Structural insights into loss of function of a pore forming toxin and its role in pneumococcal adaptation to an intracellular lifestyle

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-π interaction, governing formation of the transmembrane β-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche.     

EGFR-RAS-MAPK signaling is confined to the plasma membrane and associated endorecycling protrusions

The subcellular localization of RAS GTPases defines the operational compartment of the EGFR-ERK1/2 signaling pathway within cells. Hence, we used live-cell imaging to demonstrate that endogenous KRAS and NRAS tagged with mNeonGreen are predominantly localized to the plasma membrane. NRAS was also present in the Golgi apparatus and a tubular, plasma-membrane derived endorecycling compartment, enriched in recycling endosome markers (TERC). In EGF-stimulated cells, there was essentially no colocalization of either mNeonGreen-KRAS or mNeonGreen-NRAS with endosomal EGFR, which, by contrast, remained associated with endogenous Grb2-mNeonGreen, a receptor adaptor upstream of RAS. ERK1/2 activity was diminished by blocking cell surface EGFR with cetuximab, even after most ligand-bound, Grb2-associated EGFRs were internalized. Endogenous mCherry-tagged RAF1, an effector of RAS, was recruited to the plasma membrane, with subsequent accumulation in mNG-NRAS–containing TERCs. We propose that a small pool of surface EGFRs sustain signaling within the RAS-ERK1/2 pathway and that RAS activation persists in TERCs, whereas endosomal EGFR does not significantly contribute to ERK1/2 activity.     

Manganese increases high mannose glycoform on monoclonal antibody expressed in CHO when glucose is absent or limiting: Implications for use of alternate sugars

Alternate sugars such as galactose and fructose are metabolized at a slower rate than glucose and result in lower accumulation of lactate. While low lactate accumulation is desirable, we report that complete substitution of glucose with these sugars results in an increase in M5 high mannose glycans. Surprisingly, this increase is much higher when the culture is supplemented with manganese: for example, when cells are cultured with galactose, M5 high mannose glycan content increased from 5% at 1 nM Mn²⁺ in the basal medium to 32% with 16 µM Mn²⁺ supplementation. When galactose is supplemented with glucose maintained at low concentrations, a small reduction in high mannose glycans is seen. In control cultures with glucose, the high mannose content was however <2% in this range of Mn²⁺ concentration. By varying Mn²⁺ and glucose supplementation levels, with or without galactose, we systematically demonstrate that Mn²⁺ concentration and glucose availability, together, significantly affect the high mannose glycan content. To our knowledge, this is the first report that shows that the effect of Mn²⁺ on high mannose glycan content depends on glucose availability. At each Mn²⁺ supplementation level evaluated, galactosylation percentages were highest for cultures where galactose was supplemented with glucose at non‐limiting concentration. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:460–467, 2015     

Using GFP video to track 3D movement and conditional gene expression in free-moving flies

Background

In vivo imaging and quantification of fluorescent reporter molecules is increasingly useful in biomedical research. For example, tracking animal movement in 3D with simultaneous quantification of fluorescent transgenic reporters allows for correlations between behavior, aging and gene expression. However implementation has been hindered in the past by the complexity of operating the systems.

Results

We report significant technical improvements and user-friendly software (called FluoreScore) that enables tracking of 3D movement and the dynamics of gene expression in adult Drosophila, using two cameras and recorded GFP videos. Expression of a transgenic construct encoding eGFP was induced in free-moving adult flies using the Gene-Switch system and RU486 drug feeding. The time course of induction of eGFP expression was readily quantified from internal tissues including central nervous tissue.

Conclusions

FluoreScore should facilitate a variety of future studies involving quantification of movement behaviors and fluorescent molecules in free-moving animals.     

Association of small Rho GTPases and actin ring formation in epithelial cells during the invasion by Candida albicans

Invasion of epithelial cells is a major virulence determinant of Candida albicans ; however, the molecular events that occur during invasion are not discerned. This study is aimed to elucidate the role of the host's actin remodeling and involvement of small GTPases during invasion. Actin filaments formed a rigid ring-like structure in the rabbit corneal epithelial cell line SIRC after C. albicans invasion. During invasion, an increase in the mRNA content of Cdc42, Rac1 and RhoA GTPase was observed in SIRC cells. Immunochemical staining and expression of chimeric green fluorescent protein (GFP)-GTPases showed that all three GTPases colocalize at invasion and actin polymerization sites. This colocalization was not seen in SIRC cells expressing a GFP-tagged dominant-negative mutant of GTPases. Inhibition of invasion was observed in SIRC cells expressing dominant-negative mutants of Rac1 and RhoA GTPases. Involvement of zonula occludens-1 (ZO-1) was observed in the process of actin-mediated endocytosis of C. albicans . Actin, GTPases and ZO-1 were colocalized in epithelial cells during uptake of polymethylmethacrylate beads coated with spent medium from a C. albicans culture. The results indicate that host actin remodeling and recruitment of small GTPases occur during invasion and molecules that are shed or secreted by C. albicans are probably responsible for cytoskeletal reorganization.     

Carrier screening for beta-thalassemia during pregnancy in India: a 7-year evaluation

AIM: Premarital screening for beta-thalassemia is not widely acceptable in India; hence, we evaluated the effectiveness of antenatal screening and counseling over 7 years. METHODS: 61,935 pregnant women were screened using the single-tube osmotic fragility test during their first antenatal visit. Individuals who were positive were investigated further for diagnosis of beta-thalassemia and other abnormal hemoglobins. Spouses of carrier women were tested whenever available. Couples at risk were given the option of prenatal diagnosis. RESULTS: Only 19% of the women registered at the antenatal clinic in the first trimester of pregnancy, and 14% of the women were positive per the osmotic fragility test; 1020 beta-thalassemia heterozygotes and 213 women with other hemoglobinopathies were identified, majority being in the second and third trimesters. Seven hundred and thirteen (69%) of their husbands could be tested, and 37 couples at risk were identified. Only 15 couples had a prenatal diagnosis done. Four couples with affected fetuses opted for termination of pregnancy. The remaining couples either did not respond after counseling or the pregnancies were advanced for prenatal intervention. CONCLUSION: This first large study shows that antenatal screening is acceptable in India; however, awareness generation is still a primary requisite to make women register early at antenatal clinics and bring their spouses for screening when required.     

Red cell genetic abnormalities, beta-globin gene haplotypes, and APOB polymorphism in the Great Andamanese, a primitive Negrito tribe of Andaman and Nicobar Islands, India.

The Great Andamanese are a primitive Negrito tribe of the Andaman and Nicobar Islands, India, with a total population of 37. We studied 29 individuals from eight families from this population for abnormal hemoglobins, G6PD deficiency, DNA haplotypes, and apolipoprotein B (APOB, gene) polymorphism. Hb E was detected in five individuals, the prevalence of Hb E heterozygotes being 14.3%. One individual had beta-thalassemia trait. One female was G6PD deficient and showed the G6PD Orissa mutation. Haplotype analysis of the beta-globin gene cluster showed that the betaE chromosomes were linked to two haplotypes (- - - - - + + and + + - + + + +) representing the framework 1 gene, whereas the betaA chromosomes showed eight different haplotypic patterns corresponding to framework 1 and 3 genes. APOB polymorphism analysis showed that the 631-base-pair (bp) allele was the predominant one with a high homozygosity rate, which could be due to the higher rate of inbreeding in this isolated group. The presence of Hb E and our findings on haplotype analysis supports the hypothesis that the Great Andamanese are reasonably believed to be the surviving representatives of the Negrito race that once flourished in the entire Southeast Asian region in ancient times.