Membrane Chaperone SecDF Plays a Role in the Secretion of Listeria monocytogenes Major Virulence Factors

Listeria monocytogenes is a Gram-positive human intracellular pathogen that infects diverse mammalian cells. Upon invasion, L. monocytogenes secretes multiple virulence factors that target host cellular processes and promote infection. It has been presumed, but was not empirically established, that the Sec translocation system is the primary mediator of this secretion. Here, we validate an important role for SecDF, a component of the Sec system, in the secretion of several critical L. monocytogenes virulence factors. A ΔsecDF mutant is demonstrated to exhibit impaired membrane translocation of listeriolysin O (LLO), PlcA, PlcB, and ActA, factors that mediate L. monocytogenes phagosomal escape and spread from cell to cell. This impaired translocation was monitored by accumulation of the factors on the bacterial membrane and by reduced activity upon secretion. This defect in secretion is shown to be associated with a severe intracellular growth defect of the ΔsecDF mutant in macrophages and a less virulent phenotype in mice, despite normal growth in laboratory medium. We further show that SecDF is upregulated when the bacteria reside in macrophage phagosomes and that it is necessary for efficient phagosomal escape. Taken together, these data support the premise that SecDF plays a role as a chaperone that facilitates the translocation of L. monocytogenes virulence factors during infection.     

The dynamics of the CHO host cell protein profile during clarification and protein A capture in a platform antibody purification process

Recombinant protein products such as monoclonal antibodies (mAbs) for use in the clinic must be clear of host cell impurities such as host cell protein (HCP), DNA/RNA, and high molecular weight immunogenic aggregates. Despite the need to remove and monitor HCPs, the nature, and fate of these during downstream processing (DSP) remains poorly characterized. We have applied a proteomic approach to investigate the dynamics and fate of HCPs in the supernatant of a mAb producing cell line during early DSP including centrifugation, depth filtration, and protein A capture chromatography. The primary clarification technique selected was shown to influence the HCP profile that entered subsequent downstream steps. MabSelect protein A chromatography removed the majority of contaminating proteins, however using 2D‐PAGE we could visualize not only the antibody species in the eluate (heavy and light chain) but also contaminant HCPs. These data showed that the choice of secondary clarification impacts upon the HCP profile post‐protein A chromatography as differences arose in both the presence and abundance of specific HCPs when depth filters were compared. A number of intracellularly located HCPs were identified in protein A elution fractions from a Null cell line culture supernatant including the chaperone Bip/GRP78, heat shock proteins, and the enzyme enolase. We demonstrate that the selection of early DSP steps influences the resulting HCP profile and that 2D‐PAGE can be used for monitoring and identification of HCPs post‐protein A chromatography. This approach could be used to screen cell lines or hosts to select those with reduced HCP profiles, or to identify HCPs that are problematic and difficult to remove so that cell‐engineering approaches can be applied to reduced, or eliminate, such HCPs. Biotechnol. Bioeng. 2013; 110: 240–251. © 2012 Wiley Periodicals, Inc.     

Follow-up of children born by ICSI

The debate concerning the health of children conceived by artificial reproduction technology (ART) continues. Among these techniques, intracytoplasmic sperm injection (ICSI) is the subject of most attention. Indeed, several studies have concentrated on the evaluation of risks associated with ICSI. The publication of a few recent articles on the subject is providing an opportunity to reconsider the situation. Generally, women conceiving via ART are older, more often primipar and present increased rates of uterine pathologies compared to women conceiving naturally. Furthermore, ART pregnancies are sources of anxiety resulting in a significant increase rates of caesarean section. ART children present an increased risk of low birth rate often linked to multiple pregnancy, but this is also true for singleton pregnancy. Major studies have not revealed a significantly increased rate of malformations in ICSI children. However, sporadic observations of errors in genomic imprinting or of rare tumors in children conceived by ICSI point to a need for increased vigilance of ICSI practices. Finally, the mental development, the family and social life of ICSI children appears similar to children conceived naturally.     

Correlation between HSP90 induction kinetics in murine leukemia cells and the amount of cisplatin over a wide range of cytostatic concentrations

The induction of HSP90 in murine erythroleukemia cells, clone F4 N, by cisplatin (DDP) was examined using indirect immunofluorescence and avidin-biotin technique, and compared with cisplatin cytotoxicity. A reverse dependence of HSP90 induction time was found on a wide range of cisplatin concentrations (0.5-10 microM), which proved to be cytostatic up to 48 h of continuous treatment. Thus, the observed induction pattern of HSP90 in F4 N cells strictly correlated with their high tolerance toward DDP. This indicates that HSP90 might be responsible, at least in part, for cisplatin resistance of F4 N cells.     

