•Pneumococcal iron acquisition ATP binding cassette is abbreviated to PiaABC.•The cell-surface lipoprotein PiaA is a key component of PiaABC.•Trp63, Trp158 and Phe255, in the metal binding site of PiaA, were mutated.•Thermodynamic and kinetic study on the ferrichrome binding ability was performed.•Trp158 is a crucial residue for structure stability and ferrichrome binding of PiaA.The pathogenic Streptococcus pneumoniae (S. pneumoniae) has evolved a special mechanism such as pneumococcal iron acquisition ATP binding cassette (PiaABC) to take up siderophore-iron from its host. The cell-surface lipoprotein PiaA, a key component of PiaABC, is the primary receptor to bind ferrichrome (Fc). To study the structure-function relationship of PiaA, three conservative amino-acid residues, Trp63, Trp158 and Phe255, in the hydrophobic barrel of the metal binding site of PiaA, were individually and collectively mutated to alanine; and the resulted single-point mutants, W63A, W158A and F255A, and triple mutant W63A/W158A/F255A were characterized by using biochemical and biophysical methods. Experiments showed that wild-type PiaA (WT-PiaA) and the single-point mutant proteins bound Fc with a similar kinetics mode, but the reaction rate of W158 A was lower than that for WT-PiaA. The binding affinity of W158A toward Fc was significantly weaker than that of the WT-PiaA-Fc (wild-type PiaA bound with Fc) interaction. Furthermore, the absence of Trp158 in the protein led to a significant impact on the secondary structure of PiaA, resulting in a labile conformational structure of W158A, with impaired resistance to thermal and chemical denaturation. Collectively, Trp158 is a crucial residue for binding Fc, playing an important role in stabilizing the PiaA-Fc complex. This study revealed the critical role of the conserved tryptophan residues in Fc-binding protein PiaA, and provided valuable information for understanding the Fc transport mechanism mediated by PiaA or its homologous proteins in bacteria.PiaA is a surface-exposed lipoprotein of pneumococcal iron acquisition ATP binding cassette (PiaABC). Trp158 is a significant residue of PiaA for ferrichrome (Fc) binding and plays an important role in stabilizing the complex of PiaA and Fc in Streptococcus pneumoniae.

Streptococcus pneumoniae is a Gram-positive bacterial pathogen causing a variety of diseases, including otitis media, bacteraemia and meningitis. Although copper is an essential trace metal for bacterial growth, high intracellular levels of free-copper are toxic. Copper resistance has emerged as an important virulence determinant of microbial pathogens. In this study, we determined the minimum inhibition concentration of copper for the growth inhibition of S. pneumoniae. Two-dimensional-electrophoresis coupled with mass spectrometry was applied to identify proteins involved in copper resistance of S. pneumoniae. In total, forty-four proteins with more than 1.5-fold alteration in expression (p < 0.05) were identified. Quantitative reverse transcription PCR was used to confirm the proteomic results. Bioinformatics analysis showed that the differentially expressed proteins were mainly involved in the cell wall biosynthesis, protein biosynthesis, purine biosynthesis, pyrimidine biosynthesis, primary metabolic process, and the nitrogen compound metabolic process. Many up-regulated proteins in response to the copper treatment directly or indirectly participated in the cell wall biosynthesis, indicating that the cell wall is a critical determinant in copper resistance of S. pneumoniae.

•Ru(II) complex X-03 shows a significant antibacterial activity.•We investigated the antimicrobial mechanism of Ru(II) on S. pneumoniae.•X-03 may affect various important molecular pathways.•X-03 interferes with iron acquisition systems in S. pneumoniae.Streptococcus pneumoniae is a Gram-positive pathogen that causes a variety of infection diseases in human. In this project, we determined the antibacterial activity of a Ru(II) complex X-03 against S. pneumoniae in vitro, by comparing its toxicity to host cells A549 and HBE. We performed two-dimensional gel electrophoresis (2-DE)-based proteomic analysis to characterize the protein alterations in S. pneumoniae after treatment with X-03. In total, 50 proteins exhibiting significant differential expressions were identified. RT-PCR was used to confirm the expression differences for selected proteins. Bioinformatics analysis on the proteomic alterations suggested that Ru(II) complex X-03 may obstruct bacterial fatty acid synthesis and oxidation–reduction process to suppress the growth of S. pneumoniae. Metal-uptake experiments revealed that iron-acquisition pathway in the bacterium may be interfered by X-03. These results provide useful clues for further investigations on the mechanism of the antibacterial action of metal compounds.Biological significanceThe appearance of bacterial strains with broad antibiotic resistance is becoming an alarming global health concern. The development of novel efficient antibacterial compound is urgently needed. In the present study, we found that Ru(II) complex X-03 has a significant antibacterial activity and applied proteomic technology combined with bioinformatics analysis to investigate its antimicrobial mechanism in S. pneumoniae. Many proteins were found to be dysregulated, implicating that X-03 may affect various molecular pathways leading to the inhibition of bacterial growth. Metal-uptake experiments demonstrated that X-03 treatment reduced the iron content in the bacterium, suggesting the interference with iron acquisition systems by the complex. This disturbance in iron acquisition may directly or indirectly induce the proteomic response that involved many pathways. In addition, X-03 could selectively suppress Gram-positive bacteria but execute less cytotoxicity to Gram-negative bacteria, with almost no effect on human cells, implicating its potential to be developed as a specific antimicrobial agent. These results provide useful information for further investigations on the mechanism of the antibacterial action of metal drugs and development of efficient antibacterial drugs.

