Characterization of M-Type-Specific Pilus Expression in Group A Streptococcus.

In addition to providing a typing mechanism for group A Streptococcus (GAS) isolates (T typing), cell surface pilus production impacts GAS virulence characteristics, including adherence and immune evasion. The pilus biosynthesis genes are located in the fibronectin- and collagen-binding T-antigen (FCT) region of the genome, and nine different FCT types, encoding more than 20 different T types, have been described. GAS isolates are not uniform in their degree or pattern of pilus expression, as highlighted by pilus production being thermoregulated in isolates that harbor the FCT-type FCT-3 (e.g., M-types M3 and M49) but not in isolates that harbor FCT-2 (e.g., M-type M1). Here, we investigated the molecular basis underlying our previous finding that M3 GAS isolates produce pili in lower abundance than M1 or M49 isolates do. We discovered that, at least in part, the low pilus expression observed for M3 isolates is a consequence of the repression of pilus gene expression by the CovR/CovS two-component regulatory system and of an M3-specific mutation in the nra gene, encoding a positive regulator of pilus gene expression. We also discovered that the orthologous transcriptional regulators RofA and Nra, whose encoding genes are located within FCT-2 and FCT-3, respectively, are not functionally identical. Finally, we sequenced the genome of an M3 isolate that had naturally undergone recombinational replacement of the FCT region, changing the FCT and T types of this strain from FCT-3/T3 to FCT-2/T1. Our study furthers the understanding of strain- and type-specific variation in virulence factor production by an important human pathogen. IMPORTANCE Our ability to characterize how a pathogen infects and causes disease, and consequently our ability to devise approaches to prevent or attenuate such infections, is inhibited by the finding that isolates of a given pathogen often show phenotypic variability, for example, in their ability to adhere to host cells through modulation of cell surface adhesins. Such variability is observed between isolates of group A Streptococcus (GAS), and this study investigates the molecular basis for why some GAS isolates produce pili, cell wall-anchored adhesins, in lower abundance than other isolates do. Given that pili are being considered as potential antigens in formulations of future GAS vaccines, this study may inform vaccine design.

The RD2 Pathogenicity Island Modifies the Disease Potential of the Group A Streptococcus.

Serotype M28 isolates of the group A Streptococcus (GAS; Streptococcus pyogenes) are nonrandomly associated with cases of puerperal sepsis, a potentially life-threatening infection that can occur in women following childbirth. Previously, we discovered that the 36.3-kb RD2 pathogenicity island, which is present in serotype M28 isolates but lacking from most other isolates, promotes the ability of M28 GAS to colonize the female reproductive tract. Here, we performed a gain-of-function study in which we introduced RD2 into representative serotype M1, M49, and M59 isolates and assessed the phenotypic consequences of RD2 acquisition. All RD2-containing derivatives colonized a higher percentage of mice, and at higher CFU levels, than did the parental isolates in a mouse vaginal colonization model. However, for two additional phenotypes, survival in heparinized whole human blood and adherence to two human vaginal epithelial cell lines, there were serotype-specific differences from RD2 acquisition. Using transcriptomic comparisons, we identified that such differences may be a consequence of RD2 altering the abundance of transcripts from select core genome genes along serotype-specific lines. Our study is the first that interrogates RD2 function in GAS serotypes other than M28 isolates, shedding light on variability in the phenotypic consequences of RD2 acquisition and informing on why this mobile genetic element is not ubiquitous in the GAS population.

The group A Streptococcus accessory protein RocA: regulatory activity, interacting partners and influence on disease potential.

The group A Streptococcus (GAS) causes diseases that range from mild (e.g. pharyngitis) to severely invasive (e.g. necrotizing fasciitis). Strain- and serotype-specific differences influence the ability of isolates to cause individual diseases. At the center of this variability is the CovR/S two-component system and the accessory protein RocA. Through incompletely defined mechanisms, CovR/S and RocA repress the expression of more than a dozen immunomodulatory virulence factors. Alleviation of this repression is selected for during invasive infections, leading to the recovery of covR, covS or rocA mutant strains. Here, we investigated how RocA promotes CovR/S activity, identifying that RocA is a pseudokinase that interacts with CovS. Disruption of CovS kinase or phosphatase activities abolishes RocA function, consistent with RocA acting through the modulation of CovS activity. We also identified, in conflict with a previous study, that the RocA regulon includes the secreted protease-encoding gene speB. Finally, we discovered an inverse correlation between the virulence of wild-type, rocA mutant, covS mutant and covR mutant strains during invasive infection and their fitness in an ex vivo upper respiratory tract model. Our data inform on mechanisms that control GAS disease potential and provide an explanation for observed strain- and serotype-specific variability in RocA function.

