Growth In Human Blood Research Articles

Molecular characterization of the interaction between human IgG and the M-related proteins from Streptococcus pyogenes

Group A Streptococcal M-related proteins (Mrp) are dimeric α-helical-coiled-coil cell membrane-bound surface proteins. During infection, Mrp recruit the fragment crystallisable region of human immunoglobulin G (IgG-Fc) via their A-repeat regions to the bacterial surface, conferring upon the bacteria enhanced phagocytosis resistance and augmented growth in human blood. However, Mrps show a high degree of sequence diversity, and it is currently not known whether this diversity affects the Mrp-IgG interaction. Herein, we report that diverse Mrps all bind human IgG subclasses with nanomolar affinity, with differences in affinity which ranged from 3.7-11.1 nM for mixed IgG. Using surface plasmon resonance, we confirmed Mrps display preferential IgG-subclass binding. All Mrps were found to have a significantly weaker affinity for IgG3 (p<0.05) compared to all other IgG-subclasses. Furthermore, plasma pulldown assays analysed via western blotting revealed that all Mrp were able to bind IgG in the presence of other serum proteins at both 25°C and 37°C. Finally, we report for the first time that dimeric Mrps bind to IgG with a 1:1 stoichiometry, enhancing our understanding of this important host-pathogen interaction.

Electrochemical label-free pathogen identification for bloodstream infections diagnosis: Towards a machine learning based smart blood culture bottle

Bloodstream infections are a growing public health concern. Current pathogen identification systems are based on complex and expensive devices, intended for use in centralized laboratories. Subsequent identification requires harmful chemical reagents, specialized personnel and time. Here we describe a new approach for rapid and decentralized diagnosis of positive blood cultures using electrochemical sensors. By implementing a multi-material potentiometric platform in a blood culture bottle, we have developed a portable system for pathogen identification of Gram and of genus. Bacterial growth in human blood generates a specific label-free multiplex electrochemical fingerprint according to the detected species. Analysis of these fingerprints using homemade machine learning algorithms allow for rapid identification of the pathogen after detection (14 species and 9 genus) (GRAM= 99 % accuracy 5.75 h after detection, GENUS= 85 % accuracy 7.8 h after positivity) without further handling of the contaminated sample.

Loss of Dihydroxyacid Dehydratase Induces Auxotrophy in Bacillus anthracis.

Anthrax disease is caused by infection with the bacteria Bacillus anthracis which, if left untreated, can result in fatal bacteremia and toxemia. Current treatment for infection requires prolonged administration of antibiotics. Despite this, inhalational and gastrointestinal anthrax still result in lethal disease. By identifying key metabolic steps that B. anthracis uses to grow in host-like environments, new targets for antibacterial strategies can be identified. Here, we report that the ilvD gene, which encodes dihydroxyacid dehydratase in the putative pathway for synthesizing branched chain amino acids, is necessary for B. anthracis to synthesize isoleucine de novo in an otherwise limiting microenvironment. We observed that ΔilvD B. anthracis cannot grow in media lacking isoleucine, but growth is restored when exogenous isoleucine is added. In addition, ΔilvD bacilli are unable to utilize human hemoglobin or serum albumin to overcome isoleucine auxotrophy, but can when provided with the murine forms. This species-specific effect is due to the lack of isoleucine in human hemoglobin. Furthermore, even when supplemented with physiological levels of human serum albumin, apotransferrin, fibrinogen, and IgG, the ilvD knockout strain grew poorly relative to nonsupplemented wild type. In addition, comparisons upon infecting humanized mice suggest that murine hemoglobin is a key source of isoleucine for both WT and ΔilvD bacilli. Further growth comparisons in murine and human blood show that the auxotrophy is detrimental for growth in human blood, not murine. This report identifies ilvD as necessary for isoleucine production in B. anthracis, and that it plays a key role in allowing the bacilli to effectively grow in isoleucine poor hosts. IMPORTANCE Anthrax disease, caused by B. anthracis, can cause lethal bacteremia and toxemia, even following treatment with antibiotics. This report identifies the ilvD gene, which encodes a dihydroxyacid dehydratase, as necessary for B. anthracis to synthesize the amino acid isoleucine in a nutrient-limiting environment, such as its mammalian host. The use of this strain further demonstrated a unique species-dependent utilization of hemoglobin as an exogenous source of extracellular isoleucine. By identifying mechanisms that B. anthracis uses to grow in host-like environments, new targets for therapeutic intervention are revealed.

