A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness

Hypervirulent K. pneumoniae (hvKp) is a distinct pathotype that causes invasive community-acquired infections in healthy individuals. Hypermucoviscosity (hmv) is a major phenotype associated with hvKp characterized by copious capsule production and poor sedimentation. Dissecting the individual functions of CPS production and hmv in hvKp has been hindered by the conflation of these two properties. Although hmv requires capsular polysaccharide (CPS) biosynthesis, other cellular factors may also be required and some fitness phenotypes ascribed to CPS may be distinctly attributed to hmv. To address this challenge, we systematically identified genes that impact capsule and hmv. We generated a condensed, ordered transposon library in hypervirulent strain KPPR1, then evaluated the CPS production and hmv phenotypes of the 3,733 transposon mutants, representing 72% of all open reading frames in the genome. We employed forward and reverse genetic screens to evaluate effects of novel and known genes on CPS biosynthesis and hmv. These screens expand our understanding of core genes that coordinate CPS biosynthesis and hmv, as well as identify central metabolism genes that distinctly impact CPS biosynthesis or hmv, specifically those related to purine metabolism, pyruvate metabolism and the TCA cycle. Six representative mutants, with varying effect on CPS biosynthesis and hmv, were evaluated for their impact on CPS thickness, serum resistance, host cell association, and fitness in a murine model of disseminating pneumonia. Altogether, these data demonstrate that hmv requires both CPS biosynthesis and other cellular factors, and that hmv and CPS may serve distinct functions during pathogenesis. The integration of hmv and CPS to the metabolic status of the cell suggests that hvKp may require certain nutrients to specifically cause deep tissue infections.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

Characterization of FKGK18 as Inhibitor of Group VIA Ca2+-Independent Phospholipase A2 (iPLA2β): Candidate Drug for Preventing Beta-Cell Apoptosis and Diabetes

Ongoing studies suggest an important role for iPLA₂β in a multitude of biological processes and it has been implicated in neurodegenerative, skeletal and vascular smooth muscle disorders, bone formation, and cardiac arrhythmias. Thus, identifying an iPLA₂βinhibitor that can be reliably and safely used in vivo is warranted. Currently, the mechanism-based inhibitor bromoenol lactone (BEL) is the most widely used to discern the role of iPLA₂β in biological processes. While BEL is recognized as a more potent inhibitor of iPLA₂ than of cPLA₂ or sPLA₂, leading to its designation as a “specific” inhibitor of iPLA₂, it has been shown to also inhibit non-PLA₂ enzymes. A potential complication of its use is that while the S and R enantiomers of BEL exhibit preference for cytosol-associated iPLA₂β and membrane-associated iPLA₂γ, respectively, the selectivity is only 10-fold for both. In addition, BEL is unstable in solution, promotes irreversible inhibition, and may be cytotoxic, making BEL not amenable for in vivo use. Recently, a fluoroketone (FK)-based compound (FKGK18) was described as a potent inhibitor of iPLA₂β. Here we characterized its inhibitory profile in beta-cells and find that FKGK18: (a) inhibits iPLA₂β with a greater potency (100-fold) than iPLA₂γ, (b) inhibition of iPLA₂β is reversible, (c) is an ineffective inhibitor of α-chymotrypsin, and (d) inhibits previously described outcomes of iPLA₂β activation including (i) glucose-stimulated insulin secretion, (ii) arachidonic acid hydrolysis; as reflected by PGE2 release from human islets, (iii) ER stress-induced neutral sphingomyelinase 2 expression, and (iv) ER stress-induced beta-cell apoptosis. These findings suggest that FKGK18 is similar to BEL in its ability to inhibit iPLA₂β. Because, in contrast to BEL, it is reversible and not a non-specific inhibitor of proteases, it is suggested that FKGK18 is more ideal for ex vivo and in vivo assessments of iPLA₂β role in biological functions.     

Parthenogenetic development of dwarf surf dam,Mulinia lateralis,oocytes treated with polar body suppressing agents

Summary

Parthenogenesis following oocyte activation has been observed in a number of marine invertebrates, but the fate of parthenogenesis in bivalve mollusc embryos is unclear. We used the dwarf surf clam, Mulinia lateralis, to examine parthenogenetic development of KC1-activated oocytes using the polar body suppressing agents caffeine and heat or cytochalasin B. Development was followed by epifluorescence microscopy and flow-cytometric analysis using the DNA-specific fluorochrome DAPI. All agents suppressed polar body formation to some degree, putatively increasing the ploidy level and retaining a meiotic centrosome in the zygote; but the zygotes failed to develop normally. Failure of the zygotes to develop suggests that the meiotic centrosome is incapable of participating in mitosis in bivalves.     

