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Histamine is an active amine compound that occurs in various fermented foods that may cause adverse effects on the human health. Certain microorganisms are able to degrade histamine by an oxidative deamination reaction. Therefore, the present study aimed to quantify histamine‐forming and/or ‐degrading activity of the isolates derived from milk of goat and sheep herds, in Iran, by the capillary zone electrophoresis (CZE) method; and we evaluated the molecular characteristics of staphylococcal isolates. Among 243 staphylococcal isolates, 29 histamine‐degrading bacteria were identified. One of these isolates, identified as Staph. epidermidis, No. 605, exhibited the highest activity compared to others, degrading available histamine to 58.33% within 24 h. By polymerase chain reaction (PCR) analysis, the isolate, No. 605 that exhibited remarkable histamine‐degrading activity lacked the genes encoding coagulase and DNase, nor did it harbor any of the five classical enterotoxin genes. This is the first report to show that seven Staphylococcus species, including Staph. chromogenes, Staph. aureus, Staph. haemolyticus, Staph. epidermidis, Staph. pseudintermedius, Staph. agnetis, and Staph. hyicus, were able to degrade histamine, which were hitherto not known to have this capacity. Therefore, histamine‐degrading activity is a definite criterion to introduce fermenting organisms able to decrease histamine content in different food products.

Colistin (COL) is a cationic cyclic peptide that disrupts negatively-charged bacterial cell membranes and frequently serves as an antibiotic of last resort to combat multidrug-resistant Gram-negative bacterial infections. Emergence of the horizontally transferable plasmid-borne mobilized colistin resistance (mcr) determinant and its spread to Gram-negative strains harboring extended-spectrum β-lactamase and carbapenemase resistance genes threatens futility of our chemotherapeutic arsenal. COL is widely regarded to have zero activity againstmcr+patients based on standard antimicrobial susceptibility testing (AST) performed in enriched bacteriological growth media; consequently, the drug is withheld from patients withmcr+infections. However, these standard testing media poorly mimic in vivo physiology and omit host immune factors. Here we report previously unrecognized bactericidal activities of COL againstmcr-1+isolates ofEscherichia coli(EC),Klebsiella pneumoniae(KP), andSalmonella enterica(SE) in standard tissue culture media containing the physiological buffer bicarbonate. Moreover, COL promoted serum complement deposition on themcr-1+Gram-negative bacterial surface and synergized potently with active human serum in pathogen killing. At COL concentrations readily achievable with standard dosing, the peptide antibiotic killedmcr-1+EC, KP, and SE in freshly isolated human blood proved effective as monotherapy in a murine model ofmcr-1+EC bacteremia. Our results suggest that COL, currently ignored as a treatment option based on traditional AST, may in fact benefit patients withmcr-1+Gram negative infections based on evaluations performed in a more physiologic context. These concepts warrant careful consideration in the clinical microbiology laboratory and for future clinical investigation of their merits in high risk patients with limited therapeutic options.

Histamine is an active amine compound that occurs in various fermented foods that may cause adverse effects on the human health. Certain microorganisms are able to degrade histamine by an oxidative deamination reaction. Therefore, the present study aimed to quantify histamine-forming and/or -degrading activity of the isolates derived from milk of goat and sheep herds, in Iran, by the capillary zone electrophoresis (CZE) method; and we evaluated the molecular characteristics of staphylococcal isolates. Among 243 staphylococcal isolates, 29 histamine-degrading bacteria were identified. One of these isolates, identified as

Background The evolving antibiotic-resistant behavior of health care–associated methicillin-resistant Staphylococcus aureus (HA-MRSA) USA100 strains are of major concern. They are resistant to a broad class of antibiotics such as macrolides, aminoglycosides, fluoroquinolones, and many more. Findings The selection of appropriate antibiotic susceptibility examination media is very important. Thus, we use bacteriological (cation-adjusted Mueller-Hinton broth) as well as physiological (R10LB) media to determine the effect of vancomycin on USA100 strains. The study includes the profiling behavior of HA-MRSA USA100 D592 and D712 strains in the presence of vancomycin through various high-throughput assays. The US100 D592 and D712 strains were characterized at sub-inhibitory concentrations through growth curves, RNA sequencing, bacterial cytological profiling, and exo-metabolomics high throughput experiments. Conclusions The study reveals the vancomycin resistance behavior of HA-MRSA USA100 strains in dual media conditions using wide-ranging experiments.

Healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) USA100 strains are of major concern due to their evolving antibiotic resistant. They are resistant to a broad class of antibiotics like macrolides, aminoglycosides, fluoroquinolones, and many more. The selection of appropriate antibiotic susceptibility examination media is very important. Thus, we use bacteriological (CA-MHB) as well as physiological (R10LB) media to determine the effect of vancomycin on USA100 strains. The study includes the profiling behaviour of HA-MRSA USA100 D592 and D712 strains in the presence of vancomycin through various high-throughput assays. The US100 D592 and D712 strains were characterized at sub-inhibitory concentrations through growth curves, RNA sequencing, bacterial cytological profiling, and exo-metabolomics high throughput experiments. The study reveals the vancomycin resistance behavior of USA100 strains in dual media conditions using wide-ranging experiments.

Staphylococcus aureus strains have been continuously evolving resistance to numerous classes of antibiotics including methicillin, vancomycin, daptomycin and linezolid, compounding the enormous healthcare and economic burden of the pathogen. Cation-adjusted Mueller-Hinton broth (CA-MHB) is the standard bacteriological media for measuring antibiotic susceptibility in the clinical lab, but the use of media that more closely mimic the physiological state of the patient, e.g. mammalian tissue culture media, can in certain circumstances reveal antibiotic activities that may be more predictive of effectiveness in vivo. In the current study, we use both types of media to explore antibiotic resistance phenomena in hospital-acquired USA100 lineage methicillin-resistant, vancomycin-intermediate Staphylococcus aureus (MRSA/VISA) strain D712 via multidimensional high throughput analysis of growth rates, bacterial cytological profiling, RNA sequencing, and exo-metabolomics (HPLC and LC-MS). Here, we share data generated from these assays to shed light on the antibiotic resistance behavior of MRSA/VISA D712 in both bacteriological and physiological media., Measurement(s)Antibacterial Response • cDNA • transcription profiling assay • culture medium • organic acidTechnology Type(s)bacterial cytological profiling • RNA sequencing • liquid chromatography-tandem mass spectrometry • high-performance liquid chromatographyFactor Type(s)growth mediumSample Characteristic - OrganismStaphylococcus aureus Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.10283108

Weekly oritavancin plus ampicillin continuous infusion combination therapy was used to successfully treat a deep spine vancomycin-resistant Enterococcus faecium infection associated with hardware. Checkerboard and time-kill assays confirmed synergy between these two antibiotics. Further synergies of oritavancin and ampicillin with rifampin or the endogenous human antimicrobial peptide cathelicidin LL-37 were demonstrated.

Cation adjusted-Mueller Hinton Broth (CA-MHB) is the standard bacteriological medium utilized in the clinic for the determination of antibiotic susceptibility. However, a growing number of literature has demonstrated that media conditions can cause a substantial difference in the efficacy of antibiotics and antimicrobials. Recent studies have also shown that minimum inhibitory concentration (MIC) tests performed in standard cell culture media (e.g. RPMI and DMEM) are more indicative of in vivo antibiotic efficacy, presumably because they are a better proxy for the human host’s physiological conditions. The basis for the bacterial media dependent susceptibility to antibiotics remains undefined. To address this question, we characterized the physiological response of methicillin-resistant Staphylococcus aureus (MRSA) during exposure to sub-inhibitory concentrations of the beta-lactam antibiotic nafcillin in either CA-MHB or RPMI + 10% LB (R10LB). Here, we present high quality transcriptomic, exo-metabolomic and morphological data paired with growth and susceptibility results for MRSA cultured in either standard bacteriologic or more physiologic relevant medium., Design Type(s)replicate design • transcription profiling design • sequence analysis objectiveMeasurement Type(s)transcription profiling assay • cellular morphology • exo-metabolome • growthTechnology Type(s)RNA sequencing • fluorescence microscopy • liquid chromatography-tandem mass spectrometry • high performance liquid chromatography • Optical Density MeasurementFactor Type(s)culture medium • biological replicate • experimental conditionSample Characteristic(s)Staphylococcus aureus • culturing environment Machine-accessible metadata file describing the reported data (ISA-Tab format)

