Relating the physical properties of Pseudomonas aeruginosa lipopolysaccharides to virulence by atomic force microscopy

Lipopolysaccharides (LPS) are an important class of macromolecules that are components of the outer membrane of Gram-negative bacteria such as Pseudomonas aeruginosa. P. aeruginosa contains two different sugar chains, the homopolymer common antigen (A band) and the heteropolymer O antigen (B band), which impart serospecificity. The characteristics of LPS are generally assessed after isolation rather than in the context of whole bacteria. Here we used atomic force microscopy (AFM) to probe the physical properties of the LPS of P. aeruginosa strain PA103 (serogroup O11) in situ. This strain contains a mixture of long and very long polymers of O antigen, regulated by two different genes. For this analysis, we studied the wild-type strain and four mutants, ΔWzz1 (producing only very long LPS), ΔWzz2 (producing only long LPS), DΔM (with both the wzz1 and wzz2 genes deleted), and Wzy::GM (producing an LPS core oligosaccharide plus one unit of O antigen). Forces of adhesion between the LPS on these strains and the silicon nitride AFM tip were measured, and the Alexander and de Gennes model of steric repulsion between a flat surface and a polymer brush was used to calculate the LPS layer thickness (which we refer to as length), compressibility, and spacing between the individual molecules. LPS chains were longest for the wild-type strain and ΔWzz1, at 170.6 and 212.4 nm, respectively, and these values were not statistically significantly different from one another. Wzy::GM and DΔM have reduced LPS lengths, at 34.6 and 37.7 nm, respectively. Adhesion forces were not correlated with LPS length, but a relationship between adhesion force and bacterial pathogenicity was found in a mouse acute pneumonia model of infection. The adhesion forces with the AFM probe were lower for strains with LPS mutations, suggesting that the wild-type strain is optimized for maximal adhesion. Our research contributes to further understanding of the role of LPS in the adhesion and virulence of P. aeruginosa.     

Sepsis induces early alterations in innate immunity that impact mortality to secondary infection

Sepsis, the systemic inflammatory response to microbial infection, induces changes in both innate and adaptive immunity that presumably lead to increased susceptibility to secondary infections, multiorgan failure, and death. Using a model of murine polymicrobial sepsis whose severity approximates human sepsis, we examined outcomes and defined requirements for survival after secondary Pseudomonas aeruginosa pneumonia or disseminated Listeria monocytogenes infection. We demonstrate that early after sepsis neutrophil numbers and function are decreased, whereas monocyte recruitment through the CCR2/MCP-1 pathway and function are enhanced. Consequently, lethality to Pseudomonas pneumonia is increased early but not late after induction of sepsis. In contrast, lethality to listeriosis, whose eradication is dependent upon monocyte/macrophage phagocytosis, is actually decreased both early and late after sepsis. Adaptive immunity plays little role in these secondary infectious responses. This study demonstrates that sepsis promotes selective early, impaired innate immune responses, primarily in neutrophils, that lead to a pathogen-specific, increased susceptibility to secondary infections.     

Broad antibody mediated cross-neutralization and preclinical immunogenicity of new codon-optimized HIV-1 clade CRF02_AG and G primary isolates

Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary "street strain" isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA) to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G). Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G) and consensus tat (CRF02_AG and G) antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G.     

Russian wheat aphid (Hemiptera: Aphididae) reproduction and development on five noncultivated grass hosts

The Russian wheat aphid, Diuraphis noxia (Kurdjumov), is a small grains pest of worldwide economic importance. The Russian wheat aphid is polyphagous and may encounter differential selective pressures from noncultivated grass hosts. Aphid biotypic diversity can disrupt the progress of plant breeding programs, leading to a decreased ability to manage this pest. The goal of this research was to quantify Russian wheat aphid biotype 2 (RWA2) reproductive and development rates on five common noncultivated grass hosts to gain information about host quality, potential refuges, and sources of selection pressure. First, RWA2 reproduction was compared on crested wheatgrass (Agropyron cristatum, (L.) Gaertn.), intermediate wheatgrass (Elytrigia intermedia, (Host) Nevski), slender wheatgrass (Elymus trachycaulus, (Link) Gould ex Shinners), western wheatgrass (Pascopyrum smithi, (Rydb.) A. L?ve), and foxtail barley (Hordeum jubatum, (L.) Tesky) at 18–24°C. Second, RWA2 reproduction was compared on intermediate and crested wheatgrass at three temperature regimes 13–18°C, 18–24°C, and 24–29°C. At moderate temperatures (18–24°C), the intrinsic rate of increase values for all five hosts ranged from 0.141 to 0.199, indicating the possibility for strong population sources on all tested hosts. Aphids feeding on crested and intermediate wheatgrass at the 13–18°C temperature had lower fecundity, less nymph production days, longer generational times, and lower intrinsic rate of increase than aphids feeding at the 18–24°C temperature regime. Aphids feeding at 24–29°C did not survive long enough to reproduce. The positive intrinsic rates of increase in Russian wheat aphid on the wheatgrasses suggest that these grasses can support aphid populations at moderate to low temperatures.     