Gravel dredging alters diversity and structure of riverine fish assemblages

1. Human activities affect fish assemblages in a variety of ways. Large‐scale and long‐term disturbances such as in‐stream dredging and mining alter habitat and hydrodynamic characteristics within rivers which can, in turn, alter fish distribution. Habitat heterogeneity is decreased as the natural riffle–pool–run sequences are lost to continuous pools and, as a consequence, lotic species are displaced by lentic species, while generalist and invasive species displace native habitat specialists. Sediment and organic detritus accumulate in deep, dredged reaches and behind dams, disrupting nutrient flow and destroying critical habitat for habitat specialist species. 2. We used standard ecological metrics such as species richness and diversity, as well as stable isotope analysis of δ¹³C and δ¹⁵N, to quantify the differences in fish assemblages sampled by benthic trawls among dredged and undredged sites in the Allegheny River, Pennsylvania, U.S.A. 3. Using mixed‐effects models, we found that total catch, species richness and diversity were negatively correlated with depth (P < 0.05), while species richness, diversity and proportion of species in lithophilic (‘rock‐loving’) reproductive guilds were lower at dredged than at undredged sites (P < 0.05). 4. Principal components analysis and manova revealed that taxa such as darters in brood hider and substratum chooser reproductive guilds were predominantly associated with undredged sites along principal component axis 1 (PC1 and manova P < 0.05), while nest spawners such as catfish and open substratum spawners including suckers were more associated with dredged sites along PC2 (P < 0.05). 5. Stable isotope analysis of δ¹³C and δ¹⁵N revealed shifts from reliance on shallow water and benthic‐derived nutrients at undredged sites to reliance on phytoplankton and terrestrial detritus at deep‐water dredged sites. Relative trophic positions were also lower at dredged sites for many species; loss of benthic nutrient pathways associated with depth and dredging history is hypothesised. 6. The combination of ecological metrics and stable isotope analysis thus shows how anthropogenic habitat loss caused by gravel dredging can decrease benthic fish abundance and diversity, and that species in substratum‐specific reproductive guilds are at particular risk. The effects of dredging also manifest by altering resource use and nutrient pathways within food webs. Management and conservation decisions should therefore consider the protection of relatively shallow areas with suitable substratum for spawning for the protection of native fishes.     

Initiation of Swarming Motility by Proteus mirabilis Occurs in Response to Specific Cues Present in Urine and Requires Excess l-Glutamine

Proteus mirabilis, a leading cause of catheter-associated urinary tract infection (CaUTI), differentiates into swarm cells that migrate across catheter surfaces and medium solidified with 1.5% agar. While many genes and nutrient requirements involved in the swarming process have been identified, few studies have addressed the signals that promote initiation of swarming following initial contact with a surface. In this study, we show that P. mirabilis CaUTI isolates initiate swarming in response to specific nutrients and environmental cues. Thirty-three compounds, including amino acids, polyamines, fatty acids, and tricarboxylic acid (TCA) cycle intermediates, were tested for the ability to promote swarming when added to normally nonpermissive media. l-Arginine, l-glutamine, dl-histidine, malate, and dl-ornithine promoted swarming on several types of media without enhancing swimming motility or growth rate. Testing of isogenic mutants revealed that swarming in response to the cues required putrescine biosynthesis and pathways involved in amino acid metabolism. Furthermore, excess glutamine was found to be a strict requirement for swarming on normal swarm agar in addition to being a swarming cue under normally nonpermissive conditions. We thus conclude that initiation of swarming occurs in response to specific cues and that manipulating concentrations of key nutrient cues can signal whether or not a particular environment is permissive for swarming.     

Twelve polymorphic microsatellite loci from the Asian citrus psyllid,Diaphorina citri Kuwayama,the vector for citrus greening disease,huanglongbing

Twelve polymorphic microsatellite markers were developed from microsatellite‐enriched DNA libraries and mined from an expressed sequence tags library of Diaphorina citri, the vector of the citrus greening disease (huanglongbing). Analysis of 288 individuals from Florida, Texas, and Brazil showed that allelic diversity ranged from three to eight alleles per locus and observed and expected heterozygosities ranged from 0.014 to 0.569 and from 0.052 to 0.653, respectively. These variable microsatellite loci can provide means for assessing overall genetic variation and migration patterns for this agriculturally important pest species. This information can be used to aid in developing successful management strategies.     

