Functional Characterization of a Novel Outer Membrane Porin KpnO, Regulated by PhoBR Two-Component System in Klebsiella pneumoniae NTUH-K2044

Background

The diffusion of antibiotics through the outer membrane is primarily affected by the porin super family, changes contribute to antibiotic resistance. Recently we demonstrated that the CpxAR two-component signaling system alters the expression of an uncharacterized porin OmpC^KP, to mediate antimicrobial resistance in K. pneumoniae.

Principal Findings

In this study, functional characterization of the putative porin OmpC^KP (denoted kpnO) with respect to antimicrobial susceptibility and virulence was evaluated by generating an isogenic mutant, ΔkpnO in a clinical isolate of K. pneumoniae. Estimation of uronic acid content confirmed that ΔkpnO produced ∼2.0 fold lesser capsular polysaccharide than the wild-type. The ΔkpnO displayed higher sensitivity to hyper osmotic and bile conditions. Disruption of kpnO increased the susceptibility of K. pneumoniae to oxidative and nitrostative stress by ∼1.6 fold and >7 fold respectively. The loss of the Klebsiella porin led to an increase in the minimum inhibitory concentration of tetracycline (3-fold), nalidixic acid (4-fold), tobramycin (4-fold), streptomycin (10-fold), and spectinomycin (10-fold), which could be restored following complementation. The single deletion of kpnO reduced the survival of the pathogen by 50% when exposed to disinfectants. In Caenorhabditis elegans model, the kpnO mutant exhibited significantly (P<0.01) lower virulence. To dissect the role of PhoBR signaling system in regulating the expression of the kpnO, a phoB ^KP isogenic mutant was constructed. The phoB ^KP mutant exhibited impaired gastrointestinal stress response and decreased antimicrobial susceptibility. The mRNA levels of kpnO were found to be 4-fold less in phoB ^KP mutant compared to wild type. A regulatory role of PhoB^KP for the expression of kpnO was further supported by the specific binding of PhoB^KP to the putative promoter of kpnO.

Conclusions and Significance

Loss of PhoBR regulated porin KpnO resulted in increased antimicrobial resistance, increased susceptibility to gastrointestinal stress, and reduced virulence in K. pneumoniae NTUH-K2044.     

Isolation of a Bacteriophage Specific for a New Capsular Type of Klebsiella pneumoniae and Characterization of Its Polysaccharide Depolymerase

Background

Klebsiella pneumoniae is one of the major pathogens causing hospital-acquired multidrug-resistant infections. The capsular polysaccharide (CPS) is an important virulence factor of K. pneumoniae. With 78 capsular types discovered thus far, an association between capsular type and the pathogenicity of K. pneumoniae has been observed.

Methodology/Principal Findings

To investigate an initially non-typeable K. pneumoniae UTI isolate NTUH-K1790N, the cps gene region was sequenced. By NTUH-K1790N cps-PCR genotyping, serotyping and determination using a newly isolated capsular type-specific bacteriophage, we found that NTUH-K1790N and three other isolates Ca0507, Ca0421 and C1975 possessed a new capsular type, which we named KN2. Analysis of a KN2 CPS⁻ mutant confirmed the role of capsule as the target recognized by the antiserum and the phage. A newly described lytic phage specific for KN2 K. pneumoniae, named 0507-KN2-1, was isolated and characterized using transmission electron microscopy. Whole-genome sequencing of 0507-KN2-1 revealed a 159 991 bp double-stranded DNA genome with a G+C content of 46.7% and at least 154 open reading frames. Based on its morphological and genomic characteristics, 0507-KN2-1 was classified as a member of the Myoviridae phage family. Further analysis of this phage revealed a 3738-bp gene encoding a putative polysaccharide depolymerase. A recombinant form of this protein was produced and assayed to confirm its enzymatic activity and specificity to KN2 capsular polysaccharides. KN2 K. pneumoniae strains exhibited greater sensitivity to this depolymerase than these did to the cognate phage, as determined by spot analysis.

Conclusions/Significance

Here we report that a group of clinical strains possess a novel Klebsiella capsular type. We identified a KN2-specific phage and its polysaccharide depolymerase, which could be used for efficient capsular typing. The lytic phage and depolymerase also have potential as alternative therapeutic agents to antibiotics for treating K. pneumoniae infections, especially against antibiotic-resistant strains.     

