Gee W. Lau

A functional genomic analysis of type 3 Streptococcus pneumoniae virulence

Streptococcus pneumoniae remains a serious cause of morbidity and mortality in humans, but relatively little is known about the molecular basis of its pathogenesis. We used signature‐tagged mutagenesis together with an analysis of S. pneumoniae genome sequence to identify and characterize genes required for pathogenesis. A library of signature‐tagged mutants was created by insertion–duplication mutagenesis, and 1786 strains were analysed for their inability to survive and replicate in murine models of pneumonia and bacteraemia. One hundred and eighty‐six mutant strains were identified as attenuated, and 56 were selected for further genetic characterization based on their ability to excise the integrated plasmid spontaneously. The genomic DNA inserts of the plasmids were cloned in Escherichia coli and sequenced. These sequences were subjected to database searches, including the S. pneumoniae genome sequence, which allowed us to examine the chromosomal regions flanking these genes. Most of the insertions were in probable operons, but no pathogenicity islands were found. Forty‐two novel virulence loci were identified. Five strains mutated in genes involved in gene regulation, cation transport or stress tolerance were shown to be highly attenuated when tested individually in a murine respiratory tract infection model. Additional experiments also suggest that induction of competence for genetic transformation has a role in virulence.

Pseudomonas aeruginosa Pyocyanin Is Critical for Lung Infection in Mice

ABSTRACT Pseudomonas aeruginosa secretes copious amounts of the redox-active phenazine, pyocyanin (PCN), during cystic fibrosis lung infection. PCN has been shown to interfere with a variety of cellular processes in cultured lung epithelial cells. Here, by using two respiratory tract models of infection, we demonstrate that PCN mediates tissue damage and necrosis during lung infection.

Anaerobic killing of mucoid Pseudomonas aeruginosa by acidified nitrite derivatives under cystic fibrosis airway conditions

Mucoid, mucA mutant Pseudomonas aeruginosa cause chronic lung infections in cystic fibrosis (CF) patients and are refractory to phagocytosis and antibiotics. Here we show that mucoid bacteria perish during anaerobic exposure to 15 mM nitrite (NO2) at pH 6.5, which mimics CF airway mucus. Killing required a pH lower than 7, implicating formation of nitrous acid (HNO2) and NO, that adds NO equivalents to cellular molecules. Eighty-seven percent of CF isolates possessed mucA mutations and were killed by HNO2 (3-log reduction in 4 days). Furthermore, antibiotic-resistant strains determined were also equally sensitive to HNO2. More importantly, HNO2 killed mucoid bacteria (a) in anaerobic biofilms; (b) in vitro in ultrasupernatants of airway secretions derived from explanted CF patient lungs; and (c) in mouse lungs in vivo in a pH-dependent fashion, with no organisms remaining after daily exposure to HNO2 for 16 days. HNO2 at these levels of acidity and NO2 also had no adverse effects on cultured human airway epithelia in vitro. In summary, selective killing by HNO2 may provide novel insights into the important clinical goal of eradicating mucoid P. aeruginosa from the CF airways.

Human targets of Pseudomonas aeruginosa pyocyanin

Pseudomonas aeruginosa produces copious amounts of the redoxactive tricyclic compound pyocyanin that kills competing microbes and mammalian cells, especially during cystic fibrosis lung infection. Cross-phylum susceptibility to pyocyanin suggests the existence of evolutionarily conserved physiological targets. We screened a Saccharomyces cerevisiae deletion library to identify presumptive pyocyanin targets with the expectation that similar targets would be conserved in humans. Fifty S. cerevisiae targets were provisionally identified, of which 60% have orthologous human counterparts. These targets encompassed major cellular pathways involved in the cell cycle, electron transport and respiration, epidermal cell growth, protein sorting, vesicle transport, and the vacuolar ATPase. Using cultured human lung epithelial cells, we showed that pyocyanin-mediated reactive oxygen intermediates inactivate human vacuolar ATPase, supporting the validity of the yeast screen. We discuss how the inactivation of VATPase may negatively impact the lung function of cystic fibrosis patients.

