Regulation of nuclear-cytoplasmic shuttling and function of Family with sequence similarity 13, member A (Fam13a), by B56-containing PP2As and Akt

Recent genome-wide association studies reveal that the FAM13A gene is associated with human lung function and a variety of lung diseases, including chronic obstructive pulmonary disease, asthma, lung cancer, and pulmonary fibrosis. The biological functions of Fam13a, however, have not been studied. In an effort to identify novel substrates of B56-containing PP2As, we found that B56-containing PP2As and Akt act antagonistically to control reversible phosphorylation of Fam13a on Ser-322. We show that Ser-322 phosphorylation acts as a molecular switch to control the subcellular distribution of Fam13a. Fam13a shuttles between the nucleus and cytoplasm. When Ser-322 is phosphorylated by Akt, the binding between Fam13a and 14-3-3 is enhanced, leading to cytoplasmic sequestration of Fam13a. B56-containing PP2As dephosphorylate phospho-Ser-322 and promote nuclear localization of Fam13a. We generated Fam13a-knockout mice. Fam13a-mutant mice are viable and healthy, indicating that Fam13a is dispensable for embryonic development and physiological functions in adult animals. Intriguingly, Fam13a has the ability to activate the Wnt pathway. Although Wnt signaling remains largely normal in Fam13a-knockout lungs, depletion of Fam13a in human lung cancer cells causes an obvious reduction in Wnt signaling activity. Our work provides important clues to elucidating the mechanism by which Fam13a may contribute to human lung diseases

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 vitroand 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

Efficacy of a bacteriophage isolated from chickens as a therapeutic agent for colibacillosis in broiler chickens

The efficacy of bacteriophage EC1, a lytic bacteriophage, against Escherichia coli O78:K80, which causes colibacillosis in poultry, was determined in the present study. A total of 480 one-day-old birds were randomly assigned to 4 treatments groups, each with 4 pens of 30 birds. Birds from the control groups (groups I and II) received PBS (pH 7.4) or 10(10) pfu of bacteriophage EC1, respectively. Group III consisted of birds challenged with 10(8) cfu of E. coli O78: K80 and treated with 10(10) pfu of bacteriophage EC1 at 2 h postinfection, whereas birds from group IV were challenged with 10(8) cfu of E. coli O78: K80 only. All the materials were introduced into the birds by intratracheal inoculation. Based on the results of the present study, the infection was found to be less severe in the treated E. coli-challenged group. Mean total viable cell counts of E. coli identified on eosin methylene blue agar (designated EMB + E. coli) in the lungs were significantly lower in treated, E. coli-challenged birds than in untreated, E. coli-challenged birds on d 1 and 2 postinfection. The EMB + E. coli isolation frequency was also lower in treated birds; no E. coli was detectable in blood samples on any sampling day, and E. coli were isolated only in the liver, heart, and spleen of treated chickens at a ratio of 2/6, 1/6, and 3/6, respectively, at d 1 postinfection. The BW of birds from the E. coli-challenged group treated with bacteriophage EC1 were not significantly different from those of birds from both control groups but were 15.4% higher than those of the untreated, E. coli-challenged group on d 21 postinfection. The total mortality rate of birds during the 3-wk experimental period decreased from 83.3% in the untreated, E. coli-challenged birds (group IV) to 13.3% in birds treated with bacteriophage EC1 (group III). These results suggest that bacteriophage EC1 is effective in vivo and could be used to treat colibacillosis in chickens

Comparative Signature-Tagged Mutagenesis Identifies Pseudomonas Factors Conferring Resistance to the Pulmonary Collectin SP-A

The pulmonary collectin, surfactant protein A (SP-A), is a broad spectrum opsonin with microbicidal membrane permeabilization properties that plays a role in the innate immune response of the lung. However, the factors that govern SP-A's microbial specificity and the mechanisms by which it mediates membrane permeabilization and opsonization are not fully understood. In an effort to identify bacterial factors that confer susceptibility or resistance to SP-A, we used comparative signature-tagged mutagenesis to screen a library of 1,680 Pseudomonas aeruginosa mutants for evidence of differential pulmonary clearance in SP-A-sufficient (SP-A(+/+)) and SP-A-deficient (SP-A(−/−)) mice. Two SP-A-sensitive P. aeruginosa mutants harboring transposon insertions in genes required for salicylate biosynthesis (pch) and phosphoenolpyruvate-protein-phosphotransferase (ptsP) were recovered. The mutants were indistinguishable from the parental wild-type PA01 with regard to opsonization by SP-A, but they exhibited increased susceptibility to SP-A-mediated membrane permeabilization. These results suggest that bacterial gene functions that are required to maintain membrane integrity play crucial roles in resistance of P. aeruginosa to the permeabilizing effects of SP-A

The Anti-Sigma Factor MucA of Pseudomonas aeruginosa: Dramatic Differences of a mucA22 vs. a ΔmucA Mutant in Anaerobic Acidified Nitrite Sensitivity of Planktonic and Biofilm Bacteria in vitro and During Chronic Murine Lung Infection

Mucoid mucA22 Pseudomonas aeruginosa (PA) is an opportunistic lung pathogen of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) patients that is highly sensitive to acidified nitrite (A-NO2-). In this study, we first screened PA mutant strains for sensitivity or resistance to 20 mM A-NO2- under anaerobic conditions that represent the chronic stages of the aforementioned diseases. Mutants found to be sensitive to A-NO2- included PA0964 (pmpR, PQS biosynthesis), PA4455 (probable ABC transporter permease), katA (major catalase, KatA) and rhlR (quorum sensing regulator). In contrast, mutants lacking PA0450 (a putative phosphate transporter) and PA1505 (moaA2) were A-NO2- resistant. However, we were puzzled when we discovered that mucA22 mutant bacteria, a frequently isolated mucA allele in CF and to a lesser extent COPD, were more sensitive to A-NO2- than a truncated ΔmucA deletion (Δ157–194) mutant in planktonic and biofilm culture, as well as during a chronic murine lung infection. Subsequent transcriptional profiling of anaerobic, A-NO2--treated bacteria revealed restoration of near wild-type transcript levels of protective NO2- and nitric oxide (NO) reductase (nirS and norCB, respectively) in the ΔmucA mutant in contrast to extremely low levels in the A-NO2--sensitive mucA22 mutant. Proteins that were S-nitrosylated by NO derived from A-NO2- reduction in the sensitive mucA22 strain were those involved in anaerobic respiration (NirQ, NirS), pyruvate fermentation (UspK), global gene regulation (Vfr), the TCA cycle (succinate dehydrogenase, SdhB) and several double mutants were even more sensitive to A-NO2-. Bioinformatic-based data point to future studies designed to elucidate potential cellular binding partners for MucA and MucA22. Given that A-NO2- is a potentially viable treatment strategy to combat PA and other infections, this study offers novel developments as to how clinicians might better treat problematic PA infections in COPD and CF airway diseases

Glutathione Activates Type III Secretion System Through Vfr in Pseudomonas aeruginosa

Glutathione (GSH) is the most abundant antioxidant in all living organisms. Previously, we have shown that a deletion mutant in the glutathione synthetase gene (ΔgshB) decreases the expression of type III secretion system (T3SS) genes of Pseudomonas aeruginosa. However, the mechanism remains elusive. In this study, a comprehensive transcriptomic analysis of the GSH-deficient mutant ΔgshAΔgshB was used to elucidate the role of GSH in the pathogenesis of P. aeruginosa. The data show that the expression of genes in T3SS, type VI secretion system (T6SS) and some regulatory genes were impaired. ΔgshAΔgshB was attenuated in a mouse model of acute pneumonia, swimming and swarming motilities, and biofilm formation. Under T3SS inducing conditions, GSH enhanced the expression of T3SS in both wild-type PAO1 and ΔgshAΔgshB, but not in Δvfr. Genetic complementation of Δvfr restored the ability of GSH to induce the expression of T3SS genes. Site-directed mutagenesis based substitution of cysteine residues with alanine in Vfr protein abolished the induction of T3SS genes by GSH, confirming that GSH regulates T3SS genes through Vfr. Exposure to H2O2 decreased free thiol content on Vfr, indicating that the protein was sensitive to redox modification. Importantly, GSH restored the oxidized Vfr to reduced state. Collectively, these results suggest that GSH serves as an intracellular redox signal sensed by Vfr to upregulate T3SS expression in P. aeruginosa. Our work provides new insights into the role of GSH in P. aeruginosa pathogenesis

Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo

Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection

The Flagellum of Pseudomonas aeruginosa Is Required for Resistance to Clearance by Surfactant Protein A

Surfactant protein A (SP-A) is an important lung innate immune protein that kills microbial pathogens by opsonization and membrane permeabilization. We investigated the basis of SP-A-mediated pulmonary clearance of Pseudomonas aeruginosa using genetically-engineered SP-A mice and a library of signature-tagged P. aeruginosa mutants. A mutant with an insertion into flgE, the gene that encodes flagellar hook protein, was preferentially cleared by the SP-A(+/+) mice, but survived in the SP-A(-/-) mice. Opsonization by SP-A did not play a role in flgE clearance. However, exposure to SP-A directly permeabilized and killed the flgE mutant, but not the wild-type parental strain. P. aeruginosa strains with mutation in other flagellar genes, as well as mucoid, nonmotile isolates from cystic fibrosis patients, were also permeabilized by SP-A. Provision of the wild-type fliC gene restored the resistance to SP-A-mediated membrane permeabilization in the fliC-deficient bacteria. In addition, non-mucoid, motile revertants of CF isolates reacquired resistance to SP-A-mediated membrane permeability. Resistance to SP-A was dependent on the presence of an intact flagellar structure, and independent of flagellar-dependent motility. We provide evidence that flagellar-deficient mutants harbor inadequate amounts of LPS required to resist membrane permeabilization by SP-A and cellular lysis by detergent targeting bacterial outer membranes. Thus, the flagellum of P. aeruginosa plays an indirect but important role resisting SP-A-mediated clearance and membrane permeabilization