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We identified and characterized genomic regions of Streptococcus agalactiae that are involved in the Leloir and the tagatose-6-phosphate pathways for D-galactose catabolism. The accumulation of mutations in genes coding the Leloir pathway and the absence of these genes in a significant proportion of the strains suggest that this pathway may no longer be necessary for S. agalactiae and is heading toward extinction. In contrast, a genomic region containing genes coding for intermediates of the tagatose-6-phosphate pathway, a Gat family PTS transporter, and a DeoR/GlpR family regulator is present in the vast majority of strains. By deleting genes that code for intermediates of each of these two pathways in three selected strains, we demonstrated that the tagatose-6-phosphate pathway is their sole route for galactose catabolism. Furthermore, we showed that the Gat family PTS transporter acts as the primary importer of galactose in S. agalactiae . Finally, we proved that the DeoR/GlpR family regulator is a repressor of the tagatose-6-phosphate pathway and that galactose triggers the induction of this biochemical mechanism.IMPORTANCE S. agalactiae , a significant pathogen for both humans and animals, encounters galactose and galactosylated components within its various ecological niches. We highlighted the capability of this bacterium to metabolize D-galactose and showed the role of the tagatose-6-phosphate pathway and of a PTS importer in this biochemical process. Since S. agalactiae relies on carbohydrate fermentation for energy production, its ability to uptake and metabolize D-galactose could enhance its persistence and its competitiveness within the microbiome., Competing Interests: The authors declare no conflict of interest.

The tkt (transketolase) gene is one of the seven gene fragments used in the multilocus sequence typing (MLST) system for Streptococcus agalactiae . We discovered that the tkt_134 allele is derived from a homologous gene (which we designate tktX ) that is not present in all S. agalactiae ; all known strains that contain a match to the tkt_134 allele also contain a gene sequence that is much closer in sequence identity to the other non-tkt_134 alleles (i.e., the canonical tkt gene) in the database. Based on these data, the tkt_134 allele has been removed from the MLST database as of September 2021, and all sequence types containing tkt_134 have also been removed.IMPORTANCEMultilocus sequence typing (MLST) databases are a common good and remain important for research, medical, and epidemiological purposes. This remains true even in the context of widespread whole-genome sequencing. We discovered a contaminating allele of the tkt gene in the S. agalactiae MLST database that led to unstable, ambiguous, or erroneous MLST assignment. The allele has since been removed from the public database based on the results presented in this manuscript., Competing Interests: The authors declare no conflict of interest.

Bacteria possess the ability to develop diverse and ingenious strategies to outwit the host immune system, and proteases are one of the many weapons employed by bacteria. This study sought to identify S. agalactiae additional serine protease and determine its role in virulence. The S. agalactiae THN0901 genome features one S8 family serine peptidase B (SfpB), acting as a secreted and externally exposed entity. A S8 family serine peptidase mutant strain (ΔsfpB) and complement strain (CΔsfpB) were generated through homologous recombination. Compared to the wild-type strain THN0901, the absorption of EtBr dyes was significantly reduced (P < 0.01) in ΔsfpB, implying an altered cell membrane permeability. In addition, the ΔsfpB strain had a significantly lower survival rate in macrophages (P < 0.01) and a 61.85 % lower adhesion ability to the EPC cells (P < 0.01) compared to THN0901. In the in vivo colonization experiment using tilapia as a model, 210 fish were selected and injected with different bacterial strains at a concentration of 3 × 10 6  CFU/tail. At 6, 12, 24, 48, 72 and 96 h post-injection, three fish were randomly selected from each group and their brain, liver, spleen, and kidney tissues were isolated. Subsequently, it was demonstrated that the ΔsfpB strain exhibited a markedly diminished capacity for colonization in tilapia. Additionally, the cumulative mortality of ΔsfpB in fish after intraperitoneal injection was reduced by 19.92-23.85 %. In conclusion, the findings in this study have demonstrated that the SfpB plays a significant role in S. agalactiae cell membrane stability and immune evasion. The immune evasion is fundamental for the development and transmission of invasive diseases, the serine protease SfpB may be a promising candidate for the development of antimicrobial agents to reduce the transmission of S. agalactiae., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Anxing Li reports financial support was provided by National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Importance: Group B Streptococcus (GBS) colonizes the female reproductive tract (FRT) in one-third of women, and carriage leads to numerous adverse pregnancy outcomes including the preterm premature rupture of membranes, chorioamnionitis, and stillbirth. The presence of GBS in the FRT during pregnancy is also the largest predisposing factor for the transmission of GBS and invasive neonatal diseases, including pneumonia, sepsis, and meningitis. The factors contributing to GBS colonization are still being elucidated. Here, we show for the first time that GBS transcription is regulated by an orphan DNA cytosine methyltransferase (Dcm). Many GBS factors are regulated by Dcm, especially those involved in carbohydrate transport and metabolism. We show that GBS persistence in the FRT is dependent on the catabolism of sugars found on the vaginal mucin MUC5B. Collectively, this work highlights the regulatory importance of a DNA methyltransferase and identifies both host and bacterial factors required for GBS colonization., Competing Interests: The authors declare no conflict of interest.

