In Vivo Proteome of Pseudomonas aeruginosa in Airways of Cystic Fibrosis Patients.

Chronic airway infection with P. aeruginosa (PA) is a hallmark of cystic fibrosis (CF) disease. The mechanisms producing PA persistence in CF therapies remain poorly understood. To gain insight on PA physiology in patient airways and better understand how in vivo bacterial functioning differs from in vitro conditions, we investigated the in vivo proteomes of PA in 35 sputum samples from 11 CF patients. We developed a novel bacterial-enrichment method that relies on differential centrifugation and detergent treatment to enrich for bacteria to improve identification of PA proteome with CF sputum samples. Using two nonredundant peptides as a cutoff, a total of 1304 PA proteins were identified directly from CF sputum samples. The in vivo PA proteomes were compared with the proteomes of ex vivo-grown PA populations from the same patient sample. Label-free quantitation and proteome comparison revealed the in vivo up-regulation of siderophore TonB-dependent receptors, remodeling in central carbon metabolism including glyoxylate cycle and lactate utilization, and alginate overproduction. Knowledge of these in vivo proteome differences or others derived using the presented methodology could lead to future treatment strategies aimed at altering PA physiology in vivo to compromise infectivity or improve antibiotic efficacy.

Bacterial fitness in chronic wounds appears to be mediated by the capacity for high-density growth, not virulence or biofilm functions.

While much is known about acute infection pathogenesis, the understanding of chronic infections has lagged. Here we sought to identify the genes and functions that mediate fitness of the pathogen Pseudomonas aeruginosa in chronic wound infections, and to better understand the selective environment in wounds. We found that clinical isolates from chronic human wounds were frequently defective in virulence functions and biofilm formation, and that many virulence and biofilm formation genes were not required for bacterial fitness in experimental mouse wounds. In contrast, genes involved in anaerobic growth, some metabolic and energy pathways, and membrane integrity were critical. Consistent with these findings, the fitness characteristics of some wound impaired-mutants could be represented by anaerobic, oxidative, and membrane-stress conditions ex vivo, and more comprehensively by high-density bacterial growth conditions, in the absence of a host. These data shed light on the bacterial functions needed in chronic wound infections, the nature of stresses applied to bacteria at chronic infection sites, and suggest therapeutic targets that might compromise wound infection pathogenesis.

Gallium disrupts bacterial iron metabolism and has therapeutic effects in mice and humans with lung infections.

The lack of new antibiotics is among the most critical challenges facing medicine. The problem is particularly acute for Gram-negative bacteria. An unconventional antibiotic strategy is to target bacterial nutrition and metabolism. The metal gallium can disrupt bacterial iron metabolism because it substitutes for iron when taken up by bacteria. We investigated the antibiotic activity of gallium ex vivo, in a mouse model of airway infection, and in a phase 1 clinical trial in individuals with cystic fibrosis (CF) and chronic Pseudomonas aeruginosa airway infections. Our results show that micromolar concentrations of gallium inhibited P. aeruginosa growth in sputum samples from patients with CF. Ex vivo experiments indicated that gallium inhibited key iron-dependent bacterial enzymes and increased bacterial sensitivity to oxidants. Furthermore, gallium resistance developed slowly, its activity was synergistic with certain antibiotics, and gallium did not diminish the antibacterial activity of host macrophages. Systemic gallium treatment showed antibiotic activity in murine lung infections. In addition, systemic gallium treatment improved lung function in people with CF and chronic P. aeruginosa lung infection in a preliminary phase 1 clinical trial. These findings raise the possibility that human infections could be treated by targeting iron metabolism or other nutritional vulnerabilities of bacterial pathogens.

Determinants of Extreme ß-Lactam Tolerance in the Burkholderia pseudomallei Complex.

Slow-growing bacteria are insensitive to killing by antibiotics, a trait known as antibiotic tolerance. In this study, we characterized the genetic basis of an unusually robust ß-lactam (meropenem) tolerance seen in Burkholderia species. We identified tolerance genes under three different slow-growth conditions by extensive transposon mutant sequencing (Tn-seq), followed by single mutant validation. There were three principal findings. First, mutations in a small number of genes reduced tolerance under multiple conditions. Most of the functions appeared to be specific to peptidoglycan synthesis and the response to its disruption by meropenem action rather than being associated with more general physiological processes. The top tolerance genes are involved in immunity toward a type VI toxin targeting peptidoglycan (BTH_I0069), peptidoglycan recycling (ldcA), periplasmic regulation by proteolysis (prc), and an envelope stress response (rpoE and degS). Second, most of the tolerance functions did not contribute to growth in the presence of meropenem (intrinsic resistance), indicating that the two traits are largely distinct. Third, orthologues of many of the top Burkholderia thailandensis tolerance genes were also important in Burkholderia pseudomallei Overall, these studies show that the determinants of meropenem tolerance differ considerably depending on cultivation conditions, but that there are a few shared functions with strong mutant phenotypes that are important in multiple Burkholderia species.

Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange.

Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions and substitutions, in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selections are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic-resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires ∼2 weeks.