Evaluation of changes in the element content and biomass of invaded with Meloidogyne arenaria Tiny Tim tomato plants under NH4VO3 treatment

The parasite-host system Meloidogyne arenaria--Tiny Tim tomato plants has been studied in order to investigate the influence of the process of invasion on the chemical composition and biomass of plants. The concentrations of seven chemical elements Cu, Zn, Mg, K, Na, Mn and Fe have been determined using AAS in controls and invaded plants, and their changes have been evaluated under treatment with NH4VO3 in three different concentrations 0.01, 0.1 and 0.13 mg/100 ml H2O. The process of treatment with NH4VO3 disbalances significantly the trace element content of plants. The lowest concentration (0.01 mg NH4VO3) causes bigger changes in the concentrations of Mn, Fe and Na in non-invaded plants. The highest concentration (0.13 mg NH4VO3) balances the content of the elements back to their levels in the control plants for the elements Zn, Fe and Na. The pure effect of the process of invasion with Meloidogyne arenaria on the biomass (leaves, stems, roots and total biomass) of Tiny Tim plants is expressed in a significant increasing, mainly due to the development of the parasites. After treatment with different concentrations of NH4VO3 the decreasing in the biomass of leaves, stems and roots is observed which reflects on the total biomass of plants. The concentration of NH4VO3 eliminates the unfavourable changes not only in the chemical content of plants but also in their biomass. It could be taken into consideration as an alternative method used instead of treatment with nematocides.     

Isotopic analysis of cyanobacterial nitrogen fixation associated with subarctic lichen and bryophyte species

Dinitrogen fixation by cyanobacteria is of particular importance for the nutrient economy of cold biomes, constituting the main pathway for new N supplies to tundra ecosystems. It is prevalent in cyanobacterial colonies on bryophytes and in obligate associations within cyanolichens. Recent studies, applying interspecific variation in plant functional traits to upscale species effects on ecosystems, have all but neglected cryptogams and their association with cyanobacteria. Here we looked for species-specific patterns that determine cryptogam-mediated rates of N₂ fixation in the Subarctic. We hypothesised a contrast in N₂ fixation rates (1) between the structurally and physiologically different lichens and bryophytes, and (2) within bryophytes based on their respective plant functional types. Throughout the survey we supplied ¹⁵N-labelled N₂ gas to quantify fixation rates for monospecific moss, liverwort and lichen turfs. We sampled fifteen species in a design that captures spatial and temporal variations during the growing season in Abisko region, Sweden. We measured N₂ fixation potential of each turf in a common environment and in its field sampling site, in order to embrace both comparativeness and realism. Cyanolichens and bryophytes differed significantly in their cyanobacterial N₂ fixation capacity, which was not driven by microhabitat characteristics, but rather by morphology and physiology. Cyanolichens were much more prominent fixers than bryophytes per unit dry weight, but not per unit area due to their low specific thallus weight. Mosses did not exhibit consistent differences in N₂ fixation rates across species and functional types. Liverworts did not fix detectable amounts of N₂. Despite the very high rates of N₂ fixation associated with cyanolichens, large cover of mosses per unit area at the landscape scale compensates for their lower fixation rates, thereby probably making them the primary regional atmospheric nitrogen sink.     

Tyrosyl-DNA phosphodiesterase 1 initiates repair of apurinic/apyrimidinic sites

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of the phosphodiester linkage between the DNA 3' phosphate and a tyrosine residue as well as a variety of other DNA 3' damaged termini. Recently we have shown that Tdp1 can liberate the 3' DNA phosphate termini from apurinic/apyrimidinic (AP) sites. Here, we found that Tdp1 is more active in the cleavage of the AP sites inside bubble-DNA structure in comparison to ssDNA containing AP site. Furthermore, Tdp1 hydrolyzes AP sites opposite to bulky fluorescein adduct faster than AP sites located in dsDNA. Whilst the Tdp1 H493R (SCAN1) and H263A mutants retain the ability to bind an AP site-containing DNA, both mutants do not reveal endonuclease activity, further suggesting the specificity of the AP cleavage activity. We suggest that this Tdp1 activity can contribute to the repair of AP sites particularly in DNA structures containing ssDNA region or AP sites in the context of clustered DNA lesions.     

Human DNA polymerase λ catalyzes lesion bypass across benzo[a]pyrene-derived DNA adduct during base excision repair

The combined action of oxidative stress and genotoxic polycyclic aromatic hydrocarbons derivatives can lead to cluster-type DNA damage that includes both a modified nucleotide and a bulky lesion. As an example, we investigated the possibility of repair of an AP site located opposite a minor groove-positioned (+)-trans-BPDE-dG or a base-displaced intercalated (+)-cis-BPDE-dG adduct (BP lesion) by a BER system. Oligonucleotides with single uracil residues in certain positions were annealed with complementary oligonucleotides bearing either a cis- or trans-BP adduct. The resulting DNA duplexes contained dU either directly opposite the modified dG or shifted to adjacent 5' (-1) or 3' (+1) positions. Digestion with uracil DNA glycosylase was utilized to generate AP sites which were then hydrolyzed by APE1, and the resulting gaps were processed by DNA polymerase β (Polβ) or λ (Polλ). The AP sites in position -1 can be repaired effectively using APE1 and Polβ or Polλ. The AP sites opposite the BP lesions can be repaired using Polλ in the case of cis- but not the trans-isomeric adduct. The AP sites in position +1 are the most difficult to repair. In the case of the AP site located in position +1, the activity of Polλ does not depend on the stereoisomeric properties of the BP lesions and dCTP is the preferred inserted substrate in both cases. The capability of Polλ to introduce the correct dNTP opposite the cis-BP-dG adduct in gap filling reactions suggests that this polymerase may play a specialized role in the process of repair of these kinds of lesions.