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., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 MIC 50 /MIC 90 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 (IC 50 ) 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., (© 2021. The Author(s), under exclusive licence to the Japan Antibiotics Research Association.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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 k cat /K m 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.

Nowadays, increasing extended-spectrum β-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.

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., (Copyright © 2021 Coleman et al.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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 Å resolution 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 K D 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 β 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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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. IMPORTANCE S. 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., (Copyright © 2018 Lencina et al.)

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.

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 Mg 2+ 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.

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 Å resolution. 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.

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 Δ cas9 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., (Copyright © 2018 American Society for Microbiology.)

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.

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 β-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.

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.

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., (© 2016 APMIS. Published by John Wiley & Sons Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist.

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 MOB V1 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., (© 2016 The Authors.)

Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.

Induction of type I interferon (IFN) in response to microbial pathogens depends on a conserved cGAS-STING signaling pathway. The presence of DNA in the cytoplasm activates cGAS, while STING is activated by cyclic dinucleotides (cdNs) produced by cGAS or from bacterial origins. Here, we show that Group B Streptococcus (GBS) induces IFN-β production almost exclusively through cGAS-STING-dependent recognition of bacterial DNA. However, we find that GBS expresses an ectonucleotidase, CdnP, which hydrolyzes extracellular bacterial cyclic-di-AMP. Inactivation of CdnP leads to c-di-AMP accumulation outside the bacteria and increased IFN-β production. Higher IFN-β levels in vivo increase GBS killing by the host. The IFN-β overproduction observed in the absence of CdnP is due to the cumulative effect of DNA sensing by cGAS and STING-dependent sensing of c-di-AMP. These findings describe the importance of a bacterial c-di-AMP ectonucleotidase and suggest a direct bacterial mechanism that dampens activation of the cGAS-STING axis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Group B Streptococcus (GBS) is a leading cause of bacterial sepsis and meningitis in newborns. GBS possesses a protein with homology to the pneumococcal virulence factor, NanA, which has neuraminidase (sialidase) activity and promotes blood-brain barrier penetration. However, phylogenetic sequence and enzymatic analyses indicate the GBS NanA ortholog has lost sialidase function - and for this distinction we designate the gene and encoded protein nonA/NonA. Here we analyze NonA function in GBS pathogenesis, and through heterologous expression of active pneumococcal NanA in GBS, potential costs of maintaining sialidase function. GBS wild-type and ΔnonA strains lack sialidase activity, but forced expression of pneumococcal NanA in GBS induced degradation of the terminal sialic acid on its exopolysaccharide capsule. Deletion of nonA did not change GBS-whole blood survival or brain microvascular cell invasion. However, forced expression of pneumococcal NanA in GBS removed terminal sialic acid residues from the bacterial capsule, restricting bacterial proliferation in human blood and in vivo upon mouse infection. GBS expressing pneumococcal NanA had increased invasion of human brain microvascular endothelial cells. Thus, we hypothesize that nonA lost enzyme activity allowing the preservation of an effective survival factor, the sialylated exopolysaccharide capsule.

Unlabelled: Preterm birth increases the risk of adverse birth outcomes and is the leading cause of neonatal mortality. A significant cause of preterm birth is in utero infection with vaginal microorganisms. These vaginal microorganisms are often recovered from the amniotic fluid of preterm birth cases. A vaginal microorganism frequently associated with preterm birth is group B streptococcus (GBS), or Streptococcus agalactiae However, the molecular mechanisms underlying GBS ascension are poorly understood. Here, we describe the role of the GBS hyaluronidase in ascending infection and preterm birth. We show that clinical GBS strains associated with preterm labor or neonatal infections have increased hyaluronidase activity compared to commensal strains obtained from rectovaginal swabs of healthy women. Using a murine model of ascending infection, we show that hyaluronidase activity was associated with increased ascending GBS infection, preterm birth, and fetal demise. Interestingly, hyaluronidase activity reduced uterine inflammation but did not impact placental or fetal inflammation. Our study shows that hyaluronidase activity enables GBS to subvert uterine immune responses, leading to increased rates of ascending infection and preterm birth. These findings have important implications for the development of therapies to prevent in utero infection and preterm birth., Importance: GBS are a family of bacteria that frequently colonize the vagina of pregnant women. In some cases, GBS ascend from the vagina into the uterine space, leading to fetal injury and preterm birth. Unfortunately, little is known about the mechanisms underlying ascending GBS infection. In this study, we show that a GBS virulence factor, HylB, shows higher activity in strains isolated from cases of preterm birth than those isolates from rectovaginal swabs of healthy women. We discovered that GBS rely on HylB to avoid immune detection in uterine tissue, but not placental tissue, which leads to increased rates of fetal injury and preterm birth. These studies provide novel insight into the underlying mechanisms of ascending infection., (Copyright © 2016 Vornhagen et al.)

