Proteomics of Protein Secretion by Bacillus subtilis: Separating the “Secrets” of the Secretome

Secretory proteins perform a variety of important “remote-control” functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which ~90 extracellular proteins were identified. Analysis of these proteins disclosed various “secrets of the secretome,” such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only ~50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance.     

Rapid Species Diagnosis for Invasive Candidiasis Using Mass Spectrometry

Background

Matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF-MS) allows the identification of most bacteria and an increasing number of fungi. The potential for the highest clinical benefit of such methods would be in severe acute infections that require prompt treatment adapted to the infecting species. Our objective was to determine whether yeasts could be identified directly from a positive blood culture, avoiding the 1–3 days subculture step currently required before any therapeutic adjustments can be made.

Methodology/Principal Findings

Using human blood spiked with Candida albicans to simulate blood cultures, we optimized protocols to obtain MALDI TOF-MS fingerprints where signals from blood proteins are reduced. Simulated cultures elaborated using a set of 12 strains belonging to 6 different species were then tested. Quantifiable spectral differences in the 5000–7400 Da mass range allowed to discriminate between these species and to build a reference database. The validation of the method and the statistical approach to spectral analysis were conducted using individual simulated blood cultures of 36 additional strains (six for each species). Correct identification of the species of these strains was obtained.

Conclusions/Significance

Direct MALDI TOF-MS analysis of aliquots from positive blood cultures allowed rapid and accurate identification of the main Candida species, thus obviating the need for sub-culturing on specific media. Subsequent to this proof-of-principle demonstration, the method can be extended to other clinically relevant yeast species, and applied to an adequate number of clinical samples in order to establish its potential to improve antimicrobial management of patients with fungemia.     

Effect of Culture Conditions on Ergosterol as an Indicator of Biomass in the Aquatic Hyphomycetes

Ergosterol is a membrane component specific to fungi that can be used to estimate fungal biomass using appropriate factors of conversion. Our objectives were to determine the limits of use of ergosterol content as a measure of biomass for aquatic hyphomycetes, and to evaluate a previously established ergosterol-to-biomass conversion factor. We varied inoculum quality, growth medium, and degree of shaking of four aquatic hyphomycete species. In cultures inoculated with homogenized mycelium, we found a significant effect of shaking condition and culture age on ergosterol content. In liquid cultures with defined medium, ergosterol content reached 10 to 11 μg/mg of mycelium (dry mass) and varied by factors of 2.2 during exponential growth and 1.3 during stationary phase. The increase in ergosterol content during exponential phase could be attributed, at least in part, to rapid depletion of glucose. Oxygen availability to internal hyphae within the mycelial mass is also responsible for the differences found between culture conditions. Ergosterol concentration ranged from 0.8 to 1.6 μg/mg in static cultures inoculated with agar plugs. Ergosterol content varied by a factor of 4 in two media of different richnesses. For different combinations of these parameters, strong (r² = 0.83 to 0.98) and highly significant (P 0.001) linear relationships between ergosterol and mycelial dry mass (up to 110 mg) were observed. Overall, the ergosterol content varied by a factor of 14 (0.8 to 11 mg/g). These results suggest that care must be taken when the ergosterol content is used to compare data generated in different field environments.     

A single nucleotide change in the 5' noncoding region of Sindbis virus confers neurovirulence in rats

Two pairs of Sindbis virus (SV) variants that differ in their neuroinvasive and neurovirulent traits in mice have been isolated. Recently, we mapped the genetic determinants responsible for neuroinvasiveness in weanling mice. Here, we extend this study to newborn and adult rats and to rat neuronal cultures. Remarkably, certain aspects of the pathogenesis of these strains in rats were found to be quite distinct from the mouse model. Suckling rats were susceptible to all four isolates, and replication in the brain was observed after both intraperitoneal and intracranial (i.c.) inoculation. None of the isolates was neuroinvasive in adult rats, although all replicated after i.c. inoculation. For the isolate pair that was highly neurovirulent in mice, SVN and SVNI, only SVNI caused death after i.c. inoculation of adult rats. Similarly, only SVNI was cytotoxic for primary cultures of mature neurons. The genetic determinants responsible for the pathogenic properties of SVNI were mapped to the E2 glycoprotein and the 5' noncoding region (5'NCR). Substitution of two amino acids in SVN E2 with the corresponding residues of SVNI (Met-190 and Lys-260) led to paralysis in 3- and 5-week-old rats. More dramatically, a single substitution in the 5'NCR of SVN (G at position 8) transformed the virus into a lethal pathogen for 3-week-old rats like SVNI. In 5-week-old rats, however, this recombinant was attenuated relative to SVNI by 2 orders of magnitude. Combination of the E2 and 5'NCR determinants resulted in a recombinant with virulence properties indistinguishable from those of SVNI. These data indicate that the 5'NCR and E2 play an instrumental role in determining the age-dependent pathogenic properties of SV in rats.     