A Mobile Genetic Element Promotes the Association Between Serotype M28 Group A Streptococcus Isolates and Cases of Puerperal Sepsis.

BACKGROUND: Bacterial infections following childbirth-so-called puerperal infections-cause morbidity in 5%-10% of all new mothers. At low frequency, the infection can spread to the blood, resulting in life-threatening sepsis known as puerperal sepsis. Pathogens causing puerperal sepsis include group A Streptococcus (GAS), and epidemiological analyses have identified isolates of a single serotype, M28, as being nonrandomly associated with cases of puerperal sepsis. The genomes of serotype M28 GAS isolates harbor a 36.3-kb mobile genetic element of apparent group B Streptococcus origin, termed region of difference 2 (RD2). METHODS: The phenotypic (determined via tissue culture and a vaginal colonization model) and regulatory (determined via RNA sequencing analysis) contributions of RD2 were assessed by comparing parental, RD2 deletion mutant, and complemented mutant serotype M28 GAS strains. RESULTS: RD2 affords serotype M28 isolates an enhanced ability to adhere to human vaginal epithelial cells and to colonize the female reproductive tract in a mouse model of infection. In addition, RD2 influences the abundance of messenger RNAs from >100 core chromosomal GAS genes. CONCLUSIONS: The data are consistent with RD2 directly, via encoded virulence factors, and indirectly, via encoded regulatory proteins, modifying the virulence potential of GAS and contributing to the decades-old association of serotype M28 isolates with cases of puerperal sepsis.

Identification and Characterization of Serotype-Specific Variation in Group A Streptococcus Pilus Expression.

Isolates of a given bacterial pathogen often display phenotypic variation, and this can negatively impact public health, for example, by reducing the efficacy of preventative measures. Here, we identify that the human pathogen group A Streptococcus (GAS; Streptococcus pyogenes) expresses pili on its cell surface in a serotype-specific manner. Specifically, we show that serotype M3 GAS isolates, which are nonrandomly associated with causing particularly severe and lethal invasive infections, produce negligible amounts of pili relative to serotype M1 and M49 isolates. Performance of an interserotype transcriptome comparison (serotype M1 versus serotype M3) was instrumental in this discovery. We also identified that the transcriptional regulator Nra positively regulates pilus expression in M3 GAS isolates and that the low level of pilus expression of these isolates correlates with a low level of nra transcription. Finally, we discovered that the phenotypic consequences of low levels of pilus expression by M3 GAS isolates are a reduced ability to adhere to host cells and an increased ability to survive and proliferate in human blood. We propose that an enhanced ability to survive in human blood, in part due to reduced pilus expression, is a contributing factor in the association of serotype M3 isolates with highly invasive infections. In conclusion, our data show that GAS isolates express pili in a serotype-dependent manner and may inform vaccine development, given that pilus proteins are being discussed as possible GAS vaccine antigens.

RocA Is an Accessory Protein to the Virulence-Regulating CovRS Two-Component System in Group A Streptococcus.

Regulating gene expression during infection is critical to the ability of pathogens to circumvent the immune response and cause disease. This is true for the group A Streptococcus (GAS), a pathogen that causes both invasive (e.g., necrotizing fasciitis) and noninvasive (e.g., pharyngitis) diseases. The control of virulence (CovRS) two-component system has a major role in regulating GAS virulence factor expression. The regulator of cov (RocA) protein, which is a predicted kinase, functions in an undetermined manner through CovRS to alter gene expression and reduce invasive disease virulence. Here, we show that the ectopic expression of a truncated RocA derivative, harboring the membrane-spanning domains but not the dimerization or HATPase domain, is sufficient to complement a rocA mutant strain. Coupled with a previous bioinformatic study, the data are consistent with RocA being a pseudokinase. RocA reduces the ability of serotype M1 GAS isolates to express capsule and to evade killing in human blood, phenotypes that are not observed for M3 or M18 GAS due to isolates of these serotypes naturally harboring mutant rocA alleles. In addition, we found that varying the RocA concentration attenuates the regulatory activity of Mg2+ and the antimicrobial peptide LL-37, which positively and negatively regulate CovS function, respectively. Thus, we propose that RocA is an accessory protein to the CovRS system that influences the ability of GAS to modulate gene expression in response to host factors. A model of how RocA interacts with CovRS, and of the regulatory consequences of such activity, is presented.

Xylooligosaccharides: an economical prebiotic from agroresidues and their health benefits.