The 5'-nucleotidase S5nA is dispensable for evasion of phagocytosis and biofilm formation in Streptococcus pyogenes.

5’-nucleotidases are widespread among all domains of life. The enzymes hydrolyze phosphate residues from nucleotides and nucleotide derivatives. In some pathobiontic bacteria, 5’-nucleotidases contribute to immune evasion by dephosphorylating adenosine mono-, di-, or tri-phosphates, thereby either decreasing the concentration of pro-inflammatory ATP or increasing the concentration of anti-inflammatory adenosine, both acting on purinergic receptors of phagocytic cells. The strict human pathogen Streptococcus pyogenes expresses a surface-associated 5’-nucleotidase (S5nA) under infection conditions that has previously been discussed as a potential virulence factor. Here we show that deletion of the S5nA gene does not significantly affect growth in human blood, evasion of phagocytosis by neutrophils, formation of biofilms and virulence in an infection model with larvae of the greater wax moth Galleria mellonella in S. pyogenes serotypes M6, M18 and M49. Hence, the surface-associated 5’-nucleotidase S5nA seems dispensable for evasion of phagocytosis and biofilm formation in S. pyogenes.

Identification of Staphylococcus aureus Factors Required for Pathogenicity and Growth in Human Blood.

ABSTRACTStaphylococcus aureus is a human commensal but also has devastating potential as an opportunistic pathogen. S. aureus bacteremia is often associated with an adverse outcome. To identify potential targets for novel control approaches, we have identified S. aureus components that are required for growth in human blood. An ordered transposon mutant library was screened, and 9 genes involved specifically in hemolysis or growth on human blood agar were identified by comparing the mutants to the parental strain. Three genes (purA, purB, and pabA) were subsequently found to be required for pathogenesis in the zebrafish embryo infection model. The pabA growth defect was specific to the red blood cell component of human blood, showing no difference from the parental strain in growth in human serum, human plasma, or sheep or horse blood. PabA is required in the tetrahydrofolate (THF) biosynthesis pathway. The pabA growth defect was found to be due to a combination of loss of THF-dependent dTMP production by the ThyA enzyme and increased demand for pyrimidines in human blood. Our work highlights pabA and the pyrimidine salvage pathway as potential targets for novel therapeutics and suggests a previously undefined role for a human blood factor in the activity of sulfonamide antibiotics.

Deletion of the L-Lactate Dehydrogenase Gene ldh in Streptococcus pyogenes Leads to a Loss of SpeB Activity and a Hypovirulent Phenotype.

Streptococcus pyogenes uses lactic acid fermentation for the generation of ATP. Here, we analyzed the impact of a deletion of the L-lactate dehydrogenase gene ldh on the virulence of S. pyogenes M49. While the ldh deletion does not cause a general growth deficiency in laboratory media, the growth in human blood and plasma is significantly hampered. The ldh deletion strain is furthermore less virulent in a Galleria mellonella infection model. We show that the ldh deletion leads to a decrease in the activity of the cysteine protease SpeB, an important secreted virulence factor of S. pyogenes. The reduced SpeB activity is caused by a hampered autocatalytic activation of the SpeB zymogen into the mature SpeB. The missing SpeB activity furthermore leads to increased plasmin activation and a reduced activation of the contact system on the surface of S. pyogenes. All these effects can be reversed when ldh is reintroduced into the mutant via a plasmid. The results demonstrate a previously unappreciated role for LDH in modulation of SpeB maturation.

TSPO ligands stimulate ZnPPIX transport and ROS accumulation leading to the inhibition of P. falciparum growth in human blood.

