Patrick Fourquet

Mould Routine Identification in the Clinical Laboratory by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry

Background MALDI-TOF MS recently emerged as a valuable identification tool for bacteria and yeasts and revolutionized the daily clinical laboratory routine. But it has not been established for routine mould identification. This study aimed to validate a standardized procedure for MALDI-TOF MS-based mould identification in clinical laboratory. Materials and Methods First, pre-extraction and extraction procedures were optimized. With this standardized procedure, a 143 mould strains reference spectra library was built. Then, the mould isolates cultured from sequential clinical samples were prospectively subjected to this MALDI-TOF MS based-identification assay. MALDI-TOF MS-based identification was considered correct if it was concordant with the phenotypic identification; otherwise, the gold standard was DNA sequence comparison-based identification. Results The optimized procedure comprised a culture on sabouraud-gentamicin-chloramphenicol agar followed by a chemical extraction of the fungal colonies with formic acid and acetonitril. The identification was done using a reference database built with references from at least four culture replicates. For five months, 197 clinical isolates were analyzed; 20 were excluded because they were not identified at the species level. MALDI-TOF MS-based approach correctly identified 87% (154/177) of the isolates analyzed in a routine clinical laboratory activity. It failed in 12% (21/177), whose species were not represented in the reference library. MALDI-TOF MS-based identification was correct in 154 out of the remaining 156 isolates. One Beauveria bassiana was not identified and one Rhizopus oryzae was misidentified as Mucor circinelloides. Conclusions This works seminal finding is that a standardized procedure can also be used for MALDI-TOF MS-based identification of a wide array of clinically relevant mould species. It thus makes it possible to identify moulds in the routine clinical laboratory setting and opens new avenues for the development of an integrated MALDI-TOF MS-based solution for the identification of any clinically relevant microorganism.

Nanobacteria are mineralo fetuin complexes

“Nanobacteria” are nanometer-scale spherical and ovoid particles which have spurred one of the biggest controversies in modern microbiology. Their biological nature has been severely challenged by both geologists and microbiologists, with opinions ranging from considering them crystal structures to new life forms. Although the nature of these autonomously replicating particles is still under debate, their role in several calcification-related diseases has been reported. In order to gain better insights on this calciferous agent, we performed a large-scale project, including the analysis of “nanobacteria” susceptibility to physical and chemical compounds as well as the comprehensive nucleotide, biochemical, proteomic, and antigenic analysis of these particles. Our results definitively ruled out the existence of “nanobacteria” as living organisms and pointed out the paradoxical role of fetuin (an anti-mineralization protein) in the formation of these self-propagating mineral complexes which we propose to call “nanons.” The presence of fetuin within renal calculi was also evidenced, suggesting its role as a hydroxyapatite nucleating factor.

The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication.

The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the “Brucella-containing vacuole” (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC ι, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.

Assessment of various parameters to improve MALDI-TOF MS reference spectra libraries constructed for the routine identification of filamentous fungi

BackgroundThe poor reproducibility of matrix-assisted desorption/ionization time-of-flight (MALDI-TOF) spectra limits the effectiveness of the MALDI-TOF MS-based identification of filamentous fungi with highly heterogeneous phenotypes in routine clinical laboratories. This study aimed to enhance the MALDI-TOF MS-based identification of filamentous fungi by assessing several architectures of reference spectrum libraries.ResultsWe established reference spectrum libraries that included 30 filamentous fungus species with various architectures characterized by distinct combinations of the following: i) technical replicates, i.e., the number of analyzed deposits for each culture used to build a reference meta-spectrum (RMS); ii) biological replicates, i.e., the number of RMS derived from the distinct subculture of each strain; and iii) the number of distinct strains of a given species. We then compared the effectiveness of each library in the identification of 200 prospectively collected clinical isolates, including 38 species in 28 genera.Identification effectiveness was improved by increasing the number of both RMS per strain (p<10-4) and strains for a given species (p<10-4) in a multivariate analysis.ConclusionAddressing the heterogeneity of MALDI-TOF spectra derived from filamentous fungi by increasing the number of RMS obtained from distinct subcultures of strains included in the reference spectra library markedly improved the effectiveness of the MALDI-TOF MS-based identification of clinical filamentous fungi.