Oligosaccharides and dietary fibres are non-digestible food ingredients that preferentially stimulate the growth of prebiotic Bifidobacterium and other lactic acid bacteria in the gastro-intestinal tract. Xylooligosaccharides (XOS) provide a plethora of health benefits and can be incorporated into several functional foods. In the recent times, there has been an over emphasis on the microbial conversion of agroresidues into various value added products. Xylan, the major hemicellulosic component of lignocellulosic materials (LCMs), represents an important structural component of plant biomass in agricultural residues and could be a potent bioresource for XOS. On an industrial scale, XOS can be produced by chemical, enzymatic or chemo-enzymatic hydrolysis of LCMs. Chemical methods generate XOS with a broad degree of polymerization (DP), while enzymatic processes will be beneficial for the manufacture of food grade and pharmaceutically important XOS. Xylooligomers exert several health benefits, and therefore, have been considered to provide relief from several ailments. This review provides a brief on production, purification and structural characterization of XOS and their health benefits.

ET-traps: Potential therapeutics for preeclampsia.

Elevated endothelin-1 (ET-1) has been implicated in several diseases including preeclampsia, where it causes the induction of hypertension, oxidative stress, endoplasmic reticulum stress, microvascular dysfunction and tissue damage in different organs. ET-traps are Fc-fusion proteins with a design based on the physiological receptors of ET-1. This paper discusses the potential use of ET-traps as a therapeutic for preeclampsia. ET-traps potently bind and sequester pathologically elevated ET-1 to significantly reduce different markers of pathology to non-disease levels with no toxicity.

Coverage vs Utilization of integrated child services scheme (ICDS): A community based study in urban block of Patiala, Punjab (India).

Introduction: Integrated Child Development Services (ICDS) scheme provides a wide range of health, nutrition and education services to children, women and adolescent girls. Maternal satisfaction usually influences service uptake and utilization. Therefore, in addition to coverage, the quality of services provided and end-user satisfaction need to be assessed. Material and Methods: A cross-sectional community-level study was conducted in the urban block of the Patiala district in Punjab. A total of 54 AWCs were selected from a sample frame of 222 by applying a systematic random sampling technique. From each selected AWC, 20 mothers of registered beneficiaries (0-6 years) were randomly selected for interview. The data collected was entered and analysed using SPSS version 22. Results: The present study revealed that most of the Anganwadis (87.19%) were regularly open for the provision of various ICDS services. However, only 48.02% of children received supplementary nutrition from AWC regularly. Only 32.36% of children who were sick were referred by AWW. Satisfaction with quantity and quality of food served was found to be 61% and 45.90% among mothers, respectively. Conclusion: It was concluded that there is a need to improve the quality of services to overcome the problem of under-utilisation of services despite the wide coverage of ICDS services; regular orientation and training courses for AWWs should be conducted to ensure better service delivery to all beneficiaries.

A cross-sectional study of awareness and practices regarding thalassemia among parents of thalassemic children.

BACKGROUND: This cross-sectional study was carried out in thalassemia ward of Rajindra Hospital, Patiala, among the parents of thalassemic children to determine awareness about side effects and complications of blood transfusion therapy, other treatment options, nature of disease, and food practices of transfusion-dependent patients. The study was carried out using a predesigned questionnaire and 118 parents participated in the study. About 50.84% patients belonged to the Sikh community, 45.76% patients practiced Hindu religion, and only 3.38% of the patients were Muslim. This study shows that 87.29% parents do not know how the disease is spread. About 55.93% have no knowledge about iron-containing food should not be included in the diet of transfusion-dependent patients. About 86.44% parents believed they had no role in transmission of the disease to their child, 79.66% parents do not understand the importance of screening before marriage, and 95.76% parents do not know about alternative treatment options. This study wants to shine light about the inadequate and superficial knowledge of thalassemia among general public and how awareness of the disease will bring down the incidence rates. AIMS: The main objective of the study is to determine the degree of awareness of the disease, their knowledge of complications of blood transfusion therapy, and other treatment options among the parents of the children with thalassemia who are currently on blood transfusion therapy. SUBJECTS AND METHODS: This cross-sectional study was conducted in thalassemia ward of Rajindra Hospital, Patiala from June 2018 to November 2018. After informed verbal consent was ensured, parents of the patients were interviewed using a questionnaire as the patients received blood transfusion. Questions include prevention, progression, cause, and spread, of the disease. The questions also include side effects and complications of blood transfusion therapy and other treatment options available. STATISTICS USED: Continuous variables were summarized as mean and standard deviation and categorical variables as proportion (%). Percentage and frequency was used wherever applicable. RESULTS: Parents of about 118 patients were interviewed out of which 74.57% parents were illiterate and only 25.42% were literate. About 50.84% of the patients were Sikh, 45.76% were Hindu, and only 3.38% patients were Muslims. About 71.19% of the parents had no knowledge about the prevention of the disease, and 87.29% of the parents did not know mechanism of spread. Despite having transfusion-dependent children, only 44.07% of the parents restricted iron-containing food from the diet of their children. About 72.05% of the patients have inadequate information about risk of hepatitis B, hepatitis C, and HIV due to blood transfusions and only 21.29% of the patients understand the importance of hepatitis B vaccine. CONCLUSION: Awareness among both literate and illiterate parents was inadequate and sensitization among general public and parents of thalasemmic children should be initiated.

