Streptococcus pneumoniae Invades Erythrocytes and Utilizes Them to Evade Human Innate Immunity

Streptococcus pneumoniae, a Gram-positive bacterium, is a major cause of invasive infection-related diseases such as pneumonia and sepsis. In blood, erythrocytes are considered to be an important factor for bacterial growth, as they contain abundant nutrients. However, the relationship between S. pneumoniae and erythrocytes remains unclear. We analyzed interactions between S. pneumoniae and erythrocytes, and found that iron ion present in human erythrocytes supported the growth of Staphylococcus aureus, another major Gram-positive sepsis pathogen, while it partially inhibited pneumococcal growth by generating free radicals. S. pneumoniae cells incubated with human erythrocytes or blood were subjected to scanning electron and confocal fluorescence microscopic analyses, which showed that the bacterial cells adhered to and invaded human erythrocytes. In addition, S. pneumoniae cells were found associated with human erythrocytes in cultures of blood from patients with an invasive pneumococcal infection. Erythrocyte invasion assays indicated that LPXTG motif-containing pneumococcal proteins, erythrocyte lipid rafts, and erythrocyte actin remodeling are all involved in the invasion mechanism. In a neutrophil killing assay, the viability of S. pneumoniae co-incubated with erythrocytes was higher than that without erythrocytes. Also, H₂O₂ killing of S. pneumoniae was nearly completely ineffective in the presence of erythrocytes. These results indicate that even when S. pneumoniae organisms are partially killed by iron ion-induced free radicals, they can still invade erythrocytes. Furthermore, in the presence of erythrocytes, S. pneumoniae can more effectively evade antibiotics, neutrophil phagocytosis, and H₂O₂ killing.     

Iron Binding at Specific Sites within the Octameric HbpS Protects Streptomycetes from Iron-Mediated Oxidative Stress

The soil bacterium Streptomyces reticuli secretes the octameric protein HbpS that acts as a sensory component of the redox-signalling pathway HbpS-SenS-SenR. This system modulates a genetic response on iron- and haem-mediated oxidative stress. Moreover, HbpS alone provides this bacterium with a defence mechanism to the presence of high concentrations of iron ions and haem. While the protection against haem has been related to its haem-binding and haem-degrading activity, the interaction with iron has not been studied in detail. In this work, we biochemically analyzed the iron-binding activity of a set of generated HbpS mutant proteins and present evidence showing the involvement of one internal and two exposed D/EXXE motifs in binding of high quantities of ferrous iron, with the internal E₇₈XXE₈₁ displaying the tightest binding. We additionally show that HbpS is able to oxidize ferrous to ferric iron ions. Based on the crystal structure of both the wild-type and the mutant HbpS-D₇₈XXD₈₁, we conclude that the local arrangement of the side chains from the glutamates in E₇₈XXE₈₁ within the octameric assembly is a pre-requisite for interaction with iron. The data obtained led us to propose that the exposed and the internal motif build a highly specific route that is involved in the transport of high quantities of iron ions into the core of the HbpS octamer. Furthermore, physiological studies using Streptomyces transformants secreting either wild-type or HbpS mutant proteins and different redox-cycling compounds led us to conclude that the iron-sequestering activity of HbpS protects these soil bacteria from the hazardous side effects of peroxide- and iron-based oxidative stress.     

Chemical Interference with Iron Transport Systems to Suppress Bacterial Growth of Streptococcus pneumoniae

