Antibacterial and anti-biofilm activities of histidine kinase YycG inhibitors against Streptococcus agalactiae.

This study aims to investigate the antibacterial and anti-biofilm activities of YycG inhibitors H2-60 and H2-81 against Streptococcus agalactiae. A total of 118 nonduplicate S. agalactiae clinical isolates were collected, and the minimal inhibitory concentrations (MICs) of H2-60 and H2-81 were determined. H2-60 and H2-81 inhibit biofilm formation of S. agalactiae were detected by crystal violet staining, and against established biofilms of S. agalactiae were observed by confocal laser scanning microscope. Inhibitory effect of H2-60 and H2-81 on the phosphorylation activity of the HisKA domain of YycG' protein was measured. The MIC50/MIC90 was 3.13/6.25 µM for H2-60 and 6.25/12.5 µM for H2-81 against S. agalactiae, respectively. S. agalactiae planktonic cells can be decreased by H2-60 or H2-81 for more than 3 × log10 CFU ml-1 after 24 h treatment. Biofilm formation of 8 S. agalactiae strains (strong biofilm producers) was significantly reduced after treated with 1/4 × MIC of H2-60 or H2-81 for 24 h. H2-60 and H2-81 could reduce 45.79% and 29.56% of the adherent cells in the established biofilm of S. agalactiae after 72 h treatment, respectively. H2-60 combined with daptomycin reduced 83.63% of the adherent cells in the established biofilm of S. agalactiae, which was significantly better than that of H2-60 (45.79%) or daptomycin (55.07%) alone. The half maximal inhibitory concentrations (IC50) were 35.6 µM for H2-60 and 46.3 µM for H2-81 against the HisKA domain of YycG' protein. In conclusion, YycG inhibitors H2-60 and H2-81 exhibit excellent antibacterial and anti-biofilm activities against S. agalactiae.

Assays for hyaluronidases and heparanase using nonreducing end fluorophore-labeled hyaluronan and heparan sulfate proteoglycan.

Glycosaminoglycans (GAGs), such as hyaluronan (HA) and heparan sulfate (HS), are a large group of polysaccharides found in the extracellular matrix and on the cell surface. The turnover of these molecules is controlled by de novo synthesis and catabolism through specific endoglycosidases, which are the keys to our understanding of the homeostasis of GAGs and could hold opportunities for therapeutic intervention. Herein, we describe assays for endoglycosidases using nonreducing end fluorophore-labeled GAGs, in which GAGs were labeled via incorporation of GlcNAz by specific synthases and cycloaddition of alkyne fluorophores and then digested with corresponding endoglycosidases. Assays of various HA-specific hyaluronidases (HYALs), including PH-20 or SPAM1, and HS-specific heparanase (HPSE) are presented. We demonstrated the distinctive pH profiles, substrate specificities and specific activities of these enzymes and provided evidence that both HYAL3 and HYAL4 are authentic hyaluronidases. In addition, while all HYALs are active on high-molecular-weight HA, they are active on low-molecular-weight HA. Subsequently, we defined a new way of measuring the activities of HYALs. Our results indicate that the activities of HYALs must be under strict pH regulation. Our quantitative methods of measuring the activity GAG endoglycosidases could bring the opportunity of designing novel therapeutics by targeting these important enzymes.

Genomic insights on DNase production in Streptococcus agalactiae ST17 and ST19 strains.