Emerging antibiotic resistance among pathogenic bacteria is an issue of great clinical importance, and new approaches to therapy are urgently needed. Anacardic acid, the primary active component of cashew nut shell extract, is a natural product used in the treatment of a variety of medical conditions, including infectious abscesses. Here, we investigate the effects of this natural product on the function of human neutrophils. We find that anacardic acid stimulates the production of reactive oxygen species and neutrophil extracellular traps, two mechanisms utilized by neutrophils to kill invading bacteria. Molecular modeling and pharmacological inhibitor studies suggest anacardic acid stimulation of neutrophils occurs in a PI3K-dependent manner through activation of surface-expressed G protein-coupled sphingosine-1-phosphate receptors. Neutrophil extracellular traps produced in response to anacardic acid are bactericidal and complement select direct antimicrobial activities of the compound.

The disconnect between the genomic prediction of secondary metabolite biosynthetic potential and the observed laboratory production profile of microorganisms is well-documented. While heterologous expression of biosynthetic gene clusters (BGCs) is often seen as a potential solution to bridge this gap, it is not immune to many challenges including impaired regulation, the inability to recruit essential building blocks, and transcriptional and/or translational silence of the biosynthetic genes. Here we report the discovery, cloning, refactoring, and heterologous expression of a cryptic hybrid phenazine-type BGC (spz) from the marine actinomycete Streptomyces sp. CNB-091. Overexpression of the engineered spz pathway resulted in increased production and chemical diversity of phenazine natural products belonging to the streptophenazine family, including bioactive members containing an unprecedented N-formylglycine attachment. An atypical discrete adenylation enzyme in the spz cluster is required to introduce the formylglycine moiety and represents a phylogenetically distinct class of adenylation proteins. Bauman et al., designed and utilized promoter cassettes to transcriptionally activate the cryptic streptophenazine biosynthetic pathway from marine Streptomyces bacteria. Pathway refactoring resulted in production of over 100 compounds, including streptophenazines with a rare N-formylglycine moiety that showed enhanced antibiotic activity.

Staphylococcus aureus, a leading cause of serious human infections in both healthcare and community settings, is increasingly difficult to control due to expanding resistance to multiple antibiotic classes. Methicillin-resistant S. aureus (MRSA) strains have disseminated on a global scale and are associated with adverse patient outcomes, increased hospital stays, and significant economic costs to the healthcare system. A proximal step in S. aureus infection is colonization of the nasal mucosa, and effective strategies to decolonize high risk patients to reduce the risk of invasive infection and nosocomial spread represent an important clinical priority. With rising resistance to mupirocin, the most common antibiotic utilized for nasal MRSA decontamination, we are examining the use of pure molecular iodine (I2)-based formulations for this indication. Recently, an iodophor formulation of povidone-iodine (PVP-I) has shown significant promise for nasal MRSA decontamination by swabbing the anterior nares of patients in hospital settings, but the I2 concentration in this treatment is less than 0.01% of total iodine species present and like all providone-iodine formulations causes skin staining. Here we determine that a novel non-staining formulation of I2 combined with the safe organic emollient glycerin delivers high local concentrations of the active antimicrobial entity (I2) with minimal evaporative loss, exhibits activity at ∼1 part per million against MRSA and other important Gram-positive and -negative human pathogens. This formulation for I2 topical delivery produced similar reductions in mean bacterial burden and was associated with fewer treatment failures (