Iron is an essential nutrient for the growth of most bacteria. To obtain iron, bacteria have developed specific iron-transport systems located on the membrane surface to uptake iron and iron complexes such as ferrichrome. Interference with the iron-acquisition systems should be therefore an efficient strategy to suppress bacterial growth and infection. Based on the chemical similarity of iron and ruthenium, we used a Ru(II) complex R-825 to compete with ferrichrome for the ferrichrome-transport pathway in Streptococcus pneumoniae. R-825 inhibited the bacterial growth of S. pneumoniae and stimulated the expression of PiuA, the iron-binding protein in the ferrichrome-uptake system on the cell surface. R-825 treatment decreased the cellular content of iron, accompanying with the increase of Ru(II) level in the bacterium. When the piuA gene (SPD_0915) was deleted in the bacterium, the mutant strain became resistant to R-825 treatment, with decreased content of Ru(II). Addition of ferrichrome can rescue the bacterial growth that was suppressed by R-825. Fluorescence spectral quenching showed that R-825 can bind with PiuA in a similar pattern to the ferrichrome-PiuA interaction in vitro. These observations demonstrated that Ru(II) complex R-825 can compete with ferrichrome for the ferrichrome-transport system to enter S. pneumoniae, reduce the cellular iron supply, and thus suppress the bacterial growth. This finding suggests a novel antimicrobial approach by interfering with iron-uptake pathways, which is different from the mechanisms used by current antibiotics.     

Characterization of Streptococcus pneumoniae N-acetylglucosamine-6-phosphate deacetylase as a novel diagnostic marker

The identification of novel diagnostic markers of pathogenic bacteria is essential for improving the accuracy of diagnoses and for developing targeted vaccines. Streptococcus pneumoniae is a significant human pathogenic bacterium that causes pneumonia. N-acetylglucosamine-6-phosphate deacetylase (NagA) was identified in a protein mixture secreted by S. pneumoniae and its strong immunogenicity was confirmed in an immuno-proteomic assay against the anti-serum of the secreted protein mixture. In this study, recombinant S. pneumoniae NagA protein was expressed and purified to analyze its protein characteristics, immunospecificity, and immunogenicity, thereby facilitating its evaluation as a novel diagnostic marker for S. pneumoniae. Mass spectrometry analysis showed that S. pneumoniae NagA contains four internal disulfide bonds and that it does not undergo post-translational modification. S. pneumoniae NagA antibodies successfully detected NagA from different S. pneumoniae strains, whereas NagA from other pathogenic bacteria species was not detected. In addition, mice infected with S. pneumoniae generated NagA antibodies in an effective manner. These results suggest that NagA has potential as a novel diagnostic marker for S. pneumoniae because of its high immunogenicity and immunospecificity.     

The gene frpB2 of Helicobacter pylori encodes an hemoglobin-binding protein involved in iron acquisition

Human hemoglobin (Hb) is a metalloprotein used by pathogens as a source of iron during invasive process. It can support the Helicobacter pylori growth and several proteins are induced during iron starvation. However, the identity of those proteins remains unknown. In this work, by in silico analysis we identified FrpB2 in H. pylori genome. This protein was annotated as an iron-regulated outer membrane protein. Multiple amino acid alignment showed the motifs necessary for Hb-binding. We demonstrate the ability of FrpB2 to bind Hb by overlay experiments. In addition, the overexpression of this gene allowed the cell growth in media without free iron but supplemented with Hb. All these results support the idea that frpB2 is a gene of H. pylori involved in iron acquisition when Hb is used as a sole iron source.     

1H, 13C, 15N backbone and side chain NMR resonance assignments of the N-terminal NEAr iron transporter domain 1 (NEAT 1) of the hemoglobin receptor IsdB of Staphylococcus aureus

Staphylococcus aureus is an opportunistic pathogen that causes skin and severe infections in mammals. Critical to S. aureus growth is its ability to scavenge iron from host cells. To this effect, S. aureus has evolved a sophisticated pathway to acquire heme from hemoglobin (Hb) as a preferred iron source. The pathway is comprised of nine iron-regulated surface determinant (Isd) proteins involved in heme capture, transport, and degradation. A key protein of the heme acquisition pathway is the surface-anchored hemoglobin receptor protein IsdB, which is comprised of two NEAr transporter (NEAT) domains that act in concert to bind Hb and extract heme for subsequent transfer to downstream acquisition pathway proteins. Despite significant advances in the structural knowledge of other Isd proteins, the structural mechanisms and molecular basis of the IsdB-mediated heme acquisition process are not well understood. In order to provide more insights into the mode of function of IsdB, we have initiated NMR structural studies of the first NEAT domain of IsdB (IsdB^N1). Herein, we report the near complete ¹H, ¹³C and ¹⁵N resonance assignments of backbone and side chain atoms, and the secondary structural topology of the 148-residue IsdB NEAT 1 domain. The NMR results are consistent with the presence of eight β-strands and one α-helix characteristic of an immunoglobulin-like fold observed in other NEAT domain family proteins. This work provides a solid framework to obtain atomic-level insights toward understanding how IsdB mediates IsdB-Hb protein–protein interactions critical for heme capture and transfer.     