The First Genomic and Proteomic Characterization of a Deep-Sea Sulfate Reducer: Insights into the Piezophilic Lifestyle of Desulfovibrio piezophilus

Desulfovibrio piezophilus strain C1TLV30T is a piezophilic anaerobe that was isolated from wood falls in the Mediterranean deep-sea. D. piezophilus represents a unique model for studying the adaptation of sulfate-reducing bacteria to hydrostatic pressure. Here, we report the 3.6 Mbp genome sequence of this piezophilic bacterium. An analysis of the genome revealed the presence of seven genomic islands as well as gene clusters that are most likely linked to life at a high hydrostatic pressure. Comparative genomics and differential proteomics identified the transport of solutes and amino acids as well as amino acid metabolism as major cellular processes for the adaptation of this bacterium to hydrostatic pressure. In addition, the proteome profiles showed that the abundance of key enzymes that are involved in sulfate reduction was dependent on hydrostatic pressure. A comparative analysis of orthologs from the non-piezophilic marine bacterium D. salexigens and D. piezophilus identified aspartic acid, glutamic acid, lysine, asparagine, serine and tyrosine as the amino acids preferentially replaced by arginine, histidine, alanine and threonine in the piezophilic strain. This work reveals the adaptation strategies developed by a sulfate reducer to a deep-sea lifestyle.

Complement factor P is a ligand for the natural killer cell–activating receptor NKp46

Cross-talk between innate lymphoid cells and the alternative complement pathway protects against invasive bacterial infections. Inter-innate cooperation The different branches of the immune system work together like a well-oiled machine, but how this coordination occurs is less well understood. Narni-Mancinelli et al. have found one such mechanism—cross-talk between the alternative complement pathway and natural killer (NK) cells and innate lymphoid cells (ILCs). They report that complement factor P (CFP), a positive regulator of the alternative complement pathway, binds NKp46, which is expressed on subsets of NK cells and ILC1 and ILC3. Patients lacking CFP are more susceptible to Neisseria meningitidis infection, and in mice, this CFP protection was dependent on NKp46 and group 1 ILCs. These data suggest that ILCs and the alternative complement pathway cooperate to fight off bacterial infection. Innate lymphoid cells (ILCs) are involved in immune responses to microbes and various stressed cells, such as tumor cells. They include group 1 [such as natural killer (NK) cells and ILC1], group 2, and group 3 ILCs. Besides their capacity to respond to cytokines, ILCs detect their targets through a series of cell surface–activating receptors recognizing microbial and nonmicrobial ligands. The nature of some of these ligands remains unclear, limiting our understanding of ILC biology. We focused on NKp46, which is highly conserved in mammals and expressed by all mature NK cells and subsets of ILC1 and ILC3. We show here that NKp46 binds to a soluble plasma glycoprotein, the complement factor P (CFP; properdin), the only known positive regulator of the alternative complement pathway. Consistent with the selective predisposition of patients lacking CFP to lethal Neisseria meningitidis (Nm) infections, NKp46 and group 1 ILCs bearing this receptor were found to be required for mice to survive Nm infection. Moreover, the beneficial effects of CFP treatment for Nm infection were dependent on NKp46 and group 1 NKp46+ ILCs. Thus, group 1 NKp46+ ILCs interact with the complement pathway, via NKp46, revealing a cross-talk between two partners of innate immunity in the response to an invasive bacterial infection.

Plasmodium falciparum infection-induced changes in erythrocyte membrane proteins

Over the past decade, advances in proteomic and mass spectrometry techniques and the sequencing of the Plasmodium falciparum genome have led to an increasing number of studies regarding the parasite proteome. However, these studies have focused principally on parasite protein expression, neglecting parasite-induced variations in the host proteome. Here, we investigated P. falciparum-induced modifications of the infected red blood cell (iRBC) membrane proteome, taking into account both host and parasite proteome alterations. Furthermore, we also determined if some protein changes were associated with genotypically distinct P. falciparum strains. Comparison of host membrane proteomes between iRBCs and uninfected red blood cells using fluorescence-based proteomic approaches, such as 2D difference gel electrophoresis revealed that more than 100 protein spots were highly up-represented (fold change increase greater than five) following P. falciparum infection for both strains (i.e. RP8 and Institut Pasteur Pregnancy Associated Malaria). The majority of spots identified by mass spectrometry corresponded to Homo sapiens proteins. However, infection-induced changes in host proteins did not appear to affect molecules located at the outer surface of the plasma membrane. The under-representation of parasite proteins could not be attributed to deficient parasite protein expression. Thus, this study describes for the first time that considerable host protein modifications were detected following P. falciparum infection at the erythrocyte membrane level. Further analysis of infection-induced host protein modifications will improve our knowledge of malaria pathogenesis.