Correlation of Klebsiella pneumoniae Comparative Genetic Analyses with Virulence Profiles in a Murine Respiratory Disease Model

Klebsiella pneumoniae is a bacterial pathogen of worldwide importance and a significant contributor to multiple disease presentations associated with both nosocomial and community acquired disease. ATCC 43816 is a well-studied K. pneumoniae strain which is capable of causing an acute respiratory disease in surrogate animal models. In this study, we performed sequencing of the ATCC 43816 genome to support future efforts characterizing genetic elements required for disease. Furthermore, we performed comparative genetic analyses to the previously sequenced genomes from NTUH-K2044 and MGH 78578 to gain an understanding of the conservation of known virulence determinants amongst the three strains. We found that ATCC 43816 and NTUH-K2044 both possess the known virulence determinant for yersiniabactin, as well as a Type 4 secretion system (T4SS), CRISPR system, and an acetonin catabolism locus, all absent from MGH 78578. While both NTUH-K2044 and MGH 78578 are clinical isolates, little is known about the disease potential of these strains in cell culture and animal models. Thus, we also performed functional analyses in the murine macrophage cell lines RAW264.7 and J774A.1 and found that MGH 78578 (K52 serotype) was internalized at higher levels than ATCC 43816 (K2) and NTUH-K2044 (K1), consistent with previous characterization of the antiphagocytic properties of K1 and K2 serotype capsules. We also examined the three K. pneumoniae strains in a novel BALB/c respiratory disease model and found that ATCC 43816 and NTUH-K2044 are highly virulent (LD₅₀<100 CFU) while MGH 78578 is relatively avirulent.     

Characterization of integrative and conjugative element ICEKp1-associated genomic heterogeneity in a Klebsiella pneumoniae strain isolated from a primary liver abscess

Genomic heterogeneity has been shown to be associated with Klebsiella pneumoniae strains causing pyogenic liver abscesses (PLA) and metastatic infections. In order to explore the mechanism responsible for genomic heterogeneity in K. pneumoniae, we compared the complete genomic sequences of strains NTUH-K2044 and MGH78578. An ~76-kbp DNA fragment located adjacent to an asparagine (asn) tRNA gene was present in NTUH-K2044 but not in MGH78578. This fragment could be divided into three regions with different functions, and structurally it resembled a functional integrative and conjugative element (ICE), ICEEc1, in Escherichia coli. The 5′ region of this fragment contained genes similar to a high-pathogenicity island (HPI) of Yersinia pestis and Yersinia pseudotuberculosis. The middle region was similar to part of a large plasmid in K. pneumoniae, and the 3′ region contained genes responsible for DNA conjugative transfer. Therefore, this DNA fragment was designated ICEKp1. Precise excision and extrachromosomal circularization of ICEKp1 were detected in K. pneumoniae wild-type strain NTUH-K2044. ICEKp1 could integrate into the asn tRNA loci of the chromosome of another K. pneumoniae isolate. The prevalence of ICEKp1 was higher in PLA strains (38 of 42 strains) than in non-tissue-invasive strains (5 of 32 strains). Therefore, ICEKp1 may contribute to the transmission of the HPI and result in K. pneumoniae PLA infection-associated genomic heterogeneity.     

Evasion of IFN-γ signaling by Francisella novicida is dependent upon Francisella outer membrane protein C

Background

Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918) of F. tularensis subsp. tularensis has been identified as a virulence factor. We generated a F. novicida (F. tularensis subsp. novicida) FTN_0444 (homolog of FTT0918) fopC mutant to study the virulence-associated mechanism(s) of FTT0918.

