Enteric YaiW is a surface-exposed outer membrane lipoprotein that affects sensitivity to an antimicrobial peptide

yaiW is a previously uncharacterized gene found in enteric bacteria that is of particular interest because it is located adjacent to the sbmA gene, whose bacA ortholog is required for Sinorhizobium meliloti symbiosis and Brucella abortus pathogenesis. We show that yaiW is cotranscribed with sbmA in Escherichia coli and Salmonella enterica serovar Typhi and Typhimurium strains. We present evidence that the YaiW is a palmitate-modified surface exposed outer membrane lipoprotein. Since BacA function affects the very-long-chain fatty acid (VLCFA) modification of S. meliloti and B. abortus lipid A, we tested whether SbmA function might affect either the fatty acid modification of the YaiW lipoprotein or the fatty acid modification of enteric lipid A but found that it did not. Interestingly, we did observe that E. coli SbmA suppresses deficiencies in the VLCFA modification of the lipopolysaccharide of an S. meliloti bacA mutant despite the absence of VLCFA in E. coli. Finally, we found that both YaiW and SbmA positively affect the uptake of proline-rich Bac7 peptides, suggesting a possible connection between their cellular functions

Functional and Structural Study of the Dimeric Inner Membrane Protein SbmA

SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability.Fil: Corbalan, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Runti, Giulia. Università degli Studi di Trieste; ItaliaFil: Adler, Conrado. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Covaceuszach, Sonia. Consiglio Nazionale delle Ricerche; ItaliaFil: Ford, Robert C.. University of Manchester; Reino UnidoFil: Lamba, Doriano. Consiglio Nazionale delle Ricerche; ItaliaFil: Beis, Konstantinos. Imperial College London; Reino UnidoFil: Scocchi, Marco. Università degli Studi di Trieste; ItaliaFil: Vincent, Paula Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentin

Proteolytic Activity of Escherichia coli Oligopeptidase B Against Proline-Rich Antimicrobial Peptides.

Oligopeptidase B (OpdB) is a serine peptidase widespread among bacteria and protozoa that has emerged as a virulence factor despite its function has not yet been precisely established. By using an OpdB-overexpressing Escherichia coli strain, we found that the overexpressed peptidase makes the bacterial cells specifically less susceptible to several proline-rich antimicrobial peptides known to penetrate into the bacterial cytosol, and that its level of activity directly correlates with the degree of resistance. We established that E. coli OpdB can efficiently hydrolyze in vitro cationic antimicrobial peptides up to 30 residues in length, even though they contained several prolines, shortening them to inactive fragments. Two consecutive basic residues are a preferred cleavage site for the peptidase. In the case of a single basic residue, there is no cleavage if proline residues are present in the P1 and P2 positions. These results also indicate that cytosolic peptidases may cause resistance to antimicrobial peptides that have an intracellular mechanism of action, such as the proline-rich peptides, and may contribute to define the substrate specificity of the E. coli OpdB

Elective bone marrow transplantation in a child with X-linked hyper-IgM syndrome presenting with acute respiratory distress syndrome.

.We describe a 10-month-old boy diagnosed with X-linked hyper-IgM syndrome (XHIM) after suffering from life-threatening acute respiratory distress syndrome (ARDS) caused by Pneumocystis carinii pneumonia (PCP), although his previous clinical history and first level laboratory tests investigating immunological function did not indicate immunodeficiency. When the patient's overall condition was good, elective bone marrow transplantation from an HLA-matched older brother was performed successfully. We describe how correct diagnosis and successful treatment were made possible thanks to the involvement of a network of specialists

Functional and structural study of the dimeric inner membrane protein SbmA.

SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA.Wecreated a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics.Wealso demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability

Functional characterization of SbmA, a bacterial inner membrane transporter required for importing the antimicrobial peptide Bac7(1-35).

SbmA is an inner membrane protein of Gram-negative bacteria that is involved in the internalization of glycopeptides and prokaryotic and eukaryotic antimicrobial peptides, as well as of peptide nucleic acid (PNA) oligomers. The SbmA homolog BacA is required for the development of Sinorhizobium meliloti bacteroids within plant cells and favors chronic infections with Brucella abortus and Mycobacterium tuberculosis in mice. Here, we investigated functional features of SbmA/BacA using the proline-rich antimicrobial peptide Bac7(1-35) as a substrate. Circular dichroism and affinity chromatography studies were used to investigate the ability of SbmA to bind the peptide, and a whole-cell transport assay with fluorescently labeled peptide allowed the determination of transport kinetic parameters with a calculated Km value of 6.95 \ub1 0.89 \u3bcM peptide and a Vmax of 53.91 \ub1 3.17 nmol/min/mg SbmA. Use of a bacterial two-hybrid system coupled to SEC-MALLS (size exclusion chromatography coupled with multiangle laser light scattering) analyses established that SbmA is a homodimer in the membrane, and treatment of the cells with arsenate or ionophores indicated that the peptide transport mediated by SbmA is driven by the electrochemical gradient. Overall, these results shed light on the SbmA-mediated internalization of peptide substrates and suggest that the transport of an unknown substrate(s) represents the function of this protein