In the ongoing effort to unlock the chemical potential of marine bacteria, genetic engineering of biosynthetic gene clusters (BGCs) is increasingly used to awake or improve expression of biosynthetic genes that may lead to discovery of novel bioactive natural products. Previously, we reported the successful capture, engineering and heterologous expression of an orphan BGC from the marine actinomycete Saccharomonospora sp. CNQ-490, which resulted in the isolation of the novel lipopeptide antibiotic taromycin A. Herein we report the isolation and structure elucidation of taromycin B, the second most abundant product of the taromycin biosynthetic series, and show that taromycins A and B exhibit complex chromatographic properties indicative of interconverting conformations. Taromycins A and B display potent activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium clinical isolates, suggestive that the taromycin molecular scaffold is a promising starting point for further derivatization to produce compounds with promising antibiotic characteristics.

We recovered a non-beta-hemolytic Streptococcus pyogenes strain from a severe soft tissue infection. In this isolate, we detected a premature stop codon within the sagC gene of the streptolysin S (SLS) biosynthetic operon. Reintroduction of full-length sagC gene on a plasmid vector restored the beta-hemolytic phenotype to our clinical isolate, indicating that the point mutation in sagC accounted for loss of hemolytic activity. To the best of our knowledge, this is the first report to demonstrate that a severe soft tissue infection can be caused by a non-beta-hemolytic S. pyogenes strain lacking a functional SagC.

Summary The natural product CJ-15,801 is an inhibitor of Staphylococcus aureus , but not other bacteria. Its close structural resemblance to pantothenic acid, the vitamin precursor of coenzyme A (CoA), and its Michael acceptor moiety suggest that it irreversibly inhibits an enzyme involved in CoA biosynthesis or utilization. However, its mode of action and the basis for its specificity have not been elucidated to date. We demonstrate that CJ-15,801 is transformed by the uniquely selective S. aureus pantothenate kinase, the first CoA biosynthetic enzyme, into a substrate for the next enzyme, phosphopantothenoylcysteine synthetase, which is inhibited through formation of a tight-binding structural mimic of its native reaction intermediate. These findings reveal CJ-15,801 as a vitamin biosynthetic pathway antimetabolite with a mechanism similar to that of the sulfonamide antibiotics and highlight CoA biosynthesis as a viable antimicrobial drug target.

Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.

The aquatic zoonotic pathogen Streptococcus iniae represents a threat to the worldwide aquaculture industry and poses a risk to humans who handle raw fish. Because little is known about the mechanisms of S. iniae pathogenesis or virulence factors, we established a high-throughput system combining whole-genome pyrosequencing and transposon mutagenesis that allowed us to identify virulence proteins, including Pdi, the polysaccharide deacetylase of S. iniae, that we describe here. Using bioinformatics tools, we identified a highly conserved signature motif in Pdi that is also conserved in the peptidoglycan deacetylase PgdA protein family. A Δpdi mutant was attenuated for virulence in the hybrid striped bass model and for survival in whole fish blood. Moreover, Pdi was found to promote bacterial resistance to lysozyme killing and the ability to adhere to and invade epithelial cells. On the other hand, there was no difference in the autolytic potential, resistance to oxidative killing or resistance to cationic antimicrobial peptides between S. iniae wild-type and Δpdi. In conclusion, we have demonstrated that pdi is involved in S. iniae adherence and invasion, lysozyme resistance and survival in fish blood, and have shown that pdi plays a role in the pathogenesis of S. iniae. Identification of Pdi and other S. iniae virulence proteins is a necessary initial step towards the development of appropriate preventive and therapeutic measures against diseases and economic losses caused by this pathogen.