A UPR-independent infection-specific role for a BiP/GRP78 protein in the control of antimicrobial peptide expression in C. elegans epidermis.

The nematode C. elegans responds to infection by the fungus Drechmeria coniospora with a rapid increase in the expression of antimicrobial peptide genes. To investigate further the molecular basis of this innate immune response, we took a two-dimensional difference in-gel electrophoresis (2D-DIGE) approach to characterize the changes in host protein that accompany infection. We identified a total of 68 proteins from differentially represented spots and their corresponding genes. Through class testing, we identified functional categories that were enriched in our proteomic data set. One of these was “protein processing in endoplasmic reticulum,” pointing to a potential link between innate immunity and endoplasmic reticulum function. This class included HSP-3, a chaperone of the BiP/GRP78 family known to act coordinately in the endoplasmic reticulum with its paralog HSP-4 to regulate the unfolded protein response (UPR). Other studies have shown that infection of C. elegans can provoke a UPR. We observed, however, that in adult C. elegans infection with D. coniospora did not induce a UPR, and conversely, triggering a UPR did not lead to an increase in expression of the well-characterized antimicrobial peptide gene nlp-29. On the other hand, we demonstrated a specific role for hsp-3 in the regulation of nlp-29 after infection that is not shared with hsp-4. Epistasis analysis allowed us to place hsp-3 genetically between the Tribbles-like kinase gene nipi-3 and the protein kinase C delta gene tpa-1. The precise function of hsp-3 has yet to be determined, but these results uncover a hitherto unsuspected link between a BiP/GRP78 family protein and innate immune signaling.

Rickettsia conorii and R. prowazekii Proteome Analysis by 2DE‐MS: A Step toward Functional Analysis of Rickettsial Genomes

Abstract: In this work, we present a comparative two‐dimensional (2D) PAGE analysis of Rickettsia conorii and Rickettsia prowazekii. This analysis reveals protein spots that were either unique to or common to both strains, some of them being identified by matrix‐assisted laser desorption ionization‐time of flight mass spectrometry.

Altered Protein Networks and Cellular Pathways in Severe West Nile Disease in Mice

Background The recent West Nile virus (WNV) outbreaks in developed countries, including Europe and the United States, have been associated with significantly higher neuropathology incidence and mortality rate than previously documented. The changing epidemiology, the constant risk of (re-)emergence of more virulent WNV strains, and the lack of effective human antiviral therapy or vaccines makes understanding the pathogenesis of severe disease a priority. Thus, to gain insight into the pathophysiological processes in severe WNV infection, a kinetic analysis of protein expression profiles in the brain of WNV-infected mice was conducted using samples prior to and after the onset of clinical symptoms. Methodology/Principal Findings To this end, 2D-DIGE and gel-free iTRAQ labeling approaches were combined, followed by protein identification by mass spectrometry. Using these quantitative proteomic approaches, a set of 148 proteins with modified abundance was identified. The bioinformatics analysis (Ingenuity Pathway Analysis) of each protein dataset originating from the different time-point comparisons revealed that four major functions were altered during the course of WNV-infection in mouse brain tissue: i) modification of cytoskeleton maintenance associated with virus circulation; ii) deregulation of the protein ubiquitination pathway; iii) modulation of the inflammatory response; and iv) alteration of neurological development and neuronal cell death. The differential regulation of selected host protein candidates as being representative of these biological processes were validated by western blotting using an original fluorescence-based method. Conclusion/Significance This study provides novel insights into the in vivo kinetic host reactions against WNV infection and the pathophysiologic processes involved, according to clinical symptoms. This work offers useful clues for anti-viral research and further evaluation of early biomarkers for the diagnosis and prevention of severe neurological disease caused by WNV.