After invading red blood cells (RBCs), Plasmodium falciparum (Pf) can export its own proteins to the host membrane and activate endogenous channels that are present in the membrane of RBCs. This transport pathway involves the Voltage Dependent Anion Channel (VDAC). Moreover, ligands of the VDAC partner TranSlocator PrOtein (TSPO) were demonstrated to inhibit the growth of the parasite. We studied the expression of TSPO and VDAC isoforms in late erythroid precursors, examined the presence of these proteins in membranes of non-infected and infected human RBCs, and evaluated the efficiency of TSPO ligands in inhibiting plasmodium growth, transporting the haem analogue Zn-protoporphyrin-IX (ZnPPIX) and enhancing the accumulation of reactive oxygen species (ROS). TSPO and VDAC isoforms are differentially expressed on erythroid cells in late differentiation states. TSPO2 and VDAC are present in the membranes of mature RBCs in a unique protein complex that changes the affinity of TSPO ligands after Pf infection. TSPO ligands dose-dependently inhibited parasite growth, and this inhibition was correlated to ZnPPIX uptake and ROS accumulation in the infected RBCs. Our results demonstrate that TSPO ligands can induce Pf death by increasing the uptake of porphyrins through a TSPO2–VDAC complex, which leads to an accumulation of ROS.

The volatile metabolome of Klebsiella pneumoniae in human blood

Klebsiella pneumoniae is an important cause of bloodstream infections in critically-ill patients, with mortality exceeding 50% for infections caused by antibiotic-resistant strains. Despite its importance as a bacteremia-causing agent, there is little known about the metabolism of K. pneumoniae during growth in pure human blood.Here, we comprehensively profile the volatile metabolites produced by K. pneumoniae during growth in human blood to approximately mid-exponential (7 h) and early stationary (12 h) phases using 2D gas chromatography–time-of-flight mass spectrometry (GC×GC-TOFMS). We identified 33 volatile molecules that were significantly more abundant in K. pneumoniae cultures relative to sterile blood, of which 22 were detected in cultures only. We identified nine molecules that have not previously been reported as K. pneumoniae-associated headspace volatiles, four of which we believe to be novel bacterial-associated volatiles. We also identified a set of 17 volatile molecules that discriminate between 7 h and 12 h K. pneumoniae cultures, indicating either growth phase or cell density-associated changes in the composition of headspace volatiles.Our analysis of the volatile molecules produced by K. pneumoniae during growth in human blood using GC×GC-TOFMS has doubled the number of volatiles reported for this organism in blood-containing media, and increased the total number of K. pneumoniae-associated volatiles by 20%. The volatile molecules produced by K. pneumoniae in blood may represent novel biomarkers for the diagnosis of bacteremia.

An Extracellular Bacterial Pathogen Modulates Host Metabolism to Regulate Its Own Sensing and Proliferation

Successful infection depends on the ability of the pathogen to gain nutrients from the host. The extracellular pathogenic bacterium group A Streptococcus (GAS) causes a vast array of human diseases. By using the quorum-sensing sil system as a reporter, we found that, during adherence to host cells,GAS delivers streptolysin toxins, creating endoplasmic reticulum stress. This, in turn, increases asparagine (ASN) synthetase expression and the production of ASN. The released ASN is sensed by the bacteria, altering the expression of ∼17% of GAS genes of which about one-third are dependent on the two-component system TrxSR. The expression of the streptolysin toxins is strongly upregulated, whereas genes linked to proliferation are downregulated in ASN absence. Asparaginase, a widely used chemotherapeutic agent, arrests GAS growth in human blood and blocks GAS proliferation in a mouse model of human bacteremia. These results delineate a pathogenic pathway and propose a therapeutic strategy against GAS infections.