UnlabelledInhibitory CD33-related Siglec receptors regulate immune cell activation upon engaging ubiquitous sialic acids (Sias) on host cell surface glycans. Through molecular mimicry, Sia-expressing pathogen group B Streptococcus binds inhibitory human Siglec-9 (hSiglec-9) to blunt neutrophil activation and promote bacterial survival. We unexpectedly discovered that hSiglec-9 also specifically binds high molecular weight hyaluronan (HMW-HA), another ubiquitous host glycan, through a region of its terminal Ig-like V-set domain distinct from the Sia-binding site. HMW-HA recognition by hSiglec-9 limited neutrophil extracellular trap (NET) formation, oxidative burst, and apoptosis, defining HMW-HA as a regulator of neutrophil activation. However, the pathogen group A Streptococcus (GAS) expresses a HMW-HA capsule that engages hSiglec-9, blocking NET formation and oxidative burst, thereby promoting bacterial survival. Thus, a single inhibitory lectin receptor detects two distinct glycan "self-associated molecular patterns" to maintain neutrophil homeostasis, and two leading human bacterial pathogens have independently evolved molecular mimicry to exploit this immunoregulatory mechanism.Key messageHMW-HA is the first example of a non-sialic acid containing glycan to be recognized by CD33-related Siglecs. HMW-HA engagement of hSiglec-9 attenuates neutrophil activation. Group A Streptococcus exploits hSiglec-9 recognition via its polysaccharide HMW-HA capsule to subvert neutrophil killing.

Structural database-derived propensities for amino acids to adopt particular local protein structures, such as alpha-helix and beta-strand, have long been recognized and effectively exploited for the prediction of protein secondary structure. However, the experimental verification of database-derived propensities using mutagenesis studies has been problematic, especially for beta-strand propensities, because local structural preferences are often confounded by non-local interactions arising from formation of the native tertiary structure. Thus, the overall thermodynamic stability of a protein is not always altered in a predictable manner by changes in local structural propensity at a single position. In this study, we have undertaken an investigation of the relationship between beta-strand propensity and protein folding kinetics. By characterizing the effects of a wide variety of amino acid substitutions at two different beta-strand positions in an SH3 domain, we have found that the observed changes in protein folding rates are very well correlated to beta-strand propensities for almost all of the substitutions examined. In contrast, there is little correlation between propensities and unfolding rates. These data indicate that beta-strand conformation is well formed in the structured portion of the SH3 domain transition state, and that local structure propensity strongly influences the stability of the transition state. Since the transition state is known to be packed more loosely than the native state and likely lacks many of the non-local stabilizing interactions seen in the native state, we suggest that folding kinetics studies may generally provide an effective means for the experimental validation of database-derived local structural propensities.

Protein-protein interactions are required for most cellular functions, yet little is known about the relationship between protein-protein interaction affinity and biological activity. To investigate this issue, we engineered a series of mutants that incrementally reduced the affinity of the yeast Sho1p SH3 domain for its in vivo target, the MAP kinase kinase Pbs2p. We demonstrate a strong linear correlation between the binding energy of these mutants and quantitative in vivo outputs from the HOG high-osmolarity response pathway controlled by Sho1p. In addition, we find that reduction in binding affinity for the correct target within this pathway causes a proportional increase in misactivation of the related mating pheromone response pathway and that strong binding affinity alone does not guarantee efficient biological activity. Our experiments also indicate that a second binding surface on the Sho1p SH3 domain is required for its proper in vivo function.