Group B Streptococcus (GBS), a commensal organism, can turn into a life-threatening pathogen in neonates and elderly, or in adults with severe underlying diseases such as diabetes. We developed a vaccine targeting the GBS glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme detected at the bacterial surface, which was proven to be effective in a neonatal mouse model of infection. Since this bacterium has emerged as an important pathogen in non-pregnant adults, here we investigated whether this vaccine also confers protection in an adult susceptible and in a diabetic mouse model of infection. For immunoprotection studies, sham or immunized adult mice were infected with GBS serotype Ia and V strains, the two most prevalent serotypes isolated in adults. Sham and vaccinated mice were also rendered diabetic and infected with a serotype V GBS strain. For toxicological (pre-clinical) studies, adult mice were vaccinated three times, with three concentrations of recombinant GAPDH adjuvanted with Allydrogel, and the toxicity parameters were evaluated twenty-four hours after the last immunization. For the stability tests, the vaccine formulations were maintained at 4°C for 6 and 12 months prior immunization. The results showed that all tested doses of the vaccine, including the stability study formulations, were immunogenic and that the vaccine was innocuous. The organs (brain, blood, heart, and liver) of vaccinated susceptible or diabetic adult mice were significantly less colonized compared to those of control mice. Altogether, these results demonstrate that the GAPDH-based vaccine is safe and stable and protects susceptible and diabetic adult mice against GBS infections. It is therefore a promising candidate as a global vaccine to prevent GBS-induced neonatal and adult diseases.

In response to tissue injury, hyaluronan (HA) polymers are cleaved by host hyaluronidases, generating small fragments that ligate Toll-like receptors (TLRs) to elicit inflammatory responses. Pathogenic bacteria such as group B Streptococcus (GBS) express and secrete hyaluronidases as a mechanism for tissue invasion, but it is not known how this activity relates to immune detection of HA. We found that bacterial hyaluronidases secreted by GBS and other Gram-positive pathogens degrade pro-inflammatory HA fragments to their component disaccharides. In addition, HA disaccharides block TLR2/4 signaling elicited by both host-derived HA fragments and other TLR2/4 ligands, including lipopolysaccharide. Application of GBS hyaluronidase or HA disaccharides reduced pulmonary pathology and pro-inflammatory cytokine levels in an acute lung injury model. We conclude that breakdown of host-generated pro-inflammatory HA fragments to disaccharides allows bacterial pathogens to evade immune detection and could be exploited as a strategy to treat inflammatory diseases., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The objective of this study was to determine the lipolytic activity on milk fat of 2 bovine mastitis pathogens, that is, Staphylococcus aureus and Streptococcus agalactiae. The lipolytic activity was determined by 2 different techniques, that is, thin-layer chromatography and an extraction-titration method, in an experimental model using the most commonly occurring field strains of the 2 mastitic bacteria isolated from Swedish dairy farms. The microorganisms were inoculated into bacteria-free control milk and incubated at 37°C to reflect physiological temperatures in the mammary gland. Levels of free fatty acids (FFA) were analyzed at time of inoculation (t=0) and after 2 and 6h of incubation, showing significant increase in FFA levels. After 2h the FFA content had increased by approximately 40% in milk samples inoculated with Staph. aureus and Strep. agalactiae, and at 6h the pathogens had increased FFA levels by 47% compared with the bacteria-free control milk. Changes in lipid composition compared with the bacteria-free control were investigated at 2 and 6h of incubation. Diacylglycerols, triacylglycerols, and phospholipids increased significantly after 6h incubation with the mastitis bacteria, whereas cholesterol and sterol esters decreased. Our results suggest that during mammary infections with Staph. aureus and Strep. agalactiae, the action of lipases originating from the mastitis pathogens will contribute significantly to milk fat lipolysis and thus to raw milk deterioration., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The development of drug resistance by bacterial pathogens is a growing threat. Drug resistant infections have high morbidity and mortality rates, and treatment of these infections is a major burden on the health care system. One potential strategy to prevent the development of drug resistance would be the application of therapeutic strategies that target bacterial virulence. Hyaluronidase is virulence factor that plays a role in the ability of Gram-positive bacteria such as Staphyloccus aureus and Streptococcus agalactiae to spread in tissue. As such, this enzyme could be a target for the development of future anti-virulence therapies. To facilitate the identification of hyaluronidase inhibitors, quantitative and reproducible assays of hyaluronidase activity are required. In the present study, we developed a new mass spectrometry based bioassay for this purpose. This assay directly measures the quantity of a degradation product (3-(4-deoxy-β-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine) produced by the hyaluronidase enzyme. Validation parameters for the new assay are as follows: repeatability, <7%; intermediate precision, <10%; range, 0.78-50 μM; limit of detection, 0.29 μM; and limit of quantification, 0.78 μM. Using the new assay, the IC50 value for a published inhibitor of S. agalactiae hyaluronidase, ascorbic acyl 6-palmitate, was 8.0±1.0 μM. We also identified a new hyaluronidase inhibitor, n-cyclohexanecarbonylpentadecylamine, with an IC50 of 30.4±9.8 μM. In conclusion, we describe a new, direct, and reproducible method for assessing hyaluronidase activity using mass spectrometry that can facilitate the discovery of inhibitors., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Group B Streptococcus (GBS) expresses 3 structurally distinct pilus types (1, 2a, and 2b) identified as important virulence factors and vaccine targets. These pili are heterotrimeric polymers, covalently assembled on the cell wall by sortase (Srt) enzymes. We investigated the pilus-2b biogenesis mechanism by using a multidisciplinary approach integrating genetic, biochemical, and structural studies to dissect the role of the 2 pilus-2b-associated Srts. We show that only 1 sortase (SrtC1-2b) is responsible for pilus protein polymerization, whereas the second one (Srt2-2b) does not act as a pilin polymerase, but similarly to the housekeeping class A Srt (SrtA), it is involved in cell-wall pilus anchoring by targeting the minor ancillary subunit. Based on its function and sequence features, Srt2-2b does not belong to class C Srts (SrtCs), nor is it a canonical member of any other known family of Srts. We also report the crystal structure of SrtC1-2b at 1.9 Å resolution. The overall fold resembles the typical structure of SrtCs except for the N-terminal lid region that appears in an open conformation displaced from the active site. Our findings reveal that GBS pilus type 2b biogenesis differs significantly from the current model of pilus assembly in gram-positive pathogens., (© FASEB.)

