Proteins of Streptococcus pneumoniae Involved in Iron Acquisition

Abstract

Streptococcus pneumoniae is a human pathogen bacterium capable of using hemoglobin (Hb) and haem as a single iron source but not in presence of lactoferrin. This bacterium has developed a mechanism through the expression of several membrane proteins that bind to iron sources, between them a lipoprotein of 37 kDa called Spbhp-37 (Streptococcus pneumoniae haem-binding protein) involved in iron acquisition. The Spbhp-37 role is to maintain the viability of S. pneumoniae in presence of Hb or haem. This mechanism is relevant during the invasion of S. pneumoniae to human tissue for the acquisition of iron from hemoglobin or haem as an iron source.

1. Introduction

Iron is required for cellular growth of any bacterial species and it is known that bacterium needs an iron concentration of 10⁻⁶–10⁻⁸ M [1, 2], however, the concentration of free iron in the human body is usually 10⁻¹⁸ M [3], lower than bacterial requirements [4, 5, 6]. Therefore, human pathogens often obtain iron from alternative sources available into the body such as lactoferrin (Holo-Lf), hemoglobin (Hb) or even the haem [7]. The success of pathogens to obtain iron from host sources is based on developing different mechanisms, for instance, a direct mechanism which consists of expressing proteins attach to the membrane (termed receptors) [8, 9].

Another mechanism (known as indirect mechanism) is based on secreting siderophores or haemophores to scavenge iron then it is delivering towards a receptor protein [10]. The transportation of iron into the cytoplasm requires proteins as the ATP-binding protein cassette (ABC) [11], these mechanisms have been established in Gram-negative but not in Gram-positive bacteria.

S. pneumoniae is a bacterium Gram-positive human pathogen, which causes otitis media, sinusitis, pneumonia, meningitis or bacteriemia especially in infants and elderly persons [12, 13, 14, 15, 16]. S. pneumoniae can grow under iron-restricted medium conditions, if the growth media is supplemented with ferric and ferrous iron salts, haem or Hb [17, 18], but not when Tf, Lf or ferritin (Ft) are added [19, 20]. Moreover, S. pneumoniae expresses the Spbhp-37 lipoprotein (37 KDa) on its surface, which binds both haem and Hb. Spbhp-37 lipoprotein binds its ligand with high affinity because has a Kd of 3.57 e-7 M [21]. The interaction between Sphbp-37 lipoprotein can be inhibited with antibodies specific against this lipoprotein. These findings furthermore suggest that this lipoprotein is a receptor protein attached to the membrane of S. pneumoniae that binds haem. Despite these findings, the interaction molecular between Sphbp-37 lipoprotein and haem has not been analyzed yet and neither how spbhp-37 gene expression occurs when haem binds to the lipoprotein. To dilucidated how the lipoprotein binds haem a strategy could be designed, for instance, investigate 3D structure, the interaction between the Sphbp-37 lipoprotein and haem, or amino acid residues that interact with haem and levels of mRNA of the spbhp-37. The aim of this chapter attempt to explain how S. pneumoniae binds the iron source and the pathway developed to increase its levels of iron maybe this increase help to this pathogen to invade multiple human tissues, in the future this information could be utilized to develop new treatment allowing better control of this human pathogen.

2. S. pneumoniae iron acquisition

S. pneumoniae has the polysaccharide capsule and various virulence factors of S. pneumoniae that participate in its pathogenesis and facilitate its dissemination [22]. As any bacterium needs iron for several essential functions like the electron transport chain, energy metabolism, and many other biological functions [23], this element can be obtained from human sources such as hemoglobin (Hb), haem (from erythrocytes) ferritin (from serum and secretions) and glycoproteins (transferrin and lactoferrin) [24, 25]. Interestingly S. pneumoniae proteome does not have ferritin binding proteins (FBPs) [26] and also lacks of siderophores [27] because the large layer of the capsule avoids the presence of siderophores binding protein. To obtain iron for sources like ferritin, this pathogen has developed a smart mechanism that consists on to express PspA protein, this protein plays a key role in binding to lactoferrin at the pneumococcal surface. This mechanism could be useful in tissues like epithelial secretions where lactoferrin is the only source of iron. The expression of PspA is associated with the reduced concentration of free iron in secretions [28]. In vitro studies have showed that, the concentration of ferritin can be preserved in the culture media when a protease inhibitor (PMSF) is added, also molecular docking of pneumococcal proteases such as HtrA, ClpP, and RadA has revealed that those proteases could be inhibited by the PMSF [26]. Therefore, these observations indicated that S. pneumoniae may recruit protease-dependent pathways to obtain iron. Such pathways provide several effective strategies obviating the need for specific receptors and transporters.