The Drosophila melanogaster Toll Pathway Participates in Resistance to Infection by the Gram-Negative Human Pathogen Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa is a gram-negative pathogen that infects immunocompromised and cystic fibrosis patients. The molecular basis of the host-P. aeruginosa interaction and the effect of specific P. aeruginosa virulence factors on various components of the innate immunity pathways are largely unknown. We examine interactions between P. aeruginosa virulence factors and components of innate immunity response in the Drosophila melanogaster model system to reveal the importance of the Toll signaling pathway in resistance to infection by the P. aeruginosa human isolate PA14. Using the two PA14-isogenic mutants plcS and dsbA, we show that Drosophila loss-of-function mutants of Spatzle, the extracellular ligand of Toll, and Dorsal and Dif, two NF-κB-like transcription factors, allow increased P. aeruginosa infectivity within fly tissues. In contrast, a constitutively active Toll mutant and a loss-of-function mutant of Cactus, an IκB-like factor that inhibits the Toll signaling, reduce infectivity. Our finding that Dorsal activity is required to restrict P. aeruginosa infectivity in Drosophila provides direct in vivo evidence for Dorsal function in adult fly immunity. Additionally, our results provide the basis for future studies into interactions between P. aeruginosa virulence factors and components of the Toll signaling pathway, which is functionally conserved between flies and humans.

Regulatory Genes Controlling MPG1 Expression and Pathogenicity in the Rice Blast Fungus Magnaporthe grisea.

MPG1, a pathogenicity gene of the rice blast fungus Magnaporthe grisea, is expressed during pathogenesis and in axenic culture during nitrogen or glucose limitation. We initiated a search for regulatory mutations that would impair nitrogen metabolism, MPG1 gene expression, and pathogenicity. First, we developed a pair of laboratory strains that were highly fertile and pathogenic toward barley. Using a combinatorial genetic screen, we identified mutants that failed to utilize a wide range of nitrogen sources (e.g., nitrate or amino acids) and then tested the effect of these mutations on pathogenicity. We identified five mutants and designated them Nr- (for nitrogen regulation defective). We show that two of these mutations define two genes, designated NPR1 and NPR2 (for nitrogen pathogenicity regulation), that are essential for pathogenicity and the utilization of many nitrogen sources. These genes are nonallelic to the major nitrogen regulatory gene in M. grisea and are required for expression of the pathogenicity gene MPG1. We propose that NPR1 and NPR2 are major regulators of pathogenicity in M. grisea and may be novel regulators of nitrogen metabolism in fungi.

Pseudomonas aeruginosa biofilm infections in cystic fibrosis: insights into pathogenic processes and treatment strategies

Importance of the field: CF airway mucus can be infected by opportunistic microorganisms, notably Pseudomonas aeruginosa. Once organisms are established as biofilms, even the most potent antibiotics have little effect on their viability, especially during late-stage chronic infections. Better understanding of the mechanisms used by P. aeruginosa to circumvent host defenses and therapeutic intervention strategies is critical for advancing novel treatment strategies. Areas covered in this review: Inflammatory injury in CF lung, role of neutrophils in pathogenesis, P. aeruginosa biofilms, mucoidy and its relationship with poor airway oxygenation, mechanisms by which P. aeruginosa biofilms in the CF airway can be killed. What the reader will gain: An understanding of the processes that P. aeruginosa undergoes during CF airway disease and clues to better treat such infections in future. Take home message: The course of CF airway disease is a process involving host and microbial factors that often dictate frequency of pulmonary exacerbations, thus affecting the overall course. In the past decade significant discoveries have been made regarding the pathogenic processes used by P. aeruginosa to bypass the immune system. Many new and exciting features of P. aeruginosa now illuminate weaknesses in the organism that may render it susceptible to inexpensive compounds that force its own destruction.

Pseudomonas aeruginosa Exotoxin Pyocyanin Causes Cystic Fibrosis Airway Pathogenesis

The cystic fibrosis (CF) airway bacterial pathogen Pseudomonas aeruginosa secretes multiple virulence factors. Among these, the redox active exotoxin pyocyanin (PCN) is produced in concentrations up to 100 mumol/L during infection of CF and other bronchiectatic airways. However, the contributions of PCN during infection of bronchiectatic airways are not appreciated. In this study, we demonstrate that PCN is critical for chronic infection in mouse airways and orchestrates adaptive immune responses that mediate lung damage. Wild-type FVBN mice chronically exposed to PCN developed goblet cell hyperplasia and metaplasia, airway fibrosis, and alveolar airspace destruction. Furthermore, after 12 weeks of exposure to PCN, mouse lungs down-regulated the expression of T helper (Th) type 1 cytokines and polarized toward a Th2 response. Cellular analyses indicated that chronic exposure to PCN profoundly increased the lung population of recruited macrophages, CD4(+) T cells, and neutrophils responsible for the secretion of these cytokines. PCN-mediated goblet cell hyperplasia and metaplasia required Th2 cytokine signaling through the Stat6 pathway. In summary, this study establishes that PCN is an important P. aeruginosa virulence factor capable of directly inducing pulmonary pathophysiology in mice, consistent with changes observed in CF and other bronchiectasis lungs.