The selective inhibition of bacterial β-glucuronidases was recently shown to alleviate drug-induced gastrointestinal toxicity in mice, including the damage caused by the widely used anticancer drug irinotecan. Here, we report crystal structures of representative β-glucuronidases from the Firmicutes Streptococcus agalactiae and Clostridium perfringens and the Proteobacterium Escherichia coli, and the characterization of a β-glucuronidase from the Bacteroidetes Bacteroides fragilis. While largely similar in structure, these enzymes exhibit marked differences in catalytic properties and propensities for inhibition, indicating that the microbiome maintains functional diversity in orthologous enzymes. Small changes in the structure of designed inhibitors can induce significant conformational changes in the β-glucuronidase active site. Finally, we establish that β-glucuronidase inhibition does not alter the serum pharmacokinetics of irinotecan or its metabolites in mice. Together, the data presented advance our in vitro and in vivo understanding of the microbial β-glucuronidases, a promising new set of targets for controlling drug-induced gastrointestinal toxicity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Type 1 narcolepsy is a neurological disorder characterized by excessive daytime sleepiness and cataplexy associated with the HLA allele DQB1*06:02. Genetic predisposition along with external triggering factors may drive autoimmune responses, ultimately leading to the selective loss of hypocretin-positive neurons., Objective: The aim of this study was to investigate potential aetiological factors in Swedish cases of postvaccination (Pandemrix) narcolepsy defined by interferon-gamma (IFNγ) production from immune cells in response to molecularly defined targets., Methods: Cellular reactivity defined by IFNγ production was examined in blood from 38 (HLA-DQB1*06:02(+) ) Pandemrix-vaccinated narcolepsy cases and 76 (23 HLA-DQB1*06:02(+) and 53 HLA-DQB1*06:02(-) ) control subjects, matched for age, sex and exposure, using a variety of different antigens: β-haemolytic group A streptococcal (GAS) antigens (M5, M6 and streptodornase B), influenza (the pandemic A/H1N1/California/7/09 NYMC X-179A and A/H1N1/California/7/09 NYMC X-181 vaccine antigens, previous Flu-A and -B vaccine targets, A/H1N1/Brisbane/59/2007, A/H1N1/Solomon Islands/3/2006, A/H3N2/Uruguay/716/2007, A/H3N2/Wisconsin/67/2005, A/H5N1/Vietnam/1203/2004 and B/Malaysia/2506/2004), noninfluenza viral targets (CMVpp65, EBNA-1 and EBNA-3) and auto-antigens (hypocretin peptide, Tribbles homolog 2 peptide cocktail and extract from rat hypothalamus tissue)., Results: IFN-γ production was significantly increased in whole blood from narcolepsy cases in response to streptococcus serotype M6 (P = 0.0065) and streptodornase B protein (P = 0.0050). T-cell recognition of M6 and streptodornase B was confirmed at the single-cell level by intracellular cytokine (IL-2, IFNγ, tumour necrosis factor-alpha and IL-17) production after stimulation with synthetic M6 or streptodornase B peptides. Significantly, higher (P = 0.02) titres of serum antistreptolysin O were observed in narcolepsy cases, compared to vaccinated controls., Conclusion: β-haemolytic GAS may be involved in triggering autoimmune responses in patients who developed narcolepsy symptoms after vaccination with Pandemrix in Sweden, characterized by a Streptococcus pyogenes M-type-specific IFN-γ cellular immune response., (© 2015 The Association for the Publication of the Journal of Internal Medicine.)

