, we not only highlight the most recent synthetic phage engineering and phage product engineering studies, but also discuss a new proof-of-concept for phage-inspired antibacterial design based on the studies undertaken by our group.

Oxidative stress arises from an imbalance between the excessive accumulation of reactive oxygen species (ROS) and a cell’s capability to readily detoxify them. Although ROS are spontaneously generated during the normal oxygen respiration and metabolism, the ROS generation is usually augmented by redox-cycling agents, membrane disrupters, and bactericidal antibiotics, which contributes their antimicrobial bioactivity. It is noted that all the bacteria deploy an arsenal of inducible antioxidant defense systems to cope with the devastating effect exerted by the oxidative stress: these systems include the antioxidant effectors such as catalases and the master regulators such as OxyR. The oxidative stress response is not essential for normal growth, but critical to survive the oxidative stress conditions that the bacterial pathogens may encounter due to the host immune response and/or the antibiotic treatment. Based on these, we here define the ROS-inspired antibacterial strategies to enhance the oxidative stress of ROS generation and/or to compromise the bacterial response of ROS detoxification, by delineating the ROSgenerating antimicrobials and the core concept of the bacterial response against the oxidative stress.

Nonmammalian infection models have been exploited to understand the various aspects of host-pathogen interactions and also provided innovative research platforms for identification of virulence factors, screening for antimicrobial hits, and evaluation of antimicroial efficacy. Here we describe a relatively straightforward protocol to assess the antibacterial efficacy of bacteriophages (phages) toward the opportunistic human pathogen, Pseudomonas aeruginosa, based on the systemic infection model using the fruit fly, Drosophila melanogaster. Since phages, unlike antibacterial chemicals, can be easily and sensitively enumerated by simple assays, it is also possible to address the pharmacokinetic properties of administered phages even in this small-scale infection model.

Nonmammalian infection models have been exploited to understand the various aspects of host-pathogen interactions and also provided innovative research platforms for identification of virulence factors, screening for antimicrobial hits, and evaluation of antimicroial efficacy. Here we describe a relatively straightforward protocol to assess the antibacterial efficacy of bacteriophages (phages) toward the opportunistic human pathogen, Pseudomonas aeruginosa, based on the systemic infection model using the fruit fly, Drosophila melanogaster. Since phages, unlike antibacterial chemicals, can be easily and sensitively enumerated by simple assays, it is also possible to address the pharmacokinetic properties of administered phages even in this small-scale infection model.

Since Bacillus anthracis is a high-risk pathogen and a potential tool for bioterrorism, numerous therapeutic methods including passive immunization have been actively developed. Using a human monoclonal antibody phage display library, we screened new therapeutic antibodies for anthrax infection against protective antigen (PA) of B. anthracis. Among 5 selected clones of antibodies based on enzyme-linked immunosorbent assay (ELISA) results, 7B1 showed neutralizing activity to anthrax lethal toxin (LT) by inhibiting binding of the domain 4 of PA (PD4) to its cellular receptors. Through light chain shuffling process, we improved the productivity of 7B1 up to 25 folds. The light chain shuffled 7B1 antibody showed protective activity against LT both in vitro and in vivo. Furthermore, the antibody also conferred protection of mice from 3 × LD50 challenges of fully virulent anthrax spores. Our result expands the possibility of developing a new therapeutic antibody for anthrax cure.

The host range of a phage is determined primarily by phage-receptor interaction. Here, we profiled the host range of an RNA leviphage, PP7 that requires functional type IV pilus (TFP) in order to enter into its host bacterium, Pseudomonas aeruginosa. Out of 25 twitching-proficient P. aeruginosa strains, 4 with group I pilin and 7 with group III pilin displayed PP7-resistance. The remaining 14 possessed group II pilin, which included 10 PP7-sensitive and 4 PP7-resistant strains, suggesting that only the strains with TFP consisted of a subset of group II (hence, group IIa) pilin were susceptible to PP7. The co-expression of the PAO1 (group IIa) pilin rendered all the strains susceptible to PP7, with the exception of the 4 strains with group I pilin. Moreover, the expression of PA14 (group III) and PAK (group IIb) pilin in the PAO1 pilA mutant restored the twitching motility but not the PP7-suceptibility. Site-directed and random mutation analyses of PAO1 pilin enabled us to identify a pilin mutant (G96S) that is fully functional but resistant to PP7 infection. This is due to the lack of any phage-receptor interactions, suggesting the structural properties of the β1-β2 loop in the variable region 2 of the group II pilin might be involved in PP7 infection.