Methods and Findings

The ΔfopC strain phenotype was characterized using immunological and biochemical assays. Attenuated virulence via the pulmonary route in wildtype C57BL/6 and BALB/c mice, as well as in knockout (KO) mice, including MHC I, MHC II, and µmT (B cell deficient), but not in IFN-γ or IFN-γR KO mice was observed. Primary bone marrow derived macrophages (BMDM) prepared from C57BL/6 mice treated with rIFN-γ exhibited greater inhibition of intracellular ΔfopC than wildtype U112 strain replication; whereas, IFN-γR KO macrophages showed no IFN-γ-dependent inhibition of ΔfopC replication. Moreover, phosphorylation of STAT1 was downregulated by the wildtype strain, but not the fopC mutant, in rIFN-γ treated macrophages. Addition of N^G-monomethyl-L-arginine, an NOS inhibitor, led to an increase of ΔfopC replication to that seen in the BMDM unstimulated with rIFN-γ. Enzymatic screening of ΔfopC revealed aberrant acid phosphatase activity and localization. Furthermore, a greater abundance of different proteins in the culture supernatants of ΔfopC than that in the wildtype U112 strain was observed.

Conclusions

F. novicida FopC protein facilitates evasion of IFN-γ-mediated immune defense(s) by down-regulation of STAT1 phosphorylation and nitric oxide production, thereby promoting virulence. Additionally, the FopC protein also may play a role in maintaining outer membrane stability (integrity) facilitating the activity and localization of acid phosphatases and other F. novicida cell components.     

Role of Novel Multidrug Efflux Pump Involved in Drug Resistance in Klebsiella pneumoniae

Background

Multidrug resistant Klebsiella pneumoniae have caused major therapeutic problems worldwide due to the emergence of the extended-spectrum β-lactamase producing strains. Although there are >10 major facilitator super family (MFS) efflux pumps annotated in the genome sequence of the K. pneumoniae bacillus, apparently less is known about their physiological relevance.

Principal Findings

Insertional inactivation of kpnGH resulting in increased susceptibility to antibiotics such as azithromycin, ceftazidime, ciprofloxacin, ertapenem, erythromycin, gentamicin, imipenem, ticarcillin, norfloxacin, polymyxin-B, piperacillin, spectinomycin, tobramycin and streptomycin, including dyes and detergents such as ethidium bromide, acriflavine, deoxycholate, sodium dodecyl sulphate, and disinfectants benzalkonium chloride, chlorhexidine and triclosan signifies the wide substrate specificity of the transporter in K. pneumoniae. Growth inactivation and direct fluorimetric efflux assays provide evidence that kpnGH mediates antimicrobial resistance by active extrusion in K. pneumoniae. The kpnGH isogenic mutant displayed decreased tolerance to cell envelope stressors emphasizing its added role in K. pneumoniae physiology.

Conclusions and Significance

The MFS efflux pump KpnGH involves in crucial physiological functions besides being an intrinsic resistance determinant in K. pneumoniae.     

Mycorrhization and phosphorus nutrition affect water relations and CAM induction by drought in seedlings of Clusia minor

Background and Aims

Crassulacean acid metabolism (CAM) is currently viewed as an adaptation to water deficit. In plants of Clusia minor, which grow mostly on acidic, P-deficient soils, CAM is induced by water deficit. The symbiosis between plants and mycorrhizal fungi alleviates the symptoms of P deficiency and may influence drought resistance. Therefore, the effect of P supply, modified by three different experimental treatments, on the induction of CAM by drought in C. minor was investigated to test the hypothesis that P deficiency will produce greater CAM activity and, in addition, that treatment will modify drought tolerance.

Methods

Seedlings were grown in forest soil sterilized and inoculated with Scutellospora fulgida (SF treatment), sterilized and supplemented with P (Ph treatment) or non-sterilized and containing native mycorrhizae (Nat treatment). Leaf turgor potential (ψ_T) was determined psychrometrically, and CAM activity as nocturnal acid accumulation (ΔH⁺) by titration of dawn and dusk leaf sap.

Key Results

Plant mass and P content were higher in SF and Ph than in Nat seedlings. After 21 d of water deficit, ψ_T increased in SF, decreased in Ph and remained unchanged in Nat, and, after 7 and 14 d of water deficit, ΔH⁺ in Nat was three times higher than at the beginning of drought, whereas in SF and Ph ΔH⁺ was lower than on day 0.