Enzymatic properties of group B streptococci (GBS) serotypes from bovine milk and human routine vaginal specimens were investigated. Out of the 56 human and 66 bovine GBS, 35 and 30 could be classified serologically by a co-agglutination test with type-specific antisera, respectively. Hyaluronidase (HYAL), streptokinase (SK) and protease activities were detected using culture media. HYAL activity was observed mostly in typable human GBS, and serotypes Ia, Ic and II comprised 77.3% of the typable strains producing HYAL. Bovine GBS serotypes II, III and VII comprised 87.5% of typable bovine strains exhibiting HYAL activity. SK activity was detected only in three human GBS. Human GBS serotypes Ia, Ic, II, III, VII and almost all typable bovine GBS strains showed protease activity. β-D-glucosidase activity was frequently observed in human GBS, whereas N-acetyl-β-D-glucosaminidase activity was mostly detected in non-typable GBS from humans. These results indicate that different GBS serotypes could vary in their virulence properties, and bovine and human GBS isolates could not be differentiated by their enzyme activities. Use of the culture media appeared to be a simple-to-apply and useful method for the detection of extracellular enzyme activity such as HYAL, protease and SK.

Nisin is a 34-amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics. Nisin displays a high bactericidal activity against various Gram-positive bacteria, including some human-pathogenic strains. However, there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome. The encoded protein, NSR, cleaves off the last six amino acids of nisin, thereby reducing its bactericidal efficacy. An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method in hanging and sitting drops, resulting in crystals that diffracted X-rays to 2.8 and 2.2 Å, respectively.

Cytidine monophosphate (CMP) N-acetylneuraminic acid (NeuNAc) synthetase, which is encoded by the neuA gene, can catalyze the activation of sialic acid with CMP, and plays an important role in Streptococcus agalactiae infection pathogenesis. To study the structure and function of the S. agalactiae neuA gene, we isolated it from diseased tilapia, amplified it using polymerase chain reaction (PCR) with specific primers, and cloned it into a pMD19-T vector. The recombinant plasmid was confirmed by PCR and restriction enzyme digestion, and identified by sequencing. Molecular characterization analyses of the neuA nucleotide amino acid sequence were performed using bioinformatic tools and an online server. The results showed that the neuA nucleotide sequence contained a complete coding region, which comprised 1242 bp, encoding 413 amino acids (aa). The aa sequence was highly conserved and contained a Glyco&#95;tranf&#95;GTA&#95;type superfamily and an SGNH&#95;hydrolase superfamily conserved domain, which are related to sialic acid activation catalysis. The NeuA protein possessed many important sites related to post-translational modification, including 28 potential phosphorylation sites and 2 potential N-glycosylation sites, had no signal peptides or transmembrane regions, and was predicted to reside in the cytoplasm. Moreover, the protein had some B-cell epitopes, which suggests its potential in development of a vaccine against S. agalactiae infection. The codon usage frequency of neuA differed greatly in Escherichia coli and Homo sapiens genes, and neuA may be more efficiently expressed in eukaryotes (yeast). S. agalactiae neuA from tilapia maintains high structural homology and sequence identity with CMP-NeuNAc synthetases from other bacteria.