Elemental iron does repress transferrin, haemopexin and haemoglobin receptor expression in Haemophilusinfluenzae

The iron repressible nature of Haemophilus influenzae transferrin binding proteins suggests a regulatory role for elemental iron in their expression. The existence of a Haemophilus ferric uptake repressor (Fur) binding motif identified in the promoter region of both tbpA and tbpB further supports this hypothesis. However, a recent study using brain heart infusion growth medium suggested that transferrin binding protein synthesis in H. influenzae was haem- rather than iron-regulated. The present study re-investigates this observation and using a chemically defined medium, we demonstrate that elemental iron haem or protoporphyrin IX can each regulate Haemophilus influenzae transferrin, haemopexin and haemoglobin receptor expression.     

Mechanisms of Heme Utilization by Francisella tularensis

Francisella tularensis is a highly virulent facultative intracellular pathogen causing the severe disease tularemia in mammals. As for other bacteria, iron is essential for its growth but very few mechanisms for iron acquisition have been identified. Here, we analyzed if and how F. tularensis can utilize heme, a major source of iron in vivo. This is by no means obvious since the bacterium lacks components of traditional heme-uptake systems. We show that SCHU S4, the prototypic strain of subspecies tularensis, grew in vitro with heme as the sole iron source. By screening a SCHU S4 transposon insertion library, 16 genes were identified as important to efficiently utilize heme, two of which were required to avoid heme toxicity. None of the identified genes appeared to encode components of a potential heme-uptake apparatus. Analysis of SCHU S4 deletion mutants revealed that each of the components FeoB, the siderophore system, and FupA, contributed to the heme-dependent growth. In the case of the former two systems, iron acquisition was impaired, whereas the absence of FupA did not affect iron uptake but led to abnormally high binding of iron to macromolecules. Overall, the present study demonstrates that heme supports growth of F. tularensis and that the requirements for the utilization are highly complex and to some extent novel.     

Menaquinone biosynthesis potentiates haem toxicity in Staphylococcus aureus

Staphylococcus aureus is a pathogen that infects multiple anatomical sites leading to a diverse array of diseases. Although vertebrates can restrict the growth of invading pathogens by sequestering iron within haem, S. aureus surmounts this challenge by employing high‐affinity haem uptake systems. However, the presence of excess haem is highly toxic, necessitating tight regulation of haem levels. To overcome haem stress, S. aureus expresses the detoxification system HrtAB. In this work, a transposon screen was performed in the background of a haem‐susceptible, HrtAB‐deficient S. aureus strain to identify the substrate transported by this putative pump and the source of haem toxicity. While a recent report indicates that HrtAB exports haem itself, the haem‐resistant mutants uncovered by the transposon selection enabled us to elucidate the cellular factors contributing to haem toxicity. All mutants identified in this screen inactivated the menaquinone (MK) biosynthesis pathway. Deletion of the final steps of this pathway revealed that quinone molecules localizing to the cell membrane potentiate haem‐associated superoxide production and subsequent oxidative damage. These data suggest a model in which membrane‐associated haem and quinone molecules form a redox cycle that continuously generates semiquinones and reduced haem, both of which react with atmospheric oxygen to produce superoxide.     

