Global Proteome Analysis of Leptospira interrogans

Comparative global proteome analyses were performed on Leptospira interrogans serovar Copenhageni grown under conventional in vitro conditions and those mimicking in vivo conditions (iron limitation and serum presence). Proteomic analyses were conducted using iTRAQ and LC-ESI-tandem mass spectrometry complemented with two-dimensional gel electrophoresis and MALDI-TOF mass spec-trometry. A total of 563 proteins were identified in this study. Altered expression of 65 proteins, including upregulation of the L. interrogans virulence factor Loa22 and 5 novel proteins with homology to virulence factors found in other pathogens, was observed between the comparative conditions. Immunoblot analyses confirmed upregulation of 5 of the known or putative virulence factors in L. interrogans exposed to the in vivo-like environmental conditions. Further, ELISA analyses using serum from patients with leptospirosis and immunofluorescence studies performed on liver sections derived from L. interrogans-infected hamsters verified expression of all but one of the identified proteins during infection. These studies, which represent the first documented comparative global proteome analysis of Leptospira, demonstrated proteome alterations under conditions that mimic in vivo infection and allowed for the identification of novel putative L. interrogans virulence factors

Immunodominant Antigens of Leishmania chagasi Associated with Protection against Human Visceral Leishmaniasis

One of the most striking features of infection by Leishmania chagasi is that infection leads to a spectrum of clinical outcomes ranging from asymptomatic infection to active disease. The existence of asymptomatic infected people has served as an incentive to believe that an effective vaccine is possible, but unfortunately no successful immunological characterization of such cases was obtained. Patients recovered from visceral leishmaniasis show a similar immunological profile to asymptomatic infected individuals and both exhibit a strong cell-mediated immune response against Leishmania antigens and are resistant to disease. Since the past decade several approaches were undertaken to try to shed light on the immunological profile associated with such “resistance” to infections, notwithstanding antigenic recognition profile associated to resistance to infection was not successfully explored. In the present manuscript we describe a specific IgG recognizing pattern associated with resistant individuals (asymptomatic infected people and recovery patients to visceral leishmaniasis). These data highlight the possibility of using specific proteins in serological tests for the identification of asymptomatic infected individuals

Modulating Activity of Vancomycin and Daptomycin on the Expression of Autolysis Cell-Wall Turnover and Membrane Charge Genes in hVISA and VISA Strains

Glycopeptides are still the gold standard to treat MRSA (Methicillin Resistant Staphylococcus aureus) infections, but their widespread use has led to vancomycin-reduced susceptibility [heterogeneous Vancomycin-Intermediate-Staphylococcus aureus (hVISA) and Vancomycin-Intermediate-Staphylococcus aureus (VISA)], in which different genetic loci (regulatory, autolytic, cell-wall turnover and cell-envelope positive charge genes) are involved. In addition, reduced susceptibility to vancomycin can influence the development of resistance to daptomycin. Although the phenotypic and molecular changes of hVISA/VISA have been the focus of different papers, the molecular mechanisms responsible for these different phenotypes and for the vancomycin and daptomycin cross-resistance are not clearly understood. The aim of our study was to investigate, by real time RT-PCR, the relative quantitative expression of genes involved in autolysis (atl-lytM), cell-wall turnover (sceD), membrane charges (mprF-dltA) and regulatory mechanisms (agr-locus-graRS-walKR), in hVISA and VISA cultured with or without vancomycin and daptomycin, in order to better understand the molecular basis of vancomycin-reduced susceptibility and the modulating activity of vancomycin and daptomycin on the expression of genes implicated in their reduced susceptibility mechanisms. Our results show that hVISA and VISA present common features that distinguish them from Vancomycin-Susceptible Staphylococcus aureus (VSSA), responsible for the intermediate glycopeptide resistance i.e. an increased cell-wall turnover, an increased positive cell-wall charge responsible for a repulsion mechanism towards vancomycin and daptomycin, and reduced agr-functionality. Indeed, VISA emerges from hVISA when VISA acquires a reduced autolysis caused by a down-regulation of autolysin genes, atl/lytM, and a reduction of the net negative cell-envelope charge via dltA over-expression. Vancomycin and daptomycin, acting in a similar manner in hVISA and VISA, can influence their cross-resistance mechanisms promoting VISA behavior in hVISA and enhancing the cell-wall pathways responsible for the intermediate vancomycin resistance in VISA. Daptomycin can also induce a charge repulsion mechanism both in hVISA and VISA increasing the activity of the mprF