Survival of boll weevil （Coleoptera： Curculionidae） adults after feeding on pollens from various sources

The survival of overwintering boll weevil, Anthonomus grandis grandis （Boheman）, adults on non-cotton hosts in the Lower Rio Grande Valley （LRGV） of Texas was examined from 2001 to 2006. The success of the Boll Weevil Eradication Program, which was reintroduced into the LRGV in 2005, depends on controlling overwintering boll weevil populations. Laboratory studies were conducted using boll weevil adults that were captured in pheromone traps from September through March. The number of adults captured per trap declined significantly in the field from fall to the beginning of spring （3.5-7.0-fold）. The proportion of trapped males and females did not differ significantly. The mean weight of boll weevil adults captured in September was 13.3 mg, while those of captured adults from November to February were significantly lower and ranged from 6.7 to 7.8 mg. Our results show that boll weevil adults can feed on different plant pollens. The highest longevity occurred when adults were fed almond pollen or mixed pollens （72.6 days and 69.2 days, respectively） and the lowest when they fed on citrus pollen or a non-food source （9.7 days or 7.4 days, respectively）. The highest adult survival occurred on almond and mixed pollens [88.0%-97. 6% after 1st feeding period （10 days）, 78.0%-90.8% after 3rd feeding period （10 days）, 55. 0%-83.6% after 5th feeding period （10 days）, and 15.2%-32.4% after lOth feeding period （10 days）]. The lowest adult survival occurred on citrus pollen [52.0%-56.0% after 1st feeding period （10 days）, 13.3% after 3rd and 5th feeding periods （10 days）, and 0 after 6th feeding period （10 days）]. Pollen feeding is not a behavior restricted to adult boll weevils of a specific sex or physiological state. Understanding how boll weevil adults survive in the absence of cotton is important to ensure ultimate success of eradicating this pest in the subtropics.     

A new workerless inquiline in the Lower Attini (Hymenoptera: Formicidae), with a discussion of social parasitism in fungus‐growing ants

Ant inquilines are obligate social parasites, usually lacking a sterile worker caste, which are dependent on their hosts for survival and reproduction. Social parasites are rare among the fungus‐gardening ants (Myrmicinae: tribe Attini) and only four species are known until now, all being inquilines from the Higher Attini. We describe Mycocepurus castrator sp.n. , the first inquiline social parasite to be discovered in the Lower Attini. Our study of the parasite's behaviour and life history supports the conclusion drawn from external morphology: Mycocepurus castrator is an evolutionarily derived inquiline parasite of Mycocepurus goeldii. Inquilines are of great interest to evolutionary biology because it is debated if they originated via sympatric or allopatric speciation. We discuss the life history evolution, behaviour and morphology of socially parasitic, fungus‐growing ants.     

Membrane interactions of the synthetic N-terminal peptide of HIV-1 gp41 and its structural analogs

Structural and functional studies assessed the membrane actions of the N terminus of HIV-1 glycoprotein 41000 (gp41). Earlier site-directed mutagenesis has shown that key amino acid changes in this gp41 domain inhibit viral infection and syncytia formation. Here, a synthetic peptide corresponding to the N terminus of gp41 (FP; 23 residues, 519-541), and also FP analogs (FP520V/E with Val-->Glu at residue 520; FP527L/R with Leu-->Arg at 527; FP529F/Y with Phe-->Tyr at 529; and FPCLP1 with FP truncated at 525) incorporating these modifications were prepared. When added to human erythrocytes at physiologic pH, the lytic and aggregating activities of the FP analogs were much reduced over those with the wild-type FP. With resealed human erythrocyte ghosts, the lipid-mixing activities of the FP analogs were also substantially depressed over that with the wild-type FP. Combined with results from earlier studies, theoretical calculations using hydrophobic moment plot analysis and physical experiments using circular dichroism and Fourier transform infrared spectroscopy indicate that the diminished lysis and fusion noted for FP analogs may be due to altered peptide-membrane lipid interactions. These data confirm that the N-terminal gp41 domain plays critical roles in the cytolysis and fusion underlying HIV-cell infection.