Probing the protein interaction network of Pseudomonas aeruginosa cells by chemical cross-linking mass spectrometry.

In pathogenic Gram-negative bacteria, interactions among membrane proteins are key mediators of host cell attachment, invasion, pathogenesis, and antibiotic resistance. Membrane protein interactions are highly dependent upon local properties and environment, warranting direct measurements on native protein complex structures as they exist in cells. Here we apply in vivo chemical cross-linking mass spectrometry, to reveal the first large-scale protein interaction network in Pseudomonas aeruginosa, an opportunistic human pathogen, by covalently linking interacting protein partners, thereby fixing protein complexes in vivo. A total of 626 cross-linked peptide pairs, including previously unknown interactions of many membrane proteins, are reported. These pairs not only define the existence of these interactions in cells but also provide linkage constraints for complex structure predictions. Structures of three membrane proteins, namely, SecD-SecF, OprF, and OprI are predicted using in vivo cross-linked sites. These findings improve understanding of membrane protein interactions and structures in cells.

Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria.

Bacteria become highly tolerant to antibiotics when nutrients are limited. The inactivity of antibiotic targets caused by starvation-induced growth arrest is thought to be a key mechanism producing tolerance. Here we show that the antibiotic tolerance of nutrient-limited and biofilm Pseudomonas aeruginosa is mediated by active responses to starvation, rather than by the passive effects of growth arrest. The protective mechanism is controlled by the starvation-signaling stringent response (SR), and our experiments link SR-mediated tolerance to reduced levels of oxidant stress in bacterial cells. Furthermore, inactivating this protective mechanism sensitized biofilms by several orders of magnitude to four different classes of antibiotics and markedly enhanced the efficacy of antibiotic treatment in experimental infections.

A unique regulator controls the activation threshold of quorum-regulated genes in Pseudomonas aeruginosa.

Quorum-sensing (QS) systems allow organisms, such as the pathogen Pseudomonas aeruginosa, to link gene expression with their population density and the diffusion and flow characteristics of their environment. The leading hypotheses about QS systems' biological functions necessitate that QS-controlled gene expression be suppressed until a threshold culture density (the quorum) is reached. Despite a detailed understanding of QS in P. aeruginosa, known regulatory elements do not fully explain how the quorum threshold for gene activation is produced. Here we investigated the mechanism with a screening approach that used random gene activation. These experiments uncovered a regulator without close homologs in other species that produces the quorum expression threshold. Expression of this regulator (named QteE) reduces LasR protein stability without affecting LasR transcription or translation. QteE also independently reduces RhlR levels. Because QteE can block QS when signal levels are high, it could provide a mechanism for individual cells to exert autonomous control over their QS regulons. This unique regulator governs two central QS control points in P. aeruginosa and shapes the expression pattern thought fundamental to the biological functions of QS.

Serendipitous discovery of novel bacterial methionine aminopeptidase inhibitors.

In this article we describe the application of structural biology methods to the discovery of novel potent inhibitors of methionine aminopeptidases. These enzymes are employed by the cells to cleave the N-terminal methionine from nascent peptides and proteins. As this is one of the critical steps in protein maturation, it is very likely that inhibitors of these enzymes may prove useful as novel antibacterial agents. Involvement of crystallography at the very early stages of the inhibitor design process resulted in serendipitous discovery of a new inhibitor class, the pyrazole-diamines. Atomic-resolution structures of several inhibitors bound to the enzyme illuminate a new mode of inhibitor binding.

Development and validation of a whole-cell inhibition assay for bacterial methionine aminopeptidase by surface-enhanced laser desorption ionization-time of flight mass spectrometry.

Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP.

Physical mapping of 32 genetic markers on the Pseudomonas aeruginosa PAO1 chromosome.

The Pseudomonas aeruginosa chromosome was fractionated with the enzymes SpeI and DpnI, and genomic fragments were separated by PFGE and used for mapping a collection of 40 genes. This permitted the localization of 8 genes previously mapped and of 32 genes which had not been mapped. We showed that a careful search of databases and identification of sequences that were homologous to known genes could be used to design and synthesize DNA probes for the mapping of P. aeruginosa homologues by Southern hybridization with genomic fragments, resulting in definition of the locations of the aro-2, dapB, envA, mexA, groEL, oprH, oprM, oprP, ponA, rpoB and rpoH genetic markers. In addition, a combination of distinct DNA sources were utilized as radioactively labelled probes, including specific restriction fragments of the cloned genes (glpD, opdE, oprH, oprO, oprP, phoS), DNA fragments prepared by PCR, and single-stranded DNA prepared from phagemid libraries that had been randomly sequenced. We used a PCR approach to clone fragments of the putative yhhF, sucC, sucD, cypH, pbpB, murE, pbpC, soxR, ftsA, ftsZ and envA genes. Random sequencing of P. aeruginosa DNA from phagemid libraries and database searching permitted the cloning of sequences from the acoA, catR, hemD, pheS, proS, oprD, pyo and rpsB gene homologues. The described genomic methods permit the rapid mapping of the P. aeruginosa genome without linkage analysis.