Conclusions

P deficiency in Nat seedlings was ameliorated by inoculation or P addition. The SF and Nat seedlings showed greater tolerance of drought than Ph. P deficiency promoted the induction of CAM by drought in Nat seedlings, whereas P fertilization and mycorrhization did not. Nocturnal acid accumulation was highly and negatively correlated with plant P and N contents, indicating that P and N deficiencies are promoters of CAM in droughted plants of C. minor.Key words: Clusia minor, crassulacean acid metabolism, CAM, mycorrhiza, drought, phosphorus deficiency, nitrogen–water relations     

A method for generation phage cocktail with great therapeutic potential

Background

Bacteriophage could be an alternative to conventional antibiotic therapy against multidrug-resistant bacteria. However, the emergence of resistant variants after phage treatment limited its therapeutic application.

Methodology/Principal Findings

In this study, an approach, named “Step-by-Step” (SBS), has been established. This method takes advantage of the occurrence of phage-resistant bacteria variants and ensures that phages lytic for wild-type strain and its phage-resistant variants are selected. A phage cocktail lytic for Klebsiella pneumoniae was established by the SBS method. This phage cocktail consisted of three phages (GH-K1, GH-K2 and GH-K3) which have different but overlapping host strains. Several phage-resistant variants of Klebsiella pneumoniae were isolated after different phages treatments. The virulence of these variants was much weaker [minimal lethal doses (MLD)>1.3×10⁹ cfu/mouse] than that of wild-type K7 countpart (MLD = 2.5×10³ cfu/mouse). Compared with any single phage, the phage cocktail significantly reduced the mutation frequency of Klebsiella pneumoniae and effectively rescued Klebsiella pneumoniae bacteremia in a murine K7 strain challenge model. The minimal protective dose (MPD) of the phage cocktail which was sufficient to protect bacteremic mice from lethal K7 infection was only 3.0×10⁴ pfu, significantly smaller (p<0.01) than that of single monophage. Moreover, a delayed administration of this phage cocktail was still effective in protection against K7 challenge.

Conclusions/Significance

Our data showed that the phage cocktail was more effective in reducing bacterial mutation frequency and in the rescue of murine bacteremia than monophage suggesting that phage cocktail established by SBS method has great therapeutic potential for multidrug-resistant bacteria infection.     

Phospholipase Cε, an Effector of Ras and Rap Small GTPases,Is Required for Airway Inflammatory Response in a Mouse Model of Bronchial Asthma

Background

Phospholipase Cε (PLCε) is an effector of Ras and Rap small GTPases and expressed in non-immune cells. It is well established that PLCε plays an important role in skin inflammation, such as that elicited by phorbol ester painting or ultraviolet irradiation and contact dermatitis that is mediated by T helper (Th) 1 cells, through upregulating inflammatory cytokine production by keratinocytes and dermal fibroblasts. However, little is known about whether PLCε is involved in regulation of inflammation in the respiratory system, such as Th2-cells-mediated allergic asthma.

Methods

We prepared a mouse model of allergic asthma using PLCε ^+/+ mice and PLCε ^ΔX/ΔX mutant mice in which PLCε was catalytically-inactive. Mice with different PLCε genotypes were immunized with ovalbumin (OVA) followed by the challenge with an OVA-containing aerosol to induce asthmatic response, which was assessed by analyzing airway hyper-responsiveness, bronchoalveolar lavage fluids, inflammatory cytokine levels, and OVA-specific immunoglobulin (Ig) levels. Effects of PLCε genotype on cytokine production were also examined with primary-cultured bronchial epithelial cells.

Results

After OVA challenge, the OVA-immunized PLCε ^ΔX/ΔX mice exhibited substantially attenuated airway hyper-responsiveness and broncial inflammation, which were accompanied by reduced Th2 cytokine content in the bronchoalveolar lavage fluids. In contrast, the serum levels of OVA-specific IgGs and IgE were not affected by the PLCε genotype, suggesting that sensitization was PLCε-independent. In the challenged mice, PLCε deficiency reduced proinflammatory cytokine production in the bronchial epithelial cells. Primary-cultured bronchial epithelial cells prepared from PLCε ^ΔX/ΔX mice showed attenuated pro-inflammatory cytokine production when stimulated with tumor necrosis factor-α, suggesting that reduced cytokine production in PLCε ^ΔX/ΔX mice was due to cell-autonomous effect of PLCε deficiency.

Conclusions

PLCε plays an important role in the pathogenesis of bronchial asthma through upregulating inflammatory cytokine production by the bronchial epithelial cells.     