Exopolysaccharide-associated protein sorting in environmental organisms: the PEP-CTERM/EpsH system. Application of a novel phylogenetic profiling heuristic

BACKGROUND: Protein translocation to the proper cellular destination may be guided by various classes of sorting signals recognizable in the primary sequence. Detection in some genomes, but not others, may reveal sorting system components by comparison of the phylogenetic profile of the class of sorting signal to that of various protein families. RESULTS: We describe a short C-terminal homology domain, sporadically distributed in bacteria, with several key characteristics of protein sorting signals. The domain includes a near-invariant motif Pro-Glu-Pro (PEP). This possible recognition or processing site is followed by a predicted transmembrane helix and a cluster rich in basic amino acids. We designate this domain PEP-CTERM. It tends to occur multiple times in a genome if it occurs at all, with a median count of eight instances; Verrucomicrobium spinosum has sixty-five. PEP-CTERM-containing proteins generally contain an N-terminal signal peptide and exhibit high diversity and little homology to known proteins. All bacteria with PEP-CTERM have both an outer membrane and exopolysaccharide (EPS) production genes. By a simple heuristic for screening phylogenetic profiles in the absence of pre-formed protein families, we discovered that a homolog of the membrane protein EpsH (exopolysaccharide locus protein H) occurs in a species when PEP-CTERM domains are found. The EpsH family contains invariant residues consistent with a transpeptidase function. Most PEP-CTERM proteins are encoded by single-gene operons preceded by large intergenic regions. In the Proteobacteria, most of these upstream regions share a DNA sequence, a probable cis-regulatory site that contains a sigma-54 binding motif. The phylogenetic profile for this DNA sequence exactly matches that of three proteins: a sigma-54-interacting response regulator (PrsR), a transmembrane histidine kinase (PrsK), and a TPR protein (PrsT). CONCLUSION: These findings are consistent with the hypothesis that PEP-CTERM and EpsH form a protein export sorting system, analogous to the LPXTG/sortase system of Gram-positive bacteria, and correlated to EPS expression. It occurs preferentially in bacteria from sediments, soils, and biofilms. The novel method that led to these findings, partial phylogenetic profiling, requires neither global sequence clustering nor arbitrary similarity cutoffs and appears to be a rapid, effective alternative to other profiling methods

Actin-interacting and flagellar proteins in Leishmania spp.: Bioinformatics predictions to functional assignments in phagosome formation

Several motile processes are responsible for the movement of proteins into and within the flagellar membrane, but little is known about the process by which specific proteins (either actin-associated or not) are targeted to protozoan flagellar membranes. Actin is a major cytoskeleton protein, while polymerization and depolymerization of parasite actin and actin-interacting proteins (AIPs) during both processes of motility and host cell entry might be key events for successful infection. For a better understanding the eukaryotic flagellar dynamics, we have surveyed genomes, transcriptomes and proteomes of pathogenic Leishmania spp. to identify pertinent genes/proteins and to build in silico models to properly address their putative roles in trypanosomatid virulence. In a search for AIPs involved in flagellar activities, we applied computational biology and proteomic tools to infer from the biological meaning of coronins and Arp2/3, two important elements in phagosome formation after parasite phagocytosis by macrophages. Results presented here provide the first report of Leishmania coronin and Arp2/3 as flagellar proteins that also might be involved in phagosome formation through actin polymerization within the flagellar environment. This is an issue worthy of further in vitro examination that remains now as a direct, positive bioinformatics-derived inference to be presented

Translocation of group 1 capsular polysaccharide to the surface of Escherichia coli requires a multimeric complex in the outer membrane.

