Identification of a bacteriocin and its cognate immunity factor expressed by Moraxella catarrhalis

<p>Abstract</p> <p>Background</p> <p>Bacteriocins are antimicrobial proteins and peptides ribosomally synthesized by some bacteria which can effect both intraspecies and interspecies killing.</p> <p>Results</p> <p><it>Moraxella catarrhalis </it>strain E22 containing plasmid pLQ510 was shown to inhibit the growth of <it>M. catarrhalis </it>strain O35E. Two genes (<it>mcbA </it>and <it>mcbB</it>) in pLQ510 encoded proteins predicted to be involved in the secretion of a bacteriocin. Immediately downstream from these two genes, a very short ORF (<it>mcbC</it>) encoded a protein which had some homology to double-glycine bacteriocins produced by other bacteria. A second very short ORF (<it>mcbI</it>) immediately downstream from <it>mcbC </it>encoded a protein which had no significant similarity to other proteins in the databases. Cloning and expression of the <it>mcbI </it>gene in <it>M. catarrhalis </it>O35E indicated that this gene encoded the cognate immunity factor. Reverse transcriptase-PCR was used to show that the <it>mcbA</it>, <it>mcbB</it>, <it>mcbC</it>, and <it>mcbI </it>ORFs were transcriptionally linked. This four-gene cluster was subsequently shown to be present in the chromosome of several <it>M. catarrhalis </it>strains including O12E. Inactivation of the <it>mcbA</it>, <it>mcbB</it>, or <it>mcbC </it>ORFs in <it>M. catarrhalis </it>O12E eliminated the ability of this strain to inhibit the growth of <it>M. catarrhalis </it>O35E. In co-culture experiments involving a <it>M. catarrhalis </it>strain containing the <it>mcbABCI </it>locus and one which lacked this locus, the former strain became the predominant member of the culture after overnight growth in broth.</p> <p>Conclusion</p> <p>This is the first description of a bacteriocin and its cognate immunity factor produced by <it>M. catarrhalis</it>. The killing activity of the McbC protein raises the possibility that it might serve to lyse other <it>M. catarrhalis </it>strains that lack the <it>mcbABCI </it>locus, thereby making their DNA available for lateral gene transfer.</p

Pathogenic mechanisms and signaling pathways in Plasmodium falciparum

Plasmodium falciparum is a human intracellular parasite that is the causative agent of a deadly form of malaria. This species alone is responsible for 200 million cases of malaria annually resulting in over 1 million deaths worldwide. The excessive mortality due to P. falciparum infection is due to its ability to cause severe pathogenesis through hyperparasitemia and cytoadherence defined as the ability of infected red blood cells to adhere to host vasculature. Cytoadherence is mediated through the export of parasite proteins to the surface of the infected red blood cell (RBC). Exported proteins have been identified but the pathway for protein export is still being elucidated. Many protein coding genes in the P. falciparum genome are hypothetical and therefore still need to be studied. Random transposon mutagenesis using the piggyBac transposable element in P. falciparum has given us a library of mutants to use for forward genetic studies. In this work, we describe a novel approach for screening P. falciparum piggyBac mutants to look for differences in cytoadherence. We utilized an image-based approach in order to quantify cytoadherence in P. falciparum NF54 wild-type and thirty-four piggyBac mutants. We found cytoadherence to be affected by the expression of genes with specific gene ontologies including nucleic acid metabolism and post-translational modification. Many of these genes are annotated as hypothetical or putative and this work may result in further revelations of a role for these genes in parasite pathogenesis. We further characterized a piggyBac mutant that was increased in cytoadherent abilities as an atypical mitogen-activated protein kinase phosphatase (MKP) named PfMKP1. We were able to demonstrate phosphatase activity in a generic substrate-based assay and identify a putative substrate as mitogen-activated protein kinase 1 (Pfmap1). Furthermore, we found Pfmap1 to be differentially phosphorylated and a difference in localization (through immunofluorescence assay) in the PfMKP1 mutant line compared to the wild-type and complemented mutant. This adds to the recent work characterizing this gene as important in cell cycle progression within the erythrocytic cycle and lends to the hypothesis of a functioning mitogen-activated protein kinase (MAPK) pathway in P. falciparum for which it is currently unknown. Supplementary work focused on the development of tools to further investigate the MAPK pathway and the role for PfMKP1 as an atypical MKP of Pfmap1 in this pathway. A mass spectrometry-based technique was developed to look at Pfmap1 and its potential active state based on its phosphorylation status. This approach needs further development but the methods are described here within. In addition, the tools needed to further characterize the binding interaction between PfMKP1 and Pfmap1 were established. The mass spectrometry screen and immunofluorescence screening of Pfmap1 can further our knowledge of the MAPK pathway in P. falciparum and lead to the identification of external stimuli that can induce growth or stress response in the parasite. Taken together, the elucidation of mechanisms for cytoadherence and signal transduction pathways in the parasite can lead to new drug target identification

