You-Hee Cho

HHQ and PQS, two Pseudomonas aeruginosa quorum‐sensing molecules, down‐regulate the innate immune responses through the nuclear factor‐κB pathway

To explore whether bacterial secreted 4‐hydroxy‐2‐alkylquinolines (HAQs) can regulate host innate immune responses, we used the extracts of bacterial culture supernatants from a wild‐type (PA14) and two mutants of Pseudomonas aeruginosa that have defects in making HAQs. Surprisingly, the extract of supernatants from the P. aeruginosa pqsA mutant that does not make HAQs showed strong stimulating activity for the production of innate cytokines such as tumour necrosis factor‐α and interleukin‐6 in the J774A.1 mouse monocyte/macrophage cell line, whereas the extract from the wild‐type did not. The addition of 4‐hydroxy‐2‐heptylquinoline (HHQ) or 2‐heptyl‐3,4‐dihydroxyquinoline (PQS, Pseudomonas quinolone signal) to mammalian cell culture media abolished this stimulating activity of the extracts of supernatants from the pqsA mutant on the expression of innate cytokines in J774A.1 cells and in the primary bronchoalveolar lavage cells from C57BL/6 mice, suggesting that HHQ and PQS can suppress the host innate immune responses. The pqsA mutant showed reduced dissemination in the lung tissue compared with the wild‐type strain in a mouse in vivo intranasal infection model, suggesting that HHQ and PQS may play a role in the pathogenicity of P. aeruginosa. HHQ and PQS reduced the nuclear factor‐κB (NF‐κB) binding to its binding sites and the expression of NF‐κB target genes, and PQS delayed inhibitor of κB degradation, indicating that the effect of HHQ and PQS was mediated through the NF‐κB pathway. Our results suggest that HHQ and PQS produced by P. aeruginosa actively suppress host innate immune responses.

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.

Drosophila melanogaster-Based Screening for Multihost Virulence Factors of Pseudomonas aeruginosa PA14 and Identification of a Virulence-Attenuating Factor, HudA

ABSTRACT Pseudomonas aeruginosa is an important opportunistic human pathogen that interacts with phylogenetically diverse nonmammalian hosts, including plants, nematodes, and insects. Here, we exploited the P. aeruginosa-induced killing of the fruit fly Drosophila melanogaster as an assay system to screen for virulence-attenuated mutants of P. aeruginosa PA14. Fifteen nonredundant mutants were isolated from 4,018 random transposon (TnphoA) insertion clones, and 13 out of them (86.7%) displayed significantly reduced virulence in a murine peritonitis model as well. The TnphoA insertion sites of the 15 mutants were determined; already known virulence genes (dsbA, pvdI, fhlB, pilF, and wspF) and new virulence genes such as PA0253 (hudR), PA0369, PA2077, PA0272, PA2113, PA2965 (fabF1), and PA2002 were identified; one insertion was located at the intergenic region between PA1928 and PA1929; and the other two insertions were located in the genes (PA14_35740 and PA14_36000) within a putative genomic island, indicating a potential pathogenicity island of PA14. Further characterization of hudR, a virulence gene which encodes a MarR/SlyA family transcription factor, revealed that elevated expression of PA0254 (hudA [homologous to UbiD]) was necessary and sufficient for the virulence attenuation of the hudR mutant. The HudR protein repressed the hudAR operon by directly binding to its upstream promoter region. Collectively, these results validate the relevance of the D. melanogaster model for the high-throughput identification of new virulence factors involved in the multihost pathogenesis of P. aeruginosa.

Unusual Properties of Catalase A (KatA) of Pseudomonas aeruginosa PA14 Are Associated with Its Biofilm Peroxide Resistance

Pseudomonas aeruginosa is a ubiquitous environmental bacterium whose major catalase (KatA) is highly stable, extracellularly present, and required for full virulence as well as for peroxide resistance in planktonic and biofilm states. Here, we dismantled the function of P. aeruginosa KatA (KatA(Pa)) by comparing its properties with those of two evolutionarily related (clade 3 monofunctional) catalases from Bacillus subtilis (KatA(Bs)) and Streptomyces coelicolor (CatA(Sc)). We switched the coding region for KatA(Pa) with those for KatA(Bs) and CatA(Sc), expressed the catalases under the potential katA-regulatory elements in a P. aeruginosa PA14 katA mutant, and verified their comparable protein levels by Western blot analysis. The activities of KatA(Bs) and CatA(Sc), however, were less than 40% of the KatA(Pa) activity, suggestive of the difference in intrinsic catalatic activity or efficiency for posttranslational activity modulation in P. aeruginosa. Furthermore, KatA(Bs) and CatA(Sc) were relatively susceptible to proteinase K, whereas KatA(Pa) was highly stable upon proteinase K treatment. As well, KatA(Bs) and CatA(Sc) were undetectable in the extracellular milieu. Nevertheless, katA(Bs) and catA(Sc) fully rescued the peroxide sensitivity and osmosensitivity of the katA mutant, respectively. Both catalase genes rescued the attenuated virulence of the katA mutant in mouse acute infection and Drosophila melanogaster models. However, the peroxide susceptibility of the katA mutant in a biofilm growth state was rescued by neither katA(Bs) nor catA(Sc). Based on these results, we propose that the P. aeruginosa KatA is highly stable compared to the two major catalases from gram-positive bacteria and that its unique properties involving metastability and extracellular presence may contribute to the peroxide resistance of P. aeruginosa biofilm and presumably to chronic infections.

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.

Global gene expression analysis on the target genes of PQS and HHQ in J774A.1 monocyte/macrophage cells

We have previously shown that PQS and HHQ, two quorum sensing molecules, can down-regulate host the innate immune responses and that this is mediated through the NF-kappaB pathway. In this study, to search for a comprehensive set of genes regulated by these quorum sensing molecules, we performed a global gene expression analysis using DNA microarray in J774A.1 monocyte/macrophage cells line. The expression of these genes was confirmed by RT-PCR. We found that PQS and HHQ down-regulated the expression of genes involved in immune responses and transcription as well as other functions, some of which are downstream of NF-kappaB pathway consistent with our previous results. PQS and HHQ inhibited LPS-induced morphological change and nitric oxide production, suggesting that they inhibit macrophage activation. However, PQS and HHQ did not affect apoptosis, suggesting that their effects on immune system are not from general alteration of cell function. This study provides insight how the quorum sensing molecules influence host cells.

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.

Complete Genome Sequence of Pseudomonas aeruginosa Podophage MPK7, Which Requires Type IV Pili for Infection

ABSTRACT 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.