Streptococcus pneumoniae Arginine Synthesis Genes Promote Growth and Virulence in Pneumococcal Meningitis

Streptococcus pneumoniae (pneumococcus) is a major human pathogen causing pneumonia, sepsis and bacterial meningitis. Using a clinical phenotype based approach with bacterial whole-genome sequencing we identified pneumococcal arginine biosynthesis genes to be associated with outcome in patients with pneumococcal meningitis. Pneumococci harboring these genes show increased growth in human blood and cerebrospinal fluid (CSF). Mouse models of meningitis and pneumonia showed that pneumococcal strains without arginine biosynthesis genes were attenuated in growth or cleared, from lung, blood and CSF. Thus, S. pneumoniae arginine synthesis genes promote growth and virulence in invasive pneumococcal disease.

Study of streptococcal hemoprotein receptor (Shr) in iron acquisition and virulence of M1T1 group A streptococcus

Streptococcus pyogenes (group A streptococcus, GAS) is a human bacterial pathogen of global significance, causing severe invasive diseases associated with serious morbidity and mortality. To survive within the host and establish an infection, GAS requires essential nutrients, including iron. The streptococcal hemoprotein receptor (Shr) is a surface-localized GAS protein that binds heme-containing proteins and extracellular matrix components. In this study, we employ targeted allelic exchange mutagenesis to investigate the role of Shr in the pathogenesis of the globally disseminated serotype M1T1 GAS. The shr mutant exhibited a growth defect in iron-restricted medium supplemented with ferric chloride, but no significant differences were observed in neutrophil survival, antimicrobial peptide resistance, cell surface charge, fibronectin-binding or adherence to human epithelial cells and keratinocytes, compared with wild-type. However, the shr mutant displayed a reduction in human blood proliferation, laminin-binding capacity and was attenuated for virulence in in vivo models of skin and systemic infection. We conclude that Shr augments GAS adherence to laminin, an important extracellular matrix attachment component. Furthermore, Shr-mediated iron uptake contributes to GAS growth in human blood, and is required for full virulence of serotype M1T1 GAS in mouse models of invasive disease.

CodY-affected transcriptional gene expression of Streptococcus pyogenes during growth in human blood

In an attempt to expand the available knowledge of pathogen-host interactions during ex vivo growth of Streptococcus pyogenes (GAS) in nonimmune whole human blood, the extents to which the expression of 51 genes including regulators with known targets, established virulence factors, physiologically important transporters and metabolic enzyme genes was differentially affected in the presence or absence of a functional codY gene were determined. The results obtained by quantitative real-time PCR using the M49 strain NZ131 showed that CodY influenced GAS gene activity in a dynamic fashion, with differential responses detected for 26 genes and occasionally characterized by discordance in the blood environment compared to laboratory medium. Degenerate derivatives of the recently discovered CodY box potentially serving as a cis-regulatory element for CodY action were identified in the upstream regions of 15 genes of the NZ131 genome, and these genes featured sequence motifs identical to the NZ131 CodY box in all completely sequenced S. pyogenes genomes. As none of these genes represented a genuine virulence factor, it seems likely, therefore, that the observed differential transcription of the majority of virulence genes was caused by indirect actions of CodY as part of a regulatory network.

Role of the Mga regulon in the resistance of M type 4 Streptococcus pyogenes to phagocytosis

M type 4 Streptococcus pyogenes is a frequent cause of infections, yet little is known concerning its anti-phagocytic factors. We investigated the role of the Mga regulon on growth in blood by inactivating individual genes within this regulon. Ablation of mrp4, emm4, or sof4 reduced streptococcal growth in human blood. Inactivation of enn4 had little impact on streptococcal growth in blood. Thus, expression of mrp, emm, and sof were required for optimal resistance to phagocytosis of M type 4 S. pyogenes. M proteins from class II serotypes contribute to resistance to phagocytosis by binding C4BP, a regulator of complement activation. Our data indicate that there is an additional pathway for resistance in these streptococci that is mediated by the binding of fibrinogen. Fibrinogen was required for growth of M type 4 S. pyogenes in blood and Mrp4 was the major fibrinogen-binding protein on these bacteria. Fibrinogen–Mrp interactions prevented activation of the classical pathway, thereby contributing to the ability of these streptococci to evade phagocytosis.

Anti‐phagocytic mechanisms of Streptococcus pyogenes: binding of fibrinogen to M‐related protein

A key attribute of invasive Streptococcus pyogenes is their ability to resist phagocytosis and multiply in human blood. M-related protein (Mrp) is a major anti-phagocytic factor but the mechanism whereby it helps streptococci to evade phagocytosis has not been demonstrated. We investigated phagocytosis resistance in a strain of serotype M4 by inactivating the mrp gene and also the emm, enn, sof and sfbX genes and by analysing the effect on streptococcal growth in blood and on complement deposition on the bacterial surface. Inactivation of enn4 and sfbX4 had little impact on growth in blood, but ablation of mrp4, emm4 or sof4 reduced streptococcal growth in human blood, confirming that Mrp and Emm are required for optimal resistance to phagocytosis and providing the first indication that Sof may be an anti-phagocytic factor. Moreover, antisera against Mrp4, Emm4 and Sof4 promoted the killing of S. pyogenes, but anti-SfbX serum had no effect. Growth of S. pyogenes in blood was dependent on the presence of fibrinogen and in the absence of fibrinogen there was a twofold increase in complement deposition. Inactivation of mrp4 resulted in a loss of fibrinogen-binding and caused a twofold increase in the binding of C3b that was inhibited by Mg-EGTA. Mrp contained two fibrinogen-binding sites, one of which is within a highly conserved region. These findings indicate that Mrp-fibrinogen interactions prevent surface deposition of complement via the classical pathway, thereby contributing to the ability of these streptococci to resist phagocytosis. This may be a common mechanism for evasion of phagocytosis because Mrp is expressed by approximately half of the clinical isolates of S. pyogenes.

Respiration metabolism of Group B Streptococcus is activated by environmental haem and quinone and contributes to virulence

Group B Streptococcus (GBS) is a common constituent of the vaginal microflora, but its transmission to newborns can cause life-threatening sepsis, pneumonia and meningitis. Energy metabolism of this opportunist pathogen has been deduced to be strictly fermentative. We discovered that GBS undergoes respiration metabolism if its environment supplies two essential respiratory components: quinone and haem. Respiration metabolism led to significant changes in growth characteristics, including a doubling of biomass and an altered metabolite profile under the tested conditions. The GBS respiratory chain is inactivated by: (i) withdrawing haem and/or quinone, (ii) treating cultures with a respiration inhibitor or (iii) inactivating the cydA gene product, a subunit of cytochrome bd quinol oxidase, in all cases resulting in exclusively fermentative growth. cydA inactivation reduced GBS growth in human blood and strongly attenuated virulence in a neonatal rat sepsis model, suggesting that the animal host may supply the components that activate GBS respiration. These results suggest a role of respiration metabolism in GBS dissemination. Our findings show that environmental factors can increase the flexibility of GBS metabolism by activating a newly identified respiration chain. The need for two environmental factors may explain why GBS respiration metabolism was not found in previous studies.

Group A Streptococcus Transcriptome Dynamics during Growth in Human Blood Reveals Bacterial Adaptive and Survival Strategies

The molecular basis for bacterial responses to host signals during natural infections is poorly understood. The gram-positive bacterial pathogen group A Streptococcus (GAS) causes human mucosal, skin, and life-threatening systemic infections. During the transition from a throat or skin infection to an invasive infection, GAS must adapt to changing environments and host factors. To better understand how GAS adapts, we used transcript profiling and functional analysis to investigate the transcriptome of a wild-type serotype M1 GAS strain in human blood. Global changes in GAS gene expression occur rapidly in response to human blood exposure. Increased transcription was observed for many genes that likely enhance bacterial survival, including those encoding superantigens and host-evasion proteins regulated by a multiple gene activator called Mga. GAS also coordinately expressed genes involved in proteolysis, transport, and catabolism of oligopeptides to obtain amino acids in this protein-rich host environment. Comparison of the transcriptome of the wild-type strain to that of an isogenic deletion mutant (DeltacovR) mutated in the two-component regulatory system designated CovR-CovS reinforced the hypothesis that CovR-CovS has an important role linking key biosynthetic, catabolic, and virulence functions during transcriptome restructuring. Taken together, the data provide crucial insights into strategies used by pathogenic bacteria for thwarting host defenses and surviving in human blood.

Group A Streptococcus Transcriptome Dynamics during Growth in Human Blood Reveals Bacterial Adaptive and Survival Strategies

Group A Streptococcus Transcriptome Dynamics during Growth in Human Blood Reveals Bacterial Adaptive and Survival Strategies

Spa contributes to the virulence of type 18 group A streptococci.

Streptococcal protective antigen (Spa) is a newly described surface protein of group A streptococci that was recently shown to evoke protective antibodies (J. B. Dale, E. Y. Chiang, S. Liu, H. S. Courtney, and D. L. Hasty, J. Clin. Investig. 103:1261--1268, 1999). In this study, we have determined the complete sequence of the spa gene from type 18 streptococci. Purified, recombinant Spa protein evoked antibodies that were bactericidal against type 18 streptococci, confirming the presence of protective epitopes. Sera from patients with acute rheumatic fever contained antibodies against recombinant Spa, indicating that the Spa protein is expressed in vivo and is immunogenic in humans. To determine the role of Spa in the virulence of group A streptococci, we created a series of insertional mutants that were (i) Spa negative and M18 positive, (ii) Spa positive and M18 negative, and (iii) Spa negative and M18 negative. The mutants and the parent M18 strain (18-282) were used in assays to determine resistance to phagocytosis, growth in human blood, and mouse virulence. The results show that Spa is a virulence determinant of group A streptococci and that expression of both Spa and M18 is required for optimal virulence of type 18 streptococci.

Expression of different group A streptococcal M proteins in an isogenic background demonstrates diversity in adherence to and invasion of eukaryotic cells.

The M protein of group A streptococcus (GAS) is considered to be a major virulence factor because it renders GAS resistant to phagocytosis and allows bacterial growth in human blood. There are more than 80 known serotypes of M proteins, and protective opsonic antibodies produced during disease in humans are serotype specific. M proteins also mediate bacterial adherence to epithelial cells of skin and pharynx. GAS strains vary in the genomic organization of the mga regulon, which contains the genes encoding M and M-like proteins and other virulence factors. This diversity of organization makes it difficult to assess virulence of M proteins of different serotypes, unless they can be expressed in an isogenic background. Here, we express M proteins of different serotypes in the M protein- and protein F1-deficient GAS strain, SAM2, which also lacks M-like proteins. Genes encoding M proteins of different serotypes (emmXs) have been integrated into the SAM2 chromosome in frame with the emm6.1 promoter and its mga regulon, resulting in similar levels of emmX expression. Although SAM2 exhibits a very low level of adherence to and invasion of HEp-2 and HaCaT cells, a SAM2-derived strain expressing M6 protein adheres to and invades both cell types. In contrast, the isogenic strain expressing M18 protein adheres to both cell types, but invades with a very low efficiency. A strain expressing M3 protein adheres to both types of cells, but its invasion of HEp-2 cells is serum dependent. A GAS strain expressing M6 protein does not compete with the isogenic strain expressing M18 protein for adherence to or invasion of HaCaT cells. We conclude that M proteins of different serotypes recognize different repertoires of receptors on the surfaces of eukaryotic cells.

Spatiotemporal dynamics of clotting and pattern formation in human blood

We examined the spatial dynamics of in vitro clot growth in human blood and plasma and found that initially, a clot grows at a constant speed, then abruptly stops and becomes surrounded by an ‘inhibition zone’ in which coagulation is strongly suppressed. We also observed the formation of ‘stratified structures’ (target patterns) in which solid layers alternated with liquid plasma. These and other spatial regimes of clotting are explained in terms of two interacting concentration waves propagating without attenuation. The experimental results are consistent with a hypothesis that blood is a bi-excitable medium, a new type of excitable medium.