The mevalonate pathway is essential for the production of many important molecules in lipid biosynthesis. Inhibition of this pathway is the mechanism of statin cholesterol-lowering drugs, as well as the target of drugs to treat osteoporosis, to combat parasites, and to inhibit tumor cell growth. Unlike the human mevalonate pathway, the bacterial pathway appears to be regulated by diphosphomevalonate (DPM). Enzymes in the mevalonate pathway act to produce isopentenyl diphosphate, the product of the DPM decarboxylase reaction, utilize phosphorylated (charged) intermediates, which are poorly bioavailable. It has been shown that fluorinated DPMs (6-fluoro- and 6,6,6-trifluoro-5-diphosphomevalonate) are excellent inhibitors of the bacterial pathway; however, highly charged DPM and analogues are not bioavailable. To increase cellular permeability of mevalonate analogues, we have synthesized various prodrugs of mevalonate and 6-fluoro- and 6,6,6-trifluoromevalonate that can be enzymatically transformed to the corresponding DPM or fluorinated DPM analogues by esterases or amidases. To probe the required stabilities as potentially bioavailable prodrugs, we measured the half-lives of esters, amides, carbonates, acetals, and ketal promoieties of mevalonate and the fluorinated mevalonate analogues in human blood plasma. Stability studies showed that the prodrugs are converted to the mevalonates in human plasma with a wide range of half-lives. These studies provide stability data for a variety of prodrug options having varying stabilities and should be very useful in the design of appropriate prodrugs of mevalonate and fluorinated mevalonates.

Background: Serotype M4 group A Streptococcus lack hyaluronic acid (HA) capsule, but are capable of causing human disease. Results: Encapsulation was achieved by introducing the hasABC capsule synthesis operon in the absence of HA-degrading enzyme hyaluronate lyase (HylA). Conclusion: Capsule expression does not enhance M4 GAS virulence. Significance: We demonstrate a mutually exclusive interaction between GAS capsule and HylA expression., A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.

SummaryGroup A Streptococcus (GAS) is a leading cause of infection-related mortality in humans. All GAS serotypes express the Lancefield group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N-acetylglucosamine (GlcNAc) side chain, which is the basis of rapid diagnostic tests. No biological function has been attributed to this conserved antigen. Here we identify and characterize the GAC biosynthesis genes, gacA through gacL. An isogenic mutant of the glycosyltransferase gacI, which is defective for GlcNAc side-chain addition, is attenuated for virulence in two infection models, in association with increased sensitivity to neutrophil killing, platelet-derived antimicrobials in serum, and the cathelicidin antimicrobial peptide LL-37. Antibodies to GAC lacking the GlcNAc side chain and containing only polyrhamnose promoted opsonophagocytic killing of multiple GAS serotypes and protected against systemic GAS challenge after passive immunization. Thus, the Lancefield antigen plays a functional role in GAS pathogenesis, and a deeper understanding of this unique polysaccharide has implications for vaccine development.

All strains of group A Streptococcus (GAS), a leading cause of infection-related mortality worldwide, express group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N...

Group A Streptococcus (GAS) is a leading human pathogen, causing 650,000 severe invasive diseases annually with 25% mortality. M1 GAS is the most frequently isolated serotype from invasive disease worldwide; however, a recent occurrence of invasive infections in Australia was attributed to M4 GAS. Here, we use a combined molecular genetics and bioinformatics approach to characterize 17 clinical M4 isolates associated with this invasive disease episode. The M4 isolates were classified into 3 pulsed-field gel electrophoresis groups, and 2 multilocus sequence types. All isolates lacked hyaluronic acid (HA) capsule, a major antiphagocytic virulence factor, and whole genome sequence analysis of 2 isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Most isolates contained mutations within the covRS two-component regulator, a hallmark of hyperinvasive GAS. Transformation of a plasmid expressing hasABC into M4 GAS restored capsule production. However, encapsulation neither promoted s...

Streptococcus pyogenes is an important human pathogen, which has recently gained recognition as an intracellular microorganism during the course of severe invasive infections such as necrotizing fasciitis. Although the surface anchored M protein has been identified as a pivotal factor affecting phagosomal maturation and S. pyogenes survival within macrophages, the overall transcriptional profile required for the pathogen to adapt and persist intracellularly is as of yet unknown. To address this, the gene expression profile of S. pyogenes within human macrophages was determined and compared to that of extracellular bacteria using customized microarrays and real-time qRT-PCR. In order to model the early phase of infection involving adaptation to the intracellular compartment, samples were collected 2h post-infection. Microarray analysis revealed that the expression of 145 streptococcal genes was significantly altered in the intracellular environment. The majority of differentially regulated genes were associated with metabolic and energy-dependent processes. Key up-regulated genes in early phase intracellular bacteria were ihk and irr, encoding a two-component gene regulatory system (TCS). Comparison of gene expression of selected genes at 2h and 6h post-infection revealed a dramatic shift in response regulators over time with a down-regulation of ihk/irr genes concurring with an up-regulation of the covR/S TCS. In re-infection assays, intracellular bacteria from the 6h time point exhibited significantly greater survival within macrophages than did bacteria collected at the 2h time point. An isogenic S. pyogenes mutant deficient in ihk/irr displayed significantly reduced bacterial counts when compared to wild-type bacteria following infection of macrophages. The findings illustrate how gene expression of S. pyogenes during the intracellular life cycle is fine-tuned by temporal expression of specific two-component systems.