The current application and development of proteomic studies typically depend on the availability of sequenced genomes. Protein identification based on the detected peptides with liquid chromatography tandem mass spectrometry is limited by the absence of sequenced genomes in many nonmodel organisms. In this study, we demonstrated a new strategy based on our stable, accurate, and error-tolerant FANSe (Fast and Accurate mapping tool for Nucleotide Sequencing datasets) mapping algorithm to correct genome sequences in an iterative manner. To evaluate the efficiency of the corrected genome databases in proteomic study, MS/MS spectra of whole proteome extracted from a Bacillus pumilus strain without complete genome sequence were searched against the protein sequence databases derived from the complete reference genome sequence of a homologous bacterium and from the corrected genome sequence. The results indicated that the corrected protein sequence database could significantly facilitate peptide/protein identification. Importantly, this strategy can help to detect novel peptide variants. This strategy of genome correction will promote the development of functional proteomics in nonmodel organisms.

Gram-positive Streptococcus species are responsible for millions of cases of meningitis, bacterial pneumonia, endocarditis, erysipelas and necrotizing fasciitis. Iron is essential for the growth and survival of Streptococcus in the host environment. Streptococcus species have developed various mechanisms to uptake iron from an environment with limited available iron. Streptococcus can directly extract iron from host iron-containing proteins such as ferritin, transferrin, lactoferrin and hemoproteins, or indirectly by relying on the employment of specialized secreted hemophores (heme chelators) and small siderophore molecules (high affinity ferric chelators). This review presents the most recent discoveries in the iron acquisition system of Streptococcus species – the transporters as well as the regulators.

Streptococcus pyogenes is an important human bacterium with high pathogenicity. Heme is a major source of iron that plays a critical role in bacterial survival and virulence. In this study, heme-affinity chromatography, two-dimensional-electrophoresis and mass spectrometry were combined to identify putative heme-binding proteins and heme-regulatory proteins. In total, 68 heme-regulatory proteins and 284 putative heme-binding proteins were identified, among which 37 proteins showed expression alterations in response to heme deficiency. Bioinformatics analysis revealed that several key metabolic pathways had changed in the absence of heme, among which glycolysis was a major pathway impaired under heme-deficient conditions. New potential heme-binding proteins were successfully identified in this study providing novel clues for the study of the heme transport mechanism. Heme-binding proteins may play fundamental roles in many important biological pathways and thus contribute to bacterial pathogenicity.

Streptococcus pneumoniae is a Gram-positive pathogen responsible for pneumonia, otitis media, and meningitis. Manganese and zinc ions are essential for this bacterium, playing regulatory, structural, or catalytic roles as the critical cofactors in the bacterial proteins and metabolic enzymes. Lipoprotein PsaA has been found to mediate Mn2+ and Zn2+ transportation in Streptococcus pneumoniae. In the present work, we conducted a systemic study on the contributions from key amino acids in the metal-binding site of PsaA using various spectroscopic and biochemical methods. Our experimental data indicate that four metal-binding residues contribute unequally to the Mn2+ and Zn2+ binding, and His139 is most important for both the structural stability and metal binding of the protein. PsaA–Mn2+ has a lower thermal stability than PsaA–Zn2+, possibly due to the different coordination preferences of the metals. Kinetics analysis revealed that PsaA–Mn2+ binding is a fast first-order reaction, whereas PsaA–Zn2+ binding is a slow second-order reaction, implying that PsaA kinetically prefers binding Mn2+ to Zn2+. The present results provide complementary information for understanding the mechanisms of metal transport and bacterial virulence via lipoproteins in Streptococcus pneumoniae.

Cobalt and nickel play important roles in various biological processes. The present work focuses on the enrichment and identification of Co- and Ni-binding motifs and proteins in Gram-positive bacteria. Immobilized metal affinity column (IMAC) was used to partially enrich putative metal-binding proteins and peptides from Streptococcus pneumoniae, and then LTQ-Orbitrap mass spectrometry (MS) was applied to identify and characterize the metal-binding motifs and proteins. In total, 208 and 223 proteins were isolated by Co- and Ni-IMAC columns respectively, in which 129 proteins were present in both preparations. Based on the gene ontology (GO) analysis, the putative metal-binding proteins were found to be mainly involved in protein metabolism, gene expression regulation and carbohydrate metabolism. These putative metal-binding proteins form a highly connected network, indicating that they may synergistically work together to achieve specific biological functions. Putative Co- and Ni-binding motifs were identified with H(X)nH, M(X)nH and H(X)nM derived from the identified 51 Co-binding peptides and 66 Ni-binding peptides. Statistics of frequency of amino acids in the metal-binding motifs showed that cobalt and nickel prefer to bind histidine and methionine, but not cysteine. These results obtained by a systematic metalloproteomic approach provide important clues for the further investigation of metal homeostasis and metal-related virulence of bacteria.

Gram-positive bacteria cause a series of diseases in human, animals and plants. There has been increasing interest in efforts to investigate pathogenesis of bacteria using multiple “omic” strategies including proteomics. Proteins in different cell fractions of bacteria may play different vital roles in various physiological processes, such as adhesion, invasion, internalization, sensing, respiration, oxidative stress protection and pathogenicity. Subproteomics specifically focuses on the pre-fractionated cellular proteins and thus may be able to characterize more low-abundance molecules that are usually overlooked by the traditional whole-cell proteomics, providing comprehensive information for further investigations. This review intends to outline the current progress, challenges and future development of subproteomics in the characterization of Gram-positive bacteria. This article is part of a Special Issue entitled: Proteomics: The clinical link.Highlights► Low-abundance proteins can be specifically enriched in subproteomes. ► Subproteomics was used to characterize Gram-positive bacteria. ► The effective and specific pre-fractionation of subproteomes is the most important step. ► Further improvements in separation techniques, MS detection and bioinformatics are still needed.