Glycosaminoglycans in mammalian extracellular matrices are degraded to their constituents, unsaturated uronic (glucuronic/iduronic) acids and amino sugars, through successive reactions of bacterial polysaccharide lyase and unsaturated glucuronyl hydrolase. Genes coding for glycosaminoglycan-acting lyase, unsaturated glucuronyl hydrolase, and the phosphotransferase system are assembled into a cluster in the genome of pathogenic bacteria, such as streptococci and clostridia. Here, we studied the streptococcal metabolic pathway of unsaturated uronic acids and the structure/function relationship of its relevant isomerase and dehydrogenase. Two proteins (gbs1892 and gbs1891) of Streptococcus agalactiae strain NEM316 were overexpressed in Escherichia coli, purified, and characterized. 4-Deoxy-l-threo-5-hexosulose-uronate (Dhu) nonenzymatically generated from unsaturated uronic acids was converted to 2-keto-3-deoxy-d-gluconate via 3-deoxy-d-glycero-2,5-hexodiulosonate through successive reactions of gbs1892 isomerase (DhuI) and gbs1891 NADH-dependent reductase/dehydrogenase (DhuD). DhuI and DhuD enzymatically corresponded to 4-deoxy-l-threo-5-hexosulose-uronate ketol-isomerase (KduI) and 2-keto-3-deoxy-d-gluconate dehydrogenase (KduD), respectively, involved in pectin metabolism, although no or low sequence identity was observed between DhuI and KduI or between DhuD and KduD, respectively. Genes for DhuI and DhuD were found to be included in the streptococcal genetic cluster, whereas KduI and KduD are encoded in clostridia. Tertiary and quaternary structures of DhuI and DhuD were determined by x-ray crystallography. Distinct from KduI β-barrels, DhuI adopts an α/β/α-barrel structure as a basic scaffold similar to that of ribose 5-phosphate isomerase. The structure of DhuD is unable to accommodate the substrate/cofactor, suggesting that conformational changes are essential to trigger enzyme catalysis. This is the first report on the bacterial metabolism of glycosaminoglycan-derived unsaturated uronic acids by isomerase and dehydrogenase., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Serine-rich repeat glycoproteins (SRRPs) are highly conserved in streptococci and staphylococci. Glycosylation of SRRPs is important for bacterial adhesion and pathogenesis. Streptococcus agalactiae is the leading cause of bacterial sepsis and meningitis among newborns. Srr2, an SRRP from S. agalactiae strain COH1, has been implicated in bacterial virulence. Four genes (gtfA, gtfB, gtfC, and gtfD) located downstream of srr2 share significant homology with genes involved in glycosylation of other SRRPs. We have shown previously that gtfA and gtfB encode two glycosyltransferases, GtfA and GtfB, that catalyze the transfer of GlcNAc residues to the Srr2 polypeptide. However, the function of other glycosyltransferases in glycosylation of Srr2 is unknown. In this study, we determined that GtfC catalyzed the direct transfer of glucosyl residues to Srr2-GlcNAc. The GtfC crystal structure was solved at 2.7 Å by molecular replacement. Structural analysis revealed a loop region at the N terminus as a putative acceptor substrate binding domain. Deletion of this domain rendered GtfC unable to bind to its substrate Srr2-GlcNAc, concurrently abolished the glycosyltransferase activity of GtfC, and also altered glycosylation of Srr2. Furthermore, deletion of the corresponding regions from GtfC homologs also abolished their substrate binding and enzymatic activity, indicating that this region is functionally conserved. In summary, we have determined that GtfC is important for the glycosylation of Srr2 and identified a conserved loop region that is crucial for acceptor substrate binding from GtfC homologs in streptococci. These findings shed new mechanistic insight into this family of glycosyltransferases., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The aim of this study was to isolate and characterize streptokinase-producing β-hemolytic Streptococcus sp. from bovine milk. A total of 50 milk samples were collected randomly from different breeds of cow and goat (Vellore, Tamil Nadu, India). The samples were characterized and screened for streptokinase-producing isolates using microbial and biochemical analysis. About 97 colonies were isolated from milk samples showing hemolytic patterns of α (19.6%), β (24.7%) and γ (55.6 %). Out of 20β-hemolytic isolates, only 6 colonies (VB2, VB3, VB8, VB14, VB16, and VB17) were identified as β-hemolytic Streptococci as potent producers of streptokinase. VB2 and VB14 showed the greatest streptokinase activities of 265 U mL(-1) and 225 U mL(-1), respectively. Based on biochemical and molecular characterization, the potent isolates VB2 and VB14 were identified and confirmed as S. equinus and S. agalactiae, respectively. The identified strains were named Streptococcus equinus VIT&#95;VB2 (GenBank accession no. JX406835) and Streptococcus agalactiae VITVS5 (GenBank accession No. KF186620) The strains isolated from bovine milk provide a variance in the fibrinolytic activity on blood clots. The current study has demonstrated that the isolation of streptokinase producers from bovine milk, and the production of streptokinase from novel strain, enhanced the fibrinolytic activity. This study is the first to report that Streptococcus equinus produces streptokinase.

