In-Young Chung

Structural details of the OxyR peroxide-sensing mechanism

Significance In gram-negative bacteria, OxyR is the master peroxide sensor that regulates the transcription of defense genes in response to a low level of cellular H2O2 via a rapid kinetic reaction. In this study, we present the first, to our knowledge, full-length structures of peroxide-sensing transcription regulator OxyR together with an oxidation intermediate-mimicking structure. The structures show all of the structural features describing the tetrameric assembly and a bound H2O2 molecule near the conserved cysteine. Combining the structural results, we reveal a step-by-step molecular mechanism for OxyR from H2O2 sensing to structural changes for transcriptional activation. Our study provides a structural basis for potentially answering key questions about the role of the cysteine residue in other Cys-based sensors, even mammalian ones, in response to various oxidants. 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.

Antibacterial efficacy of temperate phage-mediated inhibition of bacterial group motilities

ABSTRACT Phage therapy against bacterial pathogens has been resurrected as an alternative and supplementary anti-infective modality. Here, we observed that bacterial group motilities were impaired in Pseudomonas aeruginosa strain PA14 lysogens for some temperate siphophages; the PA14 lysogens for DMS3 and MP22 were impaired in swarming motility, whereas the PA14 lysogen for D3112 was impaired in twitching motility. The swarming and twitching motilities of PA14 were also affected in the presence of MP22 and D3112, respectively. The in vitro killing activities of D3112 and MP22 toward PA14 did not differ, and neither did their in vivo persistence in the absence of bacterial infections in mice as well as in flies. Nevertheless, administration of D3112, not MP22, significantly reduced the mortality and the bacterial burdens in murine peritonitis-sepsis and Drosophila systemic infection caused by PA14. Taken together, we suggest that a temperate phage-mediated twitching motility inhibition might be comparably effective to control the acute infections caused by P. aeruginosa.

A phage protein that inhibits the bacterial ATPase required for type IV pilus assembly

Significance We have identified a phage-encoded protein that inhibits the bacterial ATPase PilB, which is involved in type IV pilus (TFP) biogenesis and function. This phage protein-mediated PilB dysfunction is regarded as the superinfection-exclusion maneuver of the phage toward TFP-specific phages. This study inspires an antipathogenic target based on the ATPases ubiquitously conserved in the motility and secretion machineries important in bacterial pathogenesis. 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.

Complete genome sequence of Pseudomonas aeruginosa siphophage MP1412.

ABSTRACT We report the complete genome sequence of Pseudomonas aeruginosa siphophage MP1412, which displays synteny to those of P. aeruginosa phages M6 and YuA. However, the presence of two homing endonucleases of the GIY-YIG family is unique to MP1412, suggesting their unique role in the phage life cycle of the bacterial host.

Complete Genome Sequences of Two Pseudomonas aeruginosa Temperate Phages, MP29 and MP42, Which Lack the Phage-Host CRISPR Interaction

ABSTRACT We report the complete genome sequence of two Pseudomonas aeruginosa phages MP29 and MP42. Their genomes are similar to those of P. aeruginosa temperate phages DMS3 and MP22, whose lysogens are impaired in swarming motilities, involving the host CRISPR loci. Both MP29 and MP42 lysogens, however, were proficient in swarming, suggesting the absence of the phage-host CRISPR interaction.

Superinfection exclusion reveals heteroimmunity between Pseudomonas aeruginosa temperate phages

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.

Dual promoters of the major catalase (KatA) govern distinct survival strategies of Pseudomonas aeruginosa

KatA is the major catalase required for hydrogen peroxide (H2O2) resistance and acute virulence in Pseudomonas aeruginosa PA14, whose transcription is driven from the promoter (katAp1) located at 155 nucleotide (nt) upstream of the start codon. Here, we identified another promoter (katAp2), the +1 of which was mapped at the 51u2009nt upstream of the start codon, which was responsible for the basal transcription during the planktonic culture and down-regulated upon H2O2 treatment under the control by the master regulator of anaerobiosis, Anr. To dissect the roles of the dual promoters in conditions involving KatA, we created the promoter mutants for each -10 box (p1m, p2m, and p1p2m) and found that katAp1 is required for the function of KatA in the logarithmic growth phase during the planktonic culture as well as in acute virulence, whereas katAp2 is required for the function of KatA in the stationary phase as well as in the prolonged biofilm culture. This dismantling of the dual promoters of katA sheds light on the roles of KatA in stress resistance in both proliferative and growth-restrictive conditions and thus provides an insight into the regulatory impacts of the major catalase on the survival strategies of P. aeruginosa.

Drosophila melanogaster as a polymicrobial infection model for Pseudomonas aeruginosa and Staphylococcus aureus

Non-mammalian infection models have been developed over the last two decades, which is a historic milestone to understand the molecular basis of bacterial pathogenesis. They also provide small-scale research platforms for identification of virulence factors, screening for antibacterial hits, and evaluation of antibacterial efficacy. The fruit fly, Drosophila melanogaster is one of the model hosts for a variety of bacterial pathogens, in that the innate immunity pathways and tissue physiology are highly similar to those in mammals. We here present a relatively simple protocol to assess the key aspects of the polymicrobial interaction in vivo between the human opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, which is based on the systemic infection by needle pricking at the dorsal thorax of the flies. After infection, fly survival and bacteremia over time for both P. aeruginosa and S. aureus within the infected flies can be monitored as a measure of polymicrobial virulence potential. The infection takes ~24 h including bacterial cultivation. Fly survival and bacteremia are assessed using the infected flies that are monitored up to ~60 h post-infection. These methods can be used to identify presumable as well as unexpected phenotypes during polymicrobial interaction between P. aeruginosa and S. aureus mutants, regarding bacterial pathogenesis and host immunity.

Crystal structures of the disulfide reductase DsbM from Pseudomonas aeruginosa.

In bacteria, many Dsb-family proteins play diverse roles in the conversion between the oxidized and reduced states of cysteine residues of substrate proteins. Most Dsb enzymes catalyze disulfide formation in periplasmic or secreted substrate proteins. Recently, a DsbM protein has been found in a Gram-negative bacterium, and was characterized as a cytosolic Dsb member with the conserved CXXC motif on the basis of sequence homology to the Dsb-family proteins. The protein was implicated in the reduction of the cytoplasmic redox-sensor protein OxyR in Pseudomonas aeruginosa. Here, crystal structures of DsbM from P. aeruginosa are presented, revealing that it consists of a modified thioredoxin domain containing the CXXC motif and a lid domain surrounding the CXXC motif. In a glutathione-linked structure, a glutathione molecule is linked to the CXXC motif of DsbM and is bound in an elongated cavity region in the thioredoxin domain, which is also suited for substrate peptide binding. A striking structural similarity to a human glutathione S-transferase was found in the glutathione-binding pocket. Further, biochemical evidence is presented suggesting that DsbM is directly involved in the reduction of the disulfide of Cys199 and Cys208 in OxyR, resulting in the acceleration of OxyR reduction in the absence of reactive oxygen species stress. These findings may help to expand the understanding of the diverse roles of redox-related proteins that contain the CXXC motif.