Induction of Bcl-2 expression by hepatitis B virus pre-S2 mutant large surface protein resistance to 5-fluorouracil treatment in Huh-7 cells

Background

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. Our previous studies have indicated that expression of Hepatitis B virus pre-S2 large mutant surface antigen (HBV pre-S2Δ) is associated with a significant risk of developing HCC. However, the relationship between HBV pre-S2Δ protein and the resistance of chemotherapeutic drug treatment is still unclear.

Methodology/Principal Findings

Here, we show that the expression of HBV pre-S2Δ mutant surface protein in Huh-7 cell significantly promoted cell growth and colony formation. Furthermore, HBV pre-S2Δ protein increased both mRNA (2.7±0.5-fold vs. vehicle, p = 0.05) and protein (3.2±0.3-fold vs. vehicle, p = 0.01) levels of Bcl-2 in Huh-7 cells. HBV pre-S2Δ protein also enhances Bcl-2 family, Bcl-xL and Mcl-1, expression in Huh-7 cells. Meanwhile, induction of NF-κB p65, ERK, and Akt phosphorylation, and GRP78 expression, an unfolded protein response chaperone, were observed in HBV pre-S2Δ and HBV pre-S-expressing cells. Induction of Bcl-2 expression by HBV pre-S2Δ protein resulted in resistance to 5-fluorouracil treatment in colony formation, caspase-3 assay, and cell apoptosis, and can enhance cell death by co-incubation with Bcl-2 inhibitor. Similarly, transgenic mice showed higher expression of Bcl-2 in liver tissue expressing HBV pre-S2Δ large surface protein in vivo.

Conclusion/Significance

Our result demonstrates that HBV pre-S2Δ increased Bcl-2 expression which plays an important role in resistance to 5-fluorouracil-caused cell death. Therefore, these data provide an important chemotherapeutic strategy in HBV pre-S2Δ-associated tumor.     

PI3Ks maintain the structural integrity of T-tubules in cardiac myocytes

Background

Phosphoinositide 3-kinases (PI3Ks) regulate numerous physiological processes including some aspects of cardiac function. Although regulation of cardiac contraction by individual PI3K isoforms has been studied, little is known about the cardiac consequences of downregulating multiple PI3Ks concurrently.

Methods and Results

Genetic ablation of both p110α and p110β in cardiac myocytes throughout development or in adult mice caused heart failure and death. Ventricular myocytes from double knockout animals showed transverse tubule (T-tubule) loss and disorganization, misalignment of L-type Ca²⁺ channels in the T-tubules with ryanodine receptors in the sarcoplasmic reticulum, and reduced Ca²⁺ transients and contractility. Junctophilin-2, which is thought to tether T-tubules to the sarcoplasmic reticulum, was mislocalized in the double PI3K-null myocytes without a change in expression level.

Conclusions

PI3K p110α and p110β are required to maintain the organized network of T-tubules that is vital for efficient Ca²⁺-induced Ca²⁺ release and ventricular contraction. PI3Ks maintain T-tubule organization by regulating junctophilin-2 localization. These results could have important medical implications because several PI3K inhibitors that target both isoforms are being used to treat cancer patients in clinical trials.     

Ursodeoxycholic acid is conjugated with taurine to promote secretin-stimulated biliary hydrocholeresis in the normal rat

Background & Aims

Secretin induces bicarbonate-rich hydrocholeresis in healthy individuals, but not in untreated patients with primary biliary cirrhosis (PBC). Ursodeoxycholic acid (UDCA) – the first choice treatment for PBC – restores the secretin response. Compared with humans, secretin has poor effect in experimental normal-rat models with biliary drainage, although it may elicit hydrocholeresis when the bile-acid pool is maintained. In view of the benefits of UDCA in PBC, we used normal-rat models to unravel the acute contribution of UDCA (and/or taurine-conjugated TUDCA) for eliciting the biliary secretin response.

Methods

Intravascular and/or intrabiliary administration of agonists and inhibitors was performed in normal rats with biliary monitoring. Secretin/bile-acid interplay was analyzed in 3D cultured rat cholangiocytes that formed expansive cystic structures with intralumenal hydroionic secretion.