Streptococcus agalactiae evasion from the human defense mechanisms has been linked to the production of DNases. These were proposed to contribute to the hypervirulence of S. agalactiae ST17/capsular-type III strains, mostly associated with neonatal meningitis. We performed a comparative genomic analysis between ST17 and ST19 human strains with different cell tropism and distinct DNase production phenotypes. All S. agalactiae ST17 strains, with the exception of 2211-04, were found to display DNase activity, while the opposite scenario was observed for ST19, where 1203-05 was the only DNase(+) strain. The analysis of the genetic variability of the seven genes putatively encoding secreted DNases in S. agalactiae revealed an exclusive amino acid change in the predicted signal peptide of GBS0661 (NucA) of the ST17 DNase(-), and an exclusive amino acid change alteration in GBS0609 of the ST19 DNase(+) strain. Further core-genome analysis identified some specificities (SNVs or indels) differentiating the DNase(-) ST17 2211-04 and the DNase(+) ST19 1203-05 from the remaining strains of each ST. The pan-genomic analysis evidenced an intact phage without homology in S. agalactiae and a transposon homologous to TnGBS2.3 in ST17 DNase(-) 2211-04; the transposon was also found in one ST17 DNase(+) strain, yet with a different site of insertion. A group of nine accessory genes were identified among all ST17 DNase(+) strains, including the Eco47II family restriction endonuclease and the C-5 cytosine-specific DNA methylase. None of these loci was found in any DNase(-) strain, which may suggest that these proteins might contribute to the lack of DNase activity. In summary, we provide novel insights on the genetic diversity between DNase(+) and DNase(-) strains, and identified genetic traits, namely specific mutations affecting predicted DNases (NucA and GBS0609) and differences in the accessory genome, that need further investigation as they may justify distinct DNase-related virulence phenotypes in S. agalactiae.

Functional Study of the Type II-A CRISPR-Cas System of Streptococcus agalactiae Hypervirulent Strains.

Nearly all strains of Streptococcus agalactiae, the leading cause of invasive infections in neonates, encode a type II-A clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. Interestingly, S. agalactiae strains belonging to the hypervirulent Sequence Type 17 (ST17) contain significantly fewer spacers in their CRISPR locus than other lineages, which could be the result of a less functional CRISPR-Cas system. Here, we revealed one large deletion in the ST17 cas promoter region and we evaluated its impact on the transcription of cas genes as well as the functionalities of the CRISPR-Cas system. We demonstrated that Cas9 interference is functional and that the CRISPR-Cas system of ST17 strains can still acquire new spacers, despite the absence of a regular cas promoter. We demonstrated that a promoter sequence upstream of srn036, a small RNA partially overlapping the antisense tracrRNA, is responsible for the ST17 CRISPR-Cas adaptation and interference activities.

Streptococcus agalactiae amylomaltase offers insight into the transglycosylation mechanism and the molecular basis of thermostability among amylomaltases.

Amylomaltase (AM) catalyzes transglycosylation of starch to form linear or cyclic oligosaccharides with potential applications in biotechnology and industry. In the present work, a novel AM from the mesophilic bacterium Streptococcus agalactiae (SaAM), with 18-49% sequence identity to previously reported AMs, was characterized. Cyclization and disproportionation activities were observed with the optimum temperature of 30 °C and 40 °C, respectively. Structural determination of SaAM, the first crystal structure of small AMs from the mesophiles, revealed a glycosyl-enzyme intermediate derived from acarbose and a second acarbose molecule attacking the intermediate. This pre-transglycosylation conformation has never been before observed in AMs. Structural analysis suggests that thermostability in AMs might be mainly caused by an increase in salt bridges since SaAM has a lower number of salt bridges compared with AMs from the thermophiles. Increase in thermostability by mutation was performed. C446 was substituted with A/S/P. C446A showed higher activities and higher kcat/Km values for starch in comparison to the WT enzyme. C446S exhibited a 5 °C increase in optimum temperature and the threefold increase in half-life time at 45 °C, most likely resulting from H-bonding interactions. For all enzymes, the main large-ring cyclodextrin (LR-CD) products were CD24-CD26 with CD22 as the smallest. C446S produced more CD35-CD42, especially at a longer incubation time.

Comprehensive study of antimicrobial susceptibility pattern and extended spectrum beta-lactamase (ESBL) prevalence in bacteria isolated from urine samples.

Nowadays, increasing extended-spectrum ß-lactamase (ESBL)-producing bacteria have become a global concern because of inducing resistance toward most of the antimicrobial classes and making the treatment difficult. In order to achieve an appropriate treatment option, identification of the prevalent species which generate ESBL as well as their antibiotic susceptibility pattern is essential worldwide. Hence, this study aimed to investigate the prevalence of ESBL-producing bacteria and assess their drug susceptibility in Fardis Town, Iran. A total of 21,604 urine samples collected from patients suspected to have urinary tract infection (UTI) were processed in the current study. The antimicrobial susceptibility of the isolates was tested by the disk diffusion method. The ESBL producing bacteria were determined by Double Disc Synergy Test (DDST) procedure. Bacterial growth was detected in 1408 (6.52%) cases. The most common bacterial strains causing UTI were found E. coli (72.16%), followed by K. pneumoniae (10.3%) and S. agalactiae (5.7%). Overall, 398 (28.26%) were ESBL producer. The highest ESBL production was observed in E. coli, followed by Klebsiella species. ESBL producers revealed a higher level of antibiotic resistance compared with non-ESBLs. In conclusion, ESBL production in uropathogens was relatively high. Carbapenems and Aminoglycosides were confirmed as the most effective treatment options for these bacteria.

