bPseudomonas aeruginosa PAO1 grows on a variety of polyamines as the sole source of carbon and nitrogen. Catabolism of polyamines is mediated by the ␥-glutamylation pathway, which is complicated by the existence of multiple homologous enzymes with redundant specificities toward different polyamines for a more diverse metabolic capacity in this organism. Through a series of markerless gene knockout mutants and complementation tests, specific combinations of pauABCD (polyamine utilization) genes were deciphered for catabolism of different polyamines. Among six pauA genes, expression of pauA1, pauA2, pauA4, and pauA5 was found to be inducible by diamines putrescine (PUT) and cadaverine (CAD) but not by diaminopropane. Activation of these promoters was regulated by the PauR repressor, as evidenced by constitutively active promoters in the pauR mutant. The activities of these promoters were further enhanced by exogenous PUT or CAD in the mutant devoid of all six pauA genes. The recombinant PauR protein with a hexahistidine tag at its N terminus was purified, and specific bindings of PauR to the promoter regions of most pau operons were demonstrated by electromobility shift assays. Potential interactions of PUT and CAD with PauR were also suggested by chemical cross-linkage analysis with glutaraldehyde. In comparison, growth on PUT was more proficient than that on CAD, and this observed growth phenotype was reflected in a strong catabolite repression of pauA promoter activation by CAD but was completely absent as reflected by activation by PUT. In summary, this study clearly establishes the function of PauR in control of pau promoters in response to PUT and CAD for their catabolism through the ␥-glutamylation pathway.
Erythrocyte-binding-like (EBL) proteins are known to play an important role in malaria parasite invasion of red blood cells (RBCs); however, any roles of EBL proteins in regulating host immune responses remain unknown. Here, we show that Plasmodium yoelii EBL (PyEBL) can shape disease severity by modulating the surface structure of infected RBCs (iRBCs) and host immune responses. We identified an amino acid substitution (a change of C to Y at position 741 [C741Y]) in the protein trafficking domain of PyEBL between isogenic P. yoellii nigeriensis strain N67 and N67C parasites that produce different disease phenotypes in C57BL/6 mice. Exchanges of the C741Y alleles altered parasite growth and host survival accordingly. The C741Y substitution also changed protein processing and trafficking in merozoites and in the cytoplasm of iRBCs, reduced PyEBL binding to band 3, increased phosphatidylserine (PS) surface exposure, and elevated the osmotic fragility of iRBCs, but it did not affect invasion of RBCs in vitro. The modified iRBC surface triggered PS-CD36-mediated phagocytosis of iRBCs, host type I interferon (IFN-I) signaling, and T cell differentiation, leading to improved host survival. This study reveals a previously unknown role of PyEBL in regulating host-pathogen interaction and innate immune responses, which may be explored for developing disease control strategies. IMPORTANCE Malaria is a deadly parasitic disease that continues to afflict hundreds of millions of people every year. Infections with malaria parasites can be asymptomatic, with mild symptoms, or fatal, depending on a delicate balance of host immune responses. Malaria parasites enter host red blood cells (RBCs) through interactions between parasite ligands and host receptors, such as erythrocyte-binding-like (EBL) proteins and host Duffy antigen receptor for chemokines (DARC). Plasmodium yoelii EBL (PyEBL) is known to play a role in parasite invasion of RBCs. Here, we show that PyEBL also affects disease severity through modulation of host immune responses, particularly type I interferon (IFN-I) signaling. This discovery assigns a new function to PyEBL and provides a mechanism for developing disease control strategies.
Growth of Pseudomonas aeruginosa on spermine requires a functional γ-glutamylpolyamine synthetase PauA2. Not only subjected to growth inhibition by spermine, the pauA2 mutant became more sensitive to β-lactam antibiotics in human serum. To explore PauA2 as a potential target of drug development, suppressors of the pauA2 mutant, which alleviated toxicity, were isolated from selection plates containing spermine. These suppressors share common phenotypic changes including delayed growth rate, retarded swarming motility, and pyocyanin overproduction. Genome resequencing of a representative suppressor revealed a unique C T mutation at the phoU gene that results in Ser Leu substitution and a constitutive expression of the Pho regulon. Identical phenotypes were also observed in a ΔpauA2ΔphoU double knockout mutant and complemented by the wild-type phoU gene. Accumulation of polyphosphate granules and spermine resistance in the suppressor were reversed concomitantly when expressing exopolyphosphatase PPX from a recombinant plasmid, or by the introduction of deletion alleles in pstS pstC for phosphate uptake, phoB for Pho regulation, and ppk for polyphosphate synthesis. In conclusion, this study identifies polyphosphate accumulation due to an activated Pho regulon and phosphate uptake by the phoU mutation as a potential protection mechanism against spermine toxicity.
Background Rodent malaria parasites are important models for studying host-malaria parasite interactions such as host immune response, mechanisms of parasite evasion of host killing, and vaccine development. One of the rodent malaria parasites is Plasmodium yoelii, and multiple P. yoelii strains or subspecies that cause different disease phenotypes have been widely employed in various studies. The genomes and transcriptomes of several P. yoelii strains have been analyzed and annotated, including the lethal strains of P. y. yoelii YM (or 17XL) and non-lethal strains of P. y. yoelii 17XNL/17X. Genomic DNA sequences and cDNA reads from another subspecies P. y. nigeriensis N67 have been reported for studies of genetic polymorphisms and parasite response to drugs, but its genome has not been assembled and annotated. Results We performed genome sequencing of the N67 parasite using the PacBio long-read sequencing technology, de novo assembled its genome and transcriptome, and predicted 5383 genes with high overall annotation quality. Comparison of the annotated genome of the N67 parasite with those of YM and 17X parasites revealed a set of genes with N67-specific orthology, expansion of gene families, particularly the homologs of the Plasmodium chabaudi erythrocyte membrane antigen, large numbers of SNPs and indels, and proteins predicted to interact with host immune responses based on their functional domains. Conclusions The genomes of N67 and 17X parasites are highly diverse, having approximately one polymorphic site per 50 base pairs of DNA. The annotated N67 genome and transcriptome provide searchable databases for fast retrieval of genes and proteins, which will greatly facilitate our efforts in studying the parasite biology and gene function and in developing effective control measures against malaria.
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