Results

In vivo, secretin stimulates hydrocholeresis upon UDCA/TUDCA infusion, but does not modify the intrinsic hypercholeretic effect of dehydrocholic acid (DHCA). The former effect is dependent on microtubule polymerization, and involves PKCα, PI3K and MEK pathways, as shown by colchicine (i.p.) and retrograde biliary inhibitors. In vitro, while secretin alone accelerates the spontaneous expansion of 3D-cystic structures, this effect is enhanced in the presence of TUDCA, but not UDCA or DHCA. Experiments with inhibitors and Ca²⁺-chelator confirmed that the synergistic effect of secretin plus TUDCA involves microtubules, intracellular Ca²⁺, PKCα, PI3K, PKA and MEK pathways. Gene silencing also demonstrated the involvement of the bicarbonate extruder Ae2.

Conclusions

UDCA is conjugated in order to promote secretin-stimulated hydrocholeresis in rats through Ae2, microtubules, intracellular Ca²⁺, PKCα, PI3K, PKA, and MEK.     

Phase I hydroxylated metabolites of the K2 synthetic cannabinoid JWH-018 retain in vitro and in vivo cannabinoid 1 receptor affinity and activity

Background

K2 products are synthetic cannabinoid-laced, marijuana-like drugs of abuse, use of which is often associated with clinical symptoms atypical of marijuana use, including hypertension, agitation, hallucinations, psychosis, seizures and panic attacks. JWH-018, a prevalent K2 synthetic cannabinoid, is structurally distinct from Δ⁹-THC, the main psychoactive ingredient in marijuana. Since even subtle structural differences can lead to differential metabolism, formation of novel, biologically active metabolites may be responsible for the distinct effects associated with K2 use. The present study proposes that K2''s high adverse effect occurrence is due, at least in part, to distinct JWH-018 metabolite activity at the cannabinoid 1 receptor (CB1R).

Methods/Principal Findings

JWH-018, five potential monohydroxylated metabolites (M1–M5), and one carboxy metabolite (M6) were examined in mouse brain homogenates containing CB1Rs, first for CB1R affinity using a competition binding assay employing the cannabinoid receptor radioligand [³H]CP-55,940, and then for CB1R intrinsic efficacy using an [³⁵S]GTPγS binding assay. JWH-018 and M1–M5 bound CB1Rs with high affinity, exhibiting K_i values that were lower than or equivalent to Δ⁹-THC. These molecules also stimulated G-proteins with equal or greater efficacy relative to Δ⁹-THC, a CB1R partial agonist. Most importantly, JWH-018, M2, M3, and M5 produced full CB1R agonist levels of activation. CB1R-mediated activation was demonstrated by blockade with O-2050, a CB1R-selective neutral antagonist. Similar to Δ⁹-THC, JWH-018 and M1 produced a marked depression of locomotor activity and core body temperature in mice that were both blocked by the CB1R-preferring antagonist/inverse agonist AM251.

Conclusions/Significance

Unlike metabolites of most drugs, the studied JWH-018 monohydroxylated compounds, but not the carboxy metabolite, retain in vitro and in vivo activity at CB1Rs. These observations, combined with higher CB1R affinity and activity relative to Δ⁹-THC, may contribute to the greater prevalence of adverse effects observed with JWH-018-containing products relative to cannabis.     

Loss of the SPHF homologue Slr1768 leads to a catastrophic failure in the maintenance of thylakoid membranes in Synechocystis sp. PCC 6803

Background

In cyanobacteria the photosystems are localised to, and maintained in, specialist membranes called the thylakoids. The mechanism driving the biogenesis of the thylakoid membranes is still an open question, with only two potential biogenesis factors, Vipp1 and Alb3 currently identified.

Methodology/Principal Findings

We generated a slr1768 knockout using the pGEM T-easy vector and REDIRECT. By comparing growth and pigment content (chlorophyll a fluoresence) of the Δslr1768 mutant with the wild-type, we found that Δslr1768 has a conditional phenotype; specifically under high light conditions (130 µmol m⁻² s⁻¹) thylakoid biogenesis is disrupted leading to cell death on a scale of days. The thylakoids show considerable disruption, with loss of both structure and density, while chlorophyll a density decreases with the loss of thylakoids, although photosynthetic efficiency is unaffected. Under low light (30 µmol m⁻² s⁻¹) the phenotype is significantly reduced, with a growth rate similar to the wild-type and only a low frequency of cells with evident thylakoid disruption.

Conclusions/Significance

This is the first example of a gene that affects the maintenance of the thylakoid membranes specifically under high light, and which displays a phenotype dependent on light intensity. Our results demonstrate that Slr1768 has a leading role in acclimatisation, linking light damage with maintenance of the thylakoids.     

Universal stress proteins are important for oxidative and acid stress resistance and growth of Listeria monocytogenes EGD-e in vitro and in vivo

Background

Pathogenic bacteria maintain a multifaceted apparatus to resist damage caused by external stimuli. As part of this, the universal stress protein A (UspA) and its homologues, initially discovered in Escherichia coli K-12 were shown to possess an important role in stress resistance and growth in several bacterial species.

Methods and Findings

We conducted a study to assess the role of three homologous proteins containing the UspA domain in the facultative intracellular human pathogen Listeria monocytogenes under different stress conditions. The growth properties of three UspA deletion mutants (Δlmo0515, Δlmo1580 and Δlmo2673) were examined either following challenge with a sublethal concentration of hydrogen peroxide or under acidic conditions. We also examined their ability for intracellular survival within murine macrophages. Virulence and growth of usp mutants were further characterized in invertebrate and vertebrate infection models.Tolerance to acidic stress was clearly reduced in Δlmo1580 and Δlmo0515, while oxidative stress dramatically diminished growth in all mutants. Survival within macrophages was significantly decreased in Δlmo1580 and Δlmo2673 as compared to the wild-type strain. Viability of infected Galleria mellonella larvae was markedly higher when injected with Δlmo1580 or Δlmo2673 as compared to wild-type strain inoculation, indicating impaired virulence of bacteria lacking these usp genes. Finally, we observed severely restricted growth of all chromosomal deletion mutants in mice livers and spleens as compared to the load of wild-type bacteria following infection.

Conclusion

This work provides distinct evidence that universal stress proteins are strongly involved in listerial stress response and survival under both in vitro and in vivo growth conditions.     

Mycoheterotrophy evolved from mixotrophic ancestors: evidence in Cymbidium (Orchidaceae)

Background and Aims

Nutritional changes associated with the evolution of achlorophyllous, mycoheterotrophic plants have not previously been inferred with robust phylogenetic hypotheses. Variations in heterotrophy in accordance with the evolution of leaflessness were examined using a chlorophyllous–achlorophyllous species pair in Cymbidium (Orchidaceae), within a well studied phylogenetic background.

Methods

To estimate the level of mycoheterotrophy in chlorophyllous and achlorophyllous Cymbidium, natural ¹³C and ¹⁵N contents (a proxy for the level of heterotrophy) were measured in four Cymbidium species and co-existing autotrophic and mycoheterotrophic plants and ectomycorrhizal fungi from two Japanese sites.

Key Results

δ¹³C and δ¹⁵N values of the achlorophyllous C. macrorhizon and C. aberrans indicated that they are full mycoheterotrophs. δ¹³C and δ¹⁵N values of the chlorophyllous C. lancifolium and C. goeringii were intermediate between those of reference autotrophic and mycoheterotrophic plants; thus, they probably gain 30–50 % of their carbon resources from fungi. These data suggest that some chlorophyllous Cymbidium exhibit partial mycoheterotrophy (= mixotrophy).

Conclusions

It is demonstrated for the first time that mycoheterotrophy evolved after the establishment of mixotrophy rather than through direct shifts from autotrophy to mycoheterotrophy. This may be one of the principal patterns in the evolution of mycoheterotrophy. The results also suggest that the establishment of symbiosis with ectomycorrhizal fungi in the lineage leading to mixotrophic Cymbidium served as pre-adaptation to the evolution of the mycoheterotrophic species. Similar processes of nutritional innovations probably occurred in several independent orchid groups, allowing niche expansion and radiation in Orchidaceae, probably the largest plant family.     