3. Hb-binding proteins involved in iron acquisition

Iron is also available in human sources for instance hemoglobin or haem structure within erythrocytes. S. pneumoniae expressed and secreted an Hb and haem-binding protein of 38 kDa. This protein has a multitasking function because was identified by mass spectrometry as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) a protein involved in metabolism principally. S. pneumoniae secretes GAPDH and this is protein is capable of binding two useful iron sources for this bacterium (Hb and haem). This protein could be playing a dynamic role in the success of the invasive and infective processes of this pathogen [29]. Additionally, S. pneumoniae is a pathogen capable of supporting its viability when iron sources such as Hb or haem are supplied. This bacterium can express two haem and Hb-binding proteins on its cytoplasmic membrane, whose molecular weights are 37 and 22 kDa respectively. Their respective names are Spbhp-37 and Spbhp-22 (S. pneumoniae Hb- and haem-binding proteins 37 and 22 kDa). The Hb-binding function in both proteins has been demonstrated using Hb and the respective identities of both proteins have been obtained by mass spectrometry. The amino acid sequences of both Hb-binding proteins have the motif involved in the binding of Hb or haem. Specifically, Spbhp-37 protein is founded in the surface of this pathogen. The expression of Spbhp-37 is increased when the bacterium is grown in media culture supplied with Hb this finding corroborates the importance of this protein in the Hb-binding. It could explain the mechanisms developed by S. pneumoniae to acquire iron from Hb or haem in the host, which could allow a better understanding of the biology of this bacterium.

4. Hb-iron transporters

The necessity to obtain iron in the human host has provided S. pneumoniae with a sophisticated mechanism. PiaA is a hemoglobin-binding protein localized on the surface of S. pneumoniae [27]. Moreover, Pit, Pia, and Piu have emerged as other possible iron transporters of S. pneumoniae [28]. Brown et al. reported that ABC transporter-like proteins, may be involved in iron absorption by S. pneumoniae [19, 20]. Overall, all these proteins further participate in the acquisition of this essential metal for the survival of this pathogen.

5. Which are the amino acid residues of Sphbp-37 haem-binding protein involved in the interaction with the iron source?

5.1 3D modeling of Sphbp-37 protein

To understand more about the interaction between haem-binding protein Sphbp-37 and iron source an in silico approach can be performed, for instance, 3D modeling of Sphbp-37 protein shows a globular structure (Figure 1A). This structure has nine α-helices and eleven β-sheet. After molecular dynamic simulation of 500 ns by I-TASSER server, Sphbp-37 protein stills its globular structure, showing the same number of α-helices, although the number of β-sheet was increased to thirteen (Figure 1B). Therefore the analysis of time-dependent motions of the Sphbp-37 protein by RMSD shows that this molecule gets its equilibrium after 500 ns (Figure 2A). Rg values show the protein expansion after 100 and 200 ns and a compactation at the last 20 ns (Figure 2B). RMSF value show a moderate fluctuation in some amino acid (Figure 2C).

Figure 1.

3D Modeling of Sphbp-37 protein before (A) and after (B) of 500 ns dynamic molecular simulation by I-TASSER program.

Figure 2.

RMSD analysis of Sphbp-37 protein (A). Rg values show the protein expansion after 100 and 200 ns and a compactation at the last 20 ns (B). RMSF values (C).

5.2 Interaction of Sphbp-37 protein with haem

We search the amino acids involved in the between Sphbp-37 protein and haem. The molecular dynamic simulation after 500 ns shows an interaction between haem and amino acid residues of Sphbp-37 protein: glutamic acid 152 (Glu152), glutamine 177 (Gln177), valine 178 (Val178), aspartic acid 179 (Asp179), tyrosine 180 (Tyr180), isoleucine 193 (Ile193), alanine 196 (Ala196), glutamine 197 (Gln197) and alanine 200 (Ala200) (Figure 3). We found 10 amino acids involved in the interaction of Sphbp-37 and haem.

Figure 3.

Molecular analysis between Sphbp-37 lipoprotein and haem. (A) Interaction between Sphbp-37 lipoprotein and haem. (B) The 10 amino acid residues, which are showed in green and pink color.