Delineating structures of the transition states in protein folding reactions has provided great insight into the mechanisms by which proteins fold. The most common method for obtaining this information is Φ-value analysis, which is carried out by measuring the changes in the folding and unfolding rates caused by single amino acid substitutions at various positions within a given protein. Canonical Φ-values range between 0 and 1, and residues displaying high values within this range are interpreted to be important in stabilizing the transition state structure, and to elicit this stabilization through native-like interactions. Although very successful in defining the general features of transition state structures, Φ-value analysis can be confounded when non-native interactions stabilize this state. In addition, direct information on backbone conformation within the transition state is not provided. In the work described here, we have investigated structure formation at a conserved β-bulge (with helical conformation) in the Fyn SH3 domain by characterizing the effects of substituting all natural amino acids at one position within this structural motif. By comparing the effects on folding rates of these substitutions with database-derived local structure propensity values, we have determined that this position adopts a non-native backbone conformation in the folding transition state. This result is surprising because this position displays a high and canonical Φ-value of 0.7. This work emphasizes the potential role of non-native conformations in folding pathways and demonstrates that even positions displaying high and canonical Φ-values may, nevertheless, adopt a non-native conformation in the transition state. Proteins 2012;. © 2012 Wiley Periodicals, Inc.

Group B Streptococcus (GBS) is an important human pathogen and a model system for studying the roles of bacterial glycosylation in host-microbe interactions. Sialic acid (Sia), expressed prominently in the GBS capsular polysaccharide (CPS), mimics mammalian cell surface Sia and can interact with host Sia-binding proteins to subvert immune clearance mechanisms. Our earlier work has shown that GBS partially O-acetylates CPS Sia residues and employs an intracellular O-acetylation/de-O-acetylation cycle to control the final level of this surface Sia modification. Here, we examine the effects of point mutations in the NeuD O-acetyltransferase and NeuA O-acetylesterase on specific glycosylation phenotypes of GBS, pinpointing an isogenic strain pair that differs dramatically in the degree of the O-acetyl modification (80% versus 5%) while still expressing comparable levels of overall sialylation. Using these strains, higher levels of O-acetylation were found to protect GBS CPS Sia against enzymatic removal by microbial sialidases and to impede engagement of human Siglec-9, but not to significantly alter the ability of GBS to restrict complement C3b deposition on its surface. Additional experiments demonstrated that pH-induced migration of the O-acetyl modification from the 7- to 9-carbon position had a substantial impact on GBS-Siglec-9 interactions, with 7-O-acetylation exhibiting the strongest interference. These studies show that both the degree and position of the GBS O-acetyl modification influence Sia-specific interactions relevant to the host-pathogen relationship. We conclude that native GBS likely expresses a phenotype of intermediate Sia O-acetylation to strike a balance between competing selective pressures present in the host environment.

Beta-lactam antibiotics (BLAs) are the first-line agents used against group B streptococci (GBS) infection. A clonal set of four independent, invasive GBS isolates with elevated MICs to BLAs were identified that shared a pbp2x mutation (Q557E) corresponding to a resistance-conferring pneumococcal mutation. BLA sensitivity was restored through allelic replacement or complementation with the wild-type pbp2x .