Genome Sequencing and Comparative Analysis of Klebsiella pneumoniae NTUH-K2044, a Strain Causing Liver Abscess and Meningitis

Nosocomial infections caused by antibiotic-resistant Klebsiella pneumoniae are emerging as a major health problem worldwide, while community-acquired K. pneumoniae infections present with a range of diverse clinical pictures in different geographic areas. In particular, an invasive form of K. pneumoniae that causes liver abscesses was first observed in Asia and then was found worldwide. We are interested in how differences in gene content of the same species result in different diseases. Thus, we sequenced the whole genome of K. pneumoniae NTUH-K2044, which was isolated from a patient with liver abscess and meningitis, and analyzed differences compared to strain MGH 78578, which was isolated from a patient with pneumonia. Six major types of differences were found in gene clusters that included an integrative and conjugative element, clusters involved in citrate fermentation, lipopolysaccharide synthesis, and capsular polysaccharide synthesis, phage-related insertions, and a cluster containing fimbria-related genes. We also conducted comparative genomic hybridization with 15 K. pneumoniae isolates obtained from community-acquired or nosocomial infections using tiling probes for the NTUH-K2044 genome. Hierarchical clustering revealed three major groups of genomic insertion-deletion patterns that correlate with the strains'' clinical features, antimicrobial susceptibilities, and virulence phenotypes with mice. Here we report the whole-genome sequence of K. pneumoniae NTUH-K2044 and describe evidence showing significant genomic diversity and sequence acquisition among K. pneumoniae pathogenic strains. Our findings support the hypothesis that these factors are responsible for the changes that have occurred in the disease profile over time.Klebsiella pneumoniae is a gram-negative bacterium that belongs to the gamma subdivision of the class Proteobacteria and exhibits relatively close genetic relatedness to other genera of the Enterobacteriaceae, including Escherichia, Salmonella, Shigella, and Yersinia (2). The conspicuous difference between K. pneumoniae and the other enterobacteria is the presence of a thick polysaccharide capsule, which is thought to be a significant virulence factor and to help the bacterium avoid phagocytosis (13). Infections caused by K. pneumoniae are seen throughout the world. This organism is a major cause of urinary tract infection and an important source of nosocomial infection (39). Moreover, K. pneumoniae is emerging worldwide as a major cause of bacteremia and drug-resistant infections (25, 38).The clinical pattern of K. pneumoniae infection in humans has changed since this organism was discovered (19, 20) more than 100 years ago. Until the 1960s, K. pneumoniae was an important cause of community-acquired pneumonia in the United States (8) and elsewhere. However, the incidence of this type of infection has dropped to 1 to 3% in the United States and Europe, and hospital-acquired K. pneumoniae infection now predominates (22, 39, 48). The global pattern of community-acquired K. pneumoniae bacteremia varies with geographical area (25). In the United States, Europe, Australia, and Argentina, this condition is associated with urinary tract infection, vascular catheters, and cholangitis. In Asia and South Africa, classic K. pneumoniae pneumonia still exists (25) and has remained important over the past two decades. At the same time, an invasive form of K. pneumoniae infection, which presents as primary bacteremic liver abscesses, endophthalmitis, and meningitis, has been reported almost exclusively in Asia (21), especially in Taiwan (47, 50). Although the reasons for the preponderance of this severe invasive K. pneumoniae infection in Asia are unknown, they are likely to involve both host and microbial factors.Recent studies by several groups have investigated and debated the major virulence factors of K. pneumoniae, including the magA (16) and rmpA (53) genes, capsular serotype K1 or K2 (11, 52), and even hypermucoviscosity (16, 53). In principle, other determinants may also contribute to pyogenic K. pneumoniae infection. To gather sufficient DNA sequence information for a systematic analysis of the genetic features that underlie the diverse clinical manifestations of K. pneumoniae infections, we undertook complete genome sequencing of a pathogenic strain, NTUH-K2044, which had been isolated from a Taiwanese liver abscess case (16). NTUH-K2044 is an appropriate strain because it possesses the magA and rmpA genes, belongs to capsular serotype K1, and has high virulence and hypermucoviscosity; these factors make this isolate very suitable as a model strain for genomic studies. We additionally used a genomic shotgun array (GSA) protocol developed in our laboratory (27) to compare the genome contents of NTUH-K2044 and multiple clinical isolates. The microarray data allowed us to examine the genome evolution of K. pneumoniae and to relate the various genomic signatures to the clinical patterns seen in K. pneumoniae infections.