BMP4 was associated with NSCL/P in an Asian population

Background

The Bone Morphogenetic Protein 4 gene (BMP4) is located in chromosome 14q22-q23 which has shown evidence of linkage for isolated nonsyndromic cleft lip with or without cleft palate (NSCL/P) in a genome wide linkage analysis of human multiplex families. BMP4 has been shown to play crucial roles in lip and palatal development in animal models. Several candidate gene association analyses also supported its potential risk for NSCL/P, however, results across these association studies have been inconsistent. The aim of the current study was to test for possible association between markers in and around the BMP4 gene and NSCL/P in Asian and Maryland trios.

Methodology/Principal Findings

Family Based Association Test was used to test for deviation from Mendelian assortment for 12 SNPs in and around BMP4. Nominal significant evidence of linkage and association was seen for three SNPs (rs10130587, rs2738265 and rs2761887) in 221 Asian trios and for one SNP (rs762642) in 76 Maryland trios. Statistical significance still held for rs10130587 after Bonferroni correction (corrected p = 0.019) among the Asian group. Estimated odds ratio for carrying the apparent high risk allele at this SNP was 1.61 (95%CI = 1.20, 2.18).

Conclusions

Our results provided further evidence of association between BMP4 and NSCL/P.     

Evaluation of Dynamic [(18)F]-FDG-PET Imaging for the Detection of Acute Post-Surgical Bone Infection

Diagnosing bone infection in its acute early stage is of utmost clinical importance as the failure to do so results in a therapeutically recalcitrant chronic infection that can only be resolved with extensive surgical intervention, the end result often being a structurally unstable defect requiring reconstructive procedures. [(18)F]-FDG-PET has been extensively investigated for this purpose, but the results have been mixed in that, while highly sensitive, its specificity with respect to distinguishing between acute infection and sterile inflammatory processes, including normal recuperative post-surgical healing, is limited. This study investigated the possibility that alternative means of acquiring and analyzing FDG-PET data could be used to overcome this lack of specificity without an unacceptable loss of sensitivity. This was done in the context of an experimental rabbit model of post-surgical osteomyelitis with the objective of distinguishing between acute infection and sterile post-surgical inflammation. Imaging was done 7 and 14 days after surgery with continuous data acquisition for a 90-minute period after administration of tracer. Results were evaluated based on both single and dual time point data analysis. The results suggest that the diagnostic utility of FDG-PET is likely limited to well-defined clinical circumstances. We conclude that, in the complicated clinical context of acute post-surgical or post-traumatic infection, the diagnostic utility accuracy of FDG-PET is severely limited based on its focus on the increased glucose utilization that is generally characteristic of inflammatory processes.     

Sequences in Glycoprotein gp41, the CD4 Binding Site,and the V2 Domain Regulate Sensitivity and Resistance of HIV-1 to Broadly Neutralizing Antibodies

The swarm of quasispecies that evolves in each HIV-1-infected individual represents a source of closely related Env protein variants that can be used to explore various aspects of HIV-1 biology. In this study, we made use of these variants to identify mutations that confer sensitivity and resistance to the broadly neutralizing antibodies found in the sera of selected HIV-1-infected individuals. For these studies, libraries of Env proteins were cloned from infected subjects and screened for infectivity and neutralization sensitivity. The nucleotide sequences of the Env proteins were then compared for pairs of neutralization-sensitive and -resistant viruses. In vitro mutagenesis was used to identify the specific amino acids responsible for the neutralization phenotype. All of the mutations altering neutralization sensitivity/resistance appeared to induce conformational changes that simultaneously enhanced the exposure of two or more epitopes located in different regions of gp160. These mutations appeared to occur at unique positions required to maintain the quaternary structure of the gp160 trimer, as well as conformational masking of epitopes targeted by neutralizing antibodies. Our results show that sequences in gp41, the CD4 binding site, and the V2 domain all have the ability to act as global regulators of neutralization sensitivity. Our results also suggest that neutralization assays designed to support the development of vaccines and therapeutics targeting the HIV-1 Env protein should consider virus variation within individuals as well as virus variation between individuals.