Hyaluronidase Impairs Neutrophil Function and Promotes Group B Streptococcus Invasion and Preterm Labor in Nonhuman Primates.

Invasive bacterial infections during pregnancy are a major risk factor for preterm birth, stillbirth, and fetal injury. Group B streptococci (GBS) are Gram-positive bacteria that asymptomatically colonize the lower genital tract but infect the amniotic fluid and induce preterm birth or stillbirth. Experimental models that closely emulate human pregnancy are pivotal for the development of successful strategies to prevent these adverse pregnancy outcomes. Using a unique nonhuman primate model that mimics human pregnancy and informs temporal events surrounding amniotic cavity invasion and preterm labor, we show that the animals inoculated with hyaluronidase (HylB)-expressing GBS consistently exhibited microbial invasion into the amniotic cavity, fetal bacteremia, and preterm labor. Although delayed cytokine responses were observed at the maternal-fetal interface, increased prostaglandin and matrix metalloproteinase levels in these animals likely mediated preterm labor. HylB-proficient GBS dampened reactive oxygen species production and exhibited increased resistance to neutrophils compared to an isogenic mutant. Together, these findings demonstrate how a bacterial enzyme promotes GBS amniotic cavity invasion and preterm labor in a model that closely resembles human pregnancy.IMPORTANCE Group B streptococci (GBS) are bacteria that commonly reside in the female lower genital tract as asymptomatic members of the microbiota. However, during pregnancy, GBS can infect tissues at the maternal-fetal interface, leading to preterm birth, stillbirth, or fetal injury. Understanding how GBS evade host defenses during pregnancy is key to developing improved preventive therapies for these adverse outcomes. In this study, we used a unique nonhuman primate model to show that an enzyme secreted by GBS, hyaluronidase (HylB) promotes bacterial invasion into the amniotic cavity and fetus. Although delayed immune responses were seen at the maternal-fetal interface, animals infected with hyaluronidase-expressing GBS exhibited premature cervical ripening and preterm labor. These observations reveal that HylB is a crucial GBS virulence factor that promotes bacterial invasion and preterm labor in a pregnancy model that closely emulates human pregnancy. Therefore, hyaluronidase inhibitors may be useful in therapeutic strategies against ascending GBS infection.

Relatively high rates of cefotaxime- and ceftriaxone-non-susceptible isolates among group B streptococci with reduced penicillin susceptibility (PRGBS) in Japan.

OBJECTIVES: We have previously identified group B Streptococcus (GBS) clinical isolates with reduced penicillin susceptibility (PRGBS) that were non-susceptible to cefotaxime; however, the rates of cefotaxime and ceftriaxone non-susceptibility among PRGBS isolates have never been reported. Therefore, we first determined the MICs of 22 antibacterial drugs/compounds for 74 PRGBS isolates and then determined the rates of cefotaxime and ceftriaxone non-susceptibility among these isolates. METHODS: We used 74 clinical PRGBS isolates, previously collected in Japan and confirmed to harbour relevant amino acid substitutions in PBP2X. We also used 80 penicillin-susceptible GBS (PSGBS) clinical isolates as controls. The MICs of 22 antibacterial drugs/compounds for all 154 GBS isolates were determined via microdilution and/or agar dilution methods, as recommended by the CLSI. RESULTS: The rates of non-susceptibility/resistance to ampicillin, cefotaxime, ceftriaxone and levofloxacin for the 80 PSGBS isolates were 0%, 0%, 0% and 30%, respectively, but were 15% (P = 0.0003), 28% (P < 0.0001), 36% (P < 0.0001) and 93% (P < 0.0001) for the 74 PRGBS isolates, respectively. No PRGBS isolates were identified to be non-susceptible to meropenem, doripenem, vancomycin, quinupristin/dalfopristin, daptomycin or linezolid. CONCLUSIONS: We found that cefotaxime- and ceftriaxone-non-susceptible PRGBS isolates occur at relatively high rates in Japan. Importantly, this finding suggests that the range of drugs likely to be effective in treating PRGBS infections may be limited compared with those available for PSGBS infections; therefore, clinicians should exercise care when considering drug choice and efficacy for PRGBS infections.