Pseudomonas aeruginosa Elastase Provides an Escape from Phagocytosis by Degrading the Pulmonary Surfactant Protein-A

Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute pneumonitis in immunocompromised patients and chronic lung infections in individuals with cystic fibrosis and other bronchiectasis. Over 75% of clinical isolates of P. aeruginosa secrete elastase B (LasB), an elastolytic metalloproteinase that is encoded by the lasB gene. Previously, in vitro studies have demonstrated that LasB degrades a number of components in both the innate and adaptive immune systems. These include surfactant proteins, antibacterial peptides, cytokines, chemokines and immunoglobulins. However, the contribution of LasB to lung infection by P. aeruginosa and to inactivation of pulmonary innate immunity in vivo needs more clarification. In this study, we examined the mechanisms underlying enhanced clearance of the ΔlasB mutant in mouse lungs. The ΔlasB mutant was attenuated in virulence when compared to the wild-type strain PAO1 during lung infection in SP-A+/+ mice. However, the ΔlasB mutant was as virulent as PAO1 in the lungs of SP-A-/- mice. Detailed analysis showed that the ΔlasB mutant was more susceptible to SP-A-mediated opsonization but not membrane permeabilization. In vitro and in vivo phagocytosis experiments revealed that SP-A augmented the phagocytosis of ΔlasB mutant bacteria more efficiently than the isogenic wild-type PAO1. The ΔlasB mutant was found to have a severely reduced ability to degrade SP-A, consequently making it unable to evade opsonization by the collectin during phagocytosis. These results suggest that P. aeruginosa LasB protects against SP-A-mediated opsonization by degrading the collectin.

Pseudomonas aeruginosa OxyR Is Required for Full Virulence in Rodent and Insect Models of Infection and for Resistance to Human Neutrophils

The role of the H2O2-responsive transactivator OxyR was evaluated in mouse and Drosophila models of Pseudomonas aeruginosa infection and by assays of neutrophil killing. Relative to that of wild-type bacteria, oxyR mutant viability was reduced by 99% or greater in mouse models of acute pneumonia and burn sepsis, and the oxyR mutant was more sensitive to killing by human neutrophils and was delayed in the kinetics of Drosophila melanogaster killing. During human infection by the opportunistic bacterium Pseudomonas aeruginosa, the organism faces exposure to toxic reactive oxygen intermediates generated by phagocytic cells. During the phagocytic oxidative burst, hydrogen peroxide (H2O2) is generated at high millimolar levels within the phagosomal vacuole (4). Enzymatic defenses against H2O2 in P. aeruginosa are provided by at least three catalases (KatA, KatB, and KatC) (2, 8), several probable peroxidases (refer to www.pseudomonas.com), and four established alkyl hydroperoxide reductases (AhpA, AhpB, AhpCF, and Ohr); members of the last class can also degrade H2O2 and various alkylhydroperoxides (9). The major gene product involved in endogenous H2O2 detoxification in P. aeruginosa is the 170-kDa heterotrimeric catalase KatA (8). Accordingly, katA gene expression is consistently high during vigorous aerobic growth. In fact, KatA activity is maintained at such high levels that even significant H2O2 stress triggers only a twofold increase in expression, suggesting that a high rate of KatA activity is critical for the detoxification of endogenous H2O2 produced during normal aerobic metabolism. In contrast to that of the katA gene, expression of several other oxidative stress defense genes, including katB-ankB (5), ahpB, and ahpCF, is dramatically increased upon exposure to H2O2, organic peroxides, or the redox cycling agent paraquat, suggesting that a highly specific and tightly regulated stress response exists in this organism (5, 9). We have found that such a response is governed by the 34-kDa H2O2-responsive transactivator known as OxyR in P. aeruginosa (9). P. aeruginosa lacking OxyR is exquisitely susceptible to H2O2, even though it possesses wild-type catalase activity (9). In fact, isolated colonies of oxyR mutant bacteria do not even appear on aerobic Luria-Bertani (LB) agar, because autoxidizable components in the medium itself generate ∼1.2 μM H2O2/min (3). This concentration of H2O2 has been detected in peripheral blood from human donors and is sufficient to kill these organisms (3). In this study, we tested the hypothesis that OxyR is required for the full virulence of P. aeruginosa by use of (i) a mouse intranasal model of acute pneumonia, (ii) a mouse burn sepsis model, (iii) an in vitro model of neutrophil killing, and (iv) a Drosophila melanogaster alternative model for animal infection.