The group B pathogen Streptococcus agalactiae commonly populates the human gut and urogenital tract, and is a major cause of infection-based mortality in neonatal infants and in elderly or immunocompromised adults. Nuclease A (GBS&#95;NucA), a secreted DNA/RNA nuclease, serves as a virulence factor for S. agalactiae, facilitating bacterial evasion of the human innate immune response. GBS&#95;NucA efficiently degrades the DNA matrix component of neutrophil extracellular traps (NETs), which attempt to kill and clear invading bacteria during the early stages of infection. In order to better understand the mechanisms of DNA substrate binding and catalysis of GBS&#95;NucA, the high-resolution structure of a catalytically inactive mutant (H148G) was solved by X-ray crystallography. Several mutants on the surface of GBS&#95;NucA which might influence DNA substrate binding and catalysis were generated and evaluated using an imidazole chemical rescue technique. While several of these mutants severely inhibited nuclease activity, two mutants (K146R and Q183A) exhibited significantly increased activity. These structural and biochemical studies have greatly increased our understanding of the mechanism of action of GBS&#95;NucA in bacterial virulence and may serve as a foundation for the structure-based drug design of antibacterial compounds targeted to S. agalactiae.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a conserved cytosolic enzyme, which plays a key role in glycolysis. GAPDH catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate using NAD or NADP as a cofactor. In addition, GAPDH localized on the surface of some bacteria is thought to be involved in macromolecular interactions and bacterial pathogenesis. GAPDH on the surface of group B streptococcus (GBS) enhances bacterial virulence and is a potential vaccine candidate. Here, the crystal structure of GBS GAPDH from Streptococcus agalactiae in complex with NAD is reported at 2.46 Å resolution. Although the overall structure of GBS GAPDH is very similar to those of other GAPDHs, the crystal structure reveals a significant difference in the area spanning residues 294-307, which appears to be more acidic. The amino-acid sequence of this region of GBS GAPDH is also distinct compared with other GAPDHs. This region therefore may be of interest as an immunogen for vaccine development.

We have referenced and described Streptococcus agalactiae transposable elements encoding DDE transposases. These elements belonged to nine families of insertion sequences (ISs) and to a family of conjugative transposons (TnGBSs). An overview of the physiological impact of the insertion of all these elements is provided. DDE-transposable elements affect S. agalactiae in a number of aspects of its capability to adapt to various environments and modulate the expression of several virulence genes, the scpB-lmB genomic region and the genes involved in capsule expression and haemolysin transport being the targets of several different mobile elements. The referenced mobile elements modify S. agalactiae behaviour by transferring new gene(s) to its genome, by modifying the expression of neighbouring genes at the integration site or by promoting genomic rearrangements. Transposition of some of these elements occurs in vivo, suggesting that by dynamically regulating some adaptation and/or virulence genes, they improve the ability of S. agalactiae to reach different niches within its host and ensure the 'success' of the infectious process., (© 2014 The Authors.)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme involved in glycolysis. Despite lacking the secretory signal sequence, this cytosolic enzyme has been found localized at the surface of several bacteria and fungi. As a surface protein, GAPDH exhibits various adhesive functions, thereby facilitating colonization and invasion of host tissues. Streptococcus agalactiae, also known as group B streptococcus (GBS), binds onto the host using its surface adhesins and causes sepsis and pneumonia in neonates. GAPDH is one of the surface adhesins of GBS binding to human plasminogen and is a virulent factor associated with host colonization. Although the surface-associated GAPDH has been shown to bind to a variety of host extracellular matrix (ECM) molecules in various bacteria, the molecular mechanism underlying their interaction is not fully understood. To investigate this, structural studies on GAPDH of S. agalactiae were initiated. The gapC gene of S. agalactiae NEM316 encoding GAPDH protein was cloned into pET-28a vector, overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity. The purified protein was crystallized using the hanging-drop vapour-diffusion method. The GAPDH crystals obtained in two different crystallization conditions diffracted to 2.8 and 2.6 Å resolution, belonging to two different space groups P2₁ and P2₁2₁2₁, respectively. The structure was solved by molecular replacement and structure refinement is now in progress.

Streptococcus agalactiae (Group B Streptococcus) is a commensal of the human intestine and vagina of adult women but is the leading cause of invasive infection in neonates. This Gram-positive bacterium displays a set of virulence-associated surface proteins involved in the interaction with the host, such as adhesion to host cells, invasion of tissues, or subversion of the immune system. In this study, we characterized a cell wall-localized protein as an ecto-5'-nucleoside diphosphate phosphohydrolase (NudP) involved in the degradation of extracellular nucleotides which are central mediators of the immune response. Biochemical characterization of recombinant NudP revealed a Mn(2+)-dependent ecto-5'-nucleotidase activity on ribo- and deoxyribonucleoside 5'-mono- and 5'-diphosphates with a substrate specificity different from that of known orthologous enzymes. Deletion of the gene coding the housekeeping enzyme sortase A led to the release of NudP into the culture supernatant, confirming that this enzyme is anchored to the cell wall by its non-canonical LPXTN motif. The NudP ecto-5'-nucleotidase activity is reminiscent of the reactions performed by the mammalian ectonucleotidases CD39 and CD73 involved in regulating the extracellular level of ATP and adenosine. We further demonstrated that the absence of NudP activity decreases bacterial survival in mouse blood, a process dependent on extracellular adenosine. In vivo assays in animal models of infection showed that NudP activity is critical for virulence. These results demonstrate that Group B Streptococcus expresses a specific ecto-5'-nucleotidase necessary for its pathogenicity and highlight the diversity of reactions performed by this enzyme family. These results suggest that bacterial pathogens have developed specialized strategies to subvert the mammalian immune response controlled by the extracellular nucleotide signaling pathways.