5.3 3D model of mutant Sphbp-37 protein (substitutions in 152 and 179 amino acid residues)

To investigate which amino acids of Sphbp-37 protein are involved in haem-binding, we performed a change of glu152 for alanine (glu152ala) and asp179 for alanine ala (asp179ala) (mutant Sphbp-37 protein), these amino acid directly binds the haem. The result showed that the substitution of amino acid in the position 152 and 179 by another amino acid does not allow the binding to the haem. These data shown that amino acids 152 and 179 are essential for haem or Hb-binding and participate direct binding of the iron source.

3D model of mutant Sphbp-37 protein with changes in 152 and 179 amino acid residues was analyzed by NAMD software, the result showed a globular structure inclusive after the changes, however, the binding is not preserved (Figure 4). Mutant Sphbp-37 protein is unable to bind haem these results suggest that amino acids residues of 152 and 179 positions are involved in haem binding directly.

Figure 4.

3D modeling of Sphbp-37 mutant before (A) and after (B) of 500 ns of molecular dynamic simulation by I-TASSER server. Amino acid residues of 152 position (glu) and 179 position (asp) were substituted by the ala. The globular structure was maintained.

5.4 The promoter region of the spbhp-37 gene does not fur box consensus sequences

Then, we analyzed the spbhp-37 gene and search a probable promoter sequence by the BPROM program. The analysis revealed the regions promoter −35 and −10 located upstream from the start codon (Figure 5A). The promoter sequence of the spbhp-37 gene does not align with the consensus Fur box of E. coli [30] and other Fur box-like sequences previously reported such as dhb [31], fhu [32] the fhu and sir operons from B. subtilis [33] and operons from S. aureus, with the program Jalwiew (http://www.jalview.org). The alignment showed a different sequence from those previously reported, showing only a 26% of identity when the sequence promoter of S. pneumoniae is compared with the consensus fur-box sequence of E. coli (Figure 5B). This analysis suggests that the promoter sequence of S. pneumoniae does not have fur-box and perhaps the regulation could be by a different mechanism (data in progress).

Figure 5.

Promoter predicted elements for spbhp-37 gene. (A) Schematic organization of spbhp-37 promoter, the translation-initiation codon ATG +1, −35 and − 10 regions of the promoter sequence are indicated in bold type and underlined. The box indicates the conserved fur-binding sequences. (B) Alignment of the consensus “fur box” sequences of E. coli, dhb and fhu of B. subtilis, fhu and sir of S. aureus and the spbhp-37. The differences are indicated in the boxes.

6. Discussion

S. pneumoniae is a pathogen that uses Hb and haem as only iron sources. It also expresses a lipoprotein Sphbp-37 that participates in iron acquisition binding Hb and haem [17]. This chapter presents the first effort to understand how S. pneumoniae can be stimulated for iron acquisition: under iron starvation, which could occur as in other bacterial pathogens such as Vibrio sp., Pseudomonas aeruginosa, Escherichia coli, Shigella flexneri, Bacillus subtilis [34] or with Holo-Tf stimulation such as occurs in E. histolytica [35]. Interestingly, proteins levels of the Sphbp-37 increased: first, when iron was chelated from culture media and Hb was used as an iron source, this observation was notable because in previous reports the attention was principally focused only on iron starvation, in which there is an overexpression of genes participating in iron acquisition [36]. Probably Sphbp-37 is overexpressed when S. pneumoniae requires increasing its levels of iron. Perhaps the whole mechanism involves other genes which have not related with this mechanism for instance hemolysin that lyses erythrocytes to release high amounts of Hb or haem, in this manner, it is available and could be use by S. pneumoniae. In silico analysis of 200 nucleotides upstream from the sphbp-37 start codon revealed a promoter sequence with nucleotides [37] however, no homology or identity was observed when it was compared with the fur box sequence reported for E. coli [30], dhb [31] and fhu [32]), operons of B. subtilis, the fhu and sir [33] and operons of S. aureus. This data suggests that the differences observed in the nucleotide sequences could be related with a regulation mechanism of S. pneumoniae that involves many proteins, but different to described for Gram-negative bacteria [38]. Maybe all these proteins are involved in the binding to iron, haem or Hb-binding proteins and are necessary when the pathogen invades tissue. Finally, it does not discard the possibility that this type of regulation occurs in other bacterial pathogens.

Conflict of interest

The authors declare that they have no conflict of interest.