Surface expression of the group 1 K30 capsular polysaccharide of Escherichia coli strain E69 (O9a:K30) requires Wza(K30), a member of the outer membrane auxiliary (OMA) protein family. A mutation in wza(K30) severely restricts the formation of the K30 capsular structure on the cell surface, but does not interfere with the biosynthesis or polymerization of the K30 repeat unit. Here we show that Wza(K30) is a surface-exposed outer membrane lipoprotein. Wza(K30) multimers form ring-like structures in the outer membrane that are reminiscent of the secretins of type II and III protein translocation systems. We propose that Wza(K30) forms an outer membrane pore through which the K30-capsular antigen is translocated. This is the first evidence of a potential mechanism for translocation of high molecular weight polysaccharide across the outer membrane. The broad distribution of the OMA protein family suggests a similar process for polysaccharide export in diverse Gram-negative bacteria

Identification of the catalytic nucleophile in the cellulase from Schizophyllum commune and assignment of the enzyme to Family 5, subtype 5 of the glycosidases

Differential chemical modification of the cellulase from Schizophyllum commune with [N-methyl-3H]1-ethyl-3(4-azonia-4,4-dimethylpentyl)-carbodiimide in the presence and absence of substrate identified an active site glutamate residue within the peptide: Leu-Gln-Ala-Ala-Thr-Glu-Trp-Leu-(Lys). This Glu residue is proposed to participate in binding of substrate as amino acid sequence homology studies combined with mechanism-based inhibition of the cellulase with 4',5'-epoxypentyl-beta-D-cellobioside identified a neighboring Glu residue, which conforms to the Glu-X-Gly motif of Family 5 glycosidases, as the catalytic nucleophile. These data allow the assignment of the S. commune cellulase to Family 5, subtype 5 of the glycosidases.NRC publication: Ye

Discovery of factors linked to antimony resistance in Leishmania panamensis through differential proteome analysis

The rate of treatment failure to antileishmanial chemotherapy in Latin America is up to 64%. Parasite drug resistance contributes to an unknown proportion of treatment failures. Identification of clinically relevant molecular mechanisms responsible for parasite drug resistance is critical to the conservation of available drugs and to the discovery of novel targets to reverse the resistant phenotype. We conducted comparative proteomic-based analysis of Leishmania (Viannia) panamensis lines selected in vitro for resistance to trivalent antimony (SbIII) to identify factors associated with antimony resistance. Using 2-dimensional gel electrophoresis, two distinct sub-proteomes (soluble in NP-40/urea and Triton X-114, respectively) of promastigotes of WT and SbIII-resistant lines were generated. Overall, 9 differentially expressed putative Sb-resistance factors were detected and identified by mass spectrometry. These constituted two major groups: (a) proteins involved in general stress responses and (b) proteins with highly specific metabolic and transport functions, potentially directly contributing to the Sb-resistance mechanism. Notably, the sulfur amino acid-metabolizing enzymes S-adenosylmethionine synthetase (SAMS) and S-adenosylhomocysteine hydrolase (SAHH) were over-expressed in SbIII-resistant lines and SbIII-resistant clinical isolates. These enzymes play a central role in the upstream synthesis of precursors of trypanothione, a key molecule involved in Sb-resistance in Leishmania parasites, and suggest involvement of epigenetic regulation in response to drug exposure. These data re-enforce the importance of thiol metabolism in Leishmania Sb resistance, reveal previously unrecognized steps in the mechanism(s) of Sb tolerance, and suggest a cross-talk between drug resistance, metabolism and virulence