We explored Group B Streptococcus (GBS)-induced apoptosis in human umbilical vein endothelial cells (HUVEC) and the role of phosphoramidon, a zinc metalloprotease inhibitor, in this process. GBS 90186 strain (serotype V, a blood isolate) and concentrated supernatant (CS) were used to investigate the viability and morphological alterations in HUVEC by Trypan blue uptake, electrophoresis in 2 % agarose gel and scanning electron microscopy assays. Apoptosis before and after phosphoramidon-treatment were verified by flow cytometry using annexin V-FITC labeling. Differences were considered significant when P < 0.05 using unpaired Student's t test. GBS and CS induced HUVEC death by apoptosis (76.5 and 32 %, respectively) with an increasing pro-apoptotic Bax expression and decreasing anti-apoptotic Bcl-2 expression. Caspase-3 was activated during GBS-induced endothelial apoptosis. Phosphoramidon reduced 89.3 and 100 % of GBS and CS cell death by apoptosis, respectively. Some GBS strains may induce cell death by apoptosis with involvement of metalloproteases and signaling through the intrinsic pathway of apoptosis, which may contribute to GBS survival during sepsis of adults and neonates.

Most bacteria of the genus Streptococcus are opportunistic pathogens, and some of them produce extracellular DNases, which may be important for virulence. Genome analyses of Streptococcus agalactiae (GBS) neonate isolate NEM316 revealed the presence of seven genes putatively encoding secreted DNases, although their functions, if any, are unknown. In this study, we observed that respiration growth of GBS led to the extracellular accumulation of a putative nuclease, identified as being encoded by the gbs0661 gene. When overproduced in Lactococcus lactis, the protein was found to be a divalent cation-requiring, pH-stable and heat-stable nuclease that we named Nuclease A (NucA). Substitution of the histidine(148) by alanine reduced nuclease activity of the GBS wild-type strain, indicating that NucA is the major nuclease ex vivo. We determined that GBS is able to degrade the DNA matrix comprising the neutrophil extracellular trap (NET). The nucA(H148A) mutant was impaired for this function, implicating NucA in the virulence of GBS. In vivo infection studies confirmed that NucA is required for full infection, as the mutant strain allowed increased bacterial clearance from lung tissue and decreased mortality in infected mice. These results show that NucA is involved in NET escape and is needed for full virulence., (© 2013 John Wiley & Sons Ltd.)

Objectives: Group B Streptococcus (GBS; Streptococcus agalactiae) has been regarded as uniformly susceptible to penicillins. However, we recently reported the existence of GBS with reduced penicillin susceptibility (PRGBS), with amino acid substitutions in penicillin-binding protein (PBP) 2X. Although most PRGBS show high MICs of ceftizoxime (4-64 mg/L) and cefotaxime (0.12-1 mg/L), those for strain B1 are exceptionally high (ceftizoxime MIC ≥256 mg/L and cefotaxime MIC 2 mg/L). We previously found an amino acid substitution (G539S) neighbouring the conserved K540TG motif in PBP1A in addition to the PRGBS-specific amino acid substitution Q557E in PBP2X of B1. The aim of this study was to reveal the effect of the amino acid substitutions in PBP1A and PBP2X of B1 on the high cephalosporin resistance., Methods: A ceftizoxime competition assay was performed to reveal the PBPs that are the main targets of ceftizoxime. We generated two allelic exchange mutants from β-lactam-susceptible GBS BAA-611. BAA-611 (B1PBP2X) contained the PBP2X gene derived from B1 and BAA-611 (B1PBP2X, B1PBP1A) contained both the PBP2X and the PBP1A gene derived from B1. These allelic exchange mutants and strain B1 were subjected to susceptibility testing., Results: The ceftizoxime competition assay revealed that PBP1A and PBP2X were the main targets of ceftizoxime. Although the MICs of ceftizoxime and cefotaxime for BAA-611 (B1PBP2X) were 64 and 0.5 mg/L, respectively, BAA-611 (B1PBP2X, B1PBP1A) showed high cephalosporin resistance (ceftizoxime MIC ≥256 mg/L and cefotaxime MIC 2 mg/L) comparable to B1., Conclusions: The high cephalosporin resistance of GBS was caused by amino acid substitutions in PBP1A and PBP2X.