Influence of environmental dust in transformation capacity of Streptococcus pneumoniae

S. pneumoniae, commonly known as pneumococcus, is a naturally competent Gram-positive bacterium and is the major cause of pneumonia in elderly and children in developing countries. This pathogen is associated with respiratory diseases affected by pollution. The objective of this work was determining the effect of ash and environmental dust from the burning of sugarcane on pneumococci bacterial transformation. The transformation capacity of the Pn360 pneumococci strain was performed using the assays of DNA donor of mutant for luxS gene. Thus, the transformation tests were performed in contact with dust collected in the southwestern region of Brazil (important region where burning of sugar cane is present in the agriculture). The use of degradative practices in the sugar cane agriculture in Brazil was involved in the transformation capacity of the S. pneumoniae. This phenomenon includes important consequences for public health concerning to resistance acquisition and new virulence factors of this important infection. In conclusion, we obtained important results concerning the action of environmental pollution in Streptococcus pneumoniae transformation, increasing the DNA acquisition for this pathogen.     

Structural basis for Streptococcus pneumoniae NanA inhibition by influenza antivirals zanamivir and oseltamivir carboxylate

The human pathogen Streptococcus pneumoniae is the major cause of bacterial meningitis, respiratory tract infection, septicemia, and otitis media. The bacterium expresses neuraminidase (NA) proteins that contribute to pathogenesis by cleaving sialic acids from host glycoconjugates, thereby enhancing biofilm formation and colonization. Recent in vivo experiments have shown that antiviral compounds, widely used in clinics and designed to inhibit influenza NA, significantly reduce biofilm formation and nasopharyngeal colonization of S. pneumoniae in mice. Here, we present the structural basis for the beneficial effect of these compounds against pneumococcal infection. Crystal structures of pneumococcal NanA in complex with zanamivir and oseltamivir carboxylate are discussed, correlated with measured inhibitory constants K_i, and compared with the binding modes of the inhibitors in the viral enzyme. Inhibitor structures show for the first time how clinically approved anti-influenza compounds interact with an NA of the human pathogen S. pneumoniae and give a rational explanation for their antibacterial effects.     

The Type III Secretion System-Related CPn0809 from Chlamydia pneumoniae

Chlamydia pneumoniae is an intracellular Gram-negative bacterium that possesses a type III secretion system (T3SS), which enables the pathogen to deliver, in a single step, effector proteins for modulation of host-cell functions into the human host cell cytosol to establish a unique intracellular niche for replication. The translocon proteins located at the top of the T3SS needle filament are essential for its function, as they form pores in the host-cell membrane. Interestingly, unlike other Gram-negative bacteria, C. pneumoniae has two putative translocon operons, named LcrH_1 and LcrH_2. However, little is known about chlamydial translocon proteins. In this study, we analyzed CPn0809, one of the putative hydrophobic translocators encoded by the LcrH_1 operon, and identified an ‘SseC-like family’ domain characteristic of T3S translocators. Using bright-field and confocal microscopy, we found that CPn0809 is associated with EBs during early and very late phases of a C. pneumoniae infection. Furthermore, CPn0809 forms oligomers, and interacts with the T3SS chaperone LcrH_1, via its N-terminal segment. Moreover, expression of full-length CPn0809 in the heterologous host Escherichia coli causes a grave cytotoxic effect that leads to cell death. Taken together, our data indicate that CPn0809 likely represents one of the translocon proteins of the C. pneumoniae T3SS, and possibly plays a role in the translocation of effector proteins in the early stages of infection.     

Mechanisms of iron and haem transport by Listeria monocytogenes

Abstract

Listeria monocytogenes, the causative agent of listeriosis, is a virulent foodborne Gram-positive bacterial pathogen, with 20–30% mortality. It has a broad ability to transport iron, either in the form of ferric siderophores, or by extracting it from mammalian iron binding proteins. In this review we focus on the mechanisms of ferric siderophore and haem transport into the listerial cell. Despite the fact that it does not synthesize siderophores, L. monocytogenes transports ferric siderophores in the wild environment by the actions of cytoplasmic membrane ABC-transporter systems. The bacterium acquires haem, on the other hand, by two mechanisms. At low (nanomolar) concentrations, sortase B-dependent, peptidoglycan-anchored proteins scavenge the iron porphyrin in human or animal tissues, and transfer it to the underlying ABC-transporters in the cytoplasmic membrane for uptake. At concentrations at or above 50 nM, however, haem transport becomes sortase-independent, and occurs by direct interactions of the iron porphyrin with the same ABC-transporter complexes. The architecture of the Gram-positive cell envelope plays a fundamental role in these mechanisms, and the haem acquisition abilities of L. monocytogenes are an element of its ability to cause infectious disease.     

