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Unlabelled: Sinorhizobium meliloti forms N2-fixing root nodules on alfalfa, and as a free-living bacterium, it can grow on a very broad range of substrates, including l-proline and several related compounds, such as proline betaine, trans-4-hydroxy-l-proline (trans-4-l-Hyp), and cis-4-hydroxy-d-proline (cis-4-d-Hyp). Fourteen hyp genes are induced upon growth of S. meliloti on trans-4-l-Hyp, and of those, hypMNPQ encodes an ABC-type trans-4-l-Hyp transporter and hypRE encodes an epimerase that converts trans-4-l-Hyp to cis-4-d-Hyp in the bacterial cytoplasm. Here, we present evidence that the HypO, HypD, and HypH proteins catalyze the remaining steps in which cis-4-d-Hyp is converted to α-ketoglutarate. The HypO protein functions as a d-amino acid dehydrogenase, converting cis-4-d-Hyp to Δ(1)-pyrroline-4-hydroxy-2-carboxylate, which is deaminated by HypD to α-ketoglutarate semialdehyde and then converted to α-ketoglutarate by HypH. The crystal structure of HypD revealed it to be a member of the N-acetylneuraminate lyase subfamily of the (α/β)8 protein family and is consistent with the known enzymatic mechanism for other members of the group. It was also shown that S. meliloti can catabolize d-proline as both a carbon and a nitrogen source, that d-proline can complement l-proline auxotrophy, and that the catabolism of d-proline is dependent on the hyp cluster. Transport of d-proline involves the HypMNPQ transporter, following which d-proline is converted to Δ(1)-pyrroline-2-carboxylate (P2C) largely via HypO. The P2C is converted to l-proline through the NADPH-dependent reduction of P2C by the previously uncharacterized HypS protein. Thus, overall, we have now completed detailed genetic and/or biochemical characterization of 9 of the 14 hyp genes., Importance: Hydroxyproline is abundant in proteins in animal and plant tissues and serves as a carbon and a nitrogen source for bacteria in diverse environments, including the rhizosphere, compost, and the mammalian gut. While the main biochemical features of bacterial hydroxyproline catabolism were elucidated in the 1960s, the genetic and molecular details have only recently been determined. Elucidating the genetics of hydroxyproline catabolism will aid in the annotation of these genes in other genomes and metagenomic libraries. This will facilitate an improved understanding of the importance of this pathway and may assist in determining the prevalence of hydroxyproline in a particular environment., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Apophysomyces variabilis is an emerging fungal pathogen that can cause significant infections in immunocompetent patients. We report a case of A. variabilis invasive wound infection in a 21-year-old male after a self-inflicted burn injury.

Pythium aphanidermatum is a fungus-like plant pathogen which has never been reported as a cause of human infection. We report a case of P. aphanidermatum invasive wound infection in a 21-year-old male injured during combat operations in Afghanistan.

The apical membrane antigen 1 of Plasmodium falciparum is one of the leading candidate antigens being developed as a vaccine to prevent malaria. This merozoite transmembrane protein has an ectodomain that can be divided into three subdomains (D I, D II, and D III). We have previously expressed a major portion of this ectodomain and have shown that it can induce antibodies that prevent merozoite invasion into red blood cells in an in vitro growth and invasion assay. To analyze the antibody responses directed against the individual subdomains, we constructed six different genes that express each of the domains separately (D I, D II, or D III) or in combination with another domain (D I+II, D II+III, or D I+III). These proteins were purified and used to immunize rabbits to raise construct-specific antibodies. We demonstrated that D I+II induced a significant amount of the growth-inhibitory antibodies active in the growth and invasion assay.

We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens.