A role of streptokinase in experimental post-streptococcal glomerulonephritis

Post-streptococcal glomerulonephritis (PSGN) refers to the sequela of the acute infection, caused by Streptococcus pyogenes (group A streptococcus, GAS). This pathology has been studied for a long time, and today attempts are being made to identify the products of their life activity, able to initiate an immunopathological process in kidneys. Most attention has been paid to streptokinase, the enzyme transforming blood plasminogen into plasmin, capable, together with the plasmin receptor (NAPlr), of damaging the glomerular tissue, as well as activating the complement system. The aim of the study was to consider two tasks: to study the ability of the GAS-obtained enzyme to transform plasminogen of different species into plasmin as well as to study its role in the development of PSGN in rabbits having subcutaneously implanted tissue chambers. The animals were infected by inoculating GAS cultures into the chambers. Materials and methods. GAS strains of M types 1, 12, 22 and their ska– isogenic mutants were used in the study. Purified plasminogen preparations were isolated from fresh human, rabbit or mouse plasma by using chromatographic column with Lysine Sepharose 4B. To reveal the ability of streptokinase to activate plasminogen into plasmin, its preparation at a concentration of 1 mg/ml was added to 10 ìg of purified human, rabbit or mouse plasminogen. The concentration of plasmin was defined photometrically using S-2251 (Chromogenix, USA). To reproduce PSGN, four chambers were implanted under the skin in each rabbit; after the complete wound healing animals were infected and observed for three weeks. On day 14, the animals were treated with benzylpenicillin. The kidneys from survived rabbits were subjected to immunohistology analysis. Results. During in vitro experiments, M1, M12 and M22 GAS streptokinase showed distinct functional activity on human plasminogen, transforming it into plasmin: optical density indicators at ë = 405 nm were 0.4–0.7 compared with the negative control (ОD < 0.001). Streptokinase did not activate mouse plasminogen (ОD = 0.001) and exerted quite a weak effect on transformation of the rabbit plasminogen into plasmin (ОD = 0.002). In experiments on PSGN induction in rabbits, we failed to detect streptokinase involvement, because no differences between initiation of glomerulonephritis by wild strains or ska– isogenic mutants were identified. Mutant strains deficient in the gene responsible for streptokinase synthesis but retained ability to bind rabbit and human IgG, caused morphological changes in kidney tissue, specific for PSGN. In addition, a comparative analysis of PSGN “rabbit” and “mouse” models developed by the same technology, was carried out and led to opposing conclusions regarding a role of streptokinase in pathogenesis of experimental glomerulonephritis. The role of IgG Fc-binding activity of GAS in development of experimental PSGN is discussed.

Listeria monocytogenes 10403S Alternative Sigma-54 Factor σL Has a Negative Role on Survival Ability Under Bile Exposure

Listeria monocytogenes is a Gram-positive bacterium causing listeriosis in animals and humans. To initiate a foodborne infection, L. monocytogenes has to pass through the host gastrointestinal tract (GIT). In this study, we evaluated survival abilities of L. monocytogenes 10403S wild type (WT) and its isogenic mutants in alternative sigma (σ) factor genes (i.e., sigB, sigC, sigH, and sigL) under simulated gastric, duodenal, and bile fluids. Within 10min of exposures, only bile fluid was able to significantly reduce survival ability of L. monocytogenes WT by 2 logs CFU/ml. Loss of sigL showed the greatest bile resistance among 16 strains tested, p&lt;0.0001, (i.e., WT, four single alternative σ factor mutants, six double mutants, four triple mutants, and one quadruple mutant). To further investigate the role of σL in bile response, RNA-seq was conducted to compare the transcriptional profiles among L. monocytogenes 10403S ΔBCH triple mutant (lacking sigB, sigC, and sigH genes; expressing housekeeping σA and σL) and ΔBCHL quadruple mutant (lacking all alternative sigma factor genes; expressing only σA) strains under BHI and 1% bile conditions. A total of 216 and 176 differentially expressed genes (DEGs) were identified in BHI and bile, respectively. We confirmed that mpt operon was shown to be strongly activated by σL. Interestingly, more than 80% of DEGs were found to be negatively regulated in the presence of σL. This includes PrfA regulon and its mediated genes (i.e., hly, hpt, inlB, clpP, clpE, groL, and inlC) which were downregulated in response to bile in the presence of σL. This result suggests the potential negative role of σL on bile survival, and the roles of σL and σB might be in a seesaw model prior to host cell invasion.

Investigation of the regulation of expression of E. Coli Common Pilus subunit, ECPA, of enterohaemorrhagic E. Coli 0157:H7 under acid stress

Escherichia coli (EHEC) 0157:H7 is a pathogenic bacterial species that is most commonly linked to severe diarrhea, but is also the leading cause of the potentially fatal hemolytic-uremic syndrome (HUS). In order to establish infection in the colon, EHEC must endure different stresses encountered in the gastrointestinal (GI) tract, such as acid stress in the stomach, bile salt stress in the small intestine, and short-chain fatty acid (SCFA) stress in the colon. These bacteria are likely able to use GI stresses as indicators of their location, impacting gene expression of adhesion, motility, and virulence factors. The E. coli Common Pilus (ECP) has been shown to be an important factor for EHEC adhesion to epithelial cells, which is increased after either acid or SCFA stress. It has also been demonstrated via microarray that genes of this operon are upregulated after acid stress. The aim of this study is to determine how expression of the main subunit of this structure, EcpA, is regulated upon exposure of EHEC 0157:H7 to acid or SCFA-stress. Both transcriptional and translational regulation are hypothesized to be involved. Isogenic mutants have been constructed that lacked key regulators suspected to be important for each system. Two approaches are used to determine if the predicted regulatory systems are playing a role in response to stress: observing EcpA protein expression analysis through Western blotting with anti-EcpA antibodies, and examining differences in ecp operon promoter activity in regulatory mutants. In this study Western blots reconfirmed H-NS does not modulate ecpA expression in direct response to acute acid stress. This suggests an alternate regulatory response in EHEC 0157:H7 to acute acid stress resulting in the upregulation of ecpA expression previously observed with microarray analysis.

Investigation of the regulation of expression of E. Coli Common Pilus subunit, ECPA, of enterohaemorrhagic E. Coli 0157:H7 under acid stress

Escherichia coli (EHEC) 0157:H7 is a pathogenic bacterial species that is most commonly linked to severe diarrhea, but is also the leading cause of the potentially fatal hemolytic-uremic syndrome (HUS). In order to establish infection in the colon, EHEC must endure different stresses encountered in the gastrointestinal (GI) tract, such as acid stress in the stomach, bile salt stress in the small intestine, and short-chain fatty acid (SCFA) stress in the colon. These bacteria are likely able to use GI stresses as indicators of their location, impacting gene expression of adhesion, motility, and virulence factors. The E. coli Common Pilus (ECP) has been shown to be an important factor for EHEC adhesion to epithelial cells, which is increased after either acid or SCFA stress. It has also been demonstrated via microarray that genes of this operon are upregulated after acid stress. The aim of this study is to determine how expression of the main subunit of this structure, EcpA, is regulated upon exposure of EHEC 0157:H7 to acid or SCFA-stress. Both transcriptional and translational regulation are hypothesized to be involved. Isogenic mutants have been constructed that lacked key regulators suspected to be important for each system. Two approaches are used to determine if the predicted regulatory systems are playing a role in response to stress: observing EcpA protein expression analysis through Western blotting with anti-EcpA antibodies, and examining differences in ecp operon promoter activity in regulatory mutants. In this study Western blots reconfirmed H-NS does not modulate ecpA expression in direct response to acute acid stress. This suggests an alternate regulatory response in EHEC 0157:H7 to acute acid stress resulting in the upregulation of ecpA expression previously observed with microarray analysis.

Alternative σ Factors Regulate Overlapping as Well as Distinct Stress Response and Metabolic Functions in Listeria monocytogenes under Stationary Phase Stress Condition

Listeria monocytogenes can regulate and fine-tune gene expression, to adapt to diverse stress conditions encountered during foodborne transmission. To further understand the contributions of alternative sigma (σ) factors to the regulation of L. monocytogenes gene expression, RNA-Seq was performed on L. monocytogenes strain 10403S and five isogenic mutants (four strains bearing in-frame null mutations in three out of four alternative σ factor genes, ΔCHL, ΔBHL, ΔBCL, and ΔBCH, and one strain bearing null mutations in all four genes, ΔBCHL), grown to stationary phase. Our data showed that 184, 35, 34, and 20 genes were positively regulated by σB, σL, σH, and σC (posterior probability > 0.9 and Fold Change (FC) > 5.0), respectively. Moreover, σB-dependent genes showed the highest FC (based on comparisons between the ΔCHL and the ΔBCHL strain), with 44 genes showing an FC > 100; only four σL-dependent, and no σH- or σC-dependent genes showed FC >100. While σB-regulated genes identified in this study are involved in stress-associated functions and metabolic pathways, σL appears to largely regulate genes involved in a few specific metabolic pathways, including positive regulation of operons encoding phosphoenolpyruvate (PEP)-dependent phosphotransferase systems (PTSs). Overall, our data show that (i) σB and σL directly and indirectly regulate genes involved in several energy metabolism-related functions; (ii) alternative σ factors are involved in complex regulatory networks and appear to have epistatic effects in stationary phase cells; and (iii) σB regulates multiple stress response pathways, while σL and σH positively regulate a smaller number of specific pathways.

Functional Analysis of Phenazine Biosynthesis Genes in Burkholderia spp.

Burkholderia encompass a group of ubiquitous Gram-negative bacteria that include numerous saprophytes as well as species that cause infections in animals, immunocompromised patients, and plants. Some species of Burkholderia produce colored, redox-active secondary metabolites called phenazines. Phenazines contribute to competitiveness, biofilm formation, and virulence in the opportunistic pathogen Pseudomonas aeruginosa, but knowledge of their diversity, biosynthesis, and biological functions in Burkholderia is lacking. In this study, we screened publicly accessible genome sequence databases and identified phenazine biosynthesis genes in multiple strains of the B. cepacia complex, some isolates of the B. pseudomallei clade, and the plant pathogen B. glumae. We then focused on B. lata ATCC 17760 to reveal the organization and function of genes involved in the production of dimethyl 4,9-dihydroxy-1,6-phenazinedicarboxylate. Using a combination of isogenic mutants and plasmids carrying different segments of the phz locus, we characterized three novel genes involved in the modification of the phenazine tricycle. Our functional studies revealed a connection between the presence and amount of phenazines and the dynamics of biofilm growth in flow cell and static experimental systems, but at the same time, failed to link the production of phenazines with the capacity of Burkholderia to kill fruit flies and rot onions. IMPORTANCE Although the production of phenazines in Burkholderia was first reported almost 70 years ago, the role these metabolites play in the biology of these economically important microorganisms remains poorly understood. Our results revealed that the phenazine biosynthetic pathway in Burkholderia has a complex evolutionary history, which likely involved horizontal gene transfers among several distantly related groups of organisms. The contribution of phenazines to the formation of biofilms suggests that Burkholderia, like fluorescent pseudomonads, may benefit from the unique redox-cycling properties of these versatile secondary metabolites.

L-Alanine Prototrophic Suppressors Emerge from L-Alanine Auxotroph through Stress-Induced Mutagenesis in Escherichia coli

In Escherichia coli, L-alanine is synthesized by three isozymes: YfbQ, YfdZ, and AvtA. When an E. coli L-alanine auxotrophic isogenic mutant lacking the three isozymes was grown on L-alanine-deficient minimal agar medium, L-alanine prototrophic mutants emerged considerably more frequently than by spontaneous mutation; the emergence frequency increased over time, and, in an L-alanine-supplemented minimal medium, correlated inversely with L-alanine concentration, indicating that the mutants were derived through stress-induced mutagenesis. Whole-genome analysis of 40 independent L-alanine prototrophic mutants identified 16 and 18 clones harboring point mutation(s) in pyruvate dehydrogenase complex and phosphotransacetylase-acetate kinase pathway, which respectively produce acetyl coenzyme A and acetate from pyruvate. When two point mutations identified in L-alanine prototrophic mutants, in pta (D656A) and aceE (G147D), were individually introduced into the original L-alanine auxotroph, the isogenic mutants exhibited almost identical growth recovery as the respective cognate mutants. Each original- and isogenic-clone pair carrying the pta or aceE mutation showed extremely low phosphotransacetylase or pyruvate dehydrogenase activity, respectively. Lastly, extracellularly-added pyruvate, which dose-dependently supported L-alanine auxotroph growth, relieved the L-alanine starvation stress, preventing the emergence of L-alanine prototrophic mutants. Thus, L-alanine starvation-provoked stress-induced mutagenesis in the L-alanine auxotroph could lead to intracellular pyruvate increase, which eventually induces L-alanine prototrophy.

Detection and Isolation of Emetic Bacillus cereus Toxin Cereulide by Reversed Phase Chromatography

The emetic toxin cereulide is a 1.2 kDa dodecadepsipeptide produced by the food pathogen Bacillus cereus. As cereulide poses a serious health risk to humans, sensitive and specific detection, as well as toxin purification and quantification, methods are of utmost importance. Recently, a stable isotope dilution assay tandem mass spectrometry (SIDA–MS/MS)-based method has been described, and an method for the quantitation of cereulide in foods was established by the International Organization for Standardization (ISO). However, although this SIDA–MS/MS method is highly accurate, the sophisticated high-end MS equipment required for such measurements limits the method’s suitability for microbiological and molecular research. Thus, we aimed to develop a method for cereulide toxin detection and isolation using equipment commonly available in microbiological and biochemical research laboratories. Reproducible detection and relative quantification of cereulide was achieved, employing reversed phase chromatography (RPC). Chromatographic signals were cross validated by ultraperformance liquid chromatography–mass spectrometry (UPLC–MS/MS). The specificity of the RPC method was tested using a test panel of strains that included non-emetic representatives of the B. cereus group, emetic B. cereus strains, and cereulide-deficient isogenic mutants. In summary, the new method represents a robust, economical, and easily accessible research tool that complements existing diagnostics for the detection and quantification of cereulide.

The Relative Role of Toxins A and B in the Virulence of Clotridioides difficile

Most pathogenic strains of C. difficile possess two large molecular weight single unit toxins with four similar functional domains. The toxins disrupt the actin cytoskeleton of intestinal epithelial cells leading to loss of tight junctions, which ultimately manifests as diarrhea in the host. While initial studies of purified toxins in animal models pointed to toxin A (TcdA) as the main virulence factor, animal studies using isogenic mutants demonstrated that toxin B (TcdB) alone was sufficient to cause disease. In addition, the natural occurrence of TcdA−/TcdB+ (TcdA−/B+)mutant strains was shown to be responsible for cases of C. difficile infection (CDI) with symptoms identical to CDI caused by fully toxigenic (A+/B+) strains. Identification of these cases was delayed during the period when clinical laboratories were using immunoassays that only detected TcdA (toxA EIA). Our hospital laboratory at the time performed culture as well as toxA EIA on patient stool samples. A total of 1.6% (23/1436) of all clinical isolates recovered over a 2.5-year period were TcdA−/B+ variants, the majority of which belonged to the restriction endonuclease analysis (REA) group CF and toxinotype VIII. Despite reports of serious disease due to TcdA−/B+ CF strains, these infections were typically mild, often not requiring specific treatment. While TcdB alone may be sufficient to cause disease, clinical evidence suggests that both toxins have a role in disease.

Hydrogen Peroxide Production by Streptococcus pneumoniae Results in Alpha-hemolysis by Oxidation of Oxy-hemoglobin to Met-hemoglobin

ABSTRACT Streptococcus pneumoniae and other streptococci produce a greenish halo on blood agar plates referred to as alpha-hemolysis. This phenotype is utilized by clinical microbiology laboratories to report culture findings of alpha-hemolytic streptococci, including S. pneumoniae, and other bacteria. The alpha-hemolysis halo on blood agar plates has been related to the hemolytic activity of pneumococcal pneumolysin (Ply) or, to a lesser extent, to lysis of erythrocytes by S. pneumoniae-produced hydrogen peroxide. We investigated the molecular basis of the alpha-hemolysis halo produced by S. pneumoniae. Wild-type strains TIGR4, D39, R6, and EF3030 and isogenic derivative Δply mutants produced similar alpha-hemolytic halos on blood agar plates, while cultures of hydrogen peroxide knockout ΔspxB ΔlctO mutants lacked this characteristic halo. Moreover, in the presence of catalase, the alpha-hemolysis halo was absent in cultures of the wild-type (wt) and Δply mutant strains. Spectroscopic studies demonstrated that culture supernatants of TIGR4 released hemoglobin-bound heme (heme-hemoglobin) from erythrocytes and oxidized oxy-hemoglobin to met-hemoglobin within 30 min of incubation. As expected, given Ply hemolytic activity and that hydrogen peroxide contributes to the release of Ply, TIGR4Δply and ΔspxB ΔlctO isogenic mutants had significantly decreased release of heme-hemoglobin from erythrocytes. However, TIGR4Δply that produces hydrogen peroxide oxidized oxy-hemoglobin to met-hemoglobin, whereas TIGR4ΔspxB ΔlctO failed to produce oxidation of oxy-hemoglobin. Studies conducted with all other wt strains and isogenic mutants resulted in similar findings. We demonstrated that the so-called alpha-hemolysis halo is caused by the oxidation of oxy-hemoglobin (Fe+2) to a non-oxygen-binding met-hemoglobin (Fe+3) by S. pneumoniae-produced hydrogen peroxide. IMPORTANCE There is a misconception that alpha-hemolysis observed on blood agar plate cultures of Streptococcus pneumoniae and other alpha-hemolytic streptococci is produced by a hemolysin or, alternatively, by lysis of erythrocytes caused by hydrogen peroxide. We noticed in the course of our investigations that wild-type S. pneumoniae strains and hemolysin (e.g., pneumolysin) knockout mutants produced the alpha-hemolytic halo on blood agar plates. In contrast, hydrogen peroxide-defective mutants prepared in four different strains lacked the characteristic alpha-hemolysis halo. We also demonstrated that wild-type strains and pneumolysin mutants oxidized oxy-hemoglobin to met-hemoglobin. Hydrogen peroxide knockout mutants, however, failed to oxidize oxy-hemoglobin. Therefore, the greenish halo formed on cultures of S. pneumoniae and other so-called alpha-hemolytic streptococci is caused by the oxidation of oxy-hemoglobin produced by hydrogen peroxide. Oxidation of oxy-hemoglobin to the nonbinding oxygen form, met-hemoglobin, might occur in the lungs during pneumococcal pneumonia.