Retention and Degradation of N-Glycoproteins in the Rough Endoplasmic Reticulum

Recent studies have shown that newly synthesized proteins and glycoproteins are submitted to a quality control mechanism in the rough endoplasmic reticulum (ER). In this report we present two models: One model will illustrate a transient retention in rough ER leading to a further degradation of glycoproteins in the cytosol, (soluble alkaline phosphatase expressed in Man-P-Dol deficient CHO cells lines). The second model will illustrate a strict retention of glycoproteins in rough ER without degradation nor recycling through the Golgi (E1, E2 glycoproteins of Hepatitis C virus in stably transfected UHCV-11.4 cells and in infected Hep G2 cells).In both cases, oligomannoside structures are markers of these phenomena, either as free soluble released oligomannosides in the case of degradation, or as N-linked oligomannosides for strict retention in rough ER.     

The<Emphasis Type="Italic">atspo11-1</Emphasis> mutation rescues <Emphasis Type="Italic">atxrcc3</Emphasis> meiotic chromosome fragmentation

Homologous recombination events occurring during meiotic prophase I ensure the proper segregation of homologous chromosomes at the first meiotic division. These events are initiated by programmed double-strand breaks produced by the Spo11 protein and repair of such breaks by homologous recombination requires a strand exchange activity provided by the Rad51 protein. We have recently reported that the absence of AtXrcc3, an ArabidopsisRad51 paralogue, leads to extensive chromosome fragmentation during meiosis, first visible in diplotene of meiotic prophase I. The present study clearly shows that this fragmentation results from un- or mis-repaired AtSpo11-1 induced double-strand breaks and is thus due to a specific defect in the meiotic recombination process.     

Influence of the whole-cell patch-clamp configuration on electrophysiological properties of the voltage-dependent sodium current expressed in MDA-MB-231 breast cancer cells

Voltage-gated ionic channels are known to be involved in oncogenesis. However, only a few studies describe the functional characteristics of these channels or the mechanisms by which they are involved in the proliferation and invasive processes. Breast cancer cells proliferate and migrate under the constant activation of growth factors, hormones, extracellular matrix interactions, etc. It would thus not be surprising if the activity of the ionic channels was modulated by intracellular regulation pathways such as kinases or phosphatases, which in turn can affect oncogenic properties. In the present study, we investigated some of the electrophysiological properties of the fast inward sodium current found in the breast cancer cell line MDA-MB-231 with two configurations of the patch-clamp technique. With perforated patch, a configuration which allows to keep the cytoplam intact, the mean current amplitude was lower, the relative conductance–voltage relationship was shifted to more positive potentials and the recovery from inactivation was accelerated when compared to ruptured patch, where the cytoplasm is dialysed by the intrapipette solution. There was no difference in availability–voltage (pseudo-steady-state inactivation) relationship and in time to peak of the current. These results suggest that regulation mechanisms, possibly involving kinases or phosphatases, are switched off when the cytoplasm is diluted. We propose that such a regulation can modulate the functioning of the channels even in the absence of membrane voltage changes, which in turn can affect oncogenic properties. This finding is of importance when evaluating the physiopathological role of ionic channel in cancer development.Abbreviations DMEM Dulbeccos modified Eagles medium - FBS fetal bovine serum - PP perforated patch - RP ruptured patch Presented at the Biophysical Society Meeting on Ion Channels—from structure to disease held in May 2003, Rennes, France     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides derived from 4-isothiocyanato-benzolamide

A series of sulfonamides incorporating 4-thioureido-benzolamide moieties have been prepared from aminobenzolamide and thiophosgene followed by the reaction of the thiocyanato intermediate with aliphatic/aromatic amines or hydrazines. The new derivatives have been investigated as inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), and more precisely of the cytosolic isozymes hCA I and II, as well as the tumor-associated isozyme hCA IX (all of human origin). The new compounds showed excellent inhibitory properties against all three isozymes with inhibition constants in the range of 0.6-62 nM against hCA I, 0.5-1.7 nM against hCA II and 3.2-23 nM against hCA IX, respectively. These derivatives are interesting candidates for the development of novel therapies targeting hypoxic tumors.