Pathogenic mechanisms and signaling pathways in Plasmodium falciparum

Plasmodium falciparum is a human intracellular parasite that is the causative agent of a deadly form of malaria. This species alone is responsible for 200 million cases of malaria annually resulting in over 1 million deaths worldwide. The excessive mortality due to P. falciparum infection is due to its ability to cause severe pathogenesis through hyperparasitemia and cytoadherence defined as the ability of infected red blood cells to adhere to host vasculature. Cytoadherence is mediated through the export of parasite proteins to the surface of the infected red blood cell (RBC). Exported proteins have been identified but the pathway for protein export is still being elucidated. Many protein coding genes in the P. falciparum genome are hypothetical and therefore still need to be studied. Random transposon mutagenesis using the piggyBac transposable element in P. falciparum has given us a library of mutants to use for forward genetic studies. In this work, we describe a novel approach for screening P. falciparum piggyBac mutants to look for differences in cytoadherence. We utilized an image-based approach in order to quantify cytoadherence in P. falciparum NF54 wild-type and thirty-four piggyBac mutants. We found cytoadherence to be affected by the expression of genes with specific gene ontologies including nucleic acid metabolism and post-translational modification. Many of these genes are annotated as hypothetical or putative and this work may result in further revelations of a role for these genes in parasite pathogenesis. We further characterized a piggyBac mutant that was increased in cytoadherent abilities as an atypical mitogen-activated protein kinase phosphatase (MKP) named PfMKP1. We were able to demonstrate phosphatase activity in a generic substrate-based assay and identify a putative substrate as mitogen-activated protein kinase 1 (Pfmap1). Furthermore, we found Pfmap1 to be differentially phosphorylated and a difference in localization (through immunofluorescence assay) in the PfMKP1 mutant line compared to the wild-type and complemented mutant. This adds to the recent work characterizing this gene as important in cell cycle progression within the erythrocytic cycle and lends to the hypothesis of a functioning mitogen-activated protein kinase (MAPK) pathway in P. falciparum for which it is currently unknown. Supplementary work focused on the development of tools to further investigate the MAPK pathway and the role for PfMKP1 as an atypical MKP of Pfmap1 in this pathway. A mass spectrometry-based technique was developed to look at Pfmap1 and its potential active state based on its phosphorylation status. This approach needs further development but the methods are described here within. In addition, the tools needed to further characterize the binding interaction between PfMKP1 and Pfmap1 were established. The mass spectrometry screen and immunofluorescence screening of Pfmap1 can further our knowledge of the MAPK pathway in P. falciparum and lead to the identification of external stimuli that can induce growth or stress response in the parasite. Taken together, the elucidation of mechanisms for cytoadherence and signal transduction pathways in the parasite can lead to new drug target identification

Moraxella catarrhalis Synthesizes an Autotransporter That Is an Acid Phosphatase▿

Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA) was most similar (the BLAST probability score was 10−10) to bacterial class A nonspecific acid phosphatases. The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins, whereas the C-terminal portion of MapA resembled the translocation domain of conventional autotransporters. Cloning and expression of the M. catarrhalis mapA gene in Escherichia coli confirmed the presence of acid phosphatase activity in the MapA protein. The MapA protein was shown to be localized to the outer membrane of M. catarrhalis and was not detected either in the soluble cytoplasmic fraction from disrupted M. catarrhalis cells or in the spent culture supernatant fluid from M. catarrhalis. Use of the predicted MapA translocation domain in a fusion construct with the passenger domain from another predicted M. catarrhalis autotransporter confirmed the translocation ability of this MapA domain. Inactivation of the mapA gene in M. catarrhalis strain O35E reduced the acid phosphatase activity expressed by this organism, and this mutation could be complemented in trans with the wild-type mapA gene. Nucleotide sequence analysis of the mapA gene from six M. catarrhalis strains showed that this protein was highly conserved among strains of this pathogen. Site-directed mutagenesis of a critical histidine residue (H233A) in the predicted active site of the acid phosphatase domain in MapA eliminated acid phosphatase activity in the recombinant MapA protein. This is the first description of an autotransporter protein that expresses acid phosphatase activity

Moraxella catarrhalis Expresses an Unusual Hfq Protein ▿

The Hfq protein is recognized as a global regulatory molecule that facilitates certain RNA-RNA interactions in bacteria. BLAST analysis identified a 630-nucleotide open reading frame in the genome of Moraxella catarrhalis ATCC 43617 that was highly conserved among M. catarrhalis strains and which encoded a predicted protein with significant homology to the Hfq protein of Escherichia coli. This protein, containing 210 amino acids, was more than twice as large as the Hfq proteins previously described for other bacteria. The C-terminal half of the M. catarrhalis Hfq protein was very hydrophilic and contained two different types of amino acid repeats. A mutation in the M. catarrhalis hfq gene affected both the growth rate of this organism and its sensitivity to at least two different types of stress in vitro. Provision of the wild-type M. catarrhalis hfq gene in trans eliminated these phenotypic differences in the hfq mutant. This M. catarrhalis hfq mutant exhibited altered expression of some cell envelope proteins relative to the wild-type parent strain and also had a growth advantage in a continuous flow biofilm system. The presence of the wild-type M. catarrhalis hfq gene in trans in an E. coli hfq mutant fully reversed the modest growth deficiency of this E. coli mutant and partially reversed the stress sensitivity of this E. coli mutant to methyl viologen. The use of an electrophoretic mobility shift assay showed that this M. catarrhalis Hfq protein could bind RNA derived from a gene whose expression was altered in the M. catarrhalis hfq mutant

Modular Arrangement of Allelic Variants Explains the Divergence in Moraxella catarrhalis UspA Protein Function▿

Ubiquitous surface protein A molecules (UspAs) of Moraxella catarrhalis are large, nonfimbrial, autotransporter proteins that can be visualized as a “fuzzy” layer on the bacterial surface by transmission electron microscopy. Previous studies attributed a wide array of functions and binding activities to the closely related UspA1, UspA2, and/or UspA2H protein, yet the molecular and phylogenetic relationships among these activities remain largely unexplored. To address this issue, we determined the nucleotide sequence of the uspA1 genes from a variety of independent M. catarrhalis isolates and compared the deduced amino acid sequences to those of the previously characterized UspA1, UspA2, and UspA2H proteins. Rather than being conserved proteins, we observed a striking divergence of individual UspA1, UspA2, and UspA2H proteins resulting from the modular assortment of unrelated “cassettes” of peptide sequence. The exchange of certain variant cassettes correlates with strain-specific differences in UspA protein function and confers differing phenotypes upon these mucosal surface pathogens