Crystal structure of GAPDH of Streptococcus agalactiae and characterization of its interaction with extracellular matrix molecules.

GAPDH being a key enzyme in the glycolytic pathway is one of the surface adhesins of many Gram-positive bacteria including Streptococcus agalactiae. This anchorless adhesin is known to bind to host plasminogen (PLG) and fibrinogen (Fg), which enhances the virulence and modulates the host immune system. The crystal structure of the recombinant GAPDH from S. agalactiae (SagGAPDH) was determined at 2.6 Šresolution by molecular replacement. The structure was found to be highly conserved with a typical NAD binding domain and a catalytic domain. In this paper, using biolayer interferometry studies, we report that the multifunctional SagGAPDH enzyme binds to a variety of host molecules such as PLG, Fg, laminin, transferrin and mucin with a KD value of 4.4 × 10-7 M, 9.8 × 10-7 M, 1 × 10-5 M, 9.7 × 10-12 M and 1.4 × 10-7 M respectively. The ligand affinity blots reveal that SagGAPDH binds specifically to α and ß subunits of Fg and the competitive binding ELISA assay reveals that the Fg and PLG binding sites on GAPDH does not overlap each other. The PLG binding motif of GAPDH varies with organisms, however positively charged residues in the hydrophobic surroundings is essential for PLG binding. The lysine analogue competitive binding assay and lysine succinylation experiments deciphered the role of SagGAPDH lysines in PLG binding. On structural comparison with S. pneumoniae GAPDH, K171 of SagGAPDH is being predicted to be involved in PLG binding. Further SagGAPDH exhibited enzymatic activity in the presence of Fg, PLG and transferrin. This suggests that these host molecules does not mask the active site and bind at some other region of GAPDH.

Enhanced production of streptokinase from Streptococcus agalactiae EBL-31 by response surface methodology.

Streptokinase (SK) is a fibrinolytic protein used for the treatment of cardiovascular disorders. In the present study, enhanced production of SK was achieved by determining the optimum fermentation conditions for the maximum growth of Streptococcus agalactiae EBL-31 using response surface methodology (RSM). Four process variables (pH, temperature, incubation time and inoculum size) with five levels were evaluated in 30 experimental runs. Central composite rotatable design (CCRD) was employed to predict the effect of independent variables on SK activity. The statistical evaluation by ANOVA showed that the model was fit as the effect of single factors, quadratic effects and most of the interactions among variables. The value ofR2 (0.9988) indicated the satisfactory interaction between the experimental and predicted responses. Furthermore, the model F value (902.67) and coefficient of variation (1.92) clearly showed that the model is significant (p =>0.0001). The functional activity of SK was determined by spectrophotometric analysis and maximum SK production was obtained at pH-7.0, temperature- 37.5oC, an incubation time of 36 hours and 2.5 mL inoculum size. Hence it was concluded that the optimization of culture conditions through RSM increases the production of SK by 2.01-fold. Production of SK by fermentation is an economical choice to be used for the treatment of cardiovascular diseases.

Type 2 NADH Dehydrogenase Is the Only Point of Entry for Electrons into the Streptococcus agalactiae Respiratory Chain and Is a Potential Drug Target.

