The mechanism of iron-compensation for manganese deficiency of Streptococcus pneumoniae.

Given their involvement in catalysis, infection, and biofilm formation, Fe and Mn are essential for bacterial survival and virulence. In this study, we found that Streptococcus pneumoniae (S. pneumoniae) could grow in the Mn-deficient medium (MDCM). Furthermore, findings showed that the Fe concentration in the bacterium increased when the Mn concentration decreased. In addition, it was noted that supplementing MDCM with Fe resulted in the recovery of bacterial growth. Quantitative proteomics using stable-isotope dimethyl labeling was performed to investigate the adaptive growth mechanism of S. pneumoniae under Mn-deficient conditions. It was found that the expression levels of 25 proteins were downregulated, whereas those of 54 proteins were upregulated in S. pneumoniae grown in MDCM. It was also noted that several of the downregulated proteins were involved in cell energy metabolism, amino acid synthesis, and reduction of oxidation products. More importantly, several ATP-binding cassette transporters related to Fe uptake, such as PiuA, PiaA, PitA, and SPD_1609, were overexpressed for increased Fe uptake from the MDCM. The results suggest that Mn deficiency disturbs multiple metabolic processes in S. pneumoniae. Furthermore, it causes a compensatory effect of Fe for Mn, which is beneficial for the survival of the bacterium in extreme environments. SIGNIFICANCE: The relationship between manganese and iron metabolism in S. pneumoniae has not been clearly revealed. In this paper, we suggest that Mn limitation disturbs multiple metabolic processes and evidently decreases the ATP level in the bacterium. In order to survive in this extreme environment, bacteria upregulated three type of Fe ion transporters PiuABC (heme), PiaABC (ferrichrome) and PitABC (Fe3+) to uptake enough Fe ions to response to Mn deficiency. Therefore, this study reveals a bacterial mechanism of Fe compensation for Mn, and provides new insight for investigating the relativeness of Fe and Mn metabolism of bacteria.

Evolution and molecular mechanism of PitAs in iron transport of Streptococcus species.

Iron is an essential element for almost all bacteria. The iron ATP-binding cassette (ABC) transporters located on the cell membrane affects bacterial virulence and infection. Although a variety of Fe3+-transporters have been found in bacteria, their evolutionary processes are rarely studied. Pneumococcal iron ABC transporter (PitA), a highly conserved Fe3+-transporter in most pathogenic bacteria, influences the capsule formation and virulence of bacteria. However, multiple sequence alignment revealed that PitA is expressed in four different variants in bacteria, and the structural complexity of these variants increases progressively. To more efficiently import Fe3+ ions into bacterial cells, bacteria have evolved a fused PitA from two separately expressed PitA-1 (SPD_0227) and PitA-2 (SPD_0226) proteins. Further biochemical characterization indicated that both PitA-1 and PitA-2 have weaker Fe3+-binding ability than their protein complex. More importantly, Glutathione S-Transferase (GST) pull-down and isothermal titration calorimetry (ITC) detection showed that PitA-1 and PitA-2 interact with each other via Tyr111-Leu37, Asn112-Gln38, Asn103-Leu33, and Asn103-Thr34. Further molecular dynamics (MD) simulations demonstrated that this interaction in full-length PitA is stronger than that in the two individual proteins. Deletion of PitA family genes could lead to decrease in the ability of iron acquisition and of adhesion and invasion of S. pneumoniae. Our study revealed the evolving state and molecular mechanism of Fe3+-transporter PitAs in bacteria and provided important information for understanding the iron transportation mechanism in bacteria and designing new antibacterial drugs.

Comparative Proteomics of Streptococcus pneumoniae Response to Vancomycin Treatment.

Streptococcus pneumoniae is a gram-positive pathogen that causes otitis media, pneumonia, meningitis, and other serious diseases. Vancomycin is one of the most important drugs currently used for the treatment of gram-positive bacterial infections, representing, importantly, the last line of defense against bacteria that have developed resistance to other antibiotics. While primary efforts of most investigations focused on the antibacterial mechanism of vancomycin, few studies have been performed to assess the tolerance mechanism of bacteria to vancomycin. In this work, whole cellular proteins were extracted from S. pneumoniae D39 with or without vancomycin treatment. Subsequently, differentially expressed proteins (DEPs) were identified with two-dimensional gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry (MS)/MS. In total, 27 proteins were upregulated and four proteins were downregulated in vancomycin-treated S. pneumoniae. Gene ontology analysis indicated that these DEPs were mainly involved in the nucleic acid, protein, and carbohydrate biosynthetic processes. Verification experiments with real-time quantitative polymerase chain reaction showed that the gene expression profiles were consistent with proteomic data. These new observations may serve as a valuable resource for future investigations of vancomycin tolerance mechanisms of S. pneumoniae.