The Transcription Factor AmrZ Utilizes Multiple DNA Binding Modes to Recognize Activator and Repressor Sequences of Pseudomonas aeruginosa Virulence Genes

AmrZ, a member of the Ribbon-Helix-Helix family of DNA binding proteins, functions as both a transcriptional activator and repressor of multiple genes encoding Pseudomonas aeruginosa virulence factors. The expression of these virulence factors leads to chronic and sustained infections associated with worsening prognosis. In this study, we present the X-ray crystal structure of AmrZ in complex with DNA containing the repressor site, amrZ1. Binding of AmrZ to this site leads to auto-repression. AmrZ binds this DNA sequence as a dimer-of-dimers, and makes specific base contacts to two half sites, separated by a five base pair linker region. Analysis of the linker region shows a narrowing of the minor groove, causing significant distortions. AmrZ binding assays utilizing sequences containing variations in this linker region reveals that secondary structure of the DNA, conferred by the sequence of this region, is an important determinant in binding affinity. The results from these experiments allow for the creation of a model where both intrinsic structure of the DNA and specific nucleotide recognition are absolutely necessary for binding of the protein. We also examined AmrZ binding to the algD promoter, which results in activation of the alginate exopolysaccharide biosynthetic operon, and found the protein utilizes different interactions with this site. Finally, we tested the in vivo effects of this differential binding by switching the AmrZ binding site at algD, where it acts as an activator, for a repressor binding sequence and show that differences in binding alone do not affect transcriptional regulation

Identification of Residues in the Hepatitis C Virus Core Protein That Are Critical for Capsid Assembly in a Cell-Free System

Significant advances have been made in understanding hepatitis C virus (HCV) replication through development of replicon systems. However, neither replicon systems nor standard cell culture systems support significant assembly of HCV capsids, leaving a large gap in our knowledge of HCV virion formation. Recently, we established a cell-free system in which over 60% of full-length HCV core protein synthesized de novo in cell extracts assembles into HCV capsids by biochemical and morphological criteria. Here we used mutational analysis to identify residues in HCV core that are important for capsid assembly in this highly reproducible cell-free system. We found that basic residues present in two clusters within the N-terminal 68 amino acids of HCV core played a critical role, while the uncharged linker domain between them was not. Furthermore, the aspartate at position 111, the region spanning amino acids 82 to 102, and three serines that are thought to be sites of phosphorylation do not appear to be critical for HCV capsid formation in this system. Mutation of prolines important for targeting of core to lipid droplets also failed to alter HCV capsid assembly in the cell-free system. In addition, wild-type HCV core did not rescue assembly-defective mutants. These data constitute the first systematic and quantitative analysis of the roles of specific residues and domains of HCV core in capsid formation

Pseudomonas aeruginosa pulmonary infection results in S100A8/A9-dependent cardiac dysfunction.

Pseudomonas aeruginosa (P.a.) infection accounts for nearly 20% of all cases of hospital acquired pneumonia with mortality rates >30%. P.a. infection induces a robust inflammatory response, which ideally enhances bacterial clearance. Unfortunately, excessive inflammation can also have negative effects, and often leads to cardiac dysfunction with associated morbidity and mortality. However, it remains unclear how P.a. lung infection causes cardiac dysfunction. Using a murine pneumonia model, we found that P.a. infection of the lungs led to severe cardiac left ventricular dysfunction and electrical abnormalities. More specifically, we found that neutrophil recruitment and release of S100A8/A9 in the lungs activates the TLR4/RAGE signaling pathways, which in turn enhance systemic inflammation and subsequent cardiac dysfunction. Paradoxically, global deletion of S100A8/A9 did not improve but aggravated cardiac dysfunction and mortality likely due to uncontrolled bacterial burden in the lungs and heart. Our results indicate that P.a. infection induced release of S100A8/9 is double-edged, providing increased risk for cardiac dysfunction yet limiting P.a. growth

<i>Pseudomonas aeruginosa</i> rugose small-colony variants evade host clearance, are hyper-inflammatory, and persist in multiple host environments

<div><p><i>Pseudomonas aeruginosa</i> causes devastating infections in immunocompromised individuals. Once established, <i>P</i>. <i>aeruginosa</i> infections become incredibly difficult to treat due to the development of antibiotic tolerant, aggregated communities known as biofilms. A hyper-biofilm forming clinical variant of <i>P</i>. <i>aeruginosa</i>, known as a rugose small-colony variant (RSCV), is frequently isolated from chronic infections and is correlated with poor clinical outcome. The development of these mutants during infection suggests a selective advantage for this phenotype, but it remains unclear how this phenotype promotes persistence. While prior studies suggest RSCVs could survive by evading the host immune response, our study reveals infection with the RSCV, PAO1Δ<i>wspF</i>, stimulated an extensive inflammatory response that caused significant damage to the surrounding host tissue. In both a chronic wound model and acute pulmonary model of infection, we observed increased bacterial burden, host tissue damage, and a robust neutrophil response during RSCV infection. Given the essential role of neutrophils in <i>P</i>. <i>aeruginosa</i>-mediated disease, we investigated the impact of the RSCV phenotype on neutrophil function. The RSCV phenotype promoted phagocytic evasion and stimulated neutrophil reactive oxygen species (ROS) production. We also demonstrate that bacterial aggregation and TLR-mediated pro-inflammatory cytokine production contribute to the immune response to RSCVs. Additionally, RSCVs exhibited enhanced tolerance to neutrophil-produced antimicrobials including H₂O₂ and the antimicrobial peptide LL-37. Collectively, these data indicate RSCVs elicit a robust but ineffective neutrophil response that causes significant host tissue damage. This study provides new insight on RSCV persistence, and indicates this variant may have a critical role in the recurring tissue damage often associated with chronic infections.</p></div

PAO1Δ<i>wspF</i> exhibits tolerance to neutrophil antimicrobial products.

<p>Log phase cultures of PAO1 or PAO1Δ<i>wspF</i> were treated with A) LL-37, B) H₂O₂ and C) HOCl at the labeled concentrations for 15min. CFUs were quantified before and after treatment and log fold killing determined. Data presented as mean ± SEM. ***p<0.001.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Bacterial aggregation promotes neutrophil ROS production, but exopolysaccharides alone are not sufficient.

<p>A) ePS was purified from PAO1Δ<i>pel</i>P_BAD-psl, PAO1Δ<i>psl</i>P_BAD-pel, or PAO1Δ<i>pel</i>Δ<i>psl</i> and total carbohydrate was quantified by phenol sulfuric acid assay. PAO1 levels or 10-times PAO1 levels of ePS was added to human neutrophils and the ROS response was measured with a luminol reporter. B) PAO1Δ<i>psl</i>Δ<i>pel</i>/pHERD20T and PAO1Δ<i>psl</i>Δ<i>pel</i>/pCdrAB were grown to log phase in the presence of 1% arabinose leading to the formation of CdrA-mediated aggregates in the strain containing pCdrAB. Primary human neutrophils were infected with bacteria at an MOI (1:50). C) Neutrophils were treated with supernatant collected from cultures of PAO1/pCdrAB or PAO1Δ<i>cdrA</i>/pHERD20T, and ROS was quantified. The AUC of the ROS response over 1h was calculated and normalized by CFUs of the inoculation culture to ensure identical cell numbers regardless of aggregation.</p

D-PAO1Δ<i>wspF</i> pulmonary infection leads to severe tissue damage and neutrophil infiltration compared to PAO1 infection.

<p>Mouse lungs 24 h.p.i. were stained with H&E to assess neutrophil infiltration and lung damage based on pulmonary architecture, necrosis, and suppurative inflammation. Images are representative of lungs treated or infected with A) PBS, B) PAO1, C) PAO1Δ<i>wspF</i>, D) D-PAO1Δ<i>wspF</i>. Scale bars indicate 200μm.</p

RSCVs stimulate neutrophil ROS production.

<p>A) Neutrophils were infected with log phase bacteria (MOI 1:50) or treated with PMA in the presence of a luminol reporter. Luminescence was measured for 60min (right) and the area under curve (AUC) was calculated and normalized to the PMA response (left). RLU images are representative of the ROS response from a single donors neutrophils measured in triplicate, while AUC data was collected using neutrophils from at least 3 different donors. B and C) ROS response to the CF clinical RSCV isolates CF127 and CF39s. Data is presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.</p

PAO1Δ<i>wspF</i> induces pro-inflammatory cytokine production in a TLR-dependent manner.

<p>NR-9456 wild type and NR-15632 MyD88^-/-/TRIF^-/- mouse macrophages were infected with PAO1 or PAO1Δ<i>wspF</i> for 4h. A) IL-1β and B) IL-6 was measured in cell supernatants via ELISA. ** p< 0.01. Data presented as mean ± SEM.</p