The opportunistic pathogen Streptococcus agalactiae is the major cause of meningitis and sepsis in a newborn's first week, as well as a considerable cause of pneumonia, urinary tract infections, and sepsis in immunocompromised adults. This pathogen respires aerobically if heme and quinone are available in the environment, and a functional respiratory chain is required for full virulence. Remarkably, it is shown here that the entire respiratory chain of S. agalactiae consists of only two enzymes, a type 2 NADH dehydrogenase (NDH-2) and a cytochrome bd oxygen reductase. There are no respiratory dehydrogenases other than NDH-2 to feed electrons into the respiratory chain, and there is only one respiratory oxygen reductase to reduce oxygen to water. Although S. agalactiae grows well in vitro by fermentative metabolism, it is shown here that the absence of NDH-2 results in attenuated virulence, as observed by reduced colonization in heart and kidney in a mouse model of systemic infection. The lack of NDH-2 in mammalian mitochondria and its important role for virulence suggest this enzyme may be a potential drug target. For this reason, in this study, S. agalactiae NDH-2 was purified and biochemically characterized, and the isolated enzyme was used to screen for inhibitors from libraries of FDA-approved drugs. Zafirlukast was identified to successfully inhibit both NDH-2 activity and aerobic respiration in intact cells. This compound may be useful as a laboratory tool to inhibit respiration in S. agalactiae and, since it has few side effects, it might be considered a lead compound for therapeutics development.IMPORTANCES. agalactiae is part of the human intestinal microbiota and is present in the vagina of ~30% of healthy women. Although a commensal, it is also the leading cause of septicemia and meningitis in neonates and immunocompromised adults. This organism can aerobically respire, but only using external sources of heme and quinone, required to have a functional electron transport chain. Although bacteria usually have a branched respiratory chain with multiple dehydrogenases and terminal oxygen reductases, here we establish that S. agalactiae utilizes only one type 2 NADH dehydrogenase (NDH-2) and one cytochrome bd oxygen reductase to perform respiration. NADH-dependent respiration plays a critical role in the pathogen in maintaining NADH/NAD+ redox balance in the cell, optimizing ATP production, and tolerating oxygen. In summary, we demonstrate the essential role of NDH-2 in respiration and its contribution to S. agalactiae virulence and propose it as a potential drug target.

High-resolution crystal structure of Streptococcus agalactiae glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that plays critical roles in bacterial pathogenesis in some pathogenic bacteria. In this study, the crystal structure of group B streptococcus GAPDH was determined at 1.36 Šresolution. The structure contained an asymmetric mixed holo tetramer, with two NAD ligands bound to two protomers. Further structural analysis identified interesting phosphate ion-binding sites, which shed light on its catalytic mechanism.

Mechanistic Investigations of Lysine-Tryptophan Cross-Link Formation Catalyzed by Streptococcal Radical S-Adenosylmethionine Enzymes.

Streptide is a ribosomally synthesized and post-translationally modified peptide with a unique cyclization motif consisting of an intramolecular lysine-tryptophan cross-link. Three radical S-adenosylmethionine enzymes, StrB, AgaB, and SuiB from different species of Streptococcus, have been shown to install this modification onto their respective precursor peptides in a leader-dependent fashion. Herein, we conduct detailed investigations to differentiate among several plausible mechanistic proposals, specifically addressing radical versus electrophilic addition to the indole during cross-link formation, the role of substrate side chains in binding in the enzyme active site, and the identity of the catalytic base in the reaction cycle. Our results are consistent with a radical electrophilic aromatic substitution mechanism for the key carbon-carbon bond-forming step. They also elaborate on other mechanistic features that underpin this unique and synthetically challenging post-translational modification.

Enzymatic Production of Glutathione Coupling with an ATP Regeneration System Based on Polyphosphate Kinase.

Glutathione (GSH) is an important reducing agent in the living cells. It is synthesized by a two-step reaction and requires two molecules of adenosine triphosphate (ATP) for one molecule GSH. The enzymatic cascade reaction in vitro is a promising approach to achieve a high titer and limit side reactions; although, a cost-effective phosphate donor for ATP regeneration is required. Triphosphate (PolyP(3)), tetraphosphate (PolyP(4)), and hexametaphosphate (PolyP(6)) were investigated in this study. Triphosphate inhibited the bifunctional GSH synthetase (GshF) from Streptococcus agalactiae, while no significant inhibition was observed by adding hexametaphosphate. The polyphosphate kinase from Corynebacterium glutamicum was hence investigated to use hexametaphosphate for regeneration of ATP. Further, the orthogonal experiment, which includes seven factors (buffer concentration, pH value, ADP concentration, GshF dosage, polyphosphate kinase (PPK) dosage, reaction temperature, substrate ratio of amino acid, and reaction times), indicated that the capacity of buffer is the most significant factor of the reaction conditions for enzymatic production of glutathione coupling with a PPK-based ATP regeneration system. After optimizing the Mg2+ concentration, the reaction was scaled up to 250 mL in a stirred reactor with pH feedback control to stabilize the pH value of reaction system and nitrogen protection to avoid the oxidation of product. A yield of 12.32 g/L was achieved. This work provided a potential GshF-based enzymatic way coupling the PPK-based ATP regeneration to product GSH in the optimal conditions towards cost-effectiveness at the industrial scale.