Type IV pili (TFPs) are required for bacterial twitching motility and for phage infection in the opportunistic human pathogen Pseudomonas aeruginosa. Here we describe a phage-encoded protein, D3112 protein gp05 (hereafter referred to as Tip, representing twitching inhibitory protein), whose expression is necessary and sufficient to mediate the inhibition of twitching motility. Tip interacts with and blocks the activity of bacterial-encoded PilB, the TFP assembly/extension ATPase, at an internal 40-aa region unique to PilB. Tip expression results in the loss of surface piliation. Based on these observations and the fact that many P. aeruginosa phages require TFPs for infection, Tip-mediated twitching inhibition may represent a generalized strategy for superinfection exclusion. Moreover, because TFPs are required for full virulence, PilB may be an attractive target for the development of novel antiinfectives.

Temperate siphophages (MP29, MP42, and MP48) were isolated from the culture supernatant of clinical Pseudomonas aeruginosa isolates. The complete nucleotide sequences and annotation of the phage genomes revealed the overall synteny to the known temperate P. aeruginosa phages such as MP22, D3112, and DMS3. Genome-level sequence analysis showed the conservation of both ends of the linear genome and the divergence at the previously identified dissimilarity regions (R1 to R9). Protein sequence alignment of the c repressor (ORF1) of each phage enabled us to divide the six phages into two groups: D3112 group (D3112, MP29, MP42, and MP48) and MP22 group (MP22 and DMS3). Superinfection exclusion was observed between the phages belonging to the same group, which was mediated by the specific interaction between the c repressor and the cognate operator. Based on these, we suggest that the temperate siphophages prevalent in the clinical strains of P. aeruginosa represent at least two distinct heteroimmunity groups.

OxyR, a bacterial peroxide sensor, is a LysR-type transcriptional regulator (LTTR) that regulates the transcription of defense genes in response to a low level of cellular H2O2. Consisting of an N-terminal DNA-binding domain (DBD) and a C-terminal regulatory domain (RD), OxyR senses H2O2 with conserved cysteine residues in the RD. However, the precise mechanism of OxyR is not yet known due to the absence of the full-length (FL) protein structure. Here we determined the crystal structures of the FL protein and RD of Pseudomonas aeruginosa OxyR and its C199D mutant proteins. The FL crystal structures revealed that OxyR has a tetrameric arrangement assembled via two distinct dimerization interfaces. The C199D mutant structures suggested that new interactions that are mediated by cysteine hydroxylation induce a large conformational change, facilitating intramolecular disulfide-bond formation. More importantly, a bound H2O2 molecule was found near the Cys199 site, suggesting the H2O2-driven oxidation mechanism of OxyR. Combined with the crystal structures, a modeling study suggested that a large movement of the DBD is triggered by structural changes in the regulatory domains upon oxidation. Taken together, these findings provide novel concepts for answering key questions regarding OxyR in the H2O2-sensing and oxidation-dependent regulation of antioxidant genes.

UV irradiation elicits acute inflammation in the skin by increasing proinflammatory cytokine production in keratinocytes. However, the downstream protein target(s) that link UV radiation to the activation of signaling pathways responsible for cytokine expression have not been fully elucidated. In this study, we report a novel role of transglutaminase 2 (TG2), a member of the TG enzyme family whose activities are critical for cornified envelope formation, in mediating UV-induced inflammation. Our results showed that TG2-deficient mice exhibited reduced inflammatory responses to UV irradiation, including reduced erythema, edema, dilation of blood vessels, inflammatory cell infiltration, and levels of inflammatory cytokines. Using primary mouse keratinocytes and HaCaT cells, we found that UV irradiation-induced cytokine production by activating TG2, but not by upregulating TG2 expression, and that ER calcium release triggered by the UV-induced activation of phospholipase C was required for TG2 activation. Moreover, TG2 activity enhanced p65 phosphorylation, leading to an increase in NF-κB transcriptional activity. These results indicate that TG2 is a critical mediator of cytokine expression in the UV-induced inflammatory response of keratinocytes, and suggest that TG2 inhibition might be useful for preventing UV-related skin disorders, such as photoaging and skin cancer caused by chronic UV exposure.

We report the complete genome sequence of Pseudomonas aeruginosa podophage MPK7. It displays synteny to the P. aeruginosa phages of the Phikmvlikevirus genus, which includes phiKMV and LKA1. MPK7 requires type IV pili (TFP) for infection, suggesting the role of functional TFP as the receptor for this phage genus.