Factors affecting nutritional status of Anganwadi children: A cross-sectional study.

INTRODUCTION: As per the National Family Health Survey (NFHS) 2015-16, 35.7% children below 5 years of age are underweight. In light of Malnutrition rates still remaining alarming in children, it becomes pertinent to elicit the factors that affect nutritional status of children. So, this study was undertaken. MATERIALS AND METHODS: After obtaining ethical approval from institutional ethics committee, data were collected on a pretested questionnaire. Information from mothers of 1085 children attending Anganwadi center in an urban block of Patiala was collected and analyzed. RESULTS: Among females, 35.85% were underweight, whereas the proportion for males was 28.68%. The proportion among immunized children who were underweight was 31.34%, whereas the proportion among unimmunized children was 38.91%. Those who received supplementary nutrition were also in more in numbers in normal weight range than those who did not. CONCLUSION: Gender, birth order, and immunization status of child are significantly associated with nutritional status. This study showed that prevalence of malnutrition was less among those who received supplementary nutrition as compared to ones who did not.

Phenotypic Variation in the Group A Streptococcus Due to Natural Mutation of the Accessory Protein-Encoding Gene rocA.

Populations of a bacterial pathogen, whether recovered from a single patient or from a worldwide study, are often a heterogeneous mix of genetically and phenotypically divergent strains. Such heterogeneity is of value in changing environments and arises via mechanisms such as gene gain or gene mutation. Here, we identify an isolate of serotype M12 group A Streptococcus (GAS) (Streptococcus pyogenes) that has a natural mutation in rocA, which encodes an accessory protein to the virulence-regulating two-component system CovR/CovS (CovR/S). Disruption of RocA activity results in the differential expression of multiple GAS virulence factors, including the anti-phagocytic hyaluronic acid capsule and the chemokine protease SpyCEP. While some of our data regarding RocA-regulated genes overlaps with previous studies, which were performed with isolates of alternate GAS serotypes, some variability was also observed. Perhaps as a consequence of this alternate regulatory activity, we discovered that the contribution of RocA to the ability of the M12 isolate to survive and proliferate in human blood ex vivo is opposite that previously observed in M1, M3, and M18 GAS strains. Specifically, rocA mutation reduced, rather than enhanced, survival of the isolate. Finally, we also present data from an analysis of rocA transcription and show that rocA is transcribed in both mono- and polycistronic mRNAs. In aggregate, our data provide insight into the important regulatory role of RocA and into the mechanisms and consequences of GAS phenotypic heterogeneity.IMPORTANCE This study investigates the regulatory and phenotypic consequences of a naturally occurring mutation in a strain of the bacterial pathogen the group A Streptococcus (Streptococcus pyogenes). We show that this mutation, which occurs in a regulator-encoding gene, rocA, leads to altered virulence factor expression and reduces the ability of this isolate to survive in human blood. Critically, the blood survival phenotype and the assortment of genes regulated by RocA differ compared to previous studies into RocA activity. The data are consistent with there being strain- or serotype-specific variability in RocA function. Given that phenotypic variants can lead to treatment failures and escape from preventative regimes, our data provide information with regard to a mechanism of phenotypic variation in a prevalent Gram-positive pathogen.

Applicability of recombinant ß-xylosidase from the extremely thermophilic bacterium Geobacillus thermodenitrificans in synthesizing alkylxylosides.

The ß-xylosidase encoding gene (XsidB) of the extremely thermophilic bacterium Geobacillus thermodenitrificans has been cloned and expressed in Escherichia coli. The homotrimeric recombinant XsidB is of 204.0kDa, which is optimally active at 60°C and pH 7.0 with T1/2 of 58min at 70°C. The ß-xylosidase remains unaffected in the presence of most metal ions and organic solvents. The Km [p-nitrophenyl ß-xyloside (pNPX)], Vmax and kcat values of the enzyme are 2×10(-3)M, 1250µmolesmg(-1)min(-1) and 13.20×10(5)min(-1), respectively. The enzyme catalyzes transxylosylation reactions in the presence of alcohols as acceptors. The pharmaceutically important ß-methyl-d-xylosides could be produced using pNPX as the donor and methanol as acceptor. The products of transxylosylation were identified by TLC and HPLC, and the structure was confirmed by (1)H NMR analysis. The enzyme is also useful in synthesizing transxylosylation products from the wheat bran hydrolysate.