cas9 Enhances Bacterial Virulence by Repressing the regR Transcriptional Regulator in Streptococcus agalactiae.

Clustered regularly interspaced palindromic repeats (CRISPR) and their associated cas genes have been demonstrated to regulate self-genes and virulence in many pathogens. In this study, we found that inactivation of cas9 caused reduced adhesion and intracellular survival of the piscine Streptococcus agalactiae strain GD201008-001 and significantly decreased the virulence of this strain in zebrafish and mice. Further investigation indicated that the regR transcriptional regulator was upregulated in the Δcas9 mutant. As regR mediates the repression of hyaluronidase, a critical factor involved in opening the blood-brain barrier (BBB) in mice, cas9-mediated repression of regR transcription is important for S. agalactiae to open the BBB and thereby cause meningitis in animals. This study expands our understanding of endogenous gene regulation mediated by CRISPR-Cas systems in bacteria.

Quantitative assessment of the blood-brain barrier opening caused by Streptococcus agalactiae hyaluronidase in a BALB/c mouse model.

Streptococcus agalactiae is a pathogen causing meningitis in animals and humans. However, little is known about the entry of S. agalactiae into brain tissue. In this study, we developed a BALB/c mouse model based on the intravenous injection of ß-galactosidase-positive Escherichia coli M5 as an indicator of blood-brain barrier (BBB) opening. Under physiological conditions, the BBB is impermeable to E. coli M5. In pathological conditions caused by S. agalactiae, E. coli M5 is capable of penetrating the brain through a disrupted BBB. The level of BBB opening can be assessed by quantitative measurement of E. coli M5 loads per gram of brain tissue. Further, we used the model to evaluate the role of S. agalactiae hyaluronidase in BBB opening. The inactivation of hylB gene encoding a hyaluronidase, HylB, resulted in significantly decreased E. coli M5 colonization, and the intravenous injection of purified HylB protein induced BBB opening in a dose-dependent manner. This finding verified the direct role of HylB in BBB invasion and traversal, and further demonstrated the practicability of the in vivo mouse model established in this study. This model will help to understand the S. agalactiae-host interactions that are involved in this bacterial traversal of the BBB and to develop efficacious strategies to prevent central nervous system infections.

Lysine-Tryptophan-Crosslinked Peptides Produced by Radical SAM Enzymes in Pathogenic Streptococci.

Macrocycles represent a common structural framework in many naturally occurring peptides. Several strategies exist for macrocyclization, and the enzymes that incorporate them are of great interest, as they enhance our repertoire for creating complex molecules. We recently discovered a new peptide cyclization reaction involving a crosslink between the side chains of lysine and tryptophan that is installed by a radical SAM enzyme. Herein, we characterize relatives of this metalloenzyme from the pathogens Streptococcus agalactiae and Streptococcus suis. Our results show that the corresponding enzymes, which we call AgaB and SuiB, contain multiple [4Fe-4S] clusters and catalyze Lys-Trp crosslink formation in their respective substrates. Subsequent high-resolution-MS and 2D-NMR analyses located the site of macrocyclization. Moreover, we report that AgaB can accept modified substrates containing natural or unnatural amino acids. Aside from providing insights into the mechanism of this unusual modification, the substrate promiscuity of AgaB may be exploited to create diverse macrocyclic peptides.

Crystal Structures of Group B Streptococcus Glyceraldehyde-3-Phosphate Dehydrogenase: Apo-Form, Binary and Ternary Complexes.