Utilization of hemin and hemoglobin by Vibrio vulnificus biotype 2.

The eel pathogen Vibrio vulnificus biotype 2 is able to use hemoglobin (Hb) and hemin (Hm) to reverse iron limitation. In this stud, the adjuvant effect of both compounds on eel pathogenicity has been evaluated and confirmed. Further, we have studied the heme-iron acquisition mechanism displayed by this bacterium. Whole cells were capable of binding Hb and Hm, independently of (i) iron levels in growth medium and (ii) the presence of polysaccharide capsules on bacterial surface. The Hb- and Hm-binding capacity was retained by the outer membrane protein (OMP) fraction and was abolished after proteolytic digestion of OMP samples. Western blotting (immunoblotting) of denatured OMPs revealed that two major protein bands of 36 and 32 kDa were involved in both Hm and Hb binding. The expression of these proteins was not affected by iron levels. In addition, V. vulnificus biotype 2 produced extracellular proteases, not regulated by iron, that were active against native Hb. In conclusion, the overall data suggest that the eel pathogen V. vulnificus biotype 2 can obtain iron by means of a mechanism which involves a direct interaction between the heme moiety and constitutive OMPs.     

Blood Group Antigen Recognition by a Solute-Binding Protein from a Serotype 3 Strain of Streptococcus pneumoniae

Streptococcus pneumoniae is a common bacterial pathogen that is well known for its ability to cause acute respiratory disease (pneumonia), ear infections, and other serious illnesses. This Gram-positive bacterium relies on its carbohydrate-metabolizing capabilities for full virulence in its host; however, the range of glycan targets that it can attack is presently not fully appreciated. S. pneumoniae is known to have a fucose utilization operon that in the TIGR4 strain plays a role in its virulence. Here we identify a second type of fucose utilization operon that is present in a subset of S. pneumoniae strains, including the serotype 3 strain SP3-BS71. This operon contains a transporter with a solute-binding protein, FcsSBP (fucose solute-binding protein), that interacts tightly (K_a ∼ 1 × 10⁶ M^− 1) and specifically with soluble A- and B-antigen trisaccharides but displays no selectivity between these two sugars. The structure of the FcsSBP in complex with the A-trisaccharide antigen, determined to 2.35 Å, reveals its mode of binding to the reducing end of this sugar, thus highlighting this protein's requirement for soluble blood group antigen ligands. Overall, this report exposes a heretofore unknown capability of certain S. pneumoniae strains to transport and potentially metabolize the histo-blood group antigen carbohydrates of its host.     

Analysis of a Streptococcus pneumoniae gene encoding signal peptidase I and overproduction of the enzyme

The spi gene of Streptococcus pneumoniae was cloned and its nucleotide sequence was determined. It encodes a protein of 204 amino acids that is homologous to bacterial signal peptidase I proteins. The S. pneumoniae protein contains all of the conserved amino acid sequence motifs previously identified in this enzyme from both prokaryotic and eukaryotic sources. Sequence comparisons revealed several additional motifs characteristic of the enzyme. The cloned S. pneumoniae gene complemented an Escherichia coli mutant defective in its leader peptidase gene. Expression of the spi gene in S. pneumoniae appeared to be essential for viability. The cloned gene was shown to produce a polypeptide of approximately 20 kDa. Overproduction of the S. pneumoniae spi gene in an E. coli expression system gave a native protein product, soluble in the presence of a non-ionic detergent, which should be amenable to structural determination.     

Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method

During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen Streptococcus pneumoniae and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in S. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in S. pneumoniae and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in S. pneumoniae and related organisms.