Glyceraldehyde 3-phosphate dehydrogenase or GAPDH is an evolutionarily conserved glycolytic enzyme. It catalyzes the two step oxidative phosphorylation of D-glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate using inorganic phosphate and NAD+ as cofactor. GAPDH of Group B Streptococcus is a major virulence factor and a potential vaccine candidate. Moreover, since GAPDH activity is essential for bacterial growth it may serve as a possible drug target. Crystal structures of Group B Streptococcus GAPDH in the apo-form, two different binary complexes and the ternary complex are described here. The two binary complexes contained NAD+ bound to 2 (mixed-holo) or 4 (holo) subunits of the tetrameric protein. The structure of the mixed-holo complex reveals the effects of NAD+ binding on the conformation of the protein. In the ternary complex, the phosphate group of the substrate was bound to the new Pi site in all four subunits. Comparison with the structure of human GAPDH showed several differences near the adenosyl binding pocket in Group B Streptococcus GAPDH. The structures also reveal at least three surface-exposed areas that differ in amino acid sequence compared to the corresponding areas of human GAPDH.

Detection and comparison of neuraminidase activities in human and bovine group B streptococci.

Human and bovine group B streptococcus (GBS) isolates were serotyped and amounts of released N-acetylneuraminic acid from N-acetylneuraminyl-lactose by extracellular neuraminidase were colorimetrically assessed. According to serotyping by co-agglutination method, 30 of bovine GBS and 43 of human GBS could be serotyped (ST) by monospecific antisera coated with protein A. The remaining GBS strains were designated as nontypeable (NT). The released N-acetylneuraminic acid was determined in 90.9% of bovine GBS and 47.1% of human GBS isolates. The differences between the total bovine and human GBS isolates were statistically significant (p < 0.001). In comparison with detected N-acetylneuraminic acid level in bovine and human groups, significant decrease was observed in the bovine NT group according to increased human NT (p < 0.01) and bovine ST groups (p < 0.01). However, N-acetylneuraminic acid level in bovine ST and bovine total groups significantly (p < 0.001) increased with respect to the human ST group and human total group. Neuraminidase activity was detected more frequently in bovine GBS isolates. Considerable differentiations were observed between typeable and nontypeable isolates.

Streptococcal group B integrative and mobilizable element IMESag-rpsI encodes a functional relaxase involved in its transfer.

Streptococcus agalactiae or Group B Streptococcus (GBS) are opportunistic bacteria that can cause lethal sepsis in children and immuno-compromised patients. Their genome is a reservoir of mobile genetic elements that can be horizontally transferred. Among them, integrative and conjugative elements (ICEs) and the smaller integrative and mobilizable elements (IMEs) primarily reside in the bacterial chromosome, yet have the ability to be transferred between cells by conjugation. ICEs and IMEs are therefore a source of genetic variability that participates in the spread of antibiotic resistance. Although IMEs seem to be the most prevalent class of elements transferable by conjugation, they are poorly known. Here, we have studied a GBS-IME, termed IMESag-rpsI, which is widely distributed in GBS despite not carrying any apparent virulence trait. Analyses of 240 whole genomes showed that IMESag-rpsI is present in approximately 47% of the genomes, has a roughly constant size (approx. 9 kb) and is always integrated at a single location, the 3'-end of the gene encoding the ribosomal protein S9 (rpsI). Based on their genetic variation, several IMESag-rpsI types were defined (A-J) and classified in clonal complexes (CCs). CC1 was the most populated by IMESag-rpsI (more than 95%), mostly of type-A (71%). One CC1 strain (S. agalactiae HRC) was deep-sequenced to understand the rationale underlying type-A IMESag-rpsI enrichment in GBS. Thirteen open reading frames were identified, one of them encoding a protein (MobSag) belonging to the broadly distributed family of relaxases MOBV1 Protein MobSag was purified and, by a newly developed method, shown to cleave DNA at a specific dinucleotide. The S. agalactiae HRC-IMESag-rpsI is able to excise from the chromosome, as shown by the presence of circular intermediates, and it harbours a fully functional mobilization module. Further, the mobSag gene encoded by this mobile element is able to promote plasmid transfer among pneumococcal strains, suggesting that MobSag facilitates the spread of IMESag-rpsI and that this spread would explain the presence of the same IMESag-rpsI type in GBS strains belonging to different CCs.